WO2015085654A1 - 喹唑啉衍生物 - Google Patents

喹唑啉衍生物 Download PDF

Info

Publication number
WO2015085654A1
WO2015085654A1 PCT/CN2014/001119 CN2014001119W WO2015085654A1 WO 2015085654 A1 WO2015085654 A1 WO 2015085654A1 CN 2014001119 W CN2014001119 W CN 2014001119W WO 2015085654 A1 WO2015085654 A1 WO 2015085654A1
Authority
WO
WIPO (PCT)
Prior art keywords
acid
fluorophenylamino
chloro
quinazolin
methoxyethoxy
Prior art date
Application number
PCT/CN2014/001119
Other languages
English (en)
French (fr)
Inventor
张和胜
曾广怀
陈英伟
Original Assignee
天津合美医药科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 天津合美医药科技有限公司 filed Critical 天津合美医药科技有限公司
Priority to US15/103,596 priority Critical patent/US9840494B2/en
Priority to KR1020167018546A priority patent/KR101847757B1/ko
Priority to JP2016539159A priority patent/JP6342001B2/ja
Priority to AU2014361633A priority patent/AU2014361633B2/en
Priority to EP14870139.4A priority patent/EP3081563B1/en
Priority to CA2933082A priority patent/CA2933082C/en
Publication of WO2015085654A1 publication Critical patent/WO2015085654A1/zh

