WO2015158233A1 - 2，3，4，6-四取代苯-1，5-二胺衍生物、其制法与医药上的用途 - Google Patents

2，3，4，6-四取代苯-1，5-二胺衍生物、其制法与医药上的用途 Download PDF

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WO2015158233A1
WO2015158233A1 PCT/CN2015/076451 CN2015076451W WO2015158233A1 WO 2015158233 A1 WO2015158233 A1 WO 2015158233A1 CN 2015076451 W CN2015076451 W CN 2015076451W WO 2015158233 A1 WO2015158233 A1 WO 2015158233A1
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alkyl
compound
group
aryl
formula
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PCT/CN2015/076451
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French (fr)
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兰炯
金云舟
周福生
何琦
何向宇
吕强
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上海海雁医药科技有限公司
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Priority to JP2016562498A priority Critical patent/JP6321821B2/ja
Priority to EP15780205.9A priority patent/EP3144292B1/en
Priority to US15/303,475 priority patent/US9879008B2/en
Publication of WO2015158233A1 publication Critical patent/WO2015158233A1/zh

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    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
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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
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    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
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    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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    • C07C211/00—Compounds containing amino groups bound to a carbon skeleton
    • C07C211/43—Compounds containing amino groups bound to a carbon skeleton having amino groups bound to carbon atoms of six-membered aromatic rings of the carbon skeleton
    • C07C211/44—Compounds containing amino groups bound to a carbon skeleton having amino groups bound to carbon atoms of six-membered aromatic rings of the carbon skeleton having amino groups bound to only one six-membered aromatic ring
    • C07C211/49—Compounds containing amino groups bound to a carbon skeleton having amino groups bound to carbon atoms of six-membered aromatic rings of the carbon skeleton having amino groups bound to only one six-membered aromatic ring having at least two amino groups bound to the carbon skeleton
    • C07C211/50—Compounds containing amino groups bound to a carbon skeleton having amino groups bound to carbon atoms of six-membered aromatic rings of the carbon skeleton having amino groups bound to only one six-membered aromatic ring having at least two amino groups bound to the carbon skeleton with at least two amino groups bound to carbon atoms of six-membered aromatic rings of the carbon skeleton
    • C07C211/51—Phenylenediamines
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    • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
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    • C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
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    • C07D403/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing three or more hetero rings

Definitions

  • the invention relates to the technical field of medicine, in particular to a 2,3,4,6-tetrasubstituted benzene-1,5-diamine derivative, a preparation method thereof and application as an EGFR tyrosine kinase inhibitor, and Pharmaceutical compositions and pharmaceutical compositions prepared.
  • Lung cancer is the world's highest incidence of cancer. 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.
  • the object of the present invention is to provide a novel EGFR tyrosine kinase inhibitor 2,3,4,6-tetrasubstituted benzene-1,5-diamine derivative having high EGFR T790M selective inhibition.
  • Z 1 , Z 2 , Z 3 are each independently CR 10 or N;
  • R 10 is hydrogen, hydroxy, CN, NO 2 , halogen (preferably F or Cl), -NR 11 R 12 , C 1-10 alkyl (preferably C 1-6 alkyl, more preferably C 1-3 alkyl), C 3-8 cycloalkyl (preferably C 3-6 cycloalkyl), C 3-8 cycloalkoxy (preferred) Is C 3-6 cycloalkoxy), C 2-10 alkenyl (preferably C 2-6 alkenyl, more preferably C 2-4 alkenyl), C 2-10 alkynyl (preferably C 2- 6 alkynyl, more preferably C 2-4 alkynyl), C 1-10 alkoxy (preferably C 1-6 alkoxy, more preferably C 1-3 alkoxy), -CHO, -COC 1-10 alkyl (preferably -COC 1-6 alkyl, more preferably -COC 1-3 alkyl), -COC 6-10 ary
  • Y 1 and Y 2 are each independently a divalent C 1-3 hydrocarbon group, or a methylene unit (-CH 2 -) in Y 1 or Y 2 is -C(R y R x )-, -NR y C (O)-, cyclopropylene, -C(O)NR y -, -N(R y )SO 2 -, -SO 2 N(R y )-, -S-, -S(O)-, -SO 2 -, -OC(O)-, -C(O)O-, -O-, -N(R y )- or -C(O)-substituted; wherein R y , R x are each independently Is hydrogen, halogen (preferably F or Cl), hydroxyl, CN, NO 2 , C 1-10 alkyl (preferably C 1-6 alkyl, more preferably C 1-3 alkyl), C 1-10 alkane An oxy group (
  • X is NR z , O or S;
  • R z is hydrogen, C 1-10 alkyl (preferably C 1-6 alkyl, more preferably C 1-3 alkyl), halogenated C 1-10 alkyl (preferably a halogenated C 1-6 alkyl group, more preferably a halogenated C 1-3 alkyl group) or a C 3-8 cycloalkyl group (preferably a C 3-6 cycloalkyl group);
  • n 1 , m 3 are each independently 0, 1 or 2; m 2 is 0 or 1; and m 1 , m 2 and m 3 are not 0 at the same time;
  • Ring A is a C 6-10 aryl group (such as a benzene ring), a 3 to 7 membered saturated or partially unsaturated monocyclic ring, an 8 to 10 membered saturated or partially unsaturated bicyclic ring, having 1 to 3 independently selected from nitrogen, oxygen or a 3 to 7 membered saturated or partially unsaturated monoheterocyclic ring of a hetero atom of sulfur, an 8 to 10 membered saturated or partially unsaturated heterocyclic ring having 1 to 5 hetero atoms independently selected from nitrogen, oxygen or sulfur, having 1 a 5 to 6 membered monocyclic heteroaryl ring to 3 heteroatoms independently selected from nitrogen, oxygen or sulfur, or an 8 to 10 membered bicyclic heterocyclic ring having 1 to 5 heteroatoms independently selected from nitrogen, oxygen or sulfur.
  • Aryl ring such as a benzene ring
  • R a and R b are each independently hydrogen, hydroxy, CN, NO 2 , halogen (preferably F or Cl), -NR 11 R 12 , C 1-10 alkyl (preferably C 1-6 alkyl, more Preferred is C 1-3 alkyl), C 3-8 cycloalkyl (preferably C 3-6 cycloalkyl), C 3-8 cycloalkoxy (preferably C 3-6 cycloalkoxy), C 2-10 alkenyl (preferably C 2-6 alkenyl, more preferably C 2-4 alkenyl), C 2-10 alkynyl (preferably C 2-6 alkynyl, more preferably C 2-4 Alkynyl), C 1-10 alkoxy (preferably C 1-6 alkoxy, more preferably C 1-3 alkoxy), -CHO, -COC 1-10 alkyl (preferably -COC 1 -6 alkyl, more preferably -COC 1-3 alkyl), -COC 6-10 aryl (preferably -COC 6 aryl, such as -
  • R a , R b are each independently a 5 to 6 membered monocyclic heteroaryl group having 1 to 4 hetero atoms independently selected from nitrogen, oxygen or sulfur, having 1 to 5 independently selected from nitrogen, oxygen or sulfur. 8 to 10 membered bicyclic heteroaryl (preferably 9 to 10 membered bicyclic heteroaryl) of the hetero atom; wherein the 5 to 6 membered monocyclic heteroaryl or 8 to 10 membered bicyclic heteroaryl is not present.
  • halogen preferably F or Cl
  • hydroxyl CN, NO 2
  • C 1-10 alkyl preferably C 1-6 alkyl, More preferably, it is a C 1-3 alkyl group, a halogenated C 1-10 alkyl group (preferably a halogenated C 1-6 alkyl group, more preferably a halogenated C 1-3 alkyl group), a C 1-10 alkane.
  • An oxy group (preferably a C 1-6 alkoxy group, more preferably a C 1-3 alkoxy group), a halogenated C 1-10 alkoxy group (preferably a halogenated C 1-6 alkoxy group, more Preferred is a halogenated C 1-3 alkoxy group, a C 3-8 cycloalkyl group (preferably a C 3-6 cycloalkyl group), a C 3-8 cycloalkoxy group (preferably a C 3-6 cycloalkane).
  • C 2-10 alkenyl preferably C 2-6 alkenyl, more preferably C 2-4 alkenyl
  • C 2-10 alkynyl preferably C 2-6 alkynyl, more preferably C 2-4 alkynyl
  • -CONR 11 R 12 , -C(O)OC 1-10 alkyl (preferably -C(O)OC 1-6 alkyl, more preferably -C(O)OC 1-3 alkyl), -CHO, -OC(O)C 1-10 alkyl (preferably -OC(O)C 1-6 alkyl More preferably, it is -OC(O)C 1-3 alkyl), -SO 2 C 1-10 alkyl (preferably -SO 2 C 1-6 alkyl, more preferably -SO 2 C 1-3 alkane , -SO 2 C 6-10 aryl (preferably -SO 2 C 6 aryl, such as -SO 2 -phenyl), -COC 6-10 aryl (preferably -COC 6 aryl,
  • n 1 , n 2 are each independently 0, 1 , 2 , 3, 4, 5 or 6;
  • R 1 is hydrogen or C 1-10 alkyl (preferably C 1-6 alkyl, more preferably C 1-3 alkyl);
  • R 2 , R 3 and R 5 are each independently hydrogen, halogen (preferably F or Cl), C 1-10 alkyl (preferably C 1-6 alkyl, more preferably C 1-3 alkyl) or C 1-10 alkoxy group (preferably C 1-6 alkoxy group, more preferably C 1-3 alkoxy group);
  • R 4 is hydrogen, hydroxy, -OC 1-10 alkyl, CN, NO 2 , halogen, C 3-8 cycloalkyl, C 3-8 cycloalkoxy, C 2-10 alkenyl, C 2-10 Alkynyl, -CHO, -COC 1-10 alkyl, -COC 6-10 aryl, C 6-10 aryl, -CONR 11 R 12 , -C(O)OC 1-10 alkyl, -OC ( O) C 1-10 alkyl, -SO 2 C 1-10 alkyl, -SO 2 C 6-10 aryl, tert-butoxycarbonyl, -NHC 1-10 alkyl, -N(C 1-10 alkane 2 , -N(C 1-10 alkyl)(C 3-8 cycloalkyl), -NC(O)C 1-10 alkyl, -NSO 2 C 1-10 alkyl, or 1 or a 3 to 7 membered saturated or partially unsaturated monohetero
  • R 6 and R 7 are each independently hydrogen or -CH 2 NR 13 R 14 ; wherein R 13 and R 14 are each independently hydrogen or methyl; or R 13 , R 14 and the attached nitrogen atom together form 5- 6 yuan nitrogen-containing saturated heterocyclic ring;
  • R 11 and R 12 are each independently hydrogen, C 1-10 alkyl (more preferably C 1-6 alkyl, most preferably C 1-3 alkyl), C 1-10 alkoxy (more Preferred is a C 1-6 alkoxy group, most preferably a C 1-3 alkoxy group, a C 3-8 cycloalkyl group (more preferably a C 3-6 cycloalkyl group), a C 3-8 cycloalkoxy group.
  • C 2-10 alkenyl preferably C 2-6 alkenyl, more preferably C 2-4 alkenyl
  • C 2-10 alkynyl preferably C 2-6 alkynyl, more preferably C 2-4 alkynyl
  • C 6-10 aryl preferably phenyl
  • R 11 , R 12 and the attached nitrogen atom together form a 3-6 membered nitrogen Saturated heterocycle.
  • the ring A is a 3 to 7 membered saturated or partially unsaturated monocyclic ring
  • the 8 to 10 membered saturated or partially unsaturated bicyclic ring has 1 to 3 independently selected from the group consisting of 3 to 7 members.
  • ring A when the ring A is a monocyclic, bicyclic, monoheterocyclic or bicyclic heterocyclic ring, it is selected from the group consisting of a 3 to 7 membered saturated or partially unsaturated monocyclic ring; and a 8 to 10 membered saturated or partially unsaturated group.
  • Bicyclic 3 to 7 membered saturated or partially unsaturated monoheterocyclic ring containing 1 or 2 nitrogen atoms; 8 to 10 membered saturated or partially unsaturated diheterocyclic ring containing 1, 2 or 3 nitrogen atoms; a 3 to 7 membered saturated or partially unsaturated monoheterocyclic ring of 1 or 2 oxygen or sulfur atoms; an 8 to 10 membered saturated or partially unsaturated double heterocyclic group containing 1, 2 or 3 oxygen atoms or sulfur atoms.
  • ring A is a 5- to 6-membered monocyclic heteroaryl ring or an 8- to 10-membered bicyclic heteroaryl ring
  • the following group is selected: Wherein R a1 is hydrogen, methyl or ethyl.
  • the compound of formula (I) is a compound of formula (II):
  • n 3 , n 4 are each independently 0, 1, 2 or 3, and n 3 , n 4 are not 0 at the same time;
  • W 1 is N or CR 15 ;
  • W 2 is N, O, S or CR 15 ;
  • R 15 is hydrogen, hydroxy, CN, NO 2 , halogen (preferably F or Cl), -NR 11 R 12 , C 1- 10 alkyl (preferably C 1-6 alkyl, more preferably C 1-3 alkyl), C 3-8 cycloalkyl (preferably C 3-6 cycloalkyl), C 3-8 cycloalkoxy a group (preferably C 3-6 cycloalkoxy), a C 2-10 alkenyl group (preferably a C 2-6 alkenyl group, more preferably a C 2-4 alkenyl group), a C 2-10 alkynyl group (preferably C 2-6 alkynyl, more preferably C 2-4 alkynyl), C 1-10 alkoxy (preferably C 1-6 alkoxy, more preferably C 1-3 alkoxy), C 6 a -10 aryl group (preferably a C 6 aryl group such as a phenyl
  • R 8 , R 9 , R 0 have the same meanings as defined above for R a or R b ;
  • Z 1 , Z 2 , Z 3 , R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 11 , R 12 , Y 1 , Y 2 , X, m 1 , m 2 , m 3 , n 1 , and n 2 are as defined above.
  • Y 1 and Y 2 are each independently a divalent C 1-3 hydrocarbon group, and the methylene group in Y 1 or Y 2 is not substituted.
  • the compound of the formula (I) is a formula (VII-1), a formula (VII-2), a formula (VII-3), a formula (VII-4), a formula (VII-5), a compound of the formula (VII-6), the formula (VII-7), the formula (VII-8), the formula (VII-9), and the formula (VII-10):
  • Z 1 , Z 2 , Z 3 , R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , Y 1 , Y 2 , X, m 1 , m 2 , m 3 , W 1 , W 2 , and R 0 are as defined above;
  • R 91 , R 92 , R 93 , R 94 , R 95 , R 81 , R 82 , and R 83 are as defined for R 8 and R 9 .
  • W 1 is CH or N; and W 2 is N, O, S or CH.
  • W 1 is CH or N; and W 2 is N or CH.
  • W 1 is CH; and W 2 is N, O, S or CH.
  • W 1 is CH; and W 2 is N.
  • the compound of formula (I) is a compound of formula (III):
  • Z 1 , Z 2 , Z 3 , R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , Y 2 , X, m 2 , m 3 , n 1 , n 2 , n 3 , n 4 , and R 0 are as defined above.
  • the compound of formula (I) is a compound of formula (IV):
  • Z 2 , R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , Y 2 , X, m 3 , n 1 , n 2 , n 3 , n 4 and R 0 are as defined above.
  • the compound of formula (I) is a compound of formula (V):
  • Z 2 , R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , X, m 3 , n 1 , n 2 , n 3 , n 4 , R 0 is defined as above.
  • the compound of formula (I) is a compound of formula (VI):
  • Z 2 , R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , X, m 3 , n 3 , n 4 and R 0 are as defined above.
  • Z 2 is CR 10 or N;
  • R 10 is hydrogen, hydroxy, CN, NO 2 , fluorine, chlorine, -NR 11 R 12 , C 1-3 alkyl, C 3-6 naphthenic , C 3-6 cycloalkoxy, C 1-3 alkoxy, -CHO, -COC 1-3 alkyl, -CO-phenyl, phenyl, -CONR 11 R 12 , -C(O) OC 1-3 alkyl, -OC(O)C 1-3 alkyl, -SO 2 C 1-3 alkyl, -SO 2 -phenyl or tert-butoxycarbonyl; wherein the alkyl group, the ring The alkyl group, the phenyl group, the alkoxy group are unsubstituted or substituted by one to three substituents selected from the group consisting of fluorine, chlorine, nitro, phenyl, C 1-3 alkyl, C 1-3 Alkoxy, C 3-6
  • Z 2 is CR 10 and R 10 is hydrogen, hydroxy, NO 2 , fluorine, chlorine, -NH 2 , -N(CH 3 ) 2 , C 1-3 alkyl, cyclopropyl, Cyclopropoxy, C 1-3 alkoxy, -CHO, -COCH 3 , -CO-phenyl, phenyl, -CONH 2 , -CON(CH 3 ) 2 , -C(O)OCH 3 ,- OC(O)CH 3 , -SO 2 CH 3 , -SO 2 -phenyl or tert-butoxycarbonyl; wherein the alkyl, cyclopropyl, alkoxy or phenyl group is unsubstituted or is 1 - 3 substituents selected from the group consisting of fluorine, chlorine, nitro, phenyl, methyl, methoxy, cyclopropyl, cyclopropoxy, -CONH 2 , -CON(
  • Z 2 is CR 10 and R 10 is hydroxy, NO 2 , fluorine, chlorine, -NH 2 , -N(CH 3 ) 2 , trifluoromethyl, methoxy, -CHO, - COCH 3 , -CONH 2 , -C(O)OCH 3 or -OC(O)CH 3 .
  • R 0 is hydrogen, hydroxy, C 1-3 alkyl, C 3-6 cycloalkyl, -CHO, -COC 1-3 alkyl, -CO-phenyl, phenyl, - CONR 11 R 12 , -C(O)OC 1-3 alkyl, -SO 2 C 1-3 alkyl, -SO 2 -phenyl, -S(O)C 1-3 alkyl, -S(O a phenyl group, a tert-butoxycarbonyl group; wherein the alkyl group, the cycloalkyl group, the phenyl group is unsubstituted or substituted by one to three substituents selected from the group consisting of fluorine, chlorine, hydroxyl, NO 2 , phenyl, C 1-3 alkyl, C 1-3 alkoxy, C 3-6 cycloalkyl, C 3-6 cycloalkoxy, C 2-4 alkenyl, C 2-4 alkyne
  • R 0 is a 5- to 6-membered monocyclic heteroaryl group having 1 to 4 hetero atoms independently selected from nitrogen, oxygen or sulfur, or Wherein R a1 is hydrogen, methyl or ethyl.
  • R 0 is hydrogen, hydroxy, C 1-3 alkyl, cyclopropyl, -CHO, -COC 1-3 alkyl (preferably -COCH 3 ), -CO-phenyl, benzene , -CONH 2 , -CON(CH 3 ) 2 , -C(O)OCH 3 , -SO 2 C 1-3 alkyl (preferably -SO 2 CH 3 ), -SO 2 -phenyl, -S (O) C 1-3 alkyl (preferably -S(O)CH 3 ), -S(O)-phenyl, tert-butoxycarbonyl; wherein the alkyl group, the phenyl group is unsubstituted or Substituted by 1-3 (preferably 1) substituents selected from the group consisting of fluorine, chlorine, hydroxyl, NO 2 , phenyl, methyl, methoxy, cyclopropyl, cyclopropoxy, -CONH 2
  • R 0 is hydrogen, C 1-3 alkyl, -COC 1-3 alkyl (preferably -COCH 3 ), -CO-phenyl, -SO 2 C 1-3 alkyl ( Preferred is -SO 2 CH 3 ), -SO 2 -phenyl, tert-butoxycarbonyl; wherein the alkyl group, the phenyl group is unsubstituted or is selected from the group consisting of 1-3 (preferably 1) Substituted by a substituent: fluorine, chlorine, methyl;
  • R 0 is C 1-3 alkyl, -COC 1-3 alkyl (preferably -COCH 3 ), -SO 2 C 1-3 alkyl (preferably -SO 2 CH 3 ) Wherein the alkyl group is unsubstituted or substituted with one substituent selected from the group consisting of fluorine and chlorine.
  • R 0 is a fluorine-substituted C 1-3 alkyl group (preferably a monofluoroethyl group), a -COC 1-3 alkyl group (preferably -COCH 3 ) or -SO 2 C 1-3 alkane.
  • Base preferably -SO 2 CH 3 ).
  • m 3 is 0 or 1.
  • n 3 is 1, 2 or 3; n 4 is 1 or 2.
  • n 3 is 1; n 4 is 1.
  • X is NH, N(C 1-3 alkyl), O or S.
  • Z 2 is CR 10 and R 10 is trifluoromethyl, fluoro or chloro;
  • X is NH, O or S
  • R 0 is hydrogen, C 1-3 alkyl, -COC 1-3 alkyl (preferably -COCH 3 ), -CO-phenyl, -SO 2 C 1-3 alkyl (preferably -SO 2 CH 3 And -SO 2 -phenyl, tert-butoxycarbonyl; wherein the alkyl group, the phenyl group is unsubstituted or substituted by one to three (preferably one) substituents selected from the group consisting of fluorine , chlorine, methyl; (preferably, R 0 is a fluorine-substituted C 1-3 alkyl group (preferably a monofluoroethyl group), -COC 1-3 alkyl group (preferably -COCH 3 ) or -SO 2 C 1-3 alkyl (preferably -SO 2 CH 3 ))
  • R 1 is hydrogen;
  • R 2 is methoxy;
  • R 3 , R 5 , R 6 and R 7 are each independently hydrogen;
  • R 4 is or
  • Z 2 is CR 10 and R 10 is trifluoromethyl, fluoro or chloro;
  • X is O
  • R 0 is hydrogen, C 1-3 alkyl, -COC 1-3 alkyl (preferably -COCH 3 ), -CO-phenyl, -SO 2 C 1-3 alkyl (preferably -SO 2 CH 3 And -SO 2 -phenyl, tert-butoxycarbonyl; wherein the alkyl group, the phenyl group is unsubstituted or substituted by one to three (preferably one) substituents selected from the group consisting of fluorine , chlorine, methyl; (preferably, R 0 is a fluorine-substituted C 1-3 alkyl group (preferably a monofluoroethyl group), -COC 1-3 alkyl group (preferably -COCH 3 ) or -SO 2 C 1-3 alkyl (preferably -SO 2 CH 3 ));
  • R 1 is hydrogen;
  • R 2 is methoxy;
  • R 3 , R 5 , R 6 and R 7 are each independently hydrogen;
  • R 4 is or
  • Z 2 is CR 10 and R 10 is trifluoromethyl, fluoro or chloro;
  • X is NH or O
  • R 0 is hydrogen, C 1-3 alkyl, -COC 1-3 alkyl (preferably -COCH 3 ), -CO-phenyl, -SO 2 C 1-3 alkyl (preferably -SO 2 CH 3 And -SO 2 -phenyl, tert-butoxycarbonyl; wherein the alkyl group, the phenyl group is unsubstituted or substituted by one to three (preferably one) substituents selected from the group consisting of fluorine , chlorine, methyl; (preferably, R 0 is a fluorine-substituted C 1-3 alkyl group (preferably a monofluoroethyl group), -COC 1-3 alkyl group (preferably -COCH 3 ) or -SO 2 C 1-3 alkyl (preferably -SO 2 CH 3 ));
  • R 1 is hydrogen;
  • R 2 is methoxy;
  • R 3 , R 5 , R 6 and R 7 are each independently hydrogen;
  • R 4 is or
  • Z 2 is CR 10 and R 10 is trifluoromethyl, fluoro or chloro;
  • X is O
  • R 0 is hydrogen, C 1-3 alkyl, -COC 1-3 alkyl (preferably -COCH 3 ), -CO-phenyl, -SO 2 C 1-3 alkyl (preferably -SO 2 CH 3 And -SO 2 -phenyl, tert-butoxycarbonyl; wherein the alkyl group, the phenyl group is unsubstituted or substituted by one to three (preferably one) substituents selected from the group consisting of fluorine , chlorine, methyl; (preferably, R 0 is a fluorine-substituted C 1-3 alkyl group (preferably a monofluoroethyl group), -COC 1-3 alkyl group (preferably -COCH 3 ) or -SO 2 C 1-3 alkyl (preferably -SO 2 CH 3 ));
  • R 1 is hydrogen;
  • R 2 is methoxy;
  • R 3 , R 5 , R 6 and R 7 are each independently hydrogen;
  • R 4 is or
  • X is NH or O
  • R 0 is hydrogen, C 1-3 alkyl, -COC 1-3 alkyl (preferably -COCH 3 ), -CO-phenyl, -SO 2 C 1-3 alkyl (preferably -SO 2 CH 3 And -SO 2 -phenyl, tert-butoxycarbonyl; wherein the alkyl group, the phenyl group is unsubstituted or substituted by one to three (preferably one) substituents selected from the group consisting of fluorine , chlorine, methyl; (preferably, R 0 is a fluorine-substituted C 1-3 alkyl group (preferably a monofluoroethyl group), -COC 1-3 alkyl group (preferably -COCH 3 ) or -SO 2 C 1-3 alkyl (preferably -SO 2 CH 3 ));
  • R 1 is hydrogen;
  • R 2 is methoxy;
  • R 3 , R 5 , R 6 and R 7 are each independently hydrogen;
  • R 4 is or
  • Z 2 is CR 10 and R 10 is trifluoromethyl, fluoro or chloro;
  • X is O
  • R 0 is hydrogen, C 1-3 alkyl, -COC 1-3 alkyl (preferably -COCH 3 ), -CO-phenyl, -SO 2 C 1-3 alkyl (preferably -SO 2 CH 3 And -SO 2 -phenyl, tert-butoxycarbonyl; wherein the alkyl group, the phenyl group is unsubstituted or substituted by one to three (preferably one) substituents selected from the group consisting of fluorine , chlorine, methyl; (preferably, R 0 is a fluorine-substituted C 1-3 alkyl group (preferably a monofluoroethyl group), -COC 1-3 alkyl group (preferably -COCH 3 ) or -SO 2 C 1-3 alkyl (preferably -SO 2 CH 3 ));
  • R 1 is hydrogen;
  • R 2 is methoxy;
  • R 3 , R 5 , R 6 and R 7 are each independently hydrogen;
  • X is NH, m 3 is 0; n 3 is 1; n 4 is 1;
  • Z 2 is CR 10 and R 10 is fluorine, chlorine or trifluoromethyl
  • R 0 is -COC 1-3 alkyl (preferably -COCH 3 ) or -SO 2 C 1-3 alkyl (preferably -SO 2 CH 3 );
  • R 1 is hydrogen;
  • R 2 is methoxy;
  • R 3 , R 5 , R 6 and R 7 are each independently hydrogen;
  • R 4 is: or
  • X is O, m 3 is 0 or 1; n 3 is 1, 2 or 3; n 4 is 1 or 2;
  • Z 2 is CR 10 and R 10 is fluorine, chlorine or trifluoromethyl
  • R 1 is hydrogen;
  • R 2 is methoxy;
  • R 3 , R 5 , R 6 and R 7 are each independently hydrogen;
  • R 0 is -COC 1-3 alkyl (preferably -COCH 3 ), -SO 2 C 1-3 alkyl (preferably -SO 2 CH 3 ) or monofluoro substituted C 1-3 alkyl (preferably fluoroethyl) base);
  • R 4 is selected from the group consisting of: or
  • Z 2 is CR 10 and R 10 is fluorine, chlorine or trifluoromethyl
  • R 1 is hydrogen;
  • R 2 is methoxy;
  • R 3 , R 5 , R 6 and R 7 are each independently hydrogen;
  • R 0 is -COC 1-3 alkyl (preferably -COCH 3 ), or -SO 2 C 1-3 alkyl (preferably -SO 2 CH 3 );
  • R 4 is:
  • the compound of the formula (I), R 3 and R 5 are each independently hydrogen or a C 1-10 alkyl group (preferably a C 1-6 alkyl group, more preferably a C 1-3 alkyl group). base).
  • R 1 is hydrogen
  • R 2 is a C 1-10 alkoxy group (preferably a C 1-6 alkoxy group, more preferably a C 1-3 alkoxy group, most preferably Is methoxy).
  • R 4 is hydrogen, -OC 1-10 alkyl, -N(C 1-10 alkyl) 2 , -N(C 1-10 alkyl (C 3-8 cycloalkyl), a 3 to 7 membered saturated or partially unsaturated monoheterocyclic ring containing 1 or 2 nitrogen atoms and 0, 1, 2 or 3 oxygen or sulfur atoms.
