WO2021000912A1 - 一类抑制egfr激酶的化合物及其制备方法和用途 - Google Patents

一类抑制egfr激酶的化合物及其制备方法和用途 Download PDF

Info

Publication number
WO2021000912A1
WO2021000912A1 PCT/CN2020/099916 CN2020099916W WO2021000912A1 WO 2021000912 A1 WO2021000912 A1 WO 2021000912A1 CN 2020099916 W CN2020099916 W CN 2020099916W WO 2021000912 A1 WO2021000912 A1 WO 2021000912A1
Authority
WO
WIPO (PCT)
Prior art keywords
compound
alkyl
egfr
hydrogen
cancer
Prior art date
Application number
PCT/CN2020/099916
Other languages
English (en)
French (fr)
Inventor
谢雨礼
曹刚
樊后兴
Original Assignee
微境生物医药科技(上海)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 微境生物医药科技(上海)有限公司 filed Critical 微境生物医药科技(上海)有限公司
Priority to EP20835453.0A priority Critical patent/EP3998268A4/en
Priority to KR1020217036806A priority patent/KR20220027817A/ko
Priority to CN202080005303.6A priority patent/CN114026090B/zh
Priority to BR112021024139A priority patent/BR112021024139A2/pt
Priority to CA3142071A priority patent/CA3142071A1/en
Priority to AU2020299659A priority patent/AU2020299659A1/en
Priority to JP2021578076A priority patent/JP2022539224A/ja
Priority to MX2021013982A priority patent/MX2021013982A/es
Publication of WO2021000912A1 publication Critical patent/WO2021000912A1/zh
Priority to US17/200,856 priority patent/US20210230161A1/en

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D519/00—Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/06—Peri-condensed systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/10—Spiro-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/10—Spiro-condensed systems

Definitions

  • the present invention relates to the field of biomedicine. Specifically, the present invention provides a class of EGFR kinase inhibitors, as well as preparation methods and uses thereof.
  • EGFR is (epidermal growth factor receptor) is a membrane surface receptor with tyrosine kinase activity, belonging to ERB receptors including EGFR (ERBB1, HER1), ERBB2 (HER2), ERBB3 (HER3) and ERBB4 (HER4) Body family. EGFR promotes cell division and proliferation by activating signal pathways such as MAPK and PI3K. Many solid tumors have EGFR gene amplification, overexpression or mutation, especially in patients with lung cancer.
  • the EGFR mutation rate is as high as 50%, which is much higher than the 15% mutation rate in Europe and America. Mutations in the EGFR tyrosine kinase region mainly occur in exons 18-21.
  • the first generation of EGFR inhibitors Gefitinib (Iressa, Iressa) and Erlotinib (Tarceva, Tarceva) are targeted at patients with mutations in exons 18, 19, and 21, but with their widespread clinical application, resistance to The problem of medicine has become increasingly prominent.
  • EGFR-T790M mutation is a classic drug resistance mutation, which is mostly seen in patients who are resistant to first-generation inhibitor treatment, accounting for more than 60% of clinically resistant patients.
  • Afatinib the second-generation EGFR inhibitor
  • this type of compound also has a strong effect on wild-type EFGR and has more serious dose-dependent side effects, such as skin rash and diarrhea.
  • the third-generation EGFR inhibitor AZD9291 specifically targets the T790M mutant EGFR, and is mainly used clinically to treat patients with resistance to T790M mutations.
  • EGFR exon 20 insertion mutations are the third largest type of EGFR mutations, which occur mostly in Asians, women, non-smokers, and adenocarcinoma. In the population, the clinical and pathological characteristics of the classic EGFR mutation are the same, and the mutation frequency is about 4-10%.
  • the insertion mutation of EGFR exon 20 is between 762-823 amino acids. Among the EGFR exon 20 mutations, except for T790M, which accounts for about 50%, all other mutations are insertion mutations.
  • HER2 is similar in structure to EGFR, and its exon 20 insertion mutation is located in a similar domain to the EGFR exon 20 mutation. Therefore, the molecular, biological characteristics and response to drugs of the two are very similar.
  • the exons of EGFR and HER2 gene The 20 mutation is defined as a broad exon 20 mutation.
  • lung cancer patients carrying EGFR exon 20 insertion mutations a large number of them are primary mutations, and some are resistant mutations. Except for lung cancer, about 3% of head and neck cancer patients carry EGFR exon 20 insertion mutations, and some breast cancer patients carry HER2 exon 20 insertion mutations. Therefore, targeting exon 20 insertion mutation has important clinical value for these patients.