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • 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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A61K31/4545—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring hetero atom, e.g. pipamperone, anabasine
    • 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
    • A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • This application relates to the field of medicinal chemistry.
  • EGFR is a member of the human epidermal growth factor receptor (HER) glycoprotein family, and other members include ErbB2 (HER-2), ErbB3 (HER-3), and ErbB4 (HER-4).
  • the intracellular tyrosine kinase catalyzes the phosphorylation of various substrate proteins and plays an important role in the signaling pathway of tumor cells.
  • EGFR can activate its intracellular tyrosine kinase under the stimulation of extracellular signals. The extracellular signal is transmitted to the cells and amplified to regulate cell growth, differentiation, angiogenesis and apoptosis inhibition.
  • the relationship between abnormal expression of EGFR overexpression or mutation and the growth, invasion and metastasis of malignant tumors close.
  • EGFR EGFR-mediated signaling, thereby inhibiting the growth of tumor cells, inhibiting tumor invasion into surrounding tissues, and promoting apoptosis of tumor cells. Therefore, targeted therapy for EGFR is one of the hotspots of current research. Molecular targeted therapy targeting EGFR is more effective in selective populations.
  • EGFR-targeted drugs on the market are mainly divided into monoclonal antibodies acting on the extracellular domain of EGFR and small molecule EGFR tyrosine kinase inhibitors (EGFR-TKI) acting on the EGFR intracellular tyrosine kinase binding domain.
  • EGFR-TKI small molecule EGFR tyrosine kinase inhibitors
  • Two major categories, and EGFR-TKI drugs are divided into reversible inhibitors and irreversible inhibition due to the different ways of binding drugs to EGFR tyrosine kinases.
  • Irreversible inhibitors can be irreversibly permanent with protein tyrosine kinase
  • the level of protein tyrosine kinase is continuously reduced unless a new protein tyrosine kinase is produced. Irreversible inhibitors take longer to work.
  • the bioavailability of the existing clinically developed drug afatinib is only 11.175%.
  • afatinib is not available in the 10 mg/kg dose group. effect.
  • the MTD of afatinib is 30 mg/kg (see Li D, Ambrogio L, Shimamura T, et al.
  • BIBW2992 an irreversible EGFR/HER2 inhibitor highly effective in preclinical lung cancer models. Oncogene, 2008, 27 (34): 4702-4711)), it can be seen that the therapeutic window of afatinib is very narrow.
  • One aspect of the present application relates to N-[4-(3-chloro-4-fluorophenylamino)-7-(2-methoxyethoxy)quinazolin-6-yl]-2-[1-( 2-methoxyethyl)piperidin-4-ylidene]acetamide and pharmaceutically acceptable salts thereof.
  • Another aspect of the present application relates to N-[4-(3-chloro-4-fluorophenylamino)-7-(2-methoxyethoxy)quinazolin-6-yl]-2-[1- (2-methoxyethyl)piperidin-4-ylidene]acetamide and a pharmaceutically acceptable salt thereof, wherein the acid forming the salt is selected from the group consisting of hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid , 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, methanesulfonic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonate Acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclohe
  • Still another aspect of the present application relates to N-[4-(3-chloro-4-fluorophenylamino)-7-(2-methoxyethoxy)quinazolin-6-yl]-2-[1- (2-methoxyethyl)piperidin-4-ylidene]acetamide and a pharmaceutically acceptable salt thereof, wherein the salt is selected from the group consisting of:
  • a further aspect of the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising N-[4-(3-chloro-4-fluorophenylamino)-7-(2-methoxyethoxy)quinazolin-6-yl] -2-[1-(2-Methoxyethyl)piperidin-4-ylidene]acetamide or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent or excipient.
  • a further aspect of the present application relates to the preparation of N-[4-(3-chloro-4-fluorophenylamino)-7-(2-methoxyethoxy)quinazolin-6-yl]-2-[1 -(2-Methoxyethyl)piperidin-4-ylideneacetamide or a pharmaceutically acceptable salt thereof, comprising:
  • the compound of the formula (I) is reacted with a compound of the formula (II) to give the compound N-[4-((3-chloro-4-fluorophenylamino))-7-((2-methoxy) Ethoxy))quinazolin-6-yl]-2-[1-((2-methoxyethyl))piperidin-4-ylidene]acetamide, preferably represented by formula (I)
  • the compound is converted into an activated ester, an acid chloride, an acylated imidazole or a mixed anhydride, and then reacted with a compound represented by the formula (II), more preferably a tertiary amine such as triethylamine, N-methylmorpholine, trimethylamine, pyridine or Take The substituted pyridine is used as a catalyst, and preferably the compound represented by the formula (I) is converted into an acid chloride using chlorochlorine, phosphorus trichloride, phosphorus pen
  • the compound N-[4-((3-chloro-4-fluorophenylamino))-7-((2-methoxyethoxy))quinazolin-6-yl]-2 -[1-((2-Methoxyethyl))piperidin-4-ylidene]acetamide reacts with its pharmaceutically acceptable acid to form its corresponding pharmaceutically acceptable salt
  • Another aspect of the present application relates to a method of treating or preventing a disease associated with a protein kinase comprising administering to a subject in need of such a method a therapeutically or prophylactically effective amount of N-[4-(3-chloro-4-fluorophenylamino) 7-(2-methoxyethoxy)quinazolin-6-yl]-2-[1-(2-methoxyethyl)piperidin-4-ylideneacetamide and its pharmacy Acceptable salt.
  • a further aspect of the present invention relates to a method of treating or preventing a physiological abnormality caused by overexpression of a protein tyrosine phosphorylase in a mammal, comprising administering to a mammal in need thereof a therapeutically or prophylactically effective amount of N-[4 -(3-chloro-4-fluorophenylamino)-7-(2-methoxyethoxy)quinazolin-6-yl]-2-[1-(2-methoxyethyl)per Pyridin-4-ylidene]acetamide and pharmaceutically acceptable salts thereof.
  • Another aspect of the present application relates to N-[4-(3-chloro-4-fluorophenylamino)-7-(2-methoxyethoxy)quinazolin-6-yl]-2-[1- Use of (2-methoxyethyl)piperidin-4-ylidene]acetamide and a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment or prevention of a disease associated with a protein kinase, preferably for treatment or Use in a medicament for preventing physiological abnormalities caused by excessive expression of protein tyrosine phosphorylase in a mammal.
  • Yet another aspect of the present application relates to comprising N-[4-((3-chloro-4-fluorophenylamino))-7-((2-methoxy) Ethyloxy))quinazolin-6-yl]-2-[1-((2-methoxyethyl))piperidin-4-ylidene]acetamide or a pharmaceutically acceptable salt thereof
  • a pharmaceutical composition for the preparation of a medicament for treating or preventing a disease associated with a protein kinase, preferably for the preparation of a medicament for treating or preventing a physiological abnormality caused by overexpression of a protein tyrosine phosphorylase in a mammal .
  • references to “an embodiment” or “an embodiment” or “in another embodiment” or “in certain embodiments” throughout this specification are meant to be included in the at least one embodiment.
  • the appearances of the phrase “in one embodiment” or “in an embodiment” or “in another embodiment” or “in some embodiments” are not necessarily all referring to the same embodiment.
  • the particular elements, structures, or characteristics may be combined in any suitable manner in one or more embodiments.
  • “Pharmaceutically acceptable carrier, diluent or excipient” includes, but is not limited to, any adjuvant, carrier, excipient, glidant, which has been approved by the U.S. Food and Drug Administration for use in humans or animals, Sweeteners, diluents, preservatives, dyes/colorants, flavor enhancers, surfactants, wetting agents, dispersing agents, suspending agents, stabilizers, isotonic agents, solvents or emulsifiers The composition has various forms of carriers without side effects.
  • “Pharmaceutically acceptable salt” refers to those salts which retain the biological effectiveness and properties of the free base, which are biologically or otherwise suitable and which are formed using inorganic or organic acids
  • the inorganic acid is, for example but not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc., such as, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, Alginic acid, ascorbic acid, aspartic acid, methanesulfonic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, citric acid, caproic acid, caprylic acid, carbonic acid, cinnamon Acid, citric acid, cyclohexanesulfamic acid, dodecyl sulfate, ethane-1,2-disulfonic acid, ethanesul
  • “Pharmaceutical composition” refers to a formulation of a compound of the present invention and a medium which is generally accepted in the art for delivery of a biologically activating compound to a mammal such as a human.
  • Such media include all pharmaceutically acceptable carriers, diluents or excipients.
  • “Therapeutically effective amount” means an amount of a compound of the present invention, when administered to a mammal, such as a human, sufficient to treat (as defined below) a mammalian protein tyrosine phosphorylase-mediated disease or condition, such as a human.
  • the amount of the compound of the present application which constitutes a “therapeutically effective amount” will vary depending on the compound, the state of the disease and its severity, and the age of the mammal to be treated, but those skilled in the art will be able to rely on their own knowledge and the present disclosure. The formula determines the amount of the compound of the present application.
  • treating encompasses a treatment-related disease or condition in a mammal, such as a human, having a related disease or condition, and includes:
  • disease and “disease state” may be used interchangeably or may be different, as a particular disease or condition may not have a known causative agent (and therefore cannot be explained by etiology). Therefore, it is not recognized as a disease, but is considered to be an undesired disease state or condition in which the clinician has identified or Or less specific symptoms of the series.
  • One aspect of the present application relates to N-[4-(3-chloro-4-fluorophenylamino)-7-(2-methoxyethoxy)quinazolin-6-yl]-2-[1-( 2-methoxyethyl)piperidin-4-ylidene]acetamide and pharmaceutically acceptable salts thereof.
  • Another aspect of the present application relates to N-[4-(3-chloro-4-fluorophenylamino)-7-(2-methoxyethoxy)quinazolin-6-yl]-2-[1- (2-methoxyethyl)piperidin-4-ylidene]acetamide and a pharmaceutically acceptable salt thereof, wherein the acid forming the salt is selected from the group consisting of hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid , 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, methanesulfonic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonate Acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclohe
  • Still another aspect of the present application relates to N-[4-(3-chloro-4-fluorophenylamino)-7-(2-methoxyethoxy)quinazolin-6-yl]-2-[1- (2-methoxyethyl)piperidin-4-ylidene]acetamide and a pharmaceutically acceptable salt thereof, wherein the salt is selected from the group consisting of:
  • a further aspect of the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising N-[4-(3-chloro-4-fluorophenylamino)-7-(2-methoxyethoxy)quinazolin-6-yl] -2-[1-(2-Methoxyethyl)piperidin-4-ylidene]acetamide or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent or excipient.
  • Examples of pharmaceutically acceptable carriers that can be used in the pharmaceutical compositions of the present application include, but are not limited to, any adjuvants, carriers, excipients, helpers that have been approved by the U.S. Food and Drug Administration for use in humans or animals.
  • Agents, sweeteners, diluents, preservatives, dyes/colorants, flavor enhancers, surfactants, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, solvents or emulsifiers The various forms of the carrier that make up the pharmaceutical composition without side effects.
  • the pharmaceutical compositions of the present application can be prepared as tablets, solutions, granules, patches, ointments for parenteral, transdermal, mucosal, nasal, buccal, sublingual or oral use. , capsules, aerosols or suppositories.
  • the pharmaceutical composition of the present application can be administered orally, orally, intravenously, intraperitoneally, subcutaneously, intramuscularly, nasally, by eye, or by suction. Intra, anal, vaginal or epidermal administration.
  • a further aspect of the present application relates to the preparation of N-[4-(3-chloro-4-fluorophenylamino)-7-(2-methoxyethoxy)quinazolin-6-yl]-2-[1 -(2-Methoxyethyl)piperidin-4-ylideneacetamide or a pharmaceutically acceptable salt thereof, comprising:
  • the compound of the formula (I) is reacted with a compound of the formula (II) to give the compound N-[4-((3-chloro-4-fluorophenylamino))-7-((2-methoxy) Ethoxy))quinazolin-6-yl]-2-[1-((2-methoxyethyl))piperidin-4-ylidene]acetamide, preferably represented by formula (I)
  • the compound is converted into an activated ester, an acid chloride, an acylated imidazole or a mixed anhydride, and then reacted with a compound represented by the formula (II), more preferably a tertiary amine such as triethylamine, N-methylmorpholine, trimethylamine, pyridine or The substituted pyridine is used as a catalyst, and it is preferred that the compound represented by the formula (I) is converted to an acid chloride using sulfuric acid, phosphorus trichloride, phosphorus pen
  • the compound N-[4-((3-chloro-4-fluorophenylamino))-7-((2-methoxyethoxy))quinazolin-6-yl]-2 -[1-((2-methoxyethyl))piperidin-4-ylidene]acetamide is reacted with its pharmaceutically acceptable acid to form its corresponding pharmaceutically acceptable salt;
  • the compound of formula (II)) is prepared according to the method described in U.S. Patent Publication No. US 2002/077330 A1.
  • the compound of formula (I)) is prepared as follows:
  • the aqueous layer was extracted three more times with dichloromethane, 3 L of dichloromethane each time. Discard the water layer. The organic layers were combined, dried over MgSO. The filter cake was discarded by suction; the other 2.2 L of the reaction solution was treated in the same manner as above, and the filtrate was combined, and the filtrate was concentrated by rotary evaporation. The concentrates were combined to give an oil.
  • the concentrated hydrochloric acid was slowly added dropwise to a pH of 2 at about 0 ° C, the cooling was stopped, the stirring was continued for 30 minutes at room temperature, and the mixture was filtered with suction, and the filter cake was rinsed with 500 ml of anhydrous ethanol and then drained. Filtrate The mixture was concentrated by evaporation in a water bath at 50 ° C until no liquid was evaporated to give a yellow oil, and a large amount of white crystals precipitated. After adding 1.46 L of absolute ethanol, stirring at room temperature for 15 minutes, suction filtration, and the filter cake was rinsed with 500 ml of anhydrous ethanol and then drained and stored. The filtrate was concentrated by steaming at 50 ° C until no liquid was distilled off to give a pale yellow thick porridge.
  • Recrystallization of porridge Add 1.46L of isopropanol, stir and reflux in a water bath at 85 ° C, remove a small amount of insoluble residue by hot filtration, transfer to a 3L three-necked bottle, put in a water bath at 85 ° C, turn off the heat, naturally cool, and stir overnight.
  • N-[4-(3-chloro-4-fluorophenylamino)-7-(2-methoxyethoxy)quinazolin-6-yl]-2-[1 -(2-Methoxyethyl)piperidin-4-ylidene]acetamide is reacted with an acid to give the corresponding pharmaceutically acceptable salt.
  • Illustrative examples of the acid of the pharmaceutically acceptable salt of -(2-methoxyethyl)piperidin-4-ylidene]acetamide include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid.
  • Another aspect of the present application relates to a method of treating or preventing a disease associated with a protein kinase comprising administering to a subject in need of such a method a therapeutically or prophylactically effective amount of N-[4-(3-chloro-4-fluorophenylamino) )-7-(2-methoxyethoxy)quinazolin-6-yl]-2-[1-(2-methoxy B) Basepiperidin-4-ylidene]acetamide and pharmaceutically acceptable salts thereof.
  • the disease is cancer.
  • the individual is a mammal.
  • the mammal is a human.
  • a further aspect of the present invention relates to a method of treating or preventing a physiological abnormality caused by overexpression of a protein tyrosine phosphorylase in a mammal, comprising administering to a mammal in need thereof a therapeutically or prophylactically effective amount of N-[4 -(3-chloro-4-fluorophenylamino)-7-(2-methoxyethoxy)quinazolin-6-yl]-2-[1-(2-methoxyethyl)per Pyridin-4-ylidene]acetamide and pharmaceutically acceptable salts thereof.
  • overexpression of EGFR or Her-2 causes a physiological abnormality.
  • the physiological abnormality is cancer.
  • the mammal is a human.
  • Another aspect of the present application relates to N-[4-(3-chloro-4-fluorophenylamino)-7-(2-methoxyethoxy)quinazolin-6-yl]-2-[1- Use of (2-methoxyethyl)piperidin-4-ylidene]acetamide and a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment or prevention of a disease associated with a protein kinase, preferably for treatment or Use in a medicament for preventing physiological abnormalities caused by excessive expression of protein tyrosine phosphorylase in a mammal.
  • the physiological abnormality is caused, inter alia, by overexpression of EGFR.
  • the disease that overexpresses a physiological abnormality caused by a protein tyrosine phosphorylase and is effective by a method of inhibiting protein tyrosine phosphorylase activity is cancer.
  • cancers to be treated or prevented by the salt include, but are not limited to, breast cancer, head and neck cancer, lung cancer (including non-small cell lung cancer, small cell lung cancer), colon cancer, pancreatic cancer, esophageal cancer, gastric cancer, skin cancer, and intestine Cancer, kidney cancer, bladder cancer, ovarian cancer, oral cancer, laryngeal cancer, cervical cancer, liver cancer and prostate cancer.
  • the mammal is a human.
  • a unit dose of from about 0.1 mg to about 1000 mg of N-[4-(3-chloro-4-fluorophenylamino)-7-(2-methoxyethoxy) quinazoline is administered.
  • Unit dosages as used in this application refer to a unit that can be administered to a patient and that is easy to handle and package, ie, a single dose.
  • a unit dose of from about 1 mg to about 1000 mg of N-[4-(3-chloro-4-fluorophenylamino)-7-(2-methoxyethoxy)quinazoline is administered.
  • a unit dose of from about 10 mg to about 500 mg of N-[4-(3-chloro-4-fluorophenylamino)-7-(2-methoxyethoxy)quinazoline is administered.
  • the use thereof preferably treats or prevents physiological abnormalities caused by overexpression of protein tyrosine phosphorylase in a mammal.
  • a further aspect of the present invention relates to N-[4-(3-chloro-4-fluorophenylamino)-7-(2-methoxyethoxy)quinazolin-6-yl]-2-[1- Use of (2-methoxyethyl)piperidin-4-ylidene]acetamide and a pharmaceutically acceptable salt thereof for the preparation of a medicament for treating or preventing a disease associated with a protein kinase, preferably for inhibiting lactation Use of a drug for protein tyrosine phosphorylase activity in an animal.
  • the mammal is a human.
  • Another aspect of the present application relates to N-[4-(3-chloro-4-fluorophenylamino)-7-(2-methoxyethoxy)quinazolin-6-yl]-2-[1- (2-methoxyethyl)piperidin-4-ylidene]acetamide and its pharmacy Use of a pharmaceutical composition of an acceptable salt for the preparation of a medicament for treating or preventing a disease associated with a protein kinase, preferably for the treatment or prevention of physiological abnormalities caused by overexpression of a protein tyrosine phosphorylase in a mammal Use in medicine.
  • N-[4-(3-chloro-4-fluorophenylamino)-7-(2-methoxyethoxy)quinazolin-6-yl]-2-[1 -(2-Methoxyethyl)piperidin-4-ylidene]acetamide and a pharmaceutically acceptable salt thereof are prepared for use in the treatment or prevention of EGFR and/or Her-2 in a mammal Overexpression causes the physiological abnormality.
  • the physiological abnormality is caused, inter alia, by overexpression of EGFR.
  • the disease that overexpresses a physiological abnormality caused by a protein tyrosine phosphorylase and is effective by a method of inhibiting protein tyrosine phosphorylase activity is cancer.
  • cancers to be treated or prevented include, but are not limited to, breast cancer, head and neck cancer, lung cancer (including non-small cell lung cancer, small cell lung cancer), colon cancer, pancreatic cancer, esophageal cancer, gastric cancer, skin cancer, intestinal cancer, kidney cancer, bladder cancer, ovarian cancer, oral cancer, laryngeal cancer, cervical cancer, liver cancer and prostate cancer.
  • the mammal is a human.
  • the unit dosage is from about 0.1 mg to about 1000 mg of N-[4-(3-chloro-4-fluorophenylamino)-7-(2-methoxyethoxy)quine.
  • a pharmaceutical composition of oxazoline-6-yl]-2-[1-(2-methoxyethyl)piperidin-4-ylideneacetamide or a pharmaceutically acceptable salt thereof for the preparation of a therapeutic or prophylactic For use in a drug for a protein kinase-related disease, it is preferred to use a drug for inhibiting the activity of a protein tyrosine phosphorylase in a mammal.
  • the unit dosage is from about 1 mg to about 1000 mg of N-[4-(3-chloro-4-fluorophenylamino)-7-(2-methoxyethoxy) quinazole.
  • a pharmaceutical composition of oxa-6-yl]-2-[1-(2-methoxyethyl)piperidin-4-ylideneacetamide or a pharmaceutically acceptable salt thereof for the treatment or prevention of protein kinases The use in the drug of the related disease is preferably a drug which inhibits the activity of protein tyrosine phosphorylase in a mammal.
  • the unit dosage is from about 10 mg to about 500 mg of N-[4-(3-chloro-4-fluorophenylamino)-7-(2-methoxyethoxy) quinazoline.
  • the 20 L reactor was kept under argon. 250.0 g (1.062 mol) of 2-[1-(2-methoxyethyl)-piperidin-4-ylidene acetate hydrochloride, 1.46 L of re-hydrogenated tetrahydrofuran and 1.46 ml of chromatographically pure N,N- Dimethylformamide was added to the reaction vessel, stirred, and cooled to 0 ° C. 128.1 g (1.009 mol, 86.7 ml) of oxalyl chloride was slowly added dropwise, and the process was kept stable at ⁇ 3 ° C during the dropwise addition.
  • reaction system was stirred normally by adding appropriate amount of water, the water bath was removed, stirred at room temperature for 2 hours, filtered, and the filter cake was rinsed with distilled water until The liquid was dropped to a pH of about 7, and the pale pink solid was dried to N-[4-(3-chloro-4-fluorophenylamino)-7-(2-methoxyethoxy)quinazoline-6.
  • N-[4-(3-Chloro-4-fluorophenylamino)-7-(2-methoxyethoxy)quinazolin-6-yl]-2-[1-(2-methoxy 20 g of crude ethyl) piperidine-4-ylidene]acetamide and 250 ml of isopropanol were added to a 1 L autoclave, mechanically stirred, and heated under reflux at 88 ° C for 3 hours. The heating was stopped and kept stirring overnight.
  • MS Mass spectrometry (MS) detection (instrument model: 6410B LC-MS; Agilent), MS: 544.2 [M+H] + peak.
  • MS Mass spectrometry (MS) detection (instrument model: 6410B LC-MS; Agilent) MS: [M+H] + peak at 544.1.
  • MS Mass spectrometry (MS) detection (instrument model: 6410B LC-MS; Agilent) MS: 544 [M+H-196]+ peak.
  • Dosage Drug concentration Dosing volume Number of animals Tail vein injection (iv) 20mg/kg 5mg/ml 4ml/kg 6 Stomach (ig) 20mg/kg 2.5mg/ml 8ml/kg 6
  • rats Twelve rats were randomly divided into two groups, 6 in each group. The rats were fasted for 12 hours before the administration. After taking about 0.5 ml of blank blood, they were intragastrically injected into the tail vein and injected with N-[4-(3-chloro-).
  • D is the dose
  • AUC the area under the blood concentration-time curve
  • ig means intragastric administration
  • iv means intravenous administration.
  • N-[4-(3-Chloro-4-fluorophenylamino)-7-(2-methoxyethoxy)quinazolin-6-yl]-2-[1-(2-methoxy Ethyl) piperidine-4-ylidene]acetamide dihydrochloride has a bioavailability of 56.87% and has good pharmacokinetic properties.
  • D is the dose
  • AUC the area under the blood concentration-time curve
  • ig means intragastric administration
  • iv means intravenous administration.
  • N-[4-(3-Chloro-4-fluorophenylamino)-7-(2-methoxyethoxy)quinazolin-6-yl]-2-[1-(2-methoxy Ethyl) piperidine-4-ylidene]acetamide dihydrobromide has a bioavailability of 42.76% and has good pharmacokinetic properties.
  • Experimental animals 8 Wistar rats, male, weighing 160-190 g, were purchased from the Experimental Animal Center of the Chinese Academy of Military Medical Sciences. License number: SCXK (Army) 2012-0004; Certificate No.: 0307074.
  • the total volume refers to the volume of the solution obtained after the configuration is completed.
  • mice Eight rats were fasted to avoid water before 12 hours of administration, and were randomly divided into two groups, 4 in each group. After administration of the drugs according to the dosage regimen, they were administered before and 5 minutes, 0.5 hours, and 1 hour after administration.
  • acetic acid Alcohol containing 20 ng / ml internal standard irbesartan
  • phase A 5 mM aqueous solution of ammonium formate
  • phase B methanol
  • API3000LC-MS/MS; ESI source; MRM positive ion scan, parameters are as follows:
  • D is the dose
  • AUC the area under the blood concentration-time curve
  • ig means intragastric administration
  • iv means intravenous administration.
  • N-[4-(3-Chloro-4-fluorophenylamino)-7-(2-methoxyethoxy)quinazolin-6-yl]-2-[1-(2-methoxy Ethyl)piperidin-4-ylidene]acetamide has a bioavailability of 23.9% and has good pharmacokinetic properties.
  • Experimental animals 12 SD rats, male and female, 7-10 weeks old, weighing 200-350g, purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., raised in the animal room of GLP Center, No. 27 Taiping Road .
  • the production license number of Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. is SCXK (Beijing) 2009-0002
  • Liquid phase conditions A: aqueous solution (5 mmol/L ammonium acetate, 0.2% formic acid), B: methanol; column temperature 25 ° C; injection amount: 10 ⁇ L; gradient elution. Gradient conditions: phase A: 5 mM ammonium acetate (containing 0.2% formic acid); phase B: methanol; 0 to 0.5 min, phase A 0.21 mL/min, phase B 0.09 mL/min; 0.5-1 min, phase A linearly reduces flow rate to 0mL/min, phase B linearly increased flow rate to 0.3mL/min; 1.01min, phase B flow rate increased to 0.5mL/min for 1min; 2.01min, phase A flow rate increased to 0.21mL/min, phase B flow rate decreased 0.09 mL/min, maintaining the ratio for 2 min, equilibrated to the initial flow rate ratio.
  • A aqueous solution (5 mmol/L ammonium acetate, 0.2% formic acid)
  • B m
  • Mass spectrometry conditions ion source: Turbo Ionspray (ESI+); detection mode: MRM; electrical parameters: Compound 1: m/z 544.2 - 457.1, CE (collision energy): 36.5.
  • D is the dose
  • AUC the area under the blood concentration-time curve
  • ig means intragastric administration
  • iv means intravenous administration.
  • N-[4-(3-Chloro-4-fluorophenylamino)-7-(2-methoxyethoxy)quinazolin-6-yl]-2-[1-(2-methoxy Ethyl) piperidin-4-ylidene]acetamide dimethanesulfonate has a bioavailability of 56.9% and has good pharmacokinetic properties.
  • mice male ICR mice, weighing 19-22 g, purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., animal license number SCXK (Beijing) 2007-0001
  • mice were divided into 9 groups, 1 in the first, fourth and sixth groups, 4 in the second, fifth and eighth groups, and 2 in the third, sixth and ninth groups.
  • Compound 4, Compound 2, and Compound 12 were dissolved in water for injection.
  • Compound 4 in the first to third groups of tail veins N-[4-(3-chloro-4-fluorophenylamino)-7-(2-methoxyethoxy)quinazolin-6-yl ]-2-[piperidin-4-ylidene]acetamide dihydrochloride at doses of 100 mg/kg, 150 mg/kg, 200 mg/kg, respectively. After administration Animal reactions and body weight changes were observed, and anatomical observation was performed 14 days later.
  • N-[4-((3-chloro-4-fluorophenylamino))-7-((2-methoxyethoxy))quinazolin-6-yl]-2-[1-(( 2-Methoxyethyl)) piperidin-4-ylidene]acetamide dihydrochloride is more resistant and less toxic and is a more preferred clinical choice.
  • pY-20 murine anti-phosphotyrosine antibody-HRP (horseradish peroxidase): Invitrogen, Cat. No. 03-7720
  • TMB (3,3',5,5'-tetramethylbenzidine, HRP substrate): eBioscience, Cat. No. 00-4201-56
  • a 96-well microtiter plate was coated with 0.2 mg/ml pGT (as a substrate for the enzyme) in PBS (phosphate buffer) at 4 ° C overnight, unbound pGT wash solution (containing 0.05% Tween) Wash off -20 PBS); dry at room temperature for 2 hours.
  • pGT as a substrate for the enzyme
  • a positive solvent control group (PC) containing no compound and a negative solvent control group (NC) containing no ATP and compound were used, and the inhibition rate of the compound was obtained by comparing the OD value of the administration group with the OD value of the solvent control group.
  • the coordinates are distributed in logarithmic form, and the mean inhibition rate is plotted on the ordinate.
  • the curve is fitted with Logistic 4 parameter equation.
  • the concentration of the compound corresponding to the 50% inhibition rate on the curve is the IC 50 value.
  • the final concentration of the compound is 12.5 nM, which is configured to a concentration of 50-fold concentration, ie 625 nM.
  • the 50-fold concentration of the compound working solution was diluted 4-fold with DMSO in a 96-well plate, and a total of 7 concentrations were diluted.
  • the kinase was formulated in a kinase buffer to a 2.5-fold concentration of the enzyme solution.
  • Inhibition rate % (max - conversion) / (max - min) x 100%, where max is the conversion of the DMSO control and min is the conversion of the enzyme-free control.
  • the IC 50 value was determined from the inhibition curve.
  • N-[4-(3-Chloro-4-fluorophenylamino)-7-(2-methoxyethoxy)quinazolin-6-yl]-2-[1-(2-methoxy Ethyl)piperidin-4-ylidene]acetamide and its pharmaceutically acceptable salts are highly selective tyrosine kinase irreversible inhibitors, especially for EGFR, and are more preferred clinical options.
  • Human epidermal cancer cell line A431, human non-small cell lung cancer cell line HCC827, head and neck cancer cell line Fadu and human pancreatic cancer cell line AsPC-1 purchased from the Chinese Academy of Sciences cell bank.
  • RPMI-1640 (a name for the medium): Gibco, Cat. No. 31800-022. This was a dry powder, which was formulated into a liquid medium according to the instructions, and 2 g/L of NaHCO 3 and 5.958 g/L of HEPEs were added according to the cell culture.
  • EMEM Minimum Essential Medium with Earle's salts: Gibco, Inc., Item No. 41500-034. This was a dry powder, which was formulated into a liquid medium according to the instructions, and 2.2 g/L of NaHCO 3 and 5.958 g/L of HEPEs were added according to the cell culture.
  • DMEM Dulbecco's Modified Eagle's Medium: Gibco, Cat. No. 12800-017. This was a dry powder, which was formulated into a liquid medium according to the instructions, and 2.2 g/L of NaHCO 3 and 5.958 g/L of HEPEs were added according to the cell culture.
  • F-12K Nutrient Mixture F12Ham Kaighn's Modification: Sigma-Aldrich, Inc., item number N3520. This was a dry powder, which was formulated into a liquid medium according to the instructions, and 1.5 g/L of NaHCO 3 and 2.383 g/L of HEPEs were added according to the cell culture.
  • F-12 (Ham's F-12 Nutrient Mixture): Gibco, Cat. No. 21700-075. This was a dry powder, which was formulated into a liquid medium according to the instructions, and 1.76 g/L NaHCO 3 and 2.383 g/L HEPEs were added according to the cell culture.
  • FBS fetal calf serum
  • Hyclone, Inc. item number SV30087.
  • MTT tetrazolium salt
  • Sigma article number M5655.
  • PBS phosphate buffer
  • SRB sulfonyl rhodamine
  • Sigma-Aldrich article number S9012.
  • CO 2 cell incubator Thermo, 311, 371.
  • Ultra-clean platform Donglianhal Instrument Manufacturing Co., Ltd., DL-CJ-2N type.
  • Microplate reader Bio-Rad, 680.
  • 96-well plates Inoculate 96-well cell culture plates (hereinafter referred to as 96-well plates) with a certain number of cells, add different concentrations of compounds, co-culture for a period of time (generally 3 days), and finally determine the total amount of cellular proteins in the test wells by SRB method. Or measure cell viability by MTT assay.
  • MTT assay for cell viability aspirate the medium, add basal medium containing 0.5 mg/ml MTT (generally a medium containing no fetal bovine serum (FBS) and other additives) 100 ⁇ l/well, continue to culture 3 Hour; the basal medium containing MTT was aspirated, and 100 ⁇ l/well of DMSO was added to dissolve the formazan, and the detection was carried out at a wavelength of 490 nm.
  • basal medium containing 0.5 mg/ml MTT generally a medium containing no fetal bovine serum (FBS) and other additives
  • FBS fetal bovine serum
  • the concentration of DMSO tolerant cells was determined by the DMSO tolerance test, and the compound dilution and addition methods were selected accordingly.
  • the DMSO tolerance test measures the effect of different concentrations of DMSO on cell growth. The definition of tolerance is that cell growth is affected by no more than 20%. The two final methods are described as follows:
  • Method 1 Weigh 1-2 mg of test compound into 2 mM stock solution in DMSO, dilute to 20 ⁇ M in basal medium (or adjusted according to the test) and dilute in 3-fold gradient to keep the DMSO concentration unchanged during the dilution process. Eight concentration groups were obtained; the test wells were added with 80 ⁇ l of complete medium and 20 ⁇ l of 10 times concentration of the compound working solution, and finally the volume per well was 200 ⁇ l, and the DMSO concentration was 0.1%.
  • the cell line involved is HCC827.
  • Method 2 1-2 mg of the test compound was weighed and dissolved in DMSO to a 20 mM stock solution. The initial concentration was adjusted according to the test requirements, and then diluted with DMSO for 3 times to obtain a compound solution, a total of 8 concentrations; 99 ⁇ l of complete medium and 1 ⁇ l of compound solution were added to each test well, and finally the volume per well was 200 ⁇ l, and the DMSO concentration was 0.5%.
  • the cell lines involved are A431, Fadu, and AsPC-1.
  • the mean inhibition rate and SD were obtained from the repeated test wells.
  • the compound concentration was plotted on the abscissa and in logarithmic form.
  • the mean inhibition rate was plotted on the ordinate.
  • the curve was fitted with the Logistic 4 parameter equation.
  • the 50% inhibition rate on the curve corresponds.
  • the concentration of the compound is the IC 50 value.
  • N-[4-((3-chloro-4-fluorophenylamino))-7-((2-methoxyethoxy))quinazolin-6-yl]-2-[1-(( 2-methoxyethyl)) piperidin-4-ylidene]acetamide and pharmaceutically acceptable salts thereof are Highly selective irreversible inhibitor of tyrosine kinase, especially high inhibitory activity against EGFR.
  • Tumor cells were cultured in an MEM medium containing inactivated 10% fetal calf serum, 100 U/ml penicillin, and 100 ⁇ g/ml streptomycin in a 37 ° C, 5% CO 2 incubator.
  • the tumor cells in the logarithmic growth phase were collected, and the cells were adjusted to a suitable density, and injected into the skin of nude mice 0.2 ml/body. After the tumor was formed, it was passaged three times in nude mice to be used for the establishment of the allograft model. .
  • the tumor-bearing mice were sacrificed by cervical dislocation, and the tumor pieces were taken out under sterile conditions to cut into small tumor pieces of about 2 mm ⁇ 2 mm ⁇ 2 mm, and inoculated into the nude mice with a trocar.
  • the experimental animals were randomly divided into the following 5 groups, solvent control group, N-[4-(3-chloro-4-fluorophenylamino)-7- (2-methoxyethoxy)quinazolin-6-yl]-2-[1-(2-methoxyethyl)piperidin-4-ylidene]acetamide dimethanesulfonate 20
  • the 40 and 80 mg/kg dose groups and the positive control drug Tarceva 50 mg/kg dose group, 8 experimental animals per group.
  • Each group was administered by intragastric administration, once a day for 14 days, and on the day of grouping on day 0.
  • RTV relative tumor volume
  • V 0 the tumor volume measured at the time of group administration (ie, d0)
  • V t per Tumor volume at the time of secondary measurement.
  • the relative tumor proliferation rate T/C was calculated as the relative tumor volume
  • T is the average value of the tumor volume relative to the treatment group
  • C is the average value of the solvent control group relative to the tumor volume.
  • Tumor inhibition rate (%) (average tumor weight of the solvent control group - mean tumor weight of the treatment group) / mean tumor weight of the solvent control group ⁇ 100%.
  • Percent change in body weight W n /W 0 ⁇ 100% (W n : average body weight of experimental animals in each group on day n, W 0 : average body weight of experimental animals in each group on day 0).
  • the One-Way Anova test was performed using SPSS 13.0 for statistical analysis between groups.
  • Compound 33 dose groups can inhibit tumor growth, T/C was 30.6%, 37.7% and 19.1%, respectively, showing a good dose-effect relationship.
  • the positive control group T/C was 47.3%.
  • the inhibitory rates of compound 33 dose groups on A431 human epidermal carcinoma xenograft model were 68.6%, 73.2% and 85.6%, respectively, showing a good dose-effect relationship.
  • the positive control group had a tumor inhibition rate of 62.0%.
  • N-[4-((3-chloro-4-fluorophenylamino))-7-((2-methoxyethoxy))quinazolin-6-yl]-2-[1-(( 2-Methoxyethyl)) piperidin-4-ylidene] acetamide dimethanesulfonate has good antitumor activity and a wider therapeutic window, and is a more preferred clinical choice.
  • Tumor cells were cultured in an MEM medium containing inactivated 10% fetal calf serum, 100 U/ml penicillin, and 100 ⁇ g/ml streptomycin in a 37 ° C, 5% CO 2 incubator.
  • the tumor cells in the logarithmic growth phase were collected, and the cells were adjusted to a suitable density, and injected into the skin of nude mice 0.2 ml/body. After the tumor was formed, it was passaged three times in nude mice to be used for the establishment of the allograft model. .
  • mice were sacrificed by cervical dislocation, and the tumor pieces were taken out under sterile conditions to cut into small tumor pieces of about 2 mm ⁇ 2 mm ⁇ 2 mm, and inoculated into the right scapula of the nude mice with a trocar.
  • the experimental animals were randomly divided into the following 5 groups, solvent control group, N-[4-(3-chloro-4-fluorophenylamino)-7- (2-methoxyethoxy)quinazolin-6-yl]-2-[1-(2-methoxyethyl)piperidin-4-ylidene]acetamide dimethanesulfonate 20
  • the 40 and 80 mg/kg dose groups and the positive control drug Tarceva 50 mg/kg dose group, 8 experimental animals per group.
  • Each group was administered by intragastric administration, once a day for 14 days, and on the day of grouping on day 0.
  • RTV relative tumor volume
  • V 0 the tumor volume measured at the time of group administration (ie, d0)
  • V t per Tumor volume at the time of secondary measurement.
  • the relative tumor proliferation rate T/C was calculated as the relative tumor volume
  • T is the average value of the tumor volume relative to the treatment group
  • C is the average value of the solvent control group relative to the tumor volume.
  • Tumor inhibition rate (%) (average tumor weight of the solvent control group - mean tumor weight of the treatment group) / mean tumor weight of the solvent control group ⁇ 100%.
  • Percent change in body weight W n /W 0 ⁇ 100% (W n : average body weight of experimental animals in each group on day n, W 0 : average body weight of experimental animals in each group on day 0).
  • the One-Way Anova test was performed using SPSS 13.0 for statistical analysis between groups.
  • the T/C of each dose of Compound 3 was 61.4%, 54.7% and 31.6%, respectively.
  • Each dose group had good inhibitory activity against FaDu human head and neck cancer xenograft model.
  • the tumor inhibition rates of Compound 3 in each dose group were 37.5%, 52.4% and 76.2%, respectively.
  • Compound 3 groups had good inhibitory activity against FaDu human head and neck cancer xenograft model.
  • the dose of Tarceva administered is MTD.
  • N-[4-((3-chloro-4-fluorophenylamino))-7-((2-methoxyethoxy))quinazolin-6-yl]-2-[1-(( 2-Methoxyethyl)) piperidin-4-ylidene] acetamide dimethanesulfonate has good antitumor activity and a wider therapeutic window, and is a more preferred clinical choice.
  • Tumor cells were cultured in an MEM medium containing inactivated 10% fetal calf serum, 100 U/ml penicillin, and 100 ⁇ g/ml streptomycin in a 37 ° C, 5% CO 2 incubator.
  • the tumor cells in the logarithmic growth phase were collected, and the cells were adjusted to a suitable density, and injected into the skin of nude mice 0.2 ml/body. After the tumor was formed, it was passaged three times in nude mice to be used for the establishment of the allograft model. .
  • mice were sacrificed by cervical dislocation, and the tumor pieces were taken out under sterile conditions to cut into small tumor pieces of about 2 mm ⁇ 2 mm ⁇ 2 mm, and inoculated into the right scapula of the nude mice with a trocar.
  • the experimental animals were randomly divided into the following 5 groups, solvent control group, N-[4-(3-chloro-4-fluorophenylamino)-7- (2-methoxyethoxy)quinazolin-6-yl]-2-[1-(2-methoxyethyl)piperidin-4-ylidene]acetamide dimethanesulfonate 20
  • the 40 and 80 mg/kg dose groups and the positive control drug Tarceva 50 mg/kg dose group, 8 experimental animals per group.
  • Each group was administered by intragastric administration, once a day for 14 days, and on the day of grouping on day 0.
  • the relative tumor proliferation rate T/C was calculated as the relative tumor volume, T is the average value of the tumor volume relative to the treatment group, and C is the average value of the solvent control group relative to the tumor volume.
  • T/C T RTV /C RTV ⁇ 100% (T RTV : treatment group RTV; C RTV : solvent control group RTV).
  • Tumor inhibition rate (%) (average tumor weight of the solvent control group - mean tumor weight of the treatment group) / mean tumor weight of the solvent control group ⁇ 100%.
  • Percent change in body weight W n /W 0 ⁇ 100% (W n : average body weight of experimental animals in each group on day n, W 0 : average body weight of experimental animals in each group on day 0).
  • the One-Way Anova test was performed using SPSS 13.0 for statistical analysis between groups.
  • the T/C of the 33 dose groups of the compounds were 5.0%, 1.8%, and 1.8%, respectively, showing a good dose-effect relationship.
  • the positive control group had a T/C of 5.0%.
  • the tumor inhibition rates of the 33 dose groups of the compounds were 91.7%, 95.2% and 97.4%, respectively.
  • the dose of Tarceva administered is MTD.
  • N-[4-((3-chloro-4-fluorophenylamino))-7-((2-methoxyethoxy))quinazolin-6-yl]-2-[1-(( 2-Methoxyethyl)) piperidine-4-ylidene]acetamide dimethanesulfonate is a more preferred clinical drug option with good antitumor activity and a wider therapeutic window.
  • Direct mutagen 1 Dai Kesong, Dexon (DIMA TECHNOLOGY INC; batch number: 456-2D)
  • Direct mutagen 2 sodium azide, SA (AMRESCO Inc.; batch number: 0580c509)
  • Solvent 1 dimethyl sulfoxide, DMSO (Beijing Chemical Plant; batch number: 20111209)
  • Solvent 2 Sterile water for injection (Tianjin Pharmaceutical Co., Ltd.; batch number: 11080142)
  • the genetic characteristics of the strain have been identified, including the determination of spontaneous reversion rate and the requirement of histidine. Test, crystal violet sensitivity test, UV damage excision repair deletion mutation identification test, ampicillin resistance test, tetracycline resistance test, qualified.
  • the bacterial bacterial solution frozen in liquid nitrogen was rapidly melted in a 37 ° C water bath, and 100 ⁇ L of the solution was inoculated into 20 mL of the nutrient broth, and allowed to stand at 37 ° C for 16 hours in the dark, and then taken out for the mutagenicity test.
  • Groups 1 and 2 are vehicle control groups. Groups 3-12 are the test group; groups 13-15 are the positive control group; “n” is the number of plates.
  • Compound 1 was weighed and dissolved in a quantity of DMSO to a final concentration of 15 mg/mL. This solution was filtered through a 0.22 ⁇ m filter, and 1.0 mL of the primary filtrate was discarded while filtering. This filtered test solution (15 mg/mL) was then diluted with DMSO to a solution having a concentration of 5, 1.5, 0.5 and 0.15 mg/mL.
  • Compound 3 was weighed and dissolved in a predetermined amount of sterile water for injection to a final concentration of 15 mg/mL. This solution was filtered through a 0.22 ⁇ m filter, and 1.0 mL of the primary filtrate was discarded while filtering. The filtered test solution (15 mg/mL) was then diluted with sterile water for injection into a solution having a concentration of 5, 1.5, 0.5 and 0.15 mg/mL.
  • test solution After preparation, the test solution is stored at room temperature before dosing. Dosing the remaining test solution in After the drug is added, it is treated according to medical waste.
  • Sodium azide Weigh the appropriate amount of sodium azide and dissolve it in sterile water for injection to obtain a solution with a concentration of 60 ⁇ g/mL, and filter it with a 0.22 ⁇ m sterile filter.
  • 2-Aminoguanidine An appropriate amount of 2-aminoindole was weighed and dissolved in DMSO to obtain a solution having a concentration of 30 ⁇ g/mL, which was filtered using a 0.22 ⁇ m sterile filtration membrane and used.
  • the Sprague-Dawley rat liver S9 microsome fraction used in this experiment was prepared on May 18, 2012, and the batch number was 20120518. Stored in liquid nitrogen at a protein concentration of 20.477 mg/mL, valid until May 17, 2014. Sterility testing and biological activity testing meet the test requirements.
  • the S9 mixture was prepared under aseptic conditions according to the composition ratios in the table below.
  • the S9 mixture Prior to use, the S9 mixture will be formulated under sterile conditions. The operator determines the formulation volume as needed for this test. Other solvents are formulated in accordance with our standard operating procedures. The preparation of the S9 mixture is as follows:
  • test sample or reference drug solution 0.5mL S9 mixture or pH7.4 PBS, 2.0mL top medium (containing about 0.05mM histidine, about 0.05mM organism). , about 0.6% agar, about 0.5% NaCl), Finally, 0.1 mL of the bacterial culture solution was added, and the mixture was quickly mixed on a shaking mixer, poured into the surface of the underlying medium, and gently rotated to uniformly spread the top medium on the surface of the basal medium.
  • the plate was placed on a horizontal table, and after the medium was solidified, the plate was inverted and cultured at 37 ° C for 48 hours (the strain was cultured for 72 hours except for the TA102 strain). The plates were removed and the number of colonies returned per dish was counted by the naked eye. The precipitation of the test sample was observed at the time of loading and after the completion of the culture. Each plate was individually made up of 3 plates under both activated and non-activated conditions.
  • results are expressed as the number of colonized colonies per dish, and the average number of colonies and standard deviations of each group were calculated.
  • the results are in accordance with the following 1 or 2 criteria and can be judged as positive.
  • the biological significance of the test results is first considered, and the results of the statistical tests are also referred to.
  • the background lawn is thinned and may be accompanied by a decrease in the number of revertant mutants.
  • Levene's Test is used for data homogeneity test. If the data is uniform (P>0.05), one-way analysis of variance (ANOVA) is performed; if the analysis of variance is significant (P ⁇ 0.05), Dunnett's is performed. Multiple comparisons. If the result of Levene’s Test is significant (P ⁇ 0.05), a Kruskal-wallis nonparametric test is performed. If the Kruskal-wallis nonparametric test results are significant (P ⁇ 0.05), then the Mann-Whitney U test is used for the pairwise comparison.
  • ANOVA analysis of variance
  • Compound 1 test analysis results show that the highest concentration before filtration and each concentration after filtration The test solution is between 101.77% and 104.31% of the theoretical concentration, within the acceptable range of 90%-110% and no filter has a significant effect on the solution concentration.
  • Compound 3 test The analysis results show that the highest concentration before filtration and the accuracy of each concentration of the test solution after filtration are between the theoretical concentration of 99.39%-102.89%, within the acceptable range of 90%-110% and no filter It has a significant effect on the solution concentration.
  • TA97 strain Under the condition of metabolic activation (plus S9), TA97 strain had antibacterial toxicity at 1500 ⁇ g/dose, which showed that there were fine needle colonies in the background lawn, and the number of reverting mutant colonies was significantly reduced (P ⁇ 0.05).
  • TA102 strain Under the condition of metabolic or non-metabolic activation (without S9), TA102 strain had bacteriostatic toxicity at 1500 and 500 ⁇ g/dose, which showed a significant decrease in the number of revertant colonies (P ⁇ 0.05).
  • the TA102 strain was between 15 and 150 ⁇ g/dish
  • the TA97 strain was between 15 and 500 ⁇ g/dish
  • the remaining strains were between 15 and 1500 ⁇ g/dish without an increase in the number of test-related revertant colonies.
  • P ⁇ 0.05 Some subtle but statistically significant (P ⁇ 0.05) changes in the number of revertant colonies in each dose range were considered to be normal fluctuations within the normal range.
  • the test results showed that the test samples had no protrusions to TA102 strain, TA97 strain and other strains at doses of 150 ⁇ g/dish, 500 ⁇ g/dish and 1500 ⁇ g/dish, respectively. transsexual.
  • Compound 1 and Compound 3 were not mutagenic to TA102 strain at a dose of 150 ⁇ g/tablet or less; no mutagenicity was observed for TA97 strain at a dose of 500 ⁇ g/tablet or less; at a dose of 1500 ⁇ g/tablet or less There was no mutagenicity against the TA98, TA100 and TA1535 strains. Compound 1 and Compound 3 were not mutagenic to all strains at non-bacteriostatic doses.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Medicinal Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Plural Heterocyclic Compounds (AREA)