  • alkyl, cycloalkyl, 3 to 7 membered saturated or partially unsaturated monoheterocycle may be optionally substituted with from 1 to 3 substituents selected from the group consisting of halogen, NO 2 , CN, hydroxy , -CH 2 NR 11 R 12 , -NR 11 R 12 , C 1-6 alkyl, C 3-8 cycloalkyl, C 3-8 cycloalkoxy, C 2-6 alkenyl, C 2-6 Alkynyl, C 1-6 alkoxy, -CHO, -COC 1-6 alkyl, -COC 6 aryl, C 6 aryl, -CONR 11 R 12 , -C(O)OC 1-6 alkyl , -OC(O)C 1-6 alkyl, -SO 2 C 1-6 alkyl, -SO 2 C 6 aryl, tert-butoxycarbonyl, -NHC 1-6 alkyl, -NC(O)C 1-6 alkyl,
  • R 4 is hydrogen or is selected from the group consisting of:
  • R 4 is a group selected from the group consisting of:
  • R 6 and R 7 are each independently hydrogen.
  • R 6 and R 7 are each independently hydrogen or -CH 2 NR 13 R 14 , and R 13 and R 14 are each independently hydrogen or methyl. .
  • R 6 and R 7 are each independently hydrogen or -CH 2 N R 13 R 14 , and R 13 , R 14 and the attached nitrogen atom together form 5 -6 yuan nitrogen-containing saturated heterocyclic ring.
  • the structure of the -CH 2 NR 13 R 14 is as shown in the formula (a):
  • Q is O, S, NC 1-10 alkyl or C(C 1-10 alkyl) 2 .
  • the compound of formula (I) is selected from the group consisting of
  • a pharmaceutical composition comprising a compound according to the first aspect of the present invention (such as a compound of the above formula (I), a compound of the formula (II), a compound of the formula (III), or a formula (IV) a compound, a compound of the formula (V), a compound of the formula (VI), a compound of the formula (VII-1), a compound of the formula (VII-2), a compound of the formula (VII-3), a compound of the formula (VII-4), and a formula (VII) -5) a compound, a compound of the formula (VII-6), a compound of the formula (VII-7), a compound of the formula (VII-8), a compound of the formula (VII-9), a compound of the formula (VII-10), or the above-exemplified compound) Or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a stereoisomer thereof, or a prodrug thereof, and also includes
  • a compound of the invention in general, can be administered in a suitable dosage form with one or more pharmaceutically acceptable carriers.
  • dosage forms are suitable for oral, rectal, topical, intraoral, and other parenteral administration (eg, subcutaneous, intramuscular, intravenous, etc.).
  • dosage forms suitable for oral administration include capsules, tablets, granules, and syrups and the like.
  • the compound of the present invention contained in these preparations may be a solid powder or granule; a solution or suspension in an aqueous or non-aqueous liquid; a water-in-oil or oil-in-water emulsion or the like.
  • the above dosage forms can be prepared from the active compound with one or more carriers or excipients via conventional pharmaceutical methods.
  • the above carriers need to be compatible with the active compound or other excipients.
  • commonly used non-toxic carriers include, but are not limited to, mannitol, lactose, starch, magnesium stearate, cellulose, glucose, sucrose, and the like.
  • the carrier of the agent includes water, physiological saline, aqueous dextrose, ethylene glycol, polyethylene glycol, and the like.
  • the active compound can form a solution or suspension with the above carriers.
  • compositions of the present invention are formulated, quantified, and administered in a manner consistent with medical practice.
  • the "effective amount" of a compound administered is determined by the particular condition being treated, the individual being treated, the cause of the condition, the target of the drug, and the mode of administration.
  • the third aspect of the present invention provides the compound of the first aspect of the present invention, or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a stereoisomer thereof, or a prodrug thereof, for use in preparation regulation (upregulation or A drug that down-regulates EGFR tyrosine kinase activity, or a drug for the preparation of a medicament for treating EGFR-related diseases.
  • the EGFR-related disease is cancer, diabetes, immune system disease, neurodegenerative disease or cardiovascular disease, and disease having acquired resistance during treatment with an EGFR modulator.
  • the cancer is non-small cell lung cancer, head and neck cancer, breast cancer, kidney cancer, pancreatic cancer, cervical cancer, esophageal cancer, pancreatic cancer, prostate cancer, bladder cancer, colorectal cancer, ovarian cancer, stomach cancer, brain.
  • Malignant tumors include glioblastoma and the like, or any combination thereof.
  • the acquired resistance is caused by a T790 mutation encoded by EGFR exon 20 or by a T790 mutation comprising a EGFR exon 20 encoding, such as T790M.
  • the non-small cell lung cancer is caused by a mutation in EGFR, including a sensitive mutation (such as a L858R mutation or exon 19 deletion) and a drug resistance mutation (such as an EGFR T790M mutation).
  • a sensitive mutation such as a L858R mutation or exon 19 deletion
  • a drug resistance mutation such as an EGFR T790M mutation
  • an EGFR modulator refers to a small molecule tyrosine kinase inhibitor that targets EGFR, such as gefitinib, erlotinib, ectinib, lapatinib, afatinib, and the like.
  • a fourth aspect of the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of the compound of the first aspect of the invention, or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a stereoisomer thereof Or a prodrug, and a drug selected from the group consisting of gefitinib, erlotinib, ectinib, lapatinib, XL647, NVP-AEE-788, ARRY-334543, EKB-569, BIBW2992 , HKI272, BMS-690514, CI-1033, vandetanib, PF00299804, WZ4002, cetuximab, trastuzumab, panituzumab, matuzumab, nimotuzumab, Zaluzumab, Pertuzumab, MDX-214, CDX-110, IMC-11F8, Zemab, Her2 Vaccine PX 1041, HSP
  • “Therapeutically effective amount” means an amount that is functional or active to a human and/or animal and that is acceptable to humans and/or animals.
  • a therapeutically effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a stereoisomer thereof, or a prodrug thereof, contained in the pharmaceutical composition or the pharmaceutical composition of the present invention It is preferably 0.1 mg to 5 g/kg (body weight).
  • the pharmaceutical composition can be used to treat diseases in which EGFR is abnormally expressed, such as cancer, diabetes, immune system diseases, neurodegenerative diseases or cardiovascular diseases, diseases having acquired resistance during treatment with EGFR modulators.
  • the acquired drug-resistant disease is a disease caused by a T790 mutation encoded by EGFR exon 20, or a disease caused by a T790 mutation encoded by EGFR exon 20.
  • the EGFR exon 20 encodes a T790 of T790M.
  • the compound of the formula (I) of the present invention can be used in combination with other drugs in certain diseases to achieve a desired therapeutic effect.
  • An example of a joint application is to treat advanced NSCLC.
  • the inventors have unexpectedly discovered a class of selective inhibitors of EGFR mutations through long-term and in-depth studies. In vitro experiments have shown that they can inhibit 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 wild-type EGFR enzyme and cell line A431 were relatively weakly inhibited. 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 group means a linear or branched saturated aliphatic hydrocarbon group having 1 to 10 carbon atoms.
  • It is preferably an alkyl group having 1 to 6 carbon atoms, more preferably an alkyl group having 1 to 3 carbon atoms.
  • C 2-10 alkynyl group means a straight-chain or branched unsaturated aliphatic hydrocarbon group having a carbon-carbon triple bond having 2 to 10 (preferably 2 to 6) carbon atoms.
  • C 3-8 cycloalkyl refers to a cycloalkyl group having from 3 to 8 carbon atoms.
  • Examples of the cycloalkyl group are a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, a cyclohexyl group and the like.
  • C 1-10 alkoxy means C 1-10 alkyl-O-. For example, methoxy, ethoxy, propoxy, butoxy, and the like.
  • C 3-8 cycloalkoxy means C 3-8 cycloalkyl-O-.
  • cyclopropoxy cyclobutoxy
  • cyclopentyloxy cyclohexyloxy and the like.
  • C 6-10 aryl group means an aromatic hydrocarbon group having 6 to 10 carbon atoms, such as a phenyl group, a naphthyl group or the like.
  • Halogen means fluoro, chloro, bromo or iodo.
  • the "divalent C 1-3 hydrocarbon group” means a linear or branched alkylene group, an alkenylene group and an alkynylene group.
  • alkylene means a divalent alkyl group, for example, a methylene group, an ethylene group or the like; and
  • alkenylene group means a divalent alkenyl group.
  • Methylene group is substituted means that the methylene group in the divalent C 1-3 linear or branched hydrocarbon chain can be replaced by a group as defined herein.
  • substitution for example, -CH 2 -S(O)-CH 2 -, -CH 2 -O-CH 2 -, -CH 2 -C(O)NR y -CH 2 -, -C(O)-CH 2 -CH 2 -, -CH 2 -C(R y R x )-CH 2 -, -N(R y )-CH 2 -CH 2 -, -C(R y R x )-C(R y R x )-CH 2 -etc.
  • cyclopropylene structure is:
  • Heteroaryl ring and “heteroaryl” are used interchangeably to mean having 5 to 10 ring atoms, preferably 5, 6, 9 or 10 ring atoms; sharing 6, 10 or 14 ⁇ in a ring array An electron; and a group having 1 to 5 hetero atoms in addition to a carbon atom.
  • heteroatom refers to nitrogen, oxygen or sulfur.
  • 5 to 6 membered monocyclic heteroaryl ring means a monocyclic heteroaryl ring containing 5 to 6 ring atoms, and includes, for example, but not limited to: thiophene ring, furan ring, thiazole ring, imidazole ring, evil An azole ring, a pyrrole ring, a pyrazole ring, a triazole ring, a tetrazole ring, an isoxazole ring, an oxadiazole ring, a thiadiazole ring, a pyridine ring, a pyridazine ring, a pyrimidine ring, a pyrazine ring, and the like.
  • the "8- to 10-membered bicyclic heteroaryl ring” means a bicyclic heteroaryl ring having 8 to 10 ring atoms, and includes, for example, but not limited to, a benzofuran ring, a benzothiophene ring, an anthracene ring, and an iso Anthracene ring, quinoline ring, isoquinoline ring, indazole ring, benzothiazole ring, benzimidazole ring, quinazoline ring, quinoxaline ring, porphyrin ring, pyridazine ring.
  • the 5- to 6-membered monocyclic heteroaryl ring or the 8- to 10-membered bicyclic heteroaryl ring may be selected from the group consisting of Wherein R a1 is hydrogen, methyl or ethyl.
  • partially unsaturated refers to a pi-electron system that contains one or more unsaturated bonds but does not have a complete conjugation.
  • 3 to 7 membered saturated or partially unsaturated monocyclic ring means a saturated all carbon monocyclic or partially unsaturated all carbon monocyclic ring containing from 3 to 7 ring atoms.
  • cyclopropyl ring cyclobutyl ring, cyclopentyl ring, cyclohexane, cyclohexadiene ring, cycloheptane, cycloheptatriene ring and the like.
  • 8 to 10 membered saturated or partially unsaturated bicyclic means a saturated all carbon bicyclic or partially unsaturated all carbon bicyclic ring containing from 8 to 10 ring atoms.
  • 3 to 7 membered saturated or partially unsaturated monoheterocyclic ring means a saturated monocyclic or partially unsubstituted ring containing from 3 to 7 ring atoms and one to three carbon atoms substituted with a hetero atom selected from nitrogen, oxygen or sulfur. Saturated single ring.
  • Examples of monoheterocycles include, but are not limited to, tetrahydrofuran ring, tetrahydrothiophene ring, pyrrolidinyl ring, piperidine ring, pyrroline ring, oxazolidine ring, piperazine ring, dioxolane, morpholine ring.
  • 8 to 10 membered saturated or partially unsaturated double heterocyclic ring means a saturated bicyclic or partially unsaturated group having 8 to 10 ring atoms and 1 to 5 carbon atoms substituted by a hetero atom selected from nitrogen, oxygen or sulfur. Double ring.
  • bicyclic heterocycles include, but are not limited to, tetrahydroquinoline rings, tetrahydroisoquinoline rings, decahydroquinoline rings.
  • a 3 to 7 membered saturated or partially unsaturated monoheterocyclic ring having 1 or 2 nitrogen atoms and 0 to 3 oxygen or sulfur atoms means that 1 or 2 carbon atoms are replaced by a nitrogen atom in the monoheterocyclic ring. And 0, 1, 2 or 3 carbon atoms are replaced by oxygen or sulfur atoms.
  • 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 compound of the formula (I) of the present invention can be prepared by referring to the following synthetic route, and in the specific operation, it can be The steps in the method need to be extended or merged.
  • Step 1 A compound of the formula (Ia) and a compound of the formula (Ib) can be formed into a compound of the formula (Ic) by a substitution reaction (for example, an affinity substitution reaction or the like) or a coupling reaction (such as Suzuki coupling, etc.), in the compound of the formula (Ia).
  • a substitution reaction for example, an affinity substitution reaction or the like
  • a coupling reaction such as Suzuki coupling, etc.
  • L 1 and L 2 are leaving groups including, but not limited to, triflate; chlorine, bromine, iodine; sulfonate groups such as mesylate, tosylate, p-bromobenzene a sulfonate, a p-toluenesulfonate or the like; an acyloxy group such as an acetoxy group, a trifluoroacetoxy group or the like.
  • Step 2 A compound of the formula (Ic) can be reacted with a compound of the formula (Id) by a substitution reaction or a coupling reaction to form a compound of the formula (Ie), for example, at a temperature, using a suitable catalyst (or containing a suitable ligand) or a base. And proceed with a suitable solvent. If acid catalyzed, 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 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 4 The amino compound can be condensed to the amide with the corresponding acid chloride under basic conditions or condensed with the corresponding carboxylic acid in the presence of a condensing agent.
  • the compound of the formula (VI) of the present invention can be produced by the method shown in Scheme 1.
  • Step 1 A compound of the formula (Iaa) and a compound of the formula (Iba) can be formed into a compound of the formula (Ica) by a substitution reaction (for example, an affinity substitution reaction or the like) or a coupling reaction (such as Suzuki coupling, etc.), in the compound of the formula (Iaa).
  • a substitution reaction for example, an affinity substitution reaction or the like
  • a coupling reaction such as Suzuki coupling, etc.
  • L 1 and L 2 are leaving groups including, but not limited to, triflate; chlorine, bromine, iodine; sulfonate groups such as mesylate, tosylate, p-bromobenzene a sulfonate, a p-toluenesulfonate or the like; an acyloxy group such as an acetoxy group, a trifluoroacetoxy group or the like.
  • Step 2 A compound of the formula (Ica) can be reacted with a compound of the formula (Ida) by a substitution reaction or a coupling reaction to form a compound of the formula (Iea), for example, at a temperature, using a suitable catalyst (or containing a suitable ligand) or a base. And proceed with a suitable solvent. If acid catalyzed, the catalyst can be, but is not limited to, TFA or p-toluenesulfonic acid.
  • the palladium catalyst used may be, but not limited to, Pd 2 (dba) 3 (tris(dibenzylideneacetone) dipalladium), and the ligand used may be, but not limited to, XantPhos (4, 5). - bis(diphenylphosphino)-9,9-dimethyloxaxime), the base used may be, but not limited to, cesium carbonate.
  • Step 3 conversion of the nitro compound Iea 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, Hydrogenation reduction.
  • a metal may be, but not limited to, iron powder, zinc powder
  • stannous chloride or under palladium carbon catalysis, Hydrogenation reduction.
  • Step 4 The amino compound I-f-a can be condensed to the amide under basic conditions with the corresponding acyl chloride or condensed with the corresponding carboxylic acid in the presence of a condensing agent.
  • Step 1 Deprotection of a compound of formula (I-b1) affords a compound of formula (I-b2) wherein PG in the compound of formula (I-b1) is an amino protecting group.
  • Amino protecting groups include, but are not limited to, tert-butoxycarbonyl (Boc); arylmethoxycarbonyl, benzyloxycarbonyl (Cbz) and 9-fluorenylmethoxycarbonyl (Fmoc); benzyl (Bn), trityl (Tr), 1,1-di-(4'-methoxyphenyl)methyl; trimethylsilyl (TMS) and tert-butyldimethylsilyl (TBS), and the like.
  • the method of deprotection can be referred to conventional methods in the art.
  • Compounds of formula (I-b1) are either commercially available or can be prepared by methods known to those skilled in the art.
  • Step 2 According to the difference of R 0 , a compound of the formula (I-b2) and a compound of the formula (I-b3) are subjected to acylation, coupling (such as Suzuki coupling, etc.) to form a compound of the formula (Iba).
  • HX is an electronegative nucleophilic group including, but not limited to, a hydroxyl group, an amino group, a thiol group.
  • the compound of the formula (I-b4) can be produced by a compound of the formula (Iaa) and a compound of the formula (I-b1) in a similar manner to the step 1 of the scheme 1, wherein the compound of the formula (Ica) can be obtained from the compound of the formula (I-b4).
  • the compound of the formula (VI) of the present invention can be produced by the method shown in Scheme 2.
  • the compound of the formula (Ica) can be reacted with a compound of the formula (Ija) by a substitution reaction or a coupling reaction to form a compound of the formula (VI), for example, at a certain temperature, using a suitable catalyst (or containing a suitable ligand) or a base and appropriate The solvent is carried out.
  • a suitable catalyst or containing a suitable ligand or a base and appropriate The solvent is carried out.
  • the catalyst can be, but is not limited to, TFA or p-toluenesulfonic acid.
  • the palladium catalyst used may be, but not limited to, Pd 2 (dba) 3 (tris(dibenzylideneacetone) dipalladium), BINAP(( ⁇ )-2,2'-bis-(
  • the ligand used for diphenylphosphino)-1,1'-binaphthyl can be, but is not limited to, Xantphos (4,5-bis(diphenylphosphino)-9,9-dimethyloxaxime), used
  • the base can be, but is not limited to, cesium carbonate.
  • the compound of the formula (I-j-a) is prepared by using a compound of the formula (I-d1) as a starting material, followed by an amino group protecting group reaction, an amine substitution reaction, a nitro reduction reaction, an acylation reaction and an amino deprotection reaction.
  • the above various reactions are all conventional reactions in the art.
  • Compounds of formula (I-d1) are commercially available or can be prepared by methods known to those skilled in the art (for the synthesis of compounds of formula (I-j-a) see WO2013014448A1).
  • 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).
  • LC-MS Agilent 1200 HPLC System/6140MS LC/MS mass spectrometer (manufacturer: Agilent), column WatersX-Bridge, 150 x 4.6 mm, 3.5 ⁇ m.
  • Preparative high performance liquid chromatography Waters PHW007, column XBridge C18, 4.6*150 mm, 3.5 um.
  • ISCO Combiflash-Rf75 or Rf200 automatic column analyzer Use ISCO Combiflash-Rf75 or Rf200 automatic column analyzer, Agela 4g, 12g, 20g, 40g, 80g, 120g disposable silica gel column.
  • Known starting materials can be synthesized by or according to methods known in the art, or can be purchased from ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, Accela ChemBio Inc and Dary Chemicals. the company.
  • the reactions in the examples were all carried out under a nitrogen atmosphere or an argon atmosphere; the solution in the examples was an aqueous solution.
  • the progress of the reaction can be monitored by thin layer chromatography (TLC), and the purification of the compound can be carried out by column chromatography.
  • TLC thin layer chromatography
  • the developer system for column chromatography or TLC may be selected from the group consisting of: dichloromethane and methanol systems, n-hexane and ethyl acetate systems, petroleum ether and ethyl acetate systems, and acetone systems, etc., and the volume ratio of the solvent depends on the polarity of the compound. Adjust differently.
  • DMF dimethylformamide
  • DMSO dimethyl sulfoxide
  • THF tetrahydrofuran
  • DIEA N, N-diisopropylethylamine
  • EA ethyl acetate
  • PE petroleum ether
  • BINAP (2R, 3S -2,2'-bisdiphenylphosphino-1,1'-binaphthyl
  • NBS N-bromosuccinimide
  • NCS N-chlorosuccinimide
  • Pd 2 ( Dba) 3 tris(dibenzylideneacetone)dipalladium
  • Pd(dppf)Cl 2 [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride).
  • room temperature refers to about 25 °C.
  • Step a The reaction substrate 1 (10.6 g, 58 mmol) was placed in a 500 mL one-mouth reaction flask, and a mixed solution of THF/water (100 mL / 60 mL) was added to dissolve the substrate. Ammonium chloride (15.5 g, 292 mmol) and reduced iron powder (26 g, 467 mmol) were successively added to the stirred reaction flask at room temperature, and then the reaction system was heated to 65 ° C and stirring was continued for 3 hours. The progress of the reaction was checked by TLC. After the substrate was completely reacted, excess iron powder was removed by filtration, and the filter cake was rinsed three times with ethyl acetate. The filtrate was extracted three times with ethyl acetate/water, and the organic layer was evaporated. Yield: 93%; purity: 90%; MS m/z (ESI): 142.0 [M+H] + .
  • Step b Compound 2 (8.0 g, 43 mmol) was placed in a 500 mL single-mouth reaction flask, and concentrated sulfuric acid (100 mL) was added to dissolve the substrate under constant stirring. Concentrated nitric acid (6.15 mL, 48 mmol) was slowly added dropwise to the stirred reaction flask at minus 20 ° C and kept at this temperature for 5 minutes. The progress of the reaction was checked by TLC, and after the substrate was completely reacted, it was poured into ice water. 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.
  • Step 2 The reaction substrate tert-butyl 4-fluoro-2-methoxy-5-nitrophenylcarbamic acid tert-butyl ester a2 (11.46 g, 40 mmol) was dissolved in 60 ml of N,N-dimethylacetamide N,N,N'-trimethylethylenediamine (4.90 g, 48 mmol), N,N-diisopropylethylamine (7.74 g, 60 mmol) was added, and the mixture was stirred at 90 ° C for 6 hours. The progress of the reaction was checked by TLC.
  • Step 3 tert-Butyl 4-((2-(dimethylamino)ethyl)(methyl)amino)-2-methoxy-5-nitrophenylcarbamate (3 g, 32.6 mmol) Dissolved in 200 ml of methanol and added 1.0 g of 10% Pd/C. After replacing the air with hydrogen, the hydrogen balloon was hydrogenated at room temperature, and the reaction was stirred for 1 hour. The progress of the reaction was checked by TLC. After the reaction of the substrate was completed, the filter cake was filtered with a small portion of methanol, and the filtrate was concentrated to give the desired product a4 (10.70 g, 97%). Used directly in the next step. MS m/z (ESI): 339 [M+H] + .
  • Step 4 5-Amino-4-((2-(dimethylamino)ethyl)(methyl)amino)-2-methoxyphenylcarbamic acid tert-butyl ester a4 (10.1 g, 30 mmol) Triethylamine (6.12 g, 60 mmol) was dissolved in dichloromethane (200 mL), cooled to 0 ° C, EtOAc (3. The progress of the reaction was checked by TLC. After the reaction of the substrate was completed, the mixture was washed successively with saturated aqueous sodium hydrogen carbonate and brine, dried over anhydrous sodium sulfate, filtered, ). Used directly in the next step. MS m/z (ESI): 393 [M+H] + .
  • Step 5 5-Acrylamide-4-((2-(dimethylamino)ethyl)(methyl)amino)-2-methoxyphenylcarbamic acid tert-butyl ester a5 (9.41 g, 24 mmol Dissolved in 100 ml of dichloromethane, cooled to 0 ° C, added 20 ml of trifluoroacetic acid, and stirred at room temperature under nitrogen for 18 hours. The progress of the reaction was checked by TLC, and after the reaction of the substrate was completed, the reaction solution was concentrated under reduced pressure. The residue was dissolved in EtOAc (3 mL).
  • Step 2 Compound a1 (1.96 g, 10.5 mmol), Pd 2 (dba) 3 (964 mg, 1.05 mmol), Xantphos (1.219 g, 2.11 mmol) and cesium carbonate (6.86 g, 21.0 mmol) were added to compound 4a- 2 (3 g, 10.5 mmol) in 70 ml of 1,4-dioxane solution. Stir vigorously for 20 hours at 120 ° C in a N 2 atmosphere.
  • Step 1 4-Dimethylaminopiperidine (106 mg, 0.83 mmol) and potassium carbonate (286 mg, 2.07 mmol) were added to compound 4-a (300 mg, 0.69 mmol) in 20 ml of DMF and stirred vigorously at 100 ° C for 4 h . The progress of the reaction was checked by TLC. After the substrate was completely reacted, it was extracted three times with ethyl acetate/water system. The organic layer was separated and concentrated under reduced pressure to give compound 5a-1 (400 mg, 95%), MS m/z (ESI) :544.2[M+H] + .
  • Step 2 Trifluoroacetic acid (3 mL) was added to a solution of compound 5a-1 (1 g, 1.. The progress of the reaction was checked by TLC. After the substrate was completely reacted, the excess dichloromethane was spun off under reduced pressure, diluted with water, adjusted to pH, and extracted with dichloromethane and methanol (10:1). Dry to give compound 5-a (1.1 g, 45.8%). MS m/z (ESI): 444.2 [M+H] + .
  • Step 1 Compound 1a (10 g, 54.5 mmol), potassium carbonate (13.7 g, 99.1 mmol) and N-Boc-3-hydroxyazetidine (8.58 g, 49.5 mmol) were placed in a 500 ml single-mouth reaction flask. 300 ml of acetonitrile was added, and the mixture was heated to 90 ° C to stir the reaction for 4 hours. The progress of the reaction was checked by TLC, and after the reaction of the substrate was completed, it was extracted three times with an ethyl acetate/water system, and the organic layer was separated, washed with water, washed with saturated brine and concentrated to give a crude product.
  • Step 2 The reaction substrate 7a-2 (500 mg, 1.56 mmol) was dissolved in 20 ml of dichloromethane, trifluoroacetic acid (3.5 g, 30.7 mmol) was slowly added, and the reaction was stirred at room temperature for 2 hours. The progress of the reaction was checked by TLC. After the reaction of the substrate was completed, the reaction mixture was concentrated under reduced pressure to give 800 mg of crude crude product 7a-3.
  • Step 3 The reaction substrate 7a-3 (800 mg, ca. 1.56 mmol) was dissolved in dichloromethane (25 mL). After cooling to 0 ° C, N,N-diisopropylethylamine (2.07 g, 16.02 mmol) was added. After stirring for 10 minutes, methylsulfonyl chloride (360 mg, 3.14 mmol) was added, and the mixture was stirred at room temperature for 2 hours. The progress of the reaction was checked by TLC. After the reaction of the substrate was completed, the reaction mixture was diluted with 25 ml of dichloromethane, washed with water, washed with saturated brine, and concentrated under reduced pressure.
  • Step 1 To a solution of compound 7a-2 (640 mg, 2.0 mmol) in 1,4-dioxane (10 ml), EtOAc / EtOAc. After the reaction mixture was stirred at room temperature for 3 hr, the reaction mixture was evaporated.
  • Step 2 To a solution of compound 8a-1 (440 mg, 2.0 mmol) and triethylamine (810 mg, 8.0 mmol) in dichloromethane (35 ml) Methane (5 ml) solution. The reaction mixture was stirred at 0 ° C for 1 hour. After completion of the reaction, dichloromethane and water were added and extracted with dichloromethane. The organic layer was washed with brine, dried over anhydrous sodium sulfate MS m/z (ESI): 262 [M+H] + .
  • Step 2 Trifluoroacetic acid (3.50 g, 30.70 mmol) was added to a solution of compound 14a-1 (510 mg, 1.53 mmol) in 5 ml of dichloromethane, and stirred vigorously at room temperature for 5 hours. After the completion of the reaction, the reaction mixture was evaporated to dryness mjjjjjj
  • the preparation method of the compound 18-a to the compound 21-a is the same as the preparation of the compound 14-a,
  • step 3 When preparing compound 18-a, replace 2,4,5-trichloropyrimidine in step 1 with 2,4-dichloro-5-fluoropyrimidine, step 3 The methanesulfonyl chloride is replaced with acetyl chloride.
  • Step 1 Compound a1 (186 mg, 1 mmol), Pd 2 (dba) 3 (92 mg, 0.1 mmol), BINAP (93 mg, 0.15 mmol) and cesium carbonate (652 mg, 2 mmol) were added to compound 7a-2 (320 mg, 1 mmol) ) in 11 ml of 1,4-dioxane solution. The microwave was reacted at 160 ° C for 20 hours. TLC tracks the reaction.
