WO2017008761A1 - 苯胺嘧啶衍生物及其用途 - Google Patents

苯胺嘧啶衍生物及其用途 Download PDF

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Publication number
WO2017008761A1
WO2017008761A1 PCT/CN2016/090149 CN2016090149W WO2017008761A1 WO 2017008761 A1 WO2017008761 A1 WO 2017008761A1 CN 2016090149 W CN2016090149 W CN 2016090149W WO 2017008761 A1 WO2017008761 A1 WO 2017008761A1
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WIPO (PCT)
Prior art keywords
methyl
group
amino
compound
cancer
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PCT/CN2016/090149
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English (en)
French (fr)
Inventor
朱岩
赵娜
商现星
胡远东
彭勇
张慧
刘波
罗鸿
韩永信
杨玲
徐宏江
Original Assignee
正大天晴药业集团股份有限公司
连云港润众制药有限公司
北京赛林泰医药技术有限公司
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Application filed by 正大天晴药业集团股份有限公司, 连云港润众制药有限公司, 北京赛林泰医药技术有限公司 filed Critical 正大天晴药业集团股份有限公司
Priority to CA2992317A priority Critical patent/CA2992317A1/en
Priority to CN201680041201.3A priority patent/CN107835811B/zh
Priority to AU2016292450A priority patent/AU2016292450B2/en
Priority to JP2018502096A priority patent/JP6970081B2/ja
Priority to RU2018102963A priority patent/RU2734849C2/ru
Priority to US15/745,329 priority patent/US10329277B2/en
Priority to KR1020187004698A priority patent/KR20180030190A/ko
Priority to EP16823908.5A priority patent/EP3323817B1/en
Publication of WO2017008761A1 publication Critical patent/WO2017008761A1/zh
Priority to HK18109611.0A priority patent/HK1250164A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—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
    • C07D403/04—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 directly linked by a ring-member-to-ring-member bond
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
    • C07D413/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings directly linked by a ring-member-to-ring-member bond

Definitions

  • the present application relates to an anilide derivative, a pharmaceutically acceptable salt thereof, a pharmaceutical composition thereof, and a method or use thereof for treating an EGFR-mediated disease.
  • EGFR Epithelial growth factor Receptor
  • EGF epithelial growth factor
  • EGFR belongs to the ErbB receptor family, which includes EGFR (ErbB-1), HER2/c-neu (ErbB-2), HER3 (ErbB-3) and HER4 (ErbB-4).
  • EGFR is a glycoprotein, a tyrosine kinase receptor, with a cell membrane that penetrates and has a molecular weight of 170 kDa.
  • EGFR is located on the surface of the cell membrane and is activated by binding to a ligand, including EGF and TGF ⁇ . Upon activation, EGFR is converted from a monomer to a dimer.
  • the dimer includes both the binding of two homologous receptor molecules (homologous dimerization) and also the binding of different members of the human EGF-associated receptor (HER) tyrosine kinase family (heterologous Sexual dimerization).
  • HER human EGF-associated receptor
  • EGFR cleavage activates its kinase pathways within cells, including phosphorylation of key tyrosine residues in the intracellular domain, and results in stimulation of many intracellular signaling pathways involved in cell proliferation and survival.
  • High expression or abnormal expression of EGFR is present in many solid tumors.
  • EGFR is involved in tumor cell proliferation, angiogenesis, tumor invasion, metastasis, and inhibition of apoptosis. Its possible mechanisms are: high expression of EGFR leads to enhanced downstream signaling; increased expression of mutant EGFR receptor or ligand leads to sustained activation of EGFR; enhanced role of autocrine loop; disruption of receptor downregulation; abnormal signaling Activation of the pathway, etc.
  • Overexpression of EGFR plays an important role in the progression of malignant tumors. For example, overexpression of EGFR has been found in tissues such as glial cells, kidney cancer, lung cancer, prostate cancer, pancreatic cancer, and breast cancer.
  • EGFR Abnormal expression of EGFR and Erb-B2 plays a key role in tumor transformation and growth.
  • EGFR is expressed in 50% of cases of non-small cell lung cancer (NSCLC), and its expression is associated with poor outcome.
  • NSCLC non-small cell lung cancer
  • EGFR and its family members Two small molecule inhibitors targeting EGFR, gefitinib and erlotinib, have been rapidly approved by the US FDA for the treatment of patients with advanced NSCLC who have lost response to conventional chemotherapy.
  • the T790M mutation is a point mutation in exon 20 of EGFR that produces acquired resistance to gefitinib or erlotinib treatment. Recent studies have shown that L858R combined with T790M mutation has a stronger affinity for ATP than L858R alone, while TKI is an ATP competitive kinase inhibitor, resulting in a decrease in the binding rate of TKI to the kinase domain.
  • X is selected from NR 6 or O;
  • R 1 and R 2 are independently selected from the group consisting of hydrogen, halogen, C 1-4 alkyl and cyano;
  • R 3 is selected from the group consisting of C 1-4 alkyl and C 1-4 alkoxy
  • R 4 is selected from the group consisting of [2-(dimethylamino)ethyl](methyl)amino, (2-hydroxyethyl)(methyl)amino and morpholin-4-yl;
  • R 5 is selected from the group consisting of hydrogen, C 1-4 alkyl and C 1-3 alkoxy C 1-3 alkyl;
  • R 6 is selected from the group consisting of hydrogen and C 1-4 alkyl.
  • X is selected from NR 6 or O;
  • R 1 and R 2 are independently selected from the group consisting of hydrogen, halogen, and C 1-4 alkyl;
  • R 3 is selected from C 1-4 alkoxy
  • R 4 is selected from the group consisting of [2-(dimethylamino)ethyl](methyl)amino, (2-hydroxyethyl)(methyl)amino and morpholin-4-yl;
  • R 5 is selected from the group consisting of hydrogen, C 1-4 alkyl and C 1-3 alkoxy C 1-3 alkyl;
  • R 6 is selected from the group consisting of hydrogen and C 1-4 alkyl.
  • X is selected from NR 6 or O;
  • R 1 and R 2 are independently selected from the group consisting of hydrogen, chlorine, bromine, fluorine, and methyl;
  • R 3 is a methoxy group
  • R 4 is selected from the group consisting of [2-(dimethylamino)ethyl](methyl)amino, (2-hydroxyethyl)(methyl)amino and morpholin-4-yl;
  • R 5 is selected from the group consisting of hydrogen and methoxymethyl
  • R 6 is selected from the group consisting of hydrogen and methyl.
  • the compound of formula (I) described herein includes the following compound or a pharmaceutically acceptable salt thereof:
  • compositions of the present application provide a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of formula (I), or a pharmaceutically acceptable salt thereof, disclosed herein and one or more pharmaceutically acceptable carriers or excipients.
  • the pharmaceutical compositions of the present application may further comprise one or more additional therapeutic agents.
  • Another aspect of the present application provides a method of treating an EGFR-mediated disease, the method comprising administering to a subject in need thereof a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutical combination thereof, as described herein. Things.
  • Another aspect of the present application provides the use of a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, as described herein, for the manufacture of a medicament for the treatment of an EGFR mediated disease.
  • the EGFR mediated disease is selected from the group consisting of EGFR-L858R activating mutation mediated diseases.
  • the EGFR mediated disease is selected from the group consisting of EGFR-T790M activating mutation mediated diseases. In some embodiments, the EGFR-mediated disease is selected from the group consisting of EGFR-L858R and EGFR-T790M double mutation activation mediated disease.
  • C mn means having mn carbon atoms in this moiety.
  • C 1-4 alkyl means that the alkyl group has from 1 to 4 carbon atoms.
  • C 1-4 means that the group may have 1 carbon atom, 2 carbon atoms, 3 carbon atoms or 4 carbon atoms.
  • halo or halogen means fluoro, chloro, bromo or iodo.
  • cyano refers to a -CN group.
  • alkyl refers to a straight or branched saturated aliphatic hydrocarbon group consisting of a carbon atom and a hydrogen atom, which is attached to the remainder of the molecule by a single bond.
  • alkyl groups include, but are not limited to, methyl, ethyl, propyl, 2-propyl, n-butyl, isobutyl or t-butyl, and the like.
  • alkoxy refers to a "-O-alkyl” group.
  • pharmaceutically acceptable is for those compounds, materials, compositions and/or dosage forms that are within the scope of sound medical judgment and are suitable for use in contact with human and animal tissues without Many toxic, irritating, allergic reactions or other problems or complications are commensurate with a reasonable benefit/risk ratio.
  • the pharmaceutically acceptable salt is, for example, a salt formed with an inorganic acid, a salt formed with an organic acid, or a salt formed with an acidic amino acid.
  • the pharmaceutically acceptable salts of the present application can be synthesized from the parent compound containing an acid group or a base by conventional chemical methods. In general, such salts are prepared by reacting these compounds in the form of the free base with a stoichiometric amount of the appropriate acid in water or an organic solvent or a mixture of the two.
  • Certain compounds of the present application may exist in unsolvated as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to the unsolvated forms and are included within the scope of the present application. Certain compounds of the present application may exist in polycrystalline or amorphous form.
  • Certain compounds of the present application may have asymmetric carbon atoms (optical centers) or double bonds. Racemates, diastereomers, geometric isomers, and individual isomers are included within the scope of this application.
  • the compounds of the present application may exist in specific geometric or stereoisomeric forms.
  • This application contemplates all such compounds, including the cis and trans isomers, the (-)- and (+)-p-enantiomers, the (R)- and (S)-enantiomers, diastereomeric a conformation, a (D)-isomer, a (L)-isomer, and a racemic mixture thereof, and other mixtures, such as enantiomerically or diastereomeric enriched mixtures, all of which belong to Within the scope of this application. Additional asymmetric carbon atoms may be present in the substituents such as alkyl groups. All such isomers, as well as mixtures thereof, are included within the scope of this application.
  • optically active (R)- and (S)-isomers as well as the D and L isomers can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one enantiomer of a compound of the present application is desired, it can be prepared by asymmetric synthesis or by derivatization with a chiral auxiliary wherein the resulting mixture of diastereomers is separated and the auxiliary group cleaved to provide purity. The desired enantiomer.
  • a salt of a diastereomer is formed with a suitable optically active acid or base, followed by stepping as is known in the art.
  • the diastereomeric resolution is carried out by crystallization or chromatography, and then the pure enantiomer is recovered.
  • the separation of enantiomers and diastereomers is generally accomplished by the use of chromatography using a chiral stationary phase, optionally in combination with chemical derivatization (eg, formation of an amino group from an amine). Formate).
  • the compounds of the present application may contain unnatural proportions of atomic isotopes on one or more of the atoms that make up the compound.
  • deuterium (D) tritium (3 H), iodine -125 (125 I) or C-14 (14 C) and the like. All isotopic compositional changes of the compounds of the present application, whether radioactive or not, are included within the scope of the present application.
  • pharmaceutically acceptable carrier refers to those carriers which have no significant irritation to the organism and which do not impair the biological activity and properties of the active compound.
  • “Pharmaceutically acceptable carrier” means an inert substance which, together with the active ingredient, which facilitates administration of the active ingredient, including, but not limited to, acceptable for human or animal use as permitted by the State Food and Drug Administration (eg Any of the glidants, sweeteners, diluents, preservatives, dyes/colorants, flavor enhancers, surfactants, wetting agents, dispersing agents, disintegrating agents, suspending agents, stabilizers, Isotonicity agent, solvent or emulsifier.
  • Non-limiting examples of such carriers include calcium carbonate, calcium phosphate, various sugars and various types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols, and the like.
  • excipient generally refers to the carrier, diluent and/or vehicle required to formulate an effective pharmaceutical composition.
  • an "effective amount” or “therapeutically effective amount” with respect to a pharmaceutical or pharmacologically active agent refers to a sufficient amount of a drug or agent that is non-toxic but that achieves the desired effect.
  • an "effective amount” of an active substance in a composition refers to the amount required to achieve the desired effect when used in combination with another active substance in the composition.
  • the determination of the effective amount will vary from person to person, depending on the age and general condition of the recipient, and also on the particular active substance, and a suitable effective amount in a case can be determined by one skilled in the art based on routine experimentation.
  • active ingredient refers to a chemical entity that is effective in treating a target disorder, disease or condition.
  • patient or “individual” includes humans and animals, for example, mammals (eg, primates, cows, horses, pigs, dogs, cats, mice, rats, rabbits, goats, sheep, birds, etc.).
  • mammals eg, primates, cows, horses, pigs, dogs, cats, mice, rats, rabbits, goats, sheep, birds, etc.
  • X is selected from NR 6 or O;
  • R 1 and R 2 are independently selected from the group consisting of hydrogen, halogen, C 1-4 alkyl and cyano;
  • R 3 is selected from the group consisting of C 1-4 alkyl and C 1-4 alkoxy
  • R 4 is selected from the group consisting of [2-(dimethylamino)ethyl](methyl)amino, (2-hydroxyethyl)(methyl)amino and morpholin-4-yl;
  • R 5 is selected from the group consisting of hydrogen, C 1-4 alkyl and C 1-3 alkoxy C 1-3 alkyl;
  • R 6 is selected from the group consisting of hydrogen and C 1-4 alkyl.
  • X is selected from NR 6 or O;
  • R 1 and R 2 are independently selected from the group consisting of hydrogen, halogen, and C 1-4 alkyl;
  • R 3 is selected from C 1-4 alkoxy
  • R 4 is selected from the group consisting of [2-(dimethylamino)ethyl](methyl)amino, (2-hydroxyethyl)(methyl)amino and morpholin-4-yl;
  • R 5 is selected from the group consisting of hydrogen, C 1-4 alkyl and C 1-3 alkoxy C 1-3 alkyl;
  • R 6 is selected from the group consisting of hydrogen and C 1-4 alkyl.
  • X is selected from NR 6 or O;
  • R 1 and R 2 are independently selected from the group consisting of hydrogen, chlorine, bromine, fluorine, and methyl;
  • R 3 is a methoxy group
  • R 4 is selected from the group consisting of [2-(dimethylamino)ethyl](methyl)amino, (2-hydroxyethyl)(methyl)amino and morpholin-4-yl;
  • R 5 is selected from the group consisting of hydrogen and methoxymethyl
  • R 6 is selected from the group consisting of hydrogen and methyl.
  • the compound of Formula (I) described herein includes the following compound or a pharmaceutically acceptable salt thereof:
  • the pharmaceutically acceptable salts of the compounds of Formula (I) described herein include the hydrochloride salts of the following compounds:
  • compositions of the present application provide a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of formula (I), or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers or excipients.
  • the pharmaceutical compositions of the present application may further comprise one or more additional therapeutic agents.
  • Another aspect of the present application provides a method of treating an EGFR-mediated disease, the method comprising administering to a subject in need thereof a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutical combination thereof, as described herein. Things.
  • Another aspect of the present application provides the use of a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, as described herein, for the manufacture of a medicament for the treatment of an EGFR mediated disease.
  • the EGFR mediated disease is selected from the group consisting of EGFR-L858R activating mutation mediated diseases.
  • the EGFR mediated disease is selected from the group consisting of EGFR-T790M activating mutation mediated diseases.
  • the EGFR-mediated disease is selected from the group consisting of EGFR-L858R and EGFR-T790M double mutation activation mediated diseases.
  • the EGFR-mediated disease is cancer
  • the cancer is selected from the group consisting of ovarian cancer, cervical cancer, colorectal cancer, breast cancer, pancreatic cancer, glioma, glioblastoma, Melanoma, prostate cancer, leukemia, lymphoma, non-Hodgkin's lymphoma, gastric cancer, lung cancer, hepatocellular carcinoma, gastric cancer, gastrointestinal stromal tumor, thyroid cancer, cholangiocarcinoma, endometrial cancer, kidney cancer, between Denaturing large cell lymphoma, acute myeloid leukemia, multiple myeloma, melanoma, mesothelioma;
  • the lung cancer may be selected from the group consisting of non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma.