Abstract

公开了N-[4-(3-氯-4-氟苯基氨基)-7-(2-曱氧基乙氧基)喹唑啉-6-基]-2-[1-(2-曱氧基乙基)哌啶-4-亚基]乙酰胺及其药学上可接受的盐。

Description

喹唑啉衍生物
相关申请
本申请要求2013年12月12日向中国国家知识产权局提交的第201310706058.3号发明名称为“具有抗肿瘤作用的喹唑啉衍生物及其制备和应用”的中国发明专利申请的优先权及相关权益，在此通过援引的方式将上述申请的全部内容并入本文。
领域
本申请涉及药物化学领域。
背景
EGFR是表皮生长因子受体(human epidermal growth factor receptor，HER)糖蛋白家族中的一员，其它成员还包括ErbB2(HER-2)、ErbB3(HER-3)、ErbB4(HER-4)，其位于胞内的酪氨酸激酶可以催化各种底物蛋白磷酸化，在肿瘤细胞的信号传导通路中占据十分重要的地位，EGFR在胞外信号的刺激下可以激活其胞内的酪氨酸激酶，将细胞外的信号传递到细胞内并加以放大，从而调控细胞的生长、分化、血管生成及凋亡抑制，EGFR过度表达或者突变引起的信号通路传递异常与恶性肿瘤的生长、侵袭及转移关系密切。EGFR在正常组织、癌前病变和癌组织中的表达是逐级升高的，并且EGFR的表达水平与肿瘤患者的预后密切相关。某些合成药物可以阻断EGFR介导的信号传导，从而抑制肿瘤细胞的生长、抑制肿瘤对周围组织的侵入、促进肿瘤细胞的凋亡。因此，针对EGFR的靶向治疗是目前人们研究的热点之一。以EGFR为靶点的分子靶向治疗在选择性人群中疗效较好。
目前，市场上以EGFR为靶点的药物主要分为作用于EGFR胞外区的单克隆抗体及作用于EGFR胞内酪氨酸激酶结合区的小分子EGFR酪氨酸激酶抑制剂(EGFR-TKI)两大类，而EGFR-TKI类药物又因药物与EGFR酪氨酸激酶的结合方式不同分为可逆抑制剂和不可逆抑制两种。不可逆抑制剂可以不可逆的永久的与蛋白质酪氨酸激酶结 合,持续的降低蛋白质酪氨酸激酶水平，除非有新的蛋白质酪氨酸激酶产生。不可逆抑制剂发挥药效的时间更长。但是据FDA申报资料显示，现有的临床开发药物阿法替尼生物利用度仅有11.175％；裸鼠A431人源表皮癌异体移植模型实验中，阿法替尼在10mg/kg剂量组无药效。但是阿法替尼的MTD为30mg/kg((见Li D,Ambrogio L,Shimamura T,et al.BIBW2992,an irreversible EGFR/HER2inhibitor highly effective in preclinical lung cancer models.Oncogene,2008,27(34):4702-4711))，可见阿法替尼的治疗窗很窄。
概述
本申请一方面涉及N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺及其药学上可接受的盐。
本申请另一方面涉及N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺及其药学上可接受的盐，其中形成所述盐的酸选自盐酸、氢溴酸、硫酸、硝酸、磷酸、乙酸、2,2-二氯乙酸、己二酸、褐藻酸、抗坏血酸、天冬氨酸、甲磺酸、苯磺酸、苯甲酸、4-乙酰胺基苯甲酸、樟脑酸、樟脑-10-磺酸、癸酸、己酸、辛酸、碳酸、肉桂酸、柠檬酸、环己烷基氨基磺酸、十二烷基硫酸、乙烷-1,2-二磺酸、乙烷磺酸、2-羟基乙烷磺酸、甲酸、富马酸、粘酸、龙胆酸、葡庚糖酸、葡糖酸、葡糖醛酸、谷氨酸、戊二酸、2-氧代-戊二酸、甘油磷酸、乙醇酸、马尿酸、异丁酸、乳酸、乳糖醛酸、月桂酸、马来酸、苹果酸、丙二酸、扁桃酸、甲烷磺酸、黏酸、萘-1,5-二磺酸、萘-2-磺酸、1-羟基-2-萘甲酸、烟酸、油酸、乳清酸、草酸、棕榈酸、双羟萘酸、丙酸、焦谷氨酸、丙酮酸、水杨酸、4-氨基水杨酸、乙酰水杨酸、癸二酸、硬脂酸、丁二酸、酒石酸、硫氰酸、对甲苯磺酸、三氟乙酸和十一碳烯酸。
本申请再一方面涉及N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺及其药学上可接受的盐，其中所述盐选自：
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二盐酸盐；
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二硫酸盐；
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二氢溴酸盐；
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二硝酸盐；
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二磷酸盐；
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二乙酸盐；
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二甲磺酸盐；
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二苯磺酸盐；
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二富马酸盐；
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二马来酸盐；
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二烟酸盐；
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二油酸盐；
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二草酸盐；
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二丙酸盐；
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二水杨酸盐；
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二4-氨基水杨酸盐；
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二乙酰水杨酸盐；
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二酒石酸盐；
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二对甲苯磺酸盐；
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二柠檬酸盐；
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二苹果酸盐；
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二萘-1,5-二磺酸盐；
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二癸二酸盐；和
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二L-天冬氨酸盐。
本申请又一方面涉及药物组合物，其包含N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺或其药物可接受的盐以及药学上可接受的载体、稀释剂或赋形剂。
本申请再一方面涉及制备N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺或其药学可接受的盐的方法，其包括：
将式(I)所示的化合物与式(II)所示的化合物反应得到化合物N-[4-((3-氯-4-氟苯基氨基))-7-((2-甲氧基乙氧基))喹唑啉-6-基]-2-[1-((2-甲氧基乙基))哌啶-4-亚基]乙酰胺，优选将式(I)所示的化合物转化为活化酯、酰氯、酰化咪唑或混酐后再与式(II)所示的化合物反应，更优选加入三级胺如三乙胺、N-甲基吗啡啉、三甲胺、吡啶或取 代的吡啶作为催化剂，优选式(I)所示的化合物转化为酰氯时使用二氯亚砜、三氯化磷、五氯化磷、三氯氧磷、草酰氯、三聚氰酰氯作为氯化剂；或者优选将式(I)所示的化合物转化为酸酐后再与式(II)所示的化合物反应，更优选加入吡啶、取代的吡啶如DMAP作为催化剂；
任选地，将化合物N-[4-((3-氯-4-氟苯基氨基))-7-((2-甲氧基乙氧基))喹唑啉-6-基]-2-[1-((2-甲氧基乙基))哌啶-4-亚基]乙酰胺与其药学上可接受的酸反应生成其对应的药学上可接受的盐
Figure PCTCN2014001119-appb-000001
本申请另一方面涉及治疗或预防与蛋白质激酶有关的疾病的方法，其包括向需要所述方法的个体给予治疗或预防有效量的N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺及其药学上可接受的盐。
本申请又一方面涉及治疗或预防哺乳动物体内过度表达蛋白质酪氨酸磷酰化酶引起的生理异常的方法，其包括向需要所述方法的哺乳动物给予治疗或预防有效量的N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺及其药学上可接受的盐。
本申请另一方面涉及N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺及其药学上可接受的盐在制备治疗或预防与蛋白质激酶有关的疾病的药物中的用途，优选制备用于治疗或预防哺乳动物体内过度表达蛋白质酪氨酸磷酰化酶引起的生理异常的药物中的用途。
本申请又一方面涉及包含N-[4-((3-氯-4-氟苯基氨基))-7-((2-甲氧 基乙氧基))喹唑啉-6-基]-2-[1-((2-甲氧基乙基))哌啶-4-亚基]乙酰胺或其在药学上可接受的盐的药物组合物在制备治疗或预防与蛋白质激酶有关的疾病的药物中的用途，优选制备用于治疗或预防哺乳动物体内过度表达蛋白质酪氨酸磷酰化酶引起的生理异常的药物中的用途。
详述
在以下的说明中，包括某些具体的细节以对各个公开的实施方案提供全面的理解。然而，相关领域的技术人员会认识到，不采用一个或多个这些具体的细节，而采用其它方法、部件、材料等的情况下可实现实施方案。
除非本申请中另外要求，在整个说明书和其后的权利要求书中，词语“包括”和“包含”应解释为开放式的、含括式的意义，即“包括但不限于”。
在整个本说明书中提到的“一实施方案”或“实施方案”或“在另一实施方案中”或“在某些实施方案中”意指在至少一实施方案中包括与该实施方案所述的相关的具体参考要素、结构或特征。因此，在整个说明书中不同位置出现的短语“在一实施方案中”或“在实施方案中”或“在另一实施方案中”或“在某些实施方案中”不必全部指同一实施方案。此外，具体要素、结构或特征可以任何适当的方式在一个或多个实施方案中结合。
定义
“药学上可接受的载体、稀释剂或赋形剂”包括但不限于已经被美国食品与药品管理局认可的而可用于人类或动物的任何佐剂、载体、赋形剂、助流剂、甜味剂、稀释剂、防腐剂、染料/着色剂、香味增强剂、表面活性剂、润湿剂、分散剂、助悬剂、稳定剂、等渗压剂、溶剂或乳化剂等对组成药物组合物无副作用的各种形式的载体。
“药学上可接受的盐”指保持游离碱的生物学有效性和性质的那些盐，所述酸加合盐是在生物学或其它方面合适的并且是使用无机酸或有机酸来形成的，所述无机酸例如但不限于盐酸、氢溴酸、硫酸、硝酸、磷酸等，所述有机酸例如但不限于乙酸、2,2-二氯乙酸、己二酸、 褐藻酸、抗坏血酸、天冬氨酸、甲磺酸、苯磺酸、苯甲酸、4-乙酰胺基苯甲酸、樟脑酸、樟脑-10-磺酸、癸酸、己酸、辛酸、碳酸、肉桂酸、柠檬酸、环己烷基氨基磺酸、十二烷基硫酸、乙烷-1,2-二磺酸、乙烷磺酸、2-羟基乙烷磺酸、甲酸、富马酸、粘酸、龙胆酸、葡庚糖酸、葡糖酸、葡糖醛酸、谷氨酸、戊二酸、2-氧代-戊二酸、甘油磷酸、乙醇酸、马尿酸、异丁酸、乳酸、乳糖醛酸、月桂酸、马来酸、苹果酸、丙二酸、扁桃酸、甲烷磺酸、黏酸、萘-1,5-二磺酸、萘-2-磺酸、1-羟基-2-萘甲酸、烟酸、油酸、乳清酸、草酸、棕榈酸、双羟萘酸、丙酸、焦谷氨酸、丙酮酸、水杨酸、4-氨基水杨酸、乙酰水杨酸、癸二酸、硬脂酸、丁二酸、酒石酸、硫氰酸、对甲苯磺酸、三氟乙酸、十一碳烯酸等。
“药物组合物”指本申请化合物与通常被本领域所接受的将生物活化化合物输送至诸如人类等哺乳动物的介质所形成的制剂。这样的介质包括所有药物可接受的载体、稀释剂或赋形剂。
“治疗有效量”指当对哺乳动物例如人类给药时，本申请化合物足以治疗(如下定义)哺乳动物的例如人类的蛋白质酪氨酸磷酰化酶介导的疾病或疾病状态的量。根据化合物、疾病状态及其严重性、以及待治疗哺乳动物的年龄，构成“治疗有效量”的本申请化合物的量将会不同，但是本领域的技术人员根据其自身的知识以及本公开可以依惯例确定本申请化合物的量。
本文所用的“进行治疗”或“治疗”涵盖患有相关疾病或病症的哺乳动物例如人类中治疗相关的疾病或疾病状态，并且包括：
(i)预防疾病或疾病状态在哺乳动物中发生，尤其是当该哺乳动物易感于所述疾病状态，但尚未被诊断出患有这种疾病状态时；
(ii)抑制疾病或疾病状态，即阻止其发生；或者
(iii)缓解疾病或疾病状态，即使疾病或疾病状态消退。
正如本文所用的那样，术语“疾病”和“疾病状态”可以相互交换使用，或者可以是不同的，因为特殊的疾病或疾病状态可能并没有已知的致病因子(因此不能用病因学解释)，因此其不被公认为是疾病，而是被认为是不期望的疾病状态或病症，其中临床医生已经鉴定出或多 或少的特定系列的症状。
具体实施方案
本申请一方面涉及N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺及其药学上可接受的盐。
Figure PCTCN2014001119-appb-000002
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺
本申请另一方面涉及N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺及其药学上可接受的盐，其中形成所述盐的酸选自盐酸、氢溴酸、硫酸、硝酸、磷酸、乙酸、2,2-二氯乙酸、己二酸、褐藻酸、抗坏血酸、天冬氨酸、甲磺酸、苯磺酸、苯甲酸、4-乙酰胺基苯甲酸、樟脑酸、樟脑-10-磺酸、癸酸、己酸、辛酸、碳酸、肉桂酸、柠檬酸、环己烷基氨基磺酸、十二烷基硫酸、乙烷-1,2-二磺酸、乙烷磺酸、2-羟基乙烷磺酸、甲酸、富马酸、粘酸、龙胆酸、葡庚糖酸、葡糖酸、葡糖醛酸、谷氨酸、戊二酸、2-氧代-戊二酸、甘油磷酸、乙醇酸、马尿酸、异丁酸、乳酸、乳糖醛酸、月桂酸、马来酸、苹果酸、丙二酸、扁桃酸、甲烷磺酸、黏酸、萘-1,5-二磺酸、萘-2-磺酸、1-羟基-2-萘甲酸、烟酸、油酸、乳清酸、草酸、棕榈酸、双羟萘酸、丙酸、焦谷氨酸、丙酮酸、水杨酸、4-氨基水杨酸、乙酰水杨酸、癸二酸、硬脂酸、丁二酸、酒石酸、硫氰酸、对甲苯磺酸、三氟乙酸和十一碳烯酸。
本申请再一方面涉及N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺及其药学上可接受的盐，其中所述盐选自：
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二盐酸盐；
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二硫酸盐；
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二氢溴酸盐；
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二硝酸盐；
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二磷酸盐；
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二乙酸盐；
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二甲磺酸盐；
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二苯磺酸盐；
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二富马酸盐；
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二马来酸盐；
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二烟酸盐；
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二油酸盐；
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二草酸盐；
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二丙酸盐；
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二水杨酸盐；
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二4-氨基水杨酸盐；
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二乙酰水杨酸盐；
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二酒石酸盐；
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二对甲苯磺酸盐；
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二柠檬酸盐；
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二苹果酸盐；
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二萘-1,5-二磺酸盐；
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二癸二酸盐；和
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二L-天冬氨酸盐。
本申请又一方面涉及药物组合物，其包含N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺或其药学上可接受的盐以及药物可接受的载体、稀释剂或赋形剂。
能够用于本申请药物组合物中的药物可接受的载体的实例包括但不限于已经被美国食品与药品管理局认可的而可用于人类或动物的任何佐剂、载体、赋形剂、助流剂、甜味剂、稀释剂、防腐剂、染料/着色剂、香味增强剂、表面活性剂、润湿剂、分散剂、助悬剂、稳定剂、等渗压剂、溶剂或乳化剂等对组成药物组合物无副作用的各种形式的载体。
在某些实施方案中，本申请的药物组合物可以制备为胃肠外、经皮、粘膜、鼻、口颊、舌下或经口使用的片剂、溶液剂、颗粒剂、贴剂、膏剂、胶囊剂、气雾剂或栓剂。本申请的药物组合物可以通过口服、口含、静脉注射、腹腔注射、皮下注射、肌注、滴鼻、滴眼、吸 入、肛内给药、阴道给药或表皮给药。
本申请再一方面涉及制备N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺或其药学可接受的盐的方法，其包括：
将式(I)所示的化合物与式(II)所示的化合物反应得到化合物N-[4-((3-氯-4-氟苯基氨基))-7-((2-甲氧基乙氧基))喹唑啉-6-基]-2-[1-((2-甲氧基乙基))哌啶-4-亚基]乙酰胺，优选将式(I)所示的化合物转化为活化酯、酰氯、酰化咪唑或混酐后再与式(II)所示的化合物反应，更优选加入三级胺如三乙胺、N-甲基吗啡啉、三甲胺、吡啶或取代的吡啶作为催化剂，优选式(I)所示的化合物转化为酰氯时使用二氯亚砜、三氯化磷、五氯化磷、三氯氧磷、草酰氯、三聚氰酰氯作为氯化剂；或者优选将式(I)所示的化合物转化为酸酐后再与式(II)所示的化合物反应，更优选加入吡啶、取代的吡啶如DMAP作为催化剂；
任选地，将化合物N-[4-((3-氯-4-氟苯基氨基))-7-((2-甲氧基乙氧基))喹唑啉-6-基]-2-[1-((2-甲氧基乙基))哌啶-4-亚基]乙酰胺与其药学上可接受的酸反应生成其对应的药学上可接受的盐；
Figure PCTCN2014001119-appb-000003
在某些实施方案中，根据美国专利公开号US 2002/077330 A1中描述的方法制备式((II))所示的化合物。
在某些实施方案中，如下制备式((I))所示的化合物：
将1084.2g((7.8mol))2-溴乙基甲基醚、921.0g((6mol))4-哌啶酮一水合盐酸盐、3312.0g((24mol))无水碳酸钾和3.75L N，N-二甲基乙酰胺加入20L反应釜中，室温搅拌24小时。釜内反应液共约4.4L。放出 2.2L，釜内搅拌下加入13L水，加入4L二氯甲烷搅拌10分钟，静置分液，放出二氯甲烷层。水层再用二氯甲烷萃取三次，每次用二氯甲烷3L。弃去水层。合并有机层，加入1.3kg无水硫酸镁干燥半小时，抽滤，滤饼用1L二氯甲烷淋洗。抽干弃去滤饼；另外2.2L反应液处理方式同上，合并滤液，滤液旋蒸浓缩。浓缩液合并得油状物。
减压蒸馏，收集2mmHg下82-88℃馏分，得无色透明液体1-((2-甲氧基乙基))哌啶-4-酮582.7g。
将174.1g氢化钠((4.354mol，含量60％))和5.08L二氯甲烷加入20L反应釜中，搅拌，冷却至0℃。缓慢滴加660.4g((3.628mol))膦酰基乙酸三甲酯，滴加过程釜内稳定保持≤0℃，滴加过程有大量气体产生，反应液由灰色变为白色。滴完即无气体放出，反应液为白色浆状。保持反应液≤0℃低温搅拌1小时。将577.7g((3.628mol))1-((2-甲氧基乙基))哌啶-4-酮缓慢滴加入釜中。滴加过程釜内保持≤2℃。滴完保持0℃低温搅拌15小时。
反应完全后缓慢加入1L水终止反应。停止冷却。搅拌下加入4.08L水，搅拌10分钟，静置分液。放出二氯甲烷层，水层用1.7L二氯甲烷萃取。弃水层，合并有机层，用5.08L水洗一次，1N盐酸萃取两次((3.6L+1.5L))，弃二氯甲烷层，合并盐酸层。盐酸层冷却至0℃左右，搅拌下缓慢加入1N氢氧化钠溶液至pH＝9，关闭冷却，升至室温。加入5.08L二氯甲烷萃取，弃水层，有机层用5.08L水洗一次，5.08L饱和氯化钠溶液洗一次，加入500g无水硫酸镁干燥半小时，抽滤，滤饼用0.5L二氯甲烷淋洗，滤液旋干后用油泵减压抽干，得亮黄色油状物2-[1-((2-甲氧基乙基))-哌啶-4-亚基]乙酸甲酯626.0g。
将621.0g((2.915mol))2-[1-((2-甲氧基乙基))-哌啶-4-亚基]乙酸甲酯、2.915L乙醇加入20L反应釜中，搅拌，冷却至0℃。将291.5g((7.289mol))氢氧化钠溶于0.729L水中后，冷却至10℃，保持低温，缓慢加入反应釜，加完升温至25℃，继续搅拌7小时。反应完全后将反应液冷至0℃，搅拌过夜。
保持0℃左右缓慢滴加浓盐酸至pH值为2，停止冷却，室温继续搅拌30分钟，抽滤，滤饼用500ml无水乙醇淋洗后抽干另存。滤液 50℃水浴旋蒸浓缩至无液体蒸出，得黄色油状物，并有大量白色晶体析出。加入1.46L无水乙醇，室温搅拌15分钟，抽滤，滤饼用500ml无水乙醇淋洗后抽干另存。滤液50℃旋蒸浓缩至无液体蒸出，得浅黄色浓稠粥状物。
粥状物重结晶：加入1.46L异丙醇，85℃水浴搅拌回流溶清，趁热抽滤除去少量不溶残渣，移入3L三口瓶，放入85℃水浴，关闭加热，自然降温，搅拌过夜。
冰水浴继续搅拌2小时，抽滤，滤饼用异丙醇淋洗5次，每次100ml，滤饼室温晾置2小时，45℃真空干燥，得2-[1-((2-甲氧基乙基))-哌啶-4-亚基]乙酸盐酸盐254.5g。
在某些实施方案中，N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺与酸反应得到相应的药学上可接受的盐。
能够用于本申请的制备N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺的药学上可接受的盐的酸的示例性实例包括但不限于盐酸、氢溴酸、硫酸、硝酸、磷酸、乙酸、2,2-二氯乙酸、己二酸、褐藻酸、抗坏血酸、天冬氨酸、甲磺酸、苯磺酸、苯甲酸、4-乙酰胺基苯甲酸、樟脑酸、樟脑-10-磺酸、癸酸、己酸、辛酸、碳酸、肉桂酸、柠檬酸、环己烷基氨基磺酸、十二烷基硫酸、乙烷-1,2-二磺酸、乙烷磺酸、2-羟基乙烷磺酸、甲酸、富马酸、粘酸、龙胆酸、葡庚糖酸、葡糖酸、葡糖醛酸、谷氨酸、戊二酸、2-氧代-戊二酸、甘油磷酸、乙醇酸、马尿酸、异丁酸、乳酸、乳糖醛酸、月桂酸、马来酸、苹果酸、丙二酸、扁桃酸、甲烷磺酸、黏酸、萘-1,5-二磺酸、萘-2-磺酸、1-羟基-2-萘甲酸、烟酸、油酸、乳清酸、草酸、棕榈酸、双羟萘酸、丙酸、焦谷氨酸、丙酮酸、水杨酸、4-氨基水杨酸、乙酰水杨酸、癸二酸、硬脂酸、丁二酸、酒石酸、硫氰酸、对甲苯磺酸、三氟乙酸和十一碳烯酸。
本申请另一方面涉及治疗或预防与蛋白质激酶有关的疾病的方法，其包括向需要所述方法的个体给予治疗或预防有效量的N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙 基)哌啶-4-亚基]乙酰胺及其药学上可接受的盐。
在某些实施方案中，所述疾病为癌症。
能够使用本申请的方法治疗或者预防的癌症的示例性实例包括但不限于乳腺癌、头颈癌、肺癌(包括非小细胞肺癌、小细胞肺癌)、结肠癌、胰腺癌、食管癌、胃癌和前列腺癌。
在某些实施方案中，个体为哺乳动物。
在某些实施方案中，哺乳动物为人。
在某些实施方案中，以N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺及其药学上可接受的盐作为有效成分的单位剂量为0.1mg-1000mg作为治疗或预防有效量来治疗或预防与蛋白质激酶有关的疾病。
在某些实施方案中，以N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺及其药学上可接受的盐作为有效成分的单位剂量为1mg-1000mg作为治疗或预防有效量来治疗或预防与蛋白质激酶有关的疾病。
在某些实施方案中，以N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺及其药学上可接受的盐作为有效成分的单位剂量为10mg-500mg作为治疗或预防有效量来治疗或预防与蛋白质激酶有关的疾病。