  • Step 3 Compound 22a-2 (323 mg, 0.42 mmol) was used as a starting material. Do not purify directly to the next step.
  • Step 1 To a solution of sodium hydride (220 mg, 5.49 mmol) in THF, EtOAc (EtOAc, EtOAc. After the reaction mixture was stirred at 0 ° C for 1 hour, Compound 1a (500 mg, 2.75 mmol) was added. The reaction mixture was stirred at 0 ° C for 2 hours and then at room temperature for 20 hours. After the end of the reaction, the reaction was quenched by the addition of water at 0 °C. The reaction mixture was extracted with EtOAc. EtOAc (EtOAc)EtOAc. MS m/z (ESI): 292 [M - 56 + H] + .
  • Step 2-3 The compound 23a-1 (500 mg, 1.44 mmol) was used as the starting material in the preparation of the compound 14-a in steps 2 and 3 to give 240 mg of compound 23-a, MS m/z (ESI): 326 [M+H] + .
  • the preparation method of the compound 24-a to the compound 26-a is the same as the preparation of the compound 23-a,
  • Step 1 To a solution of compound 1a (1.83 g, 10 mmol) and N,N-diisopropylethylamine (2.58 g, 20 mmol) in dichloromethane (90 ml) A solution of tert-butyl alkane-1-carboxylate (1.89 g, 11 mmol) in dichloromethane (10 mL). The reaction mixture was stirred at room temperature for 5 hours. After completion of the reaction, water and dichloromethane were added, and the organic layer was dried over anhydrous sodium sulfate. MS m/z (ESI): 319 [M+H] + .
  • Step 2 A solution of hydrochloric acid / 1,4-dioxane (10 ml, 40 mmol) was added dropwise to a solution of compound 31a-1 (638 mg, 2.0 mmol) in 1,4-dioxane (20 ml). After the reaction mixture was stirred at room temperature for 3 hours, the reaction mixture was evaporated.
  • Step 3 The compound 31a-2 is used as a raw material, and is prepared in the third step of the preparation method of the compound 7-a. Compound 31-a was obtained. MS m/z (ESI): 297 [M+H] + .
  • Step 1 Starting from the compound 1a and (S)-3-aminopiperidine-1-carboxylic acid tert-butyl ester, the preparation was carried out in the same manner as in the step 1 of the method of the compound 31-a. Compound 32a-1 was obtained. MS m/z (ESI): 347 [M+H] + .
  • Step 2-3 The compound 32a-1 is used as a raw material, and is prepared in accordance with the steps 2 and 3 in the process for producing the compound 7-a. Compound 32-a was obtained. MS m/z (ESI): 325.0 [M+H] + .
  • the compound 32a-2 was used as a raw material, and was prepared by referring to the preparation method of the compound 35-a. Compound 36-a was obtained. MS m/z (ESI): 293 [M+H] + .
  • Step 2-3 The compound 38a-1 is used as a raw material, and is prepared by sequentially referring to Step 2 and Step 3 in the preparation method of Compound 7-a. Compound 38-a was obtained. MS m/z (ESI): 325.0 [M+H] + .
  • the compound 13e is used as a raw material, and is prepared in the second step of the preparation method of the compound 8-a.
  • Step 1 2,4-Dichloro-5-(trifluoromethyl)pyrimidine (1.84 g, 8.5 mmol) and carbonic acid were added to a solution of compound 13c (1.6 g, 8.5 mmol) in EtOAc (50 mL). Potassium (2.35 g, 17 mmol) was stirred at 0 ° C for 1 h. After completion of the reaction, it was quenched with water and extracted with ethyl acetate. The title product 43a-1 (2.5 g, yield 67%) was obtained by purification by combi-flash column chromatography. MS m/z (ESI): 370 [M+H] + .
  • Step 2-3 The compound 43a-1 is used as a raw material, and is prepared by referring to steps 4 and 5 in the preparation method of the compound 13f. MS m/z (ESI): 348 [M+H] + .
  • Step 2-3 Using compound 44a-1 as a starting material, it is prepared by referring to steps 4 and 5 in the preparation method of compound 13f. MS m/z (ESI): 312 [M+H] + .
  • Step 1 Compound 47a-1 (5.0 g, 0.0292 mmol), dimethylamine hydrochloride (4.77 g, 0.0584 mmol), 2.1 g of Pd/C and 2.5 ml of acetic acid were added to 100 ml of methanol under a hydrogen atmosphere. After hydrogen exchange, the reaction was carried out at room temperature for 48 hours. After the reaction was completed, it was filtered and concentrated to give a crude product. Saturated NaHCO 3 and extracted with ethyl acetate, washed with brine, and concentrated to give the object 47a-2 (5.0g, 85% yield). MS m/z (ESI): ???[M+H] + .
  • Zinc powder (980 mg, 15.08 mmol), trimethylchlorosilane (175 mg, 1.61 mmol), 1,2-dibromoethane (303 mg, 1.61 mmol) were sequentially added to 20 ml of DMF solution, and after argon replacement, the reaction mixture was allowed to stand at room temperature. Stir for 3 minutes.
  • a solution of compound 48a-1 (3.95 g, 13.95 mmol) in 10 mL DMF
  • the above reaction solution was added to a mixture of Compound 2a (1.6 g, 10.74 mmol), Pd (dppf) Cl 2 (393 mg, 0.54 mmol), cuprous iodide (102 mg, 0.54 mmol) and 20 ml of DMF.
  • Compounds 49-a and 50-a can be prepared by referring to the compound 31-a, except that the compound 7a-1 in the step 1 is replaced with 2,4-dichloro-5-fluoropyrimidine and 2,4-di, respectively. Chloro-5-(trifluoromethyl)pyrimidine.
  • Step 1 2,4,5-Trichloropyrimidine 1a (2 g, 10.9 mmol) and cesium carbonate (10.2 g, 31.2 mmol) were added to 4-hydroxyazepane-1-carboxylic acid tert-butyl ester (2.24) g, 10.9 mmol) in 100 ml of acetonitrile solution, vigorously stirred at 85 ° C for 5 hours. After completion of the reaction, the insoluble material was filtered off, and the cake was washed with ethyl acetate. The organic layer was concentrated under reduced pressure and purified by EtOAc EtOAc (EtOAc) MS m/z (ESI): 306.0 [M - 56] + .
  • EtOAc EtOAc EtOAc
  • Step 2 Add trifluoroacetic acid (1.89 g, 16.7 mmol) to 4-(2,5-dichloropyrimidin-4-yloxy)azepane-1-carboxylic acid tert-butyl ester at 0 °C 1b (300 mg, 0.83 mmol) in 10 ml of dichloromethane was stirred vigorously at room temperature for 5 hours. After completion of the reaction, the reaction mixture was evaporated to dryness mjjjjjjj MS m/z (ESI): 262.0 [M+1] + .
  • Step 3 Add N,N-diisopropylethylamine (4 g, 30.5 mmol) to the compound 4-(2,5-dichloropyrimidin-4-yloxy)azepane at 0 °C 1c (400 mg, 1.53 mmol) in 12 ml of methylene chloride was stirred vigorously at 0 ° C for 30 min, then methanesulfonyl chloride (350 mg, 3.05 mmol) was added and vigorously stirred at 0 ° C for 2 hours.
  • Step 4 N-(5-Amino-2-((2-(dimethylamino)ethyl)(methyl)amino)-4-methoxyphenyl)acrylamide a (155 mg, 0.53 mmol), Pd 2 (dba) 3 (49 mg, 0.05 mmol), BINAP (66 mg, 0.10 mmol) and cesium carbonate (345 mg, 1.06 mmol) were added to the compound 4-(2,5-dichloropyrimidin-4-yloxy)- 1-(Methylsulfonyl)azepane 1d (180 mg, 0.53 mmol) in 5 mL 1,4-dioxane.
  • Step 2 Compound N-(5-Amino-2-(4-(dimethylamino)piperidin-1-yl)-4-methoxyphenyl)acrylamide b (250 mg, 0.80 mmol), Pd 2 (dba) 3 (80 mg, 0.08 mmol), Xantphos (100 mg, 0.16 mmol) and cesium carbonate (520 mg, 1.60 mmol) were added to the compound 4-(2-chloropyrimidin-4-yloxy)piperidin-1- tert-Butyl Formate 2b (250 mg, 0.80 mmol) in 5 mL of 1,4-dioxane solution. The mixture was vigorously stirred at 160 ° C for 30 minutes under microwave reaction.
  • Step 2 4-(2,5-dichloropyrimidin-4-yloxy)piperidine-1-carboxylic acid tert-butyl ester 3a (1.5 g, 4.32 mmol) was used as a starting material, and was carried out with reference to Step 2 in Example 1. Synthesis gave the compound 2,5-dichloro-4-(piperidin-4-yloxy)pyrimidine 3b (2.4 g, 100%). MS m/z (ESI): 248.0 [M+1] + .
  • Step 3 2,5-dichloro-4-(piperidin-4-yloxy)pyrimidine 3b (908 mg, 1.62 mmol) was used as a starting material.
  • Dichloro-4-(1-(methylsulfonyl)piperidin-4-yloxy)pyrimidine 3c (150 mg, 28%).
  • Step 4 Using 2,5-dichloro-4-(1-(methylsulfonyl)piperidin-4-yloxy)pyrimidine 3c (130 mg, 0.40 mmol) as a starting material, refer to step 4 in Example 1.
  • the synthesis was carried out to give the compound N-(5-(5-chloro-4-(1-(methylsulfonyl)piperidin-4-yloxy)pyrimidin-2-ylamino)-2-((2-( Dimethylamino)ethyl)(methyl)amino)-4-methoxyphenyl)acrylamide J-3 (75.60 mg, 33%).
  • the compound 2a (10 g, 67.11 mmol) was used as a starting material, and the synthesis was carried out by the method of Example 1, except that the 4-hydroxyazepane-1-carboxylic acid tert-butyl ester in the first step of Example 1 was changed to 4 -Hydroxypiperidine-1-carboxylic acid tert-butyl ester (12.8 g, 63.68 mmol) to give the compound N-(2-((2-(dimethylamino)ethyl)(methyl)amino)-4-methoxy 5-(4-(1-(methylsulfonyl)piperidin-4-yloxy)pyrimidin-2-ylamino)phenyl)acrylamide J-4 (93.42 mg, 33%).
  • the compound 2a (10 g, 67.11 mmol) was used as a starting material, and the synthesis was carried out by the method of Example 1, except that the 4-hydroxyazepane-1-carboxylic acid tert-butyl ester in the first step of Example 1 was replaced with tert-Butyl 4-hydroxypiperidine-1-carboxylate (12.8 g, 63.68 mmol), Compound a in step 4 of Example 1 was replaced with compound b (164 mg, 0.52 mmol) to give compound N-(2-(4) -(dimethylamino)piperidin-1-yl)-4-methoxy-5-(4-(1-(methylsulfonyl)piperidin-4-yloxy)pyrimidin-2-ylamino Phenyl) acrylamide (142.64 mg, 47%).
  • Step 1 The compound 2,4-dichloropyrimidine 2a (19.7 g, 110 mmol), cesium carbonate (65.2 g, 200 mmol) was added to 400 ml of N-Boc-3-hydroxyazetidine (17.3 g, 100 mmol). The mixture was vigorously stirred at 83 ° C for 4 hours in DMF solvent. The progress of the reaction was checked by TLC. After the substrate was completely reacted, it was extracted three times with an ethyl acetate/water system, and the organic layer was separated, washed with water, washed with saturated brine, and dried to give a crude product.
  • Step 2 The compound 4-fluoro-2-methoxy-5-nitroaniline (1.96g, 10.5mmol), Pd 2 (dba) 3 (964mg, 1.05mmol), Xantphos (1.219g, 2.11mmol) and carbonate ⁇ (6.86g, 21.0mmol) was added to the compound 3-(2-chloropyrimidin-4-yloxy)azetidin-1-carboxylic acid tert-butyl ester 6a (3g, 10.5mmol) in 70ml of 1,4- In a dioxane solution. Stir vigorously for 20 hours at 120 ° C in a N 2 atmosphere.
  • Step 3 Add N,N,N'-trimethylethylenediamine (4.08 g, 40.0 mmol) and potassium carbonate (13.8 g, 100 mmol) to the compound 3-(2-(4-fluoro-2-methoxy) 5-Bistidine-5-nitrophenylaminopyrimidin-4-yloxy)azetidin-1-carboxylic acid tert-butyl ester 6b (14.5 g, 33.3 mmol) in 170 ml of DMF, vigorously stirred at 100 ° C for 4 h . The progress of the reaction was checked by TLC. After the substrate was completely reacted, it was extracted three times with ethyl acetate/water system.
  • Step 4 Add trifluoroacetic acid (27 mL, 356.0 mmol) to the compound 3-(2-(4-(2-(dimethylamino)ethyl)(methyl)amino)-2-methoxy- 5-nitrophenylaminopyrimidin-4-yloxy)azetidin-1-carboxylic acid tert-butyl ester 6c (9.2 g, 17.8 mmol) in 250 ml of dichloromethane, stirring vigorously at room temperature 6 hour. The progress of the reaction was checked by TLC. After the substrate was completely reacted, the excess dichloromethane was spun off under reduced pressure, diluted with water, adjusted to pH, and extracted with dichloromethane and methanol (10:1).
  • Step 5 Addition of triethylamine (1.01 g, 10 mmol) to the compound N1-(4-(azetidin-3-yloxy)pyrimidin-2-yl)-N4- (at 0 ° C) 2-(Dimethylamino)ethyl)-2-methoxy-N4-methyl-5-nitrobenzene-1,4-diamine 6d (1.4 g, 1 mmol) in 10 mL dichloromethane After vigorous stirring at 0 ° C for 30 minutes, benzoyl chloride (140 mg, 1.2 mmol) was added.
  • Step 6 Add Pd/C (110 mg) to the compound (3-(2-(4-((2-(dimethylamino)ethyl)))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))
  • a solution of nitrophenylaminopyrimidin-4-yloxy)azetidin-1-yl)(phenyl)methanone 6e 650 mg, 1 mmol
  • Step 7 Add acryloyl chloride (138 mg, 1.54 mmol) and triethylamine (195 mg, 1.92 mmol) to the compound (3-(2-(5-amino-4-((2-)) (amino)ethyl)ethyl)(methyl)amino)-2-methoxyphenylamino)pyrimidin-4-yloxy)azetidin-1-yl)(phenyl)methanone 6f (630 mg, 1 mmol) in 10 ml of dichloromethane was stirred vigorously at 0 °C for 2 hours. After completion of the reaction, it was diluted with water and extracted with EtOAc EtOAc.
  • the compound 6b (14.5 g, 33.3 mmol) was used as a starting material, and the synthesis was carried out by the method of Example 6 except that the benzoyl chloride in the step 5 of Example 6 was replaced with benzenesulfonyl chloride (210 mg, 1.2 mmol).
  • benzenesulfonyl chloride 210 mg, 1.2 mmol.
  • the compound 6b (6.7 g, 15.4 mmol) was used as a starting material, and the synthesis was carried out by the method of Example 6, except that the N,N,N'-trimethylethylenediamine in the step 3 of Example 6 was changed to 4- Dimethylaminopiperidine (2.37 g, 18.5 mmol) gave the compound N-(5-(4-(1-benzoylazin-3-yloxy)pyrimidin-2-ylamino)-2-(4) -(Dimethylamino)piperidin-1-yl)-4-methoxyphenyl)acrylamide J-8 (33.46 mg, 15%).
  • the compound 6b (6.7 g, 15.4 mmol) was used as a starting material, and the synthesis was carried out by the method of Example 6, except that the N,N,N'-trimethylethylenediamine in the step 3 of Example 6 was changed to 4- Dimethylaminopiperidine (2.37 g, 18.5 mmol), benzoyl chloride in Step 5 of Example 6 was replaced with benzenesulfonyl chloride (88 mg, 0.45 mmol) to give compound N-(2-(4-(dimethyl) Amino)piperidin-1-yl)-4-methoxy-5-(4-(1-(phenylsulfonyl)azetidin-3-yloxy)pyrimidin-2-ylamino)benzene Acrylamide J-9 (78.86 mg, 34%).
  • the compound 6b (6.7 g, 15.4 mmol) was used as a starting material, and the synthesis was carried out by the method of Example 6, except that the N,N,N'-trimethylethylenediamine in the step 3 of Example 6 was changed to 4- Dimethylaminopiperidine (2.37 g, 18.5 mmol), benzoyl chloride in Step 5 of Example 6 was replaced with methanesulfonyl chloride (57 mg, 0.50 mmol) to give compound N-(2-(4-(dimethyl) Amino)piperidin-1-yl)-4-methoxy-5-(4-(1-(methylsulfonyl)azetidin-3-yloxy)pyrimidin-2-ylamino)benzene Acrylamide J-10 (93.21 mg, 45%).
  • the compound 1a (10 g, 54.5 mmol) was used as a starting material, and the synthesis was carried out by the method of Example 1, except that the 4-hydroxyazepane-1-carboxylic acid tert-butyl ester in the first step of Example 1 was replaced with N-Boc-3-hydroxyazetidine (8.58 g, 49.5 mmol) gave N-(5-(5-chloro-4-(1-(methylsulfonyl)azetidin-3- Benzyl)pyrimidin-2-ylamino)-2-((2-(dimethylamino)ethyl)(methyl)amino)-4-methoxyphenyl)acrylamide J-11 (56.2 mg, 31.5%).
  • the compound 1a (10 g, 54.5 mmol) was used as a starting material, and the synthesis was carried out by the method of Example 1, except that the 4-hydroxyazepane-1-carboxylic acid tert-butyl ester in the first step of Example 1 was replaced with N-Boc-3-hydroxyazetidine (8.58 g, 49.5 mmol), the compound a in step 4 of Example 1 was replaced with compound b (160 mg, 0.50 mmol) to give compound N-(5-(5) -Chloro-4-(1-(methylsulfonyl)azetidin-3-yloxy)pyrimidin-2-ylamino)-2-(4-(dimethylamino)piperidin-1- Base -4-methoxyphenyl) acrylamide formate J-12 (81.9 mg, 30.7%).
  • Step 1 3-Iodoazetidine-1-carboxylic acid tert-butyl ester 13a (2 g, 7.2 mmol) and cesium carbonate (4.72 g, 14.4 mmol) were added to the compound thioacetic acid (1.1 g, 14.4 mmol).
  • cesium carbonate 4.72 g, 14.4 mmol
  • the insoluble material was filtered off, and the cake was washed with ethyl acetate.
  • the organic layer was concentrated under reduced pressure and purified by EtOAc EtOAc (EtOAc) MS m/z (ESI): 232 [M+H] + .
  • Step 2 3-(acetylthio)azetidin-1-carboxylic acid tert-butyl ester 13b (0.7 g, 3.03 mmol) and potassium carbonate (0.836 g, 6.05 mmol) were added to 10 ml of methanol at 50 ° C Stir vigorously for 2 hours. After the reaction was completed, it was cooled to room temperature, adjusted to pH 2-3 with 2M aqueous hydrochloric acid solution, diluted with water, and extracted twice with ethyl acetate/water system, and the organic layer was concentrated under reduced pressure to give compound 3-indylazetidine. tert-Butyl-l-carboxylate 13c (450 mg, 78%) was used in the next step without purification. MS m/z (ESI): 190 [M+1] + .
  • Step 3 3-decylazetidine-1-carboxylic acid tert-butyl ester 13c (0.45 g, 2.38 mmol), 2,4,5-trichloropyrimidine (0.46 g, 2.5 mmol) and sodium carbonate (0.631) g, 5.96 mmol) was added to 20 ml of acetonitrile and stirred vigorously at 70 ° C for 3 hours. After completion of the reaction, the mixture was concentrated tolulululululululululululululululululululululululululululululululululululululululululululululululululululululululululululululululululululululululululululululululululululululululululululululululululululululululululululululululululululululululululululululululululululululululul
  • Step 4 3-(2,5-Dichloropyrimidin-4-ylsulfanyl)azetidin-1-carboxylic acid tert-butyl ester 13d (0.58 g, 1.73 mmol) was added to 10 mL dichloromethane. Trifluoroacetic acid (5 g, 43.9 mmol) was added dropwise at 0 ° C under stirring, and the reaction was returned to room temperature for 3 hours. After concentration, the crude compound was obtained as a crude 3-(2,5-dichloropyrimidin-4-ylthio) nitrogen. Heterocyclobutane 13e (1.5 g) was used in the next step without purification. MS m/z (ESI): 237[M+1] + .
  • Step 5 Compound 13e (1.5 g) was used as a starting material, which was synthesized by the method of Step 3 of Example 1, to give the compound 2,5-dichloro-4-(1-(methylsulfonyl)azetidin-3 -ylthio)pyrimidine 13f.
  • Step 6 Compound 13f (314 mg, 1 mmol) and compound b (318 mg, 1 mmol) were used as the starting materials, and the compound was obtained by the step 2 of Example 2 to obtain the compound N-(5-(5-chloro-4-(1-(-) Sulfosyl)azetidin-3-ylthio)pyrimidin-2-ylamino)-2-(4-(dimethylamino)piperidin-1-yl)-4-methoxyphenyl) Acrylamide J-13 (89.06 mg).
  • Step 1 3-bromoquinoline (0.31 g, 1.473 mmol), Pd 2 (dba) 3 (0.135 g, 0.1473 mmol), Xantphos (0.171 g, 0.2946 mmol) and cesium carbonate (1.92 g, 5.69 mmol)
  • Pd 2 (dba) 3 0.135 g, 0.1473 mmol
  • Xantphos 0.171 g, 0.2946 mmol
  • cesium carbonate (1.92 g, 5.69 mmol
  • Step 2 Compound 14b (80 mg, 0.15 mmol) was used as a starting material to give the crude product of compound 14c (yield: 70 mg). MS m/z (ESI): 515 [M+H] + .
  • Compound J-20 to compound J-32 are all starting from various 5-substituted or unsubstituted 2,4-dichloropyrimidines and N-Boc-3-hydroxyazetidine, and refer to Example 6
  • the method was prepared.
  • the N,N,N'-trimethylethylenediamine and benzoyl chloride in Steps 3 and 5 of Example 6 were replaced with the corresponding amine and acid chloride depending on the structure of the compound.
  • Compounds J-35, J-36 and J-37 are all starting from various 5-position chloro or unsubstituted 2,4-dichloropyrimidines and 4-hydroxyazepane-1-carboxylic acid tert-butyl esters.
  • the starting materials were prepared by the method of Example 1.
  • the methanesulfonyl chloride and compound a in Step 3 and Step 4 of Example 1 were replaced with the corresponding methanesulfonyl chloride and Compound b depending on the structure of the compound.
  • Compounds J-46, J-47, J-48, J-49, J-50, J-51 and J-52 are compounds 15-a, 16-a, 17-a, 18-a, 19-a, respectively.
  • 20-a and 21-a are prepared by reacting a raw material with compound b, and the reaction steps and conditions are as described in Example 45.
  • Step 1 A solution of compound 6d (210 mg, 0.5 mmol), 1-bromo-2-fluoroethane (75 mg, 0.6 mmol) and potassium carbonate (205 mg, 1.5 mmol) in acetonitrile (5 ml) was stirred at 50 ° C. After the completion of the reaction, the mixture was extracted with EtOAc EtOAc EtOAc. The crude product of compound 53b was obtained. Purification by Combi-flash column chromatography gave compound 53b (l. MS m/z (ESI): 464 [M+H] + .
  • Compound J-54 was prepared according to the method of Example 53 using Compound 22-a as a material.
  • Compound J-55 was prepared from Compound 46-a using the procedure of Step 2 of Example 3 and Step 1 of Example 53 in that order.
  • Compounds J-61, J-62, J-63, J-64, J-73 and J-74 are compounds 33-a, 49-a, 35-a, 50-a, 39-a and 51-a, respectively. It is prepared by reacting a raw material with compound b, and the reaction steps and conditions are as described in Example 60.
  • Step 1 Compound a1 (186 mg, 1 mmol), Pd 2 (dba) 3 (92 mg, 0.1 mmol), Xantphos (90 mg, 0.15 mmol) and cesium carbonate (652 mg, 2 mmol) were added to compound 7-a (298 mg, 1 mmol) ) in 4 ml of 1,4-dioxane solution. The microwave was reacted at 160 ° C for 20 minutes. After completion of the reaction, the mixture was filtered, washed with ethyl acetate, and then evaporated. MS m/z (ESI): 448 [M+H] + .
  • Step 2 1-Methylpiperazine (72 mg, 0.72 mmol) and potassium carbonate (99.5 mg, 0.72 mmol) were added to compound 70b (160 mg, 0.36 mmol) in 3 ml of DMF and stirred vigorously at 100 ° C for 2 h. The progress of the reaction was checked by TLC. After the substrate was completely reacted, it was extracted three times with ethyl acetate/water. The organic layer was separated and concentrated under reduced pressure to yield 180 mg of crude compound 70c. MS m/z (ESI): 528 [M+H] + .
  • Step 3-4 The compound 70c (180 mg, 0.34 mmol) was used as a material.
  • 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.
  • test substance was assayed for T790M/L858R double mutant EGFR kinase (Invitrogen, PV4879) and wild type EGFR kinase (Invitrogen, PV3872) by the z-lyte method.
  • the working concentrations of the components in the 10 uL T790M/L858R kinase reaction system were: 25 ⁇ M ATP, 0.1 (or 0.08) ng/ ⁇ L T790M/L858R kinase, 2 ⁇ M Tyr04 substrate (Invitrogen, PV3193).
  • the concentration of DMSO after addition of the compound prepared in the above examples of the present invention was 2%.
  • the working concentrations of the components in the 10 uL wild-type EGFR kinase reaction system were: 10 ⁇ M ATP, 0.8 ng/ ⁇ L wild-type EGFR kinase, and 2 ⁇ M Tyr04 substrate (Invitrogen, PV3193).
  • the concentration of DMSO after adding the test substance was 2%.
  • the 10 mM drug stock solution was diluted at room temperature with a water gradient of 4% DMSO to a final concentration of 10-0.005 [mu]M.
  • 2.5 ⁇ L of the test substance solution and 5 ⁇ L of a mixture of T790M/L858R kinase (or wild-type EGFR kinase) diluted with the reaction buffer and Tyr04 substrate were added to each well, and 2.5 ⁇ L of ATP was added to initiate the reaction.
  • the C1 well was replaced with ATP by a reaction buffer, the C2 well was not added with any drug, and the C3 well was added with a phosphorylated substrate as described in the instructions. After 60 minutes of shaking at 25 degrees on a shaker.
  • the inhibition rate calculation method (refer to the specification of Invitrogen, PV3193) is as follows:
  • Phosphorylation rate (1-((ER ⁇ C3 520nm - C3 450nm ) / ((C1 450nm - C3 450nm ) + ER ⁇ (C3 520nm - C1 520nm )))) ⁇ 100%
  • Inhibition rate (IR) (1 - (phosphorylation rate of test compound) / (phosphorylation rate of C2)) ⁇ 100%
  • the half-inhibitory concentration IC 50 was calculated using XLFIT 5.0 software (IDBS, UK). See Table 1 for the inhibitory activity and selective inhibitory activity of the enzyme.
  • the exemplified compounds of the present invention inhibit the activity of the EGFR mutant enzyme (T790M/L858R) at a concentration of 100 nM (some compounds below 10 nM), exhibiting a strong inhibitory activity against EGFR wild-type enzyme.
  • T790M WT The inhibitory activity was weak, and the compound of the present invention had a significant selective inhibitory activity against the EGFR mutant enzyme compared to the positive control BIBW2992 (Afatinib).
  • the selective inhibitory activity of the exemplified compound of the present invention against the EGFR mutant enzyme exceeds that of the comparative compound 1 (the specific structure is shown below, and can be referred to WO2013014448A1), and the selectivity is up to 21 times higher than that of the comparative compound 1.
  • the selective inhibitory activity against the EGFR mutant enzyme was significantly lower than that of the comparative compound 1 after the substitution of the substituent R 0 to the heteroaryl ring.
  • 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 minutes, 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: H1975 cells at 27,800 per ml, A431 cells and NIH3T3 cells at 33,300 per ml. The cells were seeded in a 96-well plate (BD 3072), 90 ⁇ L per well, and cultured overnight.
  • 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. 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 or selective inhibitory activities of the exemplified compounds on the cells are shown in Tables 2 and 3.
  • the exemplary compounds of the present invention inhibited the activity of H1975 cells at a concentration of 100 nM or less (some compounds below 10 nM), and showed strong inhibitory activity against EGFR mutant cells (H1975 cells), while EGFR was inhibited.