  • the pharmaceutical composition of the present application can be prepared by combining the compound of the present application or a salt thereof with a suitable pharmaceutically acceptable carrier, for example, it can be formulated into a solid, semi-solid, liquid or gaseous preparation such as a tablet, a pill, or a capsule.
  • a suitable pharmaceutically acceptable carrier for example, it can be formulated into a solid, semi-solid, liquid or gaseous preparation such as a tablet, a pill, or a capsule.
  • Typical routes of administration of a compound of the present application, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof include, but are not limited to, oral, rectal, transmucosal, enteral, or topical, transdermal, inhalation, parenteral, sublingual, Intravaginal, intranasal, intraocular, intraperitoneal, intramuscular, subcutaneous or intravenous administration.
  • the pharmaceutical composition of the present application can be produced by a method well known in the art, such as a conventional mixing method, a dissolution method, a granulation method, a sugar coating pill method, a grinding method, an emulsification method, and a freeze drying method.
  • the pharmaceutical compositions may be formulated by admixing the active compound withpharmaceutically acceptable carriers such carriers.
  • pharmaceutically acceptable carriers such carriers.
  • These carriers enable the compounds of the present application to be formulated into tablets, pills, troches, dragees, capsules, liquids, gels, slurries or suspensions for oral administration to a patient.
  • Solid oral compositions can be prepared by conventional methods of mixing, filling or tabletting. For example, it can be obtained by mixing the active compound with a solid excipient, optionally milling the resulting mixture, adding other suitable adjuvants if necessary, and then processing the mixture into granules. The core of a tablet or dragee.
  • Suitable excipients include, but are not limited to, binders, diluents, disintegrants, lubricants, glidants, sweeteners, and/or flavoring agents, and the like.
  • microcrystalline cellulose glucose solution, gum arabic, gelatin solution, sucrose and / or starch paste; talc, starch, magnesium stearate, calcium stearate and / or stearic acid; lactose, sucrose, starch, nectar Sugar alcohol, sorbitol and/or dicalcium phosphate; silica; Sodium carboxymethyl cellulose, pre-treated starch, sodium starch glycolate, alginic acid, corn starch, potato starch, methyl cellulose, agar, carboxymethyl cellulose and/or cross-linked polyvinylpyrrolidone.
  • the core of the dragee may optionally be coated according to methods well known in the ordinary pharmaceutical practice, especially using enteric coatings.
  • compositions may also be suitable for parenteral administration, such as sterile solutions, suspensions or lyophilized products in a suitable unit dosage form.
  • a compound of Formula (I) described herein, or a pharmaceutically acceptable salt thereof can be administered by any suitable route and method, such as by oral or parenteral (eg, intravenous) administration.
  • a therapeutically effective amount of a compound of formula (I) is from about 0.0001 to 20 mg/kg body weight per day, such as from 0.001 to 10 mg/kg body weight per day.
  • the dosage frequency of the compound of formula (I) is determined by the needs of the individual patient, for example, once or twice daily, or more times per day. Administration can be intermittent, for example, wherein the patient receives a daily dose of the compound of formula (I) over a period of several days, followed by a patient's daily dose of the compound of formula (I) over a period of several days.
  • the compounds of the present application can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, combinations thereof with other chemical synthesis methods, and those well known to those skilled in the art. Equivalent alternatives, specific embodiments include, but are not limited to, embodiments of the present application.
  • a portion of the compound of formula (I) of the present application can be prepared by one skilled in the art of organic synthesis by standard methods in the art using Route 1:
  • a compound of the formula (V) is obtained, wherein a compound of the formula (V) is bonded to R 6 to give a compound of the formula (VI), wherein a pyrimidine ring chlorine atom of the compound of the formula (VI) is reacted with an amino group of a benzene ring of the compound of the formula (VII) to give a formula (VIII)
  • a compound of the formula (VIII) is attached to the side chain R 4 to give a compound of the formula (IX) wherein the nitro group of the compound of the formula (IX) is reduced to an amino group and then an amide bond with the compound of the formula (XI) to give the final product of the formula (I-a).
  • those skilled in the art can perform the preparation according to the steps of the route 1 or the route 2, and according to the structure of the final product, the route 1 or the route 2 can be added, reduced or changed according to the structure of the final product.
  • the order of the steps is also within the scope of this application.
  • DMF stands for N,N-dimethylformamide
  • NMP stands for N-methylpyrrolidone
  • DCM stands for dichloromethane
  • PE stands for petroleum ether
  • EA stands for ethyl acetate
  • MeOH stands for methanol
  • Pd 2 (dba) 3 represents tris(dibenzylideneacetone)dipalladium
  • TsOH represents p-toluenesulfonic acid
  • BINAP stands for ( ⁇ )-2,2'-bis-(diphenylphosphino)-1,1'- Binaphtane.
  • N 1 -(2-Chloropyrimidin-4-yl)phenyl-1,2-diamine (2.21 g, 10 mmol) was dissolved in DMF (15 mL), EtOAc (2. After 1 hour, pour into water (50 mL) and continue stirring for 10 minutes. Filtration, washing with water (30 mL*3),
  • Step 4 1-(2-(4-Fluoro-2-methoxy-5-nitrophenylamino)pyrimidin-4-yl)-3-methyl-1H-benzo[d]imidazole-2 ( 3H)-keto-p-toluenesulfonate
  • Step 5 1-(2-(4-(Dimethylamino)ethyl)(methyl)amino)-2-methoxy-5-nitrophenylaminopyrimidin-4-yl )-3-methyl-1H-benzo[d]imidazole-2(3H)-one
  • Step 6 1-(2-(5-Amino-4-((2-(dimethylamino)ethyl)(methyl)amino)-2-methoxyphenylamino)pyrimidin-4-yl) -3-methyl-1H-benzo[d]imidazole-2(3H)-one
  • Step 7 N-(2-((2-(Dimethylamino)ethyl)(methyl)amino)-4-methoxy-5-(4-(3-methyl-2-oxo-) 2,3-dihydrogen -1H-benzo[d]imidazol-1-yl)pyrimidin-2-ylamino)phenyl)acrylamide hydrochloride
  • Step 1 1-(2-(4-Fluoro-2-methoxy-5-nitrophenylamino)pyrimidin-4-yl)-1H-benzo[d]imidazole-2(3H)-one Tosylate
  • Step 2 1-(2-(4-(Dimethylamino)ethyl)(methyl)amino)-2-methoxy-5-nitrophenylaminopyrimidin-4-yl )-1H-benzo[d]imidazole-2(3H)-one
  • Step 3 N-(2-((2-(Dimethylamino)ethyl)(methyl)amino)-4-methoxy-5-(4-(2-oxo-2,3-di) Hydrogen-1H-benzo[d]imidazol-1-yl)pyrimidin-2-ylamino)phenyl)acrylamide hydrochloride
  • N 1 -(2,5-Dichloropyrimidin-4-yl)benzene-1,2-diamine 100 mg, 0.39 mmol was dissolved in ethyl acetate (5 mL) and diisopropylethylamine ( 151 mg, 1.17 mmol), cooled in an ice water bath.
  • Triphosgene 71 mg, 0.24 mmol was added portionwise, and the mixture was warmed to room temperature and stirring was continued for one hour.
  • Saturated sodium bicarbonate solution (10 mL) was added and stirring was continued for 10 min and ethyl acetate (20 mL*2). The organic phase was washed with EtOAc EtOAc m.
  • Step 4 1-(5-Chloro-2-(4-fluoro-2-methoxy-5-nitrophenylamino)pyrimidin-4-yl)-3-methyl-1H-benzo[d] Imidazole-2(3H)-one
  • Step 5 1-(5-Chloro-2-(4-((2-dimethylamino)ethyl)(methyl)amino)-2-methoxy-5-nitrophenylamino)pyrimidine 4-yl)-3-methyl-1H-benzo[d]imidazole-2(3H)-one
  • Step 6 1-(2-(5-Amino-4-((2-(dimethylamino)ethyl)(methyl)amino)-2-methoxyphenylamino)-5-chloropyrimidine- 4- 3-methyl-1H-benzo[d]imidazole-2(3H)-one
  • Step 7 N-(5-(5-Chloro-4-(3-methyl-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-yl)pyrimidin-2- Benzyl)-2-((2-(dimethylamino)ethyl)(methyl)amino)-4-methoxyphenyl)acrylamide hydrochloride
  • Step 1 1-(5-Chloro-2-(4-fluoro-2-methoxy-5-nitrophenylamino)pyrimidin-4-yl)-1H-benzo[d]imidazole-2 (3H )-ketone
  • Step 2 1-(5-Chloro-2-(4-((2-dimethylamino)ethyl)(methyl)amino)-2-methoxy-5-nitrophenylamino)pyrimidine -4-yl)-1H-benzo[d]imidazole-2(3H)-one
  • Step 3 N-(5-(5-Chloro-4-(2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-yl)pyrimidin-2-ylamino)-2 -((2-(Dimethylamino)ethyl)(methyl)amino)-4-methoxyphenyl)acrylamide hydrochloride
  • Step 4 3-(2-(4-Fluoro-2-methoxy-5-nitrophenylamino)pyrimidin-4-yl)benzo[d]oxazole-2(3H)-one p-toluene Acid salt
  • Step 5 3-(2-(4-(2-Methylamino)ethyl)(methyl)amino)-2-methoxy-5-nitrophenylaminopyrimidin-4-yl Benzo[d]oxazole-2(3H)-one
  • Step 6 N-(2-((2-(Dimethylamino)ethyl)(methyl)amino)-4-methoxy-5-(4-(2-oxobenzo[d]) Azole-3(2H)-yl)pyrimidin-2-ylamino)phenyl)acrylamide hydrochloride
  • Step 1 1-(2-(4-(Ethylethyl))(methyl)amino)-2-methoxy-5-nitrophenylamino)pyrimidin-4-yl)-3-A Base -1H-benzo[d]imidazole-2(3H)-one
  • Step 2 N-(2-((2-hydroxyethyl)(methyl)amino)-4-methoxy-5-(4-(3-methyl-2-oxo-2,3-di) Hydrogen-1H-benzo[d]imidazol-1-yl)pyrimidin-2-ylamino)phenyl)acrylamide
  • Step 2 N-(4-Methoxy-5-(4-(3-methyl-2-oxo-2,3-dihydrobenzo[d]imidazol-1-yl)pyrimidin-2-yl Amino)-2-morpholinylphenyl)acrylamide
  • the title compound was synthesized from N 1 -(2-chloropyrimidin-4-yl)-4-methylbenzene-1,2-diamine and carbonyldiimidazole in a manner similar to that described in the step 2 of Example 1.
  • Step 3 1-(2-(4-Fluoro-2-methoxy-5-nitrophenylamino)pyrimidin-4-yl)-5-methyl-1H-benzo[d]imidazole-2 ( 3H)-keto-p-toluenesulfonate
  • Step 4 1-(2-(4-(2-Methylamino)ethyl)(methyl)amino)-2-methoxy-5-nitrophenylaminopyrimidin-4-yl )-5-Methyl-1H-benzo[d]imidazole-2(3H)-one
  • Step 5 N-(2-((2-(Dimethylamino)ethyl)(methyl)amino)-4-methoxy-5-(4-(5-methyl-2-oxo-) 2,3-Dihydrobenzo[d]imidazol-1-yl)pyrimidin-2-ylamino)phenyl)acrylamide hydrochloride
  • Step 4 5-Fluoro-1-(2-(4-fluoro-2-methoxy-5-nitrophenylamino)pyrimidin-4-yl)-3-methyl-1H-benzo[d] Imidazole-2(3H)-one p-toluenesulfonate
  • Step 5 1-(2-(4-(Dimethylamino)ethyl)(methyl)amino)-2-methoxy-5-nitrophenylaminopyrimidin-4-yl )-5-fluoro-3-methyl-1H-benzo[d]imidazole-2(3H)-one
  • Step 6 N-(2-((2-(Dimethylamino)ethyl)(methyl)amino)-5-(4-(5-fluoro-3-methyl-2-oxo-2, 3-Dihydrobenzo[d]imidazol-1-ylpyrimidin-2-ylamino)-4-methoxyphenyl)acrylamide hydrochloride
  • Step 2 1-(2-(4-(Dimethylamino)ethyl)(methyl)amino)-2-methoxy-5-nitrophenylaminopyrimidin-4-yl -5-fluoro-1H-benzo[d]imidazole-2(3H)-one
  • Step 3 N-(2-((2-(Dimethylamino)ethyl)(methyl)amino)-5-(4-(5-fluoro-2-oxo-2,3-dihydrobenzene) And [d]imidazol-1-yl)pyrimidin-2-ylamino)-4-methoxyphenyl)acrylamide hydrochloride
  • Step 1 tert-Butyl 2-((5-methoxy-4-(4-(3-methyl-2-oxo-2,3-dihydrobenzo[d]imidazol-1-yl)pyrimidine) -2-ylamino)-2nitrophenyl)(methyl)amino)ethyl(methyl)carbamate
  • Step 2 tert-Butyl 2-((2-acrylamide-5-methoxy-4-(4-(3-methyl-2-oxo-2,3-dihydrobenzo[d]imidazole-) 1-yl)pyrimidin-2-ylamino)phenyl)(methyl)amino)ethyl(methyl)carbamate
  • Step 3 N-(4-Methoxy-2-(methyl(2-(methylamino)ethyl)amino)-5-(4-(3-methyl-2-oxo-2,3) -Dihydrobenzo[d]imidazol-1-yl)pyrimidin-2-ylamino)phenyl)acrylamide hydrochloride
  • Acetyl chloride (0.3 mL, 1.7 mmol) was slowly added dropwise to an additional ice water-cooled anhydrous methanol (3 mL) and stirring was continued for one hour.
  • tert-Butyl 2-((2-acrylamide-5-methoxy-4-(4-(3-methyl-2-oxo-2,3-dihydrobenzo[d]imidazole-1-) (1) pyrimidin-2-ylamino)phenyl)(methyl)amino)ethyl(methyl)carbamate (100 mg, 0.166 mmol) was dissolved in anhydrous methanol (2 mL) and then added to methanol In solution. The system was warmed to rt. EtOAc (EtOAc)EtOAc.
  • Step 1 4-Chloro-N 1 -(2-chloropyrimidin-4-yl)benzene-1,2-diamine
  • Step 5 5-Chloro-1-(2-(4-((2-(dimethylamino)ethyl)(methyl)amino)-2-methoxy-5-nitrophenylamino)pyrimidine 4-yl)-3-methyl-1H-benzo[d]imidazole-2(3H)-one
  • Step 6 1-(2-(5-Amino-4-((2-(dimethylamino)ethyl)(methyl)amino)-2-methoxyphenylamino)pyrimidin-4-yl) -5-chloro-3-methyl-1H-benzo[d]imidazole-2(3H)-one
  • Step 7 N-(5-(4-(5-Chloro-3-methyl-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-yl)pyrimidin-2- Benzyl)-2-((2-(dimethylamino)ethyl)(methyl)amino)-4-methoxyphenyl)acrylamide hydrochloride
  • Step 1 4-Bromo-N 1 -(2-chloropyrimidin-4-yl)benzene-1,2-diamine
  • Step 5 5-Bromo-1-(2-(4-((2-(dimethylamino)ethyl)(methyl)amino)-2-methoxy-5-nitrophenylamino)pyrimidine 4-yl)-3-methyl-1H-benzo[d]imidazole-2(3H)-one
  • Step 6 1-(2-(5-Amino-4-((2-(dimethylamino)ethyl)(methyl)amino)-2-methoxyphenylamino)pyrimidin-4-yl) -5-bromo-3-methyl-1H-benzo[d]imidazole-2(3H)-one
  • Step 7 N-(5-(4-(5-Bromo-3-methyl-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-yl)pyrimidin-2- Benzyl)-2-((2-(dimethylamino)ethyl)(methyl)amino)-4-methoxyphenyl)acrylamide hydrochloride
  • EGFR, EGFR (T790M, L858R) kinase is obtained by expression purification of an insect expression system or obtained by purchasing a commercially available product.
