WO2015127872A1 - 2,4-二取代苯-1,5-二胺衍生物及其应用以及由其制备的药物组合物和药用组合物 - Google Patents

2,4-二取代苯-1,5-二胺衍生物及其应用以及由其制备的药物组合物和药用组合物 Download PDF

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WO2015127872A1
WO2015127872A1 PCT/CN2015/073044 CN2015073044W WO2015127872A1 WO 2015127872 A1 WO2015127872 A1 WO 2015127872A1 CN 2015073044 W CN2015073044 W CN 2015073044W WO 2015127872 A1 WO2015127872 A1 WO 2015127872A1
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compound
formula
mmol
reaction
methyl
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PCT/CN2015/073044
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English (en)
French (fr)
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兰炯
金云舟
周福生
谢婧
沈思达
胡毅
刘伟
吕强
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上海海雁医药科技有限公司
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Priority to KR1020167026439A priority Critical patent/KR101904632B1/ko
Priority to SG11201606615QA priority patent/SG11201606615QA/en
Priority to US15/121,320 priority patent/US10435400B2/en
Priority to MX2016011112A priority patent/MX2016011112A/es
Priority to AU2015222584A priority patent/AU2015222584B2/en
Priority to CA2940488A priority patent/CA2940488C/en
Priority to EP15754829.8A priority patent/EP3112364B1/en
Priority to CN201580002426.3A priority patent/CN105934432B/zh
Application filed by 上海海雁医药科技有限公司 filed Critical 上海海雁医药科技有限公司
Priority to NZ723151A priority patent/NZ723151A/en
Priority to JP2016554670A priority patent/JP6267806B2/ja
Priority to RU2016137615A priority patent/RU2649001C1/ru
Publication of WO2015127872A1 publication Critical patent/WO2015127872A1/zh
Priority to IL247227A priority patent/IL247227A0/en
Priority to PH12016501693A priority patent/PH12016501693A1/en

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    • 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
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    • A61K31/5375—1,4-Oxazines, e.g. morpholine
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    • C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
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    • C07D401/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
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Definitions

  • the present invention relates to the field of medical technology, and in particular to a 2,4-disubstituted phenyl-1,5-diamine derivative and its use as an EGFR tyrosine kinase inhibitor, and pharmaceutical compositions and medicaments prepared therefrom Use the composition.
  • Lung cancer is the world's highest incidence of cancer. It ranks first among all cancers in China. 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%, and such patients are more sensitive to EGFR inhibitors.
  • the first-generation EGFR inhibitors such as erlotinib have been marketed, and gefitinib is effective in such patients, which can reduce tumors in more than 60% of patients and significantly prolong the progression-free survival of patients.
  • the second-generation irreversible pan-EGFR inhibitor (Afatinib (BIBW2992)) currently on the market 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.
  • An object of the present invention is to provide a 2,4-disubstituted phenyl-1,5-diamine derivative, or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof; and the above derivative Pharmaceutical composition of the substance.
  • a second object of the present invention is to provide use of the above derivative or composition for the preparation of a medicament for modulating EGFR tyrosine kinase activity or treating an EGFR-related disease.
  • a first aspect of the invention provides a compound of formula (I), or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof:
  • A is any one selected from the group consisting of:
  • Z 2 is CR 5 or N; R 5 is hydrogen, halogen, trifluoromethyl or C 1-10 alkoxy; Z 21 is N or CH; and R 4 , R 42 and R 43 are each independently hydrogen.
  • R 41 is hydrogen, halogen, three Fluoromethyl, C 1-10 alkyl or C 1-10 alkoxy; (preferably, R 4 , R 42 , R 43 are each independently hydrogen, methyl or ethyl; R 41 is hydrogen, chlorine, Fluorine, trifluoromethyl, methyl or methoxy;)
  • R 1 ', R 2 ' and R 5 ' are each independently hydrogen, C 1-10 alkyl; (preferably, R 1 ', R 2 ' And R 5 ' are each independently hydrogen, methyl or ethyl;)
  • R 3 ', R 4 ' are each independently hydrogen.
  • R 0 is hydrogen, halogen, C 1-10 alkyl, -NR a R b or -CO-NR a R b , wherein R a and R b are each independently a C 1-10 alkyl group;
  • Z 1 is CR 6 or N; wherein R 6 is hydrogen, halogen, trifluoromethyl, methoxy or -CO 2 C 1-10 alkyl;
  • R 1 is hydrogen or selected from any of the following groups:
  • R 2 and R 3 are each independently hydrogen or -CH 2 NR 7 R 8 ; wherein (1) R 7 and R 8 are each independently hydrogen or methyl; or (2) R 7 , R 8 and are attached The nitrogen atoms together form a 5-6 membered nitrogen-containing saturated heterocyclic ring;
  • R 12 is hydrogen, halogen, C 1-10 alkyl or C 1-10 alkoxy (preferably methoxy).
  • A is any one selected from the group consisting of:
  • R 4 , R 41 , R 42 , R 43 , R 1 ', R 2 ', R 3 ', R 4 ', R 5 ', R a ' are as defined in the formula (I).
  • R 4 is hydrogen, methyl, ethyl, isopropyl, cyclopropyl, difluoromethyl or piperidinyl; and R 41 is hydrogen, chlorine, fluorine, Trifluoromethyl, methyl or methoxy; R 42 is hydrogen, methyl or ethyl; R 43 is hydrogen, methyl or ethyl.
  • R 2 and R 3 are each independently hydrogen; and R 12 is a methoxy group.
  • Z 1 is CR 6 or N; wherein R 6 is hydrogen, fluorine, chlorine, trifluoromethyl, methoxy or -CO 2 C 1-10 alkyl ;
  • Z 1 is CR 6 , wherein R 6 is hydrogen, fluorine, chlorine, trifluoromethyl, methoxy or -CO 2 CH 3 .
  • R 0 is hydrogen
  • A is any one selected from the group consisting of:
  • R 2 and R 3 are each independently hydrogen.
  • R 2 and R 3 are each independently hydrogen or -CH 2 NR 7 R 8 , and R 7 and R 8 are each independently hydrogen or methyl.
  • R 2 and R 3 are each independently hydrogen or -CH 2 NR 7 R 8 , and R 7 , R 8 and the attached nitrogen atom together form a 5-6 member. A nitrogen saturated heterocyclic ring.
  • R 2 and R 3 are each independently hydrogen or -CH 2 NR 7 R 8 , and R 7 , R 8 and the adjacent nitrogen atom together form 5-6.
  • the nitrogen-containing saturated heterocyclic ring, the structure of the 5-6 membered nitrogen-containing saturated heterocyclic ring is as shown in the formula (III):
  • X is O, S, NR 9 or CR 10 R 11 ; wherein R 9 , R 10 and R 11 are each independently hydrogen, C 1-10 alkoxy or -CO-C 1-10 alkyl .
  • the compound of formula (I) is a compound of formula (IV-1):
  • R 0 , R 1 , R 2 , R 3 and R 6 are as defined in formula (I).
  • the compound of the formula (I) is a compound of the formula: (IV-2):
  • R 0 , R 1 , R 2 and R 3 are as defined in formula (I).
  • the compound represented by the formula (I) is a compound represented by the formula (V-1):
  • R 1 , R 2 , R 3 , R 4 , R 41 and R 6 are as defined in formula (I).
  • the compound represented by the formula (I) is a compound represented by the formula (V-2):
  • R 1 , R 2 , R 3 , R 4 and R 41 are as defined in formula (I).
  • the compound represented by the formula (I) has a structure as shown in the formula (VI-1):
  • R 1 , R 2 , R 3 , R 4 , R 41 and R 6 are as defined in formula (I).
  • the compound represented by the formula (I) has a structure as shown in the formula (VI-2):
  • R 1 , R 2 , R 3 , R 4 and R 41 are as defined in formula (I).
  • the compound represented by the formula (I) has a structure as shown in the formula (VII-1):
  • R 1 , R 2 , R 3 and R 6 are as defined in formula (I).
  • the compound represented by the formula (I) has a structure as shown in the formula (VII-2):
  • R 1 , R 2 and R 3 are as defined in formula (I).
  • the compound of the formula (I) is a compound of the formula: (VIII-1):
  • R 1 ', R 2 ', R 3 ', R 4 ', R 5 ', R 1 , R 2 , R 3 , R 6 and Y are as defined in the formula (I).
  • the compound represented by the formula (I) is a compound represented by the formula (VIII-2):
  • R 1 ', R 2 ', R 3 ', R 4 ', R 5 ', R 1 , R 2 , R 3 and Y are as defined in the formula (I).
  • the compound represented by the formula (I) has a structure as shown in the formula (IX-1):
  • R 1 ', R 2 ', R 4 ', R 5 ', R 1 , R 2 , R 3 and R 6 are as defined in the formula (I).
  • the compound represented by the formula (I) has a structure as shown in the formula (IX-2):
  • R 1 ', R 2 ', R 4 ', R 5 ', R 1 , R 2 and R 3 are as defined in formula (I).
  • the compound represented by the formula (I) is a compound represented by the formula (X-1):
  • R 1 ', R 5 ', R 1 , R 2 , R 3 and R 6 are as defined in formula (I).
  • the compound represented by the formula (I) is a compound represented by the formula (X-2):
  • R 1 ', R 5 ', R 1 , R 2 and R 3 are as defined in formula (I).
  • the compound represented by the formula (I) is a compound represented by the formula (XI-1):
  • R 1 , R 2 , R 3 , R 4 , R 42 , R 43 and R 6 are as defined in the formula (I).
  • the compound represented by the formula (I) is a compound represented by the formula (XI-2):
  • R 1 , R 2 , R 3 , R 4 , R 42 and R 43 are as defined in formula (I).
  • the compound represented by the formula (I) is a compound represented by the formula (XII-1):
  • R 1 , R 2 , R 3 , R 4 , R 41 and R 6 are as defined in formula (I).
  • the compound represented by the formula (I) is a compound represented by the formula (XII-2):
  • R 1 , R 2 , R 3 , R 4 and R 41 are as defined in formula (I).
  • R 6 is hydrogen, fluorine, chlorine or trifluoromethyl.
  • R 2 and R 3 are each independently hydrogen.
  • R 1 is selected from the group consisting of:
  • R 4 , R 42 and R 43 are each independently hydrogen, C 1-10 alkyl, halogenated C 1- 10 alkyl or piperidinyl;
  • R 4 , R 42 and R 43 are each independently hydrogen, methyl, ethyl, difluoromethyl or piperidine. base.
  • R 4 is hydrogen, methyl, difluoromethyl or piperidinyl; and R 42 and R 43 are each independently hydrogen. Or methyl.
  • the compound of the formula (V-1), the formula (V-2), the formula (VI-1), the formula (VI-2), the formula (XII-1) or the formula (XII-2) R 4 is hydrogen, C 1-10 alkyl, C 3-10 cycloalkyl or halogenated C 1-10 alkyl.
  • the compound of the formula (V-1), the formula (V-2), the formula (VI-1), the formula (VI-2), the formula (XII-1) or the formula (XII-2) R 4 is hydrogen, methyl, ethyl, isopropyl, cyclopropyl or difluoromethyl.
  • R 41 is hydrogen, chlorine, fluorine, trifluoromethyl, methyl or methoxy.
  • the compound of the formula (V-1), the formula (V-2), the formula (VI-1), the formula (VI-2), the formula (XII-1) or the formula (XII-2) R 2 and R 3 are each independently hydrogen.
  • R 6 is hydrogen, fluorine, chlorine, trifluoromethyl, methoxy or -CO 2 CH 3 .
  • R 1 is selected from:
  • the compound is selected from the group consisting of:
  • a second aspect of the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the compound of the first aspect of the invention, or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof, and further comprising a pharmaceutically acceptable Accepted carrier.
  • the pharmaceutical composition comprises a therapeutically effective amount of a compound of the first aspect of the invention, such as a compound of formula (I) above, a compound of formula (IV-1), a compound of formula (IV-2) a compound of the formula (V-1), a compound of the formula (V-2), a compound of the formula (VI-1), a compound of the formula (VI-2), a compound of the formula (VII-1), a compound of the formula (VII-2), and a formula (VIII-1) a compound, a compound of the formula (VIII-2), a compound of the formula (IX-1), a compound of the formula (IX-2), a compound of the formula (X-1), a compound of the formula (X-2), and a formula (XI) A compound, or a compound of the formula (XI-2), or an exemplified compound described above, or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof; and a pharmaceutically acceptable carrier.
  • a compound of the invention can be administered in a suitable dosage form with one or more pharmaceutical carriers.
  • These 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. Active compound can be used with The carrier forms a solution or suspension.
  • 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 third aspect of the invention provides the compound of the first aspect of the invention, or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof, for use in the preparation of a modulator of EGFR tyrosine kinase activity or treatment of EGFR The application of drugs for related diseases.
  • the EGFR-related disease is cancer, diabetes, immune system disease, neurodegenerative disease or cardiovascular disease.
  • 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 glioblastoma and the like, or any combination thereof.
  • 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
  • the compound, or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof can also be used to prepare a disease for the treatment of aberrant expression of EGFR or to have acquired resistance during treatment with an EGFR modulator.
  • the application of drugs for medicinal diseases can also be used to prepare a disease for the treatment of aberrant expression of EGFR or to have acquired resistance during treatment with an EGFR modulator.
  • 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.
  • an EGFR modulator refers to a small molecule tyrosine kinase inhibitor that targets EGFR, such as gefitinib, erlotinib, ectinib, lapatinib or afatinib.
  • a fourth aspect of the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of a compound of the first aspect of the invention, or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof And one or more other drugs 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, nicotine Zumuzumab, Zalumuzumab, Pertuzumab, MDX-214, CDX-110, IMC-11F8, Zemab, Her2 Vaccine PX 1041, HSP90 Inhibitor, CNF2024
  • terapéuticaally effective amount refers to an amount that is functional or active to a human and/or animal and that is acceptable to humans and/or animals.
  • the therapeutically effective amount of the compound of the present invention, or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof, contained in the pharmaceutical composition of the present invention or the pharmaceutical composition is preferably 0.1 mg to 5 g. /kg (weight).
  • the pharmaceutical composition of the present invention can be used for the treatment of EGFR-related diseases such as cancer, diabetes, immune system diseases, neurodegenerative diseases or cardiovascular diseases, or diseases having acquired resistance during treatment with EGFR modulators.
  • EGFR-related diseases such as cancer, diabetes, immune system diseases, neurodegenerative diseases or cardiovascular diseases, or diseases having acquired resistance during treatment with EGFR modulators.
  • the acquired drug-resistant disease is caused by a T790 mutation encoded by EGFR exon 20 or by a T790 mutation encoded by EGFR exon 20.
  • the EGFR exon 20 encodes a T790 of T790M.
  • the compounds of the invention may be used in combination with other drugs in certain diseases to achieve the desired therapeutic effect.
  • An example of a joint application is to treat advanced NSCLC.
  • a therapeutically effective amount of a compound of the formula I according to the invention is used in combination with an mTOR inhibitor (for example rapamycin); or with a Met inhibitor (including the Met antibody MetMAb and the Met small molecule inhibitor PF02341066); Used in combination with an IGF1R inhibitor (eg, OSI-906); or in combination with a heat shock protein inhibitor.
  • the inventors unexpectedly discovered a class of selective inhibitors of EGFR mutations.
  • In vitro experiments showed that it inhibited the proliferation of EGFR T790M/L858R double mutant enzyme and cell line H1975 at nanomolar concentrations.
  • the EGFR-sensitive mutant cell line HCC827 (exon 19 deletion) also had a high inhibitory intensity, while the inhibition of the wild-type EGFR enzyme and the 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.
  • these compounds have low cytotoxicity in normal cell lines (such as 3T3 cells), which greatly reduces non-specific side effects and is an ideal replacement for second-generation EGFR-TKI.
  • C 1-10 alkyl refers to a straight or branched saturated aliphatic hydrocarbon group having from 1 to 10 carbon atoms.
  • C 1-10 alkoxy refers to C 1-10 alkyl-O-.
  • halogen refers to fluoro, chloro, bromo or iodo.
  • C 3-10 cycloalkyl refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent having from 3 to 10 carbon atoms, preferably a cycloalkyl group containing from 3 to 8 carbon atoms (C 3-8 cycloalkyl), most preferably the cycloalkyl group contains 3 to 6 carbon atoms (C 3-6 cycloalkyl).
  • Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptene
  • the alkenyl group, the cyclooctyl group and the like are preferably a cyclopropyl group, a cyclopentyl group or a cyclohexenyl group.
  • Polycyclic cycloalkyl groups include spiro, fused, and bridged cycloalkyl groups.
  • C 3-10 heterocycloalkyl refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent comprising from 3 to 10 ring atoms, wherein one or more ring atoms are selected from nitrogen a hetero atom of oxygen or S(O) t (wherein t is an integer from 0 to 2), but excluding the ring moiety of -OO-, -OS- or -SS-, the remaining ring atoms being carbon.
  • It preferably comprises from 3 to 8 ring atoms, wherein from 1 to 3 are heteroatoms, more preferably the heterocycloalkyl group contains from 3 to 6 ring atoms, more preferably the heterocycloalkyl group contains from 5 to 6 ring atoms.
  • monocyclic heterocyclic groups include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, pyranyl, tetrahydrofuranyl and the like.
  • Polycyclic heterocyclic groups include spiro, fused, and bridged heterocyclic groups.
  • halogenated C1-10 alkyl refers to an alkyl group substituted with 1, 2, 3, 4 or 5 halo, wherein alkyl is as defined above.
  • a halogenated C 1-10 alkyl group is preferred, a halogenated C 1-6 alkyl group is more preferred, and a halogenated C 1-3 alkyl group is most preferred.
  • 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 a significant resistance to EGFR T790M mutation (especially EGFR T790M/L858R double mutation) 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 methods of preparation of these compounds can include, but are not limited to, the procedures described below.
  • 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.
  • Step 1 The chlorine at the 4-position of the pyrimidine is reacted with a boronic acid ester or boric acid, or a suitable amine, and can be carried out at a certain temperature using a suitable catalyst and a suitable solvent.
  • a suitable catalyst can be, but is not limited to, PdCl 2 (dppf)
  • the base used can be, but is not limited to, sodium carbonate.
  • Step 2 The chlorine at the 2-position of the pyrimidine is substituted with an amine, and it can be carried out at a certain temperature using a suitable catalyst and 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.
  • Step 3 The fluorine at the 4-position of the phenyl group is substituted with an amine, and it can be carried out at a certain temperature using a suitable base and a suitable solvent.
  • the base used may be, but not limited to, potassium carbonate.
  • Step 4 conversion of the nitro compound to the corresponding amine compound can be carried out under acidic conditions with a metal (may be, but not limited to, iron powder, zinc powder) or stannous chloride; or under palladium carbon catalysis, Hydrogen reduction.
  • a metal may be, but not limited to, iron powder, zinc powder
  • stannous chloride or under palladium carbon catalysis, Hydrogen reduction.
  • Step 5 The amine compound 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 (I) and the preparation method, the drug composition and the treatment plan of the present invention can be realized by those skilled in the art by referring to the contents of the present invention and appropriately improving the process parameters. It is to be understood that all such alternatives and modifications are obvious to those skilled in the art and are considered to be included in the present invention.
  • the products, methods and applications of the present invention have been described in terms of the preferred embodiments, and it is obvious that the methods and applications described herein may be modified or appropriately modified and combined without departing from the spirit, scope and scope of the invention.
  • the techniques of the present invention are implemented and applied.
  • 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).
  • the structure and purity of the compound are determined by nuclear magnetic resonance (1H NMR) and/or liquid chromatography-mass spectrometry (LC-MS).
  • 1H NMR Bruker AVANCE-400 nuclear magnetic instrument with internal standard tetramethylsilane (TMS).
  • LC-MS Agilent 1200 HPLC System/6140 MS LC/MS (available from Agilent), column Waters X-Bridge, 150 x 4.6 mm, 3.5 [mu]m.
  • Preparative High Performance Liquid Chromatography pre-HPLC: 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.
  • the specifications for the board are 0.4mm-0.5mm.
  • silica gel Yantai Huanghai silica gel 200-300 mesh silica gel is generally used as a carrier.
  • the basic alumina column is generally prepared by using FCP200-300 mesh basic alumina as a carrier.
  • the known starting materials of the present invention can be synthesized by or according to methods known in the art, or can be used in ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, Accela ChemBio Inc and Dari Chemical Products and other companies to buy.
  • the reactions were all carried out under a nitrogen or argon atmosphere.
  • the solution means an aqueous solution.
  • DMF means dimethylformamide
  • DMSO means dimethyl sulfoxide
  • THF means tetrahydrofuran
  • DIEA means N,N-diisopropylethylamine
  • EA means ethyl acetate
  • PE means petroleum ether.
  • BINAP stands for (2R,3S)-2,2'-bisdiphenylphosphino-1,1'-binaphthyl
  • NBS stands for N-bromosuccinimide
  • NCS stands for N-chlorosuccinimide
  • Pd 2 (dba) 3 represents tris(dibenzylideneacetone)dipalladium
  • Pd(dppf)Cl 2 represents [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride.
  • room temperature refers to about 25 °C.
  • Step a Compound 1a1 (10.6 g, 58 mmol) was placed in a 500 mL reaction flask, and dissolved in a mixed solution of THF/water (100 mL / 60 mL). Ammonium chloride (15.5 g, 292 mmol) and reduced iron powder (26 g, 467 mmol) were successively added thereto with stirring at room temperature, and then the reaction system was heated to 65 ° C and stirring was continued for 3 h. The progress of the reaction was checked by TLC. After the reaction was completed, excess iron powder was removed by filtration, and the filter cake was rinsed three times with EA.
  • Step b Compound 1a2 (8.0 g, 43 mmol) was placed in a 500 mL reaction flask, and concentrated sulfuric acid (100 mL) was added to dissolve the substrate. Concentrated nitric acid (6.15 mL, 48 mmol) was slowly added dropwise with stirring at minus 20 ° C, and stirred for 5 min. The progress of the reaction was detected by TLC, and after the reaction was completed, it was poured into ice water. The ice bath was kept at minus 20 ° C, and a sodium hydroxide/water solution (150 mL / 300 mL) was slowly added to the reaction system to adjust the pH to 8-9. The reaction mixture was extracted with EA/water system three times.
  • Step 2 Compound 2a2 (11.46 g, 40 mmol) was dissolved in 60 mL of N,N-dimethylacetamide and N,N,N'-trimethylethylenediamine (4.90 g, 48 mmol), N,N- Diisopropylethylamine (7.74 g, 60 mmol) was heated to 90 ° C and stirred for 6 h. The reaction progress by TLC. After completion of the reaction, the reaction solution was cooled to room temperature, poured into ice water, extracted with ethyl acetate, washed with water, brine, dried over anhydrous Na 2 SO 4, and concentrated under reduced pressure to obtain the target product Compound 2A3 ( 12.51g, 85%). Used directly in the next step. MS m/z (ESI): 369 [M+H] + .
  • Step 3 Compound 2a3 (12 g, 32.6 mmol) was dissolved in 200 mL of methanol and 1.0 g of 10% Pd/C. After replacing the air with hydrogen, the hydrogen gas was hydrogenated at room temperature, and the reaction was stirred for 1 h. The progress of the reaction was checked by TLC. After completion of the reaction, the mixture was filtered with a celite funnel, and the filter cake was washed with a small amount of methanol, and the filtrate was concentrated to give the title compound 2a4 (10.70 g, 97%). Used directly in the next step. MS m/z (ESI): 339 [M+H] + .
  • Step 4 Compound 2a4 (10.1 g, 30 mmol) and triethylamine (6.12 g, 60 mmol) were dissolved in dichloromethane (200 mL), cooled to 0 ° C, acryloyl chloride (3.24 g, 36 mmol). Stir for 3 h. TLC was used to detect the progress of the reaction. After the reaction was completed, the mixture was washed successively with saturated aqueous sodium hydrogen carbonate solution and brine, dried over anhydrous Na 2 SO 4 , filtered, and concentrated under reduced pressure to give the title compound 2a5 (9.64 g, 82% ). Used directly in the next step. MS m/z (ESI): 393 [M+H] + .
  • Step 2 1-Methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo[2,3-b Pyridine (compound 1-3)
  • Step 4 5-Chloro-N-(4-fluoro-2-methoxy-5-nitrophenyl)-4-(1-methyl-1H-pyrrolo[2,3-b]pyridine-5 -yl)pyrimidin-2-amine (compounds 1-5)
  • Step 5 N 1 -(5-chloro-4-(1-methyl-1H-pyrrolo[2,3-b]pyridin-5-yl)pyrimidin-2-yl)-N 4 -(2-( Dimethylamino)ethyl)-2-methoxy-N 4 -methyl-5-nitrophenyl-1,4-diamine (compounds 1-6)
  • Step 6 N 4 -(5-chloro-4-(1-methyl-1H-pyrrolo[2,3-b]pyridin-5-yl)pyrimidin-2-yl)-N 1 -(2-( Dimethylamino)ethyl)-5-methoxy-N 1 -methylphenyl-1,2,4-triamine (compound 1-7)
  • Step 7 N-(5-(5-Chloro-4-(1-methyl-1H-pyrrolo[2,3-b]pyridin-5-yl)pyrimidin-2-ylamino)-2-(( 2-(Dimethylamino)ethyl)methylamino)-4-methoxyphenyl)propanamide (Compound Z-1)
  • Step 1 5-Chloro-N-(4-(4-(dimethylamino)piperidin-1-yl)-2-methoxy-5-nitrophenyl)-4-(1-methyl- 1H-pyrrolo[2,3-b]pyridin-5-yl)pyrimidin-2-amine (Compound 2-6)
  • Step 2 N 1 -(5-chloro-4-(1-methyl-1H-pyrrolo[2,3-b]pyridin-5-yl)pyrimidin-2-yl)-4-(4-(two Methylamino)piperidin-1-yl)-6-methoxyphenyl-1,3-diamine (Compound 2-7)
  • reaction substrate 2-6 110 mg, 0.205 mmol
  • ethanol 6 mL
  • Stannous chloride 138 mg, 0.615 mmol
  • TLC was used to detect the progress of the reaction.