  • Wild type cells A431 cells
  • the compounds of the present invention had significant selective inhibitory activity against EGFR mutant cell growth compared to the positive control BIBW2992.
  • the selective inhibitory activity against EGFR mutant cell growth exceeded that of Comparative Compounds 1 and 2.
  • the selectivity was increased to nearly 10 times higher than that of Comparative Compound 1.
  • the exemplified compounds of the present invention have higher IC 50 values for NIH3T3 cells than the positive control Comparative Compound 1, and thus exhibit less toxicity.
  • 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 ug / mL Aprotinin (Sigma), 10 ug / mL Leupeptin (Sigma), now available.
  • 1xPBS buffer NaCl: 0.137M, KCl: 0.0027M , Na 2 PO 4 -12H 2 O: 0.01M, KH 2 PO 4: 0.0015M, pH7.4.
  • 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 exemplary compounds of the present invention inhibited the activity of H1975 cells at a concentration of 100 nM or less (some compounds below 10 nM), and showed strong inhibitory activity against mutant cell-level targets, whereas for EGFR.
  • Wild-type cells A431 cells
  • the exemplified compounds of the invention have significant selective inhibitory activity on cell-level targets compared to the positive control BIBW2992.
  • the selective inhibitory activity against the cell-level target was up to 32-fold higher than that of the comparative compounds 1 and 2.
  • 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% aqueous sodium carboxymethylcellulose 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, 5% DMAC, 5% Solutol HS 15 and 90% saline)
  • 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 sampling time (dorsal vein administration: 0.083, 0.25, 0.5, 1 after administration, respectively, before administration). , 2, 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 At each time point, blood was collected through the tail vein, or blood was collected from the heart (final blood collection) by about 150 uL.
  • 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, 24 blood collection, a total of 7 time points), blood collection through the eyelids, or blood collection by the heart (final blood collection) about 150uL.
  • 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 examples of the present invention in rats and mice at the same dosage and mode of administration are shown in Table 5:
  • 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

本发明涉及2，3，4，6-四取代苯-1，5-二胺衍生物、其制法与医药上的用途。具体地，本发明公开了式（I）化合物或其药学上可接受的盐、立体异构体、溶剂化合物或前药，及其制备方法和应用，式中各基团的定义详见说明书。

Description

2,3,4,6-四取代苯-1,5-二胺衍生物、其制法与医药上的用途 技术领域
本发明涉及医药技术领域，特别涉及一种2,3,4,6-四取代苯-1,5-二胺衍生物及其制备方法和作为EGFR酪氨酸激酶抑制剂的应用，以及由其制备的药物组合物和药用组合物。
背景技术
肺癌是全球发病率最高的癌症，在中国肺癌发病率位居所有癌症中第一位，也是中国发病率和死亡率最高的癌症，在中国肺癌病人中，30％病人具有EGFR突变，其中L858R和外显子19缺失突变占90％以上，这类病人对EGFR抑制剂更为敏感。现有已上市第一代EGFR抑制剂例如厄洛替尼，吉非替尼对这类病人效果良好，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
本发明的目的在于提供一种新型EGFR酪氨酸激酶抑制剂2,3,4,6-四取代苯-1,5-二胺衍生物，具有较高的EGFR T790M选择抑制性。
本发明的第一方面，提供一种式(I)所示的化合物、或其药学上可接受的盐、或其溶剂化物、或其立体异构体、或其前药；
Figure PCTCN2015076451-appb-000001
式中，
Z1、Z2、Z3各自独立地为CR10或N；R10为氢、羟基、CN、NO2、卤素(优选为F或Cl)、-NR11R12、C1-10烷基(优选为C1-6烷基，更优选为C1-3烷基)、C3-8环烷基(优选为C3-6环烷基)、C3-8环烷氧基(优选为C3-6环烷氧基)、C2-10烯基(优选为C2-6烯基，更优选为C2-4烯基)、C2-10炔基(优选为C2-6炔基，更优选为C2-4炔基)、C1-10烷氧基(优选为C1-6烷氧基，更优选为C1-3烷氧基)、-CHO、-COC1-10烷基(优选为-COC1-6烷基，更优选为-COC1-3烷基)、-COC6-10芳基(优选为-COC6芳基，如-CO-苯基)、C6-10芳基(优选为C6芳基，如苯基)、-CONR11R12、-C(O)OC1-10烷基(优选为-C(O)OC1-6烷基，更优选为-C(O)OC1-3烷基)、-OC(O)C1-10烷基(优选为-OC(O)C1-6烷基，更优选为-OC(O)C1-3烷基)、-SO2C1-10烷基(优选为-SO2C1-6烷基，更优选为-SO2C1-3烷基)、-SO2C6-10芳基(优选为-SO2C6芳基，如-SO2-苯基)或叔丁氧羰基；其中，所述的烷基、环烷基、烯基、炔基、芳基、烷氧基为未取代的或被1-3个选自下组的取代基所取代：卤素(优选为F或Cl)、硝基、C6-10芳基(优选苯基)、C1-10烷基(优选为C1-6烷基，更优选为C1-3烷基)、C1-10烷氧基(优选为C1-6烷氧基，更优选为C1-3烷氧基)、C3-8环烷基(优选为C3-6环烷基)、C3-8环烷氧基(优选为C3-6环烷氧基)、C2-10烯基(优选为C2-6烯基，更优选为C2-4烯基)、C2-10炔基(优选为C2-6炔基，更优选为C2-4炔基)、-CONR11R12、-C(O)OC1-10烷基(优选为-C(O)OC1-6烷基，更优选为-C(O)OC1-3烷基)、-CHO、-OC(O)C1-10烷基(优选为-OC(O)C1-6烷基，更优选为-OC(O)C1-3烷基)、-SO2C1-10烷基(优选为-SO2C1-6烷基，更优选为-SO2C1-3烷基)、-SO2C6-10芳基(优选为-SO2C6芳基，如-SO2-苯基)、-COC6-10芳基(优选为-COC6芳基，如-CO-苯基)；
Y1、Y2各自独立地为二价C1-3烃基，或Y1或Y2中的亚甲基单元(-CH2-)被-C(RyRx)-、-NRyC(O)-、亚环丙基、-C(O)NRy-、-N(Ry)SO2-、-SO2N(Ry)-、-S-、-S(O)-、-SO2-、-OC(O)-、-C(O)O-、-O-、-N(Ry)-或-C(O)-置换；其中，Ry、Rx各自独立地为氢、卤素(优选F或Cl)、羟基、CN、NO2、C1-10烷基(优选为C1-6烷基，更优选为C1-3烷基)、C1-10烷氧基(优选为C1-6烷氧基，更优选为C1-3烷氧基)、C3-8环烷基(优选为C3-6环烷基)、C3-8环烷氧基(优选为C3-6环烷氧基)、C2-10烯基(优选为C2-6烯基，更优选为C2-4烯基)、C2-10炔基(优选为C2-6炔基，更优选为C2-4炔基)、C6-10芳基(优选为苯基)；
X为NRz、O或S；Rz为氢、C1-10烷基(优选为C1-6烷基，更优选为C1-3烷基)、卤代的C1-10烷基(优选为卤代的C1-6烷基，更优选为卤代的C1-3烷基)或C3-8环烷基(优选为C3-6环烷基)；
m1、m3各自独立地为0、1或2；m2为0或1；且m1、m2和m3不同时为0；
A环为C6-10芳基(如苯环)、3至7元饱和或部分不饱和单环、8至10元饱和或部分不饱和双环、具有1到3个独立选自氮、氧或硫的杂原子的3至7元饱和或部分不饱和单杂环、具有1到5个独立选自氮、氧或硫的杂原子的8至10元饱和或部分不饱和双杂环、具有1到3个独立选自氮、氧或硫的杂原子的5至6元单环杂芳基环、或具有1到5个独立选自氮、氧或硫的杂原子的8至10元双环杂芳基环；
Ra、Rb各自独立地为氢、羟基、CN、NO2、卤素(优选为F或Cl)、-NR11R12、C1-10烷基(优选为C1-6烷基，更优选为C1-3烷基)、C3-8环烷基(优选为C3-6环烷基)、C3-8环烷氧基(优选为C3-6环烷氧基)、C2-10烯基(优选为C2-6烯基，更优选为C2-4烯基)、C2-10炔基(优选为C2-6 炔基，更优选为C2-4炔基)、C1-10烷氧基(优选为C1-6烷氧基，更优选为C1-3烷氧基)、-CHO、-COC1-10烷基(优选为-COC1-6烷基，更优选为-COC1-3烷基)、-COC6-10芳基(优选为-COC6芳基，如-CO-苯基)、C6-10芳基(优选为C6芳基，如苯基)、-CONR11R12、-C(O)OC1-10烷基(优选为-C(O)OC1-6烷基，更优选为-C(O)OC1-3烷基)、-OC(O)C1-10烷基(优选为-OC(O)C1-6烷基，更优选为-OC(O)C1-3烷基)、-SO2C1-10烷基(优选为-SO2C1-6烷基，更优选为-SO2C1-3烷基)、-SO2C6-10芳基(优选为-SO2C6芳基，如-SO2-苯基)、-S(O)C1-10烷基(优选为-S(O)C1-6烷基，更优选为-S(O)C1-3烷基)、-S(O)C6-10芳基(优选为-S(O)C6芳基，如-S(O)-苯基)、叔丁氧羰基、-NHC1-10烷基(优选为-NHC1-6烷基，更优选为-NHC1-3烷基)、-NC(O)C1-10烷基(优选为-NC(O)C1-6烷基，更优选为-NC(O)C1-3烷基)、-NSO2C1-10烷基(优选为-NSO2C1-6烷基，更优选为-NSO2C1-3烷基)；其中，所述的烷基、环烷基、烯基、炔基、芳基、烷氧基可未取代或任选地具有1-3个选自下组的取代基：卤素(优选为F或Cl)、羟基、NO2、C6-10芳基(优选苯基)、C1-10烷基(优选为C1-6烷基，更优选为C1-3烷基)、C1-10烷氧基(优选为C1-6烷氧基，更优选为C1-3烷氧基)、C3-8环烷基(优选为C3-6环烷基)、C3-8环烷氧基(优选为C3-6环烷氧基)、C2-10烯基(优选为C2-6烯基，更优选为C2-4烯基)、C2-10炔基(优选为C2-6炔基，更优选为C2-4炔基)、-CONR11R12、-C(O)OC1-10烷基(优选为-C(O)OC1-6烷基，更优选为-C(O)OC1-3烷基)、-CHO、-OC(O)C1-10烷基(优选为-OC(O)C1-6烷基，更优选为-OC(O)C1-3烷基)、-SO2C1-10烷基(优选为-SO2C1-6烷基，更优选为-SO2C1-3烷基)、-SO2C6-10芳基(优选为-SO2C6芳基，如-SO2-苯基)、-COC6-10芳基(优选为-COC6芳基，如-CO-苯基)；
或者Ra、Rb各自独立地为具有1到4个独立选自氮、氧或硫的杂原子的5至6元单环杂芳基、具有1到5个独立选自氮、氧或硫的杂原子的8至10元双环杂芳基(优选为9至10元双环杂芳基)；其中，所述的5至6元单环杂芳基或8至10元双环杂芳基可未取代或任选地具有1-5个选自下组的取代基：卤素(优选为F或Cl)、羟基、CN、NO2、C1-10烷基(优选为C1-6烷基，更优选为C1-3烷基)、卤代的C1-10烷基(优选卤代的C1-6烷基，更优选卤代的C1-3烷基)、C1-10烷氧基(优选为C1-6烷氧基，更优选为C1-3烷氧基)、卤代的C1-10烷氧基(优选为卤代的C1-6烷氧基，更优选为卤代的C1-3烷氧基)、C3-8环烷基(优选为C3-6环烷基)、C3-8环烷氧基(优选为C3-6环烷氧基)、C2-10烯基(优选为C2-6烯基，更优选为C2-4烯基)、C2-10炔基(优选为C2-6炔基，更优选为C2-4炔基)、-CONR11R12、-C(O)OC1-10烷基(优选为-C(O)OC1-6烷基，更优选为-C(O)OC1-3烷基)、-CHO、-OC(O)C1-10烷基(优选为-OC(O)C1-6烷基，更优选为-OC(O)C1-3烷基)、-SO2C1-10烷基(优选为-SO2C1-6烷基，更优选为-SO2C1-3烷基)、-SO2C6-10芳基(优选为-SO2C6芳基，如-SO2-苯基)、-COC6-10芳基(优选为-COC6芳基，如-CO-苯基)；
n1、n2各自独立地为0、1、2、3、4、5或6；
R1为氢或C1-10烷基(优选为C1-6烷基，更优选为C1-3烷基)；
R2、R3、R5各自独立地为氢、卤素(优选F或Cl)、C1-10烷基(优选为C1-6烷基，更优选为C1-3烷基)或C1-10烷氧基(优选为C1-6烷氧基，更优选为C1-3烷氧基)；
R4为氢，羟基、-OC1-10烷基、CN、NO2、卤素、C3-8环烷基、C3-8环烷氧基、C2-10烯基、C2-10炔基、-CHO、-COC1-10烷基、-COC6-10芳基、C6-10芳基、-CONR11R12、-C(O)OC1-10烷基、-OC(O)C1-10烷基、-SO2C1-10烷基、-SO2C6-10芳基、叔丁氧羰基、-NHC1-10烷基、-N(C1-10 烷基)2、-N(C1-10烷基)(C3-8环烷基)、-NC(O)C1-10烷基、-NSO2C1-10烷基、或含1或2个氮原子以及0到3个氧或硫原子的3至7元饱和或部分不饱和单杂环(优选为5至6元饱和或部分不饱和单杂环)；其中，所述的烷基、环烷基、烯基、炔基、芳基、3至7元饱和或部分不饱和单杂环为未取代的或被1-3个选自下组的取代基所取代：卤素(优选为F或Cl)、NO2、CN、羟基、-CH2NR11R12、-NR11R12、C1-10烷基(优选为C1-6烷基，更优选为C1-3烷基)、C3-8环烷基(优选为C3-6环烷基)、C3-8环烷氧基(优选为C3-6环烷氧基)、C2-10烯基(优选为C2-6烯基，更优选为C2-4烯基)、C2-10炔基(优选为C2-6炔基，更优选为C2-4炔基)、C1-10烷氧基(优选为C1-6烷氧基，更优选为C1-3烷氧基)、-CHO、-COC1-10烷基(优选为-COC1-6烷基，更优选为-COC1-3烷基)、-COC6-10芳基(优选为-COC6芳基，如-CO-苯基)、C6-10芳基(优选为C6芳基，如苯基)、-CONR11R12、-C(O)OC1-10烷基(优选为-C(O)OC1-6烷基，更优选为-C(O)OC1-3烷基)、-OC(O)C1-10烷基(优选为-OC(O)C1-6烷基，更优选为-OC(O)C1-3烷基)、-SO2C1-10烷基(优选为-SO2C1-6烷基，更优选为-SO2C1-3烷基)、-SO2C6-10芳基(优选为-SO2C6芳基，如-SO2-苯基)、叔丁氧羰基、-NHC1-10烷基(优选为-NHC1-6烷基，更优选为-NHC1-3烷基)、-NC(O)C1-10烷基(优选为-NC(O)C1-6烷基，更优选为-NC(O)C1-3烷基)、-NSO2C1-10烷基(优选为-NSO2C1-6烷基，更优选为-NSO2C1-3烷基)、吗啉基、四氢吡咯基、哌嗪基、甲基哌嗪基；
R6和R7各自独立地为氢或-CH2NR13R14；其中，R13、R14各自独立地为氢或甲基；或R13、R14和相连的氮原子共同形成5-6元含氮饱和杂环；
其中，R11、R12各自独立地为氢、C1-10烷基(更优选为C1-6烷基，最优选为C1-3烷基)、C1-10烷氧基(更优选为C1-6烷氧基，最优选为C1-3烷氧基)、C3-8环烷基(更优选为C3-6环烷基)、C3-8环烷氧基(更优选为C3-6环烷氧基)、C2-10烯基(优选为C2-6烯基，更优选为C2-4烯基)、C2-10炔基(优选为C2-6炔基，更优选为C2-4炔基)、C6-10芳基(优选为苯基)；或者R11、R12和相连的氮原子共同形成3-6元含氮饱和杂环。
在另一优选例中，式(I)所示化合物中，A环为3至7元饱和或部分不饱和单环、8至10元饱和或部分不饱和双环、具有1到3个独立选自氮、氧或硫的杂原子的3至7元饱和或部分不饱和单杂环、具有1到5个独立选自氮、氧或硫的杂原子的8至10元饱和或部分不饱和双杂环。
在另一优选例中，A环为单环、双环、单杂环或双杂环时，选自下组：3至7元饱和或部分不饱和单环；8至10元饱和或部分不饱和双环；含1个或2个氮原子的3至7元饱和或部分不饱和单杂环；含1个、2个或3个氮原子的8至10元饱和或部分不饱和双杂环；含1个或2个氧原子或硫原子的3至7元饱和或部分不饱和单杂环；含1个、2个或3个氧原子或硫原子的8至10元饱和或部分不饱和双杂环；含1个氮原子和1个氧原子或硫原子的3至7元饱和或部分不饱和单杂环；含1个或2个氮原子和1个氧原子或硫原子8至10元饱和或部分不饱和双杂环；含1个硫原子和1个氧原子的3至7元饱和或部分不饱和单杂环；或者含1个硫原子和1个氧原子的8至10元饱和或部分不饱和双杂环。
在另一优选例中，A环为5至6元单环杂芳基环或8至10元双环杂芳基环时，选 自下组：
Figure PCTCN2015076451-appb-000002
其中，Ra1为氢、甲基或乙基。
在另一优选例中，所述式(I)化合物为式(II)所示化合物：
Figure PCTCN2015076451-appb-000003
式中，
n3、n4各自独立地为0、1、2或3，且n3、n4不同时为0；
W1为N或CR15；W2为N、O、S或CR15；R15为氢、羟基、CN、NO2、卤素(优选为F或Cl)、-NR11R12、C1-10烷基(优选为C1-6烷基，更优选为C1-3烷基)、C3-8环烷基(优选为C3-6环烷基)、C3-8环烷氧基(优选为C3-6环烷氧基)、C2-10烯基(优选为C2-6烯基，更优选为C2-4烯基)、C2-10炔基(优选为C2-6炔基，更优选为C2-4炔基)、C1-10烷氧基(优选为C1-6烷氧基，更优选为C1-3烷氧基)、C6-10芳基(优选为C6芳基，如苯基)；且当W2为O或S时，R0不存在；
R8、R9、R0的定义同上述Ra或Rb的定义；
Z1、Z2、Z3、R1、R2、R3、R4、R5、R6、R7、R11、R12、Y1、Y2、X、m1、m2、m3、n1、n2定义如上。
在另一优选例中，所述式(I)化合物中，Y1、Y2各自独立地为二价C1-3烃基，且Y1或Y2中的亚甲基未被置换。
在另一优选例中，所述式(I)化合物为式(VII-1)、式(VII-2)、式(VII-3)、式(VII-4)、式(VII-5)、式(VII-6)、式(VII-7)、式(VII-8)、式(VII-9)、式(VII-10)所示化合物：
Figure PCTCN2015076451-appb-000004
Figure PCTCN2015076451-appb-000005
上述各式中，Z1、Z2、Z3、R1、R2、R3、R4、R5、R6、R7、Y1、Y2、X、m1、m2、m3、W1、W2、R0定义如上；R91、R92、R93、R94、R95、R81、R82、R83如R8、R9的定义。
在另一优选例中，式(I)所示化合物中，W1为CH或N；W2为N、O、S或CH。
在另一优选例中，式(I)所示化合物中，W1为CH或N；W2为N或CH。
在另一优选例中，式(I)所示化合物中，W1为CH；且W2为N、O、S或CH。
在另一优选例中，式(I)所示化合物中，W1为CH；且W2为N。
在另一优选例中，所述式(I)化合物为式(III)所示化合物：
Figure PCTCN2015076451-appb-000006
式中，Z1、Z2、Z3、R1、R2、R3、R4、R5、R6、R7、R8、R9、Y2、X、m2、m3、n1、n2、n3、n4、R0定义如上。
在另一优选例中，所述式(I)化合物为式(IV)所示化合物：
Figure PCTCN2015076451-appb-000007
式中，Z2、R1、R2、R3、R4、R5、R6、R7、R8、R9、Y2、X、m3、n1、n2、n3、n4、R0定义如上。
在另一优选例中，所述式(I)化合物为式(V)所示化合物：
Figure PCTCN2015076451-appb-000008
式中，Z2、R1、R2、R3、R4、R5、R6、R7、R8、R9、X、m3、n1、n2、n3、n4、R0定义如上。
在另一优选例中，所述式(I)化合物为式(VI)所示化合物：
Figure PCTCN2015076451-appb-000009
式中，Z2、R1、R2、R3、R4、R5、R6、R7、X、m3、n3、n4、R0定义如上。
在另一优选例中，Z2为CR10或N；R10为氢、羟基、CN、NO2、氟、氯、-NR11R12、C1-3烷基、C3-6环烷基、C3-6环烷氧基、C1-3烷氧基、-CHO、-COC1-3烷基、-CO-苯基、苯基、-CONR11R12、-C(O)OC1-3烷基、-OC(O)C1-3烷基、-SO2C1-3烷基、-SO2-苯基或叔丁氧羰基；其中，所述的烷基、环烷基、苯基、烷氧基为未取代的或被1-3个选自下组的取代基所取代：氟、氯、硝基、苯基、C1-3烷基、C1-3烷氧基、C3-6环烷基、C3-6环烷氧基、-CONR11R12、-C(O)OC1-3烷基、-CHO、-OC(O)C1-3烷基、-SO2C1-3烷基、-SO2-苯基、-CO-苯基。
在另一优选例中，Z2为CR10，R10为氢、羟基、NO2、氟、氯、-NH2、-N(CH3)2、C1-3烷基、环丙基、环丙氧基、C1-3烷氧基、-CHO、-COCH3、-CO-苯基、苯基、-CONH2、-CON(CH3)2、-C(O)OCH3、-OC(O)CH3、-SO2CH3、-SO2-苯基或叔丁氧羰基；其中，所述的烷基、环丙基、烷氧基或苯基为未取代的或被1-3个选自下组的取代基所取代：氟、氯、硝基、苯基、甲基、甲氧基、环丙基、环丙氧基、-CONH2、-CON(CH3)2、-C(O)OCH3、-CHO、-OC(O)CH3、-SO2CH3、-SO2-苯基、-CO-苯基。
在另一优选例中，Z2为CR10，R10为羟基、NO2、氟、氯、-NH2、-N(CH3)2、三氟甲基、甲氧基、-CHO、-COCH3、-CONH2、-C(O)OCH3或-OC(O)CH3。
在另一优选例中，R0为氢、羟基、C1-3烷基、C3-6环烷基、-CHO、-COC1-3烷基、-CO-苯基、苯基、-CONR11R12、-C(O)OC1-3烷基、-SO2C1-3烷基、-SO2-苯基、-S(O)C1-3烷基、-S(O)-苯基、叔丁氧羰基；其中，所述的烷基、环烷基、苯基为未取代的或被1-3个选自下组的取代基所取代：氟、氯、羟基、NO2、苯基、C1-3烷基、C1-3烷氧基、C3-6环烷基、C3-6环烷氧基、C2-4烯基、C2-4炔基、-CONR11R12、-C(O)OC1-3烷基、-CHO、-OC(O)C1-3烷基、-SO2C1-3烷基、-SO2-苯基、-CO-苯基；或
R0为具有1到4个独立选自氮、氧或硫的杂原子的5至6元单环杂芳基、或为
Figure PCTCN2015076451-appb-000010
其中，Ra1为氢、甲基或乙基。
在另一优选例中，R0为氢、羟基、C1-3烷基、环丙基、-CHO、-COC1-3烷基(优选为-COCH3)、-CO-苯基、苯基、-CONH2、-CON(CH3)2、-C(O)OCH3、-SO2C1-3烷基(优选为-SO2CH3)、-SO2-苯基、-S(O)C1-3烷基(优选为-S(O)CH3)、-S(O)-苯基、叔丁氧羰基；其中，所述的烷基、苯基为未取代的或被1-3个(优选1个)选自下组的取代基所取代：氟、氯、羟基、NO2、苯基、甲基、甲氧基、环丙基、环丙氧基、-CONH2、-CON(CH3)2、-C(O)OCH3、-CHO、-OC(O)CH3、-SO2CH3、-SO2-苯基、-CO-苯基；或者R0选自：吡啶基、
Figure PCTCN2015076451-appb-000011
Figure PCTCN2015076451-appb-000012
在另一优选例中，R0为氢、C1-3烷基、-COC1-3烷基(优选为-COCH3)、-CO-苯基、-SO2C1-3烷基(优选为-SO2CH3)、-SO2-苯基、叔丁氧羰基；其中，所述的烷基、苯基为未取代的或被1-3个(优选1个)选自下组的取代基所取代：氟、氯、甲基；
在另一优选例中，R0为C1-3烷基、-COC1-3烷基(优选为-COCH3)、-SO2C1-3烷基(优 选为-SO2CH3)；其中，所述的烷基为未取代的或被1个选自下组的取代基所取代：氟、氯。
在另一优选例中，R0为一氟取代的C1-3烷基(优选一氟乙基)、-COC1-3烷基(优选-COCH3)或-SO2C1-3烷基(优选-SO2CH3)。
在另一优选例中，m3为0或1。
在另一优选例中，n3为1、2或3；n4为1或2。
在另一优选例中，n3为1；n4为1。
在另一优选例中，X为NH、N(C1-3烷基)、O或S。
在另一优选例中，式(VI)化合物中，
(i)Z2为CR10，R10为三氟甲基、氟或氯；
m3为0；n3为1；n4为1；
X为NH、O或S；
R0为氢、C1-3烷基、-COC1-3烷基(优选为-COCH3)、-CO-苯基、-SO2C1-3烷基(优选为-SO2CH3)、-SO2-苯基、叔丁氧羰基；其中，所述的烷基、苯基为未取代的或被1-3个(优选1个)选自下组的取代基所取代：氟、氯、甲基；(较佳地，R0为一氟取代的C1-3烷基(优选一氟乙基)、-COC1-3烷基(优选-COCH3)或-SO2C1-3烷基(优选-SO2CH3))
R1为氢；R2为甲氧基；R3、R5、R6和R7各自独立地为氢；
R4为
Figure PCTCN2015076451-appb-000013
或
(ii)Z2为CR10，R10为三氟甲基、氟或氯；
m3为0；n3为3；n4为2；
X为O；
R0为氢、C1-3烷基、-COC1-3烷基(优选为-COCH3)、-CO-苯基、-SO2C1-3烷基(优选为-SO2CH3)、-SO2-苯基、叔丁氧羰基；其中，所述的烷基、苯基为未取代的或被1-3个(优选1个)选自下组的取代基所取代：氟、氯、甲基；(较佳地，R0为一氟取代的C1-3烷基(优选一氟乙基)、-COC1-3烷基(优选-COCH3)或-SO2C1-3烷基(优选-SO2CH3))；
R1为氢；R2为甲氧基；R3、R5、R6和R7各自独立地为氢；
R4为
Figure PCTCN2015076451-appb-000014
或
(iii)Z2为CR10，R10为三氟甲基、氟或氯；
m3为0；n3为2；n4为2；
X为NH或O；
R0为氢、C1-3烷基、-COC1-3烷基(优选为-COCH3)、-CO-苯基、-SO2C1-3烷基(优选为-SO2CH3)、-SO2-苯基、叔丁氧羰基；其中，所述的烷基、苯基为未取代的或被1-3个(优选1个)选自下组的取代基所取代：氟、氯、甲基；(较佳地，R0为一氟取代的C1-3 烷基(优选一氟乙基)、-COC1-3烷基(优选-COCH3)或-SO2C1-3烷基(优选-SO2CH3))；