  • the ELISA, EGFR (T790M, L858R) kinase activity detection platform was established by Cisbio's homogeneous phase-resolved fluorescence (HTRF) method to determine the activity of the compound.
  • Compounds were diluted 10-fold with 100% DMSO starting at 1 ⁇ M, and 4 ⁇ l of each concentration was added to 96 ⁇ l of reaction buffer (50 mM HEPES (pH 7.0), 0.02% NaN 3 , 0.01% BSA, 0.1 mM Orthovanadate, 5 mM MgCl 2 , 50 nM SEB, 1 mM DTT), 2.5 ⁇ l was added to a 384-well plate (OptiPlate-384, PerkinElmer), then 2.5 ⁇ l of kinase was added, mixed by centrifugation, and 5 ⁇ l of ATP and TK Substrate-biotin were added to initiate the reaction.
  • reaction buffer 50 mM HEPES (pH 7.0
  • Human non-small cell lung cancer NCI-H1975 was cultured in a cell culture incubator (37 ° C, 5% CO 2 ) using RPMI-1640 medium (10% fetal bovine serum and 1% streptomycin). The cells were seeded in 96-well plates at 2000 cells per volume (volume: 195 ul) for overnight. The compound was added the next day, the compound was subjected to 3-fold serial dilution from 10 mM, 4 ⁇ l of each concentration was added to 96 ⁇ l of the medium, and then 5 ⁇ l was added to the cell culture medium (final concentration of DMSO 0.1%, v/v). After 72 hours of treatment, the medium was aspirated and 30 ⁇ l was added. (Promega) reagent, florescent signals are read on Envison (Perkin Elmer), using the IC50 values calculated IC GraphPad Prism 5.0 compounds on cell proliferation.
  • the human squamous cell carcinoma cell line A431 was cultured in a cell culture incubator (37 ° C, 5% CO 2 ) with DMEM plus 10% fetal bovine serum and 1% streptomycin.
  • the concentration of the underlying substrate was 0.6%, and after the cells were reselected with 0.3% of low melting point agar, 2000 cells per well (100 ⁇ l) were plated in a 96-well plate.
  • the compound was subjected to 3-fold serial dilution from 10 mM, and 2 ⁇ l of each concentration was added to 98 ⁇ l of the medium, and then 5.3 ⁇ l was added to the cell culture medium (final concentration of DMSO 0.1%, v/v) for one week (7 days).
  • NT not tested; AZD9291 was prepared according to Example 28 of WO2013014448.
  • the compound of the present application has a good inhibitory effect on EGFR, especially the EGFR-L858R/T790M double mutant, and the WT/DM data shows the compound of the present application. Has good selectivity.
  • the results of the experiments of the compounds of the present application in human non-small cell lung cancer NCI-H1975 and human squamous cell carcinoma cell line A431 showed good inhibitory effects.
  • test compound was administered to healthy adult male rats by single-dose intragastric administration with an adjuvant of 20% sulfobutylether- ⁇ -cyclodextrin at a dose of 10 mg/kg.
  • the animals administered by intragastric administration were fasted overnight before the experiment.
  • the fasting time was from 10 hours before the administration to 4 hours after the administration, and 0.25, 0.5, 1, 2, 4, 6, 8 and 24 after the intragastric administration. Blood is collected in hours.

Abstract

本公开涉及作为EGFR抑制剂的苯胺嘧啶衍生物或其药学上可接受的盐，具体涉及式(I)所示化合物或其药学上可接受的盐、其药物组合物、及其用于治疗EGFR介导的疾病的方法和用途。

Description

苯胺嘧啶衍生物及其用途
相关申请的交叉引用
本申请要求于2015年07月16日向中国国家知识产权局提交的第201510419018.X号中国专利申请的优先权和权益，所述申请公开的内容通过引用整体并入本文中。
技术领域
本申请涉及作为EGFR抑制剂的苯胺嘧啶衍生物、其药学上可接受的盐、其药物组合物、及其用于治疗EGFR介导的疾病的方法或用途。
背景技术
EGFR(Epidermal Growth Factor Receptor)是上皮生长因子(EGF)细胞增殖和信号传导的受体，也被称作HER1、ErbB1。EGFR属于ErbB受体家族的一种，该家族包括EGFR(ErbB-1)，HER2/c-neu(ErbB-2)，HER3(ErbB-3)和HER4(ErbB-4)。EGFR是一种糖蛋白，属于酪氨酸激酶型受体，细胞膜贯通，分子量170KDa。
EGFR位于细胞膜表面，靠与配体结合来激活，包括EGF和TGFα，激活后，EGFR由单体转化为二聚体。所述二聚体既包括两个同种受体分子的结合(同源性二聚作用)，也包括人类EGF相关性受体(HER)酪氨酸激酶家族中的不同成员的结合(异源性二聚作用)。EGFR二聚后可以激活它位于细胞内的激酶通路，包括胞内域中关键酪氨酸残基的磷酸化，并且导致对参与细胞增殖和生存的许多细胞内信号传导通路的刺激。
在许多实体肿瘤中存在EGFR的高表达或异常表达。EGFR与肿瘤细胞的增殖、血管生成、肿瘤侵袭、转移及细胞凋亡的抑制有关。其可能机制有：EGFR的高表达引起下游信号传导的增强；突变型EGFR受体或配体表达的增加导致EGFR的持续活化；自分泌环的作用增强；受体下调机制的破坏；异常信号传导通路的激活等。EGFR的过表达在恶性肿瘤的演进中起重要作用，例如，在胶质细胞、肾癌、肺癌、***癌、胰腺癌、乳腺癌等组织中都已发现有EGFR的过表达。
EGFR和Erb-B2的异常表达在肿瘤的转化与增长中起着关键性的作用。以肺癌为例，EGFR在50％的非小细胞肺癌(NSCLC)病例中有表达，而且其表达与愈后不佳相关。这两个因素使得EGFR及其家族成员成为开展靶向治疗的主要候选者。两种靶向EGFR的小分子抑制剂，吉非替尼和厄洛替尼，得到了美国FDA的快速批准用于治疗晚期NSCLC患者，所述患者对常规化疗失去了反应。
早期的临床数据表明，10％的NSCLC患者对吉非替尼和厄洛替尼有反应。分子生物学分 析表明，在多数情况下，对药物有反应的患者在编码EGFR基因上带有特定的突变：第19外显子的第747～750位氨基酸的缺失占突变的45％，还有10％的突变发生在第18和第20外显子。最常见的EGFR激活突变(L858R和delE746_A750)导致相对于野生型(WT)EGFR而言，对小分子酪氨酸激酶抑制剂(TKI)的亲和力增加、以及对三磷酸腺苷(ATP)亲和力下降。T790M突变是EGFR第20外显子中的一个点突变，其产生对吉非替尼或厄洛替尼治疗的获得性抗性。最新研究显示，L858R合并T790M突变对ATP的亲和力比单纯L858R强，而TKI是ATP竞争性激酶抑制剂，故导致TKI与激酶区结合率降低。
发明内容
本申请的第一个方面，提供了式(Ⅰ)所示化合物或者其药学上可接受的盐：
Figure PCTCN2016090149-appb-000001
其中：
X选自NR6或O；
R1、R2独立地选自氢、卤素、C1-4烷基和氰基；
R3选自C1-4烷基和C1-4烷氧基；
R4选自[2-(二甲氨基)乙基](甲基)氨基、(2-羟基乙基)(甲基)氨基和吗啉-4-基；
R5选自氢、C1-4烷基和C1-3烷氧基C1-3烷基；
R6选自氢和C1-4烷基。
在本申请的一个实施方式中，提供了式(I)的化合物或其药学上可接受的盐，其中：
X选自NR6或O；
R1、R2独立地选自氢、卤素和C1-4烷基；
R3选自C1-4烷氧基；
R4选自[2-(二甲氨基)乙基](甲基)氨基、(2-羟基乙基)(甲基)氨基和吗啉-4-基；
R5选自氢、C1-4烷基和C1-3烷氧基C1-3烷基；
R6选自氢和C1-4烷基。
在本申请的一个实施方式中，提供了式(I)的化合物或其药学上可接受的盐，其中：
X选自NR6或O；
R1、R2独立地选自氢、氯、溴、氟和甲基；
R3为甲氧基；
R4选自[2-(二甲氨基)乙基](甲基)氨基、(2-羟基乙基)(甲基)氨基和吗啉-4-基；
R5选自氢和甲氧基甲基；
R6选自氢和甲基。
在本申请的一个实施方式中，本申请所述式(Ⅰ)化合物包括如下化合物或其药学上可接受的盐：
Figure PCTCN2016090149-appb-000002
Figure PCTCN2016090149-appb-000003
本申请的另一方面提供了一种药物组合物，其包含本文公开的式(Ⅰ)化合物或其药学上可接受的盐和一种或多种药学上可接受的载体或赋形剂。任选地，本申请的药物组合物可以进一步含有一种或多种额外的治疗剂。
本申请的另一方面提供了一种治疗EGFR介导的疾病的方法，所述方法包括给予有需要的个体本申请所述的式(Ⅰ)化合物或者其药学上可接受的盐或其药物组合物。
本申请的另一个方面提供了本申请所述的式(Ⅰ)化合物或者其药学上可接受的盐或其药物组合物在制备治疗EGFR介导的疾病的药物中的用途。
在本申请的一些实施方式中，所述EGFR介导的疾病选自EGFR-L858R激活突变介导的疾病。
在本申请的一些实施方式中，所述EGFR介导的疾病选自EGFR-T790M激活突变介导的疾病。一些实施方式中，所述EGFR介导的疾病选自EGFR-L858R合并EGFR-T790M双突变激活介导的疾病。
实施方式的详细描述
定义：
除非另有说明，本文所用的下列术语和短语具有下列含义。一个特定的术语或短语在没有特别定义的情况下不应该被认为是不确定的或不清楚的，而应该按照普通的含义去理解。当本文中出现商品名时，意在指代其对应的商品或其活性成分。
本文所用的Cm-n指该部分中具有m-n个碳原子。例如，“C1-4烷基”指该烷基具有1-4个碳原子。
本文中的数字范围，是指给定范围中的各个整数。例如“C1-4”是指该基团可具有1个碳原子、2个碳原子、3个碳原子或4个碳原子。
术语“卤”或“卤素”是指氟、氯、溴或碘。
术语“氰基”指-CN基团。
术语“烷基”是指由碳原子和氢原子组成的直链或支链的饱和的脂肪烃基团，其通过单键与分子的其余部分连接。烷基的非限制性实例包括但不限于甲基、乙基、丙基、2-丙基、正丁基、异丁基或叔丁基等。
术语“烷氧基”是指“-O-烷基”基团。
术语“药学上可接受的”，是针对那些化合物、材料、组合物和/或剂型而言，它们在可靠的医学判断的范围之内，适用于与人类和动物的组织接触使用，而没有过多的毒性、刺激性、过敏性反应或其它问题或并发症，与合理的利益/风险比相称。
作为药学上可接受的盐，例如，与无机酸形成的盐、与有机酸形成的盐、与酸性氨基酸形成的盐等。
本申请的药学上可接受的盐可由含有酸根或碱基的母体化合物通过常规化学方法合成。一般情况下，这样的盐的制备方法是：在水或有机溶剂或两者的混合物中，经由游离碱形式的这些化合物与化学计量的适当酸反应来制备。
本申请的某些化合物可以以非溶剂化形式或者溶剂化形式存在，包括水合物形式。一般而言，溶剂化形式与非溶剂化的形式相当，都包含在本申请的范围之内。本申请的某些化合物可以以多晶或无定形形式存在。
本申请的某些化合物可以具有不对称碳原子(光学中心)或双键。外消旋体、非对映异构体、几何异构体和单个的异构体都包括在本申请的范围之内。
本文中消旋体、ambiscalemic、scalemic或者对映体纯的化合物的图示法来自Maehr,J.Chem.Ed.1985,62:114-120。除非另有说明，用楔形键和虚线键表示一个立体中心的绝对构型。当本文所述化合物含有烯属双键或其它几何不对称中心，除非另有规定，它们包括E、Z几何异构体。同样地，所有的互变异构形式均包括在本申请的范围之内。
本申请的化合物可以存在特定的几何或立体异构体形式。本申请设想所有的这类化合物，包括顺式和反式异构体、(-)-和(+)-对对映体、(R)-和(S)-对映体、非对映异构体、(D)-异构体、(L)-异构体，及其外消旋混合物和其他混合物，例如对映异构体或非对映体富集的混合物，所有这些混合物都属于本申请的范围之内。烷基等取代基中可存在另外的不对称碳原子。所有这些异构体以及它们的混合物，均包括在本申请的范围之内。
可以通过的手性合成或手性试剂或者其他常规技术制备光学活性的(R)-和(S)-异构体以及D和L异构体。如果想得到本申请某化合物的一种对映体，可以通过不对称合成或者具有手性助剂的衍生作用来制备，其中将所得非对映体混合物分离，并且辅助基团裂开以提供纯的 所需对映异构体。或者，当分子中含有碱性官能团(如氨基)或酸性官能团(如羧基)时，与适当的光学活性的酸或碱形成非对映异构体的盐，然后通过本领域所公知的分步结晶法或色谱法进行非对映异构体拆分，然后回收得到纯的对映体。此外，对映异构体和非对映异构体的分离通常是通过使用色谱法完成的，所述色谱法采用手性固定相，并任选地与化学衍生法相结合(例如由胺生成氨基甲酸盐)。