  • a large number of in vitro and clinical studies have shown that the most common insertion mutation in EGFR exon 20 after amino acid 764 is not sensitive to the first, second, and third-generation EGFR inhibitors that have been marketed. For such patients, the current clinical practice There is no effective treatment.
  • small molecule inhibitors are Poziotinib and Mobocertinib for the EGFR exon 20 insertion mutant protein in clinical development.
  • Poziotinib has poor selectivity to wild-type EGFR and has serious side effects. Therefore, research and development of EGFR mutation inhibitors with low toxicity and side effects, especially inhibitors specific to exon 20 insertion mutations, is imminent.
  • the compound of the present invention Compared with AZD9291 and Poziotinib, the compound of the present invention not only has better activity on T790M mutation at the enzyme and cell level, but also has good activity on EGFR20 exon insertion mutation.
  • the present invention provides a compound represented by the general formula (I) or a pharmaceutically acceptable salt thereof:
  • X is selected from N and CH;
  • R 1 is selected from hydrogen, halogen, C1-6 alkyl, C3-6 cycloalkyl, -C(O)OR 8 , and -CN;
  • R 2 is selected from C1-6 alkyl, deuterated C1-6 alkyl, C3-6 cycloalkyl, and C1-6 haloalkyl;
  • R 3 is selected from -NR 9 (CH 2) 2 NR 9 'R 9 ",
  • R 5 , R 6 , and R 7 are independently selected from hydrogen, halogen, C1-6 alkyl, C1-6 haloalkyl, C1-6 alkoxy, and CN;
  • R 8 is selected from hydrogen, C1-6 alkyl, and C1-6 haloalkyl
  • R 9 is selected from hydrogen, C1-6 alkyl, deuterated C1-6 alkyl, and C1-6 haloalkyl;
  • R 9 'and R 9 " are independently selected from hydrogen, C1-6 alkyl, C3-6 cycloalkyl, deuterated C1-6 alkyl, and C1-6 haloalkyl, or R 9 ' and R 9 " and The connected nitrogen atoms are cyclized together to form an unsubstituted or heterocycloalkyl group substituted with 1-3 selected from halogen, C1-6 alkyl, and C1-6 haloalkyl;
  • R 10 is selected from hydrogen, halo, C1-6 alkyl, and -CH 2 NR 12 'R 12 " ;
  • R 11 is selected from hydrogen, halogen, and C1-6 alkyl
  • R 12 and R 12 ' are independently selected from hydrogen, C1-6 alkyl, C1-6 haloalkyl, or R 12 ' and R 12 "are cyclized together with the connected nitrogen atom to form unsubstituted or 1-3 It is selected from halogen, C1-6 alkyl, C1-3 alkoxy, methylmercapto, methylsulfone, and C1-6 haloalkyl substituted heterocycloalkyl.
  • preferred R 1 is preferably selected from hydrogen, halogen, C1-6 alkyl, -C(O)OR 8 , and -CN; and preferred R 5 , R 6 , and R 7 are independently Selected from hydrogen and halogen.
  • the present invention provides compounds of general formula (I), which can inhibit one or more EGFR activating or drug resistance mutations, for example, T790M drug resistance mutants, exon 20 insertion activating mutants, therefore, such compounds It can be used for cancer treatment programs for patients who have developed a certain degree of resistance to existing therapies based on EGFR inhibitors.
  • the present invention provides compounds of general formula (I) that have higher inhibition of EGFR formed by activated or resistant mutants than wild-type EGFR. Because of the reduced toxicity associated with the inhibition of wild-type EGFR, such compounds are more suitable for use As a therapeutic agent, it is especially suitable for the treatment of cancer.
  • the present invention provides methods for preparing compounds of general formula (I).
  • the present invention provides a pharmaceutical combination, which contains the compound of the general formula (I) or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, excipient or diluent.
  • the present invention provides the use of a compound of general formula (I) or a pharmaceutically acceptable salt thereof in the treatment of diseases mediated by EGFR-activated or drug-resistant mutants in mammals, especially humans, especially cancer.
  • the present invention provides a method for treating diseases, especially cancer, mediated by EGFR-activated or drug-resistant mutants in mammals, especially humans, which method comprises administering a compound of general formula (I) or a pharmaceutically acceptable compound thereof to a patient Or a pharmaceutical composition comprising a therapeutically effective amount of a compound of general formula (I) and a pharmaceutically acceptable carrier, excipient or diluent.
  • the present invention provides a method for selectively inhibiting EGFR activation or drug resistance mutations compared to wild-type EGFR, the method comprising contacting a biological sample or administering to a patient a compound of general formula (I) or a pharmaceutically acceptable compound thereof The salt or its pharmaceutical composition.