R1为氢；R2为甲氧基；R3、R5、R6和R7各自独立地为氢；
R4为
Figure PCTCN2015076451-appb-000015
或
(iv)Z2为CR10，R10为三氟甲基、氟或氯；
m3为0；n3为1；n4为2；
X为O；
R0为氢、C1-3烷基、-COC1-3烷基(优选为-COCH3)、-CO-苯基、-SO2C1-3烷基(优选为-SO2CH3)、-SO2-苯基、叔丁氧羰基；其中，所述的烷基、苯基为未取代的或被1-3个(优选1个)选自下组的取代基所取代：氟、氯、甲基；(较佳地，R0为一氟取代的C1-3烷基(优选一氟乙基)、-COC1-3烷基(优选-COCH3)或-SO2C1-3烷基(优选-SO2CH3))；
R1为氢；R2为甲氧基；R3、R5、R6和R7各自独立地为氢；
R4为
Figure PCTCN2015076451-appb-000016
或
(v)Z2为CR10，R10为三氟甲基、氟或氯；
m3为0；n3为3；n4为1；
X为NH或O；
R0为氢、C1-3烷基、-COC1-3烷基(优选为-COCH3)、-CO-苯基、-SO2C1-3烷基(优选为-SO2CH3)、-SO2-苯基、叔丁氧羰基；其中，所述的烷基、苯基为未取代的或被1-3个(优选1个)选自下组的取代基所取代：氟、氯、甲基；(较佳地，R0为一氟取代的C1-3烷基(优选一氟乙基)、-COC1-3烷基(优选-COCH3)或-SO2C1-3烷基(优选-SO2CH3))；
R1为氢；R2为甲氧基；R3、R5、R6和R7各自独立地为氢；
R4为
Figure PCTCN2015076451-appb-000017
或
(vi)Z2为CR10，R10为三氟甲基、氟或氯；
m3为1；n3为1；n4为1；
X为O；
R0为氢、C1-3烷基、-COC1-3烷基(优选为-COCH3)、-CO-苯基、-SO2C1-3烷基(优选为-SO2CH3)、-SO2-苯基、叔丁氧羰基；其中，所述的烷基、苯基为未取代的或被1-3个(优选1个)选自下组的取代基所取代：氟、氯、甲基；(较佳地，R0为一氟取代的C1-3烷基(优选一氟乙基)、-COC1-3烷基(优选-COCH3)或-SO2C1-3烷基(优选-SO2CH3))；
R1为氢；R2为甲氧基；R3、R5、R6和R7各自独立地为氢；
R4为
Figure PCTCN2015076451-appb-000018
在另一优选例中，式(VI)化合物中，
(i)X为NH，m3为0；n3为1；n4为1；
Z2为CR10，R10为氟、氯或三氟甲基；
R0为-COC1-3烷基(优选-COCH3)或-SO2C1-3烷基(优选-SO2CH3)；
R1为氢；R2为甲氧基；R3、R5、R6和R7各自独立地为氢；
R4为：
Figure PCTCN2015076451-appb-000019
或
(ii)X为O，m3为0或1；n3为1、2或3；n4为1或2；
Z2为CR10，R10为氟、氯或三氟甲基；
R1为氢；R2为甲氧基；R3、R5、R6和R7各自独立地为氢；
R0为-COC1-3烷基(优选-COCH3)、-SO2C1-3烷基(优选-SO2CH3)或一氟取代的C1-3烷基(优选一氟乙基)；
R4为选自下组：或
(iii)X为S，m3为0；n3为1；n4为1；
Z2为CR10，R10为氟、氯或三氟甲基；
R1为氢；R2为甲氧基；R3、R5、R6和R7各自独立地为氢；
R0为-COC1-3烷基(优选-COCH3)、或-SO2C1-3烷基(优选-SO2CH3)；
R4为：
Figure PCTCN2015076451-appb-000021
在另一优选例中，式(I)所示化合物，R3、R5各自独立地为氢或C1-10烷基(优选为C1-6烷基，更优选为C1-3烷基)。
在另一优选例中，式(I)所示化合物中，R1为氢。
在另一优选例中，式(I)所示化合物中，R2为C1-10烷氧基(优选为C1-6烷氧基，更优选为C1-3烷氧基，最优选为甲氧基)。
在另一优选例中，式(I)所示化合物中，R4为氢、-OC1-10烷基、-N(C1-10烷基)2、-N(C1-10烷基)(C3-8环烷基)、含1或2个氮原子以及0、1、2或3个氧或硫原子的3至7元饱和或部分不饱和单杂环。其中，所述烷基、环烷基、3至7元饱和或部分不饱和单杂环可任选地被1-3个选自下组的取代基所取代：卤素、NO2、CN、羟基、-CH2NR11R12、-NR11R12、C1-6烷基、C3-8环烷基、C3-8环烷氧基、C2-6烯基、C2-6炔基、C1-6烷氧基、-CHO、-COC1-6 烷基、-COC6芳基、C6芳基、-CONR11R12、-C(O)OC1-6烷基、-OC(O)C1-6烷基、-SO2C1-6烷基、-SO2C6芳基、叔丁氧羰基、-NHC1-6烷基、-NC(O)C1-6烷基、-NSO2C1-6烷基、吗啉基、四氢吡咯基、哌嗪基、甲基哌嗪基。
在另一优选例中，式(I)所示化合物中，R4为氢，或选自下组：
Figure PCTCN2015076451-appb-000022
在另一优选例中，R4为选自下组的基团：
Figure PCTCN2015076451-appb-000023
在另一优选例中，式(I)所示化合物中，R6和R7各自独立地为氢。
在另一优选例中，式(I)所示化合物中，R6和R7各自独立地为氢或-CH2NR13R14，而且，R13、R14各自独立地为氢或甲基。
在另一优选例中，式(I)所示化合物中，R6和R7各自独立地为氢或-CH2N R13R14，而且，R13、R14和相连的氮原子共同形成5-6元含氮饱和杂环。
较佳地，所述-CH2NR13R14的结构如式(a)所示：
Figure PCTCN2015076451-appb-000024
式中，Q为O、S、NC1-10烷基或C(C1-10烷基)2。
在另一优选例中，所述式(I)化合物选自下组：
Figure PCTCN2015076451-appb-000025
Figure PCTCN2015076451-appb-000026
Figure PCTCN2015076451-appb-000027
本发明第二方面，提供了一种药物组合物，它包括如本发明第一方面所述的化合物(如上述式(I)化合物、式(II)化合物、式(III)化合物、式(IV)化合物、式(V)化合物、式(VI)化合物、式(VII-1)化合物、式(VII-2)化合物、式(VII-3)化合物、式(VII-4)化合物、式(VII-5)化合物、式(VII-6)化合物、式(VII-7)化合物、式(VII-8)化合物、式(VII-9)化合物、式(VII-10)化合物，或上述示例化合物)、或其药学可接受的盐、或其溶剂化物、或其立体异构体、或前药，并且还包括药学可接受的载体。
通常，本发明化合物或其药学可接受的盐、或其溶剂化物、或其立体异构体、或前药可以与一种或多种药用载体形成适合的剂型施用。这些剂型适用于口服、直肠给药、局部给药、口内给药以及其他非胃肠道施用(例如，皮下、肌肉、静脉等)。例如，适合口服给药的剂型包括胶囊、片剂、颗粒剂以及糖浆等。这些制剂中包含的本发明的化合物可以是固体粉末或颗粒；水性或非水性液体中的溶液或是混悬液；油包水或水包油的乳剂等。上述剂型可由活性化合物与一种或多种载体或辅料经由通用的药剂学方法制成。上述的载体需要与活性化合物或其他辅料兼容。对于固体制剂，常用的无毒载体包括但不限于甘露醇、乳糖、淀粉、硬脂酸镁、纤维素、葡萄糖、蔗糖等。用于液体制 剂的载体包括水、生理盐水、葡萄糖水溶液、乙二醇和聚乙二醇等。活性化合物可与上述载体形成溶液或是混悬液。
本发明的组合物以符合医学实践规范的方式配制，定量和给药。给予化合物的“有效量”由要治疗的具体病症、治疗的个体、病症的起因、药物的靶点以及给药方式等因素决定。
本发明第三方面，提供了本发明第一方面所述的化合物、或其药学上可接受的盐、或其溶剂化物、或其立体异构体、或前药在用于制备调控(上调或下调)EGFR酪氨酸激酶活性的药物，或制备治疗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、使用EGFR调节剂治疗期间具有获得性耐药性的疾病。
作为优选，所述癌症为非小细胞肺癌、头颈癌、乳腺癌、肾癌、胰腺癌、子宫颈癌、食道癌、胰腺癌、前列腺癌、膀胱癌、结肠直肠癌、卵巢癌、胃癌、脑恶性肿瘤包括成胶质细胞瘤等，或它们的任何组合。
作为优选，所述获得性耐药性是由EGFR外显子20编码的T790突变引起的或者是包含EGFR外显子20编码的T790突变所引起的耐药性，如T790M。
作为优选，所述非小细胞肺癌是由EGFR突变引起的，包括敏感型突变(如L858R突变或外显子19缺失)和耐药性突变(如EGFR T790M突变)。
在本发明中，EGFR调节剂是指靶向EGFR的小分子酪氨酸激酶抑制剂，如吉非替尼，厄洛替尼，埃克替尼，拉帕替尼、阿法替尼等。
本发明第四方面，提供了一种药用组合物，包括治疗有效量的本发明第一方面所述的化合物、或其药学上可接受的盐、或其溶剂化物、或其立体异构体、或前药，以及选自下组的药物：吉非替尼、厄洛替尼、埃克替尼、拉帕替尼、XL647、NVP-AEE-788、ARRY-334543、EKB-569、BIBW2992、HKI272、BMS-690514、CI-1033、凡德他尼、PF00299804、WZ4002、西妥昔单抗、曲妥珠单抗、帕尼突单抗、马妥珠单抗、尼妥珠单抗、扎鲁木单抗、帕妥珠单抗、MDX-214、CDX-110、IMC-11F8、Zemab、Her2疫苗PX 1041、HSP90抑制剂、CNF2024、坦螺旋霉素、阿螺旋霉素、IPI-504、SNX-5422、NVP-AUY922、或其组合。除本发明的化合物或其药学上可接受的盐、或其溶剂化物、或其立体异构体、或前药以外，上述药用组合物中的其他药物均为本领域技术人员熟知的抗肿瘤药物。
“治疗有效量”是指可对人和/或动物产生功能或活性的且可被人和/或动物所接受的量。
本发明的所述药物组合物或所述药用组合物中含有的本发明化合物或其药学上可接受的盐、或其溶剂化物、或其立体异构体、或其前药的治疗有效量优选为0.1mg-5g/kg(体重)。
所述的药用组合物可用于治疗EGFR异常表达的疾病，如癌症，糖尿病，免疫系统疾病，神经退行性疾病或心血管疾病、使用EGFR调节剂治疗期间具有获得性耐药性的疾病。
所述获得性耐药性的疾病是由EGFR外显子20编码的T790突变所引起的疾病，或者是包含EGFR外显子20编码的T790突变所引起的疾病。
在另一优选例中，所述的EGFR外显子20编码的T790为T790M。
本发明的式(I)化合物、或其药学上可接受的盐、或溶剂化物、或其立体异构体、或前药在某些疾病中可以与其他药物联合应用，以达到预期的治疗效果。一个联合应用的例子是用来治疗晚期NSCLC。例如，将治疗有效量的本发明式(I)所示化合物与mTOR抑制剂联用(例如雷帕霉素)；或与Met抑制剂(包括Met抗体MetMAb和Met小分子抑制剂PF02341066)联用；或与IGF1R抑制剂联用(例如OSI-906)；或与热休克蛋白抑制剂联用等。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。
具体实施方式
本发明人经过长期而深入的研究，意外地发现了一类EGFR突变选择性抑制剂，体外实验表明其在纳摩尔浓度下即可抑制EGFR T790M/L858R双突变酶和细胞株H1975的增殖，同时对EGFR敏感性突变细胞株HCC827(外显子19缺失)也有很高的抑制强度，而对野生型EGFR酶和细胞株A431的抑制则相对较弱。因此，此类结构不但可用于EGFR敏感型突变癌症的治疗，还适用于目前EGFR-TKI治疗中产生继发性耐药的病例；同时其突变选择性大大减小了因抑制野生型EGFR而产生的毒副作用，此外，该类化合物在正常细胞系(如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
“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、正丁基、异丁基、叔丁基、戊基、己基等。优选为含有1至6个碳原子的烷基，更优选的是含有1至3个碳原子的烷基。
“C2-10烯基”指具有2-10个(较佳地2-6个)碳原子的直链或支链的具有碳碳双键(C＝C)的不饱和脂族烃基。例如乙烯基、丙烯基、异丙烯基、正丁烯基、异丁烯基、戊烯基、己烯基等。
“C2-10炔基”指具有2-10个(较佳地2-6个)碳原子的直链和支链的具有碳碳三键的不饱和脂族烃基。例如乙炔基、丙炔基、正丁炔基、异丁炔基、戊炔基、己炔基等。
“C3-8环烷基”指具有3-8个碳原子的环烷基。环烷基实例有环丙基、环丁基、环戊基、环己基等。
“C1-10烷氧基”指C1-10烷基-O-。例如甲氧基、乙氧基、丙氧基、丁氧基等。
“C3-8环烷氧基”指C3-8环烷基-O-。例如环丙氧基、环丁氧基、环戊氧基、环己氧基等。
“C6-10芳基”是指具有6-10个碳原子的芳香烃基，例如苯基、萘基等。
“卤素”指氟、氯、溴或碘。
“二价C1-3烃基”是指直链或支链的亚烷基、亚烯基和亚炔基。其中，“亚烷基”是指二价烷基，例如，亚甲基、亚乙基等；“亚烯基”指二价烯基。“亚甲基被置换”是指二价C1-3直链或分支链烃链中的亚甲基可以被本文所定义的基团所置换。置换后例如，-CH2-S(O)-CH2-、-CH2-O-CH2-、-CH2-C(O)NRy-CH2-、-C(O)-CH2-CH2-、-CH2-C(RyRx)-CH2-、-N(Ry)-CH2-CH2-、-C(RyRx)-C(RyRx)-CH2-等。
术语“亚环丙基”结构为：
Figure PCTCN2015076451-appb-000028
“杂芳基环”与“杂芳基”可互换使用，是指具有5到10个环原子，优选5、6、9或10个环原子；环阵列中共享6、10或14个π电子；且除碳原子外还具有1到5个杂原子的基团。术语“杂原子”是指氮、氧或硫。
“5至6元单环杂芳基环”是指含5至6个环原子的单环杂芳基环，例如包括(但不限于)：噻吩环、呋喃环、噻唑环、咪唑环、噁唑环、吡咯环、吡唑环、三唑环、四唑环、异噁唑环、噁二唑环、噻二唑环、吡啶环、哒嗪环、嘧啶环、吡嗪环等。
“8至10元双环杂芳基环”是指含8至10个环原子的双环杂芳基环，例如包括(但不限于)：苯并呋喃环、苯并噻吩环、吲哚环、异吲哚环、喹啉环、异喹啉环、吲唑环、苯并噻唑环、苯并咪唑环、喹唑啉环、喹喔啉环、噌啉环、酞嗪环。
在本发明中，所述5至6元单环杂芳基环或8至10元双环杂芳基环可选自下组：
Figure PCTCN2015076451-appb-000029
其中，Ra1为氢、甲基或乙基。
如本文所用，“部分不饱和”是指含有一个或多个不饱和键但不具有完全共轭的π电子系统。
“3至7元饱和或部分不饱和单环”是指含3至7个环原子的饱和的全碳单环或部分不饱和的全碳单环。例如包括(但不限于)：环丙基环、环丁基环、环戊基环、环己烷、环己二烯环、环庚烷、环庚三稀环等。
“8至10元饱和或部分不饱和双环”是指含8至10个环原子的饱和的全碳双环或部分不饱和的全碳双环。“3至7元饱和或部分不饱和单杂环”是指含3至7个环原子且1到3个碳原子被选自氮、氧或硫的杂原子所取代的饱和单环或部分不饱和单环。单杂环的实例包括(但不限于)四氢呋喃环、四氢噻吩环、吡咯烷基环、哌啶环、吡咯啉环、噁唑烷环、哌嗪环、二氧戊环、吗啉环。
“8至10元饱和或部分不饱和双杂环”是指具有8至10个环原子且1到5个碳原子被选自氮、氧或硫的杂原子所取代的饱和双环或部分不饱和双环。双杂环的实例包括(但不限于)四氢喹啉环、四氢异喹啉环、十氢喹啉环。
“含1或2个氮原子以及0到3个氧或硫原子的3至7元饱和或部分不饱和单杂环”是指该单杂环中，1或2个碳原子被氮原子所置换，且0、1、2或3个碳原子被氧或硫原子所置换。
药物组合物
术语“本发明的活性物质”或“本发明的活性化合物”是指本发明式(I)化合物、或其药学上可接受的盐、或其溶剂化物、或其立体异构体、或其前药，其具有显著耐药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。
本发明式(I)化合物可以参照下述合成路线进行制备，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。
Figure PCTCN2015076451-appb-000030
步骤1：式(I-a)化合物与式(I-b)化合物可通过取代反应(例如亲和取代反应等)或偶联反应(如Suzuki偶联等)生成式(I-c)化合物，式(I-a)化合物中的L1和L2为离去基团，包括(但不限于)三氟甲磺酸酯；氯、溴、碘；磺酸酯基，如甲磺酸酯、甲苯磺酸酯、对溴苯磺酸酯、对甲苯磺酸酯等；酰氧基，如乙酰氧基、三氟乙酰氧基等。
步骤2：式(I-c)化合物可通过取代反应或偶联反应与式(I-d)化合物反应生成式(I-e)化合物，例如在一定温度下，使用合适的催化剂(或含合适的配体)或碱及适当的溶剂进行。如使用酸催化，催化剂可以是但不限于TFA或对甲苯磺酸。使用Buchwald-Hartwig胺化法，所用的钯催化剂可以是但不限于Pd2(dba)3，所用的配体可以是但不限于XantPhos(4,5-双(二苯基膦)-9,9-二甲基氧杂蒽)，所用的碱可以是但不限于碳酸铯。
步骤3：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4：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。式(I-a)化合物与式(I-b)化合物可以市购得到，或通过本领域技术人员已知的方法制备。
本发明式(VI)化合物可通过方案1所示的方法制备。
方案1
Figure PCTCN2015076451-appb-000031
方案1中，各取代基和基团如说明书中所定义。
步骤1：式(I-a-a)化合物与式(I-b-a)化合物可通过取代反应(例如亲和取代反应等)或偶联反应(如Suzuki偶联等)生成式(I-c-a)化合物，式(I-a-a)化合物中的L1和L2为离去基团，包括(但不限于)三氟甲磺酸酯；氯、溴、碘；磺酸酯基，如甲磺酸酯、甲苯磺酸酯、对溴苯磺酸酯、对甲苯磺酸酯等；酰氧基，如乙酰氧基、三氟乙酰氧基等。
步骤2：式(I-c-a)化合物可通过取代反应或偶联反应与式(I-d-a)化合物反应生成式(I-e-a)化合物，例如在一定温度下，使用合适的催化剂(或含合适的配体)或碱及适当的溶剂进行。如使用酸催化，催化剂可以是但不限于TFA或对甲苯磺酸。使用Buchwald-Hartwig胺化法，所用的钯催化剂可以是但不限于Pd2(dba)3(三(二亚苄基丙酮)二钯)，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：该硝基化合物I-e-a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4：该胺基化合物I-f-a可在碱性条件下与相应的酰氯缩合成酰胺，或者在缩合剂存在下与相应的羧酸缩合成酰胺。
方案1中，式(I-a-a)化合物可市购得到，式(I-b-a)化合物可按如下示例性方法1制得。
方法1
Figure PCTCN2015076451-appb-000032
步骤1：通过对式(I-b1)化合物进行脱保护得到式(I-b2)化合物，式(I-b1)化合物中的PG为氨基保护基。氨基保护基包括但不限于：叔丁氧基羰基(Boc)；芳基甲氧羰基，苄氧羰基(Cbz)和9-芴甲氧羰基(Fmoc)；苄基(Bn)、三苯甲基(Tr)、1,1-二-(4'-甲氧基苯基)甲基；三甲基甲硅烷基(TMS)和叔丁基二甲基甲硅烷基(TBS)等等。脱保护的方法可参考本领域常规方法。式(I-b1)化合物可市购得到，或通过本领域技术人员已知的方法制备。
步骤2：根据R0的不同，选择合适方法将式(I-b2)化合物和式(I-b3)化合物进行酰化、偶联(如Suzuki偶联等)等反应生成式(I-b-a)化合物，其中，HX为带有电负性的亲核基团，包括(但不限于)羟基、氨基、巯基。
式(I-c-a)化合物也可按如下方法制得
Figure PCTCN2015076451-appb-000033
式(I-b4)化合物可通过将式(I-a-a)化合物与式(I-b1)化合物按方案1中步骤1的类似方法进行制备，式(I-c-a)化合物可以式(I-b4)化合物为原料，按照方案1中方法1的类似方法制备。
本发明式(VI)化合物可通过方案2所示的方法制备。
方案2
Figure PCTCN2015076451-appb-000034
方案2中，各取代基和基团如说明书中所定义。
式(I-c-a)化合物可通过取代反应或偶联反应与式(I-j-a)化合物反应生成式(VI)化合物，例如在一定温度下，使用合适的催化剂(或含合适的配体)或碱及适当的溶剂进行。如使用酸催化，催化剂可以是但不限于TFA或对甲苯磺酸。使用Buchwald-Hartwig胺化法，所用的钯催化剂可以是但不限于Pd2(dba)3(三(二亚苄基丙酮)二钯)，BINAP((±)-2,2'-双-(二苯膦基)-1,1'-联萘)所用的配体可以是但不限于Xantphos(4,5-双(二苯基膦)-9,9-二甲基氧杂蒽)，所用的碱可以是但不限于碳酸铯。
方案2中，式(I-j-a)化合物可通过如下示例性方法进行制备：
Figure PCTCN2015076451-appb-000035
以式(I-d1)化合物为起始原料，依次经过氨基上保护基反应、胺取代反应、硝基还原反应、酰化反应和氨基脱保护反应来制备式(I-j-a)化合物。上述各步反应均为本领域常规反应。式(I-d1)化合物可市购得到，或通过本领域技术人员已知的方法制备(式(I-j-a)化合物的合成方法可参见WO2013014448A1)。
本发明公开的式(I)化合物以及化合物的制备方法、药物组成和治疗方案，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的产品、方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
与现有技术相比，本发明的主要优点在于：
(1)本发明化合物对EGFR T790M突变型(特别是EGFR T790M/L858R双突变型)酶和细胞具有高抑制活性，且对EGFR野生型(EGFR WT)酶和细胞具有低抑制活性，因此具有高选择抑制性。
(2)本发明化合物对EGFR双突变型酶和细胞表现高选择抑制性的同时还具有低非特异性细胞毒性。
(3)与其他已知的EGFR突变抑制剂相比，本发明化合物还显示出有利的物理性质(例如，较高的水溶性)，有利的毒性特征(例如较低的hERG阻断倾向)，有利的代谢特征(例如，较好的药代动力学特征，如生物利用度)。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
除非另行定义，本文所用的术语与本领域熟练人员所熟悉的意义相同。此外，任何与所记载内容相似或同等的方法及材料皆可应用于本发明中。
试剂与仪器
1HNMR：Bruker AVANCE-400核磁仪，内标为四甲基硅烷(TMS)；
LC-MS：Agilent 1200HPLC System/6140MS液质联用质谱仪(生产商：安捷伦)，柱子 WatersX-Bridge，150×4.6mm，3.5μm。
制备高效液相色谱(pre-HPLC)：Waters PHW007，柱子XBridge C18，4.6*150mm，3.5um。
采用ISCO Combiflash-Rf75或Rf200型自动过柱仪，Agela 4g、12g、20g、40g、80g、120g一次性硅胶柱。
已知的起始原料可以采用或按照本领域已知的方法来合成，或可以购自ABCR GmbH&Co.KG，Acros Organics，Aldrich Chemical Company，韶远化学科技(Accela ChemBio Inc)和达瑞化学品等公司。
若无特殊说明，实施例中的反应均在氮气氛或氩气氛下进行；实施例中的溶液为水溶液。
实施例中，反应进程的监测可采用薄层色谱法(TLC)，化合物纯化可采用柱层析。柱层析或TLC所用的展开剂体系可选自：二氯甲烷和甲醇体系、正己烷和乙酸乙酯体系、石油醚和乙酸乙酯体系和丙酮体系等，溶剂的体积比根据化合物的极性不同而进行调节。
DMF：二甲基甲酰胺，DMSO：二甲基亚砜，THF：四氢呋喃，DIEA：N,N-二异丙基乙胺，EA：乙酸乙酯，PE：石油醚，BINAP：(2R,3S)-2,2'-双二苯膦基-1,1'-联萘；NBS(N-溴代丁二酰亚胺)、NCS(N-氯代丁二酰亚胺)、Pd2(dba)3(三(二亚苄基丙酮)二钯)、Pd(dppf)Cl2([1,1'-双(二苯基磷)二茂铁]二氯化钯)。
如本文所用，室温是指约25℃。
化合物a1的制备
Figure PCTCN2015076451-appb-000036
步骤a：将反应底物1(10.6g,58mmol)放置于500mL的单口反应瓶中，加入THF/水(100mL/60mL)混合溶液使底物溶解。在室温下，向搅拌的反应瓶中依次加入氯化铵(15.5g,292mmol)和还原铁粉(26g,467mmol)，随后将反应体系加热至65℃并持续搅拌3小时。通过TLC检测反应进度，待底物完全反应后，过滤除去多余的铁粉，滤饼用乙酸乙酯淋洗三次。滤液用乙酸乙酯/水体系萃取三次，分离出有机层，经水洗，饱和食盐水洗涤，无水硫酸钠干燥后，减压浓缩得到化合物2(8.0g)，直接用于下一步反应。产率：93％；纯度：90％；MS m/z(ESI):142.0[M+H]+。
步骤b：将化合物2(8.0g,43mmol)放置于500mL的单口反应瓶中，在匀速搅拌下加入浓硫酸(100mL)使底物溶解。在零下20℃下，向搅拌的反应瓶中缓慢滴加浓硝酸(6.15mL,48mmol)，并保持该温度搅拌5分钟。通过TLC检测反应进度，待底物完全反应后，倒入冰水中。保持零下20℃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。中和完成后的反应液用乙酸乙酯/水体系萃取三次，分离出有机层，经水洗，饱和食盐水洗涤，无水硫酸钠干燥后，减压浓缩得到化合物a1(8.7g)，直接用于下一步反应。产率：80％；纯度：100％；MS m/z(ESI)：187.0[M+H]+；1H NMR(400MHz,DMSO-d6):δ7.34(d,J＝7.8Hz,1H),7.04(d,J＝13.4Hz,1H),5.25(brs,2H),3.90(s,3H)。
化合物a的制备
Figure PCTCN2015076451-appb-000037
步骤1：将4-氟-2-甲氧基-5-硝基苯胺a1(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18小时。通过TLC检测反应进度，待底物反应完全后，减压浓缩反应液，柱层析[PE:EA＝80:20]分离纯化得到目标产物a2(12.56g，73％)。MS m/z(ESI):285[M-H]+。
步骤2：将反应底物叔丁基4-氟-2-甲氧基-5-硝基苯基氨基甲酸叔丁酯a2(11.46g，40mmol)溶于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6小时。通过TLC检测反应进度，待底物反应完全后，将反应液冷却至室温，倒入冰水中，用乙酸乙酯萃取，饱和食盐水洗涤，无水硫酸钠干燥，减压浓缩得目标产物a3(12.51g，85％)。直接用于下一步反应。MS m/z(ESI):369[M+H]+。
步骤3：将4-((2-(二甲基氨基)乙基)(甲基)氨基)-2-甲氧基-5-硝基苯基氨基甲酸叔丁酯a3(12g，32.6mmol)溶于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1小时。通过TLC检测反应进度，待底物反应完全后，经砂芯漏斗抽滤，以少量甲醇洗涤滤饼，将滤液浓缩得到目标产物a4(10.70g，97％)。直接用于下一步反应。MS m/z(ESI):339[M+H]+。
步骤4：将5-氨基-4-((2-(二甲基氨基)乙基)(甲基)氨基)-2-甲氧基苯基氨基甲酸叔丁酯a4(10.1g，30mmol)和三乙胺(6.12g，60mmol)溶于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3小时。通过TLC检测反应进度，待底物反应完全后，以饱和碳酸氢钠水溶液、饱和食盐水顺次洗涤，无水硫酸钠干燥，抽滤，经减压浓缩得到目标产物a5(9.64g，82％)。直接用于下一步反应。MS m/z(ESI):393[M+H]+。
步骤5：将5-丙烯酰胺基-4-((2-(二甲基氨基)乙基)(甲基)氨基)-2-甲氧基苯基氨基甲酸叔丁酯a5(9.41g，24mmol)溶解在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18小时。通过TLC检测反应进度，待底物反应完全后，减压浓缩反应液。将残留物以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，无水硫酸钠干燥，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N-(5-氨基-2-((2-(二甲基氨基)乙基)(甲基)氨基)-4-甲氧苯基)丙烯酰胺a(3.26g，46.5％)。MS m/z(ESI):293[M+H]+。
化合物b的制备
Figure PCTCN2015076451-appb-000038
化合物4-a的制备
Figure PCTCN2015076451-appb-000039