本申请的化合物可以在一个或多个构成该化合物的原子上包含非天然比例的原子同位素。例如，氘(D)，氚(3H)，碘-125(125I)或C-14(14C)等。本申请的化合物的所有同位素组成的变换，无论放射性与否，都包括在本申请的范围之内。
术语“药学上可接受的载体”是指对有机体无明显刺激作用，而且不会损害该活性化合物的生物活性及性能的那些载体。“药学上可接受的载体”是指与活性成份一同给药的、有利于活性成份给药的惰性物质，包括但不限于国家食品药品监督管理局许可的可接受的用于人或动物(例如家畜)的任何助流剂、增甜剂、稀释剂、防腐剂、染料/着色剂、矫味增强剂、表面活性剂、润湿剂、分散剂、崩解剂、助悬剂、稳定剂、等渗剂、溶剂或乳化剂。所述载体的非限制性实例包括碳酸钙、磷酸钙、各种糖和各类淀粉、纤维素衍生物、明胶、植物油和聚乙二醇等。关于载体的其他信息，可以参考Remington:The Science and Practice of Pharmacy,21st Ed.,Lippincott,Williams&Wilkins(2005)，该文献的内容通过引用的方式并入本文。
术语“赋形剂”通常是指配制有效的药物组合物所需要载体、稀释剂和/或介质。
针对药物或药理学活性剂而言，术语“有效量”或“治疗有效量”是指无毒的但能达到预期效果的药物或药剂的足够用量。对于本申请中的口服剂型，组合物中一种活性物质的“有效量”是指与该组合物中另一种活性物质联用时为了达到预期效果所需要的用量。有效量的确定因人而异，取决于受体的年龄和一般情况，也取决于具体的活性物质，个案中合适的有效量可以由本领域技术人员根据常规试验确定。
术语“活性成分”、“治疗剂”，“活性物质”或“活性剂”是指一种化学实体，它可以有效地治疗目标紊乱、疾病或病症。
术语“患者”或“个体”包括人和动物，例如，哺乳动物(如灵长类动物，牛，马，猪，狗，猫，小鼠，大鼠，兔，山羊，绵羊以及禽类等)。
具体实施方式
本申请的第一个方面，提供了式(Ⅰ)所示化合物或者其药学上可接受的盐：
Figure PCTCN2016090149-appb-000004
其中：
X选自NR6或O；
R1、R2独立地选自氢、卤素、C1-4烷基和氰基；
R3选自C1-4烷基和C1-4烷氧基；
R4选自[2-(二甲氨基)乙基](甲基)氨基、(2-羟基乙基)(甲基)氨基和吗啉-4-基；
R5选自氢、C1-4烷基和C1-3烷氧基C1-3烷基；
R6选自氢和C1-4烷基。
在本申请的一个实施方式中，提供了式(I)的化合物或其药学上可接受的盐，其中：
X选自NR6或O；
R1、R2独立地选自氢、卤素和C1-4烷基；
R3选自C1-4烷氧基；
R4选自[2-(二甲氨基)乙基](甲基)氨基、(2-羟基乙基)(甲基)氨基和吗啉-4-基；
R5选自氢、C1-4烷基和C1-3烷氧基C1-3烷基；
R6选自氢和C1-4烷基。
在本申请的一个实施方式中，提供了式(I)的化合物或其药学上可接受的盐，其中：
X选自NR6或O；
R1、R2独立地选自氢、氯、溴、氟和甲基；
R3为甲氧基；
R4选自[2-(二甲氨基)乙基](甲基)氨基、(2-羟基乙基)(甲基)氨基和吗啉-4-基；
R5选自氢和甲氧基甲基；
R6选自氢和甲基。
在本申请的一些实施方式中，本申请所述式(Ⅰ)化合物包括如下化合物或其药学上可接受的盐：
Figure PCTCN2016090149-appb-000005
在本申请的一些实施方式中，本申请所述式(Ⅰ)化合物的药学上可接受的盐包括如下化合物的盐酸盐：
Figure PCTCN2016090149-appb-000006
本申请的另一方面提供了一种药物组合物，其包含式(Ⅰ)化合物或其药学上可接受的盐和一种或多种药学上可接受的载体或赋形剂。本申请的药物组合物可以进一步含有一种或多种额外的治疗剂。
本申请的另一方面提供了一种治疗EGFR介导的疾病的方法，所述方法包括给予有需要的个体本申请所述的式(Ⅰ)化合物或者其药学上可接受的盐或其药物组合物。
本申请的另一个方面提供了本申请所述的式(Ⅰ)化合物或者其药学上可接受的盐或其药物组合物在制备治疗EGFR介导的疾病的药物中的用途。
在本申请的一些实施方式中，所述EGFR介导的疾病选自EGFR-L858R激活突变介导的疾病。
在本申请的一些实施方式中，所述EGFR介导的疾病选自EGFR-T790M激活突变介导的疾病。
在本申请的一些实施方式中，所述EGFR介导的疾病选自EGFR-L858R合并EGFR-T790M双突变激活介导的疾病。
在本申请的一些实施方式中，所述EGFR介导的疾病是癌症；所述癌症选自卵巢癌、***、结肠直肠癌、乳腺癌、胰腺癌、胶质瘤、胶质母细胞瘤、黑色素瘤、***癌、白血病、淋巴瘤、非霍奇金淋巴瘤、胃癌、肺癌、肝细胞癌、胃癌、胃肠道间质瘤、甲状腺癌、胆管癌、子宫内膜癌、肾癌、间变性大细胞淋巴瘤、急性髓细胞白血病、多发性骨髓瘤、黑色素瘤、间皮瘤；所述肺癌可以选自非小细胞肺癌、小细胞肺癌、肺腺癌、肺鳞癌。
本申请的药物组合物可通过将本申请的化合物或其盐与适宜的药学上可接受的载体组合而制备，例如可配制成固态、半固态、液态或气态制剂，如片剂、丸剂、胶囊剂、粉剂、颗粒剂、膏剂、乳剂、悬浮剂、溶液剂、栓剂、注射剂、吸入剂、凝胶剂或微球及气溶胶等。
给予本申请的化合物或其药物可接受的盐或其药物组合物的典型途径包括但不限于口服、直肠、透黏膜、经肠给药，或者局部、经皮、吸入、肠胃外、舌下、***内、鼻内、眼内、腹膜内、肌内、皮下或静脉内给药。
本申请的药物组合物可以采用本领域众所周知的方法制造，如常规的混合法、溶解法、制粒法、制糖衣药丸法、磨细法、乳化法和冷冻干燥法等。
对于口服给药，可以通过将活性化合物与本领域熟知的药学上可接受的载体混合来配制该药物组合物。这些载体能使本申请的化合物被配制成片剂、丸剂、锭剂、糖衣剂、胶囊剂、液体、凝胶剂、浆剂或悬浮剂等，用于对患者的口服给药。
可以通过常规的混合、填充或压片方法来制备固体口服组合物。例如，可通过下述方法获得：将所述的活性化合物与固体赋形剂混合，任选地碾磨所得的混合物，如果需要则加入其它合适的辅剂，然后将该混合物加工成颗粒，得到了片剂或糖衣剂的核心。适合的辅料包括但不限于：粘合剂、稀释剂、崩解剂、润滑剂、助流剂、甜味剂和/或矫味剂等。如微晶纤维素、葡萄糖溶液、***胶浆、明胶溶液、蔗糖和/或淀粉糊；滑石、淀粉、硬脂酸镁、硬脂酸钙和/或硬脂酸；乳糖、蔗糖、淀粉、甘露糖醇、山梨糖醇和/或磷酸二钙；二氧化硅；交 联羧甲基纤维素钠、预交化淀粉、淀粉羟乙酸钠、藻酸、玉米淀粉、马铃薯淀粉、甲基纤维素、琼脂、羧甲基纤维素和/或交联聚乙烯吡咯烷酮等。可以根据通常药物实践中公知的方法任选地对糖衣剂的核心进行包衣，尤其使用肠溶包衣。
药物组合物还可适用于肠胃外给药，如合适的单位剂型的无菌溶液剂、混悬剂或冻干产品。
在一些实施方式中，本文所述的式(Ⅰ)化合物或其药学上可接受的盐可以通过任何适用的途径和方法给药，例如通过口服或肠胃外(例如，静脉内)给药。式(Ⅰ)化合物的治疗有效量为从约0.0001到20mg/Kg体重/天，例如从0.001到10mg/Kg体重/天。
在一些实施方式中，式(Ⅰ)化合物的剂量频率由患者个体的需求决定，例如，每天1次或2次，或每天更多次。给药可以是间歇性的，例如，其中在若干天的期间内，患者接受式(Ⅰ)化合物的每日剂量，接着在若干天的期间，患者不接受式(Ⅰ)化合物的每日剂量。
本申请的化合物可以通过本领域技术人员所熟知的多种合成方法来制备，包括下面列举的具体实施方式、其与其他化学合成方法的结合所形成的实施方式以及本领域技术上人员所熟知的等同替换方式，具体的实施方式包括但不限于本申请的实施例。
本申请具体实施方式的化学反应是在合适的溶剂中完成的，所述的溶剂须适合于本申请的化学变化及其所需的试剂和物料。为了获得本申请的化合物，有时需要本领域技术人员在已有实施方式的基础上对合成步骤或者反应流程进行修改或选择。
在一个具体的实施方式中，本申请的部分式(Ⅰ)所示化合物可以由有机合成领域技术人员通过路线1用本领域的标准方法来制备：
Figure PCTCN2016090149-appb-000007
从式(Ⅱ)化合物和式(Ⅲ)化合物出发，先发生式(Ⅱ)化合物的苯环氨基和式(Ⅲ)化合物的嘧啶氯原子的取代反应，然后式(Ⅳ)化合物构建羰基并形成环结构得式(Ⅴ)化合物，式(Ⅴ)化合物与R6连接得式(Ⅵ)化合物，式(Ⅵ)化合物的嘧啶环氯原子与式(Ⅶ)化合物苯环上氨基反应得式(Ⅷ)化合物，式(Ⅷ)化合物与侧链R4连接得式(Ⅸ)化合物，式(Ⅸ)化合物的硝基还原得氨基然后与式(Ⅺ)化合物成酰胺键得终产物式(Ⅰ-a)化合物。
本申请的部分式(Ⅰ)所示化合物也可以由有机合成领域技术人员通过路线2用本领域的标准方法来制备：
Figure PCTCN2016090149-appb-000008
从式(Ⅻ)化合物和式(Ⅲ)化合物出发，先发生式(Ⅻ)化合物的苯环氨基和式(Ⅲ)化合物的嘧啶氯原子的取代反应，然后式(ⅩⅢ)化合物脱除甲基得式(ⅩⅣ)化合物，式(ⅩⅣ)化合物构建羰基并形成环结构得式(ⅩⅤ)化合物，式(ⅩⅤ)化合物的嘧啶环氯原子与式(Ⅶ)化合物苯环上氨基发生取代反应得式(ⅩⅥ)化合物，式(ⅩⅥ)化合物与侧链R4连接得式(ⅩⅦ)化合物，式(ⅩⅦ)化合物的硝基还原得氨基然后与式(Ⅺ)化合物成酰胺键得终产物式(Ⅰ-b)化合物。
在本申请的部分实施方式中，本领域的技术人员可以依据而非严格按照路线1或路线2的步骤进行制备，根据终产物的结构，可以对路线1或路线2进行增加、减少或者改变各个步骤的顺序，这也属于本申请的范围。
为清楚起见，本申请进一步用实施例来阐述。但是实施例不局限于定义或者指定发明范围。
本申请所使用的所有溶剂是市售的，无需进一步纯化即可使用。涉及对水和/或氧气敏感的实验的所有操作都在预干燥玻璃仪器中于氮气氛下进行。除非另有说明，所有原料均为商业原料，并且在使用前未作进一步纯化。本申请所用柱层析采用的是青岛海洋化工所生产的 硅胶(200-300目)。薄层色谱采用默克公司(E.Merck)预涂色谱板(硅胶60PF254，0.25毫米)。核磁共振光谱分析使用的仪器为瓦里安VNMRS-400共振光谱仪，化学位移以四甲基硅烷(TMS＝δ0.00)为内标，核磁共振氢谱数据的记录格式为：质子数，峰型(s，单峰；d，双重峰；t，三重峰；q，四重峰；m，多重峰)，耦合常数(以赫兹Hz为单位)。
本申请采用下述缩略词：DMF代表N,N-二甲基甲酰胺；NMP代表N-甲基吡咯烷酮；DCM代表二氯甲烷；PE代表石油醚；EA代表乙酸乙酯；MeOH代表甲醇；Pd2(dba)3代表三(二亚苄基丙酮)二钯；TsOH代表对甲苯磺酸；BINAP代表(±)-2,2'-双-(二苯膦基)-1,1'-联萘。
化合物经手工或者
Figure PCTCN2016090149-appb-000009
软件命名，市售化合物采用供应商目录名称。
实施例
下面的具体实施例，其目的是使本领域的技术人员能更清楚地理解和实施本申请。它们不应该被认为是对本申请范围的限制，而只是本申请的示例性说明和典型代表。
实施例1  N-(2-((2-(二甲基氨基)乙基)(甲基)氨基)-4-甲氧基-5-(4-(3-甲基-2-氧代-2,3-二氢-1H-苯并[d]咪唑-1-基)嘧啶-2-基氨基)苯基)丙烯酰胺盐酸盐。
Figure PCTCN2016090149-appb-000010
步骤1：N1-(2-氯嘧啶-4-基)苯-1,2-二胺
Figure PCTCN2016090149-appb-000011
将邻苯二胺(3.24g,30mmol)和2,4-二氯嘧啶(4.47g,30mmol)分散于无水乙醇(60mL)中，加入二异丙基乙基胺(7.74g,60mmol)，加热回流3小时。真空浓缩去除溶剂，残余物溶于二氯甲烷(100mL)中，水洗，饱和食盐水洗，真空浓缩去除溶剂。残余物经柱层析(EA:PE＝1:2)分离得标题所示化合物(5.32g,80％)。
1H NMR(CDCl3):δ8.08(1H,d,J＝5.6Hz),7.20-7.12(2H,m),6.85-6.78(2H,m),6.74(1H,s),6.24(1H,d,J＝5.6Hz),3.82(2H,br)。
步骤2：1-(2-氯嘧啶-4-基)-1H-苯并[d]咪唑-2(3H)-酮
Figure PCTCN2016090149-appb-000012
将N1-(2-氯嘧啶-4-基)苯基-1,2-二胺(2.21g,10mmol)溶于DMF(15mL)中，加入羰基二咪唑(2.43g,15mmol)，室温搅拌1小时，倾入水(50mL)中，继续搅拌10分钟。抽滤，水洗(30mL*3)，干燥得标题所示化合物(2.23g,90％)。
1H NMR(DMSO-d6):δ11.64(1H,br),8.78(1H,d,J＝5.6Hz),8.43(1H,d,J＝5.6Hz),8.26(1H,d,J＝7.6Hz),7.22-7.10(3H,m)。
步骤3：1-(2-氯嘧啶-4-基)-3-甲基-1H-苯并[d]咪唑-2(3H)-酮
Figure PCTCN2016090149-appb-000013
将1-(2-氯嘧啶-4-基)-1H-苯并[d]咪唑-2(3H)-酮(600mg,2.43mmol)分散于无水DMF(10mL)中，冰水浴冷却。加入氢化钠(116mg,60％,2.90mmol)，搅拌1小时，滴加碘甲烷(345mg,2.43mmol)，继续搅拌1小时。将反应液倾入水(50mL)中，搅拌30分钟，抽滤，水洗(30mL*3)，干燥得标题所示化合物(459mg,72％)。
1H NMR(DMSO-d6):δ8.79(1H,d,J＝5.6Hz),8.44(1H,d,J＝6.0Hz),8.29(1H,d,J＝8.0Hz),7.30-7.28(2H,m),7.24-7.19(1H,m),3.39(3H,s)。
步骤4：1-(2-(4-氟-2-甲氧基-5-硝基苯基氨基)嘧啶-4-基)-3-甲基-1H-苯并[d]咪唑-2(3H)-酮对甲苯磺酸盐
Figure PCTCN2016090149-appb-000014
1-(2-氯嘧啶-4-基)-3-甲基-1H-苯并[d]咪唑-2(3H)-酮(459mg,1.76mmol)，4-氟-2-甲氧基-5-硝基苯胺(360mg,1.93mmol)和对甲苯磺酸一水合物(551mg,2.89mmol)分散于2-戊醇(10mL)中，反应在105℃下搅拌过夜。冷却后抽滤，滤饼用少量2-戊醇洗三次，干燥 得标题所示化合物(440mg,43％)。
1H NMR(CDCl3):δ10.95(1H,br),8.49(1H,d,J＝7.6Hz),8.39(1H,d,J＝7.2Hz),8.21(1H,d,J＝7.2Hz),7.87(2H,d,J＝8.4Hz),7.68(1H,d,J＝8.4Hz),7.28-7.23(2H,m),7.04(2H,d,J＝7.6Hz),6.91-6.85(2H,m),3.92(3H,s),3.46(3H,s),2.38(3H,s)。
步骤5：1-(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)-3-甲基-1H-苯并[d]咪唑-2(3H)-酮
Figure PCTCN2016090149-appb-000015
将1-(2-(4-氟-2-甲氧基-5-硝基苯基氨基)嘧啶-4-基)-3-甲基-1H-苯并[d]咪唑-2(3H)-酮对甲苯磺酸盐(440mg,0.76mmol)溶于NMP(5mL)中，加入二异丙基乙基胺(206mg,1.59mmol)和N1,N1,N2-三甲基乙烷-1,2-二胺(116mg,1.14mmol)，反应在85℃下搅拌过夜。反应液冷却后倾入水(50mL)中，抽滤，少量甲醇冲洗，干燥得标题所示化合物(326mg,88％)。