  • the cancer mentioned in the present invention can be selected from hepatocellular carcinoma, lung cancer, pancreatic cancer, breast cancer, cervical cancer, endometrial cancer, colorectal cancer, gastric cancer, lung cancer, nasopharyngeal cancer, ovarian cancer, prostate cancer, leukemia, Lymphoma, non-Hodgkin's lymphoma, myeloma, glioma, glioblastoma, melanoma, gastrointestinal stromal tumor (GIST), thyroid cancer, cholangiocarcinoma, kidney cancer, anaplastic large cell lymphoma , Acute myeloid leukemia (AML), multiple myeloma, mesothelioma.
  • AML Acute myeloid leukemia
  • the present invention provides a method for preparing a compound of general formula (I), which comprises the following steps:
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 and R 7 have the same meaning as in the above general formula (I).
  • intermediate 1 is obtained by substitution reaction under the action of a base.
  • Intermediate 1 and intermediate 2 are substituted or coupled to obtain compound (c).
  • Compound (c) is Nucleophilic substitution gives compound (d), compound (d) nitro is reduced to give compound (e), compound (e) is acylated to give compound (I); or intermediate 1 and intermediate 2'are substituted or coupled The reaction directly yields compound (I).
  • R 2 , R 3 and R 4 have the same meanings in the above general formula (I).
  • the compound (g) is obtained by etherification reaction, the nitro group of compound (g) is reduced to obtain compound (h), and compound (h) is acylated to obtain compound ( i), compound (i) is nitrated to obtain compound (j), and then deprotected to obtain intermediate 2.
  • the etherification reaction is carried out under the action of a strong base.
  • the strong base includes but is not limited to sodium hydride, potassium hydride, sodium hydroxide, and hydroxide. Potassium, sodium ethoxide, sodium methoxide; methods for reducing the nitro group use conventional reducing agents known in the art, including but not limited to iron powder, zinc powder, sodium sulfide, H 2 / PtO 2 ; the upper protecting group or deprotecting group is Use conventional methods well-known in the art, and proceed under appropriate acidic or alkaline conditions.
  • halogen refers to fluorine, chlorine, bromine or iodine.
  • C1-6 alkyl group refers to a straight or branched chain alkyl group containing 1 to 6 carbon atoms, and preferably a straight or branched chain alkyl group containing 1 to 4 carbon atoms.
  • Branched chain means that one or more alkyl groups of 1 to 4 carbon atoms, such as methyl, ethyl, or propyl, are connected to a straight chain alkyl group.
  • Preferred C1-6 alkyl groups include but are not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl and the like.
  • Deuterated alkyl refers to the replacement of one or more hydrogen atoms in an alkyl group with deuterium. For example, all three hydrogen atoms in a methyl group are replaced by deuterium to become a deuterated methyl group CD 3 .
  • C1-6 haloalkyl refers to a C1-6 alkyl group as defined above that contains one or more halogen atom substituents.
  • C3-6 cycloalkyl refers to a non-aromatic monocyclic or polycyclic group containing 3 to 6 carbon atoms, preferably 3 to 6 carbon atoms.
  • Preferred monocyclic C3-6 cycloalkyl groups include but are not limited to cyclopropyl, cyclopentyl, cyclohexyl and the like.
  • C1-6 alkoxy refers to a C1-6 alkyl-O- group that is bonded to the parent moiety through an oxygen, wherein the C1-6 alkyl group is as described above.
  • Preferred C1-6 alkoxy groups include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy and n-butoxy.
  • any of the aforementioned functional groups of the present invention may be unsubstituted or substituted by the substituents described in the present invention.
  • substituted refers to the replacement of one or more hydrogen atoms on a designated atom with a group selected from the designated group, provided that the normal valence of the designated atom is not exceeded, and the substitution produces a stable Compound.
  • the combination of the substituents and/or variables is allowed only when the combination forms a stable compound.
  • the present invention also includes pharmaceutically acceptable salts of compounds of general formula (I).
  • pharmaceutically acceptable salt refers to a relatively non-toxic acid addition salt or base addition salt of the compound of the present invention.
  • the acid addition salt is a salt formed by a compound of general formula (I) of the present invention and a suitable inorganic or organic acid. These salts can be prepared in the final separation and purification process of the compound, or a purified general formula (I) can be used. ) The compound is prepared by reacting its free base form with a suitable organic acid or inorganic acid.