  • the reaction solution was concentrated to dryness and extracted three times with EA/water system. The organic layer was separated, washed with water, washed with saturated brine and dried over anhydrous Na 2 SO 4 . The filtrate was concentrated under reduced pressure to give crude crystals (yield: ⁇ /RTI>
  • Step 3 N-(5-(5-Chloro-4-(1-methyl-1H-pyrrolo[2,3-b]pyridin-5-yl)pyrimidin-2-yl)-2-(4) -(Dimethylamino)piperidin-1-yl)-4-methoxyphenyl)acrylamide (Compound Z-2)
  • N-methylpiperazine (126 mg, 1.26 mmol) and potassium carbonate (261 mg, 1.89 mmol) were added to compound 1-5 (200 mg, 0.47 mmol) in 4 mL of DMF and stirred vigorously at 100 ° C for 2 h. TLC was used to detect the progress of the reaction. After the reaction was completed, 10 mL of water was added, and the mixture was extracted three times with EA/water system. The organic layer was separated and concentrated under reduced pressure to give compound 3-6 (200 mg, 84%) MS m/z (ESI): 509.2 [M+H] + .
  • Step 2 N 1 -(5-chloro-4-(1-methyl-1H-pyrrolo[2,3-b]pyridin-5-yl)pyrimidin-2-yl)-6-methoxy-4 -(4-methylpiperazin-1-yl)phenyl-1,3-diamine (Compound 3-7)
  • Step 3 N-(5-(5-Chloro-4-(1-methyl-1H-pyrrolo[2,3-b]pyridin-5-yl)pyrimidin-2-ylamino)-4-methoxy 2-(4-methylpiperazin-1-yl)phenyl)acrylamide (compound Z-3)
  • reaction substrate 4-3 (3.8 g, 14.6 mmol) and 2,4-dichloropyrimidine (2.6 g, 17.5 mmol) were placed in a 250 mL reaction flask, and acetonitrile (130 mL) and sodium carbonate solution (22 mL, 2M) were added. . After the reaction system was replaced with argon three times with air, Pd(dppf)Cl 2 (0.55 g, 0.73 mmol) was added, and then the air was replaced with nitrogen three times. The reaction was heated to 85 ° C and stirring was continued for 3 h. LC-MS was used to detect the progress of the reaction. After the reaction was completed, it was cooled to room temperature, and a large amount of solid was precipitated.
  • Step 4 N-(4-Fluoro-2-methoxy-5-nitrophenyl)-4-(1-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl) Pyrimidin-2-amine (compound 4-5)
  • reaction substrate 4-4 (420 mg, 1.7 mmol) and 4-fluoro-2-methoxy-5-nitroaniline (316 mg, 1.7 mmol) were placed in a 100 mL sealed reaction flask, and isobutanol was added. 40 mL) partially dissolved the substrate. Then p-toluenesulfonic acid (807 mg, 4.25 mmol) was added, and the reaction system was kept under heating at 130 ° C for 16 h. After the reaction was completed, it was cooled to room temperature, and a large amount of solid was precipitated in the solution. The filter cake was filtered through a Buchner funnel and the residue was purified by refluxing to give the title compound 4-5 (524 mg, 78%). Purity: 47%. MS m/z (ESI): 396.2 [M+H] +
  • Step 5 N-(4-(4-(Dimethylamino)piperidin-1-yl)-2-methoxy-5-nitrophenyl)-4-(1-methyl-1H-pyridyl Zoxao[3,4-b]pyridin-5-yl)pyrimidin-2-amine (Compound 4-6)
  • reaction substrate 4-5 (146 mg, 0.25 mmol) and potassium carbonate (104 mg, 0.75 mmol) were placed in a 25 mL reaction flask, and DMF (10 mL) was added to dissolve the substrate completely. Then, 4-dimethylaminopiperidine (42 mg, 0.325 mmol) was added and the reaction was maintained at 100 ° C for 2 h. The reaction progress by TLC. After completion of the reaction, the reaction solution was extracted three times with EA / water, the organic layer was separated, washed with water, saturated brine, dried over Na 2 SO 4 dried, and concentrated under reduced pressure to give the title compound 4- 6 (120 mg, 98%). MS m/z (ESI): 504.2 [M+H] + .
  • Step 6 4-(4-(Dimethylamino)piperidin-1-yl)-6-methoxy-N 1 -(4-(1-methyl-1H-pyrazolo[3,4- b] Pyridin-5-yl)pyrimidin-2-yl)benzene-1,3-diamine (Compound 4-7)
  • Step 7 N-(2-(4-(Dimethylamino)piperidin-1-yl)-4-methoxy-5-(4-(1-methyl-1H-pyrazolo[3,4] -b]pyridine-5-yl) Pyrimidin-2-ylamino)phenyl)acrylamide (compound Z-4)
  • the compound 4-7 (113 mg, 0.24 mmol) was used as a starting material, and the crude product was subjected to the reversed step of step 7 in the same procedure.
  • Step 1 N-(2-Methoxy-4-(4-methylpiperazin-1-yl)-5-nitrophenyl)-4-(1-methyl-1H-pyrazolo[3 ,4-b]pyridin-5-yl)pyrimidin-2-amine (Compound 5-6)
  • Step 2 6-Methoxy-N 1 -(4-(1-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)pyrimidin-2-yl)-4-(4 -methylpiperazin-1-yl)phenyl-1,3-diamine (compound 5-7)
  • Step 3 N-(4-Methoxy-5-(4-(1-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)pyrimidin-2-ylamino)-2 -(4-methylpiperazin-1-yl)phenyl)acrylamide (Compound Z-5)
  • Step 1 N 1 -(2-(Dimethylamino)ethyl)-5-methoxy-N 1 -methyl-N 4 -(4-(1-methyl-1H-pyrazolo[3] ,4-b]pyridin-5-yl)pyrimidin-2-yl)-2-nitrophenyl-1,4-diamine (Compound 6-6)
  • Step 2 N 1 -(2-(Dimethylamino)ethyl)-5-methoxy-N 1 -methyl-N 4 -(4-(1-methyl-1H-pyrazolo[3] ,4-b]pyridin-5-yl)pyrimidin-2-yl)benzene-1,2,4-triamine (compound 6-7)
  • Step 3 N-(2-((2-(Dimethylamino)ethyl)(methyl)amino)-4-methoxy-5-(4-(1-methyl-1H-pyrazole) [3,4-b]pyridin-5-yl)pyrimidin-2-ylamino)phenyl)acrylamide (Compound Z-6)
  • Step 3 5-Chloro-N-(4-(4-(dimethylamino)piperidin-1-yl)-2-methoxy-5-nitrophenyl)-4-morpholinium pyrimidine-2- Amine (Compound 7-4)
  • Step 4 N-(5-Chloro-4-morpholinan-2-yl)-4-(4-(dimethylamino)piperidin-1-yl)-6-methoxybenzene-1,3- Diamine (compound 7-5)
  • Step 5 N-(5-((5-Chloro-4-morpholinan-2-yl)amino)-2-(4-(dimethylamino)piperidin-1-yl)-4-methoxybenzene Acrylamide (Compound Z-7) formate
  • Step 1 5-Chloro-N-(2-methoxy-4-(4-methylpiperazin-1-yl)-5-nitrophenyl)-4-morpholinium pyrimidine-2-amine (compound) 8-4)
  • Step 2 N-(5-Chloro-4-morpholinium-2-yl)-6-methoxy-4-(4-methylpiperazin-1-yl)benzene-1,3-diamine ( Compound 8-5)
  • Step 3 N-(5-((5-Chloro-4-morpholinan-2-yl)amino)-4-methoxy-2-(4-methylpiperazin-1-yl)phenyl) Acrylamide (Compound Z-8)
  • Step 1 N-(5-Chloro-4-morpholinimidin-2-yl)-N-(2-(dimethylamino)ethyl)-2-methoxy-N-methyl-5-nitro Benzene-1,4-diamine (compound 9-4)
  • Step 2 N-(5-Chloro-4-morpholinimidin-2-yl)-N-(2-(dimethylamino)ethyl)-5-methoxy-N-methylbenzene-1,2 , 4-triamine (compound 9-5)
  • Step 3 N-(5-((5-Chloro-4-morpholinan-2-yl)amino)-2-((2-(dimethylamino)ethyl)methylamino)-4-methoxy Phenyl)acrylamide (compound Z-9)
  • Step 2 N-(4-Fluoro-2-methoxy-5-nitrophenyl)-4-(quinolin-3-yl)pyrimidin-2-amine (Compound 10-3)
  • Step 5 N-(4-(3-(Dimethylamino)azetidin-1-yl)-2-methoxy-5-nitrophenyl)-4-(quinoline-3- Pyrimidine-2-amine (compound 10-7)
  • Step 6 4-(3-(Dimethylamino)azetidin-1-yl)-6-methoxy-N 1-(4-(quinolin-3-yl)pyrimidin-2-yl Benzene-1,3-diamine (compound 10-8)
  • Step 7 N-(2-(3-(Dimethylamino)azetidin-1-yl)-4-methoxy-5-((4-(quinolin-3-yl)pyrimidine- 2-yl)amino)phenyl)acrylamide (compound Z-10)
  • Step 1 (R)-N-(4-(3-(Dimethylamino)pyrrolidin-1-yl)-2-methoxy-5-nitrophenyl)-4-(quinoline-3 -yl)pyrimidin-2-amine (compound 11-7)
  • Step 2 (R)-4-(3-(Dimethylamino)pyrrolidin-1-yl)-6-methoxy-N 1 -(4-(quinolin-3-yl)pyrimidin-2- Benzo-1,3-diamine (compound 11-8)
  • Step 3 (R)-N-(2-(3-(Dimethylamino)pyrrolidin-1-yl)-4-methoxy-5-((4-(quinolin-3-yl)pyrimidine) -2-yl)amino)phenyl)acrylamide (Compound Z-11)
  • Step 1 3-(2-Chloropyrimidin-4-yl)quinoline (Compound 10-2, step 1 according to Example 10)
  • Step 5 4-(4-Amino-5-methoxy-2-nitrophenyl)-5,6-dihydropyridine-1(2H)-carboxylic acid tert-butyl ester (Compound 12-7)
  • Step 6 4-(5-Methoxy-2-nitro-4-((4-(quinolin-3-yl)pyrimidin-2-yl)amino)phenyl)-5,6-dihydropyridine -1(2H)-tert-butyl formate (Compound 12-8)
  • Step 7 N-(2-Methoxy-5-nitro-4-(1,2,3,6-tetrahydropyridin-4-yl)phenyl)-4-(quinolin-3-yl) Pyrimidin-2-amine (compound 12-9)
  • Step 8 N-(2-Methoxy-4-(1-methyl-1,2,3,6-tetrahydropyridin-4-yl)-5-nitrophenyl)-4-(quinoline- 3-yl)pyrimidin-2-amine (compound 12-10)
  • Step 9 6-Methoxy-4-(1-methyl-1,2,3,6-tetrahydropyridin-4-yl)-N 1 -(4-(quinolin-3-yl)pyrimidine- 2-yl)benzene-1,3-diamine (compound 12-11)
  • Step 10 N-(4-Methoxy-2-(1-methyl-1,2,3,6-tetrahydropyridin-4-yl)-5-((4-(quinolin-3-yl) Pyrimidine-2-yl)amino)phenyl)propanamide (Compound Z-12)
  • Step 2 N-(4-Fluoro-2-methoxy-5-nitrophenyl)-5-methoxy-4-(quinolin-3-yl)pyrimidin-2-amine (Compound 13-3 )
  • Step 3 N 1 -(2-(Dimethylamino)ethyl)-5-methoxy-N 4 -(5-methoxy-4-(quinolin-3-yl)pyrimidin-2-yl )-N-methyl-2-nitrobenzene-1,4-diamine (compound 13-4)
  • Step 4 N 1 -(2-(Dimethylamino)ethyl)-5-methoxy-N 4 -(5-methoxy-4-(quinolin-3-yl)pyrimidin-2-yl )-N 1 -methylbenzene-1,2,4-triamine (compound 13-5)
  • Step 5 N-(2-((2-(Dimethylamino)ethyl)(methyl)amino)-4-methoxy-5-(5-methoxy-4-(quinoline-3) -yl)pyrimidin-2-ylamino)phenyl)acrylamide (Compound Z-13)
  • Step 1 4-Chloro-N-(4-fluoro-2-methoxy-5-nitrophenyl)-1,3,5-triazin-2-amine (Compound 14-2)
  • Step 2 N-(4-Fluoro-2-methoxy-5-nitrophenyl)-4-(quinolin-3-yl)-1,3,5-triazin-2-amine (Compound 14 -3)
  • reaction substrate compound 14-2 (1.2 g, 4 mmol), 3-quinoline boronic acid (5.8 g, 34 mmol), potassium carbonate (1.11 g, 8 mmol) was placed in a 250 mL reaction flask, and ethylene glycol dimethyl ether was added ( 20 mL) and water (5 mL). After the reaction system was replaced with nitrogen three times with air, Pd(dppf)Cl 2 (300 mg, 0.4 mmol) was added, and then the air was replaced with nitrogen three times. The reaction system was heated to 100 ° C and stirring was continued for 4 h. The progress of the reaction was checked by TLC, and after completion of the reaction, it was concentrated under reduced pressure to give a crude material. Purification by Combi-Flash column chromatography gave the title compound 14-3 (700 mg, 45%). MS m/z (ESI): 393.1 [M+H] + .
  • Step 3 N 1 -(2-(Dimethylamino)ethyl)-5-methoxy-N 1 -methyl-2-nitro-N 4 -(4-(quinolin-3-yl) -1,3,5-triazin-2-yl)benzene-1,4-diamine (Compound 14-4)
  • reaction substrate compound 14-3 400 mg, 1 mmol
  • N,N,N'-trimethylethylenediamine 156 mg, 1.5 mmol
  • cesium carbonate 670 mg, 2 mmol
  • Methyl carboxamide 6 mL
  • TLC TLC was used to detect the progress of the reaction.
  • the reaction mixture was diluted with water and filtered to give a crude product which was purified by Combi-Flash column chromatography to afford compound 14-4 (200 mg, 42%).
  • Step 4 N 1 -(2-(Dimethylamino)ethyl)-5-methoxy-N-methyl-N 4 -(4-(quinolin-3-yl)-1,3,5 -triazin-2-yl)benzene-1,2,4-triamine (compound 14-5)
  • Step 5 N-(2-((2-(Dimethylamino)ethyl)(methyl)amino)-4-methoxy-5-(4-(quinolin-3-yl)-1, 3,5-triazin-2-yl Amino)phenyl)acrylamide (Compound Z-14)
  • Step 1 N-(4-(4-(Dimethylamino)piperidin-1-yl)-2-methoxy-5-nitrophenyl)-4-(quinolin-3-yl)- 1,3,5-triazin-2-amine (Compound 15-4)
  • Step 2 4-(4-(Dimethylamino)piperidin-1-yl)-6-methoxy-N 1 -(4-(quinolin-3-yl)-1,3,5-tri Pyrazin-2-yl)benzene-1,3-diamine (compound 15-5)
  • the compound 15-4 (100 mg, 0.2 mmol) was used as a starting material, and the compound was obtained by the step 6 of Example 1 to give the compound compound 15-5 (90 mg).
  • Step 3 N-(2-(4-(Dimethylamino)piperidin-1-yl)-4-methoxy-5-(4-(quinolin-3-yl)-1,3,5 -triazin-2-ylamino)phenyl)acrylamide (Compound Z-15)
  • the compound 15-5 (90 mg, 0.2 mmol) was used as a starting material, and the crude product obtained was purified by Prep-HPLC column chromatography to obtain the target compound Z-15 (2.09 mg, 2%, FA salt). Purity: 100.0%. MS m/z (ESI): 525.3 [M+H] + .
  • Step 2 N-(4-Fluoro-2-methoxy-5-nitrophenyl)-4-(1-methyl-1H-pyrrolo[2,3-b]pyridin-5-yl)pyrimidine -2-amine (compound 16-3)
  • Step 3 N 1 -(2-(Dimethylamino)ethyl)-5-methoxy-N-methyl-N 4 -(4-(1-methyl-1H-pyrrolo[2,3 -b]pyridin-5-yl)pyrimidin-2-yl)-2-nitrobenzene-1,4-diamine (compound 16-4)
  • Step 4 N 1- (2-(Dimethylamino)ethyl)-5-methoxy-N-methyl-N 4 -(4-(1-methyl-1H-pyrrolo[2,3 -b]pyridin-5-yl)pyrimidin-2-yl)benzene-1,2,4-triamine (compound 16-5)
  • Step 5 N-(2-((2-(Dimethylamino)ethyl)(methyl)amino)-4-methoxy-5-((4-(1-methyl-1H-pyrrole) [2,3-b]pyridin-5-yl)pyrimidin-2-yl)amino)phenyl)acrylamide (Compound Z-16)
  • Step 2 N-(4-Fluoro-2-methoxy-5-nitrophenyl)-4-(1-methyl-1H-pyrazol-4-yl)pyrimidin-2-amine (Compound 17- 3)
  • Step 3 N-(4-(4-(Dimethylamino)piperidin-1-yl)-2-methoxy-5-nitrophenyl)-4-(1-methyl-1H-pyridyl Zin-4-yl)pyrimidin-2-amine (Compound 17-4)
  • Step 4 4-(4-(Dimethylamino)piperidin-1-yl)-6-methoxy-N 1-(4-(1-methyl-1H-pyrazol-4-yl)pyrimidine -2-yl)benzene-1,3-diamine (compound 17-5)
  • the compound 17-4 (70 mg, 0.155 mmol) was used as a starting material to give the crude compound 17-5 (60 mg, 92%).
  • Step 5 N-(2-(4-(Dimethylamino)piperidin-1-yl)-4-methoxy-5-((4-(1-methyl-1H-pyrazole-4-) Pyrimido-2-yl)amino)phenyl)acrylamide (Compound Z-17)
  • Step 1 N 1 -(2-(Dimethylamino)ethyl)-5-methoxy-N-methyl-N 4-(4-(1-methyl-1H-pyrazol-4-yl) Pyrimidine-2-yl)-2-nitrobenzene-1,4-diamine (Compound 18-4)
  • Step 2 N 1 -(2-(Dimethylamino)ethyl)-5-methoxy-N-methyl-N 4-(4-(1-methyl-1H-pyrazol-4-yl) Pyrimidine-2-yl)benzene-1,2,4-triamine (compound 18-5)
  • the compound 18-4 (110 mg, 0.258 mmol) was used as a starting material, which was obtained by the step 6 of Example 1 to give the crude compound 18-5 (110 mg, 100%).
  • Step 3 N-(2-((2-(Dimethylamino)ethyl)(methyl)amino)-4-methoxy-5-((4-(1-methyl-1H-pyrazole) 4-yl)pyrimidin-2-yl)amino)phenyl)acrylamide (Compound Z-18)
  • the compound 18-5 (110 mg, 0.278 mmol) was used as a starting material, and the crude product was purified by preparative chromatography. The obtained product was purified by a preparative plate. The ratio of DCM:MeOH was 10:1. 18 (10.0 mg, 8.2%).
  • Step 3 N 1 -(5-chloro-4-(1-methyl-1H-pyrazol-4-yl)pyrimidin-2-yl)-N 4 -(2-(dimethylamino)ethyl) -2-methoxy-N 4 -methyl-5-nitrobenzene-1,4-diamine (compound 19-4)
  • Step 4 N 4 -(5-chloro-4-(1-methyl-1H-pyrazol-4-yl)pyrimidin-2-yl)-N 1 -(2-(dimethylamino)ethyl) -5-methoxy-N-methylbenzene-1,2,4-triamine (Compound 19-5)
  • Step 5 N-(5-((5-Chloro-4-(1-methyl-1H-pyrazol-4-yl)pyrimidin-2-yl)amino)-2-((2-(dimethyl) Amino)ethyl)(methyl)amino)-4-methoxyphenyl)acrylamide (Compound Z-19)
  • 2,4-Dichloro-5-fluoropyrimidine (2.0 g, 7.8 mmol), PdCl 2 (dppf) 2 (0.57 g, 0.78 mmol) and sodium carbonate (1.64 g, 15.6 mmol) were added to the compound 1-methyl -5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo[2,3-b]pyridine (2.0 g, 7.8 Methyl, commercially available) a mixed solution of 20 mL of acetonitrile and 4 mL of water. Stir vigorously for 2 h at 80 ° C under a nitrogen atmosphere.
  • Step 2 5-Fluoro-N-(4-fluoro-2-methoxy-5-nitrophenyl)-4-(1-methyl-1H-pyrrolo[2,3-b]pyridine-5 -yl)pyrimidin-2-amine (compound 20-3)
  • Step 3 N 1 -(2-(Dimethylamino)ethyl)-N 4 -(5-fluoro-4-(1-methyl-1H-pyrrolo[2,3-b]pyridine-5- Pyrimidin-2-yl)-5-methoxy-N-methyl-2-nitrobenzene-1,4-diamine (compound 20-4)
  • Step 4 N 1 -(2-(Dimethylamino)ethyl)-N 4 -(5-fluoro-4-(1-methyl-1H-pyrrolo[2,3-b]pyridine-5- Pyrimidin-2-yl)-5-methoxy-N 1-methylbenzene-1,2,4-triamine (compound 20-5)
  • Step 5 N-(2-((2-(Dimethylamino)ethyl)(methyl)amino)-5-((5-fluoro-4-(1-methyl-1H-pyrrolo[2] ,3-b]pyridin-5-yl)pyrimidin-2-yl)amino)-4-methoxyphenyl)acrylamide (Compound Z-20)
  • Compound Z-20 (11.9 mg, 3.4%) was obtained.
  • Step 2 N-(4-Fluoro-2-methoxy-5-nitrophenyl)-5-methoxy-4-(1-methyl-1H-pyrrolo[2,3-b]pyridine -5-yl)pyrimidin-2-amine (compound 21-3)
  • Step 3 N 1 -(2-(Dimethylamino)ethyl)-5-methoxy-N 4 -(5-methoxy-4-(1-methyl-1H-pyrrolo[2, 3-b]pyridin-5-yl)pyrimidin-2-yl)-N 1 -methyl-2-nitrobenzene-1,4-diamine (compound 21-4)
  • Step 4 N 1 -(2-(Dimethylamino)ethyl)-5-methoxy-N 4 -(5-methoxy-4-(1-methyl-1H-pyrrolo[2, 3-b]pyridin-5-yl)pyrimidin-2-yl)-N 1 -methylbenzene-1,2,4-triamine (compound 21-5)
  • Step 5 N-(2-((2-(Dimethylamino)ethyl)(methyl)amino)-4-methoxy-5-((5-methoxy-4-(1-methyl) -1H-pyrrolo[2,3-b]pyridazol-5-yl)pyrimidin-2-yl)amino)phenyl)acrylamide (Compound Z-21)
  • Z-21 (59 mg, 15.6%).
  • Step 2 N-(4-Fluoro-2-methoxy-5-nitrophenyl)-4-(1-methyl-1H-pyrrolo[2,3-b]pyridin-5-yl)- 5-(trifluoromethyl)pyrimidin-2-amine (compound 22-3)
  • Step 3 N 1 -(2-(Dimethylamino)ethyl)-5-methoxy-N-methyl-N 4-(4-(1-methyl-1H-pyrrolo[2,3 -b]pyridine-5-yl)-5-(trifluoro)pyrimidin-2-yl)-2-nitrobenzene-1,4-diamine (compound 22-4)
  • the compound 22-3 (370 mg, 0.62 mmol) was used as the starting material, and the compound was obtained by the procedure of the step 5 of Example 1 to obtain the compound 22-4 (300 mg, 90%) MS m/z (ESI): 544.2 [M+H] + .
  • Step 4 N 1 -(2-(Dimethylamino)ethyl)-5-methoxy-N-methyl-N 4-(4-(1-methyl-1H-pyrrolo[2,3 -b]pyridine-5-yl)-5-(trifluoro)pyrimidin-2-yl)benzene-1,2,4-triamine (compound 22-5)
  • Step 5 N-(2-((2-(Dimethylamino)ethyl)(methyl)amino)-4-methoxy-5-((4-(1-methyl-1H-pyrrole) [2,3-b]pyridin-5-yl)-5-(trifluoromethyl)pyrimidin-2-yl)amino)phenyl)acrylamide (Compound Z-22)
  • Step 2 5-Chloro-N-(4-fluoro-2-methoxy-5-nitrophenyl)-4-(1,4-oxazepan-4-yl)pyrimidin-2-amine (Compound 23-3)
  • Step 3 N 1 -(5-chloro-4-(1,4-oxazepan-4-yl)pyrimidin-2-yl)-N 4 -(2-(dimethylamino)ethyl) -2-methoxy-N 4 -methyl-5-nitrobenzene 1,4-diamine (compound 23-4)
  • Step 4 N 4 -(5-chloro-4-(1,4-oxazepan-4-yl)pyrimidin-2-yl)-N 1 -(2-(dimethylamino)ethyl) -5-methoxy-N-methylbenzene-1,2,4-triamine (compound 23-5)
  • Step 5 N-(5-((5-Chloro-4-(1,4-oxazepan-4-yl)pyrimidin-2-yl)amino)-2-((2-(dimethyl) Amino)ethyl)(methyl)amino)-4-methoxyphenyl)acrylamide (Compound Z-23)
  • Step 2 4-((1S,4S)-2-oxa-5-azabicyclo[2.2.1]heptane-5-yl)-5-chloro-N-(4-fluoro-2-methoxy 5--5-nitrophenyl)pyrimidine-2-amine (Compound 24-3)
  • Step 3 N 1 -(4-((1S,4S)-2-oxa-5-azabicyclo[2.2.1]heptane-5-yl)-5-chloropyrimidin-2-yl)-N 4- -2-(Dimethylamino)ethyl)yl-2-methoxy-N 4 -methyl-5-nitrobenzene-1,4-diamine (Compound 24-4)
  • Step 4 N 4 -(4-((1S,4S)-2-oxa-5-azabicyclo[2.2.1]heptane-5-yl)-5-chloropyrimidin-2-yl)-N 1- (2-(Dimethylamino)ethyl)-5-methoxy-N 1 -methylbenzene-1,2,4-triamine (Compound 24-5)
  • Step 5 N-(5-(4-((1S,4S)-2-oxa-5 ⁇ -azabicyclo[2.2.1]heptan-5-yl)-5-chloropyridin-2-yl) Amino)-2-((2-(dimethylamino)ethyl)(methyl)amino)-4-methoxyphenyl)acrylamide (Compound Z-24)
  • the target compounds in Examples 25-78, 87 and 88 are represented by the formula (IV-1), wherein the substituents R 0 , R 2 and R 3 are all hydrogen, and the remaining substituents A, R 1 and R 6 As shown in the table below.