1H NMR(CDCl3):δ8.92(1H,s),8.51(1H,d,J＝5.6Hz),8.27(1H,d,J＝7.6Hz),7.82(1H,d,J＝5.6Hz),7.47(1H,s),7.29-7.19(1H,m),7.17-7.13(1H,m),7.04(1H,d,J＝7.6Hz),6.69(1H,s),3.98(3H,s),3.47(3H,s),3.27(2H,t,J＝7.2Hz),2.89(3H,s),2.88(2H,t,J＝7.2Hz),2.26(6H,s)。
步骤6：1-(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)-3-甲基-1H-苯并[d]咪唑-2(3H)-酮
Figure PCTCN2016090149-appb-000016
将1-(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)-3-甲基-1H-苯并[d]咪唑-2(3H)-酮(326mg,0.66mmol)溶于甲醇(10mL)中，加入Pd/C(10％,30mg)，氢气置换三次，体系在氢气氛下搅拌过夜，抽滤，产品易于氧化，所得滤液快速真空浓缩，直接投入下步反应。
步骤7：N-(2-((2-(二甲基氨基)乙基)(甲基)氨基)-4-甲氧基-5-(4-(3-甲基-2-氧代-2,3-二氢 -1H-苯并[d]咪唑-1-基)嘧啶-2-基氨基)苯基)丙烯酰胺盐酸盐
Figure PCTCN2016090149-appb-000017
将上步反应所得1-(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)-3-甲基-1H-苯并[d]咪唑-2(3H)-酮溶于无水二氯甲烷(10mL)中，加入二异丙基乙基胺(129mg,1.00mmol)，冰水浴冷却。将溶有丙烯酰氯(60mg,0.66mmol)的无水二氯甲烷(2mL)溶液缓慢滴加入体系中，用时15分钟。继续搅拌15分钟后，将反应液倾入石油醚(50mL)中，搅拌10分钟。抽滤，石油醚冲洗滤饼。所得粗品经柱层析(DCM:MeOH＝20:1)分离得标题所示化合物(164mg,两步合并收率45％)。
1H NMR(DMSO-d6):δ10.15(1H,br),9.72(1H,br),8.70(1H,s),8.41(1H,d,J＝5.6Hz),8.16-8.12(2H,m),7.67(1H,d,J＝5.6Hz),7.22-712(2H,m),6.99-6.92(3H,m),6.19(1H,dd,J＝2.0Hz,17.2Hz),5.68(1H,dd,J＝2.0Hz,10.4Hz),3.77(3H,s),3.34(3H,s),3.28(4H,br),2.72(6H,s),2.60(3H,s)。
实施例2  N-(2-((2-(二甲基氨基)乙基)(甲基)氨基)-4-甲氧基-5-(4-(2-氧代-2,3-二氢苯并[d]咪唑-1-基)嘧啶-2-基氨基)苯基)丙烯酰胺盐酸盐
Figure PCTCN2016090149-appb-000018
步骤1：1-(2-(4-氟-2-甲氧基-5-硝基苯基氨基)嘧啶-4-基)-1H-苯并[d]咪唑-2(3H)-酮对甲苯磺酸盐
Figure PCTCN2016090149-appb-000019
以类似于实施例1步骤4中所描述的方法由1-(2-氯嘧啶-4-基)-1H-苯并[d]咪唑-2(3H)-酮 和4-氟-2-甲氧基-5-硝基苯胺合成标题所示化合物。
1H NMR(DMSO-d6):δ11.47(1H,s),9.12(1H,s),8.62(1H,d,J＝8.4Hz),8.50(1H,d,J＝5.6Hz),8.16-8.14(1H,m),7.82(1H,d,J＝5.6Hz),7.47-7.40(3H,m),7.15-7.09(3H,m),7.04(1H,d,J＝7.6Hz),6.95-6.91(1H,m),3.96(3H,s),2.27(3H,s)。
步骤2：1-(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)-1H-苯并[d]咪唑-2(3H)-酮
Figure PCTCN2016090149-appb-000020
以类似于实施例1步骤5中所描述的方法由1-(2-(4-氟-2-甲氧基-5-硝基苯基氨基)嘧啶-4-基)-1H-苯并[d]咪唑-2(3H)-酮对甲苯磺酸盐合成标题所示化合物。
1H NMR(DMSO-d6):δ11.39(1H,s),8.72(1H,s),8.42(1H,d,J＝5.6Hz),8.12-8.03(2H,m),7.68(1H,d,J＝5.6Hz),7.11-7.07(1H,m),7.02-7.00(1H,m),6.88-6.84(1H,m),6.81(1H,s),3.87(3H,s),3.28(2H,t,J＝6.8Hz),2.85(3H,s),2.46(2H,t,J＝6.8Hz),2.15(6H,s)。
步骤3：N-(2-((2-(二甲基氨基)乙基)(甲基)氨基)-4-甲氧基-5-(4-(2-氧代-2,3-二氢-1H-苯并[d]咪唑-1-基)嘧啶-2-基氨基)苯基)丙烯酰胺盐酸盐
Figure PCTCN2016090149-appb-000021
以类似于实施例1步骤6，7中所描述的方法由1-(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)-1H-苯并[d]咪唑-2(3H)-酮合成标题所示化合物。
1H NMR(MeOD):δ8.46(1H,d,J＝5.6Hz),8.28(1H,s),8.14(1H,d,J＝8.4Hz),7.70(1H,d,J＝5.6Hz),7.12-7.06(2H,m),7.03-7.99(1H,m),6.97(1H,s),6.51-6.38(2H,m),5.83-5.80(1H,m),3.97(3H,s),3.45(2H,br),3.21(2H,br),2.81(6H,s),2.71(3H,s)。
实施例3  N-(5-(5-氯-4-(3-甲基-2-氧代-2,3-二氢-1H-苯并[d]咪唑-1-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)丙烯酰胺盐酸盐
Figure PCTCN2016090149-appb-000022
步骤1：N1-(2,5-二氯嘧啶-4-基)苯-1,2-二胺
Figure PCTCN2016090149-appb-000023
以类似于实施例1步骤1中所描述的方法由2,4,5-三氯嘧啶和邻苯二胺合成标题所示化合物。
1H NMR(CDCl3):δ8.18(1H,s),7.46(1H,dd,J＝1.6Hz,7.2Hz),7.15-7.11(2H,m),6.91-6.86(2H,m),3.67(2H,br)。
步骤2：1-(2,5-二氯嘧啶-4-基)-1H-苯并[d]咪唑-2(3H)-酮
Figure PCTCN2016090149-appb-000024
将N1-(2,5-二氯嘧啶-4-基)苯-1,2-二胺(100mg,0.39mmol)溶于乙酸乙酯(5mL)中，加入二异丙基乙基胺(151mg,1.17mmol)，冰水浴冷却。分批加入三光气(71mg,0.24mmol)，自然升至室温，继续搅拌1小时。加入饱和碳酸氢钠溶液(10mL)，继续搅拌10分钟，乙酸乙酯萃取(20mL*2)，合并有机相。有机相用饱和食盐水洗，真空浓缩去除溶剂得标题所示化合物(105mg,95％)。
1H NMR(DMSO-d6):δ11.44(1H,br),9.16(1H,s),7.20-7.04(4H,m)。
步骤3：1-(2,5-二氯嘧啶-4-基)-3-甲基-1H-苯并[d]咪唑-2(3H)-酮
Figure PCTCN2016090149-appb-000025
以类似于实施例1步骤3中所描述的方法由1-(2,5-二氯嘧啶-4-基)-1H-苯并[d]咪唑-2(3H)- 酮合成标题所示化合物。
1H NMR(CDCl3):δ8.80(1H,s),7.27-7.23(2H,m),7.18-7.16(1H,m),7.06(1H,d,J＝8.0Hz),3.48(3H,s)。
步骤4：1-(5-氯-2-(4-氟-2-甲氧基-5-硝基苯基氨基)嘧啶-4-基)-3-甲基-1H-苯并[d]咪唑-2(3H)-酮
Figure PCTCN2016090149-appb-000026
将1-(2,5-二氯嘧啶-4-基)-3-甲基-1H-苯并[d]咪唑-2(3H)-酮(50mg,0.17mmol)、4-氟-2-甲氧基-5-硝基苯胺(63mg,0.34mmol)、BINAP(11mg,0.017mmol)和碳酸铯(110mg,0.34mmol)分散于无水甲苯(5mL)中，氮气鼓泡20分钟后加入Pd2(dba)3(8mg,0.008mmol)。体系在微波反应器中(100W,100℃)反应1小时，真空浓缩去除溶剂。所得残余物经柱层析(DCM to DCM:EA＝20:1)分离得到标题所示化合物(54mg,72％)。
1H NMR(CDCl3):9.31(1H,s),8.02(1H,s),8.55(1H,d,J＝8.2Hz),7.33(1H,d,J＝13.6Hz),7.25(1H,d,J＝7.2Hz),7.21-7.16(2H,m),6.78(1H,d,J＝12.0Hz),3.97(3H,s),3.43(3H,s)。
步骤5：1-(5-氯-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)-3-甲基-1H-苯并[d]咪唑-2(3H)-酮
Figure PCTCN2016090149-appb-000027
以类似于实施例1步骤5中所描述的方法由1-(5-氯-2-(4-氟-2-甲氧基-5-硝基苯基氨基)嘧啶-4-基)-3-甲基-1H-苯并[d]咪唑-2(3H)-酮合成标题所示化合物。
1H NMR(CDCl3):δ8.88(1H,s),8.59(1H,s),7.69(1H,s),7.25-7.19(2H,m),7.16-7.14(1H,m),7.05(1H,d,J＝7.2Hz),6.65(1H,s),3.95(3H,s),3.49(3H,s)3.25(2H,t,J＝7.2Hz),2.56(3H,s),2.54(2H,t,J＝7.2Hz),2.25(6H,s)。
步骤6：1-(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)-5-氯嘧啶-4- 基)-3-甲基-1H-苯并[d]咪唑-2(3H)-酮
Figure PCTCN2016090149-appb-000028
将1-(5-氯-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)-3-甲基-1H-苯并[d]咪唑-2(3H)-酮(320mg,0.61mmol)、铁粉(139mg,2.48mmol)和氯化铵(50mg,0.93mmol)分散于乙醇/水(8mL/4mL)的混合溶液中。体系在80℃下剧烈搅拌3小时，冷却后过滤，真空去除有机溶剂。所得物加入水(20mL)，乙酸乙酯萃取(20mL*3)。所得有机相用饱和食盐水洗，真空浓缩去除溶剂得到标题所示化合物，直接用于下步反应。
步骤7：N-(5-(5-氯-4-(3-甲基-2-氧代-2,3-二氢-1H-苯并[d]咪唑-1-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)丙烯酰胺盐酸盐
Figure PCTCN2016090149-appb-000029
以类似于实施例1步骤7中所描述的方法由1-(5-氯-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)-3-甲基-1H-苯并[d]咪唑-2(3H)-酮合成标题所示化合物。
1H NMR(DMSO-d6):δ10.15(1H,br),9.68(1H,br),9.05(1H,s),8.66(1H,s),8.11(1H,s),7.23-7.20(1H,m),7.15-7.09(2H,m),7.05-6.99(2H,m),6.89(1H,s),6.41(1H,dd,J＝2.0Hz,16.8Hz),5.70(1H,dd,J＝2.0Hz,10.0Hz),3.79(3H,s),3.35(3H,s),3.28(2H,br),2.65(6H,s),2.55(5H,s)。
实施例4  N-(5-(5-氯-4-(2-氧代-2,3-二氢-1H-苯并[d]咪唑-1-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)丙烯酰胺盐酸盐
Figure PCTCN2016090149-appb-000030
步骤1：1-(5-氯-2-(4-氟-2-甲氧基-5-硝基苯基氨基)嘧啶-4-基)-1H-苯并[d]咪唑-2(3H)-酮
Figure PCTCN2016090149-appb-000031
以类似于实施例3步骤4中所描述的方法由1-(2,5-二氯嘧啶-4-基)-1H-苯并[d]咪唑-2(3H)-酮和4-氟-2-甲氧基-5-硝基苯胺合成标题所示化合物。
1H NMR(CDCl3):9.24(1H,d,J＝8.0Hz),8.68(1H,s),8.45(1H,s),7.83(1H,s),7.34(1H,s),7.22-7.10(3H,m),6.78(1H,d,J＝12.0Hz),4.02(3H,s)。
步骤2：1-(5-氯-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)-1H-苯并[d]咪唑-2(3H)-酮
Figure PCTCN2016090149-appb-000032
1H NMR(CDCl3):δ8.94(1H,s),8.66(1H,s),8.38(1H,br),7.84(1H,s),7.23-7.13(4H,m),4.05(3H,s),3.56(2H,br),3.08(2H,br),2.89(3H,s)2.69(6H,s)。
以类似于实施例1步骤5中所描述的方法由1-(5-氯-2-(4-氟-2-甲氧基-5-硝基苯基氨基)嘧啶-4-基)-1H-苯并[d]咪唑-2(3H)-酮合成标题所示化合物。
步骤3：N-(5-(5-氯-4-(2-氧代-2,3-二氢-1H-苯并[d]咪唑-1-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)丙烯酰胺盐酸盐
Figure PCTCN2016090149-appb-000033
以类似于实施例3步骤6,7中所描述的方法由1-(5-氯-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)-1H-苯并[d]咪唑-2(3H)-酮合成标题所示化合物。
1H NMR(CDCl3):δ12.20(1H,br),9.35(1H,br),9.17(1H,br),8.64(1H,s),8.41(1H,br),7.69(1H,s),7.21-7.16(1H,m),7.13-7.07(3H,m),6.67(1H,s),6.42(1H,dd,J＝1.6Hz,16.8Hz),5.71(1H,d,J＝11.6Hz),3.84(3H,s),3.24(2H,br),3.08(2H,br),2.72(9H,br)。
实施例5  N-(2-((2-(二甲基氨基)乙基)(甲基)氨基)-4-甲氧基-5-(4-(2-氧代苯并[d]恶唑-3(2H)-基)嘧啶-2-基氨基)苯基)丙烯酰胺盐酸盐
Figure PCTCN2016090149-appb-000034
步骤1：2-氯-N-(2-甲氧基苯基)嘧啶-4-胺
Figure PCTCN2016090149-appb-000035
以类似于实施例1步骤1中所描述的方法由邻甲氧基苯胺和2,4-二氯嘧啶合成标题所示化合物。
1H NMR(CDCl3):δ8.14(1H,d,J＝5.6Hz),7.83(1H,br),7.27(1H,m),7.17-7.13(1H,m),7.04-6.99(1H,m),6.96-6.94(1H,m),6.63-6.62(1H,d,J＝6.0Hz),3.89(3H,s)。
步骤2：2-(2-氯嘧啶-4-基氨基)苯酚