  • Representative acid addition salts include hydrobromide, hydrochloride, sulfate, bisulfate, sulfite, acetate, oxalate, valerate, oleate, palmitate, stearic acid Salt, laurate, borate, benzoate, lactate, phosphate, hydrogen phosphate, carbonate, bicarbonate, toluate, citrate, maleate, fumarate Acid salt, succinate, tartrate, benzoate, methanesulfonate, p-toluenesulfonate, gluconate, lactobionate and lauric acid sulfonate, etc.
  • the base addition salt is a salt formed by a compound of general formula (I) of the present invention with a suitable inorganic or organic base, including, for example, salts formed with alkali metals, alkaline earth metals, and quaternary ammonium cations, such as sodium salts, lithium salts, Potassium salt, calcium salt, magnesium salt, tetramethylammonium salt, tetraethylammonium salt, etc.; amine salt, including salt with ammonia (NH 3 ), primary ammonia, secondary ammonia, or tertiary amine, such as methylamine salt , Dimethylamine salt, trimethylamine salt, triethylamine salt, ethylamine salt, etc.
  • a suitable inorganic or organic base including, for example, salts formed with alkali metals, alkaline earth metals, and quaternary ammonium cations, such as sodium salts, lithium salts, Potassium salt, calcium salt, magnesium salt, te
  • the enzyme activity experiment proved that the compound of the present invention has good activity on the exon 20 insertion activating mutant enzyme; through the cell experiment, it can be used for activating mutations, namely exon 20 insertion activating mutant cells, drug-resistant tumor cells and wild
  • the in vitro proliferation inhibition experiment of EGFR human skin cells proved that the compound of the present invention has a good proliferation inhibitory effect on activating mutation or drug resistance mutation tumor cells, but the proliferation inhibitory effect on wild-type EGFR cancer cells is weak and has good Selective.
  • the compounds of the present invention can be used as drugs for treating diseases or conditions mediated by the activity of EGFR-activated or resistant mutants, particularly tumors such as cancer.
  • the cancer includes, but is not limited to, for example, hepatocellular carcinoma, lung cancer, head and neck cancer, pancreatic cancer, breast cancer, cervical cancer, endometrial cancer, colorectal cancer, stomach cancer, lung cancer, nasopharyngeal cancer, ovarian cancer, prostate cancer, Leukemia, lymphoma, non-Hodgkin's lymphoma, myeloma, glioma, glioblastoma, melanoma, gastrointestinal stromal tumor (GIST), thyroid cancer, cholangiocarcinoma, renal cancer, anaplastic large cell lymphoma Tumors, acute myeloid leukemia (AML), multiple myeloma, mesothelioma, especially for the mutation of the 790 threonine of epidermal growth factor receptor to methionine (EGFR T790M) and the activating mutation that is the insertion type of exon 20 Tumor types with activating mutations have better applications.
  • Figure 1 is a graph showing the effect of compound 1 on the survival rate of PC9 cell brain in situ nude mice.
  • the mass spectrum was recorded with a liquid chromatography-mass spectrometer (LC-MS) (Agilent 6120B single quadrupole liquid chromatography-mass spectrometer).
  • NMR spectrum such as hydrogen spectrum ( 1 H), carbon spectrum ( 13 C), phosphorus spectrum ( 31 P) and fluorine spectrum ( 19 F), etc.
  • LC-MS liquid chromatography-mass spectrometer
  • NMR spectrum such as hydrogen spectrum ( 1 H), carbon spectrum ( 13 C), phosphorus spectrum ( 31 P) and fluorine spectrum ( 19 F), etc.
  • deuterated solvents such as deuterated chloroform, deuterated methanol, deuterated water or deuterated dimethyl sulfoxide, and use the deuterated solvent peak as the reference standard.
  • the unit of chemical shift ⁇ is ppm, and the unit of coupling constant (J) is Hertz (Hz, Hertz).
  • the coupling split peak in the NMR spectrum is expressed as: broad singlet (brs), singlet (s), doublet ( d), double doublet (dd), triplet (t), quartet (q) and multiplet (m).
  • 3-Chloropropionyl chloride (653g, 1eq) was dissolved in 6.5L of dichloromethane, and the raw material (783.6g, 1.05eq) was added dropwise under a dry ice ethanol bath to cause a large amount of solids to precipitate, and the internal temperature was maintained at 0-10 degrees. After the addition is complete, stir for 0.5h, add imidazole (405g, 1.01eq) in batches (increasingly obvious), and maintain the internal temperature at 0-10 degrees. After stirring for 1 hour, the reaction is complete, the reaction solution is poured into dilute hydrochloric acid, and the liquid is separated. The organic phase is concentrated until a large amount of solids precipitate out.