本申请又一方面涉及治疗或预防哺乳动物体内过度表达蛋白质酪氨酸磷酰化酶引起的生理异常的方法，其包括向需要所述方法的哺乳动物给予治疗或预防有效量的N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺及其药学上可接受的盐。
在某些实施方案中，由EGFR或Her-2过度表达引起生理异常。
在某些实施方案中，生理异常为癌症。
能够使用本申请的方法治疗或者预防的癌症的示例性实例包括但不限于乳腺癌、头颈癌、肺癌(包括非小细胞肺癌、小细胞肺癌)、结肠癌、胰腺癌、食管癌、胃癌和前列腺癌。
在某些实施方案中，所述哺乳动物为人。
在某些实施方案中，以N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺及其药学上可接受的盐作为有效成分的单位剂量为0.1mg-1000mg作为治疗或预防有效量来治疗或预防哺乳动物体内过度表达蛋白质酪氨酸磷酰化酶引起的生理异常。
在某些实施方案中，以N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺及其药学上可接受的盐作为有效成分的单位剂量为1mg-1000mg作为治疗或预防有效量来治疗或预防哺乳动物体内过度表达蛋白质酪氨酸磷酰化酶引起的生理异常。
在某些实施方案中，以N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺及其药学上可接受的盐作为有效成分的单位剂量为10mg-500mg作为治疗或预防有效量来治疗或预防哺乳动物体内过度表达蛋白质酪氨酸磷酰化酶引起的生理异常。
本申请另一方面涉及N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺及其药学上可接受的盐在制备治疗或预防与蛋白质激酶有关的疾病的药物中的用途，优选制备用于治疗或预防哺乳动物体内过度表达蛋白质酪氨酸磷酰化酶引起的生理异常的药物中的用途。
在某些实施方案中，N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺及其药学上可接受的盐在制备用于治疗或预防哺乳动物体内由EGFR和/或Her-2过度表达引起所述生理异常。在某些实施方案中，尤其是由EGFR过度表达引起所述生理异常。
在某些实施方案中，过度表达蛋白质酪氨酸磷酰化酶引起的生理异常及通过抑制蛋白质酪氨酸磷酰化酶活性的方法有效的疾病，为癌症。
能够使用本申请的N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺及其药学上可接 受的盐治疗或预防的癌症的示例性实例包括但不限于乳腺癌、头颈癌、肺癌(包括非小细胞肺癌、小细胞肺癌)、结肠癌、胰腺癌、食管癌、胃癌、皮肤癌、肠癌、肾癌、膀胱癌、卵巢癌、口腔癌、喉癌、宫颈癌、肝癌和前列腺癌。
在某些实施方案中，哺乳动物是人。
在某些实施方案中，给予单位剂量为约0.1mg至约1000mg的N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺或其药学上可接受的盐来治疗或预防与蛋白质激酶有关的疾病的药物中的用途，优选治疗或预防哺乳动物体内过度表达蛋白质酪氨酸磷酰化酶引起的生理异常。本申请中所述的单位使用剂量如无特殊标注均指：能施用于患者并且易于操作和包装的单元，即单个剂量。
在某些实施方案中，给予单位剂量为约1mg至约1000mg的N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺或其药学上可接受的盐来治疗或预防与蛋白质激酶有关的疾病的药物中的用途，优选治疗或预防哺乳动物体内过度表达蛋白质酪氨酸磷酰化酶引起的生理异常。
在某些实施方案中，给予单位剂量为约10mg至约500mg的N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺或其药学上可接受的盐来制备治疗与蛋白质激酶有关的疾病的药物中的用途，优选治疗或预防哺乳动物体内过度表达蛋白质酪氨酸磷酰化酶引起的生理异常。
本申请又一方面涉及N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺及其药学上可接受的盐在制备治疗或预防与蛋白质激酶有关的疾病的药物中的用途，优选制备用于抑制哺乳动物体内蛋白质酪氨酸磷酰化酶活性的药物中的用途。
在某些实施方案中，哺乳动物是人。
本申请另一方面涉及N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺及其药学上 可接受的盐的药物组合物在制备治疗或预防与蛋白质激酶有关的疾病的药物中的用途，优选制备用于治疗或预防哺乳动物体内过度表达蛋白质酪氨酸磷酰化酶引起的生理异常的药物中的用途。
在某些实施方案中，N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺及其药学上可接受的盐的药物组合物在制备用于治疗或预防哺乳动物体内由EGFR和/或Her-2过度表达引起所述生理异常。在某些实施方案中，尤其是由EGFR过度表达引起所述生理异常。
在某些实施方案中，过度表达蛋白质酪氨酸磷酰化酶引起的生理异常及通过抑制蛋白质酪氨酸磷酰化酶活性的方法有效的疾病，为癌症。
能够使用本申请的N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺及其药学上可接受的盐的药物组合物治疗或预防的癌症的示例性实例包括但不限于乳腺癌、头颈癌、肺癌(包括非小细胞肺癌、小细胞肺癌)、结肠癌、胰腺癌、食管癌、胃癌、皮肤癌、肠癌、肾癌、膀胱癌、卵巢癌、口腔癌、喉癌、宫颈癌、肝癌和前列腺癌。
在某些实施方案中，哺乳动物是人。
在某些实施方案中，给予单位剂量为含有约0.1mg至约1000mg的N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺或其药学上可接受的盐的药物组合物来制备治疗或预防与蛋白质激酶有关的疾病的药物中的用途，优选制备抑制哺乳动物体内蛋白质酪氨酸磷酰化酶活性的药物的用途。
在某些实施方案中，给予单位剂量为含有约1mg至约1000mg的N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺或其药学上可接受的盐的药物组合物来治疗或预防与蛋白质激酶有关的疾病的药物中的用途，优选抑制哺乳动物体内蛋白质酪氨酸磷酰化酶活性的药物的用途。
在某些实施方案中，给予单位剂量为含有约10mg至约500mg的N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲 氧基乙基)哌啶-4-亚基]乙酰胺或其药学上可接受的盐的药物组合物来制备治疗或预防与蛋白质激酶有关的疾病的药物中的用途，优选制备抑制哺乳动物体内蛋白质酪氨酸磷酰化酶活性的药物的用途。
下文中，本公开将通过如下实施例进行详细解释以便更好地理解本公开的各个方面及其优点。然而，应当理解，以下的实施例是非限制性的而且仅用于说明本公开的某些实施方案。
实施例
实施例1
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺
20L反应釜保持通氩气。将250.0g(1.062mol)2-[1-(2-甲氧基乙基)-哌啶-4-亚基]乙酸盐酸盐、1.46L重蒸四氢呋喃和1.46ml色谱纯N,N-二甲基甲酰胺加入反应釜，搅拌，冷却至0℃，缓慢滴加128.1g(1.009mol，86.7ml)草酰氯，滴加过程釜内稳定保持≤3℃。滴毕停止冷却，停止通氩气，25℃搅拌3小时，得2-[1-(2-甲氧基乙基)-哌啶-4-亚基]乙酰氯盐酸盐的四氢呋喃溶液。
将有2-[1-(2-甲氧基乙基)-哌啶-4-亚基]乙酰氯盐酸盐的四氢呋喃溶液的反应釜保持通氩气，搅拌，冷却至0℃以下。将256.7g(0.708mol)N4-(3-氯-4-氟苯基)-7-(2-甲氧基乙氧基)喹唑啉-4,6-二胺溶于1.46L N-甲基吡咯烷酮，滤液缓慢滴入釜中，保持釜内稳定在0℃以下。滴完后继续搅拌1小时，停止冷却，自然升至25℃搅拌过夜。
TLC监测反应是否完全。反应完全后，反应液冷却至0℃左右，缓慢滴加2.9L水，釜内溶清透明。反应液抽滤除去残渣后移入10L圆底瓶，40℃水浴，机械搅拌。滴加5N氢氧化钠溶液至pH值约为10，很快有固体析出并迅速增多，加入适量水使反应体系正常搅拌，撤去水浴，室温搅拌2小时，抽滤，滤饼用蒸馏水淋洗至滴下液体pH值约为7，烘干得浅粉色固体为N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺粗产品。
将N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺粗产品20g和异丙醇250ml加入1L釜中混合，机械搅拌，88℃加热回流3小时，停止加热，保持搅拌过夜。抽滤，滤饼用异丙醇淋洗四次，每次250ml，抽干并晾干，得产品为N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺。
质谱(MS)检测(仪器型号：6410B LC-MS；Agilent)，MS:544.2[M+H]+峰。
核磁共振氢谱检测(1H-NMR)(仪器型号：VARIAN INOVA 500MHz；测定条件：溶剂DMSO)得如下结果：
Figure PCTCN2014001119-appb-000004
序号 化学位移δ(ppm) 多重性 质子数 相应的质子
a 3.225 s 3 1
b 3.42-3.444 t 2 2
c 2.488 s 4 3，4
d 2.549 s 2 5
e 2.286 s 2 6
f 2.995 s 2 7
g 6.091 s 1 9
h 3.337 s 3 11
i 3.777-3.795 t 2 12
j 4.333-4.351 t 2 13
k 7.292 s 1 15
l 8.507 s 1 17
m 8.853 s 1 20
n 8.103-8.121 m 1 23
o 7.385-7.421 t 1 26
p 7.774-7.806 m 1 27
q 9.255 s 1 CONH
r 9.764 s 1 NH
核磁共振碳谱检测(13C-NMR)(仪器型号：VARIAN INOVA 500MHz；测定条件：溶剂DMSO)得如下结果：
Figure PCTCN2014001119-appb-000005
碳原子序号 化学位移 碳原子类型
1 58.695 CH3
2 70.815 CH2
3 57.257 CH2
4 54.911 CH2
5 55.670 CH2
6 36.958 CH2
7 29.397 CH2
8 156.491 C
9 117.538 CH
10 165.284 C
11 59.000 CH3
12 70.677 CH2
13 69.048 CH2
14 155.217 C
15 108.261 CH
16 149.434 C
17 154.431 CH
18 157.418 C
19 109.653 C
20 116.417 CH
21 128.143 C
22 137.512-137.539 C
23 124.206 CH
24 119.266，119.415 C
25 152.856，154.786 C
26 117.016，117.187 CH
27 123.058-123.112 CH
实施例2
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二甲磺酸盐
290.0gN-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺和4.5L异丙醇加入20L反应釜，40℃加热搅拌。
135ml蒸馏水和128.1g甲磺酸混合液，缓慢滴加入釜中，釜内澄清，滴毕再搅拌5分钟，停止搅拌，放出反应液，过滤，取水：异丙醇＝3：100(体积比)混合液150ml洗釜后混入反应液，抽滤除去残渣，滤液移入10L圆底瓶中，再取的水：异丙醇＝3：100(体积比)混合液150ml洗滤瓶，并入圆底瓶中，室温搅拌过夜。析出固体，抽滤，滤饼用异丙醇淋洗4次，每次250ml，抽干，35℃真空干燥24小时，得N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二甲磺酸盐356.1g，收率90.7％。
质谱(MS)检测(仪器型号：6410B LC-MS；Agilent)MS:544.1处[M+H]+峰。
核磁共振氢谱(1H-NMR)(仪器型号：VARIAN INOVA 500MHz；测定条件：溶剂D2O)结果如下：
Figure PCTCN2014001119-appb-000006
Figure PCTCN2014001119-appb-000007
核磁共振碳谱(13C-NMR)(仪器型号：VARIAN INOVA 500MHz；测定条件：溶剂D2O)检测结果如下：
Figure PCTCN2014001119-appb-000008
碳原子序号 化学位移 碳原子类型
1 58.550 CH3
2 65.501 CH2
3 55.754 CH2
4 52.671 CH2
5 53.102 CH2
6 32.308 CH2
7 25.647 CH2
8 149.720 C
9 119.160 CH
10 165.849 C
11 58.420 CH3
12 69.712 CH2
13 69.128 CH2
14 129.254 C
15 112.030 CH
16 136.322 C
17 149.018 CH
18 157.315 C
19 155.129 C
20 99.807 CH
21 106.510 C
22 132.862，132.889 C
23 124.607 CH
24 120.228，120.377 C
25 154.424，156.396 C
26 116.672，116.848 CH
27 122.944，123.005 CH
甲磺酸上甲基 38.648 CH3
实施例3
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二硫酸盐
10gN-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺和155mL异丙醇加入500mL玻璃瓶中，40℃加热搅拌。
5ml蒸馏水和3.3ml浓硫酸混合，缓慢滴加入玻璃瓶中，玻璃瓶内澄清，滴毕再搅拌5分钟，停止搅拌，放出反应液，过滤，取水：异丙醇＝3：100(体积比)混合液5ml洗玻璃瓶后混入反应液，抽滤除去残渣，滤液移入500mL玻璃瓶中，再取水：异丙醇＝3：100(体积比)混合液5ml洗滤瓶，并入圆底瓶中，室温搅拌过夜。析出固体，抽滤，滤饼用异丙醇淋洗4次，每次155ml，抽干，35℃真空干燥24小时，得N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二硫酸盐。
质谱(MS)检测(仪器型号：6410B LC-MS；Agilent)MS:544处[M+H-196]+峰。
实施例4-24
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺其他酸的盐
按照实施例2的制备方法将N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺分别与盐酸、氢溴酸、硝酸、磷酸、乙酸、苯磺酸、富马酸、马来酸、烟酸、油酸、草酸、丙酸、水杨酸、4-氨基水杨酸、乙酰水杨酸、酒石酸、对甲苯磺酸、柠檬酸、苹果酸、萘-1,5-二磺酸、癸二酸、L-天冬氨酸反应制
得如下产物：
Figure PCTCN2014001119-appb-000009
Figure PCTCN2014001119-appb-000010
以D2O为溶剂，使用Bruker AV400，得到化合物4至化合物25的1H NMR数据如下：
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二盐酸盐
2.50(s,1H),2.52(s,1H),2.61(d,1H),2.94(t,1H),3.02(t,1H),3.30(s,2H),3.32(s,3H),3.39(s,3H),3.59(s,1H),3.61(s,1H),3.66(s,1H),3.69(d,2H),3.81(s,2H),4.18(s,2H),5.98(s,1H),6.84(s,1H),6.95(t,1H),7.20(t,1H),7.45(t,1H),8.27(s,1H),8.34(s,1H).
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二氢溴酸盐
2.48(s,1H),2.53(s,1H),2.63(d,1H),2.90(t,1H),3.00(t,1H),3.31(s,2H),3.35(s,3H),3.37(s,3H),3.55(s,1H),3.62(s,1H),3.64(s,1H),3.67(d,2H),3.85(s,2H),4.15(s,2H),5.94(s,1H),6.80(s,1H), 6.97(t,1H),7.25(t,1H),7.49(t,1H),8.31(s,1H),8.38(s,1H).
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二硝酸盐
2.49(s,1H),2.54(s,1H),2.62(d,1H),2.93(t,1H),3.03(t,1H),3.33(s,2H),3.36(s,3H),3.38(s,3H),3.54(s,1H),3.67(s,1H),3.69(s,1H),3.70(d,2H),3.88(s,2H),4.16(s,2H),5.97(s,1H),6.83(s,1H),6.96(t,1H),7.23(t,1H),7.46(t,1H),8.33(s,1H),8.36(s,1H).
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二磷酸盐
2.52(s,1H),2.56(s,1H),2.66(d,1H),2.95(t,1H),3.02(t,1H),3.35(s,2H),3.36(s,3H),3.39(s,3H),3.54(s,1H),3.65(s,1H),3.69(s,1H),3.72(d,2H),3.88(s,2H),4.16(s,2H),5.98(s,1H),6.83(s,1H),6.91(t,1H),7.23(t,1H),7.48(t,1H),8.30(s,1H),8.38(s,1H).
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二乙酸盐
2.08(s,6H),2.54(s,1H),2.56(s,1H),2.69(d,1H),2.95(t,1H),3.07(t,1H),3.35(s,2H),3.36(s,3H),3.37(s,3H),3.54(s,1H),3.62(s,1H),3.69(s,1H),3.74(d,2H),3.88(s,2H),4.16(s,2H),5.99(s,1H),6.83(s,1H),6.91(t,1H),7.26(t,1H),7.44(t,1H),8.30(s,1H),8.39(s,1H).
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二苯磺酸盐
2.51(s,1H),2.56(s,1H),2.60(d,1H),2.94(t,1H),3.07(t,1H),3.32(s,2H),3.36(s,3H),3.39(s,3H),3.54(s,1H),3.62(s,1H),3.70(s,1H),3.74(d,2H),3.85(s,2H),4.16(s,2H),6.01(s,1H),6.83(s,1H),6.93(t,1H),7.27(t,1H),7.31-7.42(m,2H),7.46(t,1H),7.54-7.93(m,8H),8.32(s,1H),8.35(s,1H).
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二富马酸盐
2.53(s,1H),2.55(s,1H),2.60(d,1H),2.95(t,1H),3.07(t,1H), 3.33(s,2H),3.36(s,3H),3.39(s,3H),3.54(s,1H),3.67(s,1H),3.71(s,1H),3.74(d,2H),3.86(s,2H),4.18(s,2H),6.03(s,1H),6.83(s,1H),6.95(t,1H),6.96(s,2H),7.04(s,2H),7.28(t,1H),7.47(t,1H),8.32(s,1H),8.38(s,1H).
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二马来酸盐
2.50(s,1H),2.57(s,1H),2.61(d,1H),2.97(t,1H),3.08(t,1H),3.34(s,2H),3.37(s,3H),3.39(s,3H),3.54(s,1H),3.66(s,1H),3.71(s,1H),3.77(d,2H),3.86(s,2H),4.18(s,2H),6.02(s,1H),6.28(s,2H),6.30(s,2H),6.84(s,1H),6.95(t,1H),7.26(t,1H),7.44(t,1H),8.33(s,1H),8.39(s,1H).
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二烟酸盐
2.47(s,1H),2.52(s,1H),2.62(d,1H),2.94(t,1H),3.06(t,1H),3.38(s,2H),3.39(s,3H),3.40(s,3H),3.56(s,1H),3.67(s,1H),3.68(s,1H),3.70(d,2H),3.85(s,2H),4.16(s,2H),5.94(s,1H),6.83(s,1H),6.95(t,1H),7.23(t,1H),7.43(t,1H),7.50(m,2H),8.17(m,2H),8.32(s,1H),8.38(s,1H),8.79(m,2H),9.04(m,2H).
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二油酸盐
0.96(t,6H),1.27-1.31(m,28H),1.33-1.35(m,12H),1.53-1.58(m,4H),1.94-1.98(m,8H),2.23(t,4H),2.49(s,1H),2.53(s,1H),2.61(d,1H),2.93(t,1H),3.01(t,1H),3.34(s,2H),3.36(s,3H),3.39(s,3H),3.55(s,1H),3.65(s,1H),3.68(s,1H),3.71(d,2H),3.88(s,2H),4.17(s,2H),5.41(s,2H),5.45(s,2H),5.98(s,1H),6.83(s,1H),6.95(t,1H),7.23(t,1H),7.45(t,1H),8.32(s,1H),8.37(s,1H).
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二草酸盐
2.48(s,1H),2.52(s,1H),2.62(d,1H),2.95(t,1H),3.03(t,1H),3.35(s,2H),3.36(s,3H),3.37(s,3H),3.56(s,1H),3.67(s,1H),3.69(s, 1H),3.72(d,2H),3.88(s,2H),4.16(s,2H),5.99(s,1H),6.83(s,1H),6.93(t,1H),7.24(t,1H),7.46(t,1H),8.30(s,1H),8.38(s,1H).
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二丙酸盐
1.09(t,6H),2.27(q,4H),2.45(s,1H),2.57(s,1H),2.65(d,1H),2.95(t,1H),3.05(t,1H),3.32(s,2H),3.36(s,3H),3.39(s,3H),3.56(s,1H),3.67(s,1H),3.69(s,1H),3.70(d,2H),3.88(s,2H),4.16(s,2H),6.01(s,1H),6.84(s,1H),6.93(t,1H),7.25(t,1H),7.46(t,1H),8.31(s,1H),8.37(s,1H).
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二水杨酸盐
2.47(s,1H),2.59(s,1H),2.65(d,1H),2.94(t,1H),3.05(t,1H),3.31(s,2H),3.36(s,3H),3.39(s,3H),3.55(s,1H),3.67(s,1H),3.68(s,1H),3.72(d,2H),3.85(s,2H),4.16(s,2H),6.03(s,1H),6.84(s,1H),6.97(t,1H),6.95-7.04(m,4H),7.23(t,1H),7.46(t,1H),7.48-7.96(m,4H),8.30(s,1H),8.38(s,1H).
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二4-氨基水杨酸盐
2.50(s,1H),2.61(s,1H),2.66(d,1H),2.93(t,1H),3.05(t,1H),3.32(s,2H),3.36(s,3H),3.37(s,3H),3.58(s,1H),3.67(s,1H),3.69(s,1H),3.75(d,2H),3.85(s,2H),4.16(s,2H),6.05(s,1H),6.11-6.24(m,4H),6.88(s,1H),6.98(t,1H),7.25(t,1H),7.47(t,1H),7.68-7.74(m,2H),8.34(s,1H),8.36(s,1H).
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二乙酰水杨酸盐
2.11(s,6H),2.49(s,1H),2.51(s,1H),2.65(d,1H),2.92(t,1H),3.03(t,1H),3.31(s,2H),3.34(s,3H),3.39(s,3H),3.57(s,1H),3.67(s,1H),3.68(s,1H),3.75(d,2H),3.89(s,2H),4.17(s,2H),6.03(s,1H),6.82(s,1H),6.98(t,1H),7.27(t,1H),7.21-7.30(m,4H),7.42(t,1H),7.78-8.09(m,4H),8.31(s,1H),8.36(s,1H).
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二酒石酸盐