  • the target compound in Examples 79-86 is represented by the formula (IV-2), wherein the substituents R 0 , R 2 and R 3 are all hydrogen, and the remaining substituents A and R 1 are shown in the following table.
  • Compounds Z-83 to Z-86 were prepared in the same manner as in Example 14 using differently substituted boric acid esters or boric acid and 2,4-dichloro-1,3,5-triazine as starting materials.
  • Step 1 1-Methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole under a nitrogen atmosphere (1.1 g, 5.25 mmol), a mixture of compound 19-1 (745 mg, 4 mmol), Pd(dppf)Cl 2 (109.7 mg, 0.15 mmol) and sodium carbonate solution (5 mL, 2M) in acetonitrile (30 mL) Stir at °C for 6 h.
  • Step 2 Compound 4a (159 mg, 0.5 mmol), compound 89-2 (114 mg, 0.5 mmol), Pd 2 (dba) 3 (45.75 mg, 0.05 mmol) and Xantphos (28.4 mg, 0.05 mmol) of 1,4
  • cesium carbonate 487.5 mg, 0.5 mmol
  • EA 50 ml ⁇ 2
  • water 10 ml
  • the preparation method was the same as that in Example 89 except that the compound 89-1 in the step 1 of Example 89 was replaced with the compound 91-1, and the compound 3a in the step 2 was replaced with the compound 2a to obtain the title compound Z-91 (22 mg, yield. Rate 11%). It is a yellow solid.
  • Example 93 N-(2-((2-(Dimethylamino)ethyl)(methyl)amino)-5-(5-fluoro-4-(1-methyl-1H-pyrazole-4) Of -yl)pyrimidin-2-ylamino)-4-methoxyphenyl)acrylamide (Z-93)
  • Step 1 To a solution of 4-hydroxypiperidine-1-carboxylic acid tert-butyl ester (2.0 g, 10 mmol) in dichloromethane (20 mL) A solution of the sulfonyl chloride (1.73 g, 15 mmol) in dichloromethane (5 mL). After the reaction mixture was stirred for IH, the reaction mixture was poured into ice water, extracted with methylene chloride, the organic phase was washed with brine, dried over anhydrous Na 2 SO 4, and concentrated to give 2.5g of crude product 96-2 without purification Next step; MS m/z (ESI): 223 [M - 56] + .
  • Step 3 Compound 19-1 (549 mg, 3.0 mmol) and compound 96-3 (754 mg, 2.0 mmol) were used as the starting material. %), MS m/z (ESI): 398 [M+H] + .
  • Step 4 1,4-Dioxane (Compound 96-4 (298 mg, 0.75 mmol), Compound 2a (159 mg, 0.5 mmol), cesium carbonate (326 mg, 1.0 mmol) and Xantphos (58 mg, 0.1 mmol) 2 ml) of the solution was added Pd 2 (dba) 3 (46 mg, 0.05 mmol), and the reaction solution was replaced with nitrogen three times. After microwave reaction at 160 ° C for 0.5 h, the reaction mixture was filtered and concentrated to give a crude product. The crude product was purified by EtOAc EtOAcjjjjjjj MS m/z (ESI): 680.3 [M+H] + .
  • Step 5 To a solution of the compound 96-5 (240 mg, 0.35 mmol) in dichloromethane (10 ml), a mixture of hydrochloric acid / 1,4-dioxane, and the mixture was stirred at room temperature for 2 h, and Na 2 was added. The pH of the CO 3 was adjusted to 9, and dichloromethane was added. The organic phase was washed with hydrochloric acid and concentrated to give a crude material. The obtained crude product was purified by preparative liquid chromatography to afford 20 mg of title compound MS m/z (ESI): 580 [M+H] + .
  • Step 2 To a solution of compound 98-2 (100 mg, 0.44 mmol) in 1,4-dioxane (6 ml), Compound 3a (140 mg, 0.4 mmol), Pd 2 (dba) 3 (41 mg, 0.044 mmol) BINAP (55 mg, 0.088 mmol) and cesium carbonate (286 mg, 0.88 mmol). The reaction solution was microwaved at 130 ° C for 30 minutes.
  • Step 1 To the acetonitrile (100 ml) was added the compound 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole 101-1 ( 4.0g, 0.21mol), sodium difluorochloroacetate (3.77g 0.25mol), 15-crown-5 (103.8g, 0.65mol), heated to reflux under nitrogen atmosphere, stirred for 24h, cooled to room temperature, poured into the reaction solution water (50mL) of quenched and extracted with EA (100ml X 3), dried with saturated brine (20ml) Na 2 SO 4, filtered, and the filtrate was spin-dried to give compound 101-2 (4.1g, 80% yield) White solid. MS m/z (ESI):245.1 [M+H] + .
  • Step 2 To a reaction flask containing tetrakistriphenylphosphine palladium (0.264 g, 2.58 x 10 -4 mol) was added dimethoxyethane dissolved in compound 101-2 (2.1 g, 8.9 x 10 -3 mol).
  • Step 3 The compound 101-3 and the compound 3a were used as a starting material, and the crude product was purified by preparative liquid chromatography to give the title compound Z-101 as a yellow solid.
  • Example 102 N-(5-(5-Chloro-4-(1H-pyrazol-4-yl)pyrimidin-2-ylamino)-2-(4-(dimethylamino)piperidin-1- Of 4-(4-methoxyphenyl)acrylamide (Z-102)
  • Step 1 To a solution of compound 101-1 (5.0 g, 25.8 mmol) and 2,3-dihydropyran (4.33 g, 51.5 mmol) in dichloromethane (100 ml) The reaction mixture was stirred at 40 ° C for 2 h. After completion of the reaction, the reaction mixture was washed with EtOAc EtOAc EtOAc. ] + .
  • Step 2 Compound 19-1 (4.5 g, 25 mmol) and Compound 102-2 (7.0 g, 25 mmol) were used as the starting materials.
  • the crude product was obtained by Combi-flash column chromatography. Purification of the title product 102-3 (850 mg, yield 10%). MS m/z (ESI): 299 [M+H] + .
  • Step 3 Compound 102-3 (300 mg, 1 mmol) and compound 3a (320 mg, 1 mmol) were used as the starting material, and the crude product 102-4 (60 mg, yield 70%) was obtained. Purification was carried out directly to the next step, MS m/z (ESI): 581[M+H] + .
  • Step 4 To a solution of the compound 102-4 (700 mg, 1 mmol) in dichloromethane (5 ml) The product was isolated and purified by preparative liquid chromatography to afford title compound Z-102 (80 mg, yield 60%) as a yellow solid.
  • Examples 103 to 127 are represented by the formula (IV-1), wherein the substituents R 0 , R 2 and R 3 are all hydrogen, and the remaining substituents A, R 1 and R 6 are shown in the following table. .
  • Comparative Compounds 2 and 3 were prepared in the same manner as in Example 1 using the corresponding boron ester and 5-substituted or unsubstituted 2,4-dichloropyrimidine as starting materials, respectively.
  • 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 working concentrations of the components in the 10 ⁇ L T790M/L858R kinase reaction system were: 25 ⁇ M ATP, 0.08 (or 0.1) ng/ ⁇ L EGFR T790M/L858R kinase, 2 ⁇ M Tyr04 substrate (Invitrogen, PV3193, the same below).
  • the concentration of DMSO after addition of the compound prepared in the above examples of the present invention was 2%.
  • the working concentrations of the components in the 10 ⁇ L EGFR WT kinase reaction system were: 10 ⁇ M ATP, 0.8 ng/ ⁇ L EGFR WT kinase, and 2 ⁇ M Tyr04 substrate.
  • the concentration of DMSO after adding the test substance was 2%.
  • the 10 mM stock solution of the test compound was dissolved at room temperature and diluted with a water gradient of 4% DMSO to a final concentration of 10-0.005 ⁇ M.
  • 2.5 ⁇ L of the test substance solution and 5 ⁇ L of a mixture of EGFR T790M/L858R kinase (or EGFR WT 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 with a reaction buffer, no drug was added to the C2 well, and the phosphorylated substrate was added to the C3 well as described.
  • the inhibition rate is calculated 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). See Tables 1 to 3 for the inhibitory activity of the enzyme and the selection of the inhibitory activity.
  • the exemplified compounds of the present invention exhibited strong inhibitory activity against EGFR mutant enzymes (T790M/L858R and L858R) and inhibitory activity against EGFR wild-type enzyme (T790M WT).
  • the compounds of the present invention have significant selective inhibitory activity against EGFR mutant enzymes compared to the positive control BIBW2992 (Afatinib).
  • the selective inhibitory activity of the exemplified compound of the present invention against the EGFR mutant enzyme exceeded that of the comparative compound 1 (the specific structure is shown below, and can be referred to WO2013014448A1), and the selectivity was up to 18 times higher than that of the comparative compound 1.
  • Test Example 2 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 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 min, the supernatant was discarded, and the cells were suspended with fresh medium. The cells were then counted and the cells were diluted to the following concentrations: A431 and H1975 cells per mL 2.78 million, NIH3T3 per mL 333,000. The cells were seeded in a 96-well plate (BD 3072), 90 ⁇ L per well, and cultured overnight.
  • BD 3072 96-well plate
  • A431 cell culture medium 10% FBS (Gibco, 10099-141) DMEM (Hyclone SH30243.01B);
  • NIH3T3 cell culture medium 10% FBS (Gibco, 10099-141) DMEM (Hyclone SH30243.01B);
  • H1975 cell culture medium 10% FBS (Gibco, 10099-141) RPMI-1640 (Hyclone SH30809.01B);
  • 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 (about 10-20 min). 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 inhibitory activity or selective inhibitory activity of the exemplified compounds on the cells is shown in Tables 4 to 7.
  • the exemplified compounds of the present invention showed strong inhibitory activity against EGFR mutant cells (H1975 cells), and showed weak inhibitory activity against EGFR wild-type cells (A431 cells), and Compared to the positive control BIBW2992, the compounds of the present invention have significant selective inhibitory activity against EGFR mutant cell growth. And most of the exemplified compounds have selective inhibitory activity against EGFR mutant cell growth over Comparative Compounds 1, 2 and 3. The selectivity was up to nearly 4 times higher than that of Comparative Compound 1. However, it was found that after replacing A with a pyridine ring or a quinoline ring, the activity of H1975 cells and the selective inhibitory activity against cell growth were significantly reduced.
  • the exemplary compounds of the invention have higher IC 50 values NIH3T3 cells, thus showing less toxic.
  • Test Example 3 Determination of EGFR T790M inhibitor cell activity 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% NP-40, 20 mM Tris (pH 8.0), 137 mM NaCl, 10% glycerol, 1 mM NaVO 3 , 2 mM EDTA.
  • Cell lysate cell lysis buffer + 10 ⁇ g/mL aprotinin (Sigma), 10 ⁇ g/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% Tween-20.
  • Antibody dilutions were detected: 20 mM Tris, 137 mM NaCl, 0.05% Tween-20, 0.1% BSA, pH 7.2-7.4.
  • Blocking solution PBS buffer containing 1% BSA.
  • ELISA kits R&D DYC3570, R&D DYC1095E and R&D DYC1095BE.
  • H1975 cells were seeded at a density of 1 ⁇ 10 4 /well into 96-well plates at 90 ⁇ l of 10% FBS per well, 1640 medium, and cultured overnight at 37 ° C, 5% 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 were seeded at a density of 1 ⁇ 10 4 /well into a 96-well plate, and 90 ⁇ l of each well was cultured in DMEM medium containing 10% FBS at 37 ° C, 5% CO 2 overnight.
  • 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 exemplified compounds of the present invention have significant selective inhibitory activity against cell-level targets compared to the positive control BIBW2992. Moreover, compared with the comparative compounds 1, 2 and 3, the selective inhibitory activity against the cell-level target was increased up to 90-fold. However, it was found that after replacing A with a pyridine ring or a quinoline ring, the selective inhibitory activity against H1975 cell activity and cell-level target was significantly reduced, and even no inhibitory activity was selected.
  • Test Example 4 In vivo test in rats or mice
  • the drug concentration in plasma at different times after intragastric administration and intravenous administration of the compound of the present invention was determined by LC/MS/MS method, and the pharmacokinetic behavior of the compound of the present invention in rats or mice was investigated. To evaluate its pharmacokinetic characteristics.
  • Test animals healthy adult male SD rats (body weight 200-300 g, 6 fasting) or male CD1 mice (body weight 20-30 g, 18 free drinking water and diet), provided by Slark;
  • SD rats were given dorsal vein (1 mg/kg, 5 mL/kg, 5% DMAC (dimethylacetamide), 5% Solutol HS 15 (polyethylene glycol stearate 15) And 90% saline and intragastric administration (20 mg/kg, 10 mL/kg, 0.5% CMC-Na aqueous solution); administration of CD1 mouse tail vein (1 mg/kg, 5 mL/kg, 5% DMAC, 5%) Solutol HS 15 and 90% Saline) and intragastric administration (5 mg/kg, 10 mL/kg, 0.5% CMC-Na in water)
  • Blood sample collection First, select the animals that meet the experimental requirements before the administration, and weigh the markers. Before the blood sample is collected, the rats or mice are bound, and each rat is administered at a predetermined blood collection time point (intravenous administration: 0.083, 0.25, 0.5, 1, 2 after administration, respectively, before administration). , 4,8,24h blood collection, a total of 9 time points; intragastric administration: before administration, 0.083, 0.25, 0.5, 1, 2, 4, 8, 24h after administration, blood collection, a total of 9 time Point), blood was collected through the tail vein, or blood was collected by the heart (final blood collection) about 150 ⁇ L.