  • LiAlH 4 (48g, 1.27mol) was dissolved in THF (1L), and 6-fluoro-8-nitro-3,4-dihydroquinoline-2(1H)-one (89g, 0.42mol) in THF was added in batches (100mL) suspension, keep the internal temperature 5-10 degrees. After the addition is complete, it will naturally return to 12 degrees and stir for 0.5h.
  • 6-Fluoro-8-amino-1,2,3,4-tetrahydroquinoline (166g, 1mol) is dissolved in THF (1L), and triphosgene (118g, 0.4mol) in THF (300mL) suspension is added dropwise , Keep the internal temperature 5-10 degrees.
  • Step 6 Synthesis of 1-(2-chloropyrimidin-4-yl)-8-fluoro-5,6-dihydro-4H-imidazole[4,5,1-ij]quinoline-2(1H)-one
  • Step 4 Synthesis of 1-(2-chloropyrimidin-4-yl)-5,6-dihydro-4H-imidazole[4,5,1-ij]quinolin-2(1H)-one
  • Step 1 Synthesis of N 1 -(2-(dimethylamino)ethyl)-5-methoxy-N 1 -methyl-2-nitrobenzene-1,4-diamine
  • Step 2 Synthesis of tert-butyl (4-((2-(dimethylamino)ethyl)(methyl)amino)-2-methoxy-5-nitrophenyl)carbamate
  • Step 3 Synthesis of tert-butyl (5-amino-4-((2-(dimethylamino)ethyl)(methyl)amino)-2-methoxyphenyl)carbamate
  • Step 4 Synthesis of tert-butyl(5-acrylamido-4-((2-(dimethylamino)ethyl)(methyl)amino)-2-methoxyphenyl)carbamate
  • Step 5 Synthesis of N-(5-amino-2-((2-(dimethylamino)ethyl)(methyl)amino)-4-methoxyphenyl)acrylamide
  • Step 6 N2-(2-(dimethylamino)ethyl)-N2-methyl-3-nitro-6-(2,2,2-trifluoroethoxy)-2,5-diamine synthesis
  • 6-Chloro-2-trifluoroethoxy-5-nitropyridin-3-amine (950mg, 3.5mmol) was dissolved in acetonitrile (15mL), K 2 CO 3 (967mg, 7mmol) was added at room temperature, N, N , N'-trimethylethylenediamine (643mg, 6.3mmol), stirred overnight in an oil bath at 80 degrees.
  • Step 7 N2-(2-(Dimethylamino)ethyl)-N2-methyl-3-nitro-5-di-tert-butoxycarbonylamino-6-(2,2,2-trifluoroethoxy )-2-amine synthesis
  • Step 8 N2-(2-(Dimethylamino)ethyl)-N2-methyl-5-di-tert-butoxycarbonylamino-6-(2,2,2-trifluoroethoxy)-2,3 -Synthesis of Diamine
  • Step 9 N-(5-Di-tert-butoxycarbonylamino-2-((2-(dimethylamino)ethyl)(methyl)amino)-6-(2,2,2-trifluoroethoxy) )Pyridin-3-yl)acrylamide synthesis
  • Step 10 N-(5-amino-2-((2-(dimethylamino)ethyl)(methyl)amino)-6-(2,2,2-trifluoroethoxy)pyridine-3- Base) Synthesis of acrylamide
  • N-(5-Di-tert-butoxycarbonylamino-2-((2-(dimethylamino)ethyl)(methyl)amino)-6-(2,2,2-trifluoroethoxy)pyridine- 3-yl)acrylamide (318 mg, 0.57 mmol) was dissolved in DCM (20 mL), methanesulfonic acid (1.63 g, 5.7 mmol) was added dropwise under an ice-water bath, and the mixture was naturally returned to room temperature and stirring was continued for 2.5 h.
  • Example 1 N-(2-((2-(dimethylamino)ethyl)(methyl)amino)-5-((4-(8-fluoro-2-oxo-5,6-dihydro -4H-imidazole[4,5,1-ij]quinoline-1(2H)-yl)pyrimidin-2-yl)amino)-4-methoxyphenyl)acrylamide
  • Test Example 1 Inhibition of wild-type EGFR, HER2, HER4 and mutant EGFR kinase activity
  • Use Echo 550 to transfer 10 nL of the diluted compound to each well of the reaction plate (784075, Greiner), seal the reaction plate with a sealing film, and centrifuge at 1000 g for 1 minute; prepare 2X Enzyme with 1X kinase reaction buffer, and transfer it to the reaction plate Add 5 ⁇ L of 2X Enzyme to each well, seal the plate with a sealing film, and centrifuge at 1000g for 30 seconds, and leave it at room temperature for 10 minutes; prepare 2X TK-substrate-biotin and ATP mixture with 1X kinase reaction buffer, add 5 ⁇ L of TK to the reaction plate -Substrate-biotin/ATP mixture, seal the plate with a sealing membrane and centrifuge at 1000g for 30 seconds, react at room temperature for 40 minutes; prepare 2X Sa-XL 665 and TK-antibody-Cryptate mixture with HTRF detection buffer, add 10 ⁇ L to each well Sa-XL 665/TK-antibody-Cryptate mixture, centr
  • test results show that, compared with AZD9291, the above compound has high activity against EGFR kinase with exon 20 insertion mutation or point mutation, and other compounds of the present invention also have good activity.