2.53(s,1H),2.56(s,1H),2.61(d,1H),2.97(t,1H),3.05(t,1H),3.33(s,2H),3.37(s,3H),3.39(s,3H),3.54(s,1H),3.69(s,1H),3.71(s,1H),3.75(d,2H),3.91(s,2H),4.18(s,2H),4.51(s,4H),5.99(s,1H),6.82(s,1H),6.98(t,1H),7.23(t,1H),7.48(t,1H),8.31(s,1H),8.38(s,1H).
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二对甲苯磺酸盐
2.33(s,6H),2.50(s,1H),2.52(s,1H),2.61(d,1H),2.91(t,1H),3.05(t,1H),3.30(s,2H),3.31(s,3H),3.35(s,3H),3.57(s,1H),3.63(s,1H),3.71(s,1H),3.74(d,2H),3.86(s,2H),4.16(s,2H),6.05(s,1H),6.84(s,1H),6.91(t,1H),7.25(t,1H),7.31-7.42(m,4H),7.45(t,1H),7.74-7.93(m,4H),8.31(s,1H),8.37(s,1H).
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二柠檬酸盐
2.47(s,1H),2.50(s,1H),2.60(d,1H),2.64(s,8H),2.92(t,1H),3.05(t,1H),3.31(s,2H),3.35(s,3H),3.37(s,3H),3.59(s,1H),3.62(s,1H),3.75(s,1H),3.78(d,2H),3.88(s,2H),4.11(s,2H),6.02(s,1H),6.85(s,1H),6.90(t,1H),7.24(t,1H),7.45(t,1H),8.31(s,1H),8.41(s,1H).
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二苹果酸盐
2.47(s,1H),2.53(q,2H),2.56(s,1H),2.63(d,1H),2.78(q,2H),2.95(t,1H),3.04(t,1H),3.35(s,2H),3.38(s,3H),3.39(s,3H),3.53(s,1H),3.69(s,1H),3.71(s,1H),3.73(d,2H),3.85(s,2H),4.12(s,2H),4.42(q,2H),5.92(s,1H),6.85(s,1H),6.92(t,1H),7.22(t,1H),7.47(t,1H),8.33(s,1H),8.43(s,1H).
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二萘-1,5-二磺酸盐
2.48(s,1H),2.58(s,1H),2.62(d,1H),2.97(t,1H),3.00(t,1H),3.33(s,2H),3.37(s,3H),3.38(s,3H),3.54(s,1H),3.66(s,1H),3.77(s,1H),3.79(d,2H),3.88(s,2H),4.10(s,2H),5.95(s,1H),6.80(s,1H),6.93(t,1H),7.24(t,1H),7.48(t,1H),7.63-7.98(m,12H),8.30(s,1H),8.40(s,1H).
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二癸二酸盐
1.27-1.31(m,16H),1.54-1.58(m,8H),2.23(t,8H),2.54(s,1H),2.61(s,1H),2.65(d,1H),2.94(t,1H),3.02(t,1H),3.32(s,2H),3.36(s,3H),3.39(s,3H),3.53(s,1H),3.67(s,1H),3.78(s,1H),3.81(d,2H),3.85(s,2H),4.13(s,2H),5.97(s,1H),6.83(s,1H),6.93(t,1H),7.22(t,1H),7.46(t,1H),8.32(s,1H),8.42(s,1H).
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二L-天冬氨酸盐
2.50(s,1H),2.60(q,2H),2.58(s,1H),2.67(d,1H),2.85(q,2H),2.97(t,1H),3.04(t,1H),3.32(s,2H),3.35(s,3H),3.37(s,3H),3.55(s,1H),3.68(s,1H),3.75(s,1H),3.73(d,2H),3.82(q,2H),3.87(s,2H),4.14(s,2H),5.97(s,1H),6.88(s,1H),6.91(t,1H),7.2(t,1H),7.48(t,1H),8.34(s,1H),8.47(s,1H).
以下实验过程使用的化合物及其简称：
化合物1：N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺；
化合物2：N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二盐酸盐；
化合物3：N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二甲磺酸盐；
化合物4：N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[哌啶-4-亚基]乙酰胺二盐酸盐，结构式如下：
Figure PCTCN2014001119-appb-000011
化合物5：N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[哌啶-4-亚基]乙酰胺二硫酸盐；
化合物6：N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二硝酸盐；
化合物7：N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二磷酸盐；
化合物8：N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二苯磺酸盐；
化合物9：N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二富马酸盐；
化合物10：N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二马来酸盐；
化合物11：N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二对甲苯磺酸盐；
化合物12：N-[4-(3-乙炔苯基氨基-7-(2-乙氧基)喹唑啉-6-基]-2-[1-甲基哌啶-4-亚基]乙酰胺二盐酸盐，结构式如下：
Figure PCTCN2014001119-appb-000012
生物学实施例
生物学实施例1
大鼠体内生物利用度实验
实验一N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二盐酸盐大鼠体内生物利用度实验
1.实验动物：
Wistar大鼠12只，雄性，体重200-220g.购于北京维通利华实验动物技术有限公司。许可证编号：SCXK(京)2007-0001。
2.试验药物的配制：
2.1称取N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二盐酸盐30mg，加6ml超纯水配制成5mg/ml的样品液，供静脉给药使用。
2.2称N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二盐酸盐30mg，加12ml超纯水配制成2.5mg/ml的样品液，供灌胃给药使用。
3.实验方案：
给药方式 给药剂量 药物浓度 给药体积 动物数
尾静脉注射(iv) 20mg/kg 5mg/ml 4ml/kg 6只
灌胃(ig) 20mg/kg 2.5mg/ml 8ml/kg 6只
4.实验方法：
4.1HPLC/MS条件：
4.1.1HPLC条件：色谱柱：十八烷基键合硅胶为填充剂(4.6mm×50mm，1.8μm)，Agilent；流动相：甲醇：5mM乙酸铵(PH4.0)(60：40)；流速：1ml/min；柱温：40℃
4.1.2MS条件：
源参数：
Figure PCTCN2014001119-appb-000013
MRM模式检测
离子：544.3→457.1(N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二盐酸盐)
559.3→440.3(内标，阿托伐他汀半钙盐)
驻留时间：80；裂解电压：180；碰撞能：25；
4.2标线的建立：
称取约1mg的N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二盐酸盐，配制成1mg/ml的甲醇溶液，逐级稀释成50ng/ml、100ng/ml、200ng/ml、500ng/ml、1μg/ml、2μg/ml、5μg/ml、10μg/ml、20μg/ml、50μg/ml、100μg/ml的标准溶液，分别吸取10μl至1.5ml离心管中，加入10μl内标(1μg/ml的Lipitor甲醇溶液)，加入100μl的大鼠血浆，涡旋混匀，加入200μl乙腈沉蛋白，涡旋1min。16000r/min离心8min，吸取上清液，直接进样10μl测定。
4.3血样的处理：
12只大鼠随机分为2组，每组6只，给药前12h禁食不禁水，各取约0.5ml空白血后，分别灌胃、尾静脉注射N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二盐酸盐(20mg/kg)，给药后不同时间点眼眶静脉丛采血约0.5ml至肝素化离心管中(尾静脉给药采血点为：5min、15min、0.5h、1h、2h、3h、4h、6h、8h、12h、18h、24h、30h；灌胃给药采血点为：5min、10min、20min、30min、45min、1h、2h、3h、4h、6h、8h、12h、18h、24h、30h)，8000r/min离心10min，吸取100μl血浆至1.5ml离心管中，加入10μl内标(1μg/ml的阿托伐他汀半钙盐甲醇溶液)，涡旋混匀，加入200μl乙腈沉蛋白，涡旋1min。16000r/min离心8min，吸取上清液，直接进样10μl测定。
5.结果
5.1以样品浓度的自然对数为横坐标，以样品与内标峰面积比值的自然对数为纵坐标建立标准曲线为：y＝1.008x-4.149；R2＝0.998
5.2各时间点血药浓度(ng/ml)见下表：
5.2.1静脉给药结果
Figure PCTCN2014001119-appb-000014
Figure PCTCN2014001119-appb-000015
5.2.2灌胃给药结果：
Figure PCTCN2014001119-appb-000016
Figure PCTCN2014001119-appb-000017
5.2.3结论
生物利用度计算方法：F＝(AUCig×Div)/(AUCiv×Dig)×100％
式中D为剂量，AUC：血药浓度-时间曲线下面积，ig表示灌胃给药，iv表示静脉注射给药。
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二盐酸盐生物利用度为56.87％，且具有较好的药代动力学性质。
实验二N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二氢溴酸盐大鼠体内生物利用度实验
1.实验动物：
Wistar大鼠12只，雌雄各半，体重160-190g，购于北京维通利华实验动物技术有限公司。许可证编号：SCXK(京)2007-0001。
2.给药方案：
Figure PCTCN2014001119-appb-000018
3.实验方法：
3.1动物给药及血样处理：
12只大鼠给药前12h禁食不禁水，随机分为2组，每组6只，以给药方案下方式分别给予药物后，均于给药前及给药后5min、15min、0.5h、1h、2h、3h、4h、6h、8h、10h、24h、30h眼眶静脉丛采血约0.5ml至肝素化离心管中，8000r/min离心10min，吸取100μl血浆，加10μl内标(1μg/ml的阿托伐他汀半钙盐甲醇溶液)，涡旋混匀，加200μl乙腈沉淀蛋白，涡旋2min，13000r/min离心10min，吸取上清 液，进样20μl测定。
3.2标准曲线的建立：
分别吸取10μlN-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二氢溴酸盐不同浓度甲醇溶液(50ng/ml、100ng/ml、250ng/ml、500ng/ml、1μg/ml、2.5μg/ml、5μg/ml、10μg/ml、25μg/ml、50μg/ml、100μg/ml)至1.5ml离心管，均加100μl大鼠空白血浆，涡旋混匀，加10μl内标(1μg/ml的阿托伐他汀半钙盐甲醇溶液)，加200μl乙腈沉淀蛋白，涡旋2min，13000r/min离心10min，吸取上清液，进样20μl测定。以样品与内标血药浓度比为横坐标，样品与内标峰面积比为纵坐标建立标准曲线。
3.3HPLC/MS条件同实验一。
4、结果：
DAS2.0(统计矩)计算主要药代参数及口服生物利用度结果
Figure PCTCN2014001119-appb-000019
5.结论
生物利用度计算方法：F＝(AUCig×Div)/(AUCiv×Dig)×100％
式中D为剂量，AUC：血药浓度-时间曲线下面积，ig表示灌胃给药，iv表示静脉注射给药。
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二氢溴酸盐生物利用度为42.76％，且具有较好的药代动力学性质。
实验三N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺生物利用度实验
1.实验动物：Wistar大鼠8只，雄性，体重160-190g，购于中国人民解放军军事医学科学院实验动物中心。许可证编号：SCXK(军)2012-0004；合格证号：0037074。
2.给药方案：
Figure PCTCN2014001119-appb-000020
预制剂制备：
1)准确称取化合物于10ml玻璃试管中；
2)用移液枪吸取总体积2％的DMSO，用移液枪枪头慢慢地贴着离心管内壁旋转，分几次注入DMSO，将离心管壁上吸附的化合物溶解，当化合物全部转移到底部后，涡旋溶解，置于80℃水浴中；
3)再加入总体积6％的聚氧乙烯35-蓖麻油，涡旋混合；
4)再加入总体积92％的0.9％氯化钠注射液涡旋混合均匀，得澄清溶液，60分钟内使用。
注：总体积是指配置完成后所得到的溶液体积。
3.实验方法：
3.1动物给药及血样处理：
8只大鼠给药前12h禁食不禁水，随机分为2组，每组4只，以给药方案下方式分别给予药物后，均于给药前及给药后5min、0.5h、1h、2h、3h、4h、5h、6h及8h眼眶静脉丛采血约0.5ml至肝素化离心管中，8000r/min离心10min，吸取100μl血浆，加200μl 0.2％醋酸甲 醇(含20ng/ml内标厄贝沙坦)沉淀蛋白，涡旋2min，13000r/min离心10min，吸取上清液，进样20μl测定。
3.2LC-MS测定条件：
(1)色谱条件：
色谱柱：phenomenex Synergi Polar-RP(150*4.6mm，2.5μm)
流速：1ml/min；柱温：40℃
流动相：A相：5mM甲酸铵水溶液；B相：甲醇，梯度如下：
时间(min) A相 B相
0 60 40
3 15 85
4 15 85
4.5 60 40
8.5 60 40
(2)质谱条件：
API3000LC-MS/MS；ESI源；MRM正离子扫描，参数如下：
化合物 离子对 CE(碰撞能量)
化合物1 544.1→457.2 40
内标(阿托伐他汀半钙盐) 429.2→207.1 35
4、结果：
DAS2.0(统计矩)计算主要药代参数及口服生物利用度结果
Figure PCTCN2014001119-appb-000021
Figure PCTCN2014001119-appb-000022
5.结论
生物利用度计算方法：F＝(AUCig×Div)/(AUCiv×Dig)×100％
式中D为剂量，AUC：血药浓度-时间曲线下面积，ig表示灌胃给药，iv表示静脉注射给药。
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺生物利用度为23.9％，且具有较好的药代动力学性质。
实验四N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二甲磺酸盐大鼠体内生物利用度实验
1.实验动物：12只SD大鼠，雌雄各半，7-10周龄，体重200-350g，购于北京维通利华实验动物技术有限公司，饲养于太平路27号院GLP中心动物房。北京维通利华实验动物技术有限公司的生产许可证号为SCXK(京)2009-0002
2.给药方案
Figure PCTCN2014001119-appb-000023
3.实验方法：
3.1动物给药及血样处理：
各组动物按给药方案给药后，于1min、5min、15min、30min及1h、2h、3h、4h、6h、8h、12h、24h用玻璃毛细管通过眼眶静脉采血0.5mL左右至肝素化管中，轻轻上下振摇，3000rpm 4℃离心10min。分离血浆，取50μL置于5mL离心管中，加入50μL 50％甲 醇水溶液，以及50μL噻吩诺啡-D4同位素内标，混合均匀后，加入甲醇：乙腈＝1：3(体积比)的溶液，涡旋振30s，充分混合均匀，3000rpm离心5min后取上清500μL，再加入500μL 20％甲醇溶液等倍稀释，涡旋振荡30s，充分混合均匀后13000rpm下离心5min，取10μL进样，进行LC/MS/MS分析。
3.2LC-MS测定条件：
色谱柱：Agela Venusil AQ-C18,5μm 2.1*50mm；S/N:AQ-2105060029；
液相条件：A：水溶液(5mmol/L醋酸铵，0.2％甲酸)、B：甲醇；柱温25℃；进样量：10μL；梯度洗脱。梯度条件：A相：5mM醋酸铵(含0.2％甲酸)；B相：甲醇；0～0.5min，A相0.21mL/min，B相0.09mL/min；0.5-1min，A相线性降低流速至0mL/min，B相线性升高流速至0.3mL/min；1.01min，B相流速升至0.5mL/min，保持1min；2.01min，A相流速升至0.21mL/min，B相流速降至0.09mL/min，保持该比例2min，平衡至初始流速比例。
质谱条件：离子源：Turbo Ionspray(ESI+)；检测模式：MRM；电参数：化合物1:m/z 544.2–457.1,CE(碰撞能量):36.5。
4、结果：
DAS2.0(统计矩)计算主要药代参数及口服生物利用度结果大鼠单次静脉推注5.0mg/kg药动学统计矩参数(n＝6)
Figure PCTCN2014001119-appb-000024
大鼠单次灌胃5.0mg/kg药动学统计矩参数(n＝6)
Figure PCTCN2014001119-appb-000025
5、结论
生物利用度计算方法：F＝(AUCig×Div)/(AUCiv×Dig)×100％
式中D为剂量，AUC：血药浓度-时间曲线下面积，ig表示灌胃给药，iv表示静脉注射给药。
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二甲磺酸盐生物利用度为56.9％，且具有较好的药代动力学性质。
生物学实施例2
小鼠单次灌胃MTD实验
1.实验动物：雄性ICR小鼠，体重19-22g，购于北京维通利华实验动物技术有限公司，动物许可证号SCXK(京)2007-0001
2.实验方案：取小鼠21只，分为9组，第一、四、六组1只，第二、五、八组4只，第三、六、九组2只。取化合物4、化合物2、化合物12溶于注射用水。第一支至三组尾静脉给药化合物4：N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[哌啶-4-亚基]乙酰胺二盐酸盐，剂量分别为100mg/kg，150mg/kg，200mg/kg。给药后 观察动物反应及体重变化，14天后处死解剖观察。第四至六组尾静脉给药化合物2：N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二盐酸盐，剂量分别为100mg/kg，150mg/kg，200mg/kg。给药后观察动物反应及体重变化，14天后处死解剖观察。第七至九组尾静脉给药化合物12：化合物12：N-[4-(3-乙炔苯基氨基-7-(2-乙氧基)喹唑啉-6-基]-2-[1-甲基哌啶-4-亚基]乙酰胺二盐酸盐，剂量分别为100mg/kg，150mg/kg，200mg/kg。给药后观察动物反应及体重变化，14天后处死解剖观察。
3、实验结果：
化合物4小鼠死亡情况及解剖现象
Figure PCTCN2014001119-appb-000026
化合物2小鼠死亡情况及解剖现象
Figure PCTCN2014001119-appb-000027
化合物12小鼠死亡情况及解剖现象
Figure PCTCN2014001119-appb-000028
根据初步药代动力学研究发现，化合物4和化合物12在胃肠道聚集，口服生物利用度很低。
N-[4-((3-氯-4-氟苯基氨基))-7-((2-甲氧基乙氧基))喹唑啉-6-基]-2-[1-((2-甲氧基乙基))哌啶-4-亚基]乙酰胺二盐酸盐耐受性更好，毒性更低，是更优选的临床用药选择。
生物学实施例3
对EGFR的酪氨酸激酶活性的抑制作用
1.主要试剂材料
EGFR：Invitrogen公司，货号PV3872
pGT(多聚谷氨酸-酪氨酸)：Sigma-Aldrich公司，货号P0275
pY-20(鼠抗磷酸化酪氨酸抗体-HRP(辣根过氧化物酶))：Invitrogen公司，货号03-7720
TMB(3,3’,5,5’-四甲基联苯胺，HRP底物)：eBioscience公司，货号00-4201-56
96孔微孔板：Nunc公司，货号442404
2.主要仪器设备
酶标仪：Bio-Rad公司，680型
96孔板洗板机：Bio-Rad公司，1575型
3.试验方法
3.1基本方法
实验方法参考文献J.Moyer,E.Barbacci,K.Iwata,et al.Induction of apoptosis and cell cycle arrest by CP-358,774,an inhibitor of epidermal growth factor receptor tyrosine kinase.Cancer Research.1997,57:4838-4848.，并在该文献的基础上进行调整。实验方法简要描述如下：
3.1.1用0.2mg/ml溶于PBS(磷酸盐缓冲液)的pGT(作为酶的底物)4℃过夜包被96孔微孔板，未结合的pGT用洗液(含0.05％吐温-20的PBS)洗去；室温晾干2小时。
3.1.2酶反应在50μl的50mM HEPEs(N-(2-羟乙基)哌嗪-N’-2-乙磺酸)，pH 7.5，0.01％BRIJ-35(聚环氧乙烯月桂酰醚)，10mM MgCl2，1mM EGTA(乙二醇双(2-氨基乙基醚)四乙酸)，含一定浓度ATP(三磷酸腺苷)及激酶的体系中进行，室温反应30分钟，激酶可催化ATP末端的磷酸基团转移到pGT的酪氨酸残基上，使其磷酸化。
3.1.3加入终浓度1％的SDS(十二烷基硫酸钠)终止反应，用pY-20识别磷酸化的酪氨酸残基，该抗体末端的HRP使TMB显色，再加入等量的2N H2SO4，终止反应，在450nm下测其OD值，OD值与pGT酪氨酸残基的磷酸化程度成正相关。
3.2激酶和底物ATP的浓度摸索参考文献：Invitrogen公司的“Optimization of a LanthaScreen Kinase assay for EGFR(ErbB1)”(https://tools.lifetechnologies.com/content/sfs/manuals/EGFR_L  anthaScreen_activity_Europium.pdf公开)进行，最终浓度的选择标准是：激酶浓度使产物的生成量在系统检测线性范围内，ATP浓度接近Km(米氏常数)值。
3.3化合物活性检测
3.3.1取化合物适量，用DMSO(二甲基亚砜)溶解得到2mM储备液，再用DMSO稀释成最高试验浓度的50倍浓度的工作液并4倍梯度稀释，一共7个浓度。
3.3.2向44μl的反应体系(50mM HEPEs，pH 7.5，0.01％BRIJ-35，10mM MgCl2，1mM EGTA，含44.4ng/ml EGFR，终浓度40ng/ml中加入1μl 50倍浓度的化合物工作液，混匀。加入5μl溶于H2O的10倍浓度的ATP(此ATP浓度在EGFR试验中为100μM，在Her2试验中为400μM)以启动酶反应，室温反应30分钟。
3.3.3同时设不含化合物的阳性溶剂对照组(PC)和不含ATP及化合物的阴性溶剂对照组(NC)，给药组OD值与溶剂对照组OD值比较后得到化合物的抑制率。计算方法如下：抑制率＝[1-(试验值-NC均值)/(PC均值-NC均值)]×100％，由复孔求得抑制率均值及SD(标准偏差)，以化合物浓度为横坐标并以对数形式分布，抑制率均值为纵坐标，作图，以Logistic 4参数方程拟合曲线，曲线上50％抑制率对应的化合物浓度即为IC50值。
EGFR试验条件
Figure PCTCN2014001119-appb-000029
4.试验结果
对EGFR的抑制活性(IC50：nM)
Figure PCTCN2014001119-appb-000030
Figure PCTCN2014001119-appb-000031
生物学实施例4
对突变型EGFR(L858R，L858R/T790M)酪氨酸激酶活性的抑制作用
1.主要试剂材料
EGFR(L858R)：Invitrogen公司，货号PR7447A
EGFR(L858R/T790M)：Invitrogen公司，货号PR8911A
P22(多肽底物)：吉尔生化公司，货号112393
96孔板：Corning公司，货号3365
384孔板：Corning公司，货号3573
2.主要仪器设备
Caliper工作站
3.试验方法
3.1试验基本条件
Figure PCTCN2014001119-appb-000032
3.2配制激酶缓冲液：50mM HEPES,pH 7.5，0.0015％Brij-35，10mM MgCl2，2mM DTT(二硫苏糖醇)
3.3配制终止液：100mM HEPES,pH 7.5，0.015％Brij-35，0.2％Coating Reagent#3(包被试剂3，Caliper工作站配备)，50mM EDTA(乙二胺四乙酸)