Figure PCTCN2016090149-appb-000036
将2-氯-N-(2-甲氧基苯基)嘧啶-4-胺(100mg,0.42mmol)溶于无水二氯甲烷(3mL)中，冰水浴冷却。缓慢滴加三溴化硼的二氯甲烷溶液(2.5mL,2.12mmol)，自然升至室温，继续搅拌2小时。加入饱和氯化铵溶液淬灭反应，二氯甲烷萃取(20mL*3)，合并有机相并用饱和食盐水洗，真空去除溶剂得标题所示化合物(84mg,89％)。
1H NMR(DMSO-d6):δ9.85(1H,s),9.31(1H,s),8.07(1H,d,J＝6.0Hz),7.57(1H,br),7.05-7.01(1H,m),6.93(1H,d,J＝8.0Hz),6.84-6.80(1H,m),6.66(1H,br)。
步骤3：3-(2-氯嘧啶-4-基)苯并[d]恶唑-2(3H)-酮
Figure PCTCN2016090149-appb-000037
以类似于实施例3步骤2中所描述的方法由2-(2-氯嘧啶-4-基氨基)苯酚合成标题所示化合物。
1H NMR(DMSO-d6):δ8.87(1H,d,J＝5.6Hz),8.25-8.19(2H,m),7.49(1H,d,J＝7.6Hz),7.39-7.30(2H,m)。
步骤4：3-(2-(4-氟-2-甲氧基-5-硝基苯基氨基)嘧啶-4-基)苯并[d]恶唑-2(3H)-酮对甲苯磺酸盐
Figure PCTCN2016090149-appb-000038
以类似于实施例1步骤4中所描述的方法由3-(2-氯嘧啶-4-基)苯并[d]恶唑-2(3H)-酮合成标题所示化合物。
1H NMR(DMSO-d6):δ10.83(1H,br),9.23(1H,s),8.59(1H,d,J＝5.6Hz),8.24(1H,br),7.64(1H,d,J＝5.6Hz),7.47-7.43(4H,m),7.31-7.24(1H,m),7.19-7.14(1H,m),7.11(2H,d,J＝8.0Hz),3.97(3H,s),2.28(3H,s)。
步骤5：3-(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)苯并[d]恶唑-2(3H)-酮
Figure PCTCN2016090149-appb-000039
以类似于实施例1步骤5中所描述的方法由3-(2-(4-氟-2-甲氧基-5-硝基苯基氨基)嘧啶-4-基)苯并[d]恶唑-2(3H)-酮对甲苯磺酸盐合成标题所示化合物。
1H NMR(CDCl3):δ8.86(1H,s),9.23(1H,s),8.57(1H,d,J＝5.6Hz),8.24-8.22(1H,m),7.74(1H,d,J＝5.6Hz),7.48(1H,s),7.29-7.25(2H,m),6.72(1H,s),4.01(3H,s),3.29(2H,t,J＝7.2Hz),2.89(3H,s),2.57(2H,t,J＝7.2Hz),2.27(6H,s)。
步骤6：N-(2-((2-(二甲基氨基)乙基)(甲基)氨基)-4-甲氧基-5-(4-(2-氧代苯并[d]恶唑-3(2H)-基)嘧啶-2-基氨基)苯基)丙烯酰胺盐酸盐
Figure PCTCN2016090149-appb-000040
以类似于实施例1步骤6，7中所描述的方法由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)苯并[d]恶唑-2(3H)-酮合成标题所示化合物。
1H NMR(CDCl3):δ12.21(1H,br),9.51(1H,br),9.22(1H,s),8.55(1H,d,J＝5.6Hz),8.25(1H,d,J＝8.0Hz),7.51(1H,s),7.26(1H,s),7.23-7.16(4H,m),6.72(1H,s),6.31(1H,dd,J＝2.0Hz,16.8Hz),5.67(1H,dd,J＝2.0Hz,10.4Hz,),3.90(3H,s),3.29(2H,br),3.13(2H,br),2.76(9H,br)。
实施例6  N-(2-((2-羟基乙基)(甲基)氨基)-4-甲氧基-5-(4-(3-甲基-2-氧代-2,3-二氢-1H-苯并[d]咪唑-1-基)嘧啶-2-基氨基)苯基)丙烯酰胺
Figure PCTCN2016090149-appb-000041
步骤1：1-(2-(4-((2-羟基乙基)(甲基)氨基)-2-甲氧基-5-硝基苯基氨基)嘧啶-4-基)-3-甲基 -1H-苯并[d]咪唑-2(3H)-酮
Figure PCTCN2016090149-appb-000042
以类似于实施例1步骤5中所描述的方法由2-(甲基氨基)乙醇合成标题所示化合物。
1H NMR(CDCl3):δ8.97(1H,s),8.52(1H,d,J＝6.0Hz),8.26(1H,J＝7.2Hz),7.83(1H,d,J＝5.6Hz),7.56(1H,s),7.27-7.23(1H,m),7.18-7.16(1H,m),7.04(1H,J＝7.2Hz),6.70(1H,s),4.00(3H,s),3.79-3.76(2H,m),3.48(3H,s),3.40(2H,t,J＝7.2Hz),2.84(3H,s)。
步骤2：N-(2-((2-羟基乙基)(甲基)氨基)-4-甲氧基-5-(4-(3-甲基-2-氧代-2,3-二氢-1H-苯并[d]咪唑-1-基)嘧啶-2-基氨基)苯基)丙烯酰胺
Figure PCTCN2016090149-appb-000043
以类似于实施例1步骤6，7中所描述的方法由1-(2-(4-((2-羟基乙基)(甲基)氨基)-2-甲氧基-5-硝基苯基氨基)嘧啶-4-基)-3-甲基-1H-苯并[d]咪唑-2(3H)-酮合成标题所示化合物。
1H NMR(CDCl3):δ9.30(1H,s),9.03(1H,s),8.52(1H,d,J＝5.6Hz),8.32(1H,d,J＝8.0Hz),7.80(1H,d,J＝5.6Hz),7.46(1H,s),7.18-7.14(1H,m),7.08-7.03(1H,m),7.01-6.95(1H,m),6.77(1H,s),6.34-6.32(2H,m),5.65-5.63(1H,m),3.87(3H,s),3.77-3.74(2H,m),3.45(3H,s),2.99(2H,t,J＝4.8Hz),3.75(3H,s)。
实施例7  (E)-N-(2-((2-(二甲基氨基)乙基)(甲基)氨基)-4-甲氧基-5-(4-(3-甲基-2-氧代-2,3-二氢-1H-苯并[d]咪唑-1-基)嘧啶-2-基氨基)苯基)-4-甲氧基丁-2-烯酰胺
Figure PCTCN2016090149-appb-000044
以类似于实施例1步骤7中所描述的方法由1-(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)-3-甲基-1H-苯并[d]咪唑-2(3H)-酮和(E)-4-甲氧基丁-2-烯酰氯合成标题所示化合物。
1H NMR(CDCl3):δ12.32(1H,br),9.24(1H,br),9.17(1H,br),8.52(1H,d,J＝5.6Hz),8.31(1H,d,J＝8.0Hz),7.80(1H,d,J＝5.6Hz),7.49(1H,br),7.19-7.10(2H,m),6.99-6.96(2H,m),6.90-6.84(1H,m),6.72(1H,s),4.14(2H,d,J＝3.2Hz),3.90(3H,s),3.45(3H,s),3.40(3H,s),3.38-3.35(2H,m),3.12-3.08(2H,m),2.80(6H,s),2.74(3H,s)。
实施例8  N-(4-甲氧基-5-(4-(3-甲基-2-氧代-2,3-二氢苯并[d]咪唑-1-基)嘧啶-2-基氨基)-2-吗啉基苯基)丙烯酰胺
Figure PCTCN2016090149-appb-000045
步骤1：1-(2-(2-甲氧基-4-吗啉-5-硝基苯基氨基)嘧啶-4-基)-3-甲基-1H-苯并[d]咪唑-2(3H)-酮
Figure PCTCN2016090149-appb-000046
以类似于实施例1步骤5中所描述的方法由1-(2-(4-氟-2-甲氧基-5-硝基苯基氨基)嘧啶-4-基)-3-甲基-1H-苯并[d]咪唑-2(3H)-酮对甲苯磺酸盐和吗啉合成标题所示化合物。
1H NMR(CDCl3):δ9.10(1H,s),8.54(1H,d,J＝5.6Hz),8.27(1H,d,J＝8.0Hz),7.85(1H,d,J＝5.6Hz),7.59(1H,s),7.27-7.24(1H,m),7.19-7.17(1H,m),7.05(1H,d,J＝8.0Hz),6.65(1H,s),4.04(3H,s),3.91-3.89(4H,m),3.49(3H,s),3.10-3.08(4H,m)。
步骤2：N-(4-甲氧基-5-(4-(3-甲基-2-氧代-2,3-二氢苯并[d]咪唑-1-基)嘧啶-2-基氨基)-2-吗啉基苯基)丙烯酰胺
Figure PCTCN2016090149-appb-000047
以类似于实施例1步骤6，7中所描述的方法由1-(2-(2-甲氧基-4-吗啉-5-硝基苯基氨基)嘧啶-4-基)-3-甲基-1H-苯并[d]咪唑-2(3H)-酮合成标题所示化合物。
1H NMR(CDCl3):δ9.37(1H,s),8.55(1H,d,J＝5.6Hz),8.48(1H,s),8.34(1H,d,J＝8.0Hz),7.81(1H,d,J＝5.6Hz),7.46(1H,s),7.20-7.16(1H,m),7.10-7.06(1H,m),7.01-6.99(1H,m),6.79(1H,s),6.36-6.22(2H,m),5.75-5.72(1H,m),3.91-3.88(7H,m),3.46(3H,s),2.90(4H,t,J＝4.8Hz)。
实施例9  N-(2-((2-(二甲基氨基)乙基)(甲基)氨基)-4-甲氧基-5-(4-(5-甲基-2-氧代-2,3-二氢苯并[d]咪唑-1-基)嘧啶-2-基氨基)苯基)丙烯酰胺盐酸盐
Figure PCTCN2016090149-appb-000048
步骤1：N1-(2-氯嘧啶-4-基)-4-甲基苯-1,2-二胺
Figure PCTCN2016090149-appb-000049
以类似于实施例1步骤1中所描述的方法由4-甲基苯-1,2-二胺和2,4-二氯嘧啶合成标题所示化合物。
1H NMR(CDCl3):δ8.06(1H,d,J＝6.0Hz),7.00(1H,d,J＝8.0Hz),6.77(1H,s),6.66(1H,s),6.62(1H,d,J＝8.0Hz),6.21(1H,d,J＝6.0Hz),3.79(2H,br),2.31(3H,s)。
步骤2：1-(2-氯嘧啶-4-基)-5-甲基-1H-苯并[d]咪唑-2(3H)-酮
Figure PCTCN2016090149-appb-000050
以类似于实施例1步骤2中所描述的方法由N1-(2-氯嘧啶-4-基)-4甲基苯-1,2-二胺和羰基二咪唑合成标题所示化合物。
1H NMR(CDCl3):δ8.62(1H,d,J＝5.6Hz),8.47(1H,d,J＝5.6Hz),8.34(1H,d,J＝8.4Hz),7.97(1H,s),7.03(1H,d,J＝8.4Hz),6.91(1H,s),2.42(3H,s)。
步骤3：1-(2-(4-氟-2-甲氧基-5-硝基苯基氨基)嘧啶-4-基)-5-甲基-1H-苯并[d]咪唑-2(3H)-酮对甲苯磺酸盐
Figure PCTCN2016090149-appb-000051
以类似于实施例1步骤4中所描述的方法由1-(2-氯嘧啶-4-基)-5-甲基-1H-苯并[d]咪唑-2(3H)-酮和4-氟-2-甲氧基-5-硝基苯胺合成标题所示化合物。
1H NMR(DMSO-d6):δ11.39(1H,s),9.04(1H,s),8.61(1H,d,J＝8.4Hz),8.49(1H,d,J＝5.6Hz),8.04-8.02(1H,m),7.82(1H,d,J＝5.6Hz),7.49-7.41(3H,m),7.11(2H,d,J＝8.0Hz),6.93(1H,s),6.87(1H,s),6.76-6.74(1H,m),3.97(3H,s),2.33(3H,s),2.29(3H,s)。
步骤4：1-(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)-5-甲基-1H-苯并[d]咪唑-2(3H)-酮
Figure PCTCN2016090149-appb-000052
以类似于实施例1步骤5中所描述的方法由1-(2-(4-氟-2-甲氧基-5-硝基苯基氨基)嘧啶-4-基)-5-甲基-1H-苯并[d]咪唑-2(3H)-酮对甲苯磺酸盐和二异丙基乙基胺合成标题所示化合物。
1H NMR(CDCl3):δ8.92(1H,s),8.50(1H,d,J＝5.6Hz),8.30(1H,br),8.11(1H,d,J＝8.0 Hz),7.78(1H,d,J＝5.6Hz),7.45(1H,s),6.94(1H,d,J＝8.0Hz),6.89(1H,s),6.70(1H,s),3.98(3H,s),3.31(2H,t,J＝6.8Hz),2.88(3H,s),2.61(2H,t,J＝6.8Hz),2.40(3H,s),2.31(6H,s)。
步骤5：N-(2-((2-(二甲基氨基)乙基)(甲基)氨基)-4-甲氧基-5-(4-(5-甲基-2-氧代-2,3-二氢苯并[d]咪唑-1-基)嘧啶-2-基氨基)苯基)丙烯酰胺盐酸盐
Figure PCTCN2016090149-appb-000053
以类似于实施例1步骤6，7中所描述的方法由1-(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)-5-甲基-1H-苯并[d]咪唑-2(3H)-酮合成标题所示化合物。
1H NMR(MeOD):δ8.32(1H,d,J＝6.0Hz),7.91(1H,d,J＝8.0Hz),7.61(1H,d,J＝5.6Hz),6.87-6.84(2H,m),6.78(1H,s),6.70(1H,d,J＝8.0Hz),6.40-6.26(2H,m),5.73-5.70(1H,m),3.86(3H,s),3.26(2H,br),2.93(2H,br),2.61(3H,s),2.59(6H,s),2.22(3H,s)。
实施例10  N-(2-((2-(二甲基氨基)乙基)(甲基)氨基)-5-(4-(5-氟-3-甲基-2-氧代-2,3-二氢苯并[d]咪唑-1-基)嘧啶-2-基氨基)-4-甲氧基苯基)丙烯酰胺盐酸盐
Figure PCTCN2016090149-appb-000054
步骤1：N1-(2-二氯嘧啶-4-基)-4-氟苯-1,2-二胺
Figure PCTCN2016090149-appb-000055
以类似于实施例1步骤1中所描述的方法由2,4-二氯嘧啶和4-氟苯-1,2-二胺合成标题所示化合物。
1H NMR(CDCl3):δ8.06(1H,d,J＝6.0Hz),7.06-7.03(1H,m),6.71(1H,s),6.52-6.44(2H,m),6.14(1H,d,J＝6.0Hz),3.94(2H,br)。
步骤2：1-(2-氯嘧啶-4-基)-5-氟-1H-苯并[d]咪唑-2(3H)-酮
Figure PCTCN2016090149-appb-000056
以类似于实施例1步骤2中所描述的方法由N1-(2-二氯嘧啶-4-基)-4-氟苯-1,2-二胺和羰基二咪唑合成标题所示化合物。
1H NMR(DMSO-d6):δ11.81(1H,s),8.77(1H,d,J＝6.0Hz),8.40(1H,d,J＝6.0Hz),8.24-8.21(1H,m),7.03-6.96(2H,m)。
步骤3：1-(2-氯嘧啶-4-基)-5-氟-3-甲基-1H-苯并[d]咪唑-2(3H)-酮
Figure PCTCN2016090149-appb-000057
以类似于实施例1步骤3中所描述的方法由1-(2-氯嘧啶-4-基)-5-氟-1H-苯并[d]咪唑-2(3H)-酮合成标题所示化合物。