  • Test Example 2 For human skin cancer cells (A431, wild-type EGFR), human lung cancer cells (H1975, EGFR T790M drug-resistant mutation), Ba/F 3 (overexpressing EGFR D770_N771insSVD, exon 20 insertion mutant EGFR Pro-B cells), Ba/F 3 (pro-B cells overexpressing V769_D770insASV with exon 20 insertion mutant EGFR) proliferation inhibition
  • a control group (adding culture fluid and cells, no drugs) and a blank group (only adding culture fluid, no cells and drugs).
  • a control group (adding culture fluid and cells, no drugs) and a blank group (only adding culture fluid, no cells and drugs).
  • 5% CO 2 at 37°C for 72 hours, during which time the cell proliferation and drug precipitation were observed under an inverted microscope.
  • Aspirate 80 ⁇ L of fresh culture medium per well then add 10 ⁇ L/well of MTT solution (5mg/ml, 0.5% MTT) to each well, continue to incubate for 4h, add 50 ⁇ L/well of triple solution (10% SDS-5% isobutanol) -0.01mol/L HCl), cultured overnight in a CO 2 incubator.
  • Measure the OD570 value with a microplate reader, calculate the percentage of inhibition compared with the control group, and then calculate the IC 50 of the drug inhibiting cell proliferation.
  • Table 5 The results are shown in Table 5:
  • the test results show that the above compounds have strong proliferation of Ba/F 3 (expressing and dependent on EGFR exon 20 insertion mutations including D770_N771insSVD, V769_D770insASV growing proB cells), human lung cancer cells (H1975, EGFR T790M drug-resistant mutation) Inhibition, the proliferation inhibitory effect on human skin cancer cells (A431, wild-type EGFR) is weak, that is, compared with AZD9291, the compound of the present invention maintains the T790M drug-resistant mutation activity while greatly improving the dependence on EGFR 20
  • the activity of Ba/F3 cells proliferated with exon insertion mutation activity, and at the same time has good selectivity relative to wild-type EGFR.
  • Other compounds of the present invention also have good activity and selectivity.
  • Test Example 3 on the effect of tumor growth in mouse subcutaneous PDX and CDX models
  • Nude mice were inoculated with tumor masses of LU-01-0493, LU-01-0426 and LU-0387 carrying EGFR exon 20 insertion mutations or H1975 cells carrying EGFR T790M.
  • the group is divided into groups, and compound 1 or poziotinib is administered orally once a week.
  • Tumor volume and mouse body weight were measured twice a week, and on the 28th day (LU-01-0493, LU-01-0426 and LU-0387) or 21st day (H1975) at the end of administration.
  • TGI tumor growth inhibition rate
  • compound 1 is safer and more effective. It has stronger activity to inhibit tumor growth in vivo carrying EGFR exon 20 insertion mutation or EGFR T790M mutation. At an effective dose, it has an effect on mouse body weight. Smaller.
  • mice were injected with luciferase-labeled 3*10 5 PC9 cells carrying EGFR Del 19 in situ in the brain. Three days after injection, they were randomly divided into groups according to fluorescence intensity and body weight for administration. Compound 1 was administered daily to evaluate the survival rate of the mice. Except for the naturally dead mice, a weight loss of more than 20% indicates that the mice are near death. According to animal welfare, the mice will be euthanized and included in the death of the mice.
  • mice in the control group died within 28 days after administration, and all mice survived to 28 days after administration of compound 1, indicating that compound 1 can inhibit the death of mice induced by brain tumors , Suggesting that compound 1 can enter the brain and inhibit tumor growth in the brain.
  • the compound of the present invention has a high inhibitory activity on the kinase with exon 20 insertion mutation or point mutation at the enzyme level, and the inhibition is greatly improved at the cell level.
  • the activity of Ba/F3 cells proliferated with proton insertion mutation activity, and at the same time has good selectivity relative to wild-type EGFR.
  • compound 1 shows strong tumor-inhibiting activity. Compared with poziotinib, compound 1 is safer and more effective.