步骤1：将化合物2a(19.7g，110mmol)，碳酸铯(65.2g，200mmol)加入到N-Boc-3-羟基氮杂环丁烷(17.3g，100mmol)的400ml的DMF溶剂中，83℃下剧烈搅拌4小时。通过TLC检测反应进度，待底物完全反应后，用乙酸乙酯/水体系萃取三遍，分离出有机层，经水洗，饱和食盐水洗涤，旋干后得到的粗产品。经Combi-flash柱层析[PE:EA＝100:0～40:60]纯化后得化合物4a-2(25g，88％)。MS m/z(ESI):286.1[M+H]+。
步骤2：将化合物a1(1.96g，10.5mmol)，Pd2(dba)3(964mg，1.05mmol)，Xantphos(1.219g，2.11mmol)和碳酸铯(6.86g，21.0mmol)加入到化合物4a-2(3g，10.5mmol)的70ml 1,4-二氧六环溶液中。120℃下，在N2氛围中剧烈搅拌20小时。反应结束后，过滤，乙酸乙酯洗涤，滤液减压浓缩后得粗产品，经Combi-flash柱层析[PE:EA＝100:0～20:80]纯化后得化合物4-a(2.86g，62％)。MS m/z(ESI):436.2[M+H]+。
化合物5-a的制备
Figure PCTCN2015076451-appb-000040
步骤1：将4-二甲氨基哌啶(106mg，0.83mmol)和碳酸钾(286mg，2.07mmol)加入到化合物4-a(300mg，0.69mmol)的20ml的DMF中，100℃下剧烈搅拌4h。通过TLC检测反应进度，待底物完全反应后，用乙酸乙酯/水体系萃取三次，分离出有机层，减压浓缩得到化合物5a-1(400mg，95％)，MS m/z(ESI):544.2[M+H]+。
步骤2：将三氟乙酸(3mL)加入到化合物5a-1(1g，1.84mmol)的30ml的二氯甲烷溶液中，室温下剧烈搅拌6小时。通过TLC检测反应进度，待底物完全反应后，减压下旋 掉多余的二氯甲烷，加水稀释，调节pH至碱性，用二氯甲烷和甲醇(10:1)萃取，将有机相旋干，得化合物5-a(1.1g，45.8％)。MS m/z(ESI):444.2[M+H]+。
化合物7-a的制备
Figure PCTCN2015076451-appb-000041
步骤1：将化合物1a(10g,54.5mmol)、碳酸钾(13.7g,99.1mmol)和N-Boc-3-羟基氮杂环丁烷(8.58g,49.5mmol)置于500ml单口反应瓶中，加入300ml乙腈,加热至90℃搅拌反应4小时。通过TLC检测反应进度,待底物反应完全后,用乙酸乙酯/水体系萃取三遍,分离出有机层,经水洗,饱和食盐水洗涤,浓缩得到粗产品。经combi-flash柱层析[PE:EA＝100:0～40:60]分离纯化得到目标产物7a-2(11.6g,73.2％)。MS m/z(ESI):320.1[M+H]+。
步骤2：将反应底物7a-2(500mg,1.56mmol)溶于20ml二氯甲烷，缓慢加入三氟乙酸(3.5g,30.7mmol)，在室温下搅拌反应2小时。通过TLC检测反应进度，待底物反应完全后，减压浓缩反应液得到800mg目标粗产物7a-3，直接用于下一步反应。
步骤3：将反应底物7a-3(800mg,约1.56mmol)溶于25ml二氯甲烷，冷却至0℃后，加入N,N-二异丙基乙基胺(2.07g,16.02mmol)。搅拌10分钟后，加入甲基磺酰氯(360mg,3.14mmol)，升至室温搅拌反应2小时。通过TLC检测反应进度，待底物反应完全后，加入二氯甲烷25ml稀释反应液，经水洗，饱和食盐水洗涤，减压浓缩得到粗产品。以combi-flash柱层析[PE:EA＝100:0～75:25]分离纯化得到目标产物7-a(362mg,77.8％)。MS m/z(ESI):298.0[M+H]+。
化合物8-a的制备
Figure PCTCN2015076451-appb-000042
步骤1：室温下，向化合物7a-2(640mg，2.0mmol)的1,4-二氧六环(10ml)溶液中滴加盐酸/1,4-二氧六环(10ml，40mmol)溶液。反应混合物室温搅拌3小时后，减压浓缩反应液得486mg化合物8a-1的粗品，直接用于下一步反应。
步骤2：0℃下，向化合物8a-1(440mg，2.0mmol)和三乙胺(810mg，8.0mmol)的二氯甲烷(35ml)溶液中滴加乙酰氯(165mg，2.1mmol)的二氯甲烷(5ml)溶液。反应混合物在0℃下搅拌1小时，反应结束后，加二氯甲烷和水并用二氯甲烷萃取。有机相用盐水洗涤，无水硫酸钠干燥，减压浓缩得300mg粗品，粗品经combi-flash柱层析分离纯化得到目标产物8-a(148.8mg，产率57％)。MS m/z(ESI):262[M+H]+。
化合物14-a的制备
Figure PCTCN2015076451-appb-000043
步骤1：将化合物1a(3g，16.36mmol)、(R)-3-羟基吡咯烷-1-甲酸叔丁酯(3.1g，16.56mmol)和碳酸铯(8g，24.55mmol)加入30ml乙腈中，反应混合物在80℃下搅拌3小时。用TLC板和LC-MS跟踪反应。反应结束后，将反应液过滤，用二氯甲烷洗涤，浓缩滤液得粗产品，粗产品经combiflash(PE:EA＝95:5-80:20)纯化得到化合物14a-1(3.31g，产率60.0％)。MS m/z(ESI):278[M-56+H]+。
步骤2：在0℃下,将三氟乙酸(3.50g,30.70mmol)加入到化合物14a-1(510mg，1.53mmol)的5ml的二氯甲烷溶液中,室温下剧烈搅拌5小时。反应结束后,将反应液在减压下蒸干,得化合物14a-2粗产品831mg，产物直接用于下一步。
步骤3：在0℃下,将N,N-二异丙基乙胺(1.92g,14.86mmol)加入到化合物14a-2(831mg，1.53mmol)的25ml二氯甲烷溶液中,0℃下剧烈搅拌30分钟后加入甲磺酰氯(344mg,3.00mmol),0℃下剧烈搅拌2小时。反应结束后,加水稀释,用二氯甲烷/水体系萃取三次,有机层减压浓缩,将得到的粗产品经combiflash(PE:EA＝100:0-50:50)纯化得到化合物14-a(302mg，产率70.1％)。MS m/z(ESI):312[M+H]+。
化合物15-a、16-a、17-a的制备
Figure PCTCN2015076451-appb-000044
化合物18-a至化合物21-a的制备
化合物18-a至化合物21-a的制备方法同化合物14-a的制备，不同的是，
制备化合物18-a时，将步骤1中2,4,5-三氯嘧啶换成2,4-二氯-5-氟嘧啶，将步骤3 中甲磺酰氯换成乙酰氯。
制备化合物19-a时，将步骤1中2,4,5-三氯嘧啶换成2,4-二氯-5-氟嘧啶，将(R)-叔丁基-3-羟基吡咯烷-1-甲酸叔丁酯换成(S)-叔丁基-3-羟基吡咯烷-1-甲酸叔丁酯，将步骤3中甲磺酰氯换成乙酰氯。
制备化合物20-a时，将步骤1中2,4,5-三氯嘧啶换成2,4-二氯-5-氟嘧啶。
制备化合物21-a时，将步骤1中2,4,5-三氯嘧啶换成2,4-二氯-5-氟嘧啶，将(R)-3-羟基吡咯烷-1-甲酸叔丁酯换成(S)-3-羟基吡咯烷-1-甲酸叔丁酯。
Figure PCTCN2015076451-appb-000045
化合物22-a的制备
Figure PCTCN2015076451-appb-000046
步骤1：将化合物a1(186mg，1mmol)，Pd2(dba)3(92mg，0.1mmol)，BINAP(93mg，0.15mmol)和碳酸铯(652mg，2mmol)加入到化合物7a-2(320mg，1mmol)的11ml 1,4-二氧六环溶液中。160℃下微波反应20小时。TLC跟踪反应。反应结束后，过滤，乙酸乙酯洗涤，滤液减压浓缩后得粗产品，经Combi-flash柱层析[PE:EA＝90:10～50:50]纯化后得化合物22a-1(185.5mg，产率39.5％)。MS m/z(ESI):370[M-100+H]+。
步骤2：将4-二甲氨基哌啶(106mg，0.83mmol)和碳酸钾(152mg，1.10mmol)加入到化合物22a-1(371mg，0.55mmol)的6ml的DMF中，100℃下剧烈搅拌1h。通过TLC检测反应进度，待底物完全反应后，用乙酸乙酯/水体系萃取三次，分离出有机层，减压浓缩得到粗产品，经Combi-flash柱层析[PE:EA＝100:0～85:15]纯化后得化合物22a-2(323mg，产率100％)。MS m/z(ESI):578.3[M+H]+。
步骤3：以化合物22a-2(323mg，0.42mmol)为原料，参照化合物5-a制备方法的步骤2制备，得216mg化合物22-a。不纯化直接下一步。
化合物23-a的制备
Figure PCTCN2015076451-appb-000047
步骤1：0℃下，向氢化钠(220mg，5.49mmol)的THF溶液中加入(R)-3-羟基哌啶-1-甲酸叔丁酯(550mg，2.75mmol)。反应混合物在0℃下搅拌1小时后，向其中加入化合物1a(500mg，2.75mmol)。反应混合物在0℃下搅拌2小时后再在室温下搅拌20小时。反应结束后，0℃下加入水淬灭反应。反应液用乙酸乙酯萃取，用饱和氯化钠溶液和水洗涤，浓缩得粗产品，粗产品经combiflash(PE:EA＝80:20)纯化得到500mg化合物23a-1。MS m/z(ESI):292[M-56+H]+。
步骤2-3：以化合物23a-1(500mg，1.44mmol)为原料参照化合物14-a的制备方法中的步骤2和3制备，得240mg化合物23-a，MS m/z(ESI):326[M+H]+。
化合物24-a至化合物26-a的制备
化合物24-a至化合物26-a的制备方法同化合物23-a的制备，不同的是，
制备化合物24-a时，将步骤1中(R)-3-羟基哌啶-1-甲酸叔丁酯换成(S)-3-羟基哌啶-1-甲酸叔丁酯。
制备化合物25-a时，将步骤1中化合物7a-1换成2,4-二氯-5-氟嘧啶。
制备化合物26-a时，将步骤3中甲磺酰氯换成乙酰氯。
Figure PCTCN2015076451-appb-000048
化合物31-a的制备
Figure PCTCN2015076451-appb-000049
步骤1：室温下，向化合物1a(1.83g，10mmol)和N,N-二异丙基乙胺(2.58g，20mmol)的二氯甲烷(90ml)溶液中滴加3-氨基氮杂环丁烷-1-羧酸叔丁酯(1.89g，11mmol)的二氯甲烷(10ml)溶液。反应混合物室温下搅拌5小时。反应结束后，加水和二氯甲烷萃取，有机相经无水硫酸钠干燥后减压浓缩得2.85g粗产品31a-1，不经纯化直接下一步反应。MS m/z(ESI):319[M+H]+。
步骤2：室温下，向化合物31a-1(638mg，2.0mmol)的1,4-二氧六环(20ml)溶液中滴加盐酸/1,4-二氧六环(10ml，40mmol)溶液。反应混合物室温搅拌3小时后，减压浓缩反应液得680mg化合物31a-2的粗品，直接用于下一步反应。
步骤3：以化合物31a-2为原料，参照化合物7-a的制备方法中步骤3制备。得化合物31-a。MS m/z(ESI):297[M+H]+。
化合物32-a的制备
Figure PCTCN2015076451-appb-000050
步骤1：以化合物1a和(S)-3-氨基哌啶-1-甲酸叔丁酯为原料，参照化合物31-a制法中的步骤1制备。得化合物32a-1。MS m/z(ESI):347[M+H]+。
步骤2-3：以化合物32a-1为原料，参照化合物7-a制法中的步骤2、3制备。得化合物32-a。MS m/z(ESI):325.0[M+H]+。
化合物33-a、34-a的制备
Figure PCTCN2015076451-appb-000051
化合物35-a的制备
Figure PCTCN2015076451-appb-000052
将化合物31a-2(584mg，2.0mmol)、1-溴-2-氟乙烷(280mg，2.2mmol)和碳酸钾(1.1g，8.0mmol)的乙腈(40ml)溶液在50℃下封管搅拌过夜。反应混合物加水和乙酸乙酯萃取，有机相用无水硫酸钠干燥，减压浓缩得粗产品。经combi-flash柱层析[PE:EA＝100:0～90:10]分离纯化得到目标产物35-a(71mg，产率13.4％)。MS m/z(ESI):265[M+H]+。
化合物36-a的制备
Figure PCTCN2015076451-appb-000053
以化合物32a-2为原料，参照化合物35-a的制备方法制备。得化合物36-a。MS m/z(ESI):293[M+H]+。
化合物37-a的制备
Figure PCTCN2015076451-appb-000054
化合物38-a的制备
Figure PCTCN2015076451-appb-000055
步骤1：向4-氨基哌啶-1-甲酸叔丁酯(550mg，2.75mmol)的乙腈(10ml)溶液中加入化合物1a(500mg，2.75mmol)和碳酸钾(758mg，5.49mmol)，反应混合物在90℃下搅拌2小时，反应结束后过滤浓缩。经combi-flash柱层析[PE:EA＝80:20]分离纯化得到700mg目标产物38a-1，MS m/z(ESI):347.2[M+H]+。
步骤2-3：以化合物38a-1为原料，依次参照化合物7-a的制备方法中步骤2和步骤3进行制备。得化合物38-a。MS m/z(ESI):325.0[M+H]+。
化合物39-a的制备
Figure PCTCN2015076451-appb-000056
将化合物31-a(500mg，1.68mmol)的5ml二甲基甲酰胺溶液0℃下逐滴加入到钠氢(80mg，2mmol)的10ml二甲基甲酰胺溶液中，0℃搅拌1小时后，室温搅拌下逐滴加入碘甲烷(286mg，2mmol)，室温搅拌3小时。反应结束，加入冰水，二氯甲烷和水萃取，有机相无水硫酸钠干燥，过滤减压浓缩得到粗品，经Combi-flash柱层析纯化得到固体化合物39-a(260mg)，直接用于下一步反应。产率：49.8％，MS m/z(ESI):311[M+H]+。
化合物40-a的制备
Figure PCTCN2015076451-appb-000057
以化合物13e为原料，参照化合物8-a的制备方法中步骤2制备。MS m/z(ESI):278[M+H]+。
化合物41-a的制备
Figure PCTCN2015076451-appb-000058
以化合物13c和2,4-二氯-5-氟嘧啶为起始原料，依次参照化合物13f的制备方法中的步骤3、4和5制备化合物41-a。MS m/z(ESI):298[M+H]+。
化合物42-a的制备
Figure PCTCN2015076451-appb-000059
以化合物41a-2为原料，参照化合物8-a的制备方法中步骤2制备。MS m/z(ESI):262[M+H]+。
化合物43-a的制备
Figure PCTCN2015076451-appb-000060
步骤1：0℃下，向化合物13c(1.6g，8.5mmol)的乙腈(50ml)溶液中加入2,4-二氯-5-(三氟甲基)嘧啶(1.84g，8.5mmol)和碳酸钾(2.35g，17mmol)，反应混合物在0℃下搅拌1小时。反应结束后加水淬灭，用乙酸乙酯萃取，浓缩有机相得到粗产品。经combi-flash柱层析分离纯化得到目标产物43a-1(2.5g，产率67％)。MS m/z(ESI):370[M+H]+。
步骤2-3：以化合物43a-1为原料，参照化合物13f制备方法中步骤4和5制备。MS m/z(ESI):348[M+H]+。
化合物44-a的制备
Figure PCTCN2015076451-appb-000061
步骤1：将化合物1a(1g，5.45mmol)、3-(羟甲基)氮杂环丁烷-1-甲酸(1g，5.34mmol)和碳酸铯(2.67g，8.19mmol)加入15ml乙腈中，反应混合物80℃下搅拌3小时，反应结束后过滤，用二氯甲烷洗涤，浓缩，经combi-flash柱层析[PE:EA＝100:0～80:20]分离纯化得到目标产物44a-1(1.06g，产率58.18％)。MS m/z(ESI):278[M-55]+。
步骤2-3：以化合物44a-1为原料，参照化合物13f制备方法中步骤4和5制备。MS m/z(ESI):312[M+H]+。
化合物45-a的制备
Figure PCTCN2015076451-appb-000062
化合物46-a的制备
Figure PCTCN2015076451-appb-000063
以化合物27a-2和化合物b为原料，参考化合物J-27的制备方法制备得化合物46-a。MS m/z(ESI):586.3[M+H]+。
化合物47-a的制备
Figure PCTCN2015076451-appb-000064
步骤1：在氢气氛下，将化合物47a-1(5.0g，0.0292mmol)、二甲胺盐酸盐(4.77g，0.0584mmol)、2.1g Pd/C和2.5ml醋酸加入到100ml甲醇中，氢气置换后室温反应48小时。反应结束后过滤，浓缩得粗产品。加饱和NaHCO3和乙酸乙酯萃取，盐水洗涤，浓缩得目标物47a-2(5.0g，产率85％)。MS m/z(ESI):201[M+H]+。
步骤2：0℃下，向化合物47a-2(5.0g，0.025mmol)的二氯甲烷(100ml)溶液中加入HCl/1,4-二氧六环溶液(4M)。反应混合物室温搅拌3小时。反应结束后浓缩，得目标物47-a(3.5g，82％)。MS m/z(ESI):173[M+H]+。
化合物48-a的制备
Figure PCTCN2015076451-appb-000065
将锌粉(980mg，15.08mmol)、三甲基氯硅烷(175mg，1.61mmol)、1,2-二溴乙烷(303mg，1.61mmol)依次加入20mlDMF溶液中，氩气置换后，反应混合物室温搅拌3分钟。加入化合物48a-1(3.95g，13.95mmol)的10ml DMF溶液，室温搅拌1.5小时。将上述反应液加入到化合物2a(1.6g，10.74mmol)、Pd(dppf)Cl2(393mg，0.54mmol)、碘化亚铜(102mg，0.54mmol)和20mlDMF的混合物中。氩气置换后100℃封管反应16小时。反应结束后过滤，浓缩，加二氯甲烷溶解，用饱和碳酸氢钠和盐水溶液洗涤，浓缩，经combi-flash柱层析分离纯化得到目标产物48-a。MS m/z(ESI):214[M-55]+。
化合物49-a和50-a的制备
化合物49-a和50-a可参照化合物31-a的方法制备，不同的是将步骤1中的化合物7a-1分别换成2,4-二氯-5-氟嘧啶和2,4-二氯-5-(三氟甲基)嘧啶。
Figure PCTCN2015076451-appb-000066
化合物51-a和52-a的制备
Figure PCTCN2015076451-appb-000067
实施例1N-(5-(5-氯-4-(1-(甲磺酰基)氮杂环庚烷-4-基氧基)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(J-1)的制备
Figure PCTCN2015076451-appb-000068
步骤1：将2,4,5-三氯嘧啶1a(2g，10.9mmol)和碳酸铯(10.2g，31.2mmol)加入到4-羟基氮杂环庚烷-1-羧酸叔丁酯(2.24g，10.9mmol)的100ml乙腈溶液中，85℃下剧烈搅拌5小时。反应结束后，滤去不溶物，以乙酸乙酯洗涤滤饼。有机层减压浓缩，经combiflash纯化得到化合物4-(2,5-二氯嘧啶-4-基氧基)氮杂环庚烷-1-羧酸叔丁酯1b(200mg，5％)。MS m/z(ESI):306.0[M-56]+。
步骤2：在0℃下，将三氟乙酸(1.89g，16.7mmol)加入到4-(2,5-二氯嘧啶-4-基氧基)氮杂环庚烷-1-甲酸叔丁酯1b(300mg，0.83mmol)的10ml的二氯甲烷溶液中，室温下剧烈搅拌5小时。反应结束后，将反应液在减压下蒸干，得到化合物4-(2,5-二氯嘧啶-4-基氧基)氮杂环庚烷1c(380mg)，产物直接用于下一步。MS m/z(ESI):262.0[M+1]+。
步骤3：在0℃下，将N,N-二异丙基乙胺(4g，30.5mmol)加入到化合物4-(2,5-二氯嘧啶-4-基氧基)氮杂环庚烷1c(400mg，1.53mmol)的12ml二氯甲烷溶液中，0℃下剧烈搅拌30分钟后加入甲磺酰氯(350mg，3.05mmol)，0℃下剧烈搅拌2小时。反应结束后，加水稀释，用二氯甲烷/水体系萃取三次，有机层减压浓缩，经combiflash纯化得到化合物4-(2,5-二氯嘧啶-4-基氧基)-1-(甲基磺酰基)氮杂环庚烷1d(360mg，70％)。MS m/z(ESI): 362.0[M+1]+。
步骤4：将N-(5-氨基-2-((2-(二甲基氨基)乙基)(甲基)氨基)-4-甲氧苯基)丙烯酰胺a(155mg，0.53mmol)，Pd2(dba)3(49mg，0.05mmol)，BINAP(66mg，0.10mmol)和碳酸铯(345mg，1.06mmol)加入到化合物4-(2,5-二氯嘧啶-4-基氧基)-1-(甲基磺酰基)氮杂环庚烷1d(180mg，0.53mmol)的5ml 1,4-二氧六环溶液中。在微波反应下，160℃下剧烈搅拌30分钟。反应结束后，过滤，滤液用乙酸乙酯/水体系萃取三次，有机相减压浓缩后得粗产品，经制备液相分离纯化得化合物N-(5-(5-氯-4-(1-(甲基磺酰基)氮杂环庚烷-4-基氧基)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J-1(84.24mg)。MS m/z(ESI):596.3[M+1]+；1HNMR(400MHz，DMSO-d6)δ10.12(s,1H),8.75(s,1H),8.29(s,1H),8.26(s,1H),8.25(s,1H),6.99(s,1H),6.44(dd,J＝16.9,10.1Hz,1H),6.23(dd,J＝16.9,1.8Hz,1H),5.75(dd,J＝10.1,1.7Hz,1H),5.46(dd,J＝7.6,3.8Hz,1H),3.83(s,3H),3.39-3.32(m,1H),3.27(t,J＝6.3Hz,3H),2.90(d,J＝6.0Hz,2H),2.87(s,3H),2.68(s,3H),2.37(t,J＝5.6Hz,2H),2.24(s,6H),2.13-2.03(m,1H),1.98-1.79(m,4H),1.72-1.58(m,1H)。
实施例2 4-(2-(5-丙烯酰胺基-4-(4-(二甲基氨基)哌啶-1-基)-2-甲氧基苯基氨基)嘧啶-4-基氧基)哌啶-1-甲酸叔丁酯(J-2)的制备
Figure PCTCN2015076451-appb-000069
步骤1：将2,4-二氯嘧啶2a(10g，67.11mmol)和碳酸铯(39.8g，122.09mmol)加入到叔丁基-4-羟基哌啶-1-甲酸叔丁酯(12.8g，63.68mmol)的300ml DMF溶液中，80℃下剧烈搅拌8小时。反应结束后，加水稀释，用乙酸乙酯/水体系萃取三次，水和饱和氯化钠洗三次，有机层减压浓缩后，经combiflash纯化[PE:EA＝100:0～50:50]得到化合物4-(2-氯嘧啶-4-基氧基)哌啶-1-甲酸叔丁酯2b(7.6g，40％)。MS m/z(ESI):314.1[M+1]+。
步骤2：将化合物N-(5-氨基-2-(4-(二甲基氨基)哌啶-1-基)-4-甲氧基苯基)丙烯酰胺b(250mg，0.80mmol)，Pd2(dba)3(80mg，0.08mmol)，Xantphos(100mg，0.16mmol)和碳酸铯(520mg，1.60mmol)加入到化合物4-(2-氯嘧啶-4-基氧基)哌啶-1-甲酸叔丁酯2b(250mg，0.80mmol)的5ml 1,4-二氧六环溶液中。在微波反应下，160℃下剧烈搅拌30分钟。反应结束后，过滤，滤液用乙酸乙酯/水体系萃取三次，有机相减压浓缩后得粗产品，经制备液相分离纯化得4-(2-(5-丙烯酰胺基-4-(4-(二甲基氨基)哌啶-1-基)-2-甲氧基苯基氨基)嘧啶-4-基氧基)哌啶-1-甲酸叔丁酯J-2(34.91mg，7％)。MS m/z(ESI):596.3[M+1]+；1HNMR(400MHz，DMSO-d6)δ9.02(s,1H),8.66(s,1H),8.15(d,J＝5.6Hz,1H),7.90(s,1H),6.83(s,1H),6.68(dd,J＝17.0,10.2Hz,1H),6.25-6.15(m,2H),5.71(d,J＝ 10.2Hz,1H),5.34(s,1H),3.85(s,3H),3.71(d,J＝13.3Hz,2H),3.01(d,J＝11.6Hz,4H),2.65(t,J＝10.6Hz,2H),2.22(s,6H),2.17(s,1H),1.94(d,J＝8.9Hz,2H),1.82(d,J＝11.0Hz,2H),1.68(d,J＝9.3Hz,2H),1.49(d,J＝9.6Hz,2H),1.40(s,9H)。
实施例3N-(5-(5-氯-4-(1-(甲基磺酰基)哌啶-4-基氧基)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(J-3)的制备
Figure PCTCN2015076451-appb-000070
步骤1：在0℃下，将4-羟基哌啶-1-甲酸叔丁酯(10g，49.8mmol)加入到氢化钠(2.4g，59.8mmol)的200ml的THF溶液中，0℃下搅拌1h后，加入2,4,5-三氯嘧啶1a(10g，54.7mmol)，室温下剧烈搅拌8小时。反应结束后，加水稀释，用乙酸乙酯/水体系萃取三次，水和饱和氯化钠洗三次，有机层减压浓缩后，经combiflash纯化[PE:EA＝100:0～50:50]得到化合物4-(2,5-二氯嘧啶-4-基氧基)哌啶-1-甲酸叔丁酯3a(7.6g，52％)。MS m/z(ESI):292.0[M+1]+。
步骤2：以4-(2,5-二氯嘧啶-4-基氧基)哌啶-1-甲酸叔丁酯3a(1.5g，4.32mmol)为原料，参照实施例1中的步骤2进行合成，得到化合物2,5-二氯-4-(哌啶-4-基氧基)嘧啶3b(2.4g，100％)，产物直接用于下一步。MS m/z(ESI):248.0[M+1]+。
步骤3：以2,5-二氯-4-(哌啶-4-基氧基)嘧啶3b(908mg，1.62mmol)为原料，参照实施例1中的步骤3进行合成，得到化合物2,5-二氯-4-(1-(甲基磺酰基)哌啶-4-基氧基)嘧啶3c(150mg，28％)。MS m/z(ESI):326.0[M+1]+。
步骤4：以2,5-二氯-4-(1-(甲基磺酰基)哌啶-4-基氧基)嘧啶3c(130mg，0.40mmol)为原料，参照实施例1中的步骤4进行合成，得化合物N-(5-(5-氯-4-(1-(甲基磺酰基)哌啶-4-基氧基)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J-3(75.60mg，33％)。MS m/z(ESI):582.2[M+1]+；1HNMR(400MHz，DMSO-d6)δ9.92(s,1H),8.72(s,1H),8.27(d,J＝9.5Hz,2H),8.19(s,1H),6.99(s,1H),6.54(dd,J＝16.9,10.1Hz,1H),6.26(dd,J＝16.9,1.8Hz,1H),5.77(dd,J＝10.2,1.7Hz,1H),5.47-5.32(m,1H),3.84(s,3H),3.40-3.34(m,2H),3.03(dt,J＝11.8,7.3Hz,4H),2.87(s,3H),2.66(s,5H),2.41(s,6H),2.10-2.00(m,2H),1.84-1.72(m,2H)。
实施例4N-(2-((2-(二甲基氨基)乙基)(甲基)氨基)-4-甲氧基-5-(4-(1-(甲基磺酰基)哌啶-4-基氧基)嘧啶-2-基氨基)苯基)丙烯酰胺(J-4)的制备
Figure PCTCN2015076451-appb-000071
以化合物2a(10g，67.11mmol)为原料，参照实施例1的方法进行合成，不同的是将实施例1步骤1中4-羟基氮杂环庚烷-1-羧酸叔丁酯换成4-羟基哌啶-1-甲酸叔丁酯(12.8g，63.68mmol)，得化合物N-(2-((2-(二甲基氨基)乙基)(甲基)氨基)-4-甲氧基-5-(4-(1-(甲基磺酰基)哌啶-4-基氧基)嘧啶-2-基氨基)苯基)丙烯酰胺J-4(93.42mg，33％)。MS m/z(ESI):548.3[M+1]+；1HNMR(400MHz,DMSO-d6)δ10.15(s,1H),8.96(s,1H),8.17(d,J＝5.6Hz,1H),7.94(s,1H),7.00(s,1H),6.40(dd,J＝16.9,10.0Hz,1H),6.25(td,J＝6.6,2.0Hz,2H),5.76(dd,J＝10.0,1.9Hz,1H),5.45-5.32(m,1H),3.85(s,3H),3.38(dd,J＝11.6,5.6Hz,2H),3.02(t,J＝9.1Hz,2H),2.86(d,J＝9.0Hz,5H),2.70(s,3H),2.28(t,J＝5.7Hz,2H),2.19(s,6H),2.09-2.01(m,2H),1.77-1.66(m,2H)。