1H NMR(DMSO-d6):δ8.77(1H,d,J＝5.6Hz),8.38(1H,d,J＝5.6Hz),8.23-8.19(1H,m),7.29(1H,d,J＝8.8Hz),7.04-7.00(1H,m),3.36(3H,s)。
步骤4：5-氟-1-(2-(4-氟-2-甲氧基-5-硝基苯基氨基)嘧啶-4-基)-3-甲基-1H-苯并[d]咪唑-2(3H)-酮对甲苯磺酸盐
Figure PCTCN2016090149-appb-000058
以类似于实施例1步骤4中所描述的方法由1-(2-氯嘧啶-4-基)-5-氟-3-甲基-1H-苯并[d]咪唑-2(3H)-酮和4-氟-2-甲氧基-5-硝基苯胺合成标题所示化合物，直接用于下步反应。
步骤5：1-(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)-5-氟-3-甲基-1H-苯并[d]咪唑-2(3H)-酮
Figure PCTCN2016090149-appb-000059
以类似于实施例1步骤5中所描述的方法由5-氟-1-(2-(4-氟-2-甲氧基-5-硝基苯基氨基)嘧啶-4-基)-3-甲基-1H-苯并[d]咪唑-2(3H)-酮对甲苯磺酸盐合成标题所示化合物。
1H NMR(CDCl3):δ8.86(1H,s),8.50(1H,d,J＝5.6Hz),8.24-8.21(1H,m),7.81(1H,d,J＝5.6Hz),7.42(1H,s),6.86-6.81(1H,m),6.76(1H,dd,J＝2.8Hz,8.0Hz),6.69(1H,s),3.98(3H,s),3.44(3H,s),3.29(2H,t,J＝7.2Hz),2.89(3H,s),2.57(2H,t,J＝7.2Hz),2.27(6H,s)。
步骤6：N-(2-((2-(二甲基氨基)乙基)(甲基)氨基)-5-(4-(5-氟-3-甲基-2-氧代-2,3-二氢苯并[d]咪唑-1-基)嘧啶-2-基氨基)-4-甲氧基苯基)丙烯酰胺盐酸盐
Figure PCTCN2016090149-appb-000060
以类似于实施例1步骤6，7中所描述的方法由1-(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)-5-氟-3-甲基-1H-苯并[d]咪唑-2(3H)-酮合成标题所示化合物。
1H NMR(DMSO-d6):δ10.26(1H,s),9.78(1H,s),8.74(1H,s),8.43(1H,d,J＝6.0Hz),8.17(2H,s),7.70(1H,d,J＝6.0Hz),7.22(1H,dd,J＝2.4Hz,8.8Hz),7.06-6.99(1H,m),6.97(1H,s),6.79-6.75(1H,m),6.21(1H,dd,J＝2.0Hz,16.8Hz),5.71-5.68(1H,m),3.80(3H,s),3.63-3.57(2H,m),3.34(3H,s),3.16-3.09(2H,m),2.75(6H,s),2.63(3H,s)。
实施例11  N-(2-((2-(二甲基氨基)乙基)(甲基)氨基)-5-(4-(5-氟-2-氧代-2,3-二氢苯并[d]咪唑-1-基)嘧啶-2-基氨基)-4-甲氧基苯基)丙烯酰胺盐酸盐
Figure PCTCN2016090149-appb-000061
步骤1：5-氟-1-(2-(4-氟-2-甲氧基-5-硝基苯基氨基)嘧啶-4-基)-1H-苯并[d]咪唑-2(3H)-酮对甲苯磺酸盐
Figure PCTCN2016090149-appb-000062
以类似于实施例1步骤4中所描述的方法由1-(2-氯嘧啶-4-基)-5-氟-1H-苯并[d]咪唑-2(3H)-酮和4-氟-2-甲氧基-5-硝基苯胺合成标题所示化合物。
1H NMR(DMSO-d6):δ11.68(1H,s),9.24(1H,s),8.64(1H,d,J＝8.0Hz),8.51(1H,d,J＝6.0Hz),8.23(1H,br),7.83(1H,d,J＝6.0Hz),7.48(2H,d,J＝8.0Hz),7.43(1H,d,J＝8.0Hz),7.12(2H,d,J＝8.0Hz),6.94-6.91(1H,m),6.80-6.75(1H,m),3.98(3H,s),2.29(3H,s)。
步骤2：1-(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)-5-氟-1H-苯并[d]咪唑-2(3H)-酮
Figure PCTCN2016090149-appb-000063
以类似于实施例1步骤5中所描述的方法由5-氟-1-(2-(4-氟-2-甲氧基-5-硝基苯基氨基)嘧啶-4-基)-1H-苯并[d]咪唑-2(3H)-酮对甲苯磺酸盐合成标题所示化合物。
1H NMR(DMSO-d6):δ8.83(1H,s),8.47(1H,d,J＝5.6Hz),8.26(1H,s),8.23-8.17(1H,m),7.77-7.68(2H,m),6.99-6.94(2H,m),6.77-6.72(1H,m),3.99(3H,s),3.63(2H,m),3.19-3.15(2H,m),2.88(3H,s),2.66(6H,s)。
步骤3：N-(2-((2-(二甲基氨基)乙基)(甲基)氨基)-5-(4-(5-氟-2-氧代-2,3-二氢苯并[d]咪唑-1-基)嘧啶-2-基氨基)-4-甲氧基苯基)丙烯酰胺盐酸盐
Figure PCTCN2016090149-appb-000064
以类似于实施例1步骤6，7中所描述的方法由1-(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)-5-氟-1H-苯并[d]咪唑-2(3H)-酮合成标题所示化合物。
1H NMR(CD3OD):δ8.45(1H,dd,J＝2.4Hz,5.6Hz),8.17-8.12(1H,m),7.73-7.69(2H,m),7.22(1H,d,J＝8.0Hz),6.95(1H,d,J＝3.6Hz),6.88-6.83(1H,m),6.77-6.71(1H,m),6.44-6.42(1H,m),5.84-5.81(1H,m),3.97(3H,s),3.49-3.46(2H,m),3.23(2H,m),2.84(6H,s),2.71(3H,s)。
实施例12  N-(4-甲氧基-2-(甲基(2-(甲基氨基)乙基)氨基)-5-(4-(3-甲基-2-氧代-2,3-二氢苯并[d]咪唑-1-基)嘧啶-2-基氨基)苯基)丙烯酰胺盐酸盐
Figure PCTCN2016090149-appb-000065
步骤1：叔丁基2-((5-甲氧基-4-(4-(3-甲基-2-氧代-2,3-二氢苯并[d]咪唑-1-基)嘧啶-2-基氨基)-2硝基苯基)(甲基)氨基)乙基(甲基)氨基甲酸酯
Figure PCTCN2016090149-appb-000066
以类似于实施例1步骤5中所描述的方法由1-(2-(4-氟-2-甲氧基-5-硝基苯基氨基)嘧啶-4-基)-3-甲基-1H-苯并[d]咪唑-2(3H)-酮对甲苯磺酸盐和2-(甲基氨基)乙基氨基甲酸叔丁酯合成 标题所示化合物。
1H NMR(CDCl3):δ8.94(1H,s),8.51(1H,d,J＝5.6Hz),8.26(1H,d,J＝7.2Hz),7.80(1H,d,J＝5.6Hz),7.48(1H,s),7.24-7.22(1H,m),7.17-7.15(1H,m),7.03(1H,d,J＝7.2Hz),6.76(1H,s),4.01(3H,s),3.47-3.37(5H,m),3.31-3.23(2H,m),2.91(3H,s),2.83(3H,s),1.44(9H,s)。
步骤2：叔丁基2-((2-丙烯酰胺-5-甲氧基-4-(4-(3-甲基-2-氧代-2,3-二氢苯并[d]咪唑-1-基)嘧啶-2-基氨基)苯基)(甲基)氨基)乙基(甲基)氨基甲酸酯
Figure PCTCN2016090149-appb-000067
以类似于实施例1步骤6，7中所描述的方法由叔丁基2-((5-甲氧基-4-(4-(3-甲基-2-氧代-2,3-二氢苯并[d]咪唑-1-基)嘧啶-2-基氨基)-2硝基苯基)(甲基)氨基)乙基(甲基)氨基甲酸酯合成标题所示化合物。
1H NMR(CDCl3):δ9.37(1H,br),8.67(1H,br),8.54(1H,d,J＝5.6Hz),8.34(1H,d,J＝7.6Hz),7.79(1H,d,J＝5.6Hz),7.43(1H,s),7.19-7.15(1H,m),7.08-7.04(1H,m),6.98(1H,d,J＝7.6Hz),6.79(1H,s),6.35(2H,d,J＝2.4Hz),5.69-5.72(1H,m),3.89(3H,s),3.45(3H,s),3.40-3.37(2H,m),3.00-2.85(2H,m),2.85(3H,s),2.70(3H,s),1.47(9H,s)。
步骤3：N-(4-甲氧基-2-(甲基(2-(甲基氨基)乙基)氨基)-5-(4-(3-甲基-2-氧代-2,3-二氢苯并[d]咪唑-1-基)嘧啶-2-基氨基)苯基)丙烯酰胺盐酸盐
Figure PCTCN2016090149-appb-000068
将乙酰氯(0.3mL,1.7mmol)缓慢滴加到外置冰水浴冷却的无水甲醇(3mL)中，继续搅拌1小时。将叔丁基2-((2-丙烯酰胺-5-甲氧基-4-(4-(3-甲基-2-氧代-2,3-二氢苯并[d]咪唑-1-基)嘧啶-2-基氨基)苯基)(甲基)氨基)乙基(甲基)氨基甲酸酯(100mg,0.166mmol)分散于无水甲醇(2mL)中，然后加入到前述氯化氢甲醇溶液中。体系自然升至室温并搅拌过夜，真空浓缩去除溶剂，硅胶柱层析(DCM:MeOH＝20:1)得标题所示化合物(78mg,93％)。
1H NMR(CD3OD):δ8.45(1H,d,J＝5.6Hz),8.19(2H,d,J＝6.0Hz),7.77(1H,d,J＝5.6Hz), 7.21-7.16(2H,m),7.09-7.05(1H,m),6.95(1H,s),6.50-6.36(2H,m),5.80(1H,dd,J＝2.0Hz,6.0Hz),3.96(3H,s),3.45(3H,s),3.35(4H,br),2.73(3H,s),2.72(3H,s)。
实施例13  N-(2-((2-(二甲基氨基)乙基)(甲基)氨基)-4-甲氧基-5-(4-(3-甲基-2-氧代-2,3-二氢-1H-苯并[d]咪唑-1-基)嘧啶-2-基氨基)苯基)丙烯酰胺
Figure PCTCN2016090149-appb-000069
将实施例1步骤6所得1-(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)-3-甲基-1H-苯并[d]咪唑-2(3H)-酮(82g)加入THF(800mL)和水(80mL)中，搅拌溶解，滴加3-氯丙酰氯(24.8g)。TLC显示原料消失后，加入三乙胺(358.2g)，升温至65℃反应。反应结束后，将反应液浓缩至干，加入1L二氯甲烷溶解，水(500mL)分液两次，收集有机相浓缩得到88g粗品。所得粗品经柱层析(DCM:MeOH＝20:1)分离得标题所示化合物62.5g。
ESI-MS[M+H]+:517.2677。
1H NMR(DMSO-d6):δ10.05(1H,s),8.67(1H,s),8.5(1H,s),8.44(1H,d,J＝5.6Hz),8.12(1H,d,J＝7.6Hz),7.13(2H,m),6.9(1H,t,J＝6.4Hz),7.7(1H,d,J＝5.6Hz),7.05(1H,s),6.4(1H,dd,J＝10.15Hz,16.9Hz),6.21(1H,dd,J＝1.6Hz,16.9Hz),5.72(1H,brd,J＝11.50Hz),3.77(3H,s),3.35(3H,s),2.91(2H,t,J＝5.65Hz),2.75(3H,s),2.34(2H,t,J＝5.7Hz),2.21(6H,s)。
实施例14  N-(5-(4-(5-氯-3-甲基-2-氧代-2,3-二氢-1H-苯并[d]咪唑-1-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)丙烯酰胺盐酸盐
Figure PCTCN2016090149-appb-000070
步骤1：4-氯-N1-(2-氯嘧啶-4-基)苯-1,2-二胺
Figure PCTCN2016090149-appb-000071
以类似于实施例1步骤1中所描述的方法由2,4-二氯嘧啶和4-氯-1,2-苯二胺合成标题所示化合物。
1H NMR(DMSO-d6):δ9.14(1H,s),8.05(1H,d,J＝6.0Hz),7.06(1H,d,J＝8.4Hz),6.80(1H,s),6.56(1H,dd,J＝2.8Hz,8.0Hz),6.35(1H,s),5.32(2H,br s)。
步骤2：5-氯-1-(2-氯嘧啶-4-基)-1H-苯并[d]咪唑-2(3H)-酮
Figure PCTCN2016090149-appb-000072
以类似于实施例1步骤2中所描述的方法由4-氯-N1-(2-氯嘧啶-4-基)苯-1,2-二胺和N,N'-羰基二咪唑合成标题所示化合物。
1H NMR(DMSO-d6):δ11.79(1H,br s),8.79(1H,d,J＝5.6Hz),8.39(1H,d,J＝6.0Hz),8.21(1H,d,J＝8.8Hz),7.23(1H,dd,J＝2.0Hz,8.4Hz),7.12(1H,s)。
步骤3：5-氯-1-(2-氯嘧啶-4-基)-3-甲基-1H-苯并[d]咪唑-2(3H)-酮
Figure PCTCN2016090149-appb-000073
以类似于实施例1步骤3中所描述的方法由5-氯-1-(2-氯嘧啶-4-基)-1H-苯并[d]咪唑-2(3H)-酮合成标题所示化合物。
1H NMR(DMSO-d6):δ8.78(1H,d,J＝5.6Hz),8.38(1H,d,J＝5.6Hz),8.19(1H,d,J＝8.8Hz),7.45(1H,s),7.23(1H,d,J＝8.8Hz),3.36(3H,s)。
步骤4：5-氯-1-(2-(4-氟-2-甲氧基-5-硝基苯基氨基)嘧啶-4-基)-3-甲基-1H-苯并[d]咪唑-2(3H)-酮
Figure PCTCN2016090149-appb-000074
以类似于实施例1步骤4中所描述的方法由5-氯-1-(2-氯嘧啶-4-基)-3-甲基-1H-苯并[d]咪唑-2(3H)-酮和4-氟-2-甲氧基-5-硝基苯胺合成标题所示化合物。
1H NMR(DMSO-d6):9.23(1H,s),8.61(1H,d,J＝8.0Hz),8.54(1H,d,J＝5.6Hz),8.22(1H,d,J＝8.0Hz),7.82(1H,d,J＝5.6Hz),7.44-7.41(2H,m),7.02(1H,dd,J＝2.0Hz,8.4Hz),3.97(3H,s),3.37(3H,s)。
步骤5：5-氯-1-(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)-3-甲基-1H-苯并[d]咪唑-2(3H)-酮
Figure PCTCN2016090149-appb-000075
以类似于实施例1步骤5中所描述的方法由5-氯-1-(2-(4-氟-2-甲氧基-5-硝基苯基氨基)嘧啶-4-基)-3-甲基-1H-苯并[d]咪唑-2(3H)-酮合成标题所示化合物。
1H NMR(CDCl3):δ8.84(1H,s),8.49(1H,d,J＝5.6Hz),8.19(1H,d,J＝8.4Hz),7.79(1H,d,J＝5.6Hz),7.42(1H,s),7.09(1H,dd,J＝2.4Hz,8.8Hz),7.00(1H,s),6.69(1H,s),3.98(3H,s),3.43(3H,s)3.29(2H,t,J＝7.2Hz),2.89(3H,s),2.56(2H,t,J＝7.2Hz),2.27(6H,s)。