  • Each of the administered mice was administered at a predetermined blood sampling time (tail vein administration at 0.083, 0.25, 0.5, 1, 2, 4, 8, 24 blood collection for a total of 8 time points; intragastric administration: at 0.25 , 0.5, 1, 2, 4, 8, 24h blood collection, a total of 7 time points), blood collection through the eyelids, or blood collection by the heart (final blood collection) about 150 ⁇ L.
  • the blood was transferred to a 1.5 mL tube pre-added to K 2 EDTA.
  • the collected blood samples were placed on wet ice, centrifuged for 5 min (2000 g, 4 ° C), and the plasma was taken out. The whole process was completed within 15 min after blood collection. All samples need to be stored in a -70 ° C refrigerator until sample analysis.
  • the drug concentration was determined by LC/MS/MS method.
  • the pharmacokinetic properties of the compounds of some embodiments of the present invention in the same dosage and mode of administration, and the pharmacokinetic parameters in rats and mice are shown in Table 9:
  • the exemplified compounds of the present invention have good pharmacological absorption, have significant pharmacological absorption effects, and exhibit good bioavailability.

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Abstract

本发明提供了一类对EGFR酪氨酸激酶具有抑制作用的2,4-二取代苯-1,5-二胺衍生物及其药学上可接受的盐、立体异构体、溶剂化物或前药。式中各基团的定义详见说明书。此外，本发明还公开了该化合物的药物组合物、药用组合物及其应用。

Description

2,4-二取代苯-1,5-二胺衍生物及其应用以及由其制备的药物组合物和药用组合物 技术领域
本发明涉及医药技术领域，特别涉及一种2,4-二取代苯-1,5-二胺衍生物及其作为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抑制剂将不再起效，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。
目前开发上市的第二代不可逆pan-EGFR抑制剂(Afatinib(BIBW2992))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抑制剂耐药病人对这类药物产生应答。
为了提高对耐药EGFR T790M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
本发明的目的是提供一种2,4-二取代苯-1,5-二胺衍生物，或其药学上可接受的盐、立体异构体、溶剂化物或其前药；以及包含上述衍生物的药物组合物。
本发明的第二个目的是提供上述衍生物或组合物在制备调节EGFR酪氨酸激酶活性或治疗EGFR相关疾病的药物中的应用。
本发明第一方面提供了一种结构如式(I)所示的化合物，或其药学上可接受的盐、立体异构体、溶剂化物或其前药：
Figure PCTCN2015073044-appb-000001
式(I)中，A为选自下组中的任意一种：
Figure PCTCN2015073044-appb-000002
其中，Z2为CR5或N；R5为氢、卤素、三氟甲基或C1-10烷氧基；Z21为N或CH；R4、 R42、R43各自独立地为氢或C1-10烷基、C3-10环烷基、C3-10杂环烷基(如哌啶基)、或卤代的C1-10烷基；R41为氢、卤素、三氟甲基、C1-10烷基或C1-10烷氧基；(优选地，R4、R42、R43各自独立地为氢、甲基或乙基；R41为氢、氯、氟、三氟甲基、甲基或甲氧基；)R1'、R2'和R5'各自独立地为氢、C1-10烷基；(优选地，R1'、R2'和R5'各自独立地为氢、甲基或乙基；)R3'、R4'各自独立地为氢、C1-10烷基，或R3'与R4'相连形成桥环结构；(优选地，R3'、R4'各自独立地为氢、甲基或乙基；或者R3'与R4'相连为-CH2-；)Y为O、CH2、NRa'；其中Ra'为氢、甲基、叔丁氧羰基、乙酰基、甲磺酰基；n为0或1；
R0为氢、卤素、C1-10烷基、-NRaRb或-CO-NRaRb，其中，Ra、Rb各自独立地为C1-10烷基；
Z1为CR6或N；其中，R6为氢、卤素、三氟甲基、甲氧基或-CO2C1-10烷基；
R1为氢，或选自下组中的任意一种：
Figure PCTCN2015073044-appb-000003
R2和R3各自独立地为氢或-CH2NR7R8；其中，(1)R7、R8各自独立地为氢或甲基；或(2)R7、R8和相连的氮原子共同形成5-6元含氮饱和杂环；
R12为氢、卤素、C1-10烷基或C1-10烷氧基(优选地为甲氧基)。
在另一优选例中，式(I)中，A为选自下组中的任意一种：
Figure PCTCN2015073044-appb-000004
其中，n、R4、R41、R42、R43、R1'、R2'、R3'、R4'、R5'、Ra'如式(I)中所定义。
在另一优选例中，式(I)中，R4为氢、甲基、乙基、异丙基、环丙基、二氟甲基或哌啶基；R41为氢、氯、氟、三氟甲基、甲基或甲氧基；R42为氢、甲基或乙基；R43为氢、甲基或乙基。
在另一优选例中，式(I)中，R2和R3各自独立地为氢；且R12为甲氧基。
在另一优选例中，式(I)中，Z1为CR6或N；其中，R6为氢、氟、氯、三氟甲基、甲氧基或-CO2C1-10烷基；
在另一优选例中，式(I)中，Z1为CR6，其中，R6为氢、氟、氯、三氟甲基、甲氧基或-CO2CH3。
在另一优选例中，式(I)中，R0为氢。
在另一优选例中，式(I)中，A为选自下组中的任意一种：
Figure PCTCN2015073044-appb-000005
和
Figure PCTCN2015073044-appb-000006
这里，
Figure PCTCN2015073044-appb-000007
和
Figure PCTCN2015073044-appb-000008
可以分别是
Figure PCTCN2015073044-appb-000009
和
Figure PCTCN2015073044-appb-000010
在另一优选例中，式(I)中，R2和R3各自独立地为氢。
在另一优选例中，式(I)中，R2和R3各自独立地为氢或-CH2NR7R8，且R7、R8各自独立地为氢或甲基。
在另一优选例中，式(I)中，R2和R3各自独立地为氢或-CH2NR7R8，且R7、R8和相连的氮原子共同形成5-6元含氮饱和杂环。
在另一优选例中，式(I)中，R2和R3各自独立地为氢或-CH2NR7R8，且R7、R8和相邻的氮原子共同形成5-6元含氮饱和杂环，所述5-6元含氮饱和杂环的结构如式(Ⅲ)所示：
Figure PCTCN2015073044-appb-000011
式中，X为O、S、NR9或CR10R11；其中，R9、R10、R11各自独立地为氢、C1-10烷氧基或-CO-C1-10烷基。
在另一优选例中，式(I)所示的化合物为结构如式(IV-1)所示化合物：
Figure PCTCN2015073044-appb-000012
式中，A、R0、R1、R2、R3和R6如式(I)中所定义。
在另一优选例中，式(I)所示的化合物为结构如式(IV-2)所示的化合物：
Figure PCTCN2015073044-appb-000013
式中，A、R0、R1、R2和R3如式(I)中所定义。
在另一优选例中，式(I)所示的化合物为结构如式(V-1)所示的化合物：
Figure PCTCN2015073044-appb-000014
式中，R1、R2、R3、R4、R41和R6如式(I)中所定义。
在另一优选例中，式(I)所示的化合物为结构如式(V-2)所示的化合物：
Figure PCTCN2015073044-appb-000015
式中，R1、R2、R3、R4和R41如式(I)中所定义。
在另一优选例中，式(I)所示的化合物为结构如式(VI-1)所示：
Figure PCTCN2015073044-appb-000016
式中，R1、R2、R3、R4、R41和R6如式(I)中所定义。
在另一优选例中，式(I)所示的化合物为结构如式(VI-2)所示：
Figure PCTCN2015073044-appb-000017
式中，R1、R2、R3、R4和R41如式(I)中所定义。
在另一优选例中，式(I)所示的化合物为结构如式(VII-1)所示：
Figure PCTCN2015073044-appb-000018
式中，R1、R2、R3和R6如式(I)中所定义。
在另一优选例中，式(I)所示的化合物为结构如式(VII-2)所示：
Figure PCTCN2015073044-appb-000019
式中，R1、R2和R3如式(I)中所定义。
在另一优选例中，式(I)所示的化合物为结构如式(VIII-1)所示的化合物：
Figure PCTCN2015073044-appb-000020
式中，R1'、R2'、R3'、R4'、R5'、R1、R2、R3、R6和Y如式(I)中所定义。
在另一优选例中，式(I)所示的化合物为结构如式(VIII-2)所示的化合物：
Figure PCTCN2015073044-appb-000021
式中，R1'、R2'、R3'、R4'、R5'、R1、R2、R3和Y如式(I)中所定义。
在另一优选例中，式(I)所示的化合物为结构如式(IX-1)所示：
Figure PCTCN2015073044-appb-000022
式中，R1'、R2'、R4'、R5'、R1、R2、R3和R6如式(I)中所定义。
在另一优选例中，式(I)所示的化合物为结构如式(IX-2)所示：
Figure PCTCN2015073044-appb-000023
式中，R1'、R2'、R4'、R5'、R1、R2和R3如式(I)中所定义。
在另一优选例中，式(I)所示的化合物为结构如式(X-1)所示的化合物：
Figure PCTCN2015073044-appb-000024
式中，R1'、R5'、R1、R2、R3和R6如式(I)中所定义。
在另一优选例中，式(I)所示的化合物为结构如式(X-2)所示的化合物：
Figure PCTCN2015073044-appb-000025
式中，R1'、R5'、R1、R2和R3如式(I)中所定义。
在另一优选例中，式(I)所示的化合物为结构如式(XI-1)所示的化合物：
Figure PCTCN2015073044-appb-000026
式中，R1、R2、R3、R4、R42、R43和R6如式(I)中所定义。
在另一优选例中，式(I)所示的化合物为结构如式(XI-2)所示的化合物：
Figure PCTCN2015073044-appb-000027
式中，R1、R2、R3、R4、R42和R43如式(I)中所定义。
在另一优选例中，式(I)所示的化合物为结构如式(XII-1)所示的化合物：
Figure PCTCN2015073044-appb-000028
式中，R1、R2、R3、R4、R41和R6如式(I)中所定义。
在另一优选例中，式(I)所示的化合物为结构如式(XII-2)所示的化合物：
Figure PCTCN2015073044-appb-000029
式中，R1、R2、R3、R4和R41如式(I)中所定义。
在另一优选例中，式(XI-1)化合物中，R6为氢、氟、氯、三氟甲基。
在另一优选例中，式(XI-1)或式(XI-2)化合物中，R2和R3各自独立地为氢。
在另一优选例中，式(XI-1)或式(XI-2)化合物中，R1选自：
Figure PCTCN2015073044-appb-000030
在另一优选例中，式(XI-1)或式(XI-2)化合物中，R4、R42、R43各自独立地为氢、C1-10烷基、卤代的C1-10烷基或哌啶基；
在另一优选例中，式(XI-1)或式(XI-2)化合物中，R4、R42、R43各自独立地为氢、甲基、乙基、二氟甲基或哌啶基。
在另一优选例中，式(XI-1)或式(XI-2)化合物中，R4为氢、甲基、二氟甲基或哌啶基；R42、R43各自独立地为氢或甲基。
在另一优选例中，式(V-1)、式(V-2)、式(VI-1)、式(VI-2)、式(XII-1)或式(XII-2)化合物中，R4为氢、C1-10烷基、C3-10环烷基或卤代的C1-10烷基。
在另一优选例中，式(V-1)、式(V-2)、式(VI-1)、式(VI-2)、式(XII-1)或式(XII-2)化合物中，R4为氢、甲基、乙基、异丙基、环丙基或二氟甲基。
在另一优选例中，式(V-1)、式(V-2)、式(VI-1)、式(VI-2)、式(XII-1)或式(XII-2)化合物中，R41为氢、氯、氟、三氟甲基、甲基或甲氧基。
在另一优选例中，式(V-1)、式(V-2)、式(VI-1)、式(VI-2)、式(XII-1)或式(XII-2)化合物中，R2和R3各自独立地为氢。
在另一优选例中，式(V-1)、式(VI-1)或式(XII-1)化合物中，R6为氢、氟、氯、三氟甲基、甲氧基或-CO2CH3。
在另一优选例中，式(V-1)、式(V-2)、式(VI-1)、式(VI-2)、式(XII-1)或式(XII-2)化合物中，R1选自：
Figure PCTCN2015073044-appb-000031
Figure PCTCN2015073044-appb-000032
在另一优选例中，所述的化合物选自下组：
Figure PCTCN2015073044-appb-000033
Figure PCTCN2015073044-appb-000034
Figure PCTCN2015073044-appb-000035
Figure PCTCN2015073044-appb-000036
本发明第二方面提供了一种药物组合物，它包括本发明第一方面所述的化合物，或其药学可接受的盐、立体异构体、溶剂化物或其前药，并且还包括药学可接受的载体。
在另一优选例中，所述药物组合物包括治疗有效量的本发明第一方面所述的化合物，如上述式(I)化合物、式(IV-1)化合物、式(IV-2)化合物、式(V-1)化合物、式(V-2)化合物、式(VI-1)化合物、式(VI-2)化合物、式(VII-1)化合物、式(VII-2)化合物、式(VIII-1)化合物、式(VIII-2)化合物、式(IX-1)化合物、式(IX-2)化合物、式(X-1)化合物、式(X-2)化合物、式(XI-1)化合物、或式(XI-2)化合物、或上述示例化合物，或其药学上可接受的盐、立体异构体、溶剂化物或其前药；以及药学可接受的载体。
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、立体异构体、溶剂化物或其前药可用于制备调控EGFR酪氨酸激酶活性或治疗EGFR相关疾病的药物中的应用。
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。
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)。
作为优选，所述的化合物，或其药学上可接受的盐、立体异构体、溶剂化物或其前药还可用于制备治疗EGFR异常表达的疾病或在使用EGFR调节剂治疗期间具有获得性耐药性的疾病的药物中的应用。
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。
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、拉帕替尼或阿法替尼。
本发明第四方面提供了一种药用组合物，其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、立体异构体、溶剂化物或其前药，以及选自下组药物中的一种或多种其它药物：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、立体异构体、溶剂化物或其前药以外，上述药用组合物中的其它药物均为本领域技术人员熟知的抗肿瘤药物。
如本文所用，“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、立体异构体、溶剂化物或其前药的治疗有效量优选为0.1mg-5g/kg(体重)。
本发明的药用组合物可用于治疗EGFR相关疾病，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，或使用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，或者是包含EGFR外显子20编码的T790突变所引起的。
在另一优选例中，所述的EGFR外显子20编码的T790为T790M。
本发明所述的化合物在某些疾病中可以与其它药物联合应用，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。
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
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
如本文所用，“C1-10烷基”是指具有1至10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、丁基、异丁基、仲丁基、叔丁基、戊基、己基、庚基、辛基、壬基、癸基等。
如本文所用，“C1-10烷氧基”指C1-10烷基-O-。例如甲氧基、乙氧基、丙氧基、异丙氧基、丁氧基、异丁氧基、仲丁氧基、叔丁氧基等。
如本文所用，“卤素”指氟、氯、溴或碘。
如本文所用，“C3-10环烷基”指包括3至10个碳原子饱和或部分不饱和单环或多环环状烃取代基，优选环烷基包含3至8个碳原子(C3-8环烷基)，最优选环烷基包含3至6个碳原子(C3-6环烷基)。单环环烷基的非限制性实施例包含环丙基、环丁基、环戊基、环戊烯基、环己基、环己烯基、环己二烯基、环庚基、环庚三烯基、环辛基等，优选环丙基、环戊基、环己烯基。多环环烷基包括螺环、稠环和桥环的环烷基。
如本文所用，“C3-10杂环烷基”指饱和或部分不饱和单环或多环环状烃取代基，其包括3至10个环原子，其中一个或多个环原子选自氮、氧或S(O)t(其中t是整数0至2)的杂原子，但不包括-O-O-、-O-S-或-S-S-的环部分，其余环原子为碳。优选包括3至8个环原子，其中1～3个是杂原子，更优选杂环烷基包含3至6个环原子，更优选杂环烷基包含5至6个环原子。单环杂环基的非限制性实施例包含吡咯烷基、哌啶基、哌嗪基、吗啉基、硫代吗啉基、高哌嗪基、吡喃基、四氢呋喃基等。多环杂环基包括螺环、稠环和桥环的杂环基。
如本文所用，“卤代的C1-10烷基”指烷基被1、2、3、4或5个卤素取代，其中烷基的定义如上所述。优选卤代C1-10烷基，更优选卤代C1-6烷基，最优选卤代C1-3烷基。包括(但不限于)一氯乙基、二氯甲基、1,2－二氯乙基、一溴乙基、一氟乙基、一氟甲基、二氟甲基、三氟甲基等。
活性成分
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，其具有显著耐药EGFR T790M突变(特别是EGFR T790M/L858R双突变)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。
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 PCTCN2015073044-appb-000037
步骤1：嘧啶4位的氯与硼酸酯或者硼酸，或者合适的胺反应，需在一定温度下，使用合适的催化剂及适当的溶剂可以进行。使用Suzuki方法，所用的钯催化剂可以是但不限于PdCl2(dppf)，所用的碱可以是但不限于碳酸钠。
步骤2：嘧啶2位的氯以胺取代，需在一定温度下，使用合适的催化剂及适当的溶剂才能进行。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：苯基4位的氟以胺取代，需在一定温度下，使用合适的碱及适当的溶剂才能进行。所用的碱可以是但不限于碳酸钾。
步骤4：该硝基化合物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：该胺基化合物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。
以上各步骤中的反应均是本领域技术人员已知的常规反应。
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)。
为了使本领域技术人员更好地理解本发明的技术方案，下面结合具体实施例对本发明作进一步的详细说明。
试剂与仪器
本发明中，化合物的结构和纯度通过核磁共振(1HNMR)和/或液质联用质谱(LC-MS)来确定。1HNMR：Bruker AVANCE-400核磁仪，内标为四甲基硅烷(TMS)。LC-MS：Agilent 1200HPLC System/6140MS液质联用质谱仪(购自安捷伦)，柱子WatersX-Bridge,150×4.6mm,3.5μm。制备高效液相色谱(pre-HPLC)：用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。
薄层层析硅胶板使用烟台黄海HSGF254或青岛GF254硅胶板，薄层色谱法(TLC)，检测反应使用的硅胶板采用的规格是0.15mm-0.2mm，薄层色谱法分离纯化产品使用的硅胶板采用的规格是0.4mm-0.5mm。硅胶一般使用烟台黄海硅胶200-300目硅胶为载体。碱性氧化铝柱一般使用国药层析用FCP200-300目碱性氧化铝为载体。
本发明的已知的起始原料可以采用或按照本领域已知的方法来合成，或可以于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处购买。
实施例中无特殊说明，反应均在氮气或氩气氛下进行。实施例中无特殊说明，溶液是指水溶液。
如本文所用，DMF表示二甲基甲酰胺，DMSO表示二甲基亚砜，THF表示四氢呋喃，DIEA表示N,N-二异丙基乙胺，EA表示乙酸乙酯，PE表示石油醚。BINAP表示(2R,3S)-2,2'-双二苯膦基-1,1'-联萘，NBS表示N-溴代丁二酰亚胺，NCS表示N-氯代丁二酰亚胺，Pd2(dba)3表示三(二亚苄基丙酮)二钯，Pd(dppf)Cl2表示[1,1'-双(二苯基磷)二茂铁]二氯化钯。
如本文所用，室温指的是约25℃。
化合物1a的制备：
Figure PCTCN2015073044-appb-000038
步骤a:将化合物1a1(10.6g,58mmol)置于500mL反应瓶中，加入THF/水(100mL/60mL)混合溶液溶解。在室温下，边搅拌边依次加入氯化铵(15.5g,292mmol)和还原铁粉(26g,467mmol)，随后将反应体系加热至65℃并持续搅拌3h。TLC检测反应进度，待反应完成后，过滤除去多余铁粉，滤饼用EA淋洗三次。滤液用EA/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Na2SO4干燥后，减压浓缩得到化合物1a2(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1a2(8.0g,43mmol)置于500mL反应瓶中，匀速搅拌下加入浓硫酸(100mL)使底物溶解。在零下20℃下，边搅拌边缓慢滴加浓硝酸(6.15mL,48mmol)，并保温 搅拌5min。TLC检测反应进度，待反应完成后，倒入冰水中。保持零下20℃冰浴，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。然后反应液用EA/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Na2SO4干燥后，减压浓缩得到化合物1a(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)。
中间体2a的制备
Figure PCTCN2015073044-appb-000039
步骤1：将化合物1a(11.16g，60mmol)溶于150mL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18h。TLC检测反应进度，待反应完成后，减压浓缩反应液，柱层析[PE:EA＝80:20]分离纯化得到目标产物化合物2a2(12.56g，73％)。MS m/z(ESI):285[M-H]+。
步骤2：将化合物2a2(11.46g，40mmol)溶于60mL 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℃搅拌反应6h。TLC检测反应进度，待反应完成后，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Na2SO4干燥，减压浓缩得目标产物化合物2a3(12.51g，85％)。直接用于下一步反应。MS m/z(ESI):369[M+H]+。
步骤3：将化合物2a3(12g，32.6mmol)溶于200mL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1h。TLC检测反应进度，待反应完成后，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化合物2a4(10.70g，97％)。直接用于下一步反应。MS m/z(ESI):339[M+H]+。
步骤4：将化合物2a4(10.1g，30mmol)和三乙胺(6.12g，60mmol)溶于200mL二氯甲烷，冷却至0℃，加入丙烯酰氯(3.24g，36mmol)，在氮气保护下，室温搅拌3h。TLC检测反应进度，待反应完成后，以饱和碳酸氢钠水溶液、饱和食盐水顺次洗涤，无水Na2SO4干燥，抽滤，经减压浓缩得到目标产物化合物2a5(9.64g，82％)。直接用于下一步反应。MS m/z(ESI):393[M+H]+。
步骤5：将化合物2a5(9.41g，24mmol)溶解在100mL二氯甲烷中，冷却到0℃，加入20mL三氟乙酸，在氮气保护下室温搅拌18h。TLC检测反应进度，待反应完成后，减压浓缩反应液。将残留物以300mL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Na2SO4干燥，抽滤，减压浓缩得到粗品。经柱层析[DCM:MeOH＝10:1]纯化得到目标产 物化合物2a(3.26g，46.5％)。MS m/z(ESI):293[M+H]+。
中间体3a的制备
Figure PCTCN2015073044-appb-000040
中间体4a的制备
实施例1:N-(5-(5-氯-4-(1-甲基-1H-吡咯并[2,3-b]吡啶-5-基)嘧啶-2-胺基)-2-((2-(二甲氨)乙基)甲基胺基)-4-甲氧基苯基)丙酰胺(化合物Z-1)
Figure PCTCN2015073044-appb-000042
步骤1：5-溴-1-甲基-1H-吡咯并[2,3-b]吡啶(化合物1-2)
在0℃下，将化合物5-溴-1H-吡咯并[2,3-b]吡啶(5g，25.4mmol，市购)的DMF溶液加入到氢化钠(4.32g，30.5mmol)的40mLDMF中，0℃下剧烈搅拌0.5h后，加入碘甲烷(1.22g，30.5mmol)，室温下剧烈搅拌3h。TLC检测反应进度，待反应完成后，将反应体系倒入冰水中。待固体完全析出后，过滤，固体用二氯甲烷和甲醇(10:1)溶解，水洗3次，干燥，分离出有机层，减压浓缩得到化合物1-2(5.4g，99％)，产物直接用于下一步。MS m/z(ESI):211.0[M+H]+。
步骤2：1-甲基-5-(4,4,5,5-四甲基-1,3,2-二氧杂硼烷-2-基)-1H-吡咯并[2,3-b]吡啶(化合物1-3)
将双联频哪醇硼酸酯(11.26g，43.6mmol)，PdCl2(dppf)(814mg，1.11mmol)和乙酸钾(6.53g，32.7mmol)加入到化合物1-2(4.6g,21.8mmol)的50mL1,4-二氧六环溶液中。90℃下，在N2氛围中剧烈搅拌8h。反应结束后，过滤，滤液浓缩，得粗产品化合物1-3(13.9g)，产物直接用于下一步。MS m/z(ESI):259.1[M+H]+。
步骤3：5-(2,5-二氯嘧啶-4-基)-1-甲基-1H-吡咯并[2,3-b]吡啶(化合物1-4)