3.4化合物稀释：
3.4.1配制50倍浓度的的化合物：例如化合物的检测终浓度为12.5nM,配置成50倍浓度的浓度，即625nM。在96孔板上用DMSO对50倍浓度的化合物工作液进行4倍稀释，共稀释7个浓度。
3.4.2转移5倍浓度的化合物到反应板：从上述96孔板的每一孔取10μl到另一块96孔板中，加入90μl激酶缓冲液。从上述96孔板中取出5μl到一块384孔反应板。因此，384孔反应板中就有5μl的10％DMSO溶解的5倍浓度的化合物。阴性对照孔中加入5μl250mM的EDTA。
3.5激酶反应
3.5.1将激酶用激酶缓冲液配制成2.5倍浓度的酶溶液。
3.5.2将多肽和ATP用激酶缓冲液配制成2.5倍浓度的底物溶液。
3.5.3往384孔反应板中加入10μl的2.5倍浓度的酶溶液，室温下孵育10分钟。
3.5.4往384孔反应板中加入10μl的2.5倍浓度的底物溶液，28℃下孵育1小时。
3.5.5加25μl终止液终止反应。
3.6Caliper读取底物转化率数据。
3.7计算抑制率：把转化率转化成抑制率数据。
抑制率％＝(max-转化率)/(max-min)×100％，其中max是指DMSO对照的转化率，min是指无酶对照的转化率。由抑制曲线求得IC50值。
对突变型EGFR的抑制活性(IC50：nM)
Figure PCTCN2014001119-appb-000033
N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺及其药学可接受的盐是高选择性的酪氨酸激酶不可逆抑制剂，尤其是对EGFR的高抑制活性，是更优选的临床用药选择。
生物学实施例5
体外对人源肿瘤细胞的抑制作用
1.试验细胞及主要试剂
人表皮癌细胞系A431，人非小细胞肺癌细胞系HCC827、头颈部癌细胞系Fadu和人胰腺癌细胞系AsPC-1：购自中国科学院细胞库。
RPMI-1640(一种培养基名称)：Gibco公司，货号31800-022。此为干粉，按照说明书配制成液体培养基，并根据细胞培养需要添加2g/L NaHCO3，5.958g/L HEPEs。
EMEM(Minimum Essential Medium with Earle’s salts)：Gibco公司，货号41500-034。此为干粉，按照说明书配制成液体培养基，并根据细胞培养需要添加2.2g/L NaHCO3，5.958g/L HEPEs。
DMEM(Dulbecco’s Modified Eagle’s Medium)：Gibco公司，货号12800-017。此为干粉，按照说明书配制成液体培养基，并根据细胞培养需要添加2.2g/L NaHCO3，5.958g/L HEPEs。
F-12K(Nutrient Mixture F12Ham Kaighn’s Modification)：Sigma-Aldrich公司，货号N3520。此为干粉，按照说明书配制成液体培养基，并根据细胞培养需要添加1.5g/L NaHCO3，2.383g/L HEPEs。
F-12(Ham’s F-12Nutrient Mixture)：Gibco公司，货号21700-075。此为干粉，按照说明书配制成液体培养基，并根据细胞培养需要添加1.76g/L NaHCO3，2.383g/L HEPEs。
FBS(胎牛血清)：Hyclone公司，货号SV30087。
MTT(四氮唑盐)：Sigma公司，货号M5655。以PBS(磷酸盐缓冲液)制成5mg/ml储备液。
SRB(磺酰罗丹明)：Sigma-Aldrich公司，货号S9012。以1％乙酸制成0.4％(重量/体积)的工作液。
2.主要仪器设备
CO2细胞培养箱：Thermo公司，311型，371型。
超净台：东联哈尔仪器制造有限公司，DL-CJ-2N型。
酶标仪：Bio-Rad公司，680型。
96孔板洗板机：Bio-Rad公司，1575型。
3.试验方法
3.1细胞培养基配方(完全培养基)一般根据中国科学院细胞库以及ATCC(美国模式培养物集存库)提供的配方，结合基础培养基的配方对添加物进行调整。
3.2细胞培养方法：完全培养基，37℃，5％CO2，饱和湿度培养细胞至对数生长期(贴壁细胞一般接近完全融合)，收集细胞以进行后续试验(贴壁细胞先用胰酶/EDTA消化以去贴壁)。
3.3以一定数量的细胞接种96孔细胞培养板(以下简称96孔板)，加入不同浓度的化合物，共培养一段时间(一般为3天)，最后以SRB法测定试验孔中细胞蛋白总量，或以MTT法测细胞活力。
3.3.1由细胞生长曲线试验确定细胞的接种浓度。该试验完全模拟化合物抑制试验过程，用不同浓度的细胞接种96孔板，合适的浓度应该是：在没有化合物干扰的情况下，在试验的时间跨度内保持对数生长的最大(或接近最大)可接种浓度。
3.3.2SRB法测细胞蛋白总量：吸走孔中培养基，加入10％三氯乙酸固定细胞1小时以上；去除三氯乙酸，用H2O洗净，加0.4％SRB染色15-30分钟；除去多余SRB，用1％乙酸洗净；加100μl 10mM Tris(三羟甲基氨基甲烷)水溶液使与蛋白结合的SRB溶解，在570nm波长下进行检测。
3.3.3MTT法检测细胞活力：吸走培养基，加入含0.5mg/ml MTT的基础培养基(一般指不含胎牛血清(FBS)及其他添加物的培养基)100μl/孔，继续培养3小时；吸走含MTT的基础培养基，加入DMSO100μl/孔使甲瓒溶解，在490nm波长进行检测。
3.3.4SRB和MTT两种方法的选择：人表皮癌细胞系A431实验、头颈部癌细胞系Fadu实验和人胰腺癌细胞系AsPC-1实验使用MTT法，人非小细胞肺癌细胞系HCC827实验使用SRB法。
3.4稀释并添加化合物：由DMSO耐受试验确定细胞耐受DMSO的浓度，并以此选择化合物稀释和添加方法。DMSO耐受试验测定不同浓度DMSO对细胞生长的影响。对耐受的定义是细胞生长所受影响不超过20％。最终确定的两种方法描述如下：
方法一：称取受试化合物1-2mg以DMSO溶解成2mM储液，以基础培养基稀释至20μM(或根据试验需要调整)并以3倍梯度稀释，保持稀释过程中DMSO浓度1％不变，得到8个浓度组；试验孔加入80μl完全培养基和20μl 10倍浓度的化合物工作液，最终每孔体积为200μl，DMSO浓度0.1％。涉及细胞系有HCC827。
方法二：称取受试化合物1-2mg，以DMSO溶解成20mM储液。根据试验需要调整起始浓度，再以DMSO进行3倍梯度稀释，得到化合物溶液，一共8个浓度；每个试验孔加入99μl完全培养基和1μl化合物溶液，最终每孔体积为200μl，DMSO浓度为0.5％。涉及细胞系有A431、Fadu、AsPC-1。
3.5化合物抑制活性计算：试验包含化合物试验组，不含化合物的阳性溶剂对照组(PC)和不含细胞及化合物的阴性溶剂对照组(NC)，抑制率＝[1-(试验值-NC均值)/(PC均值-NC均值)]×100％。由重复试验孔求得抑制率均值及SD，以化合物浓度为横坐标并以对数形式分布，抑制率均值为纵坐标作图，以Logistic 4参数方程拟合曲线，曲线上50％抑制率对应的化合物浓度即为IC50值。
人肿瘤细胞系培养基配方及细胞接种浓度
Figure PCTCN2014001119-appb-000034
Figure PCTCN2014001119-appb-000035
注：-表示没有
4.试验结果
4.1对人表皮癌细胞系A431的抑制作用
对人表皮癌细胞系A431的抑制活性(IC50:μM)
Figure PCTCN2014001119-appb-000036
4.2对人非小细胞肺癌细胞系HCC827的抑制作用
对人非小细胞肺癌细胞系HCC827的抑制活性(IC50：μM)
Figure PCTCN2014001119-appb-000037
4.3对人头颈部癌细胞系Fadu的抑制作用
对人头颈部癌细胞系Fadu的抑制活性(IC50:μM)
Figure PCTCN2014001119-appb-000038
4.4对人胰腺癌细胞系AsPC-1的抑制作用
对人胰腺癌细胞系AsPC-1的抑制活性(IC50:μM)
Figure PCTCN2014001119-appb-000039
N-[4-((3-氯-4-氟苯基氨基))-7-((2-甲氧基乙氧基))喹唑啉-6-基]-2-[1-((2-甲氧基乙基))哌啶-4-亚基]乙酰胺及其药学可接受的盐是 高选择性的酪氨酸激酶不可逆抑制剂，尤其是对EGFR的高抑制活性。
生物学实施例6
对A431人源表皮癌异体移植模型的药效学活性评价
1.试验方法
细胞培养：
用含有灭活的10％胎牛血清、100U/ml的青霉素和100μg/ml的链霉素的MEM培养基在37℃、5％CO2的培养箱中培养肿瘤细胞。收集处于对数生长期的肿瘤细胞，并将细胞调整至合适密度，注射于裸鼠皮下0.2ml/只，待肿瘤形成后，经裸鼠体内传代3次以上方能用于异体移植模型的建立。
肿瘤的接种与分组：
将上述荷瘤鼠脱颈处死，无菌条件下取出瘤块切割成2mm×2mm×2mm左右的小瘤块，用套管针接种到裸鼠皮下。当荷瘤裸鼠肿瘤体积长到约150±50mm3时，将实验动物随机分为以下5组，溶剂对照组、N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二甲磺酸盐20、40和80mg/kg剂量组及阳性对照药Tarceva 50mg/kg剂量组，每组8只实验动物。各组均为灌胃给药，每天给药1次，连续给药14天，分组当天为第0天。
2.实验终点与数据处理
肿瘤体积的计算公式为：体积＝0.5×长径×短径2。根据测量的结果计算出相对肿瘤体积(relative tumor volume，RTV)，计算公式为：RTV＝Vt/V0，其中V0为分组给药时(即d0)测量的肿瘤体积，Vt为每次测量时的肿瘤体积。以相对肿瘤体积计算相对肿瘤增殖率T/C，T为治疗组相对肿瘤体积的平均值，C为溶剂对照组相对肿瘤体积的平均值。计算公式如下：T/C＝TRTV/CRTV×100％(TRTV：治疗组RTV；CRTV：溶剂对照组RTV)。抑瘤率(％)＝(溶剂对照组平均瘤重-治疗组平均瘤重)/溶剂对照组平均瘤重×100％。体重变化百分率＝Wn/W0×100％(Wn：第n天各组实验动物平均体重，W0：第0天各组实验动物平均体重)。
3.统计学分析
应用SPSS13.0进行One-Way Anova检验，进行组间统计学分析。
4.实验结果
实验结束时各组肿瘤体积、相对肿瘤体积及T/C
Figure PCTCN2014001119-appb-000040
肿瘤体积指标与相对肿瘤体积指标中，**p<0.01、***p<0.001与溶剂对照组相比；
各组肿瘤重量及抑瘤率
Figure PCTCN2014001119-appb-000041
“—”表示不填写或无有效数据；***p<0.001与溶剂对照组相比
5、实验结论
化合物33个剂量组均可以抑制肿瘤的生长，T/C分别为30.6％、37.7％和19.1％，呈现出较好的量-效关系，阳性对照组T/C为47.3％。
化合物33个剂量组对A431人源表皮癌异体移植模型的抑瘤率分别为68.6％、73.2％和85.6％，呈现出较好的量-效关系，阳性对照组抑瘤率为62.0％。
化合物33个剂量组实验动物体重均无明显降低，且通过对实验动物的观察未见明显异常，提示N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙 氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二甲磺酸盐各剂量不会对实验动物产生明显的毒性。Tarceva给药剂量为MTD。
N-[4-((3-氯-4-氟苯基氨基))-7-((2-甲氧基乙氧基))喹唑啉-6-基]-2-[1-((2-甲氧基乙基))哌啶-4-亚基]乙酰胺二甲磺酸盐具有良好的抗肿瘤活性，治疗窗更宽，是更优选的临床用药选择。
生物学实施例7
对FaDu人源头颈部癌异体移植模型的药效学活性评价
1.试验方法
细胞培养：
用含有灭活的10％胎牛血清、100U/ml的青霉素和100μg/ml的链霉素的MEM培养基在37℃、5％CO2的培养箱中培养肿瘤细胞。收集处于对数生长期的肿瘤细胞，并将细胞调整至合适密度，注射于裸鼠皮下0.2ml/只，待肿瘤形成后，经裸鼠体内传代3次以上方能用于异体移植模型的建立。
肿瘤的接种与分组：
将上述荷瘤鼠脱颈处死，无菌条件下取出瘤块切割成2mm×2mm×2mm左右的小瘤块，用套管针接种到裸鼠右侧肩胛部皮下。当荷瘤裸鼠肿瘤体积长到约120±50mm3时，将实验动物随机分为以下5组，溶剂对照组、N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二甲磺酸盐20、40和80mg/kg剂量组及阳性对照药Tarceva 50mg/kg剂量组，每组8只实验动物。各组均为灌胃给药，每天给药1次，连续给药14天，分组当天为第0天。
2.实验终点与数据处理
肿瘤体积的计算公式为：体积＝0.5×长径×短径2。根据测量的结果计算出相对肿瘤体积(relative tumor volume，RTV)，计算公式为：RTV＝Vt/V0，其中V0为分组给药时(即d0)测量的肿瘤体积，Vt为每次测量时的肿瘤体积。以相对肿瘤体积计算相对肿瘤增殖率T/C，T为治疗组相对肿瘤体积的平均值，C为溶剂对照组相对肿瘤体积的平均值。计算公式如下： T/C＝TRTV/CRTV×100％(TRTV：治疗组RTV；CRTV：溶剂对照组RTV)。抑瘤率(％)＝(溶剂对照组平均瘤重-治疗组平均瘤重)/溶剂对照组平均瘤重×100％。体重变化百分率＝Wn/W0×100％(Wn：第n天各组实验动物平均体重，W0：第0天各组实验动物平均体重)。
3.统计学分析
应用SPSS13.0进行One-Way Anova检验，进行组间统计学分析。
4.实验结果
实验结束时各组肿瘤体积、相对肿瘤体积及T/C
Figure PCTCN2014001119-appb-000042
**p<0.01、***p<0.001与溶剂对照组相比；##p<0.01与阳性对照组相比。
各组肿瘤重量及抑瘤率
Figure PCTCN2014001119-appb-000043
“—”表示不填写或无有效数据；**p<0.01、***p<0.001与溶剂对照组相比；##p<0.01与阳性对照组相比。
5、实验结论
化合物3各剂量组的T/C分别为61.4％、54.7％和31.6％，各剂量组对FaDu人源头颈部癌异体移植模型均有着较好的抑制活性。
化合物3各剂量组的抑瘤率分别为37.5％、52.4％和76.2％，化合物3各剂量组对FaDu人源头颈部癌异体移植模型均有着较好的抑制活性。
化合物3各剂量组实验动物体重均无明显降低，且通过对实验动物的观察未见明显异常，提示化合物3各剂量不会对实验动物产生明显的毒性。Tarceva给药剂量为MTD。
N-[4-((3-氯-4-氟苯基氨基))-7-((2-甲氧基乙氧基))喹唑啉-6-基]-2-[1-((2-甲氧基乙基))哌啶-4-亚基]乙酰胺二甲磺酸盐具有良好的抗肿瘤活性，治疗窗更宽，是更优选的临床用药选择。
生物学实施例8
对HCC827人源非小细胞肺癌异体移植模型的药效学评价
1.试验方法
细胞培养：
用含有灭活的10％胎牛血清、100U/ml的青霉素和100μg/ml的链霉素的MEM培养基在37℃、5％CO2的培养箱中培养肿瘤细胞。收集处于对数生长期的肿瘤细胞，并将细胞调整至合适密度，注射于裸鼠皮下0.2ml/只，待肿瘤形成后，经裸鼠体内传代3次以上方能用于异体移植模型的建立。
肿瘤的接种与分组：
将上述荷瘤鼠脱颈处死，无菌条件下取出瘤块切割成2mm×2mm×2mm左右的小瘤块，用套管针接种到裸鼠右侧肩胛部皮下。当荷瘤裸鼠肿瘤体积长到约150±50mm3时，将实验动物随机分为以下5组，溶剂对照组、N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二甲磺酸盐20、40和80mg/kg剂量组及阳性对照药Tarceva 50mg/kg剂量组，每组8只实验动物。各组均为灌胃给药，每天给药1次，连续给药14天，分组当天为第0天。
2.实验终点与数据处理
肿瘤体积的计算公式为：体积＝0.5×长径×短径2。根据测量的结果计 算出相对肿瘤体积(relative tumor volume，RTV)，计算公式为：RTV＝Vt/V0，其中V0为分组给药时(即d0)测量的肿瘤体积，Vt为每次测量时的肿瘤体积。以相对肿瘤体积计算相对肿瘤增殖率T/C，T为治疗组相对肿瘤体积的平均值，C为溶剂对照组相对肿瘤体积的平均值。计算公式如下：T/C＝TRTV/CRTV×100％(TRTV：治疗组RTV；CRTV：溶剂对照组RTV)。抑瘤率(％)＝(溶剂对照组平均瘤重-治疗组平均瘤重)/溶剂对照组平均瘤重×100％。体重变化百分率＝Wn/W0×100％(Wn：第n天各组实验动物平均体重，W0：第0天各组实验动物平均体重)。
3.统计学分析
应用SPSS13.0进行One-Way Anova检验，进行组间统计学分析。
4.实验结果
实验结束时各组肿瘤体积、相对肿瘤体积及T/C
Figure PCTCN2014001119-appb-000044
***p<0.001与溶剂对照组相比
各组肿瘤重量及抑瘤率
Figure PCTCN2014001119-appb-000045
“—”表示不填写或无有效数据；***p<0.001与溶剂对照组相比
5.实验结论
化合物33个剂量组的T/C分别为5.0％、1.8％和1.8％，呈现出较好的量-效关系，阳性对照组T/C为5.0％。
化合物33个剂量组的抑瘤率分别为91.7％、95.2％和97.4％。
化合物3各剂量组实验动物体重均无明显降低且通过对实验动物的观察未见明显异常，提示化合物3各剂量均不会对实验动物产生明显的毒性。Tarceva给药剂量为MTD。
N-[4-((3-氯-4-氟苯基氨基))-7-((2-甲氧基乙氧基))喹唑啉-6-基]-2-[1-((2-甲氧基乙基))哌啶-4-亚基]乙酰胺二甲磺酸盐是具备良好的的抗肿瘤活性，治疗窗更宽，是更优选的临床用药选择。
生物学实施例8
对鼠伤寒沙门氏菌的诱变性试验
1.材料与方法：
化合物1：N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺
化合物3：N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二甲磺酸盐
直接诱变剂1：敌克松，Dexon(DIMA TECHNOLOGY INC；批号：456-2D)
直接诱变剂2：叠氮钠，SA(AMRESCO Inc.；批号：0580c509)
间接诱变剂：2-氨基蒽，2-AA(SIGMA-ALDRICH INC.；批号：STBB1901V)
菌株：组氨酸营养缺陷型鼠伤寒沙门氏菌突变株TA97、TA98、TA100、TA1535、TA102。(中国医学科学院实验动物研究所，液氮中冷冻保存)
溶媒1：二甲基亚砜，DMSO(北京化工厂；批号：20111209)
溶媒2：无菌注射用水(天津药业焦作有限公司；批号：11080142)
2.菌株遗传特性鉴定
已对菌株进行遗传特性鉴定，包括自发回变率的测定、组氨酸需 求试验、结晶紫敏感试验、紫外损伤切除修复缺失突变鉴定试验、氨苄青霉素抗性试验、四环素抗性试验，经鉴定合格。
3.菌株增菌培养
将液氮冷冻保存的细菌菌液，37℃水浴速融后，取100μL接种于20mL的营养肉汤内，置37℃避光静置培养16小时后，取出用于本诱变性试验。
4.实验方法
4.1试验分组
Figure PCTCN2014001119-appb-000046
Figure PCTCN2014001119-appb-000047
注：组别1、2为溶媒对照组。组别3-12为供试品组；组别13-15为阳性对照组；“n”为平皿数。
4.2供试品配制
称取化合物1溶解在一定量DMSO中使其终浓度为15mg/mL。将该溶液用0.22μm滤膜进行过滤，过滤时弃去初滤液1.0mL。然后将此过滤后的供试品溶液(15mg/mL)使用DMSO梯度稀释成浓度为5、1.5、0.5和0.15mg/mL的溶液。
称取化合物3溶解在一定量灭菌注射用水中使其终浓度为15mg/mL。将该溶液用0.22μm滤膜进行过滤，过滤时弃去初滤液1.0mL。然后将此过滤后的供试品溶液(15mg/mL)使用灭菌注射用水梯度稀释成浓度为5、1.5、0.5和0.15mg/mL的溶液。
4.3供试品浓度分析
过滤前将15mg/mL供试品溶液留样2份，每份体积0.5mL。供试品配制完成后将过滤后各浓度供试品溶液留样2份，每份体积0.5mL，室温保存，HPLC法进行浓度准确度分析。
HPLC检测方法：用十八烷基硅烷键合硅胶为填充剂(4.6mm×250mm，5μm)；以0.3％的乙酸水溶液(v/v，用氨水调节pH值至8.10±0.05)-甲醇-乙腈(1:7)＝48:52(v/v)，流速为每分钟1ml，柱温为40℃，检测波长为254nm，运行时间30分钟。
4.4供试品配制溶液保存与处置
配制后供试品溶液加药前于室温保存。加药剩余的供试品溶液在 加药结束后按照医疗垃圾进行处理。
4.5阳性对照品配制
敌克松：称取适量敌克松，用灭菌注射用水溶解，得到浓度为250μg/mL的溶液，用0.22μm除菌滤膜过滤后使用。
叠氮钠：称取适量叠氮钠用灭菌注射用水溶解，得到浓度为60μg/mL的溶液，用0.22μm除菌滤膜过滤后使用。
2-氨基蒽：称取适量2-氨基蒽溶于DMSO中得到浓度为30μg/mL的溶液，用0.22μm除菌滤膜过滤后使用。
4.6S9混合液的制备及配制
本试验使用的Sprague-Dawley大鼠肝脏S9微粒体组分，制备日期：2012年5月18日，批号为20120518。保存在液氮中，蛋白浓度为20.477mg/mL，有效期至2014年5月17日。无菌检测和生物活性检测符合试验要求。S9混合液按照下表的组成比例在无菌条件下配制。
用之前，S9混合液将在无菌条件下配制。操作人员根据本试验的需要确定配制体积。其它溶剂按照本中心的标准操作规程进行配制。S9混合液的配制参照下表：
Figure PCTCN2014001119-appb-000048
4.6试验操作
将顶层培养基加热融化后，45℃水浴平衡待用。
取相应数量的10mL玻璃试管，依次加入0.1mL供试品或对照品药液，0.5mLS9混合液或pH7.4的PBS，2.0mL顶层培养基(含有约0.05mM组氨酸、约0.05mM生物素、约0.6％琼脂、约0.5％NaCl)， 最后加入0.1mL细菌培养液，迅速在振荡混合器上混匀，倒入底层培养基表面，轻轻旋转，将顶层培养基均匀地铺在基础培养基表面。
将平皿置水平桌面上，待培养基凝固后，倒置平皿，37℃培养48小时(除TA102菌株外，该菌株培养72小时)。取出平皿，肉眼计数每皿的回变菌落数。加样时以及培养结束后均对供试品沉淀现象进行观察。每个组别分别在活化和非活化条件下各做3个平皿。
5.结果判定
结果表示为每皿的回变菌落数，并计算各组的平均回变菌落数和标准差。结果符合以下1条或2条标准，可判定为阳性。结果判定时首先考虑试验结果的生物学意义，同时参考统计学检验结果。
1)至少在一个菌株上，在有或无代谢活化条件下，回复突变菌落数出现剂量依赖性增加。
2)在有或无代谢活化条件下，一个或多个剂量组的回复突变菌落数出现显著增加，并且可以重复。
根据下述准则判断供试品对菌株是否有抑菌毒性：
1)背景菌苔变薄，同时可能会伴有回复突变菌落数减少。
2)背景菌苔缺失，即细菌生长完全被抑制。
3)出现针尖状非回复突变细小菌落(通常伴有背景菌苔缺失)。
6.数据处理方法
统计分析采用双尾分析，统计学显著水平设在P≤0.05。回复突变菌落数统计平均数和标准差。
数据将按下列过程分析：首先进行Levene’s Test进行数据均一性检验，如果数据均一(P>0.05)，则进行单因素方差分析检验(ANOVA)；如果方差分析显著(P≤0.05)，则进行Dunnett’s多重比较。如果Levene’s Test的结果显著(P≤0.05)，则进行Kruskal-wallis非参数检验。如果Kruskal-wallis非参数检验结果显著(P≤0.05)，则进一步采用Mann-Whitney U检验进行两两比较。
7.结果
7.1供试品分析结果
化合物1试验：分析结果表明，过滤前最高浓度以及过滤后各浓 度供试品溶液在理论浓度的101.77％-104.31％之间，在90％-110％可接受范围内且未见滤膜对溶液浓度有明显影响。
化合物3试验：分析结果表明，过滤前最高浓度以及过滤后各浓度供试品溶液的准确度在理论浓度99.39％-102.89％之间，在90％-110％可接受范围内且未见滤膜对溶液浓度有明显影响。
7.2背景菌苔及菌落形态观察
代谢活化(加S9)条件下，TA97菌株在1500μg/皿剂量下有抑菌毒性，表现为背景菌苔出现针尖样细小菌落，回复突变菌落数明显减少(P≤0.05)；
代谢或非代谢活化(不加S9)条件下，TA102菌株在1500和500μg/皿剂量下有抑菌毒性，表现为回复突变菌落数明显减少(P≤0.05)；
其余菌株各剂量代谢或非代谢活化条件下均未见明显抑菌毒性。
7.3沉淀现象观察
各菌株非代谢活化条件下在150-1500μg/皿剂量范围内，代谢活化条件下在500和1500μg/皿剂量时，体系中加入供试品溶液后出现白色混浊，加入顶层后500和1500μg/皿剂量下仍然浑浊，表明此剂量下培养体系中有沉淀析出，其余组别无沉淀现象。培养结束时各剂量组均无沉淀现象。
7.4菌落计数
试验结果显示，溶媒对照组在代谢活化或非代谢活化条件下，各菌株的自发回复突变菌落数均在本实验室正常范围内。阳性对照敌克松和叠氮钠组在非代谢活化条件下以及2-氨基蒽组在代谢活化条件下其回复突变菌落数均明显增多(P≤0.05)，明显超过溶媒对照组回复突变菌落数的2倍以上，表现出预期的阳性结果。
TA102菌株在15至150μg/皿剂量之间，TA97菌株在15至500μg/皿剂量之间以及其余菌株在15至1500μg/皿剂量之间均无供试品相关回复突变菌落数升高。各菌株在相应剂量范围内一些细微但有统计学意义(P≤0.05)的回复突变菌落数变化被认为是正常范围内的正常波动。试验结果表明供试品分别在小于等于150μg/皿、500μg/皿以及1500μg/皿剂量下相应对TA102菌株、TA97菌株以及其余菌株不具有致突 变性。
8.结论
在本试验条件下，化合物1、化合物3在小于等于150μg/皿剂量下对TA102菌株无致突变性；在小于等于500μg/皿剂量下对TA97菌株无致突变性；在小于等于1500μg/皿剂量下对TA98、TA100和TA1535菌株无致突变性。化合物1、化合物3在非抑菌毒性剂量下对所有菌株均无致突变性。
N-[4-((3-氯-4-氟苯基氨基))-7-((2-甲氧基乙氧基))喹唑啉-6-基]-2-[1-((2-甲氧基乙基))哌啶-4-亚基]乙酰胺及N-[4-((3-氯-4-氟苯基氨基))-7-((2-甲氧基乙氧基))喹唑啉-6-基]-2-[1-((2-甲氧基乙基))哌啶-4-亚基]乙酰胺二甲磺酸盐无Ames毒性，是更优选的临床用药选择。
从前述中可以理解，尽管为了示例性说明的目的描述了本发明的具体实施方案，但是在不偏离本发明的精神和范围的条件下，本领域所述技术人员可以作出各种变形或改进。这些变形或修改都应落入本申请所附权利要求的范围。