步骤6：1-(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)-5-氯-3-甲基-1H-苯并[d]咪唑-2(3H)-酮
Figure PCTCN2016090149-appb-000076
将5-氯-1-(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)-3-甲基-1H-苯并[d]咪唑-2(3H)-酮(1.00g)和锌粉(1.24g,18.97mmol)分散于二氯甲烷/ 甲醇(15mL/15mL)的混合溶液中，室温下滴加20mL饱和氯化铵溶液，搅拌十分钟后过滤，滤液中加入水(30mL)，二氯甲烷萃取(30mL*3)。所得有机相用饱和食盐水洗，真空浓缩去除溶剂得到标题所示化合物，直接用于下步反应。
步骤7：N-(5-(4-(5-氯-3-甲基-2-氧代-2,3-二氢-1H-苯并[d]咪唑-1-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)丙烯酰胺盐酸盐
Figure PCTCN2016090149-appb-000077
以类似于实施例1步骤7中所描述的方法由1-(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)-5-氯-3-甲基-1H-苯并[d]咪唑-2(3H)-酮合成标题所示化合物。
1H NMR(DMSO-d6):δ10.42(1H,br s),9.82(1H,s),8.78(1H,s),8.44(1H,d,J＝5.6Hz),8.18(1H,s),8.13(1H,s),7.66(1H,d,J＝5.6Hz),7.38(1H,d,J＝2.4Hz),7.13-7.06(1H,m),6.99-6.96(2H,m),6.20(1H,dd,J＝2.0Hz,16.8Hz),5.69(1H,dd,J＝2.0Hz,10.0Hz),3.80(3H,s),3.36-3.30(7H,m),2.75(6H,s),2.63(3H,s)。
实施例15  N-(5-(4-(5-溴-3-甲基-2-氧代-2,3-二氢-1H-苯并[d]咪唑-1-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)丙烯酰胺盐酸盐
Figure PCTCN2016090149-appb-000078
步骤1：4-溴-N1-(2-氯嘧啶-4-基)苯-1,2-二胺
Figure PCTCN2016090149-appb-000079
以类似于实施例1步骤1中所描述的方法由2,4-二氯嘧啶和4-溴-1,2-苯二胺合成标题所示化合物。
1H NMR(DMSO-d6):δ9.13(1H,s),8.05(1H,d,J＝6.0Hz),7.01(1H,d,J＝8.8Hz),6.94(1H,d,J＝2.4Hz),6.69(1H,dd,J＝2.0Hz,8.4Hz),6.38(1H,s),5.31(2H,br s)。
步骤2：5-溴-1-(2-氯嘧啶-4-基)-1H-苯并[d]咪唑-2(3H)-酮
Figure PCTCN2016090149-appb-000080
以类似于实施例1步骤2中所描述的方法由4-溴-N1-(2-氯嘧啶-4-基)苯-1,2-二胺和N,N'-羰基二咪唑合成标题所示化合物。
1H NMR(DMSO-d6):δ11.81(1H,br s),8.79(1H,d,J＝5.6Hz),8.40(1H,d,J＝5.6Hz),8.17(1H,d,J＝8.8Hz),7.37(1H,dd,J＝2.0Hz,8.8Hz),7.26(1H,d,J＝2.0Hz)。
步骤3：5-溴-1-(2-氯嘧啶-4-基)-3-甲基-1H-苯并[d]咪唑-2(3H)-酮
Figure PCTCN2016090149-appb-000081
以类似于实施例1步骤3中所描述的方法由5-溴-1-(2-氯嘧啶-4-基)-1H-苯并[d]咪唑-2(3H)-酮合成标题所示化合物。
1H NMR(DMSO-d6):δ8.81(1H,d,J＝6.0Hz),8.42(1H,d,J＝8.0Hz),8.19(1H,d,J＝8.4Hz),7.60(1H,d,J＝2.0Hz),7.41(1H,dd,J＝2.0Hz,8.4Hz),3.40(3H,s)。
步骤4：5-溴-1-(2-(4-氟-2-甲氧基-5-硝基苯基氨基)嘧啶-4-基)-3-甲基-1H-苯并[d]咪唑-2(3H)-酮
Figure PCTCN2016090149-appb-000082
以类似于实施例1步骤4中所描述的方法由5-溴-1-(2-氯嘧啶-4-基)-3-甲基-1H-苯并[d]咪唑-2(3H)-酮和4-氟-2-甲氧基-5-硝基苯胺合成标题所示化合物。
1H NMR(DMSO-d6):9.22(1H,s),8.61(1H,d,J＝8.4Hz),8.54(1H,d,J＝5.6Hz),8.17(1H,d,J＝8.0Hz),7.82(1H,d,J＝5.6Hz),7.53(1H,d,J＝2.0Hz),7.43(1H,d,J＝13.2Hz), 7.44-7.41(2H,m),7.14(1H,dd,J＝2.0Hz,13.2Hz),3.97(3H,s),3.37(3H,s)。
步骤5：5-溴-1-(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)-3-甲基-1H-苯并[d]咪唑-2(3H)-酮
Figure PCTCN2016090149-appb-000083
以类似于实施例1步骤5中所描述的方法由5-溴-1-(2-(4-氟-2-甲氧基-5-硝基苯基氨基)嘧啶-4-基)-3-甲基-1H-苯并[d]咪唑-2(3H)-酮合成标题所示化合物。
1H NMR(CDCl3):δ8.85(1H,s),8.50(1H,d,J＝5.6Hz),8.15(1H,d,J＝8.4Hz),7.79(1H,d,J＝6.0Hz),7.42(1H,s),7.24(1H,dd,J＝2.0Hz,8.4Hz),7.15(1H,d,J＝2.0Hz),6.69(1H,s),3.98(3H,s),3.44(3H,s)3.30(2H,t,J＝6.8Hz),2.90(3H,s),2.58(2H,t,J＝7.2Hz),2.28(6H,s)。
步骤6：1-(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)-5-溴-3-甲基-1H-苯并[d]咪唑-2(3H)-酮
Figure PCTCN2016090149-appb-000084
将5-溴-1-(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)-3-甲基-1H-苯并[d]咪唑-2(3H)-酮(1.00g)和锌粉(1.24g,18.97mmol)分散于二氯甲烷/甲醇(15mL/15mL)的混合溶液中，室温下滴加20mL饱和氯化铵溶液，搅拌十分钟后过滤，滤液中加入水(30mL)，二氯甲烷萃取(30mL*3)。所得有机相用饱和食盐水洗，真空浓缩去除溶剂得到标题所示化合物，直接用于下步反应。
步骤7：N-(5-(4-(5-溴-3-甲基-2-氧代-2,3-二氢-1H-苯并[d]咪唑-1-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)丙烯酰胺盐酸盐
Figure PCTCN2016090149-appb-000085
以类似于实施例1步骤7中所描述的方法由1-(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)-5-溴-3-甲基-1H-苯并[d]咪唑-2(3H)-酮合成标题所示化合物。
1H NMR(DMSO-d6):δ10.23(1H,br s),9.82(1H,br s),8.75(1H,s),8.44(1H,d,J＝5.2Hz),8.20(1H,s),8.05(1H,s),7.65(1H,d,J＝5.2Hz),7.49(1H,d,J＝2.0Hz),7.08(1H,d,J＝8.4Hz),7.00-6.96(2H,m),6.21(1H,dd,J＝2.0Hz,17.2Hz),5.71(1H,dd,J＝1.6Hz,10.4Hz),3.80(3H,s),3.36(3H,s),3.29-3.24(2H,m),3.16(3H,s),2.76-3.68(2H,m),2.65(6H,s)。
体外活性试验
1.体外酶学检测方法
EGFR,EGFR(T790M,L858R)激酶通过昆虫表达***表达纯化得到或购买市售产品得到。
采取Cisbio公司的匀相时间分辨荧光(HTRF)方法建立了EGFR,EGFR(T790M,L858R)的激酶活性检测平台，进行化合物活性的测定。将化合物从1μM开始用100％DMSO进行10倍梯度稀释，每个浓度取4μl加入到96μl的反应缓冲液中(50mM HEPES(pH 7.0),0.02％NaN3,0.01％BSA,0.1mM Orthovanadate，5mM MgCl2,50nM SEB，1mM DTT)，取2.5μl加入到384孔板(OptiPlate-384,PerkinElmer)，然后加入2.5μl的激酶，离心混匀，再加入5μl的ATP和TK Substrate-biotin启动反应。将384孔板放于孵育箱中23℃反应一定时间后，加入5μl的Eu3+-Cryptate标记的TK-Antibody，5μl的streptavidin-XL665终止反应。在孵育箱中孵育1小时后，在Envision(PerkinElmer)上读取荧光值。化合物的IC50值使用GraphPad Prism 5.0软件计算得到。
2.细胞增殖试验
人非小细胞肺癌NCI-H1975在细胞培养箱(37℃，5％CO2)中用RPMI-1640培养基(加入10％胎牛血清和1％青链霉素)进行培养。按照每孔2000个细胞(体积：195ul)接种96孔板培养过夜。第二天加入化合物，化合物从10mM进行3倍梯度稀释，每个浓度取4μl加入到96μl的培养基中，然后取5μl加入到细胞培养液中(DMSO终浓度为0.1％，v/v)， 处理72小时后，吸去培养基并加入30μl
Figure PCTCN2016090149-appb-000086
(Promega)试剂，在Envison(Perkin Elmer)上读取荧光信号，使用GraphPad Prism 5.0计算化合物对细胞增殖抑制的IC50值。
人皮肤鳞癌细胞系A431在细胞培养箱(37℃，5％CO2)中用DMEM加10％胎牛血清和1％的青链霉素进行培养。在化合物的检测中，底层基质浓度为0.6％,细胞用0.3％的低熔点琼脂重选后，2000个细胞每孔(100μl)铺于96孔板中。化合物从10mM进行3倍梯度稀释，每个浓度取2μl加入到98μl的培养基中，然后取5.3μl加入到细胞培养液中(DMSO终浓度为0.1％，v/v)，处理一周(7天)后，加入20μl
Figure PCTCN2016090149-appb-000087
(Promega)试剂，37℃孵育4个小时，在Envison(Perkin Elmer)上读取荧光信号，使用GraphPad Prism 5.0计算化合物对细胞增殖抑制的IC50值。
表1：生物活性列表
Figure PCTCN2016090149-appb-000088
NT：未测试；AZD9291根据WO2013014448实施例28制得。
从上述实验结果可知，酶活性方面，本申请化合物针对EGFR，尤其是EGFR-L858R/T790M双突变型具有良好的抑制效果，并且WT/DM数据显示出本申请化合物 具有良好的选择性。细胞活性方面，本申请化合物在人非小细胞肺癌NCI-H1975和人皮肤鳞癌细胞系A431的实验结果显示出良好的抑制效果。
药代动力学评价
受试化合物使用健康成年雄性大鼠，单剂量灌胃给药，辅剂为20％的磺丁基醚-β-环糊精，剂量为10mg/kg。灌胃给药的动物在实验前禁食过夜，禁食时间从给药前10小时至给药后4小时，在灌胃给药后0.25、0.5、1、2、4、6、8和24小时采血。通过眼眶静脉丛采集约0.3mL全血，放于肝素抗凝管中，样品于4℃、4000rpm离心5min，血浆转移至离心管中，并放于-80℃保存直到分析。血浆样品中受试品浓度分析使用非确证的液相色谱-串联质谱联用方法(LC-MS/MS)。个体动物的血浆浓度-时间数据用WinNonlin(专业版，版本6.3；Pharsight公司)软件进行分析。非房室模型被用于浓度分析。计算受试化合物的药代动力学参数。
表2.
Figure PCTCN2016090149-appb-000089

Claims (12)

  1. 式(I)所示化合物或者其药学上可接受的盐：
    Figure PCTCN2016090149-appb-100001
    其中：
    X选自NR6或O；
    R1、R2独立地选自氢、卤素、C1-4烷基和氰基；
    R3选自C1-4烷基和C1-4烷氧基；
    R4选自[2-(二甲氨基)乙基](甲基)氨基、(2-羟基乙基)(甲基)氨基和吗啉-4-基；
    R5选自氢、C1-4烷基和C1-3烷氧基C1-3烷基；
    R6选自氢和C1-4烷基。
  2. 如权利要求1所述的式(I)所示化合物或者其药学上可接受的盐，其特征在于：
    X选自NR6或O；
    R1、R2独立地选自氢、卤素和C1-4烷基；
    R3选自C1-4烷氧基；
    R4选自[2-(二甲氨基)乙基](甲基)氨基、(2-羟基乙基)(甲基)氨基和吗啉-4-基；
    R5选自氢、C1-4烷基和C1-3烷氧基C1-3烷基；
    R6选自氢和C1-4烷基。
  3. 如权利要求1所述的式(I)所示化合物或者其药学上可接受的盐，其特征在于：
    X选自NR6或O；
    R1、R2独立地选自氢、氯、溴、氟和甲基；
    R3为甲氧基；
    R4选自[2-(二甲氨基)乙基](甲基)氨基、(2-羟基乙基)(甲基)氨基和吗啉-4-基；
    R5选自氢和甲氧基甲基；
    R6选自氢和甲基。
  4. 下式表示的化合物：
    Figure PCTCN2016090149-appb-100002
    或者其药学上可接受的盐。
  5. 下式表示的化合物的盐酸盐：
    Figure PCTCN2016090149-appb-100003
  6. 药物组合物，其包含权利要求1-3任一项所述的式(I)化合物或其药学上可接受的盐、或者权利要求4或5所述化合物以及一种或多种药学上可接受的载体或赋形剂。
  7. 权利要求6所述的药物组合物，其进一步含有一种或多种额外的治疗剂。
  8. 治疗EGFR介导的疾病的方法，所述方法包括给予有需要的个体权利要求6-7所述的药物组合物或权利要求1-5任一项所述的化合物或其药学上可接受的盐。
  9. 权利要求1-5任一项所述的化合物或其药学上可接受的盐或者权利要求6或7所述的药物组合物在制备治疗EGFR介导的疾病的药物中的用途。
  10. 根据权利要求8所述的方法或权利要求9所述的用途，其中所述EGFR介导的疾病选自EGFR-L858R激活突变、EGFR-T790M激活突变或者EGFR-L858R合并EGFR-T790M双突变激活介导的疾病。
  11. 根据权利要求8所述的方法或权利要求9所述的用途，其中所述EGFR介导的疾病包括癌症。
  12. 根据权利要求11所述的方法或用途，其中所述癌症选自卵巢癌、***、结肠直肠癌、乳腺癌、胰腺癌、胶质瘤、胶质母细胞瘤、黑色素瘤、***癌、白血病、淋巴瘤、非霍奇金淋巴瘤、胃癌、肺癌、肝细胞癌、胃癌、胃肠道间质瘤、甲状腺癌、胆管癌、子宫内膜癌、肾癌、间变性大细胞淋巴瘤、急性髓细胞白血病、多发性骨髓瘤、黑色素瘤和间皮瘤；任选地，所述肺癌包括非小细胞肺癌、小细胞肺癌、肺腺癌或肺鳞癌。
PCT/CN2016/090149 2015-07-16 2016-07-15 苯胺嘧啶衍生物及其用途 WO2017008761A1 (zh)

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