将2,4,5-三氯嘧啶(4.93g，26.2mmol)，PdCl2(dppf)(1.66g，2.26mmol，市购)和23mL的2.0mol/L的碳酸钠溶液加入到化合物1-3(13.9g,21.8mmol)的50mL乙腈溶液中。85℃下，在N2氛围中剧烈搅拌4h。反应结束后，加水稀释，用EA/水体系萃取，水洗3次，干燥，有机层减压浓缩得粗产品。经Combi-flash柱层析[PE:EA＝100:0-20:80]纯化后得化合物1-4(2.16g，36％)。MS m/z(ESI):279.0[M+H]+。
步骤4：5-氯-N-(4-氟-2-甲氧基-5-硝基苯基)-4-(1-甲基-1H-吡咯并[2,3-b]吡啶-5-基)嘧啶-2-胺(化合物1-5)
将化合物4-氟-2-甲氧基-5-硝基苯胺(1.13g，6.09mmol)，Pd2(dba)3(558mg，0.61mmol)，Xantphos(705mg，1.22mmol)和碳酸铯(3.97g，12.2mmol)加入到化合物1-4(1.70g,6.09mmol)的20mL1,4-二氧六环溶液中。120℃下，在N2氛围中剧烈搅拌5h。反应结束后，过滤，滤液减压浓缩后得粗产品，经Combi-flash柱层析[PE:EA＝100:0-0:100]纯化后得化合物1-5(420mg，16％)。MS m/z(ESI):429.0[M+H]+。
步骤5：N1-(5-氯-4-(1-甲基-1H-吡咯并[2,3-b]吡啶-5-基)嘧啶-2-基)-N4-(2-(二甲氨基)乙基)-2-甲氧基-N4-甲基-5-硝基苯基-1,4-二胺(化合物1-6)
将N,N,N'-三甲基乙二胺(72mg，0.70mmol)和碳酸钾(193mg，1.40mmol)加入到化合物1-5(200mg,0.47mmol)的4mL的DMF中，100℃下剧烈搅拌2h。TLC检测反应进度，待反应完成后，加入10mL水，用EA/水体系萃取三次，分离出有机层，减压浓缩得到化合物1-6(240mg，90％)MS m/z(ESI):511.3[M+H]+。
步骤6：N4-(5-氯-4-(1-甲基-1H-吡咯并[2,3-b]吡啶-5-基)嘧啶-2-基)-N1-(2-(二甲氨基)乙基)-5-甲氧基-N1-甲基苯基-1,2,4-三胺(化合物1-7)
将Pd/C(20mg)加入到化合物1-6(100mg,0.20mmol)的20mL甲醇溶液中。室温下，在H2氛围中剧烈搅拌4h。反应结束后，过滤，滤液浓缩，得化合物1-7(80mg，83％)，产物直接用于下一步。MS m/z(ESI):481.2[M+H]+。
步骤7：N-(5-(5-氯-4-(1-甲基-1H-吡咯并[2,3-b]吡啶-5-基)嘧啶-2-胺基)-2-((2-(二甲氨)乙基)甲基胺基)-4-甲氧基苯基)丙酰胺(化合物Z-1)
在0℃下，将丙烯酰氯(17mg,0.17mmol)与三乙胺(28mg,0.25mmol)加入到化合物1-7(80mg,0.17mmol)的2mL二氯甲烷溶液中，0℃下剧烈搅拌2h。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Z-1(2.39mg，6％)。MS m/z(ESI):534.9[M+H]+；1H NMR(400MHz,DMSO-d6)δ10.11(s,1H),8.87-8.75(m,2H),8.57(d,J＝17.2Hz,3H),7.62(d,J＝3.5Hz,1H),7.00(s,1H),6.57(d,J＝3.4Hz,1H),6.44-6.35(m,1H),6.29(d,J＝16.7Hz,1H),5.77(d,J＝12.0Hz,1H),3.87(s,3H),3.82(s,3H),2.85(s,2H),2.70(s,3H),2.29(d,J＝5.7Hz,2H),2.19(s,6H)。
实施例2：N-(5-(5-氯-4-(1-甲基-1H-吡咯并[2,3-b]吡啶-5-基)嘧啶-2-胺基)-2-(4-(二甲氨基)哌啶-1-基)-4-甲氧基苯基)丙烯酰胺(化合物Z-2)
Figure PCTCN2015073044-appb-000043
步骤1：5-氯-N-(4-(4-(二甲氨基)哌啶-1-基)-2-甲氧基-5-硝基苯基)-4-(1-甲基-1H-吡咯并[2,3-b]吡啶-5-基)嘧啶-2-胺(化合物2-6)
将化合物1-5(260mg,0.607mmol)和碳酸钾(251mg,1.822mmol)置于25mL反应瓶中，加入DMF(10mL)使底物部分溶解。之后加入N,N-二甲氨基哌啶(85.4mg,0.667mmol)并保持反应体系在70℃条件下加热3h。TLC检测反应进度，待反应完成后，反应液用EA/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Na2SO4干燥后，减压浓缩得到粗产物2-6，220mg，产率68％。MS M/Z(ESI)537.2[M+H]+。
步骤2：N1-(5-氯-4-(1-甲基-1H-吡咯并[2,3-b]吡啶-5-基)嘧啶-2-基)-4-(4-(二甲氨基)哌啶-1-基)-6-甲氧基苯基-1,3-二胺(化合物2-7)
将反应底物2-6(110mg,0.205mmol)置于25mL反应瓶中，加入乙醇(6mL)。在室温条件下，向搅拌的反应瓶中加入氯化亚锡(138mg,0.615mmol)并保持反应体系在60℃条件下加热2h。TLC检测反应进度，待反应完成后，反应液浓缩旋干，用EA/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Na2SO4干燥后。滤液减压浓缩得到粗产物2-7(80mg，76.9％)，直接用于下一步反应。
步骤3：N-(5-(5-氯-4-(1-甲基-1H-吡咯并[2,3-b]吡啶-5-基)嘧啶-2-胺基)-2-(4-(二甲氨基)哌啶-1-基)-4-甲氧基苯基)丙烯酰胺(化合物Z-2)
在0℃下，将丙烯酰氯(17.0mg,0.188mmol)与DIEA(60.8mg,0.471mmol)加入到化合物2-7(80mg,0.157mmol)的3mLTHF溶液中，0℃下剧烈搅拌2h。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Z-2(9.0mg，10％)。MS m/z(ESI):560.8[M+H]+；1HNMR(400MHz,DMSO-d6)δ8.98(s,1H),8.75-8.74(d,J＝4.0Hz,1H)，8.48-8.53(m,4H),7.62-7.61(d,J＝4.0Hz,1H),6.84(s,1H),6.71-6.57(m,2H),6.29-6.25(d,J＝16.0Hz,1H),5.77-5.74(d,J＝12.0Hz,1H),3.87(s,3H),3.82(s,3H),3.05-3.02(d,J＝12.0Hz,2H),2.69-2.63(m,2H),2.30-2.19(m,7H),1.91-1.83(m,2H),1.70-1.68(m,2H)。
实施例3:N-(5-(5-氯-4-(1-甲基-1H-吡咯并[2,3-b]吡啶-5-基)嘧啶-2-胺基)-4-甲氧基-2-(4-甲基哌嗪-1-基)苯基)丙烯酰胺(化合物Z-3)
Figure PCTCN2015073044-appb-000044
步骤1：5-氯-N-(2-甲氧基-4-(4-甲基哌嗪-1-基)-5-硝基苯基)-4-(1-甲基-1H-吡咯并[2,3-b]吡啶-5-基)嘧啶-2-胺(化合物3-6)
将N-甲基哌嗪(126mg，1.26mmol)和碳酸钾(261mg，1.89mmol)加入到化合物1-5(200mg,0.47mmol)的4mL的DMF中，100℃下剧烈搅拌2h。TLC检测反应进度，待反应完成后，加入10mL水，用EA/水体系萃取三次，分离出有机层，减压浓缩得到化合物3-6(200mg，84％)MS m/z(ESI):509.2[M+H]+。
步骤2：N1-(5-氯-4-(1-甲基-1H-吡咯并[2,3-b]吡啶-5-基)嘧啶-2-基)-6-甲氧基-4-(4-甲基哌嗪-1-基)苯基-1,3-二胺(化合物3-7)
以化合物3-6(200mg,0.39mmol)为原料，参照实施例1中步骤6进行合成，得化合物3-7(140mg，75％)，产物直接用于下一步。MS m/z(ESI):479.1[M+H]+。
步骤3：N-(5-(5-氯-4-(1-甲基-1H-吡咯并[2,3-b]吡啶-5-基)嘧啶-2-胺基)-4-甲氧基-2-(4-甲基哌嗪-1-基)苯基)丙烯酰胺(化合物Z-3)
以化合物3-7(140mg,0.29mmol)为原料，参照实施例1中步骤7进行合成，得化合物Z-3(1.60mg，1％)。MS m/z(ESI):533.2[M+H]+；1HNMR(400MHz,DMSO-d6)δ8.98(s,1H),8.76(s,1H),8.51(d,J＝32.1Hz,3H),7.63(s,1H),6.86(s,1H),6.63(d,J＝28.9Hz,2H),6.27(d,J＝16.2Hz,1H),5.76(d,J＝9.1Hz,1H),5.33(s,1H),3.88(s,3H),3.84(s,3H),2.86(s,4H),2.55(s,2H),2.26(s,3H),1.99(s,2H)。
实施例4：N-(2-(4-(二甲氨基)哌啶-1-基)-4-甲氧基-5-(4-(1-甲基-1H-吡唑并[3,4-b]吡啶-5-基)嘧啶-2-基氨基)苯基)丙烯酰胺(化合物Z-4)
Figure PCTCN2015073044-appb-000045
步骤1：5-溴-1-甲基-1H-吡唑并[3,4-b]吡啶(化合物4-2)
将氢化钠(720mg,30mmol)置于100mL反应瓶中，向反应瓶中加入无水THF(40mL)。0℃条件下搅拌5min，将反应底物5-溴-1H-吡唑并[3,4-b]吡啶(4.0g,20mmol，市购)溶于THF(20mL)中，通过恒压滴液漏斗缓慢滴加到反应瓶中，搅拌30min，向反应体系中滴加碘甲烷(1.6mL,26mmol)。滴加完毕，缓慢升温至室温反应，搅拌过夜。TLC检测反应进度，待反应完 成后，向反应体系中加入冰水10mL猝灭反应，减压浓缩蒸除THF，残留物经二氯甲烷(60mL)和水(20mL×3)萃取，有机层经无水Na2SO4干燥、减压浓缩得4.1g褐色固体，经Combi-flash柱层析[PE:EA＝10:90-40:60]分离纯化得目标化合物4-2(3.1g，73％)。MS m/z(ESI):211.9[M+H]+
步骤2：1-甲基-5-(4,4,5,5-四甲基-1,3,2-二氧杂环戊硼烷-2-基)-1H-吡唑并[3,4-b]吡啶(化合物4-3)
将化合物4-2(3.1g,14.6mmol)和双联频哪醇硼酸酯(11.1g,43.8mmol)置于250mL反应瓶中，加入DMF(150mL)和乙酸钾(4.3g,43.8mmol)。反应体系用氩气置换空气三次后，加入Pd(dppf)Cl2(0.55g,0.73mmol)，之后再用氮气置换空气三次。将反应体系加热至90℃，并且持续搅拌8h。LC-MS检测反应进度，待反应完成后，抽滤，滤饼经EA(30mL×3)洗涤，滤液减压浓缩蒸除DMF得到化合物4-3(13.5g)，褐色固体，直接用于下一步反应。MS m/z(ESI)：260.2[M+H]+
步骤3：5-(2-氯嘧啶-4-基)-1-甲基-1H-吡唑并[3,4-b]吡啶(化合物4-4)
将反应底物4-3(3.8g,14.6mmol)和2,4-二氯嘧啶(2.6g,17.5mmol)置于250mL反应瓶中，加入乙腈(130mL)和碳酸钠溶液(22mL,2M)。反应体系用氩气置换空气三次后，加入Pd(dppf)Cl2(0.55g,0.73mmol)，之后再用氮气置换空气三次。将反应体系加热至85℃，并且持续搅拌3h。LC-MS检测反应进度，待反应完成后，冷却至室温，析出大量固体，抽滤，滤饼经EA(10mL×3)洗涤，烘干至恒重得2.2g黄色固体，滤液经EA和水萃取，干燥浓缩后，再经Combi-flash柱层析[PE:EA＝10:90-70:30]分离纯化得0.6g黄色固体，将固体合并得目标化合物4-4(2.8g，78％)。MS m/z(ESI)：246.1[M+H]+
步骤4：N-(4-氟-2-甲氧基-5-硝基苯基)-4-(1-甲基-1H-吡唑并[3,4-b]吡啶-5-基)嘧啶-2-胺(化合物4-5)
将反应底物4-4(420mg,1.7mmol)和4-氟-2-甲氧基-5-硝基苯胺(316mg,1.7mmol)置于100mL的封管反应瓶中，加入异丁醇(40mL)使底物部分溶解。之后加入对甲苯磺酸(807mg,4.25mmol)，并保持反应体系在130℃条件下封管加热16h。待反应结束后，冷却至室温，溶液中析出大量固体。布氏漏斗过滤得到滤饼，经过乙醇回流打浆后，得到目标化合物4-5(524mg，78％)，直接用于下一步反应。纯度：47％。MS m/z(ESI):396.2[M+H]+
步骤5：N-(4-(4-(二甲基氨基)哌啶-1-基)-2-甲氧基-5-硝基苯基)-4-(1-甲基-1H-吡唑并[3,4-b]吡啶-5-基)嘧啶-2-胺(化合物4-6)
将反应底物4-5(146mg,0.25mmol)和碳酸钾(104mg,0.75mmol)置于25mL反应瓶中，加入DMF(10mL)使底物完全溶解。之后加入4-二甲氨基哌啶(42mg,0.325mmol)并保持反应体系在100℃条件下加热反应2h。TLC检测反应进度，待反应完成后，反应液用EA/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Na2SO4干燥后，减压浓缩得到目标化合物4-6(120mg，98％)。MS m/z(ESI):504.2[M+H]+。
步骤6：4-(4-(二甲基氨基)哌啶-1-基)-6-甲氧基-N1-(4-(1-甲基-1H-吡唑并[3,4-b]吡啶-5-基)嘧啶-2-基基)苯-1,3-二胺(化合物4-7)
以化合物4-6(120mg,0.24mmol)为原料，参照实施例1中的步骤6进行合成，得到化合物4-7(113mg)，直接用于下一步反应。MS m/z(ESI):474.3[M+H]+。
步骤7：N-(2-(4-(二甲氨基)哌啶-1-基)-4-甲氧基-5-(4-(1-甲基-1H-吡唑并[3,4-b]吡啶-5-基) 嘧啶-2-基氨基)苯基)丙烯酰胺(化合物Z-4)
以化合物4-7(113mg,0.24mmol)为原料，参照实施例1中步骤7进行合成，得到的粗产物经Prep-TLC柱层析[DCM:MeOH:NH4OH＝9:1:0.1]纯化后，得到目标化合物Z-4(6mg，5％)。MS m/z(ESI):528.3[M+H]+；1HNMR(400MHz,DMSO-d6)δ9.33(s,1H),9.22(s,1H),9.04(s,1H),8.80(s,1H),8.51-8.50(d,J＝5.6Hz,2H),8.13(s,1H),7.53-7.51(d,J＝5.6Hz,1H),6.86(s,1H),6.74-6.67(m,1H),6.32-6.28(d,J＝17.0Hz,2H),5.79-5.67(d,J＝13.4Hz,1H),4.24(s,3H),3.86(s,3H),3.33(s,1H),3.05-3.03(d,J＝10.7Hz,2H),2.70-2.64(t,J＝11.7Hz,3H),2.22(s,6H),1.85-1.82(d,J＝11.3Hz,2H),1.70-1.67(d,J＝12.1Hz,2H)。
实施例5：N-(4-甲氧基-5-(4-(1-甲基-1H-吡唑并[3,4-b]吡啶-5-基)嘧啶-2-基氨基)-2-(4-甲基哌嗪-1-基)苯基)丙烯酰胺(化合物Z-5)
Figure PCTCN2015073044-appb-000046
步骤1：N-(2-甲氧基-4-(4-甲基哌嗪-1-基)-5-硝基苯基)-4-(1-甲基-1H-吡唑并[3,4-b]吡啶-5-基)嘧啶-2-胺(化合物5-6)
将化合物4-5(146mg,0.25mmol)和碳酸钾(104mg,0.75mmol)置于25mL反应瓶中，加入DMF(10mL)使底物完全溶解。之后加入N-甲基哌嗪(32mg,0.325mmol)并保持反应体系在100℃条件下加热反应2h。TLC检测反应进度，待反应完成后，反应液用EA/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Na2SO4干燥后，减压浓缩得到目标化合物5-6，直接用于下一步反应。纯度：88％。MS m/z(ESI):476.2[M+H]+
步骤2：6-甲氧基-N1-(4-(1-甲基-1H-吡唑并[3,4-b]吡啶-5-基)嘧啶-2-基)-4-(4-甲基哌嗪-1-基)苯基-1,3-二胺(化合物5-7)
以化合物5-6(119mg,0.25mmol)为原料，参照实施例1中步骤6进行合成，得到化合物5-7，直接用于下一步反应。纯度：59％。MS m/z(ESI):446.2[M+H]+。
步骤3：N-(4-甲氧基-5-(4-(1-甲基-1H-吡唑并[3,4-b]吡啶-5-基)嘧啶-2-基氨基)-2-(4-甲基哌嗪-1-基基)苯基)丙烯酰胺(化合物Z-5)
以化合物5-7(111mg,0.25mmol)为原料，参照实施例1中步骤7进行合成，得到的粗产物经Prep-TLC柱层析[DCM:MeOH:NH4OH＝9:1:0.1]纯化后得到目标化合物Z-5(8mg，6％)。纯度：96.5％。MS m/z(ESI):500.2[M+H]+；1HNMR(400MHz,DMSO-d6)δ9.33(s,1H),9.21(s,1H),9.03(s,1H),8.79(s,1H),8.51-8.50(d,J＝5.2Hz,2H),8.12(s,1H),7.52-7.51(d,J＝5.2Hz,1H),6.88(s,1H),6.68-6.61(m,1H),6.31-6.27(d,J＝16.9Hz,2H),5.78-5.76(d,J＝9.9Hz,1H),4.24(s,3H),3.87(s,3H),2.86(s,4H),2.53-2.49(d,J＝13.6Hz,4H),2.25(s,3H)。
实施例6：N-(2-((2-(二甲基氨基)乙基)(甲基)氨基)-4-甲氧基-5-(4-(1-甲基-1H-吡唑并[3,4-b]吡啶-5-基)嘧啶-2-基氨基)苯基)丙烯酰胺(化合物Z-6)
Figure PCTCN2015073044-appb-000047
步骤1：N1-(2-(二甲基氨基)乙基)-5-甲氧基-N1-甲基-N4-(4-(1-甲基-1H-吡唑并[3,4-b]吡啶-5-基)嘧啶-2-基)-2-硝基苯基-1,4-二胺(化合物6-6)
将化合物4-5(146mg,0.25mmol)和碳酸钾(104mg,0.75mmol)置于25mL反应瓶中，加入DMF(10mL)使底物完全溶解。之后加入N,N,N'-三甲基乙二胺(34mg,0.325mmol)并保持反应体系在100℃条件下加热反应2h。TLC检测反应进度，待反应完成后，反应液用EA/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Na2SO4干燥后，减压浓缩得到目标产物化合物6-6，直接用于下一步反应。纯度：94％。MS m/z(ESI):478.2[M+H]+
步骤2：N1-(2-(二甲基氨基)乙基)-5-甲氧基-N1-甲基-N4-(4-(1-甲基-1H-吡唑并[3,4-b]吡啶-5-基)嘧啶-2-基基)苯-1,2,4-三胺(化合物6-7)
以化合物6-6(119mg,0.25mmol)为原料，参照实施例1中步骤6进行合成，得到化合物6-7，直接用于下一步反应。纯度：83％。MS m/z(ESI):448.2[M+H]+。
步骤3：N-(2-((2-(二甲基氨基)乙基)(甲基)氨基)-4-甲氧基-5-(4-(1-甲基-1H-吡唑并[3,4-b]吡啶-5-基)嘧啶-2-基氨基)苯基)丙烯酰胺(化合物Z-6)
以化合物6-7(111mg,0.25mmol)为原料，参照实施例1中步骤7进行合成，得到的粗产物经Prep-HPLC分离纯化后得到目标产品化合物Z-6(1mg,1％)。纯度：96.5％。MS m/z(ESI):502.2[M+H]+；1HNMR(400MHz,DMSO-d6)δ10.13(s,1H),9.35(s,1H),9.27(s,1H),9.12(s,1H),8.52-8.51(d,J＝5.3Hz,1H),8.50(s,2H),8.20(s,1H),8.14(s,1H),7.54-7.53(d,J＝5.2Hz,1H),7.03(s,1H),6.50-6.44(d,J＝10.0Hz,1H),6.34-6.29(d,J＝17.3Hz,1H),5.80-5.78(d,J＝10.6Hz,1H),4.24(s,3H),3.87(s,3H),2.92-2.90(d,J＝5.4Hz,2H),2.69(s,3H),2.39-2.36(t,J＝5.4Hz,2H),2.25(s,6H)。
实施例7:N-(5-((5-氯-4-吗啉嘧啶-2-基)氨基)-2-(4-(二甲氨基)哌啶-1-基)-4-甲氧苯基)丙烯酰胺(化合物Z-7)甲酸盐
Figure PCTCN2015073044-appb-000048
步骤1：4-(2,5-二氯嘧啶-4-基)吗啉(化合物7-2)
在25℃下，将化合物2,4,5-三氯嘧啶(5g，27.0mmol)加入到25mLTHF中，加入三乙胺(5.5g，54.0mmol)，将吗啉(2.37g，27.0mmol)滴加至反应液中，室温下搅拌1h。TLC检测反应进度，待反应完成后，加入水和EA，分离出有机层，有机层经饱和食盐水系，干燥，减压浓缩得到化合物化合物7-2(5.2g)，产物直接用于下一步。MS m/z(ESI):234.0[M+H]+
步骤2：5-氯-N-(4-氟-2-甲氧基-5-硝基苯基)-4-吗啉嘧啶-2-胺(化合物7-3)
将化合物7-2(1.0g，4.2mmol)，4-氟-2-甲氧基-5-硝基苯胺(0.83g，4.4mmol)和对甲苯磺酸(2.57g，15.0mmol)加入到10mL1,4-二氧六环溶液中。110℃下，在N2氛围中搅拌8h。反应结束后，过滤，用EA洗滤饼，将滤饼于50℃下烘干，得粗产品化合物7-3(1.16g)，产物直接用于下一步。MS m/z(ESI):284.1[M+H]+
步骤3：5-氯-N-(4-(4-(二甲氨基)哌啶-1-基)-2-甲氧基-5-硝基苯基)-4-吗啉嘧啶-2-胺(化合物7-4)
将化合物7-3(0.1g，0.26mmol)，4-二甲氨基哌啶(0.033g，0.26mmol)和碳酸钾(0.072g，0.52mmol)加入到DMF溶液中。100℃下，在N2氛围中搅拌2h。反应结束后，加水稀释，用EA/水体系萃取，水洗3次，干燥，有机层减压浓缩得粗产品化合物7-4(0.21g)，产物直接用于下一步。MS m/z(ESI):492.2[M+H]+；
步骤4：N-(5-氯-4-吗啉嘧啶-2-基)-4-(4-(二甲氨基)哌啶-1-基)-6-甲氧基苯-1,3-二胺(化合物7-5)
将化合物7-4(0.2g，0.41mmol)，铁粉(0.51g，8.1mmol)和氯化铵(0.11g，2.0mmol)加入到16mLTHF和水的混合溶液中。65℃下，在N2氛围中剧烈搅拌3h。反应结束后，过滤，滤液经EA萃取，水洗，干燥，减压浓缩得化合物7-5(0.19g)，产物直接用于下一步。MS m/z(ESI):462.2[M+H]+
步骤5：N-(5-((5-氯-4-吗啉嘧啶-2-基)氨基)-2-(4-(二甲氨基)哌啶-1-基)-4-甲氧苯基)丙烯酰胺(化合物Z-7)甲酸盐
在0℃下，将丙烯酰氯(40mg,0.43mmol)与三乙胺(50mg,0.49mmol)加入到化合物7-5(190mg,0.41mmol)的5mL二氯甲烷溶液中，0℃下剧烈搅拌2h。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Z-7的甲酸盐(4.27mg,五步收率3.1％)。MS m/z(ESI):562.2[M+H]+；1HNMR(400MHz,DMSO-d6)δ8.98(s,1H),8.57(s,1H),8.28(s,1H),8.06(s,1H),7.83(s,1H),6.80(s,1H),6.65(dd,J＝17.0,10.1Hz,1H),6.21(d,J＝17.0Hz,1H),5.72(d,J＝10.3Hz,1H),3.84(s,3H),3.65(d,J＝7.6Hz,8H),3.02(d,J＝11.0Hz,2H),2.64(t,J＝11.0Hz,2H),2.27(overlap,7H),1.84(d,J＝10.7Hz,2H),1.68(d,J＝9.6Hz,2H)。
实施例8：N-(5-((5-氯-4-吗啉嘧啶-2-基)氨基)-4-甲氧基-2-(4-甲基哌嗪-1-基)苯基)丙烯酰胺(化合物Z-8)
Figure PCTCN2015073044-appb-000049
步骤1：5-氯-N-(2-甲氧基-4-(4-甲基哌嗪-1-基)-5-硝基苯基)-4-吗啉嘧啶-2-胺(化合物8-4)
将化合物7-3(0.5g，1.3mmol)，N-甲基哌嗪(0.13g，1.3mmol)和碳酸钾(0.36g，2.6mmol)加入到DMF溶液中。100℃下，在N2氛围中搅拌2h。反应结束后，加水稀释，用EA/水体系萃取，水洗3次，干燥，有机层减压浓缩得粗产品化合物8-4(0.6g)，产物直接用于下一步。MS m/z(ESI):464.1[M+H]+；
步骤2：N-(5-氯-4-吗啉嘧啶-2-基)-6-甲氧基-4-(4-甲基哌嗪-1-基)苯-1,3-二胺(化合物8-5)
以化合物8-4(0.6g，1.3mmol)为原料，参照实施例7中步骤4进行合成，得化合物8-5(0.56g)，产物直接用于下一步。MS m/z(ESI):434.2[M+H]+
步骤3：N-(5-((5-氯-4-吗啉嘧啶-2-基)氨基)-4-甲氧基-2-(4-甲基哌嗪-1-基)苯基)丙烯酰胺(化合物Z-8)
以化合物8-5(0.56g,1.3mmol)为原料，参照实施例1中步骤7进行合成，得到的粗产物经制备液相分离纯化后得化合物Z-8(44.51mg，三步收率7.0％)。MS m/z(ESI):488.2[M+H]+；1HNMR(400MHz,CDCl3)δ9.26(s,1H),8.46(s,1H),7.94(s,1H),7.43(s,1H),6.69(s,1H),6.29(d,J＝16.8Hz,1H),6.18(dd,J＝16.8,10.0Hz,1H),5.66(dd,J＝10.0,1.3Hz,1H),3.78(s,3H),3.76(s,8H),2.82(d,J＝4.2Hz,4H),2.55(d,J＝13.4Hz,4H),2.32(s,3H)。
实施例9：N-(5-((5-氯-4-吗啉嘧啶-2-基)氨基)-2-((2-(二甲氨基)乙基)甲氨基)-4-甲氧基苯基)丙烯酰胺(化合物Z-9)
Figure PCTCN2015073044-appb-000050
步骤1：N-(5-氯-4-吗啉嘧啶-2-基)-N-(2-(二甲氨基)乙基)-2-甲氧基-N-甲基-5-硝基苯-1,4-二胺(化合物9-4)
将化合物7-3(0.5g，1.3mmol)，N,N,N'-三甲基乙二胺(0.26g，2.6mmol)和碳酸钾(0.36g，2.6mmol)加入到DMF溶液中。100℃下，在N2氛围中搅拌2h。反应结束后，加水稀释，用EA/水体系萃取，水洗3次，干燥，有机层减压浓缩得粗产品化合物9-4(0.6g)，产物直接用于下一步。MS m/z(ESI):466.1[M+H]+。
步骤2：N-(5-氯-4-吗啉嘧啶-2-基)-N-(2-(二甲氨基)乙基)-5-甲氧基-N-甲基苯-1,2,4-三胺(化合物9-5)
以化合物9-4(0.6g，1.3mmol)为原料，参照实施例7中步骤4进行合成，得化合物化合物9-5(0.56g)，产物直接用于下一步。MS m/z(ESI):436.2[M+H]+。
步骤3：N-(5-((5-氯-4-吗啉嘧啶-2-基)氨基)-2-((2-(二甲氨基)乙基)甲氨基)-4-甲氧基苯基)丙烯酰胺(化合物Z-9)
以化合物9-5(0.50g,1.2mmol)为原料，参照实施例1中步骤7进行合成，得到的粗产物经制备液相分离纯化后得化合物Z-9(77.19mg，三步收率12.1％)。MS m/z(ESI):490.2[M+H]+；1HNMR(400MHz,CDCl3)δ10.00(s,1H),9.29(s,1H),7.94(s,1H),7.44(s,1H),6.69(s,1H),6.31(dd,J＝16.9,1.8Hz,1H),6.21(d,J＝9.6Hz,1H),5.59(dd,J＝10.0,1.8Hz,1H),3.78(d,J＝ 7.3Hz,9H),2.79(s,2H),2.62(s,3H),2.18(s,6H),1.53(s,4H)。
实施例10：N-(2-(3-(二甲基氨基)氮杂环丁烷-1-基)-4-甲氧基-5-((4-(喹啉-3-基)嘧啶-2-基)氨基)苯基)丙烯酰胺(化合物Z-10)
Figure PCTCN2015073044-appb-000051
步骤1:3-(2-氯嘧啶-4-基)喹啉(化合物10-2)
将化合物2,4-二氯嘧啶(5.0g,34mmol，市购)和喹啉-3-硼酸(5.8g,34mmol，市购)加入到乙腈(120mL)和碳酸钠溶液(50mL,2M)中。反应体系用氮气置换空气三次后，加入Pd(dppf)Cl2(494mg,0.68mmol)，之后再用氮气置换空气三次。将反应体系加热至80℃，并且持续搅拌6h。TLC检测反应进度，待反应完成后，加水稀释，析出灰色固体，布氏漏斗过滤，滤饼经水洗后，烘干至恒重，得到化合物化合物10-2(4.6g,57％)，直接用于下一步反应。MS m/z(ESI):242.7[M+H]+
步骤2:N-(4-氟-2-甲氧基-5-硝基苯基)-4-(喹啉-3-基)嘧啶-2-胺(化合物10-3)
将4-氟-2-甲氧基-5-硝基苯胺(2.2g,12mmol)和化合物10-2(2.9g,12mmol)加入对甲苯磺酸(p-TSA)(2.1g,12mmol)40ml正丁醇溶液中，130℃条件下封管加热6h。待反应结束后，冷却至室温，溶液中析出大量固体。布氏漏斗过滤得到滤饼，经过乙醇回流打浆后，得到化合物10-3(4.3g，64％)，直接用于下一步反应。MS m/z(ESI):392.0[M+H]+
步骤3：叔丁基3-(二甲基氨基)氮杂环丁烷-1-甲酸叔丁酯(化合物10-5)
将化合物3-氧氮杂环丁烷-1-羧酸叔丁酯(2.0g,11.7mmol，市购)和二甲胺盐酸盐(1.3g,17mmol)加入100ml乙酸(1ml)的甲醇溶液中，加入钯碳(1.4g),氢气换气三次后在氢气环境下搅拌5h，反应结束后过滤掉剩余钯碳，将滤液减压浓缩后得到化合物化合物10-5(2.2g,80％),直接用于下一步反应。MS m/z(ESI):200.1[M+H]+