Claims (17)

  1. N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺及其药学上可接受的盐。
  2. N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺及其药学上可接受的盐，其中形成所述盐的酸选自盐酸、氢溴酸、硫酸、硝酸、磷酸、乙酸、2,2-二氯乙酸、己二酸、褐藻酸、抗坏血酸、天冬氨酸、甲磺酸、苯磺酸、苯甲酸、4-乙酰胺基苯甲酸、樟脑酸、樟脑-10-磺酸、癸酸、己酸、辛酸、碳酸、肉桂酸、柠檬酸、环己烷基氨基磺酸、十二烷基硫酸、乙烷-1,2-二磺酸、乙烷磺酸、2-羟基乙烷磺酸、甲酸、富马酸、粘酸、龙胆酸、葡庚糖酸、葡糖酸、葡糖醛酸、谷氨酸、戊二酸、2-氧代-戊二酸、甘油磷酸、乙醇酸、马尿酸、异丁酸、乳酸、乳糖醛酸、月桂酸、马来酸、苹果酸、丙二酸、扁桃酸、甲烷磺酸、黏酸、萘-1,5-二磺酸、萘-2-磺酸、1-羟基-2-萘甲酸、烟酸、油酸、乳清酸、草酸、棕榈酸、双羟萘酸、丙酸、焦谷氨酸、丙酮酸、水杨酸、4-氨基水杨酸、乙酰水杨酸、癸二酸、硬脂酸、丁二酸、酒石酸、硫氰酸、对甲苯磺酸、三氟乙酸和十一碳烯酸。
  3. N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺及其药学上可接受的盐，其中所述盐选自：
    N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二盐酸盐；
    N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二硫酸盐；
    N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二氢溴酸盐；
    N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二硝酸盐；
    N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二磷酸盐；
    N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二乙酸盐；
    N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二甲磺酸盐；
    N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二苯磺酸盐；
    N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二富马酸盐；
    N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二马来酸盐；
    N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二烟酸盐；
    N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二油酸盐；
    N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二草酸盐；
    N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二丙酸盐；
    N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二水杨酸盐；
    N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二4-氨基水杨酸盐；
    N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二乙酰水杨酸盐；
    N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二酒石酸盐；
    N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二对甲苯磺酸盐；
    N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二柠檬酸盐；
    N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二苹果酸盐；
    N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二萘-1,5-二磺酸盐；
    N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二癸二酸盐；和
    N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺二L-天冬氨酸盐。
  4. 药物组合物，其包含权利要求1至3中任一权利要求所述的N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺或其药学上可接受的盐以及药物可接受的载体、稀释剂或赋形剂。
  5. 制备N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺或其药学可接受的盐的方法，其包括：
    将式(I)所示的化合物与式(II)所示的化合物反应得到化合物N-[4-((3-氯-4-氟苯基氨基))-7-((2-甲氧基乙氧基))喹唑啉-6-基]-2-[1-((2-甲氧基乙基))哌啶-4-亚基]乙酰胺，优选将式(I)所示的化合物转化为活化酯、酰氯、酰化咪唑或混酐后再与式(II)所示的化合物反应，更优选加入三级胺如三乙胺、N-甲基吗啡啉、三甲胺、吡啶或取代的吡啶作为催化剂，优选式(I)所示的化合物转化为酰氯时使用二氯亚砜、三氯化磷、五氯化磷、三氯氧磷、草酰氯、三聚氰酰氯作为氯化剂；或者优选将式(I)所示的化合物转化为酸酐后再与式(II)所示的化合物反应，更优选加入吡啶、取代的吡啶如DMAP作为催化剂；
    任选地，将化合物N-[4-((3-氯-4-氟苯基氨基))-7-((2-甲氧基乙氧基))喹唑啉-6-基]-2-[1-((2-甲氧基乙基))哌啶-4-亚基]乙酰胺与其药学 上可接受的酸反应生成其对应的药学上可接受的盐；
    Figure PCTCN2014001119-appb-100001
  6. 治疗或预防与蛋白质激酶有关的疾病的方法，其包括向需要所述方法的个体给予治疗或预防有效量的权利要求1至3中任一权利要求所述的N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺及其药学上可接受的盐或权利要求4的药物组合物。
  7. 如权利要求6所述的方法，其中所述疾病为癌症。
  8. 如权利要求7所述的方法，其中所述癌症选自乳腺癌、头颈癌、肺癌(包括非小细胞肺癌、小细胞肺癌)、结肠癌、胰腺癌、食管癌、胃癌和前列腺癌。
  9. 如权利要求6所述的方法，其中所述个体为哺乳动物。
  10. 如权利要求9所述的方法，其中所述哺乳动物为人。
  11. 如权利要求6至11中任一权利要求所述的方法，其中所述治疗或预防有效量为N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺及其药学上可接受的盐作为有效成分的单位剂量为0.1mg-1000mg。
  12. 治疗或预防哺乳动物体内过度表达蛋白质酪氨酸磷酰化酶引起的生理异常的方法，其包括向需要所述方法的哺乳动物给予治疗或预防有效量的权利要求1至3中任一权利要求所述的N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺及其药学上可接受的盐或权利要求4的药物组合物。
  13. 如权利要求12所述的方法，其中由EGFR或Her-2过度表达引起所述生理异常。
  14. 如权利要求13所述的方法，其中所述生理异常为癌症。
  15. 如权利要求14所述的方法，其中所述癌症选自乳腺癌、头颈癌、肺癌(包括非小细胞肺癌、小细胞肺癌)、结肠癌、胰腺癌、食管癌、胃癌和前列腺癌。
  16. 如权利要求12所述的方法，其中所述哺乳动物为人。
  17. 如权利要求12至16中任一权利要求所述的方法，其中所述治疗或预防有效量为N-[4-(3-氯-4-氟苯基氨基)-7-(2-甲氧基乙氧基)喹唑啉-6-基]-2-[1-(2-甲氧基乙基)哌啶-4-亚基]乙酰胺及其药学上可接受的盐作为有效成分的单位剂量为0.1mg-1000mg。
PCT/CN2014/001119 2013-12-12 2014-12-12 喹唑啉衍生物 WO2015085654A1 (zh)