实施例5N-(2-(4-(二甲基氨基)哌啶-1-基)-4-甲氧基-5-(4-(1-(甲基磺酰基)哌啶-4-基氧基)嘧啶-2-基氨基)苯基)丙烯酰胺(J-5)的制备
Figure PCTCN2015076451-appb-000072
以化合物2a(10g，67.11mmol)为原料，参照实施例1的方法进行合成，不同的是将实施例1步骤1中的4-羟基氮杂环庚烷-1-羧酸叔丁酯换成4-羟基哌啶-1-甲酸叔丁酯(12.8g，63.68mmol)，将实施例1步骤4中的化合物a换成化合物b(164mg，0.52mmol)，得化合物N-(2-(4-(二甲基氨基)哌啶-1-基)-4-甲氧基-5-(4-(1-(甲基磺酰基)哌啶-4-基氧基)嘧啶-2-基氨基)苯基)丙烯酰胺(142.64mg，47％)。MS m/z(ESI):574.3[M+1]+；1HNMR(400MHz,DMSO-d6)δ9.01(s,1H),8.67(s,1H),8.16(d,J＝5.6Hz,1H),7.92(s,1H),6.84(s,1H),6.67(dd,J＝16.9,10.1Hz,1H),6.29-6.19(m,2H),5.75(d,J＝10.3Hz,1H),5.31(s,1H),3.85(s,3H),3.44-3.35(m,2H),3.33(s,3H),3.01(s,4H),2.87(s,3H),2.66(t,J＝10.7Hz,2H),2.23(s,6H),2.20-2.14(m,1H),2.05(d,J＝11.2Hz,2H),1.84(d,J＝10.4Hz,2H),1.78-1.62(m,4H)。
实施例6N-(5-(4-(1-苯甲酰基氮杂-3-基氧基)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(J-6)的制备
Figure PCTCN2015076451-appb-000073
Figure PCTCN2015076451-appb-000074
步骤1：将化合物2，4-二氯嘧啶2a(19.7g，110mmol)，碳酸铯(65.2g，200mmol)加入到N-Boc-3-羟基氮杂环丁烷(17.3g，100mmol)的400ml的DMF溶剂中，83℃下剧烈搅拌4小时。通过TLC检测反应进度，待底物完全反应后，用乙酸乙酯/水体系萃取三遍，分离出有机层，经水洗，饱和食盐水洗涤，旋干后得到的粗产品。经Combi-flash柱层析[PE:EA＝100:0～40:60]纯化后得化合物3-(2-氯嘧啶-4-基氧基)氮杂环丁烷-1-甲酸叔丁酯6a(25g，88％)。MS m/z(ESI):286.1[M+1]+。
步骤2：将化合物4氟-2-甲氧基-5-硝基苯胺(1.96g，10.5mmol)，Pd2(dba)3(964mg，1.05mmol)，Xantphos(1.219g，2.11mmol)和碳酸铯(6.86g，21.0mmol)加入到化合物3-(2-氯嘧啶-4-基氧基)氮杂环丁烷-1-甲酸叔丁酯6a(3g，10.5mmol)的70ml 1,4-二氧六环溶液中。120℃下，在N2氛围中剧烈搅拌20小时。反应结束后，过滤，乙酸乙酯洗涤，滤液减压浓缩后得粗产品，经Combi-flash柱层析[PE:EA＝100:0～20:80]纯化后得化合物3-(2-(4-氟-2-甲氧基-5-硝基苯基氨基)嘧啶-4-基氧基)氮杂环丁烷-1-甲酸叔丁酯6b(2.86g，62％)。MS m/z(ESI):436.2M+1]+。
步骤3：将N,N,N’-三甲基乙二胺(4.08g，40.0mmol)和碳酸钾(13.8g，100mmol)加入到化合物3-(2-(4-氟-2-甲氧基-5-硝基苯基氨基)嘧啶-4-基氧基)氮杂环丁烷-1-甲酸叔丁酯6b(14.5g，33.3mmol)的170ml的DMF中，100℃下剧烈搅拌4h。通过TLC检测反应进度，待底物完全反应后，用乙酸乙酯/水体系萃取三次，分离出有机层，减压浓缩得到化合物3-(2-(4-((2-(二甲基氨基)乙基)(甲基)氨基)-2-甲氧基-5-硝基苯基氨基)嘧啶-4-基氧基)氮杂环丁烷-1-甲酸叔丁酯6c(9.2g，54％)，MS m/z(ESI):518.3[M+1]+。
步骤4：将三氟乙酸(27mL，356.0mmol)加入到化合物3-(2-(4-((2-(二甲基氨基)乙基)(甲基)氨基)-2-甲氧基-5-硝基苯基氨基)嘧啶-4-基氧基)氮杂环丁烷-1-甲酸叔丁酯6c(9.2g，17.8mmol)的250ml的二氯甲烷溶液中，室温下剧烈搅拌6小时。通过TLC检测反应进度，待底物完全反应后，减压下旋掉多余的二氯甲烷，加水稀释，调节pH至碱性，用二氯甲烷和甲醇(10:1)萃取，将有机相旋干，得化合物N1-(4-(氮杂环丁烷-3-基氧基)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6d(5.2g，70％)。MS m/z(ESI):418.2[M+1]+。
步骤5：在0℃下，将与三乙胺(1.01g，10mmol)加入到化合物N1-(4-(氮杂环丁烷 -3-基氧基)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6d(1.4g，1mmol)的10ml二氯甲烷溶液中，0℃下剧烈搅拌30分钟后加入苯甲酰氯(140mg，1.2mmol)。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化合物(3-(2-(4-((2-(二甲基氨基)乙基)(甲基)氨基)-2-甲氧基-5-硝基苯基氨基)嘧啶-4-基氧基)氮杂环丁烷-1-基)(苯基)甲酮6e(660mg，99％)。产物直接用于下一步。MS m/z(ESI):522.3[M+1]+。
步骤6：将Pd/C(110mg)加入到化合物(3-(2-(4-((2-(二甲基氨基)乙基)(甲基)氨基)-2-甲氧基-5-硝基苯基氨基)嘧啶-4-基氧基)氮杂环丁烷-1-基)(苯基)甲酮6e(650mg，1mmol)的50ml甲醇溶液中。室温下，在H2氛围中剧烈搅拌4小时。反应结束后，过滤，滤液浓缩，得化合物(3-(2-(5-氨基-4-((2-(二甲基氨基)乙基)(甲基)氨基)-2-甲氧基苯基氨基)嘧啶-4-基氧基)氮杂环丁烷-1-基)(苯基)甲酮6f(630mg，99％)，产物直接用于下一步。MS m/z(ESI):492.3[M+1]+。
步骤7：在0℃下，将丙烯酰氯(138mg，1.54mmol)与三乙胺(195mg，1.92mmol)加入到化合物(3-(2-(5-氨基-4-((2-(二甲基氨基)乙基)(甲基)氨基)-2-甲氧基苯基氨基)嘧啶-4-基氧基)氮杂环丁烷-1-基)(苯基)甲酮6f(630mg，1mmol)的10ml二氯甲烷溶液中，0℃下剧烈搅拌2小时。反应结束后，加水稀释，用二氯甲烷/水体系萃取三次，有机层减压浓缩得到粗产物。经制备液相分离纯化得化合物N-(5-(4-(1-苯甲酰基氮杂-3-基氧基)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J-6(70.67mg，13％)。MS m/z(ESI):545.8[M+1]+；1HNMR(400MHz,DMSO-d6)δ10.14(s,1H),8.93(s,1H),8.22(d,J＝5.6Hz,1H),8.04(s,1H),7.59(d,J＝7.1Hz,2H),7.50(t,J＝7.3Hz,1H),7.42(t,J＝7.4Hz,2H),6.97(s,1H),6.35(d,J＝5.6Hz,1H),6.34–6.27(m,1H),6.18(dd,J＝16.9,2.1Hz,1H),5.76–5.66(m,2H),4.50(d,J＝25.3Hz,2H),4.35(s,1H),4.05(s,1H),3.83(s,3H),2.82(d,J＝3.9Hz,2H),2.66(s,3H),2.23(s,2H),2.14(s,6H)。
实施例7N-(2-((2-(二甲基氨基)乙基)(甲基)氨基)-4-甲氧基-5-(4-(1-(苯基磺酰基)氮杂环丁烷-3-基氧基)嘧啶-2-基氨基)苯基)丙烯酰胺(J-7)的制备
Figure PCTCN2015076451-appb-000075
以化合物6b(14.5g，33.3mmol)为原料，参照实施例6的方法进行合成，不同的是将实施例6步骤5中的苯甲酰氯换成苯磺酰氯(210mg，1.2mmol)，化合物N-(2-((2-(二甲基氨基)乙基)(甲基)氨基)-4-甲氧基-5-(4-(1-(苯基磺酰基)氮杂环丁烷-3-基氧基)嘧啶-2-基氨基)苯基)丙烯酰胺J-7(44.30mg，7％)。MS m/z(ESI):582.3[M+1]+；1H NMR(400MHz,DMSO-d6)δ9.98(s,1H),8.65(s,1H),8.15(s,1H),8.17(d,J＝5.6Hz,1H),7.79(m,3H),7.69(t,J＝7.5Hz,2H),6.98(s,1H),6.82-6.36(m,1H),6.25(d,J＝16.7Hz,1H),6.12(d,J＝5.6Hz,1H),5.74(d,J＝11.8Hz,1H),5.29(s,1H),4.09(t,J＝ 15.6Hz,2H),3.83(s,3H),3.59(m,2H),2.99(s,2H),2.67(s,3H),2.50-2.14(m,8H)。
实施例8N-(5-(4-(1-苯甲酰基氮杂-3-基氧基)嘧啶-2-基氨基)-2-(4-(二甲基氨基)哌啶-1-基)-4-甲氧苯基)丙烯酰胺(J-8)的制备
Figure PCTCN2015076451-appb-000076
以化合物6b(6.7g，15.4mmol)为原料，参照实施例6的方法进行合成，不同的是将实施例6步骤3中的N,N,N’-三甲基乙二胺换成4-二甲氨基哌啶(2.37g，18.5mmol)，得化合物N-(5-(4-(1-苯甲酰基氮杂-3-基氧基)嘧啶-2-基氨基)-2-(4-(二甲基氨基)哌啶-1-基)-4-甲氧苯基)丙烯酰胺J-8(33.46mg，15％)。MS m/z(ESI):572.8[M+1]+；1H NMR(400MHz,DMSO-d6)δ8.95(s,1H),8.58(s,1H),8.20(d,J＝5.6Hz,1H),8.04(s,1H),7.65-7.58(m,2H),7.51(t,J＝7.3Hz,1H),7.44(t,J＝7.4Hz,2H),6.79(s,1H),6.62(dd,J＝16.9,10.2Hz,1H),6.33(d,J＝5.6Hz,1H),6.17(dd,J＝16.9,1.9Hz,1H),5.68(d,J＝10Hz,1H),5.63(s,1H),4.49(d,J＝19.8Hz,2H),4.33(s,1H),4.03(d,J＝7.7Hz,1H),3.82(s,3H),2.95(d,J＝12.6Hz,2H),2.69-2.53(m,2H),2.22(s,6H),2.16(t,J＝10.9Hz,1H),1.81(d,J＝11.1Hz,2H),1.66(d,J＝9.6Hz,2H)。
实施例9N-(2-(4-(二甲基氨基)哌啶-1-基)-4-甲氧基-5-(4-(1-(苯基磺酰基)氮杂环丁烷-3-基氧基)嘧啶-2-基氨基)苯基)丙烯酰胺(J-9)的制备
Figure PCTCN2015076451-appb-000077
以化合物6b(6.7g，15.4mmol)为原料，参照实施例6的方法进行合成，不同的是将实施例6步骤3中的N,N,N’-三甲基乙二胺换成4-二甲氨基哌啶(2.37g，18.5mmol)，将实施例6步骤5中的苯甲酰氯换成苯磺酰氯(88mg，0.45mmol)，得到化合物N-(2-(4-(二甲基氨基)哌啶-1-基)-4-甲氧基-5-(4-(1-(苯基磺酰基)氮杂环丁烷-3-基氧基)嘧啶-2-基氨基)苯基)丙烯酰胺J-9(78.86mg，34％)。MS m/z(ESI):608.3[M+1]+；1H NMR(400MHz,DMSO-d6)δ8.96(s,1H),8.41(s,1H),8.12(s,2H),7.75(m,5H),6.73(d,J＝60.1Hz,2H),6.17(d,J＝58.5Hz,2H),5.73(d,J＝20.8Hz,1H),5.25(s,1H),4.08(d,J＝7.2Hz,2H),3.82(d,J＝14.9Hz,3H),3.59(s,2H),3.03(s,2H),2.66(s,2H),2.26(s,7H),1.85(s,2H),1.72(s,2H)。
实施例10N-(2-(4-(二甲基氨基)哌啶-1-基)-4-甲氧基-5-(4-(1-(甲基磺酰基)氮杂环 丁烷-3-基氧基)嘧啶-2-基氨基)苯基)丙烯酰胺(J-10)的制备
Figure PCTCN2015076451-appb-000078
以化合物6b(6.7g，15.4mmol)为原料，参照实施例6的方法进行合成，不同的是将实施例6步骤3中的N,N,N’-三甲基乙二胺换成4-二甲氨基哌啶(2.37g，18.5mmol)，将实施例6步骤5中的苯甲酰氯换成甲磺酰氯(57mg，0.50mmol)，得到化合物N-(2-(4-(二甲基氨基)哌啶-1-基)-4-甲氧基-5-(4-(1-(甲基磺酰基)氮杂环丁烷-3-基氧基)嘧啶-2-基氨基)苯基)丙烯酰胺J-10(93.21mg，45％)。MS m/z(ESI):546.3[M+1]+；1H NMR(400MHz,DMSO-d6)δ9.01(s,1H),8.51(s,1H),8.20(d,J＝4.5Hz,1H),8.10(s,1H),6.83(s,1H),6.76-6.59(m,1H),6.30(d,J＝5.6Hz,1H),6.26(d,J＝16.4Hz,2H),5.74(d,J＝9.9Hz,1H),5.45(s,1H),4.23-4.14(t,J＝7.8Hz,2H),3.95-3.89(m,2H),3.83(s,3H),3.04(s,2H),3.01(s,3H),2.66(t,J＝10.8Hz,2H),2.24(s,7H),1.82(d,J＝12.4Hz,2H),1.69(m,2H)。
实施例11N-(5-(5-氯-4-(1-(甲基磺酰基)氮杂环丁烷-3-基氧基)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(J-11)的制备
Figure PCTCN2015076451-appb-000079
以化合物1a(10g，54.5mmol)为原料，参照实施例1的方法进行合成，不同的是将实施例1步骤1中的4-羟基氮杂环庚烷-1-羧酸叔丁酯换成N-Boc-3-羟基氮杂环丁烷(8.58g，49.5mmol)，得到N-(5-(5-氯-4-(1-(甲基磺酰基)氮杂环丁烷-3-基氧基)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J-11(56.2mg，31.5％)。MS m/z(ESI):553.6[M+H]+；1H NMR(400MHz,CDCl3):δ12.37(s,1H),9.50(s,1H),9.14(s,1H),8.08(s,1H),7.29-7.31(m,1H),6.63(s,1H),6.37-6.41(d,J＝16.4Hz,1H),5.99(s,1H),5.67-5.70(dd,J＝11.6Hz,2Hz,1H),4.27-4.31(t,J＝8Hz,2H),4.00-4.04(q,J＝4.6Hz,2H),3.85(s,3H),3.27-3.29(m,2H),3.07-3.09(m,2H),2.82(s,3H),2.77(s,3H),2.76(s,3H),2.71(s,3H)。
实施例12N-(5-(5-氯-4-(1-(甲基磺酰基)氮杂环丁烷-3-基氧基)嘧啶-2-基氨基)-2-(4-(二甲基氨基)哌啶-1-基)-4-甲氧苯基)丙烯酰胺甲酸盐(J-12)的制备
Figure PCTCN2015076451-appb-000080
以化合物1a(10g，54.5mmol)为原料，参照实施例1的方法进行合成，不同的是将实施例1步骤1中的4-羟基氮杂环庚烷-1-羧酸叔丁酯换成N-Boc-3-羟基氮杂环丁烷(8.58g，49.5mmol)，将实施例1步骤4中的化合物a换成化合物b(160mg，0.50mmol)，得化合物N-(5-(5-氯-4-(1-(甲基磺酰基)氮杂环丁烷-3-基氧基)嘧啶-2-基氨基)-2-(4-(二甲基氨基)哌啶-1-基)-4-甲氧苯基)丙烯酰胺甲酸盐J-12(81.9mg，30.7％)。MS m/z(ESI):580.2[M+H]+；1H NMR(400MHz，DMSO-d6):δ9.02(s,1H),8.43(s,1H),8.34(s,1H),8.28(s,1H),8.16(s,1H),6.83(s,1H),6.63-6.70(dd,J＝17.2Hz,10.8Hz,1H),6.24-6.29(dd,J＝17.2Hz,2Hz,1H),5.72-5.75(d,J＝11.6Hz,1H),5.42-5.45(m,1H),4.15-4.19(t,J＝8Hz,2H),3.93-3.97(q,J＝4.8Hz,2H),3.81(s,3H),3.12-3.16(m,2H),3.02(s,3H),2.66-2.71(t,J＝10.8Hz,2H),2.54-2.59(m,1H),2.42(s,6H),1.89-1.92(d,J＝11.2Hz,2H),1.72-1.80(m,2H)。
实施例13N-(5-(5-氯-4-(1-(甲基磺酰基)氮杂环丁烷-3-基硫代)嘧啶-2-基氨基)-2-(4-(二甲基氨基)哌啶-1-基)-4-甲氧苯基)丙烯酰胺(J-13)的制备
Figure PCTCN2015076451-appb-000081
步骤1：将3-碘氮杂环丁烷-1-甲酸叔丁酯13a(2g，7.2mmol)和碳酸铯(4.72g，14.4mmol)加入到化合物硫代乙酸(1.1g，14.4mmol)的10ml N，N-二甲基甲酰胺溶液中，70℃下剧烈搅拌4小时。反应结束后，滤去不溶物，以乙酸乙酯洗涤滤饼。有机层减压浓缩，经combiflash纯化得到化合物3-(乙酰硫基)氮杂环丁烷-1-甲酸叔丁酯13b(700mg，43％)。MS m/z(ESI):232[M+H]+。
步骤2：将3-(乙酰硫基)氮杂环丁烷-1-甲酸叔丁酯13b(0.7g，3.03mmol)和碳酸钾(0.836g，6.05mmol)加入到10ml甲醇中，50℃下剧烈搅拌2小时。反应结束后，冷却到室温，用2M的盐酸水溶液调pH值至2-3，加水稀释，用乙酸乙酯/水体系萃取二次，有机层减压浓缩得化合物3-巯基氮杂环丁烷-1-甲酸叔丁酯13c(450mg，78％)，不纯化直接用于下一步。MS m/z(ESI):190[M+1]+。
步骤3：将3-巯基氮杂环丁烷-1-羧酸叔丁酯13c(0.45g，2.38mmol)，2,4,5-三氯嘧 啶(0.46g，2.5mmol)和碳酸钠(0.631g，5.96mmol)加入到20ml乙腈中，70℃下剧烈搅拌3小时。反应结束后，冷却过滤浓缩，经combiflash纯化得到化合物3-(2,5-二氯嘧啶-4-基硫基)氮杂环丁烷-1-甲酸叔丁酯13d(580mg，72％)。MS m/z(ESI):337[M+1]+。
步骤4：将3-(2,5-二氯嘧啶-4-基硫基)氮杂环丁烷-1-甲酸叔丁酯13d(0.58g，1.73mmol)加入到10ml二氯甲烷中，在0℃和搅拌的条件下滴加三氟乙酸(5g，43.9mmol)，回到室温反应3小时结束，浓缩后得到化合物粗品3-(2,5-二氯嘧啶-4-基硫基)氮杂环丁烷13e(1.5g)，不游离纯化直接用于下一步反应。MS m/z(ESI):237[M+1]+。
步骤5：以化合物13e(1.5g)为原料，参照实施例1步骤3的方法合成，得到化合物2,5-二氯-4-(1-(甲基磺酰基)氮杂环丁烷-3-基硫代)嘧啶13f。MS m/z(ESI):314[M+1]+。
步骤6：以化合物13f(314mg，1mmol)和化合物b(318mg，1mmol)为原料，参照实施例2的步骤2合成，得化合物N-(5-(5-氯-4-(1-(甲基磺酰基)氮杂环丁烷-3-基硫代)嘧啶-2-基氨基)-2-(4-(二甲基氨基)哌啶-1-基)-4-甲氧苯基)丙烯酰胺J-13(89.06mg)。MS m/z(ESI):596[M+1]+；1HNMR(400MHz，DMSO-d6)δ9.94(s,1H),9.05(s,1H),8.68(s,1H),8.25(s,1H),8.23(s,1H),8.15(s,1H),6.85(s,1H),6.69(dd,J＝16.4,10.0Hz,1H),6.28(d,J＝16.0Hz,1H),5.75(d,J＝11.6Hz,1H),4.44(f,1H),4.08(br,s,2H),3.84-3.72(m,5H),3.40-3.20(m,1H),3.16(d,J＝11.6Hz,2H),3.00(s,3H),2.81(d,J＝4.8Hz,6H),2.75(t,J＝11.6Hz,2H),2.05(d,J＝9.6Hz,2H),1.85-2.00(m,2H)。
实施例14N-(2-((2-(二甲基氨基)乙基)(甲基)氨基)-4-甲氧基-5-(4-(1-(喹啉-3-基)氮杂环丁烷-3-基氧基)嘧啶-2-基氨基基)苯基)丙烯酰胺(J-14)的制备
Figure PCTCN2015076451-appb-000082
步骤1：将3-溴喹啉(0.31g，1.473mmol)，Pd2(dba)3(0.135g，0.1473mmol)，Xantphos(0.171g，0.2946mmol)和碳酸铯(1.92g，5.69mmol)加入到化合物6d(1.0g，1.473mmol)的50ml 1,4-二氧六环溶液中。120℃下，在N2氛围中剧烈搅拌20小时。反应结束后，过滤，乙酸乙酯洗涤，滤液减压浓缩后得粗产品，经Combi-flash柱层析[PE:EA＝100:0～20:80]纯化后得化合物14b(0.42g，52.8％)。MS m/z(ESI):545[M+H]+。
步骤2：以化合物14b(80mg，0.15mmol)为原料，参照实施例6中步骤6进行制备，得化合物14c的粗产品70mg，不经纯化直接下一步。MS m/z(ESI):515[M+H]+。
步骤3：以化合物14c(70mg，0.11mmol)为原料，参照实施例6中步骤7进行制备，得到的粗产品经制备液相分离纯化得标题化合物J-14(13mg，产率16.9％)。MS m/z(ESI):569[M+H]+；1H NMR(400MHz,CDCl3)δ10.12(s,1H),9.51(s,1H),8.33(d,J＝2.6Hz,1H),8.19(d,J＝5.6Hz,1H),7.99–7.92(m,1H),7.65(s,1H),7.60(dd,J＝5.9,3.4Hz,1H),7.42(dd,J＝6.3,3.3Hz,2H),6.93(s,1H),6.78(s,1H),6.25(d,J＝5.6Hz,1H),6.18 (d,J＝15.6Hz,2H),5.36(s,1H),4.57–4.50(m,2H),4.10(dd,J＝8.6,4.0Hz,2H),3.89(s,3H),2.88(s,2H),2.71(s,3H),2.22(s,6H),2.01(s,2H)。
实施例15-19
化合物J-15、J-16和J-17以化合物6d为起始原料，参照实施例14的方法进行制备，不同的是将步骤1中3-溴喹啉分别换成5-溴-1-甲基-1H-吡唑并[3,4-b]吡啶、5-溴-1-甲基-1H-吡咯并[2,3-b]吡啶和3-溴吡啶。
化合物J-18和J-19以化合物5-a为起始原料，参照实施例14的方法进行制备，不同的是将步骤1中3-溴喹啉分别换成5-溴-1-甲基-1H-吡咯并[2,3-b]吡啶和5-溴-1-甲基-1H-吡唑并[3,4-b]吡啶。
Figure PCTCN2015076451-appb-000083
Figure PCTCN2015076451-appb-000084
实施例20-34
化合物J-20至化合物J-32均以各种5位取代或未取代的2,4-二氯嘧啶与N-Boc-3-羟基氮杂环丁烷为起始原料，参照实施例6的方法进行制备。根据化合物结构的不同，将实施例6中步骤3和步骤5中的N,N,N’-三甲基乙二胺和苯甲酰氯换成相应的胺和酰氯。
化合物J-33、J-34、J-75分别以化合物45-a、44-a和52-a为原料，与化合物b反应制得，反应步骤和条件参照实施例2中步骤2。
化合物J-76的制备方法同化合物J-12类似。
Figure PCTCN2015076451-appb-000085
Figure PCTCN2015076451-appb-000086
Figure PCTCN2015076451-appb-000087
Figure PCTCN2015076451-appb-000088
Figure PCTCN2015076451-appb-000089
实施例35-37
化合物J-35、J-36和J-37均以各种5位氯代或未取代的2,4-二氯嘧啶与4-羟基氮杂环庚烷-1-羧酸叔丁酯为起始原料，参照实施例1的方法进行制备。根据化合物结构的不同，将实施例1中步骤3和步骤4中的甲磺酰氯和化合物a换成相应的甲磺酰氯和化合物b。
Figure PCTCN2015076451-appb-000090
实施例38-39
化合物J-38和J-39参照实施例3的方法制备，根据化合物结构的不同，将实施例3中步骤3和步骤4中的甲磺酰氯和化合物a换成相应的乙酰氯和化合物b。
Figure PCTCN2015076451-appb-000091
实施例40(R)-N-(5-(5-氯-4-(1-(甲基磺酰基)哌啶-3-基氧基)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(J-40)的制备
Figure PCTCN2015076451-appb-000092
向化合物23-a(109mg，0.38mmol)和化合物a(120mg，0.38mmol)的4ml 1,4-二氧六环溶液中加入Pd2(dba)3(35mg，0.04mmol)、BINAP(47mg，0.08mmol)和碳酸铯(247mg，0.75mmol)。反应混合物140℃下微波反应20分钟。反应结束后，过滤反应混合物，用二氯甲烷洗涤，滤液减压浓缩后得粗产品，经制备液相分离纯化得93mg标题化合物J-40。MS m/z(ESI):582.2[M+H]+；1HNMR(400MHz，DMSO-d6)δ10.09(s,1H),8.72(s,1H),8.29(s,1H),8.27(s,1H),8.23(s,1H),6.98(s,1H),6.50–6.36(m,1H),6.25(dd,J＝16.9,2.0Hz,1H),5.74(dd,J＝10.0,2.0Hz,1H),5.41(s,1H),3.83(s,3H),3.36(d,J＝5.4Hz,2H),3.23–3.09(m,2H),2.88(s,5H),2.67(d,J＝11.6Hz,3H),2.38(s,2H),2.25(d,J＝1.8Hz,6H),1.97–1.88(m,1H),1.86–1.71(m,2H),1.60(s,1H)。
实施例41-44
化合物J-41、J-42、J-43、J-44分别以化合物23-a、24-a、25-a、26-a、为原料与化合物b反应制得，反应步骤和条件参照实施例40。
Figure PCTCN2015076451-appb-000093
Figure PCTCN2015076451-appb-000094
实施例45(R)-N-(5-(5-氯-4-(1-(甲基磺酰基)吡咯烷-3-基氧基)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(J-45)的制备
Figure PCTCN2015076451-appb-000095
将化合物b(159mg，0.48mmol)，Pd2(dba)3(44mg，0.048mmol)，Xantphos(42mg，0.073mmol)、碳酸铯(315mg，0.97mmol)和化合物14-a(150mg，0.48mmol)加入到5ml 1,4-二氧六环溶液中。氩气置换后120℃下封管搅拌过夜。LC-Ms监测反应，反应结束后，过滤，用二氯甲烷洗涤，滤液浓缩后得粗产品，经制备液相分离纯化得标题化合物J-45(63.77mg，产率20.7％)。MS m/z(ESI):594.3[M+H]+；1HNMR(400MHz，DMSO-d6)δ9.00(s,1H),8.49(s,1H),8.26(s,2H),8.23(s,1H),6.82(s,1H),6.66(dd,J＝16.9,10.3Hz,1H),6.22(d,J＝17.0Hz,1H),5.79(s,1H),5.73(d,J＝11.6Hz,1H),3.83(s,3H),3.57(dd,J＝12.3,4.4Hz,2H),3.03(d,J＝11.2Hz,2H),2.90(s,3H),2.64(d,J＝11.0Hz,2H),2.23(d,J＝13.0Hz,9H),1.84(d,J＝11.3Hz,2H),1.68(d,J＝8.9Hz,2H)。
实施例46-52
化合物J-46、J-47、J-48、J-49、J-50、J-51和J-52分别以化合物15-a、16-a、17-a、18-a、19-a、20-a和21-a为原料与化合物b反应制得，反应步骤和条件参照实施例45。
Figure PCTCN2015076451-appb-000096
Figure PCTCN2015076451-appb-000097
实施例53N-(2-((2-(二甲基氨基)乙基)(甲基)氨基)-5-(4-(1-(2-氟乙基)氮杂环丁烷-3-基氧基)嘧啶-2-基氨基)-4-甲氧基苯基)丙烯酰胺(J-53)的制备