步骤4：3-(二甲基氨基)氮杂环丁烷(化合物10-6)盐酸盐
将盐酸的1,4-二氧六环溶液(10ml)加入到15ml化合物10-5(2.2g，11mmol)中，常温搅拌3h。反应结束减压浓缩得到化合物化合物10-6(1.3g,90％)，直接用于下一步反应。MS m/z(ESI):100.1[M+H]+
步骤5：N-(4-(3-(二甲基氨基)氮杂环丁烷-1-基)-2-甲氧基-5-硝基苯基)-4-(喹啉-3-基)嘧啶-2-胺(化合物10-7)
将化合物10-6(160mg.1.16mmol)和碳酸钾(350mg,2.3mmol)加入到化合物10-3(300mg,0.58mmol)的4ml DMF溶液中，100度下搅拌2h，TLC检测反应进度，待反应完成后，加入10mL水，用EA/水体系萃取三次，分离出有机层，减压浓缩得到化合物化合物10-7(130mg,50％),直接用于下一步反应。MS m/z(ESI):472.2[M+H]+
步骤6：4-(3-(二甲基氨基)氮杂环丁烷-1-基)-6-甲氧基-N 1-(4-(喹啉-3-基)嘧啶-2-基)苯-1,3-二胺(化合物10-8)
以化合物10-7(130mg,0.27mmol)为原料，参照实施例7中步骤4进行合成，得化合物化合物10-8(80mg.70％)，产物直接用于下一步。MS m/z(ESI):441.2[M+H]+。
步骤7：N-(2-(3-(二甲基氨基)氮杂环丁烷-1-基)-4-甲氧基-5-((4-(喹啉-3-基)嘧啶-2-基)氨基)苯基)丙烯酰胺(化合物Z-10)
以化合物10-8(80mg,0.17mmol)为原料，参照实施例1中步骤7进行合成，得到的粗产物经制备液相分离纯化后得化合物Z-10(30.69mg，30％)。MS m/z(ESI):496.2[M+H]+,1HNMR(400MHz,CDCl3)δ9.40(d,J＝2.0Hz,1H),9.32(s,1H),9.16(s,1H),8.45(d,J＝5.1Hz,1H),8.25(s,1H),8.06(d,J＝8.5Hz,1H),7.68(dd,J＝18.2,10.0Hz,2H),7.54-7.43(m,2H),7.22(d,J＝5.2Hz,1H),6.49-6.37(m,2H),6.33(d,J＝10.1Hz,1H),5.73(d,J＝10.2Hz,1H),3.92-3.78(m,5H),3.58(t,J＝6.5Hz,2H),3.07(d,J＝6.3Hz,1H),2.13(d,J＝17.8Hz,6H)。
实施例11：(R)-N-(2-(3-(二甲基氨基)吡咯烷-1-基)-4-甲氧基-5-((4-(喹啉-3-基)嘧啶-2-基)氨基)苯基)丙烯酰胺(化合物Z-11)
Figure PCTCN2015073044-appb-000052
步骤1：(R)-N-(4-(3-(二甲基氨基)吡咯烷-1-基)-2-甲氧基-5-硝基苯基)-4-(喹啉-3-基)嘧啶-2-胺(化合物11-7)
将(R)-N，N-二甲基吡咯烷-3-胺(80mg，0.70mmol，市购)和碳酸钾(193mg，1.40mmol)加入到化合物化合物10-3(200mg,0.47mmol)的4mL的DMF中，100℃下剧烈搅拌2h。TLC检测反应进度，待反应完成后，加入10mL水，用EA/水体系萃取三次，分离出有机层，减压浓缩得到化合物化合物11-7(180mg，79％)MS m/z(ESI):486.2[M+H]+。
步骤2：(R)-4-(3-(二甲基氨基)吡咯烷-1-基)-6-甲氧基-N1-(4-(喹啉-3-基)嘧啶-2-基)苯-1,3-二胺(化合物11-8)
以化合物11-7(180mg,0.37mmol)为原料，参照实施例1中步骤6进行合成，得化合物化合物11-8(150mg，89％)，产物直接用于下一步。MS m/z(ESI):456.2[M+H]+。
步骤3：(R)-N-(2-(3-(二甲基氨基)吡咯烷-1-基)-4-甲氧基-5-((4-(喹啉-3-基)嘧啶-2-基)氨基)苯基)丙烯酰胺(化合物Z-11)
以化合物11-8(150mg,0.3mmol)为原料，参照实施例1中步骤7进行合成，得到的粗产物经制备液相分离纯化后得化合物Z-11(23.58mg，9.87％)。MS m/z(ESI):509.9[M+H]+；1HNMR(400MHz,DMSO-d6):δ9.67(1s,1H)，9.40(1s,1H),8.59(d,J＝4Hz,1H),8.39(s,1H),8.31(s,1H),8.07(d,J＝8Hz,1H),7.70(m,2H),7.50(m,1H),7.27(m,1H),6.74(s,1H),6.35(m,2H),5.73(d,J＝8Hz,1H),3.83(s,3H),3.05(m,4H),1.16(m,1H),2.24(s,6H),2.05(m,2H)。
实施例12：N-(4-甲氧基-2-(1-甲基-1,2,3,6-四氢吡啶-4-基)-5-((4-(喹啉-3-基)嘧啶-2-基)胺基)苯基)丙酰胺(化合物Z-12)
Figure PCTCN2015073044-appb-000053
步骤1：3-(2-氯嘧啶-4-基)喹啉(化合物10-2，按照实施例10的步骤1)
步骤2：4-溴-2-甲氧基-1-硝基苯(化合物12-4)
在室温条件下，将化合物12-3(10.0g,46mmol，市购)溶解于150mL无水甲醇中，加入甲醇钠(4.37g，81mmol)，加热至回流并剧烈搅拌8h。TLC检测反应进度，待反应完成后，加水稀释，用EA/水体系萃取，水洗3次，干燥，有机层减压浓缩得粗产品化合物12-4(9.5g)，产物直接用于下一步。MS m/z(ESI):231.0[M+H]+
步骤3：4-溴-2-甲氧基苯胺(化合物12-5)
在室温条件下，将还原铁粉(4.48g,80mmol)和氯化铵(2.65g，50mmol)加入到化合物12-4(2.3g,10.0mmol)的60mLTHF/水(THF与水的体积比为2:1)混合溶液中。65℃下，剧烈搅拌4h。反应结束后，过滤，滤液加水稀释，用EA/水体系萃取，水洗3次，干燥，有机层减压浓缩得粗产品化合物12-5(2.0g)，产物直接用于下一步。MS m/z(ESI):201.9[M+H]+
步骤4：4-溴-2-甲氧基-5-硝基苯胺(化合物12-6)
在-20℃条件下，将硝酸钾(217mg，2.25mmol)加入到化合物12-5(400mg,2.0mmol)的3.5mL浓硫酸溶液中。-20℃下，剧烈搅拌5min。反应结束后，加水稀释，用EA/水体系萃取，水洗3次，干燥，有机层减压浓缩得粗产品化合物12-6(300mg)，产物直接用于下一步。MSm/z(ESI):246.9[M+H]+。
步骤5：4-(4-氨基-5-甲氧基-2-硝基苯基)-5,6-二氢吡啶-1(2H)-羧酸叔丁酯(化合物12-7)
将化合物12-6(100mg,0.40mmol)，Pd(PPh3)4(24mg，0.02mmol)和0.5mL的2.0mol/L的碳酸钠溶液加入到化合物4-(4,4,5,5-四甲基-1,3,2-二氧硼戊环-2-基)-5,6-二氢吡啶-1(2H)-甲酸叔丁酯(125mg,0.40mmol)，的5mL1,4-二氧六环溶液中。100℃下，在N2氛围中剧烈搅拌4h。反应结束后，过滤，滤液减压浓缩后得粗产品，经Combi-flash柱层析[PE:EA＝100:0-0:100]纯化后得化合物12-7(50mg，35％)。MS m/z(ESI):350.1[M+H]+
步骤6：4-(5-甲氧基-2-硝基-4-((4-(喹啉-3-基)嘧啶-2-基)氨基)苯基)-5,6-二氢吡啶-1(2H)-甲酸叔丁酯(化合物12-8)
将化合物12-7(500mg，1.43mmol)，Pd2(dba)3(131mg，0.143mmol)，Xantphos(165mg，0.286mmol)和碳酸铯(925mg，2.86mmol)加入到化合物10-2(345mg,1.43mmol)的30mL1,4-二氧六环溶液中。100℃下，在N2氛围中剧烈搅拌5h。反应结束后，过滤，滤液减压浓缩后得粗产品，经Combi-flash柱层析[DCM:MeOH＝100:0-0:100]纯化后得化合物12-8(600mg，76％)。MS m/z(ESI):545.0[M+H]+
步骤7：N-(2-甲氧基-5-硝基-4-(1,2,3,6-四氢吡啶-4-基)苯基)-4-(喹啉-3-基)嘧啶-2-胺(化合物12-9)
在室温下，向化合物化合物12-8(150mg,0.27mmol)的二氯甲烷(10ml)溶液滴加1ml三氟乙酸，滴加完毕后室温下搅拌3h。反应液用饱和碳酸氢钠水溶液中和，分离有机相，水相用二氯甲烷萃取(2*10ml)，合并有机相，用饱和食盐水(10ml)洗涤，无水Na2SO4干燥，过滤，减压浓缩得化合物化合物12-9(115mg,93％)，产物直接用于下一步反应。MSm/z(ESI):455.2[M+H]+。
步骤8：N-(2-甲氧基-4-(1-甲基-1,2,3,6-四氢吡啶-4-基)-5-硝基苯)-4-(喹啉-3-基)嘧啶-2-胺(化合物12-10)
在室温下，向化合物化合物12-9(115mg,0.25mmol)的甲醇(5ml)溶液中加入多聚甲醛(45mg,0.5mmol)和醋酸硼氢化钠(106mg,0.5mmol),室温下继续搅拌3h后，加入20mL二氯甲烷，有机相依次用水洗(10ml)，饱和食盐水洗(10ml)，无水Na2SO4干燥，过滤，减压浓缩得化合物化合物12-10(95mg,81％)，产物直接用于下一步反应。MSm/z(ESI):469.2[M+H]+。
步骤9：6-甲氧基-4-(1-甲基-1,2,3,6-四氢吡啶-4-基)-N1-(4-(喹啉-3-基)嘧啶-2-基)苯-1,3-二胺(化合物12-11)
以化合物12-10(95mg,0.2mmol)为原料，参照实施例7中步骤4进行合成，得化合物化合物12-11(74mg,84％)，产物直接用于下一步反应。MS m/z(ESI):439.2[M+H]+。
步骤10：N-(4-甲氧基-2-(1-甲基-1,2,3,6-四氢吡啶-4-基)-5-((4-(喹啉-3-基)嘧啶-2-基)胺基)苯基)丙酰胺(化合物Z-12)
以化合物12-11(74mg,0.17mmol)为原料，参照实施例1中步骤7合成，得到的粗产物经制备液相分离后得化合物Z-12(14mg,17％)。MS m/z(ESI):493.2[M+H]+。
实施例13：N-(2-((2-(二甲基氨基)乙基)(甲基)氨基)-4-甲氧基-5-(5-甲氧基-4-(喹啉-3-基)嘧啶-2-基氨基)苯基)丙烯酰胺(Z-13)
Figure PCTCN2015073044-appb-000054
步骤1：3-(2-氯-5-甲氧基嘧啶-4-基)喹啉(化合物13-2)
将喹啉-3-硼酸(2.93g，16.9mmol)，PdCl2(dppf)(250mg，0.34mmol)和50mL的2.0mol/L的碳酸钠溶液加入到化合物13-1(3g,16.9mmol，市购)的80mL乙腈溶液中。95℃下，在N2氛围中剧烈搅拌8h。反应结束后，倒入大量的冷水中，至固体完全析出。过滤，用水充分洗涤，干燥后得化合物化合物13-2(4g，100％)。MS m/z(ESI):272.7[M+H]+。
步骤2：N-(4-氟-2-甲氧基-5-硝基苯基)-5-甲氧基-4-(喹啉-3-基)嘧啶-2-胺(化合物13-3)
将化合物4-硝基-2-甲氧基-5-硝基苯胺(343mg，1.85mmol)，Pd2(dba)3(169mg，0.18mmol)，Xantphos(214mg，0.37mmol)和碳酸铯(1.2g，3.69mmol)加入到化合物化合物13-2(500mg，1.85mmol)的9mL1,4-二氧六环溶液中。120℃下，在N2氛围中剧烈搅拌5h。反应结束后，过滤，滤液减压浓缩后得粗产品，经Combi-flash柱层析[PE:EA＝100:0-0:100]纯化后得化合物化合物13-3(180mg，65％)。MS m/z(ESI):422.1[M+H]+。
步骤3：N1-(2-(二甲基氨基)乙基)-5-甲氧基-N4-(5-甲氧基-4-(喹啉-3-基)嘧啶-2-基)-N-甲基-2-硝基苯-1，4-二胺(化合物13-4)
将N,N,N'-三甲基乙二胺(65mg，0.64mmol)和碳酸钾(193mg，1.29mmol)加入到化合物化合物13-3(180mg,0.43mmol)的3mL的DMF中，100℃下剧烈搅拌2h。TLC检测反应进度，待反应完成后，加入10mL水，用EA/水体系萃取三次，分离出有机层，减压浓缩得到化合物化合物13-4(200mg，91％)。MS m/z(ESI):504.3[M+H]+。
步骤4：N1-(2-(二甲基氨基)乙基)-5-甲氧基-N4-(5-甲氧基-4-(喹啉-3-基)嘧啶-2-基)-N1-甲基苯-1,2,4-三胺(化合物13-5)
以化合物13-4(200mg,0.40mmol)为原料，参照实施例1中步骤6合成，得化合物化合物13-5(100mg，92％)，产物直接用于下一步。MS m/z(ESI):474.1[M+H]+。
步骤5：N-(2-((2-(二甲基氨基)乙基)(甲基)氨基)-4-甲氧基-5-(5-甲氧基-4-(喹啉-3-基)嘧啶-2-基氨基)苯基)丙烯酰胺(化合物Z-13)
以化合物13-5(100mg,0.21mmol)为原料，参照实施例1中步骤7合成，得到的粗产物经制备液相分离纯化后得化合物Z-13(4.55mg，4％)。MS m/z(ESI):527.8[M+H]+；1HNMR(400MHz,DMSO-d6)δ9.57(s,1H),9.21(d,J＝16.7Hz,2H),8.57(s,1H),8.28-8.19(m,2H),8.07(d,J＝8.2Hz,1H),7.96(s,1H),7.85(t,J＝7.4Hz,1H),7.65(t,J＝7.3Hz,1H),7.02(s,1H),6.61- 6.47(m,1H),6.32(d,J＝17.1Hz,1H),5.80(d,J＝10.8Hz,1H),3.97(s,3H),3.88(s,3H),2.94(s,2H),2.68(s,3H),2.45(s,2H),2.30(s,6H)。
实施例14:N-(2-((2-(二甲基氨基)乙基)(甲基)氨基)-4-甲氧基-5-(4-(喹啉-3-基)-1,3,5-三嗪-2-基氨基)苯基)丙烯酰胺(Z-14)
Figure PCTCN2015073044-appb-000055
步骤1:4-氯-N-(4-氟-2-甲氧基-5-硝基苯基)-1,3,5-三嗪-2-胺(化合物14-2)
将化合物1a(200mg,1.1mmol，市购)置于250mL反应瓶中，加入THF(10mL)使底物溶解。在80℃下，向搅拌的反应瓶中加入4-氟-2-甲氧基-5-硝基苯胺(200mg,1.1mmol)的THF(30mL)溶液，保持温度滴加50min。滴加完毕后继续反应10min。TLC检测反应进度，待反应完成后，减压浓缩得到粗品。经Combi-Flash柱层析纯化得到目标产品化合物14-2(110mg,34％)。MS m/z(ESI):300.0[M+H]+。
步骤2:N-(4-氟-2-甲氧基-5-硝基苯基)-4-(喹啉-3-基)-1,3,5-三嗪-2-胺(化合物14-3)
将反应底物化合物14-2(1.2g,4mmol)，3-喹啉硼酸(5.8g,34mmol)，碳酸钾(1.11g,8mmol)置于250mL反应瓶中，加入乙二醇二甲醚(20mL)和水(5mL)。反应体系用氮气置换空气三次后，加入Pd(dppf)Cl2(300mg,0.4mmol)，之后再用氮气置换空气三次。将反应体系加热至100℃，并且持续搅拌4h。TLC检测反应进度，待反应完成后，减压浓缩得到粗品。经Combi-Flash柱层析纯化得到目标产品化合物14-3(700mg,45％)。MS m/z(ESI):393.1[M+H]+。
步骤3:N1-(2-(二甲基氨基)乙基)-5-甲氧基-N1-甲基-2-硝基-N4-(4-(喹啉-3-基)-1,3,5-三嗪-2-基)苯-1,4-二胺(化合物14-4)
将反应底物化合物14-3(400mg,1mmol)，N,N,N'-三甲基乙二胺(156mg,1.5mmol)和碳酸铯(670mg,2mmol)置于50mL反应瓶中，加入二甲氨基甲酰胺(6mL)。反应体系用氮气置换空气三次后，将反应体系加热至80℃，并且持续搅拌4h。TLC检测反应进度，待反应完成后，加水稀释反应液，过滤得粗品，经Combi-Flash柱层析纯化得到目标产品化合物14-4(200mg,42％)。MS m/z(ESI):475.2[M+H]+。
步骤4:N1-(2-(二甲基氨基)乙基)-5-甲氧基-N-甲基-N4-(4-(喹啉-3-基)-1,3,5-三嗪-2-基)苯-1,2,4-三胺(化合物14-5)
以化合物14-4(190mg,0.4mmol)为原料，参照实施例1中步骤6合成，得到化合物化合物14-5(180mg)，直接用于下一步反应。MS m/z(ESI):445.2[M+H]+。
步骤5:N-(2-((2-(二甲基氨基)乙基)(甲基)氨基)-4-甲氧基-5-(4-(喹啉-3-基)-1,3,5-三嗪-2-基 氨基)苯基)丙烯酰胺(化合物Z-14)
以化合物14-5(180mg,0.41mmol)为原料，参照实施例1中步骤7合成，得到的粗产物经Prep-TLC纯化后得到目标产品化合物Z-14(2.01mg,1％)。纯度：94.99％。MS m/z(ESI):499.3[M+H]+。
实施例15：N-(2-(4-(二甲基氨基)哌啶-1-基)-4-甲氧基-5-(4-(喹啉-3-基)-1,3,5-三嗪-2-基氨基)苯基)丙烯酰胺(Z-15)
Figure PCTCN2015073044-appb-000056
步骤1:N-(4-(4-(二甲基氨基)哌啶-1-基)-2-甲氧基-5-硝基苯基)-4-(喹啉-3-基)-1,3,5-三嗪-2-胺(化合物15-4)
将化合物14-3(100mg,0.25mmol)，4-二甲氨基哌啶(66mg,0.51mmol)和碳酸钾(140mg,1.02mmol)置于20mL反应瓶中，加入二甲氨基甲酰胺(4mL)。反应体系用氮气置换空气三次后，将反应体系加热至100℃，并且持续搅拌4h。TLC检测反应进度，待反应完成后，反应液用EA/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Na2SO4干燥后，减压浓缩得到粗产物。经Combi-Flash柱层析纯化得到目标产品化合物15-4(110mg,43％)。MS m/z(ESI):501.2[M+H]+。
步骤2:4-(4-(二甲基氨基)哌啶-1-基)-6-甲氧基-N1-(4-(喹啉-3-基)-1,3,5-三嗪-2-基)苯-1,3-二胺(化合物15-5)
以化合物15-4(100mg,0.2mmol)为原料，参照实施例1中步骤6合成，得到化合物化合物15-5(90mg)，直接用于下一步反应。MS m/z(ESI):471.3[M+H]+。
步骤3:N-(2-(4-(二甲基氨基)哌啶-1-基)-4-甲氧基-5-(4-(喹啉-3-基)-1,3,5-三嗪-2-基氨基)苯基)丙烯酰胺(化合物Z-15)
以化合物15-5(90mg,0.2mmol)为原料，参照实施例1中步骤7合成，得到的粗产物经Prep-HPLC柱层析纯化后得到目标产品化合物Z-15(2.09mg,2％,FA salt)。纯度：100.0％。MS m/z(ESI):525.3[M+H]+。
实施例16：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)嘧啶-2-基)氨基)苯基)丙烯酰胺(化合物Z-16)
Figure PCTCN2015073044-appb-000057
Figure PCTCN2015073044-appb-000058
步骤1：5-(2-氯嘧啶-4-基)-1-甲基-1H-吡咯并[2,3-b]吡啶(化合物16-2)
将化合物16-1(2.0g，13.5mmol)，PdCl2(dppf)(1.04g，1.35mmol)和13.5mL的2.0mol/L的碳酸钠溶液加入到化合物1-甲基-5-(4,4,5,5-四甲基-1,3,2-二氧杂硼烷-2-基)-1H-吡咯并[2,3-b]吡啶(8.6g,13.5mmol)的50mL乙腈溶液中。80℃下，在N2氛围中剧烈搅拌4h。反应结束后，加水稀释，用EA/水体系萃取，水洗3次，干燥，有机层减压浓缩得粗产品。经Combi-flash柱层析[PE:EA＝100:0-20:80]纯化后得化合物化合物16-2(2.1g，36％)。MSm/z(ESI):245.0[M+H]+。
步骤2：N-(4-氟-2-甲氧基-5-硝基苯基)-4-(1-甲基-1H-吡咯并[2,3-b]吡啶-5-基)嘧啶-2-胺(化合物16-3)
将4-氟-2-甲氧基-5-硝基苯胺(1.6g,8.6mmol)和化合物16-2(2.1g,8.6mmol)加入对甲苯磺酸(3.7g,21.5mmol)20ml异丁醇溶液中，130℃条件下封管加热6h。待反应结束后，冷却至室温，溶液中析出大量固体。布氏漏斗过滤得到滤饼，经过乙醇回流打浆后，得到化合物16-3(1.2g，44％)，直接用于下一步反应。MS m/z(ESI):429.0[M+H]+。
步骤3：N1-(2-(二甲基氨基)乙基)-5-甲氧基-N-甲基-N4-(4-(1-甲基-1H-吡咯并[2,3-b]吡啶-5-基)嘧啶-2-基)-2-硝基苯-1,4-二胺(化合物16-4)
以化合物16-3(370mg,0.62mmol)为原料，参照实施例1中步骤5合成，得到化合物化合物16-4(300mg，90％)。MS m/z(ESI):477.2[M+H]+。
步骤4：N1-(2-(二甲基氨基)乙基)-5-甲氧基-N-甲基-N4-(4-(1-甲基-1H-吡咯并[2,3-b]吡啶-5-基)嘧啶-2-基基)苯-1,2,4-三胺(化合物16-5)
以化合物16-4(300mg，0.6mmol)为原料，参照实施例7中步骤4合成，得化合物化合物16-5(200mg)，产物直接用于下一步。MS m/z(ESI):447.2[M+H]+。
步骤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-吡咯并[2,3-b]吡啶-5-基基)嘧啶-2-基)氨基)苯基)丙烯酰胺(化合物Z-16)
以化合物16-5(200mg,0.44mmol)为原料，参照实施例1中步骤7合成，得到的粗产物经制备液相分离纯化后得化合物Z-16(23mg，10％)。MS m/z(ESI):501.3[M+H]+；1HNMR(400MHz,CDCl3)δ10.04(s,1H),9.78(s,1H),9.20(s,1H),8.90(d,J＝2.0Hz,1H),8.41(d,J＝5.2Hz,1H),7.70(s,1H),7.15(dd,J＝6.9,4.4Hz,2H),6.73(s,1H),6.59(d,J＝3.4Hz,1H),6.49-6.42(m,1H),6.30(d,J＝10.1Hz,1H),5.65(d,J＝10.1Hz,1H),3.84(d,J＝13.7Hz,6H),2.86-2.77(m,2H),2.64(s,3H),2.22(d,J＝5.8Hz,2H),2.19(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-1H-吡唑-4-基)嘧啶-2-基)氨基)苯基)丙烯酰胺(Z-17)
Figure PCTCN2015073044-appb-000059
步骤1：2-氯-4-(1-甲基-1H-吡唑-4-基)嘧啶(化合物17-2)
将化合物16-1(745mg,5mmol)和甲基-4-吡唑频哪醇硼酸酯(1.092g,5.25mmol，市购)置于100mL反应瓶中，加入乙腈(30mL)和碳酸钠溶液(5mL,2M)。反应体系用氮气置换空气三次后，加入Pd(dppf)Cl2(109.7mg,0.15mmol)，之后再用氮气置换空气三次。将反应体系加热至110℃，并且持续搅拌6h。TLC检测反应进度，待反应完成后，加水稀释，反应液用EA/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Na2SO4干燥后，减压浓缩得到粗产物1.1g。经Combi-Flash柱层析[EA:PE＝1:5-1:2]纯化得到目标产品化合物17-2(600mg，93％)。MS m/z(ESI):195.1[M+H]+。
步骤2：N-(4-氟-2-甲氧基-5-硝基苯基)-4-(1-甲基-1H-吡唑-4-基)嘧啶-2-胺(化合物17-3)
将化合物17-2(500mg,2.577mmol)和4-氟-2-甲氧基-5-硝基苯胺(479g,2.577mmol)置于50mL的封管反应瓶中，加入正丁醇(12mL)使底物部分溶解。之后加入对甲苯磺酸(1.108g,6.44mmol)，并保持反应体系在130℃条件下封管加热5h。待反应结束后，冷却至室温，溶液中析出大量固体。布氏漏斗过滤得到滤饼，经过乙醇回流打浆后，得到目标产物化合物17-3(600mg，67.8％)，直接用于下一步反应。
步骤3：N-(4-(4-(二甲基氨基)哌啶-1-基)-2-甲氧基-5-硝基苯基)-4-(1-甲基-1H-吡唑-4-基)嘧啶-2-胺(化合物17-4)
将化合物17-3(70mg,0.203mmol)和碳酸钾(84mg,0.609mmol)置于25mL反应瓶中，加入DMF(5mL)使底物部分溶解。之后加入N,N-二甲氨基哌啶(31.2mg,0.244mmol)并保持反应体系在70℃条件下加热2h。TLC检测反应进度，待反应完成后，反应液用EA/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Na2SO4干燥后，减压浓缩得到化合物17-4(70mg，76％)。MS m/z(ESI):453.2[M+H]+。
步骤4：4-(4-(二甲基氨基)哌啶-1-基)-6-甲氧基-N 1-(4-(1-甲基-1H-吡唑-4-基)嘧啶-2-基)苯-1,3-二胺(化合物17-5)
以化合物17-4(70mg,0.155mmol)为原料，参照实施例1中步骤6合成，得到粗产物化合物17-5(60mg，92％)，直接用于下一步反应。
步骤5：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)嘧啶-2-基)氨基)苯基)丙烯酰胺(化合物Z-17)
以化合物17-5(50mg,0.118mmol)为原料，参照实施例1中步骤7合成，得到的粗产物经制备板[DCM:MeOH＝10:1]纯化后得到目标产品化合物Z-17(30mg，44％)。MS m/z(ESI):477.2[M+H]+；1HNMR(400MHz,DMSO-d6):δ9.14(s,1H),9.05(s,1H),8.81(s,1H),8.36-8.35(d,J＝4.0Hz,1H),8.21(s,1H),7.76(s,1H),7.10-7.09(d,J＝4.0Hz,1H),6.86(s,1H),6.76-6.69(m,1H),6.33-6.29(d,J＝16.0Hz,1H),5.80-5.77(d,J＝12.0Hz,1H),3.92(s,3H),3.88(s,3H),3.03-3.00(m,2H),2.69-2.64(m,2H),2.27-2.15(m,7H),1.85-1.82(m,2H),1.73-1.68(m,2H)。
实施例18：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)嘧啶-2-基)氨基)苯基)丙烯酰胺(Z-18)
Figure PCTCN2015073044-appb-000060
步骤1：N1-(2-(二甲基氨基)乙基)-5-甲氧基-N-甲基-N 4-(4-(1-甲基-1H-吡唑-4-基)嘧啶-2-基)-2-硝基苯-1，4-二胺(化合物18-4)
将化合物17-3(130mg,0.378mmol，制备方法参照实施例17步骤2)和碳酸钾(156mg,1.133mmol)置于50mL反应瓶中，加入DMF(5mL)使底物部分溶解。之后加入N,N,N'-三甲基乙二胺(57.8mg,0.567mmol)并保持反应体系在70℃条件下加热3h。TLC检测反应进度，待反应完成后，反应液用EA/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Na2SO4干燥后，减压浓缩得到粗产物化合物18-4(110mg，68％)。MSm/z(ESI):427.2[M+H]+。
步骤2：N1-(2-(二甲基氨基)乙基)-5-甲氧基-N-甲基-N 4-(4-(1-甲基-1H-吡唑-4-基)嘧啶-2-基)苯-1,2,4-三胺(化合物18-5)
以化合物18-4(110mg,0.258mmol)为原料，参照实施例1中步骤6合成，得到粗产物化合物18-5(110mg，100％)，直接用于下一步反应。
步骤3：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)嘧啶-2-基)氨基)苯基)丙烯酰胺(化合物Z-18)
以化合物18-5(110mg,0.278mmol)为原料，参照实施例1中步骤7合成，得到的粗产物经制备板纯化，DCM:MeOH体积比为10:1，纯化后得到目标产品化合物Z-18(10.0mg，8.2％)。MS m/z(ESI):451.1[M+H]+；1HNMR(400MHz,DMSO-d6):δ10.18(s,1H),9.42(s,1H),8.90(s,1H),8.38-8.37(d,J＝4.0Hz,1H),8.23(s,1H),7.79(s,1H),7.12-7.11(d,J＝4.0Hz,1H),7.03(s,1H),6.46-6.32(m,2H),5.82-5.76(m,1H),3.93(s,3H),3.86(s,3H),2.97-2.93(m,2H),2.68(s,3H),2.08-2.47(m,8H)。
实施例19：N-(5-((5-氯-4-(1-甲基-1H-吡唑-4-基)嘧啶-2-基)氨基)-2-((2-(二甲基氨基)乙基)(甲基)氨基)-4-甲氧苯基)丙烯酰胺(Z-19)
Figure PCTCN2015073044-appb-000061
步骤1：2,5-二氯-4-(1-甲基-1H-吡唑-4-基)嘧啶(化合物19-2)