Priority Applications (6)

Application Number Priority Date Filing Date Title
US15/103,596 US9840494B2 (en) 2013-12-12 2014-12-12 Quinazoline derivative
KR1020167018546A KR101847757B1 (ko) 2013-12-12 2014-12-12 퀴나졸린 유도체
JP2016539159A JP6342001B2 (ja) 2013-12-12 2014-12-12 キナゾリン誘導体
AU2014361633A AU2014361633B2 (en) 2013-12-12 2014-12-12 Quinazoline derivative
EP14870139.4A EP3081563B1 (en) 2013-12-12 2014-12-12 Quinazoline derivative and salts thereof for use in the treatment of cancer
CA2933082A CA2933082C (en) 2013-12-12 2014-12-12 Quinazoline derivative

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201310706058 2013-12-12
CN201310706058.3 2013-12-12

Publications (1)

Publication Number Publication Date
WO2015085654A1 true WO2015085654A1 (zh) 2015-06-18

Family

ID=53370561

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2014/001119 WO2015085654A1 (zh) 2013-12-12 2014-12-12 喹唑啉衍生物

Country Status (8)

Country Link
US (1) US9840494B2 (zh)
EP (1) EP3081563B1 (zh)
JP (1) JP6342001B2 (zh)
KR (1) KR101847757B1 (zh)
CN (2) CN109265441A (zh)
AU (1) AU2014361633B2 (zh)
CA (1) CA2933082C (zh)
WO (1) WO2015085654A1 (zh)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020077330A1 (en) 2000-08-26 2002-06-20 Frank Himmelsbach Bicyclic heterocycles, pharmaceutical compositions containing them, their use, and processes for preparing them
CN101100466A (zh) * 2006-07-05 2008-01-09 天津和美生物技术有限公司 不可逆蛋白质酪氨酸磷酰化酶抑制剂及其制备和应用
WO2012104206A1 (de) * 2011-02-01 2012-08-09 Boehringer Ingelheim International Gmbh 9-[4-(3-chlor-2-fluor-phenylamino)-7-methoxy-chinazolin-6- yloxy]-1,4-diaza-spiro[5.5]undecan-5-on dimaleat, dessen verwendung als arzneimittel und dessen herstellung

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2631813A1 (en) 2005-12-12 2007-06-21 Boehringer Ingelheim International Gmbh Bicyclic heterocycles, medicaments containing said compounds, use thereof, and method for the production thereof
US8198301B2 (en) * 2006-07-05 2012-06-12 Hesheng Zhang Quinazoline and quinoline derivatives as irreversibe protein tyrosine kinase inhibitors
KR101434164B1 (ko) 2006-09-11 2014-08-26 쿠리스 인코퍼레이션 아연 결합 부분을 함유한 퀴나졸린 계열 ｅｇｆｒ 억제제
CN102020639A (zh) 2009-09-14 2011-04-20 上海恒瑞医药有限公司 6-氨基喹唑啉或3-氰基喹啉类衍生物、其制备方法及其在医药上的应用
AU2011264209B2 (en) * 2010-06-09 2014-08-07 Tianjin Hemay Oncology Pharmaceutical Co., Ltd Cyanoquinoline derivatives
CN102898386B (zh) 2011-07-27 2015-07-29 上海医药集团股份有限公司 喹唑啉衍生物、其制备方法、中间体、组合物及其应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020077330A1 (en) 2000-08-26 2002-06-20 Frank Himmelsbach Bicyclic heterocycles, pharmaceutical compositions containing them, their use, and processes for preparing them
CN101100466A (zh) * 2006-07-05 2008-01-09 天津和美生物技术有限公司 不可逆蛋白质酪氨酸磷酰化酶抑制剂及其制备和应用
WO2012104206A1 (de) * 2011-02-01 2012-08-09 Boehringer Ingelheim International Gmbh 9-[4-(3-chlor-2-fluor-phenylamino)-7-methoxy-chinazolin-6- yloxy]-1,4-diaza-spiro[5.5]undecan-5-on dimaleat, dessen verwendung als arzneimittel und dessen herstellung

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
J. MOYER; E. BARBACCI; K. IWATA ET AL.: "Induction of apoptosis and cell cycle arrest by CP-358,774, an inhibitor of epidermal growth factor receptor tyrosine kinase", CANCER RESEARCH, vol. 57, 1997, pages 4838 - 4848, XP001009787
LI D; AMBROGIO L; SHIMAMURA T ET AL.: "BIBW2992, an irreversible EGFR/HER2 inhibitor highly effective in preclinical lung cancer models", ONCOGENE, vol. 27, no. 34, 2008, pages 4702 - 4711, XP008162734, DOI: doi:10.1038/onc.2008.109

Also Published As

Publication number Publication date
US9840494B2 (en) 2017-12-12
KR20160096183A (ko) 2016-08-12
CN104710407B (zh) 2018-07-06
CN109265441A (zh) 2019-01-25
JP6342001B2 (ja) 2018-06-13
EP3081563A1 (en) 2016-10-19
CN104710407A (zh) 2015-06-17
US20160311796A1 (en) 2016-10-27
AU2014361633B2 (en) 2017-07-20
EP3081563A4 (en) 2017-05-17
EP3081563B1 (en) 2018-09-19
KR101847757B1 (ko) 2018-05-28
CA2933082C (en) 2018-06-26
AU2014361633A1 (en) 2016-06-30
CA2933082A1 (en) 2015-06-18
JP2016540020A (ja) 2016-12-22

Similar Documents

Publication Publication Date Title
EP2716633B1 (en) Quinazoline derivative as tyrosine-kinase inhibitor, preparation method therefor and application thereof
US10231973B2 (en) Salts of quinazoline derivative and method for preparing the same
KR20180021743A (ko) 프테리디논 유도체인 표피 생장 인자 수용체 억제제의 응용
CN112010839B (zh) 靶向丝/苏氨酸激酶抑制剂的晶型
MX2015003276A (es) Profarmacos de aminoquinazolina inhibidora de cinasa.
CA2723989C (en) Fumarate salt of 4-(3-chloro-2-fluoroanilino)-7-methoxy-6-{[1-(n-methylcarbamoylmethyl)piperidin-4-yl]oxy}quinazoline
US20220204518A1 (en) Pyrimido[5,4-b]pyrrolizin compound, optical isomer thereof, preparation method therefor and use thereof
CN111499583A (zh) 喹唑啉衍生物及其作为抗肿瘤药物的应用
WO2015085654A1 (zh) 喹唑啉衍生物
CN112358435B (zh) 取代芳并杂环类化合物、制备方法和抑制ulk1及抗肿瘤用途
EP3632912B1 (en) Pyridoquinazoline derivatives useful as protein kinase inhibitors
CN111410667A (zh) 新型(1,2,4)三唑并(1,5-a)吡啶基磷氧化物及其用途
CN114026090B (zh) 一类抑制egfr激酶的化合物及其制备方法和用途
CN114634453B (zh) 喹唑啉衍生物制备方法及其应用
CN108299419B (zh) 一种新型egfr激酶抑制剂的几种新晶型及其制备方法
WO2023093859A1 (zh) Axl激酶抑制剂的盐、其制备方法和用途
WO2023010354A1 (zh) 一种具有egfr抑制活性的小分子化合物及其制备方法与应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 14870139

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2933082

Country of ref document: CA

Ref document number: 2016539159

Country of ref document: JP

Kind code of ref document: A

REEP Request for entry into the european phase

Ref document number: 2014870139

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 2014870139

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 15103596

Country of ref document: US

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2014361633

Country of ref document: AU

Date of ref document: 20141212

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 20167018546

Country of ref document: KR

Kind code of ref document: A