Figure PCTCN2015076451-appb-000098
步骤1：将化合物6d(210mg，0.5mmol)，1-溴-2-氟乙烷(75mg，0.6mmol)和碳酸钾(205mg，1.5mmol)的乙腈(5ml)溶液在50℃下封管搅拌过夜，反应结束后，用二氯甲烷/水体系萃取三遍，分离出有机层，经饱和食盐水洗涤，无水硫酸钠干燥后，浓缩。得到化合物53b的粗产品。经Combi-flash柱层析纯化后得化合物53b(115mg，产率50％)。MS m/z(ESI):464[M+H]+。
步骤2-3：以化合物53b为原料，参照实施例6中步骤6和步骤7进行制备，经制备液相分离纯化得标题化合物J-53(35mg，产率31％)，MS m/z(ESI):488[M+H]+；1H NMR(400MHz,CDCl3)δ10.02(s,1H),9.38(s,1H),8.17-8.16(d,J＝5.6Hz,1H),7.49(s,1H),6.74(s,1H),6.47(s,1H),6.43(s,1H),6.20-6.19(d,J＝5.6Hz,1H),5.71-5.68(d,J＝11.9Hz,2H),4.52-4.50(t,J＝4.9Hz,1H),4.40-4.38(t,J＝4.9Hz,1H),3.86(s,3H),3.84-3.80(t,J＝8.2Hz,2H),3.41(s,2H),2.93(s,2H),2.85-2.77(m,2H),2.70(s,3H),2.34(s,8H)。
实施例54-55
化合物J-54以化合物22-a为原料，参照实施例53的方法制备。化合物J-55以化合物46-a为原料，依次参照实施例3步骤2和实施例53步骤1方法制备。
Figure PCTCN2015076451-appb-000099
Figure PCTCN2015076451-appb-000100
实施例56-59
化合物J-56、J-57、J-58和J-59分别以化合物40-a、41-a、42-a和43-a为原料与化合物b反应制得，反应步骤和条件参照实施例13。
Figure PCTCN2015076451-appb-000101
Figure PCTCN2015076451-appb-000102
实施例60N-(5-(5-氯-4-(1-(甲基磺酰基)氮杂环丁烷-3-基氨基)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(J-60)的制备
Figure PCTCN2015076451-appb-000103
在氩气氛围下，在室温下，向化合物b(143mg，0.45mmol)、化合物31-a(135mg，0.45mmol)、Xantphos(52mg，0.09mmol)和碳酸铯(293mg，0.9mmol)的1,4-二氧六环(10ml)溶液中加入Pd2(dba)3(41mg，0.045mmol)，反应混合物在160℃下微波反应15分钟。反应结束后过滤，减压浓缩滤液得粗产品，经制备液相分离纯化得标题化合物J-60(44.03mg，产率15.6％)。MS m/z(ESI):579[M+H]+；1HNMR(400MHz,DMSO-d6)δ8.99(s,1H),8.66(s,1H),8.23(s,1H),7.96(s,1H),7.72-7.71(d,J＝6.6Hz,1H),7.69(s,1H),6.80(s,1H),6.69-6.62(dd,J1＝10.2Hz,J2＝17.0Hz,1H),6.30-6.26(d,J＝16.8Hz,1H),5.73-5.70(d,J＝10.2Hz,1H),4.99-4.94(m,1H),4.05-4.01(t,J＝8.0Hz,2H),3.96-3.92(t,J＝6.9Hz,2H),3.83(s,3H),3.02-2.99(m,5H),2.67-2.62(t,J＝10.1Hz,2H),2.26(s,7H),1.85-1.82(d,J＝11.1Hz,2H),1.73-1.66(m,2H)。
实施例61-64和实施例73
化合物J-61、J-62、J-63、J-64、J-73和J-74分别以化合物33-a、49-a、35-a、50-a、39-a和51-a为原料与化合物b反应制得，反应步骤和条件参照实施例60。
Figure PCTCN2015076451-appb-000104
Figure PCTCN2015076451-appb-000105
实施例65N-(5-(5-氯-4-(1-(甲基磺酰基)哌啶-3-基氨基)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(J-65)的制备
Figure PCTCN2015076451-appb-000106
向化合物32-a(150mg，0.46mmol)的1,4-二氧六环溶液中加入化合物b(147mg，0.46mmol)，Pd2(dba)3(43mg，0.046mmol)，BINAP(58mg，0.092mmol)和碳酸铯(301mg，0.922mmol)，在微波反应下，130℃下搅拌25分钟。反应结束后，过滤，浓缩反应混合物后得粗产品，经制备液相分离纯化得标题化合物J-65(28.56mg)。MS m/z(ESI):607.3[M+H]+。1H NMR(400MHz,DMSO-d6)δ8.95(s,1H),8.58(s,1H),8.21(s,1H),7.95(s,1H),7.67(s,1H),6.80(s,1H),6.74(d,J＝8.3Hz,1H),6.65(dd,J＝16.9,10.1Hz,1H),6.23(d,J＝16.9Hz,1H),5.72(d,J＝11.4Hz,1H),4.28(s,1H),3.84(s,3H),3.47(d,J＝7.9Hz,1H),3.35(d,J＝11.1Hz,1H),3.03(s,2H),2.83(s,3H),2.76(dd,J＝17.4,7.5Hz,2H),2.72–2.58(m,2H),2.40(s,6H),1.88(d,J＝10.2Hz,3H),1.64(dd,J＝40.4,31.7Hz,6H)。
实施例66-69
化合物J-66、J-67、J-68和J-69分别以化合物34-a、37-a、36-a和38-a为原料与化合物b反应制得，反应步骤和条件参照实施例65。
Figure PCTCN2015076451-appb-000107
Figure PCTCN2015076451-appb-000108
实施例70N-(5-(5-氯-4-(1-(甲基磺酰基)氮杂环丁烷-3-基氧基)嘧啶-2-基氨基)-4-甲氧基-2-(4-甲基哌嗪-1-基)苯基)丙烯酰胺(J-70)的制备
Figure PCTCN2015076451-appb-000109
步骤1：将化合物a1(186mg，1mmol)，Pd2(dba)3(92mg，0.1mmol)，Xantphos(90mg，0.15mmol)和碳酸铯(652mg，2mmol)加入到化合物7-a(298mg，1mmol)的4ml 1,4-二氧六环溶液中。160℃下，微波反应20分钟。反应结束后，过滤，乙酸乙酯洗涤，滤液减压浓缩后得粗产品，经Combi-flash柱层析纯化后得化合物70b(160mg，产率35.8％)。MS m/z(ESI):448[M+H]+。
步骤2：将1-甲基哌嗪(72mg，0.72mmol)和碳酸钾(99.5mg，0.72mmol)加入到化合物70b(160mg，0.36mmol)的3ml的DMF中，100℃下剧烈搅拌2h。通过TLC检测反应进度，待底物完全反应后，用乙酸乙酯/水体系萃取三次，分离出有机层，减压浓缩得180mg粗产品化合物70c，不纯化直接下一步。MS m/z(ESI):528[M+H]+。
步骤3-4：以化合物70c(180mg，0.34mmol)为原料，参照实施例6中步骤6和步骤 7进行制备。得标题化合物J-70(74.5mg，产率37％)。MS m/z(ESI):552[M+H]+；1HNMR(400MHz,DMSO-d6)δ9.01(s,1H),8.43(s,1H),8.31(s,1H),8.29(s,1H),6.85(s,1H),6.61(dd,J＝17.0,10.2Hz,1H),6.25(d,J＝16.9Hz,1H),5.74(d,J＝11.7Hz,1H),5.50–5.38(m,1H),4.25–4.14(m,2H),3.96(dd,J＝9.8,4.7Hz,2H),3.83(s,3H),3.03(s,3H),2.86(t,J＝4.6Hz,4H),2.52(s,4H),2.26(s,3H)。
实施例71-72
化合物J-71、J-72参照实施例70的方法制备。不同的是将实施例70步骤2中的1-甲基哌嗪分别换成2-(二甲基氨基)乙醇和化合物47-a。
Figure PCTCN2015076451-appb-000110
对比化合物2的制备
Figure PCTCN2015076451-appb-000111
以化合物48-a和化合物a1为原料，依次参照化合物4-a、化合物6d和实施例14的方法制备。MS m/z(ESI):518.3[M+H]+。1H NMR(400MHz,CDCl3)δ10.02(s,1H),9.63(s,1H),8.41(d,J＝5.0Hz,1H),8.28(d,J＝2.7Hz,1H),7.65(d,J＝5.6Hz,2H),6.91(d,J＝2.7Hz,1H),6.75(s,1H),6.71(d,J＝5.1Hz,1H),6.45(d,J＝15.6Hz,2H),5.70(d,J＝11.7Hz,1H),4.40–4.35(m,2H),4.26(t,J＝6.7Hz,2H),4.19(s,3H),4.09–4.01(m,1H),3.85(s,3H),2.88(s,2H),2.67(s,3H),2.26(s,6H),1.73(s,2H)。
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T790M/L858R双突变型EGFR激酶(Invitrogen，PV4879)、野生型EGFR激酶(Invitrogen，PV3872)。
10uL T790M/L858R激酶反应体系中各组分的工作浓度为：25μM ATP，0.1(或0.08)ng/μL T790M/L858R激酶，2μM Tyr04底物(Invitrogen，PV3193)。加入本发明上述实施例制备的化合物(即待测物)后DMSO的浓度为2％。
10uL野生型EGFR激酶反应体系中各组分的工作浓度为：10μM ATP，0.8ng/μL野生型EGFR激酶，2μM Tyr04底物(Invitrogen，PV3193)。加入待测物后DMSO的浓度为2％。
室温溶解10mM的药物储存液经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T790M/L858R激酶(或野生型EGFR激酶)与Tyr04底物的混合物，再加入2.5μL的ATP启动反应。其中C1孔用反应缓冲液代替ATP，C2孔不加入任何药物，C3孔按说明书描述加入磷酸化的底物。在25度摇床避光反应60分钟后。加入5μL Development Reagent B(Invitrogen，用TR-FRET稀释缓冲液进行稀释)，于室温摇床反应60分钟。在VictorX5荧光酶标仪 (PerkinElmer)上读板，测定激发波长为405nm、发射波长为450nm和520nm的光吸收(例如，C3520nm表示C3孔在520nm的读值)。
抑制率计算方法(参照Invitrogen，PV3193的说明书)如下：
1、ER＝Coumarin Emission(450nm)/Fluorescein Emission(520nm)
2、磷酸化率＝(1-((ER×C3520nm-C3450nm)/((C1450nm-C3450nm)+ER×(C3520nm-C1520nm))))×100％
3、抑制率(IR)＝(1-(测试化合物的磷酸化率)/(C2的磷酸化率))×100％
用XLFIT 5.0软件(英国IDBS公司)拟合计算半数抑制浓度IC50。对酶的抑制活性和选择抑制活性结果参见表1。
表1酶抑制活性与选择抑制活性
Figure PCTCN2015076451-appb-000112
Figure PCTCN2015076451-appb-000113
由表1可知，本发明的示例化合物在100nM浓度(部分化合物在10nM以下)以下即可抑制EGFR突变型酶(T790M/L858R)的活性，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21倍。而研究发现将取代基R0换成杂芳基环后，对EGFR突变型酶的选择抑制活性与对比化合物1相比明显降低。
Figure PCTCN2015076451-appb-000114
对比化合物1
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。
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3分钟后弃上清，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：H1975细胞每毫升2.78万，A431细胞和NIH3T3细胞每毫升3.33万。将细胞种入96孔板(BD 3072)，每孔90μL，培养过夜。
A431细胞培养基为：10％FBS(Gibco，10099-141)DMEM(Hyclone SH30243.01B)；
NIH3T3细胞培养基为：10％FBS(Gibco，10099-141)DMEM(Hyclone SH30243.01B)；
H1975细胞培养基为：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)，并每孔加入10ul药品到细胞培养板内其中DMSO终浓度为0.5％。
加药后将细胞放入培养箱，培养72小时后，每孔加入10μL的5mg/ml的MTT(Sigma，M5655)溶液，然后将96孔板放入37℃5％CO2培养箱孵育4小时。
再在2000rpm，5min的条件下离心平板，移除上清后，每孔加入150μL DMSO，并在摇床中震荡平板至所有结晶紫溶解。最后使用酶标仪测定492nm光吸收，使用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2和表3。
表2化合物对细胞抑制活性和选择性
Figure PCTCN2015076451-appb-000115
Figure PCTCN2015076451-appb-000116
表3化合物对NIH3T3细胞的毒性测试结果
Figure PCTCN2015076451-appb-000117
由表2可知，本发明的示例化合物在100nM浓度及以下(部分化合物在10nM以下)即可抑制H1975细胞的活性，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和2。选择性最高比对比化合物1提高至近10倍。而研究发现将氮杂环直接与嘧啶环连接和/或取代基R0换成杂芳基环后，对H1975细胞活性和对细胞生长的选择抑制活性明显降低。
由表3可知，与阳性对照物对比化合物1相比，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，1mM NaVO3，2mM EDTA。
细胞裂解液：细胞裂解缓冲液+10ug/mL抑肽酶(Aprotinin)(Sigma)，10ug/mL亮抑蛋白肽酶(Leupeptin)(Sigma)，现配现用。
1xPBS缓冲液：NaCl:0.137M，KCl:0.0027M，Na2PO4-12H2O:0.01M，KH2PO4:0.0015M，pH7.4。
洗涤缓冲液：含有0.05％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4。
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OD EGF、OD药物、OD细胞。
3.3数据分析
抑制率(％)＝100％×(OD EGF-OD药物)/(OD EGF-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，结果见表4。
表4细胞活性ELISA法测定结果
Figure PCTCN2015076451-appb-000118
Figure PCTCN2015076451-appb-000119
从表4可以看出，本发明的示例化合物在100nM浓度及以下(部分化合物在10nM以下)即可抑制H1975细胞的活性，对突变型细胞水平靶点表现出较强的抑制活性，而对EGFR野生型细胞(A431细胞)水平靶点表现出较弱抑制活性。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的选择抑制活性。并且与对比化合物1和2相比，对细胞水平靶点的选择抑制活性最高提高至32倍。而研究发现将氮杂环直接与嘧啶环连接和/或取代基R0换成杂芳基环后，对H1975细胞活性和细胞水平靶点的选择抑制活性提高不大，甚至明显降低。
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％羧甲基纤维素钠水溶液)；给予CD1小鼠尾静脉给药(1mg/kg,5mL/kg，5％DMAC,5％Solutol HS 15和90％盐水)和灌胃给药(5mg/kg,10mL/kg，5％DMAC,5％Solutol HS 15和90％盐水)
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(足背静脉给药：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150uL。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0.25,0.5,1,2,4,8,24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150uL。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5所示：
表5化合物在大鼠和小鼠体内药代动力学参数
Figure PCTCN2015076451-appb-000120
从表5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 (14)

  1. 一种式(I)所示的化合物，或其药学上可接受的盐、或其溶剂化物、或其立体异构体、或其前药：
    Figure PCTCN2015076451-appb-100001
    式中，
    Z1、Z2、Z3各自独立地为CR10或N；R10为氢、羟基、CN、NO2、卤素、-NR11R12、C1-10烷基、C3-8环烷基、C3-8环烷氧基、C2-10烯基、C2-10炔基、C1-10烷氧基、-CHO、-COC1-10烷基、-COC6-10芳基、C6-10芳基、-CONR11R12、-C(O)OC1-10烷基、-OC(O)C1-10烷基、-SO2C1-10烷基、-SO2C6-10芳基或叔丁氧羰基；其中，所述的烷基、环烷基、烯基、炔基、芳基、烷氧基为未取代的或被1-3个选自下组的取代基所取代：卤素、硝基、C6-10芳基、C1-10烷基、C1-10烷氧基、C3-8环烷基、C3-8环烷氧基、C2-10烯基、C2-10炔基、-CONR11R12、-C(O)OC1-10烷基、-CHO、-OC(O)C1-10烷基、-SO2C1-10烷基、-SO2C6-10芳基、-COC6-10芳基；
    Y1、Y2各自独立地为二价C1-3烃基，或Y1或Y2中的亚甲基被-C(RyRx)-、-NRyC(O)-、亚环丙基、-C(O)NRy-、-N(Ry)SO2-、-SO2N(Ry)-、-S-、-S(O)-、-SO2-、-OC(O)-、-C(O)O-、-O-、-N(Ry)-或-C(O)-置换；其中，Ry、Rx各自独立地为氢、卤素、羟基、CN、NO2、C1-10烷基、C1-10烷氧基、C3-8环烷基、C3-8环烷氧基、C2-10烯基、C2-10炔基或C6-10芳基；
    X为NRz、O或S；Rz为氢、C1-10烷基、卤代的C1-10烷基或C3-8环烷基；
    m1、m3各自独立地为0、1或2；m2为0或1；且m1、m2和m3不同时为0；
    A环为3至7元饱和或部分不饱和单环、具有1到3个独立选自氮、氧或硫的杂原子的3至7元饱和或部分不饱和单杂环；
    Ra、Rb各自独立地为氢、羟基、CN、NO2、卤素、-NR11R12、C1-10烷基、C3-8环烷基、C3-8环烷氧基、C2-10烯基、C2-10炔基、C1-10烷氧基、-CHO、-COC1-10烷基、-COC6-10芳基、C6-10芳基、-CONR11R12、-C(O)OC1-10烷基、-OC(O)C1-10烷基、-SO2C1-10烷基、-SO2C6-10芳基、-S(O)C1-10烷基、-S(O)C6-10芳基、叔丁氧羰基、-NHC1-10烷基、-NC(O)C1-10烷基、-NSO2C1-10烷基；其中，所述的烷基、环烷基、烯基、炔基、芳基、烷氧基为未取代的或被1-3个选自下组的取代基所取代：卤素、羟基、NO2、C6-10芳基、C1-10烷基、C1-10烷氧基、C3-8环烷基、C3-8环烷氧基、C2-10烯基、C2-10炔基、-CONR11R12、-C(O)OC1-10烷基、-CHO、-OC(O)C1-10烷基、-SO2C1-10烷基、-SO2C6-10芳基、-COC6-10芳基；
    或者Ra、Rb各自独立地为具有1到4个独立选自氮、氧或硫的杂原子的5至6元单环杂芳基、具有1到5个独立选自氮、氧或硫的杂原子的8至10元双环杂芳基；其中，所述 的5至6元单环杂芳基或8至10元双环杂芳基为未取代的或被1-5个选自下组的取代基所取代：卤素、羟基、CN、NO2、C1-10烷基、卤代的C1-10烷基、C1-10烷氧基、卤代的C1-10烷氧基、C3-8环烷基、C3-8环烷氧基、C2-10烯基、C2-10炔基、-CONR11R12、-C(O)OC1-10烷基、-CHO、-OC(O)C1-10烷基、-SO2C1-10烷基、-SO2C6-10芳基、-COC6-10芳基；
    n1、n2各自独立地为0、1、2、3、4、5或6；
    R1为氢或C1-10烷基；
    R2、R3、R5各自独立地为氢、卤素、C1-10烷基或C1-10烷氧基；
    R4为氢，羟基、-OC1-10烷基、CN、NO2、卤素、C3-8环烷基、C3-8环烷氧基、C2-10烯基、C2-10炔基、-CHO、-COC1-10烷基、-COC6-10芳基、C6-10芳基、-CONR11R12、-C(O)OC1-10烷基、-OC(O)C1-10烷基、-SO2C1-10烷基、-SO2C6-10芳基、叔丁氧羰基、-NHC1-10烷基、-N(C1-10烷基)2、-N(C1-10烷基)(C3-8环烷基)、-NC(O)C1-10烷基、-NSO2C1-10烷基、或含1或2个氮原子以及0到3个氧或硫原子的3至7元饱和或部分不饱和单杂环；其中，所述的烷基、环烷基、烯基、炔基、芳基、3至7元饱和或部分不饱和单杂环为未取代的或被1-3个选自下组的取代基所取代：卤素、NO2、CN、羟基、-CH2NR11R12、-NR11R12、C1-10烷基、C3-8环烷基、C3-8环烷氧基、C2-10烯基、C2-10炔基、C1-10烷氧基、-CHO、-COC1-10烷基、-COC6-10芳基、C6-10芳基、-CONR11R12、-C(O)OC1-10烷基、-OC(O)C1-10烷基、-SO2C1-10烷基、-SO2C6-10芳基、叔丁氧羰基、-NHC1-10烷基、-NC(O)C1-10烷基、-NSO2C1-10烷基、吗啉基、四氢吡咯基、哌嗪基、甲基哌嗪基；
    R6和R7各自独立地为氢或-CH2NR13R14；其中，R13、R14各自独立地为氢或甲基；或R13、R14和相连的氮原子共同形成5-6元含氮饱和杂环；
    其中，R11、R12各自独立地为氢、C1-10烷基、C1-10烷氧基、C3-8环烷基、C3-8环烷氧基、C2-10烯基、C2-10炔基、C6-10芳基；或者R11、R12和相连的氮原子共同形成3-6元含氮饱和杂环。
  2. 如权利要求1所述的化合物、或其药学上可接受的盐、或其溶剂化物、或其前药，其中，R4为氢，或选自下组：
    Figure PCTCN2015076451-appb-100002
  3. 如权利要求1所述的化合物、或其药学上可接受的盐、或其溶剂化物、或其立体异构 体、或其前药，其中，所述式(I)化合物为式(II)所示化合物：
    Figure PCTCN2015076451-appb-100003
    式中，
    n3、n4各自独立地为0、1、2或3，且n3、n4不同时为0；
    W1为N或CR15；W2为N、O、S或CR15；R15为氢、羟基、CN、NO2、卤素、-NR11R12、C1-10烷基、C3-8环烷基、C3-8环烷氧基、C2-10烯基、C2-10炔基、C1-10烷氧基、C6-10芳基；且当W2为O或S时，R0不存在；
    R8、R9、R0的定义同权利要求1中Ra或Rb的定义；
    Z1、Z2、Z3、R1、R2、R3、R4、R5、R6、R7、R11、R12、Y1、Y2、X、m1、m2、m3、n1、n2如权利要求1所定义。
  4. 如权利要求3所述的化合物、或其药学上可接受的盐、或其溶剂化物、或其立体异构体、或其前药，其中，所述式(I)化合物为式(III)所示化合物：
    Figure PCTCN2015076451-appb-100004
    式中，Z1、Z2、Z3、R1、R2、R3、R4、R5、R6、R7、R8、R9、Y2、X、m2、m3、n1、n2、n3、n4、R0如权利要求3所定义。
  5. 如权利要求3所述的化合物、或其药学上可接受的盐、或其溶剂化物、或其立体异构体、或其前药，其中，所述式(I)化合物为式(IV)所示化合物：
    Figure PCTCN2015076451-appb-100005
    式中，Z2、R1、R2、R3、R4、R5、R6、R7、R8、R9、Y2、X、m3、n1、n2、n3、n4、R0如权利要求3所定义。
  6. 如权利要求3所述的化合物、或其药学上可接受的盐、或其溶剂化物、或其立体异构体、或其前药，其中，所述式(I)化合物为式(VI)所示化合物：
    Figure PCTCN2015076451-appb-100006
    式中，Z2、R1、R2、R3、R4、R5、R6、R7、X、m3、n3、n4、R0如权利要求3所定义。
  7. 如权利要求3所述的化合物、或其药学上可接受的盐、或其溶剂化物、或其立体异构体、或其前药，其中，R3、R5各自独立地为氢。
  8. 如权利要求3所述的化合物、或其药学上可接受的盐、或其溶剂化物、或其立体异构体、或其前药，其中，R1为氢。
  9. 如权利要求3所述的化合物、或其药学上可接受的盐、或其溶剂化物、或其立体异构体、或其前药，其中，R2为甲氧基。
  10. 如权利要求3所述的化合物、或其药学上可接受的盐、或其溶剂化物、或其立体异构体、或其前药，其中，R6和R7各自独立地为氢。
  11. 如权利要求1所述的化合物、或其药学上可接受的盐、或其溶剂化物、或其立体异构体、或其前药，其中，所述式(I)化合物选自下组：
    Figure PCTCN2015076451-appb-100007
    Figure PCTCN2015076451-appb-100008
    Figure PCTCN2015076451-appb-100009
  12. 一种药物组合物，所述组合物包括权利要求1至11中任一项所述的化合物、或其药学上可接受的盐、或其溶剂化物、或其立体异构体、或其前药；以及药学可接受的载体。
  13. 权利要求1至11中任一项所述的化合物、或其药学上可接受的盐、或其溶剂化物、或其立体异构体、或其前药在制备调控(上调或下调)EGFR酪氨酸激酶活性或者治疗EGFR相关疾病的药物中的应用。
  14. 一种药用组合物，所述药用组合物包括权利要求1-11中任一项所述的化合物、或其药学上可接受的盐、或其立体异构体、或其溶剂化物、或其前药，以及选自下组药物中的一种或多种：吉非替尼、厄洛替尼、埃克替尼、拉帕替尼、XL647、NVP-AEE-788、ARRY-334543、EKB-569、BIBW2992、HKI272、BMS-690514、CI-1033、凡德他尼、PF00299804、WZ4002、西妥昔单抗、曲妥珠单抗、帕尼突单抗、马妥珠单抗、尼妥珠单抗、扎鲁木单抗、帕妥珠单抗、MDX-214、CDX-110、IMC-11F8、Zemab、Her2疫苗PX 1041、HSP90抑制剂、CNF2024、坦螺旋霉素、阿螺旋霉素、IPI-504、SNX-5422、NVP-AUY922、或其组合。
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EP3144292B1 (en) 2020-08-26
CN104974140B (zh) 2017-11-24
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EP3144292A1 (en) 2017-03-22
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