以2,4,5三氯嘧啶(1.32g,7.21mmol)和甲基-4-吡唑频哪醇硼酸酯(1.5g,7.21mmol)为原料，参照实施例17中步骤1合成，得到的粗产物经Combi-Flash柱层析(EA:PE＝1:5)纯化后得到化合物19-2(1.4g，84.8％)。MS m/z(ESI):229.3[M+H]+。
步骤2：5-氯-N-(4-氟-2-甲氧基-5-硝基苯基)-4-(1-甲基-1H-吡唑-4-基)嘧啶-2-胺(化合物19-3)
将化合物19-2(1.1g，4.8mmol)，Pd2(dba)3(439mg，0.48mmol)，Xantphos(277mg，0.48mmol)和碳酸铯(4.68g，14.4mmol)加入到4-氟-2-甲氧基-5-硝基苯胺(892mg,4.8mmol)的30mL1,4-二氧六环溶液中。100℃下，在N2氛围中剧烈搅拌5h。反应结束后，过滤，滤液减压浓缩后得粗产品，经Combi-flash柱层析[PE:EA＝100:0-0:100]纯化后得到化合物化合物19-3(600mg，33.1％)。MS m/z(ESI):379.3[M+H]+。
步骤3：N1-(5-氯-4-(1-甲基-1H-吡唑-4-基)嘧啶-2-基)-N4-(2-(二甲基氨基)乙基)-2-甲氧基-N4-甲基-5-硝基苯-1,4-二胺(化合物19-4)
以化合物19-3(600mg,1.587mmol)为原料，参照实施例1中步骤5合成，得到粗产物化合物19-4(400mg,57.7％)，直接下一步。MS m/z(ESI):461.1[M+H]+。
步骤4：N4-(5-氯-4-(1-甲基-1H-吡唑-4-基)嘧啶-2-基)-N1-(2-(二甲基氨基)乙基)-5-甲氧基-N-甲基苯-1，2,4-三胺(化合物19-5)
以化合物19-4(200mg,0.435mmol)为原料，参照实施例2中步骤2合成，得到粗产物化合物19-5(200mg)，直接用于下一步反应。MS m/z(ESI):431.3[M+H]+。
步骤5：N-(5-((5-氯-4-(1-甲基-1H-吡唑-4-基)嘧啶-2-基)氨基)-2-((2-(二甲基氨基)乙基)(甲基)氨基)-4-甲氧苯基)丙烯酰胺(化合物Z-19)
以化合物19-5(200mg,0.465mmol)为原料，参照实施例1中步骤7合成，得到的粗产物经Prep-HPLC柱层析纯化后得到目标产品化合物Z-19(23mg,10％)。MS m/z(ESI):485.21HNMR(400MHz,DMSO-d6)δ10.21(s,1H),9.11(s,1H),8.99(s,1H),8.46(s,1H),8.37(s,1H),8.07(s,1H),7.04(s,1H),6.29-6.47(m,2H),5.79-5.81(m,1H),3.97(s,3H),3.87(s,3H),2.89(m,2H),2.70(s,3H),2.15-2.39(m,8H)。
实施例20：N-(2-((2-(二甲基氨基)乙基)(甲基)氨基)-5-((5-氟-4-(1-甲基-1H-吡咯并[2,3-b]吡啶-5-基)嘧啶-2-基)氨基)-4-甲氧苯基)丙烯酰胺(Z-20)
Figure PCTCN2015073044-appb-000062
步骤1：5-(2-氯-5-氟嘧啶-4-基)-1-甲基-1H-吡咯并[2,3-b]吡啶(化合物20-2)
将2,4-二氯-5-氟嘧啶(2.0g,7.8mmol)，PdCl2(dppf)2(0.57g,0.78mmol)和碳酸钠(1.64g,15.6mmol)加入到化合物1-甲基-5-(4,4,5,5-四甲基-1,3,2-二氧杂硼烷-2-基)-1H-吡咯并[2,3-b]吡啶(2.0g,7.8mmol，市购)的20mL乙腈和4mL水的混合溶液中。80℃下，在氮气氛围中剧烈搅拌2h。反应结束后，过滤，滤液减压浓缩后得粗产品，经Combi-flash柱层析[PE:EA＝100:0-60:40]纯化后得化合物化合物20-2(1.12g，55％)。MS m/z(ESI):263.1[M+H]+。
步骤2：5-氟-N-(4-氟-2-甲氧基-5-硝基苯基)-4-(1-甲基-1H-吡咯并[2,3-b]吡啶-5-基)嘧啶-2-胺(化合物20-3)
以化合物20-2(1.5g,5.46mmol)为原料，参照实施例19中步骤2合成，不同的是，120℃下，在N2氛围中剧烈搅拌21h，得到的粗产品经Combi-flash柱层析(PE:EA＝50:50)纯化后得化合物化合物20-3(0.8g，45％)。MS m/z(ESI):413.1[M+H]+。
步骤3：N1-(2-(二甲基氨基)乙基)-N4-(5-氟-4-(1-甲基-1H-吡咯并[2,3-b]吡啶-5-基)嘧啶-2-基)-5-甲氧基-N-甲基-2-硝基苯-1,4-二胺(化合物20-4)
以化合物20-3(300mg,1.18mmol)为原料，参照实施例1中步骤5合成，得到化合物化合物20-4(330mg，92％)，产物直接用于下一步反应。MS m/z(ESI):495.2[M+H]+。
步骤4：N1-(2-(二甲基氨基)乙基)-N4-(5-氟-4-(1-甲基-1H-吡咯并[2,3-b]吡啶-5-基)嘧啶-2-基)-5-甲氧基-N 1-甲基苯-1,2,4-三胺(化合物20-5)
以化合物20-4(330mg,0.67mmol)为原料，参照实施例1中步骤6合成，得化合物20-5(317mg，100％)，产物直接用于下一步。MS m/z(ESI):465.2[M+H]+。
步骤5：N-(2-((2-(二甲基氨基)乙基)(甲基)氨基)-5-((5-氟-4-(1-甲基-1H-吡咯并[2,3-b]吡啶-5-基基)嘧啶-2-基)氨基)-4-甲氧苯基)丙烯酰胺(化合物Z-20)
以化合物20-5(317mg,0.68mmol)为原料，参照实施例1中步骤7合成，得到的粗产物经制备板层析[DCM:MeOH:NH4OH＝90:10:1]分离纯化后得化合物Z-20(11.9mg，3.4％)。MS m/z(ESI):519.2[M+H]+；1HNMR(400MHz,CDCl3)＝9.92(br.s.,1H),9.64(s,1H),9.07(d,J＝9.2Hz,2H),8.31(d,J＝2.8Hz,1H),7.66(s,1H),7.14(d,J＝1.6Hz,1H),6.70(s,1H),6.59(d,J＝1.6Hz,1H),6.42(br.s.,2H),5.66-5.63(m,1H),3.86(s,3H),3.81(s,3H),2.87(br.s.,2H),2.63(s,3H),2.25(br.s.,8H)。
实施例21：N-(2-((2-(二甲基氨基)乙基)(甲基)氨基)-4-甲氧基-5-((5-甲氧基-4-(1-甲基-1H- 吡咯并[2,3-b]吡啶唑-5-基)嘧啶-2-基)氨基)苯基)丙烯酰胺(Z-21)
Figure PCTCN2015073044-appb-000063
步骤1：5-(2-氯-5-甲氧基嘧啶-4-基)-1-甲基-1H-吡咯并[2,3-b]吡啶(化合物21-2)
将2,4-二氯-5-甲氧基嘧啶(1.25g,7.0mmol)，PdCl2(dppf)2(0.5g,0.7mmol)和碳酸钠(1.5g,14.0mmol)加入到化合物1-甲基-5-(4,4,5,5-四甲基-1,3,2-二氧杂硼烷-2-基)-1H-吡咯并[2,3-b]吡啶(1.8g,7.0mmol，市购)的20mL乙腈和4mL水的混合溶液中。80℃下，在N2氛围中剧烈搅拌2h。反应结束后，过滤，滤液减压浓缩后得粗产品，经Combi-flash柱层析[PE:EA＝100:0-60:40]纯化后得化合物化合物21-2(1.54g，80％)。MS m/z(ESI):275.1[M+H]+。
步骤2：N-(4-氟-2-甲氧基-5-硝基苯基)-5-甲氧基-4-(1-甲基-1H-吡咯并[2,3-b]吡啶-5-基)嘧啶-2-胺(化合物21-3)
以化合物21-2(1.5g,5.46mmol)为原料，参照实施例19中步骤2合成，不同的是，120℃下，在N2氛围中剧烈搅拌21h，得粗产品经Combi-flash柱层析[PE:EA＝50:50]纯化后得化合物化合物21-3(1.2g，52％)。MS m/z(ESI):425.2[M+H]+。
步骤3：N1-(2-(二甲基氨基)乙基)-5-甲氧基-N4-(5-甲氧基-4-(1-甲基-1H-吡咯并[2,3-b]吡啶-5-基)嘧啶-2-基)-N1-甲基-2-硝基苯-1,4-二胺(化合物21-4)
以化合物21-3(500mg,1.18mmol)为原料，参照实施例1中步骤5合成，得到化合物化合物21-4(620mg，100％)，产物直接用于下一步反应。MS m/z(ESI):507.3[M+H]+。
步骤4：N1-(2-(二甲基氨基)乙基)-5-甲氧基-N4-(5-甲氧基-4-(1-甲基-1H-吡咯并[2,3-b]吡啶-5-基)嘧啶-2-基)-N1-甲基苯-1,2,4-三胺(化合物21-5)
以化合物化合物21-4(500mg,0.99mmol)为原料，参照实施例1中步骤6合成，得化合物化合物21-5(332mg，70.3％)，产物直接用于下一步。MS m/z(ESI):477.2[M+H]+。
步骤5：N-(2-((2-(二甲基氨基)乙基)(甲基)氨基)-4-甲氧基-5-((5-甲氧基-4-(1-甲基-1H-吡咯并[2,3-b]吡啶唑-5-基)嘧啶-2-基)氨基)苯基)丙烯酰胺(化合物Z-21)
以化合物21-5(332mg,0.7mmol)为原料，参照实施例1中步骤7合成，得到的粗产物经制备板层析[DCM:MeOH:HN4OH＝90:10:1]分离纯化后得化合物Z-21(59mg，15.6％)。MS m/z(ESI):531.2[M+H]+；1HNMR(400MHz,CDCl3)：9.83(br.s.,1H),9.63(s,1H),9.13(d,J＝1.6Hz,1H),8.93(s,1H),8.23(s,1H),7.52(s,1H),7.11(d,J＝3.6Hz,1H),6.67(s,1H),6.53(d,J＝3.2Hz,1H),6.48-6.34(m,2H),5.63(d,J＝11.6Hz,1H),3.84(s,3H),3.81(s,3H),3.80(s,3H),2.87(br.s.,2H),2.61(s,3H),2.37(br.s.,2H),2.28(br.s.,6H)。
实施例22：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)-5-(三氟甲基)嘧啶-2-基)氨基)苯基)丙烯酰胺(Z-22)
Figure PCTCN2015073044-appb-000064
步骤1：5-(2-氯-5-(三氟甲基)嘧啶-4-基)-1-甲基-1H-吡咯并[2,3-b]吡啶(化合物22-2)
将2,4-二氯-5-(三氟甲基)嘧啶(1.0g，6.5mmol)，PdCl2(dppf)(0.5g，0.65mmol)和6.5mL的2.0mol/L的碳酸钠溶液加入到化合物1-甲基-5-(4,4,5,5-四甲基-1,3,2-二氧杂硼烷-2-基)-1H-吡咯并[2,3-b]吡啶(4.3g,6.5mmol)的25mL乙腈溶液中。80℃下，在N2氛围中剧烈搅拌4h。反应结束后，加水稀释，用EA/水体系萃取，水洗3次，干燥，有机层减压浓缩得粗产品。经Combi-flash柱层析[PE:EA＝100:0-20:80]纯化后得化合物化合物22-2(0.6g，36％)。MSm/z(ESI):313.0[M+H]+。
步骤2：N-(4-氟-2-甲氧基-5-硝基苯基)-4-(1-甲基-1H-吡咯并[2,3-b]吡啶-5-基)-5-(三氟甲基)嘧啶-2-胺(化合物22-3)
将4-氟-2-甲氧基-5-硝基苯胺(760mg,4.1mmol)和化合物22-2(1.0g,4.1mmol)加入对甲苯磺酸(p-TSA)(1.7g,10.5mmol)20ml异丁醇溶液中，130℃条件下封管加热6h。待反应结束后，冷却至室温，溶液中析出大量固体。布氏漏斗过滤得到滤饼，经过乙醇回流打浆后，得到化合物22-3。(740mg，44％)，直接用于下一步反应。MS m/z(ESI):463.1[M+H]+。
步骤3：N1-(2-(二甲基氨基)乙基)-5-甲氧基-N-甲基-N 4-(4-(1-甲基-1H-吡咯并[2,3-b]吡啶-5-基)-5-(三氟基)嘧啶-2-基)-2-硝基苯-1,4-二胺(化合物22-4)
以化合物22-3(370mg,0.62mmol)为原料，参照实施例1中步骤5合成，得到化合物化合物22-4(300mg，90％)MS m/z(ESI):544.2[M+H]+。
步骤4：N1-(2-(二甲基氨基)乙基)-5-甲氧基-N-甲基-N 4-(4-(1-甲基-1H-吡咯并[2,3-b]吡啶-5-基)-5-(三氟基)嘧啶-2-基)苯-1,2,4-三胺(化合物22-5)
以化合物22-4(300mg，0.6mmol)为原料，参照实施例7中步骤4合成，得化合物化合物22-5(200mg)，产物直接用于下一步。MS m/z(ESI):514.2[M+H]+。
步骤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-吡咯并[2,3-b]吡啶-5-基)-5-(三氟甲基)嘧啶-2-基)氨基)苯基)丙烯酰胺(化合物Z-22)
以化合物22-5(200mg,0.44mmol)为原料，参照实施例1中步骤7合成，得到的粗产物经制备液相分离纯化后得化合物Z-22(44.9mg，20％)。MS m/z(ESI):568.8[M+H]+；1HNMR(400MHz,CDCl3)δ10.03(s,1H),9.60(s,1H),8.76(s,1H),8.56(s,1H),7.85(s,1H),7.16(s,2H),6.72(s,1H),6.52(s,1H),6.42(d,J＝16.8Hz,1H),6.24(dd,J＝17.0,10.1Hz,1H),5.64(d,J＝ 10.2Hz,1H),3.83(d,J＝24.8Hz,6H),2.79(s,2H),2.63(s,3H),2.20(d,J＝12.4Hz,8H)。
实施例23：N-(5-((5-氯-4-(1,4-氧氮杂环庚-4-基)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(Z-23)
Figure PCTCN2015073044-appb-000065
步骤1：4-(2,5-二氯嘧啶-4-基)-1,4-氧氮杂庚环(化合物23-2)
在0℃下，将化合物2,4,5-三氯嘧啶(0.3g，1.65mmol)加入到10mLTHF中，加入三乙胺(0.49g，4.9mmol)，将高吗啉(0.22g，1.65mmol，市购)加至反应液中，室温下搅拌1h。TLC检测反应进度，待反应完成后，加入水和EA，分离出有机层，有机层经饱和食盐水系，干燥，减压浓缩得到化合物化合物23-2(0.5g)，产物直接用于下一步。MS m/z(ESI):248.0[M+H]+。
步骤2：5-氯-N-(4-氟-2-甲氧基-5-硝基苯基)-4-(1,4-氧氮杂环庚-4-基)嘧啶-2-胺(化合物23-3)
将化合物23-2(0.5g，2.0mmol)，4-氟-2-甲氧基-5-硝基苯胺(0.37g，2.0mmol)和对甲苯磺酸(0.87g，5.0mmol)加入到10mL1,4-二氧六环溶液中。110℃下，在N2保护下搅拌8h。反应结束后，过滤，用EA洗滤饼，将滤饼于50℃下烘干，得粗产品化合物化合物23-3(0.45g)，产物直接用于下一步。MS m/z(ESI):398.1[M+H]+。
步骤3：N1-(5-氯-4-(1,4-氧氮杂环庚-4-基)嘧啶-2-基)-N4-(2-(二甲基氨基)乙基)-2-甲氧基-N4-甲基-5-硝基苯1,4-二胺(化合物23-4)
以化合物23-3(0.45g，1.1mmol)为原料，参照实施例1中步骤5合成，得粗产品化合物23-4(0.4g)，产物直接用于下一步。MS m/z(ESI):480.2[M+H]+。
步骤4：N4-(5-氯-4-(1,4-氧氮杂环庚-4-基)嘧啶-2-基)-N1-(2-(二甲基氨基)乙基)-5-甲氧基-N-甲基苯-1，2，4-三胺(化合物23-5)
以化合物23-4(0.4g，0.83mmol)为原料，参照实施例7中步骤4合成，得化合物化合物23-5(0.25g)，产物直接用于下一步。MS m/z(ESI):450.2[M+H]+。
步骤5：N-(5-((5-氯-4-(1,4-氧氮杂环庚-4-基)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(化合物Z-23)
以化合物23-5(0.25g,0.55mmol)为原料，参照实施例1中步骤7合成，得到的粗产物经制备液相分离纯化后得化合物Z-23(69.8mg，8.4％)。MS m/z(ESI):504.2[M+H]+；1HNMR(400MHz,CDCl3)δ9.99(s,1H),9.18(s,1H),7.89(s,1H),7.32(s,1H),6.68(s,1H),6.30(dd,J＝17.0, 1.9Hz,1H),6.20(d,J＝9.9Hz,1H),5.58(dd,J＝10.0,1.9Hz,1H),4.03-3.98(m,2H),3.96(t,J＝6.2Hz,2H),3.83-3.79(m,2H),3.78(s,3H),3.72-3.66(m,2H),2.84-2.75(m,2H),2.61(s,3H),2.19(s,2H),2.17(s,6H),1.98(dd,J＝11.9,5.9Hz,2H)。
实施例24：N-(5-((4-((1S，4S)-2-氧杂-5α-氮杂双环[2.2.1]庚烷-5-基)-5-氯吡啶-2-基)氨基)-2-((2-(二甲基氨基)乙基)(甲基)氨基)-4-甲氧苯基)丙烯酰胺(Z-24)
Figure PCTCN2015073044-appb-000066
步骤1：(1S，4S)-5-(2,5-二氯嘧啶-4-基)-2-氧杂-5-氮杂双环[2.2.1]庚烷(化合物24-2)
在0℃下，将化合物2,4,5-三氯嘧啶(0.3g，1.65mmol)加入到10mLTHF中，加入三乙胺(0.5g，4.9mmol)，将桥环吗啉(0.22g，1.65mmol，市购)滴加至反应液中，室温下搅拌1h。TLC检测反应进度，待反应完成后，加入水和EA，分离出有机层，有机层经饱和食盐水系，干燥，减压浓缩得到化合物化合物24-2(0.36g)，产物直接用于下一步。MS m/z(ESI):246.0[M+H]+。
步骤2：4-((1S，4S)-2-氧杂-5-氮杂双环[2.2.1]庚烷-5-基)-5-氯-N-(4-氟-2-甲氧基-5-硝基苯基)嘧啶-2-胺(化合物24-3)
以化合物24-2(0.36g，1.4mmol)为原料，参照实施例23中步骤2合成，得粗产品化合物化合物24-3(0.32g)，产物直接用于下一步。MS m/z(ESI):396.1[M+H]+。
步骤3：N1-(4-((1S，4S)-2-氧杂-5-氮杂双环[2.2.1]庚烷-5-基)-5-氯嘧啶-2-基)-N4-2-(二甲基氨基)乙基)基-2-甲氧基-N4-甲基-5-硝基苯-1,4-二胺(化合物24-4)
以化合物24-3(0.32g，0.81mmol)为原料，参照实施例1中步骤5合成，得粗产品化合物24-4(0.31g)，产物直接用于下一步。MS m/z(ESI):478.1[M+H]+。
步骤4：N4-(4-((1S，4S)-2-氧杂-5-氮杂双环[2.2.1]庚烷-5-基)-5-氯嘧啶-2-基)-N1-(2-(二甲基氨基)乙基)基-5-甲氧基-N1-甲基苯-1,2,4-三胺(化合物24-5)
以化合物24-4(0.31g，0.65mmol)为原料，参照实施例7中步骤4合成，得化合物化合物24-5(0.22g)，产物直接用于下一步。MS m/z(ESI):448.2[M+H]+。
步骤5：N-(5-(4-((1S，4S)-2-氧杂-5α-氮杂双环[2.2.1]庚烷-5-基)-5-氯吡啶-2-基)氨基)-2-((2-(二甲基氨基)乙基)(甲基)氨基)-4-甲氧苯基)丙烯酰胺(化合物Z-24)
以化合物24-5(0.2g,0.44mmol)为原料，参照实施例1中步骤7合成，得到的粗产物经制备液相分离纯化后得化合物Z-24(60.1mg，7.3％)。MS m/z(ESI):502.2[M+H]+；1HNMR(400 MHz,DMSO-d6)δ10.09(s,1H),8.85(s,1H),7.96(s,1H),7.67(s,1H),6.96(s,1H),6.37(dd,J＝16.9,10.0Hz,1H),6.22(dd,J＝16.9,2.1Hz,1H),5.73(dd,J＝10.0,2.1Hz,1H),5.32(s,1H),4.61(s,1H),3.88(d,J＝10.5Hz,1H),3.85(s,3H),3.81(d,J＝7.6Hz,1H),3.73(d,J＝7.5Hz,1H),3.59(d,J＝10.5Hz,1H),2.85(t,J＝5.2Hz,2H),2.68(s,3H),2.25(t,J＝5.9Hz,2H),2.18(s,6H),1.90(d,J＝9.9Hz,1H),1.78(d,J＝9.7Hz,1H)。
实施例25-78、87、88：
Figure PCTCN2015073044-appb-000067
实施例25-78、87和88中的目标化合物如式(IV-1)所示，其中，取代基R0、R2和R3都为氢，其余的取代基A、R1、R6如下表所示。
通用步骤：化合物Z-25至Z-56、Z-87、Z-88以不同取代的硼酸酯或者硼酸、5位取代的2,4-二氯嘧啶为原料，参照实施例1的类似的方法进行制备。
化合物Z-57至Z-78以不同胺、5位取代的2,4-二氯嘧啶为原料，参照实施例7的类似的方法进行制备。
Figure PCTCN2015073044-appb-000068
Figure PCTCN2015073044-appb-000069
Figure PCTCN2015073044-appb-000070
Figure PCTCN2015073044-appb-000071
Figure PCTCN2015073044-appb-000072
Figure PCTCN2015073044-appb-000073
实施例79-86
Figure PCTCN2015073044-appb-000074
实施例79-86中的目标化合物如式(IV-2)所示，其中，取代基R0、R2和R3都为氢，其余的取代基A、R1如下表所示。
通用步骤：化合物Z-79至Z-82以不同的胺、2,4-二氯-1,3,5-三嗪为原料，参照实施例23 的类似的方法进行制备。
化合物Z-83至Z-86以不同取代的硼酸酯或者硼酸、以及2,4-二氯-1,3,5-三嗪为原料，参照实施例14的类似的方法进行制备。
Figure PCTCN2015073044-appb-000075
实施例89：N-(5-(5-氯-4-(1-甲基-1H-吡唑-4-基)嘧啶-2-基氨基)-2-(4-(二甲基氨基)哌啶-1-基)-4-甲氧基苯基)丙烯酰胺(Z-89)的制备
Figure PCTCN2015073044-appb-000076
步骤1：在氮气气氛下，将1-甲基-4-(4,4,5,5-四甲基-1,3,2-二氧硼戊环-2-基)-1H-吡唑(1.1g，5.25mmol)、化合物19-1(745mg，4mmol)、Pd(dppf)Cl2(109.7mg，0.15mmol)和碳酸钠溶液(5mL，2M)的混合物的乙腈(30mL)溶液在85℃下搅拌6h。待反应完成后，加水淬灭，向反应混合物中加入EA(150ml)分层后，水相用EA(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[PE:EA＝5:1-2:1]分离纯化得0.6g目标产物89-2。MS m/z(ESI)：229[M+H]+。步骤2：向化合物3a(159mg，0.5mmol)、化合物89-2(114mg，0.5mmol)、Pd2(dba)3(45.75mg，0.05mmol)和Xantphos(28.4mg，0.05mmol)的1,4-二氧六环(4ml)溶液中加入碳酸铯(487.5mg，0.5mmol)，反应化合物在100℃下搅拌3h后，向反应混合物中加入EA(50ml×2)和水(10ml)，有机相用水(10ml×3)洗涤后经Na2SO4干燥，浓缩得到粗产品， 将所得粗产品经制备液相分离纯化得标题化合物Z-89(19mg，4％)，MS m/z(ESI):511.2[M+H]+；1HNMR(400MHz,CDCl3)δ9.50(s,1H),9.19(s,1H),8.63(s,1H),8.40(s,1H),8.24(s,1H),7.66(s,1H),6.69(s,1H),6.28(ddd,J＝26.8,16.9,5.7Hz,2H),5.69(dd,J＝9.9,1.6Hz,1H),4.00(s,3H),3.81(s,3H),2.96(d,J＝12.1Hz,2H),2.66(t,J＝10.9Hz,2H),2.15(dd,J＝9.3,5.5Hz,1H),2.04-1.91(m,2H),1.62(s,2H)。
实施例90：N-(2-(4-(二甲基氨基)哌啶-1-基)-5-(5-氟-4-(1-甲基-1H-吡唑-4-基)嘧啶-2-基氨基)-4-甲氧基苯基)丙烯酰胺(Z-90)的制备
Figure PCTCN2015073044-appb-000077
制备方法同实施例89，不同的是将实施例89步骤1中的化合物19-1换成化合物90-1，标题化合物Z-90(42mg，产率9％)。MS m/z(ESI):495.0[M+H]+；1HNMR(400MHz，DMSO-d6)δ9.05(1s,1H),9.01(1s,1H),8.80(s,1H),8.48(d,J＝4Hz,1H),8.18(s,1H),7.89(s,1H),6.87(s,1H),6.73(m,1H),6.32(m,1H),5.78(t,1H),3.97(s,3H),3.88(s,3H),3.01(d,J＝12Hz,2H),2.66(t,2H),2.19(s,6H),2.16(m,1H),1.84(m,2H),1.71(m,2H)。
实施例91：N-(5-(5-氯-4-(1,3,5-三甲基-1H-吡唑-4-基)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(Z-91)的制备
Figure PCTCN2015073044-appb-000078
制备方法同实施例89，不同的是将实施例89步骤1中的化合物89-1换成化合物91-1，步骤2中的化合物3a换成化合物2a，得标题化合物Z-91(22mg，产率11％)。为黄色固体。MS m/z(ESI):513.2[M+H]+；1HNMR(400MHz,CDCl3)δ9.32(br.s.,1H),8.43(s,1H),7.54(s,1H),6.66(s,1H),6.35(d,J＝16.0Hz,1H),5.62(d,J＝12.0Hz,1H),3.78(s,3H),3.70(s,3H),2.92(br.s.,2H),2.63(s,3H),2.34(br.s.,6H),2.21(s,3H),2.19(s,3H),1.60(br.s.,2H)。
实施例92：N-(5-(5-氯-4-(1,3,5-三甲基-1H-吡唑-4-基)嘧啶-2-基氨基)-2-(4-(二甲基氨基)哌啶-1-基)基-4-甲氧基苯基)丙烯酰胺(Z-92)的制备
Figure PCTCN2015073044-appb-000079
实施例93：N-(2-((2-(二甲基氨基)乙基)(甲基)氨基)-5-(5-氟-4-(1-甲基-1H-吡唑-4-基)嘧啶-2-基氨基)-4-甲氧基苯基)丙烯酰胺(Z-93)的制备
Figure PCTCN2015073044-appb-000080
实施例94：N-(5-(5-氯-4-(1,3-二甲基-1H-吡唑-4-基)嘧啶-2-基氨基)-2-((2-(二甲基氨基)乙基)(甲基)氨基)基-4-甲氧基苯基)丙烯酰胺(Z-94)的制备
Figure PCTCN2015073044-appb-000081
制备方法同实施例91，不同的是将实施例91步骤1中化合物91-1换成化合物94-1，得标题化合物Z-94(13mg，2.6％)，MS m/z(ESI):499.2[M+H]+；1HNMR(400MHz，DMSO-d6)δ10.13(s,1H),8.46(d,J＝3.1Hz,3H),8.40(s,1H),7.00(s,1H),6.41(dd,J＝16.7,10.0Hz,1H),6.22(dd,J＝16.9,2.0Hz,1H),5.75(dd,J＝10.1,1.9Hz,1H),3.81(d,J＝17.7Hz,6H),2.90(s,2H),2.72(s,3H),2.33(s,2H),2.21(d,J＝24.6Hz,9H)。
实施例95：N-(5-(5-氯-4-(1,3-二甲基-1H-吡唑-4-基)嘧啶-2-基氨基)-2-(4-(二甲基氨基)哌啶-1-基)-4-甲氧基苯基)丙烯酰胺(Z-96)的制备
Figure PCTCN2015073044-appb-000082
实施例96：N-(5-(5-氯-4-(1-(哌啶-4-基)-1H-吡唑-4-基)嘧啶-2-基氨基)-2-(4-(二甲基氨基)哌啶-1-基)-4-甲氧基苯基)丙烯酰胺(Z-96)的制备
Figure PCTCN2015073044-appb-000083
步骤1：0℃下，向4-羟基哌啶-1-甲酸叔丁酯(2.0g，10mmol)的二氯甲烷(20ml)溶液中加入三乙胺(2.0g，20mmol)，再滴加甲基磺酰氯(1.73g，15mmol)的二氯甲烷(5ml)溶液。反应混合物搅拌1h后，将反应混合物倒入冰水中，用二氯甲烷萃取，有机相用盐水洗涤后经无水Na2SO4干燥，浓缩，得到2.5g粗产品96-2，未经纯化直接下一步；MS m/z(ESI):223[M-56]+。
步骤2：向4-(4,4,5,5-四甲基-1,3,2-二氧硼戊环-2-基)-1H-吡唑(97mg，0.5mmol)的DMF溶液中加入化合物96-2(134mg，0.5mmol)和碳酸铯(245mg，0.75mmol)，反应混合物在90℃下搅拌12-16h，反应液冷却至室温，加水稀释。用EA萃取，合并的有机相用盐水洗涤，无水Na2SO4干燥，浓缩得到粗产品，粗产品经Combi-flash柱层析[PE:EA＝100:0-50:50]分离纯化得目标产物96-3(2g，产率44％)，MS m/z(ESI):378.2[M+1]+。