  • the mouse brain orthotopic PC9 model also shows that compound 1 can enter the brain and inhibit tumors in the brain Grow.
  • Other compounds of the present invention also have a strong ability to inhibit tumor growth in vivo.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Hematology (AREA)
  • Oncology (AREA)
  • Epidemiology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Nitrogen Condensed Heterocyclic Rings (AREA)
  • Nitrogen And Oxygen Or Sulfur-Condensed Heterocyclic Ring Systems (AREA)

Abstract

本发明提供了通式（I）所述化合物及其药学上可接受的盐以及其制备方法与应用，该类化合物能选择性抑制突变形态的表皮生长因子受体（EGFR）的活性，具有良好的抑制作用和癌细胞增殖抑制作用，从而可用作肿瘤和相关疾病的治疗。式（I）

Description

一类抑制EGFR激酶的化合物及其制备方法和用途
本申请要求申请日为2019年7月4日的中国专利申请CN2019106002291的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明涉及生物医药领域，具体地，本发明提供了一类EGFR激酶抑制剂，及其制备方法和用途。
背景技术
EGFR是(epidermal growth factor receptor)是一种具有酪氨酸激酶活性的膜表面受体，隶属于包括EGFR(ERBB1，HER1),ERBB2(HER2)，ERBB3(HER3)和ERBB4(HER4)的ERB受体家族。EGFR通过激活MAPK、PI3K等信号通路来促进细胞分裂增殖。许多实体肿瘤中都存在EGFR基因的扩增、过表达或突变，肺癌病人尤为突出。
尤其是东亚地区的肺癌病人中，EGFR突变的比例高达50％，远高于欧美15％的突变比例。EGFR酪氨酸激酶区域的突变主要发生在18-21外显子。第一代EGFR抑制剂Gefitinib(易瑞沙,Iressa)和Erlotinib(特罗凯,Tarceva)是针对外显子18,19,和21突变的病人，但是随着其在临床上的广泛应用，耐药问题日益突出。EGFR-T790M突变为经典的耐药性突变，大多见于一代抑制剂治疗后耐药的病人，约占临床耐药病人的60％以上。第二代EGFR抑制剂Afatinib属于不可逆抑制剂，对EGFR-T790M突变有效，但是此类化合物对野生型的EFGR作用也较强，存在较为严重的剂量依赖性副作用，如皮疹和腹泻。第三代EGFR抑制剂AZD9291特异性的靶向T790M突变型EGFR，临床上主要用于治疗耐药产生T790M突变的病人。
Figure PCTCN2020099916-appb-000001
除了上述的外显子18，19，21经典EGFR突变和T790M耐药突变外，EGFR外显子20插入突变是EGFR突变的第三大类型，多发生于亚裔、女性、非吸烟、腺癌人群，与经典EGFR突变的临床及病理特征相同，突变的频率约为4-10％。EGFR外显子20的插入突变位于762-823氨基酸之间，在EGFR外显子20突变之中，除去占据约50％的T790M以外，其它突变均为插入突变。除了EGFR之外，大约2％的非小细胞肺癌病人携带HER2基因突变，其中90％以上的突变为外显子20插入突变。HER2与EGFR结构类似，其外显子20插入突变位于与EGFR外显子20突变相似的结构域，因此两者的分子、生物学特征和对药物响应非常相似，EGFR和HER2基因的外显子20突变被定义为广义的20外显子突变。目前己发现122种EGFR20外显子插入突变，最常见突变是Asp770_Asn771ins，其次为Va1769_Asp770ins、A1a767_Va1769和Ser768_Asp770，它们具有相似的插入序列。HER2基因20外显子插入突变类型较少，最常见的是A775_G776insYVMA点位，该突变占比接近70％。EGFR或HER2 20外显子突变均导致EGFR或HER2不依赖配体的持续强烈激活。