步骤3：以化合物19-1(549mg，3.0mmol)和化合物96-3(754mg，2.0mmol)为原料，参照实施例89中步骤1进行制备，得到目标物96-4(580mg，产率73％)，MS m/z(ESI)：398[M+H]+。
步骤4：向化合物96-4(298mg，0.75mmol)、化合物2a(159mg，0.5mmol)、碳酸铯(326mg，1.0mmol)和Xantphos(58mg，0.1mmol)的1,4-二氧六环(2ml)溶液中加入Pd2(dba)3(46mg，0.05mmol)，反应液经氮气置换三次，在160℃下微波反应0.5h后，将反应液过滤并浓缩，得粗产品。粗产品经Combi-flash柱层析[PE:EA＝100:0-0:100]分离纯化得标题化合物96-5(250mg)，为黄色固体。MS m/z(ESI)：680.3[M+H]+。
步骤5：室温下，向化合物96-5(240mg，0.35mmol)的二氯甲烷(10ml)溶液中加入盐酸/1,4- 二氧六环的混合液，反应混合物室温搅拌2h，加入Na2CO3调PH为9，加入二氯甲烷。有机相用盐酸洗涤，浓缩得到粗产品。将所得粗产品经制备液相分离纯化得20mg标题化合物Z-96，为黄色固体。MS m/z(ESI):580[M+H]+。
实施例97：N-(5-(5-氯-4-(1,5-二甲基-1H-吡唑-4-基)嘧啶-2-基氨基)-2-(4-(二甲基氨基)哌啶-1-基)-4-甲氧基苯基)丙烯酰胺(Z-97)的制备
Figure PCTCN2015073044-appb-000084
制备方法同实施例89，不同的是将实施例89步骤1中化合物89-1换成化合物97-1，得标题化合物Z-97(56.5mg，产率26％)，为黄色固体。MS m/z(ESI):525.2[M+H]+；1HNMR(400MHz，DMSO-d6)δ8.99(s,1H),8.44(s,1H),8.39(s,1H),8.20(s,1.5H),7.99(s,1H),6.81(s,1H),6.66(dd,J＝16.8,10.0Hz,1H),6.22(dd,J＝16.8Hz,2.0Hz,1H),5.73(d,J＝10.4Hz,1H),3.79(s,3H),3.76(s,3H),3.07(d,J＝11.0Hz,2H),2.68(t,J＝11.0Hz,2H),2.46(br.s.,1H),2.37(s,6H),2.34(s,3H),1.89(d,J＝10.5Hz,2H),1.75(t,J＝11.0Hz,2H)。
实施例98：N-(5-(5-氯-4-(1-甲基-1H-吡咯-3-基)嘧啶-2-基氨基)-2-(4-(二甲基氨基)哌啶-1-基)-4-甲氧基苯基)丙烯酰胺(Z-98)的制备
Figure PCTCN2015073044-appb-000085
步骤1：以化合物19-1(400mg，2.2mmol)和1-甲基-3-(4,4,5,5-四甲基-1,3,2-二氧硼戊环-2-基)-1H-吡咯(250mg，2.0mmol)为原料，参照实施例89中步骤1进行制备，得到的粗产品经Combi-flash柱层析[PE:EA＝3：1]分离纯化得目标产物98-2(80mg，产率17.6％)。MSm/z(ESI):228[M+H]+。
步骤2：向化合物98-2(100mg，0.44mmol)的1,4-二氧六环(6ml)溶液中加入化合物3a(140mg，0.4mmol)、Pd2(dba)3(41mg，0.044mmol)、BINAP(55mg，0.088mmol)和碳酸铯(286mg，0.88mmol)。将反应液在130℃下微波反应30分钟。将反应液过滤后减压浓缩得到粗产品，将所得粗产品经制备液相分离纯化得94.47mg标题化合物Z-98，为黄色粉末。MSm/z(ESI):510.3[M+H]+；1HNMR(400MHz，DMSO-d6)δ9.06(s,1H),8.80(s,1H),8.34(s,1H),8.28(s,1H),8.06(s,1H),7.92(s,1H),6.98(s,1H),6.84(dd,J＝5.6,3.2Hz,2H),6.71(dd,J＝ 16.9,10.2Hz,1H),6.29(dd,J＝17.0,1.6Hz,1H),5.77(d,J＝11.5Hz,1H),3.87(s,3H),3.74(s,3H),3.05(d,J＝11.4Hz,2H),2.68(t,J＝11.0Hz,2H),2.42(t,J＝11.1Hz,1H),2.34(s,6H),1.89(d,J＝10.6Hz,2H),1.79-1.67(m,2H)。
实施例99：N-(5-(5-氯-4-(1-甲基-1H-吡咯-3-基)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(Z-99)的制备
Figure PCTCN2015073044-appb-000086
实施例100：N-(5-(5-氯-4-(1-甲基-1H-吡唑-4-基)嘧啶-2-基氨基)-4-甲氧基-2-(4-甲基哌嗪-1-基)苯基)丙烯酰胺(Z-100)的制备
Figure PCTCN2015073044-appb-000087
以化合物4a(100mg，0.34mmol)和化合物89-2(80mg，0.34mmol)为原料，参照实施例89步骤2的方法进行合成，得标题化合物Z-100(200mg，产率70％)；MS m/z(ESI):483[M+H]+；1HNMR(400MHz,CDCl3)δ9.51(s,1H),9.10(s,1H),8.41(s,2H),8.26(s,1H),8.20(s,1H),7.70(s,1H),6.73(s,1H),6.43-6.32(m,1H),6.26(d,J＝10.0Hz,1H),5.82-5.65(m,1H),3.99(s,3H),3.83(s,3H),3.03(s,8H),2.62(s,4H)。
实施例101：N-(5-(5-氯-4-(1-(二氟甲基)-1H-吡唑-4-基)嘧啶-2-基氨基)-2-(4-(二甲基氨基)哌啶-1-基)-4-甲氧基苯基)丙烯酰胺(Z-101)的制备
Figure PCTCN2015073044-appb-000088
步骤1：向乙腈(100ml)加入化合物4-(4,4,5,5-四甲基-1,3,2-二氧硼戊环-2-基)-1H-吡唑101-1(4.0g，0.21mol)，二氟氯乙酸钠(3.77g 0.25mol)，15-冠-5(103.8g，0.65mol)，氮气保护下 加热到回流，搅拌24h，冷却至室温，反应液倒入水(50mL)中淬灭，用EA(100ml X 3)萃取，饱和食盐水(20ml)洗涤，Na2SO4干燥，过滤，滤液旋干得到化合物101-2(4.1g，产率80％)白色固体。MS m/z(ESI):245.1[M+H]+。
步骤2：向装有四三苯基膦钯(0.264g，2.58x10-4mol)的反应瓶中加入溶有化合物101-2(2.1g，8.9x10-3mol)的二甲氧基乙烷(20ml)溶液，溶有化合物19-1(1.4g，8.0x10-3mol)的二甲氧基乙烷(20ml)溶液和溶有碳酸钠(1.8g，1.72x10-2mol)的水溶液(11ml)液，氮气保护下，加热至100℃，搅拌过夜，降至室温，水(50ml)淬灭反应液，EA(3X100ml)萃取，饱和食盐水(30ml)洗涤，Na2SO4干燥，过滤浓缩得到油状物，柱层析(硅胶；300-400目)，石油醚：EA＝30：1洗脱得到化合物101-3(870mg，收率30％)，白色固体。MS m/z(ESI):265.0[M+H]+，纯度＝96.08％(UV214)；1HNMR(400MHz，DMSO)δ:9.21(s，1H)，8.95(s，1H)，8.56(s，1H)，7.94(t，J＝58Hz,1H)。
步骤3：以化合物101-3和化合物3a为原料，参照实施例89中步骤2进行合成，所得粗产品经制备液相分离纯化得标题化合物Z-101，为黄色固体。MS m/z(ESI):547[M+H]+；1HNMR(400MHz，DMSO-d6)δ9.28(s,1H),9.06(s,1H),8.76(s,1H),8.65(s,1H),8.55(s,1H),8.28(d,J＝3.6Hz,2H),7.92(t,J＝58.6Hz,1H),6.87(s,1H),6.70(dd,J＝17.0,10.2Hz,1H),6.29(dd,J＝16.9,1.7Hz,1H),5.87-5.65(m,1H),3.86(s,3H),3.04(d,J＝11.6Hz,2H),2.68(t,J＝10.9Hz,2H),2.26(s,7H),1.85(d,J＝10.5Hz,2H),1.71(d,J＝8.9Hz,2H)。
实施例102:N-(5-(5-氯-4-(1H-吡唑-4-基)嘧啶-2-基氨基)-2-(4-(二甲基氨基)哌啶-1-基)-4-甲氧基苯基)丙烯酰胺(Z-102)的制备
Figure PCTCN2015073044-appb-000089
步骤1：搅拌下，向化合物101-1(5.0g，25.8mmol)和2,3-二氢吡喃(4.33g，51.5mmol)的二氯甲烷(100ml)溶液中加入DTSA(463mg，2.6mmol)，反应混合物在40℃下搅拌2h。反应结束后，反应液用水洗涤，EA萃取后，减压浓缩得到粗产品102-2(7.0g，60％)；不经纯化直接下一步，MS m/z(ESI):279[M+H]+。
步骤2：以化合物19-1(4.5g，25mmol)和化合物102-2(7.0g，25mmol)为原料，参照实施例89中步骤1进行制备，得到的粗产品经Combi-flash柱层析分离纯化得目标产物102-3(850mg，产率10％)。MS m/z(ESI)：299[M+H]+。
步骤3：以化合物102-3(300mg，1mmol)和化合物3a(320mg，1mmol)为原料，参照实施例89中步骤2进行制备，得粗产品102-4(60mg，产率70％)；不经纯化直接下一步，MSm/z(ESI):581[M+H]+。
步骤4：搅拌下，向化合物102-4(700mg，1mmol)的二氯甲烷(5ml)溶液中加入盐酸/1,4-二氧六环溶液(2ml)，反应液室温下搅拌4h，得粗产品，将所得粗产品经制备液相分离纯化得标题化合物Z-102(80mg，产率60％)，为黄色固体。MS m/z(ESI):497[M+H]+；1HNMR(400MHz,CDCl3)δ9.55(s,1H),8.92(s,2H),8.70(s,1H),8.35(s,1H),7.72(s,1H),6.76(s,1H),6.32(dt,J＝17.0,12.9Hz,2H),5.76(d,J＝9.9Hz,1H),3.89(s,3H),3.04(d,J＝11.9Hz,2H),2.73(t,J＝11.0Hz,2H),2.36(s,6H),2.24(d,J＝7.4Hz,1H),2.05(d,J＝11.5Hz,2H),1.70(s,2H)。
实施例103-127
实施例103至127中的目标化合物如式(IV-1)所示，其中，取代基R0、R2和R3都为氢，其余的取代基A、R1、R6如下表所示。通用步骤：化合物Z-103至Z-127以不同取代的硼酸酯或者硼酸、5位取代的2,4-二氯嘧啶为原料，参照实施例1的类似方法进行制备。
Figure PCTCN2015073044-appb-000090
Figure PCTCN2015073044-appb-000091
Figure PCTCN2015073044-appb-000092
Figure PCTCN2015073044-appb-000093
Figure PCTCN2015073044-appb-000094
对比例
对比化合物2和3分别以相应的硼酯和5位取代或未取代的2,4-二氯嘧啶为原料，参照实施例1的类似方法制备。
Figure PCTCN2015073044-appb-000095
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可购自Invitrogen。
利用z-lyte方法测定待测物对双突变型EGFR激酶(EGFR T790M/L858R激 酶)(Invitrogen，PV4879)、野生型EGFR激酶(EGFR WT)(Invitrogen，PV3872)活性的抑制作用。
10μL T790M/L858R激酶反应体系中各组分的工作浓度为：25μM ATP,0.08(或0.1)ng/μLEGFR T790M/L858R激酶,2μM Tyr04底物(Invitrogen，PV3193，下同)。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2％。
10μL EGFR WT激酶反应体系中各组分的工作浓度为：10μM ATP,0.8ng/μL EGFR WT激酶,2μM Tyr04底物。加入待测物后DMSO的浓度为2％。
测试方法：
室温溶解10mM的待测化合物储存液经4％DMSO的水梯度稀释至终浓度10-0.005μM。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EGFR T790M/L858R激酶(或EGFR WT激酶)与Tyr04底物的混合物，再加入2.5μL的ATP启动反应。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。
在室温摇床反应60min后。加入5μL Development Reagent B(Invitrogen)，于室温摇床反应60min。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。对酶的抑制活性和选择抑制活性结果参见表1至表3。
表1 酶的抑制活性
化合物编号 T790M/L858R(IC50/nM) EGFR WT(IC50/nM)
Z-1 3 25
Z-2 17 272
Z-4 14 45
Z-5 43 105
Z-6 4 10
Z-16 1 4
Z-20 2 4
Z-21 4 9
Z-22 9 57
Z-25 12 61
Z-26 27 114
Z-27 4 19
Z-29 38 118
Z-32 2 4
Z-37 5 89
Z-38 16 35
Z-39 4 33
Z-40 13 233
Z-41 9 20
Z-44 8 30
Z-54 0.5 2
Z-87 3 8
Z-88 3 11
对比化合物1 2 3
表2 酶的选择抑制活性
化合物编号 对酶的选择抑制活性[IC50(EGFR WT)/IC50(T790M/L858R)]
Z-1 8.33
Z-2 16
Z-3 7.27
Z-4 3.2
Z-5 2.4
Z-6 2.5
Z-16 4
Z-20 2
Z-21 2.25
Z-22 6.3
Z-25 5.1
Z-26 4.2
Z-27 4.8
Z-29 3.1
Z-32 2
Z-37 17.8
Z-38 2.2
Z-39 8.25
Z-40 17.9
Z-41 2.2
Z-44 3.8
Z-54 4
Z-87 2.7
Z-88 3.7
对比化合物1 1.5
表3 酶抑制活性与选择抑制活性
Figure PCTCN2015073044-appb-000096
Figure PCTCN2015073044-appb-000097
从表1、表2和表3可以看出，本发明的示例化合物对EGFR突变型酶(T790M/L858R和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，与阳性对照物BIBW2992(Afatinib)相比，本发明的化合物对EGFR突变型酶具有明显的选择抑制活性。并且本发明示例化合物对EGFR突变型酶的选择抑制活性超过了对比化合物1(具体结构如下所示，并可参见WO2013014448A1)，选择性最高比对比化合物1提高了18倍。
Figure PCTCN2015073044-appb-000098
测试例2：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。
2.1测试方法：
首先，移除培养基并加入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3min后弃上清，并用新鲜的培养基悬浮细胞。然后细胞计数，并稀释细胞至以下浓度：A431和H1975细胞每mL 2.78万，NIH3T3每mL 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：10％FBS(Gibco，10099-141)DMEM(Hyclone SH30243.01B)；
NIH3T3细胞培养基：10％FBS(Gibco，10099-141)DMEM(Hyclone SH30243.01B)；
H1975细胞培养基：10％FBS(Gibco，10099-141)RPMI-1640(Hyclone SH30809.01B)；
取20μL10mM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10X药品，再加入无血清培养基(终浓度为：10，3.333，1.111，0.370，0.123，0.041，0.014，0.005μM)，并加入每孔10μL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72h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4h。
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(约10-20min)。最后使用酶标仪测定492nm光吸收，使用XLFIT 5.0软件(英国IDBS公司)计算IC50。示例化合物对细胞的抑制活性或选择抑制活性见表4至表7。
表4 化合物对细胞生长抑制活性和选择性
Figure PCTCN2015073044-appb-000099
表5 化合物对NIH3T3细胞的毒性测试结果
化合物 IC50/nM 化合物 IC50/nM
Z-1 6587 Z-32 >10000
Z-3 8158 Z-33 >10000
Z-16 >10000 Z-38 >10000
Z-25 6906 Z-39 8887
Z-26 >10000 Z-41 >10000
Z-27 7964 Z-44 8877
Z-30 5793 对比化合物1 3552
表6 化合物对细胞生长的抑制活性和选择性
Figure PCTCN2015073044-appb-000100
Figure PCTCN2015073044-appb-000101
表7 化合物对NIH3T3细胞的毒性测试结果
Figure PCTCN2015073044-appb-000102
从表4和表6可以看出，本发明的示例化合物对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，与阳性对照物BIBW2992相比，本发明的化合物对EGFR突变型细胞生长具有明显的选择抑制活性。并且大部分示例化合物对EGFR突变型细胞生长的选择抑制活性均超过了对比化合物1、2和3。选择性最高比对比化合物1提高至近4倍。而研究发现将A换成吡啶环或喹啉环后，对H1975细胞活性和对细胞生长的选择抑制活性明显降低。
从表5和表7可以看出，本发明的示例化合物对NIH3T3细胞具有较高的IC50值，因此显示出较小的毒性。
测试例3：EGFR T790M抑制剂细胞活性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％NP-40，20mM Tris(pH 8.0)，137mM NaCl，10％glycerol，1mMNaVO3，2mM EDTA。
细胞裂解液：细胞裂解缓冲液+10μg/mL抑肽酶(Aprotinin)(Sigma)，10μg/mL亮抑蛋白肽酶(Leupeptin)(Sigma)，现配现用。
1x PBS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％Tween-20的PBS缓冲液。
检测抗体稀释液：20mM Tris，137mM NaCl，0.05％Tween-20，0.1％BSA，pH 7.2-7.4。
封闭液：含有1％BSA的PBS缓冲液。
ELISA试剂盒：R&D DYC3570，R&D DYC1095E和R&D DYC1095BE。
二、H1975细胞
2.1H1975细胞处理和裂解液制备
(1)将H1975细胞以1×104/孔的密度种到96孔板中，每孔90微升10％FBS,1640培养基，37℃、5％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％，37℃、5％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。
2.2ELISA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8。
三、A431细胞
3.1A431细胞的处理和测试步骤
(1)将A431细胞以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，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。
3.2ELISA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8。
表8 细胞活性ELISA法测定结果
Figure PCTCN2015073044-appb-000103
Figure PCTCN2015073044-appb-000104
从表8可以看出，与阳性对照物BIBW2992相比，本发明的示例化合物对细胞水平靶点具有明显的选择抑制活性。并且与对比化合物1、2和3相比，对细胞水平靶点的选择抑制活性最高提高至90倍。而研究发现将A换成吡啶环或喹啉环后，对H1975细胞活性和细胞水平靶点的选择抑制活性明显降低，甚至没有选择抑制活性。
从体外酶、细胞生长抑制实验显示，本发明化合物对EGFR突变型酶、细胞表现出较强的抑制活性，而对EGFR野生型酶、细胞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。
测试例4：大鼠或小鼠体内试验
应用LC/MS/MS法测定了大鼠或小鼠分别灌胃和静注给予实施例化合物后不同时刻血浆中的药物浓度，研究本发明化合物在大鼠或小鼠体内的药代动力学行为，评价其药动学特征。
实验方案：
试验动物：健康成年雄性SD大鼠(体重200-300g，6只，禁食)或雄性CD1小鼠(体重20-30g，18只，自由饮水和饮食)，由斯莱克公司提供；
给药方式与剂量：给予SD大鼠足背静脉给药(1mg/kg，5mL/kg，5％DMAC(二甲基乙酰胺),5％Solutol HS 15(聚乙二醇硬脂酸脂15)和90％盐水和灌胃给药(20mg/kg，10mL/kg，0.5％CMC-Na水溶液)；给予CD1小鼠尾静脉给药(1mg/kg,5mL/kg，5％DMAC,5％Solutol HS 15和90％Saline)和灌胃给药(5mg/kg,10mL/kg，0.5％CMC-Na水溶液)
血样采集：首先对给药前挑选符合实验要求的动物，称重标记。采集血样前，绑定大鼠或小鼠，每一只给药的大鼠在预定的采血时间点(静脉给药：分别于给药前，给药后的0.083,0.25,0.5,1,2,4,8,24h采血,共9个时间点；灌胃给药：分别于给药前，给药后的0.083,0.25,0.5,1,2,4,8,24h采血,共9个时间点)，通过尾静脉采血，或经心脏采血(终末采血)约150μL。每一只给药的小鼠在预定的采血时间点(尾静脉给药于0.083,0.25,0.5,1,2,4,8,24采血,共8个时间点；灌胃给药：于0.25,0.5,1,2,4,8,24h采血,共7个时间点)，通过眼眶采血，或经心脏采血(终末采血)约150μL。血液转移至预先加入K2EDTA的1.5mL试管中。采完的血样放在湿冰上，离心5min(2000g,4℃)，取出血浆，整个过程在采血后15min内完成。所有的样品都需要存放于-70℃冰箱直到样品分析。
应用LC/MS/MS法测定药物浓度，本发明部分实施例化合物在相同剂量和给药方式下，大鼠和小鼠体内的药代动力学性质参数如表9所示：
表9 化合物在大鼠和小鼠体内药代动力学参数
Figure PCTCN2015073044-appb-000105
从表9可以看出，本发明示例化合物的药代吸收好，具有明显的药代吸收效果，同时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 (10)

  1. 一种结构如式(I)所示的化合物，或其药学上可接受的盐、立体异构体、溶剂化物或其前药，
    Figure PCTCN2015073044-appb-100001
    式中，
    A为选自下组中的任意一种：
    Figure PCTCN2015073044-appb-100002
    其中，Z2为CR5或N；R5为氢、卤素、三氟甲基或C1-10烷氧基；
    Z21为N或CH；
    R4、R42、R43各自独立地为氢或C1-10烷基、C3-10环烷基、C3-10杂环烷基、或卤代的C1-10烷基；
    R41为氢、卤素、三氟甲基、C1-10烷基或C1-10烷氧基；
    R0为氢、卤素、C1-10烷基、-NRaRb或-CO-NRaRb，其中Ra、Rb各自独立地为C1-10烷基；
    Z1为CR6或N；其中，R6为氢、卤素、三氟甲基、甲氧基或-CO2C1-10烷基；
    R1为氢，或选自下组中的任意一种：
    Figure PCTCN2015073044-appb-100003
    R2和R3各自独立地为氢或-CH2NR7R8；其中，(1)R7、R8各自独立地为氢或甲基；或(2)R7、R8和相连的氮原子共同形成5-6元含氮饱和杂环；
    R12为氢、卤素、C1-10烷基或C1-10烷氧基。
  2. 如权利要求1所述的化合物，或其药学上可接受的盐、立体异构体、溶剂化物或其前 药，其中，A为选自下组中的任意一种：
    Figure PCTCN2015073044-appb-100004
    其中，R4、R41、R42、R43如权利要求1中所定义。
  3. 如权利要求1所述的化合物，或其药学上可接受的盐、立体异构体、溶剂化物或其前药，其中，所述式(I)化合物为式(IV-1)或式(IV-2)所示化合物：
    Figure PCTCN2015073044-appb-100005
    式(IV-1)中，A、R0、R1、R2、R3和R6如权利要求1中所定义；
    式(IV-2)中，A、R0、R1、R2和R3如权利要求1中所定义。
  4. 如权利要求1所述的化合物，或其药学上可接受的盐、立体异构体、溶剂化物或其前药，其中，所述式(I)化合物为式(V-1)、式(V-2)、式(VI-1)、式(VI-2)、式(XI-1)、式(XI-2)、式(XII-1)或式(XII-2)所示化合物：
    Figure PCTCN2015073044-appb-100006
    式(V-1)中，R1、R2、R3、R4、R41和R6如权利要求1中所定义；
    式(V-2)中，R1、R2、R3、R4和R41如权利要求1中所定义；
    Figure PCTCN2015073044-appb-100007
    式(VI-1)中，R1、R2、R3、R4、R41和R6如权利要求1中所定义；
    式(VI-2)中，R1、R2、R3、R4和R41如权利要求1中所定义；
    Figure PCTCN2015073044-appb-100008
    式(XI-1)中，R1、R2、R3、R4、R42、R43和R6如权利要求1中所定义；
    式(XI-2)中，R1、R2、R3、R4、R42和R43如权利要求1中所定义；
    Figure PCTCN2015073044-appb-100009
    式(XII-1)中，R1、R2、R3、R4、R41和R6如权利要求1中所定义；
    式(XII-2)中，R1、R2、R3、R4和R41如权利要求1中所定义。
  5. 如权利要求1所述的化合物，或其药学上可接受的盐、立体异构体、溶剂化物或其前药，其中，所述式(I)化合物为选自下组的化合物：
    Figure PCTCN2015073044-appb-100010
    Figure PCTCN2015073044-appb-100011
    Figure PCTCN2015073044-appb-100012
    Figure PCTCN2015073044-appb-100013
  6. 一种药物组合物，所述药物组合物包括：权利要求1-5中任一权利要求所述的化合物，或其药学上可接受的盐、立体异构体、溶剂化物或其前药；以及药学可接受的载体。
  7. 权利要求1-5中任一权利要求所述的化合物，或其药学上可接受的盐、立体异构体、溶剂化物或其前药在制备调控EGFR酪氨酸激酶或者治疗EGFR相关疾病的药物中的应用。
  8. 如权利要求7所述的应用，其中，所述的EGFR相关疾病选自下组：癌症、糖尿病、免疫系统疾病、神经退行性疾病、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。
  9. 如权利要求8所述的应用，其中，所述获得性耐药性的疾病是由EGFR外显子20编码的T790突变所引起的或者是包含EGFR外显子20编码的T790突变所引起的。
  10. 一种药用组合物，所述药用组合物包括：权利要求1-5中任一权利要求所述的化合物、或其药学上可接受的盐、立体异构体、溶剂化物或其前药；以及其它药物，所述其它药物为选自下组的一种或多种：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、或其组合。
PCT/CN2015/073044 2014-02-25 2015-02-13 2,4-二取代苯-1,5-二胺衍生物及其应用以及由其制备的药物组合物和药用组合物 WO2015127872A1 (zh)

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