在携带EGFR外显子20插入突变肺癌病人中，大量是原发性突变，也有部分为耐药性突变。除了肺癌以外，约3％的头颈癌患者携带EGFR外显子20插入突变，部分乳腺癌病人带有HER2外显子20插入突变。因此靶向外显子20插入突变对于这些病人具有重要的临床价值。然而大量的体外和临床研究表明EGFR外显子20插入突变中最为常见的氨基酸764位之后的插入突变对已经上市的一，二，三代EGFR抑制剂并不敏感，对于此类病人，临床上目前尚无有效治疗手段。目前，进入临床开发针对EGFR外显子20插入突变蛋白得到小分子抑制剂有Poziotinib和Mobocertinib。Poziotinib对野生型EGFR选择性较差，毒副作用大。因此，研究开发低毒副作用EGFR突变抑制剂，尤其是特异性针对外显子20插入突变的抑制剂迫在眉睫。本发明的化合物与AZD9291和Poziotinib相比，在酶和细胞水平，不仅对T790M突变具有更好的活性，而且对EGFR20外显子插入突变也具有很好的活性。
发明内容
本发明提供了一种通式(I)所示化合物或其药学上可接受的盐：
Figure PCTCN2020099916-appb-000002
其中：
X选自N和CH；
R 1选自氢，卤素，C1-6烷基，C3-6环烷基，-C(O)OR 8，和-CN；
R 2选自C1-6烷基，氘代C1-6烷基，C3-6环烷基，和C1-6卤代烷基；
R 3选自-NR 9(CH 2) 2NR 9’R 9”，
Figure PCTCN2020099916-appb-000003
Figure PCTCN2020099916-appb-000004
R 4为
Figure PCTCN2020099916-appb-000005
R 5，R 6，和R 7独立地选自氢，卤素，C1-6烷基，C1-6卤代烷基，C1-6烷氧基，和CN；
R 8选自氢，C1-6烷基，和C1-6卤代烷基；
R 9选自氢，C1-6烷基，氘代C1-6烷基，和C1-6卤代烷基；
R 9’和R 9”独立地选自氢，C1-6烷基，C3-6环烷基，氘代C1-6烷基，和C1-6卤代烷基，或R 9’和R 9”与所相连的氮原子一起环合形成未取代或被1-3个选自卤素，C1-6烷基，和C1-6卤代烷基取代的杂环烷基；
R 10选自氢，卤素，C1-6烷基，和-CH 2NR 12’R 12”；
R 11选自氢，卤素，和C1-6烷基；和
R 12和R 12’独立地选自氢，C1-6烷基，C1-6卤代烷基，或R 12’和R 12”与所相连的氮原子一起环合形成未取代或被1-3个选自卤素，C1-6烷基，C1-3烷氧基，甲巯基，甲砜基，和C1-6卤代烷基取代的杂环烷基。
通式(I)中，优选的R 1优选选自氢,卤素，C1-6烷基，-C(O)OR 8，和-CN；和优选的R 5，R 6，和R 7独立地选自氢和卤素。
本发明提供通式(I)化合物，能够抑制一种或者多种EGFR激活型或耐药性突变，例如，T790M耐药性突变体、20号外显子插入激活突变体，因此，这类化合物就可用于对基于EGFR抑制剂现有疗法已产生一定程度耐药性患者的癌症治疗方案。
本发明提供通式(I)化合物，对激活或耐药性突变体形成的EGFR具有比野生型EGFR更高的抑制，因为与野生型EGFR抑制相关的毒性降低，所以这类化合物更适合于用作治疗剂，尤其适用于癌症的治疗。
本发明提供通式(I)化合物的制备方法。
本发明提供药物组合，它含有上述发明通式(I)化合物或其药学上可接受的盐、以及药学上可接受的载体、赋形剂或稀释剂。
本发明提供通式(I)化合物或其药学上可接受的盐在治疗哺乳动物尤其人类由EGFR激活型或耐药性突变体介导的疾病，特别是癌症方面的应用。
本发明提供一种治疗哺乳动物尤其人类由EGFR激活型或耐药性突变体介导的疾病、特别是癌症的方法，所述方法包括对患者施用通式(I)化合物或其药学上可接受的盐、或包括治疗有效量的通式(I)化合物和药物可接受的载体、赋形剂或稀释剂的药物组合物。
本发明提供一种相比于野生型EGFR选择性地抑制EGFR激活型或耐药性突变的方法，所述方法包括使生物样品接触或向患者投与通式(I)化合物或其药学上可接受的盐或其药物组合物。
本发明所提及癌症，可以选自肝细胞癌、肺癌、胰腺癌、乳腺癌、宫颈癌、子宫内膜癌、结肠直肠癌、胃癌、肺癌、鼻咽癌、卵巢癌、前列腺癌、白血病、淋巴瘤、、非霍奇金淋巴瘤骨髓瘤、胶质瘤、胶质母细胞瘤、黑色素瘤、胃肠道基质瘤(GIST)、甲状腺癌、胆管癌、肾癌、间变性大细胞淋巴瘤、急性髓细胞白血病(AML)、多发性骨髓瘤、间皮瘤。
在本发明中，具体优选的通式(I)化合物或其药学上可接受的盐，所述的化合物包括如下：
表1
Figure PCTCN2020099916-appb-000006
Figure PCTCN2020099916-appb-000007
Figure PCTCN2020099916-appb-000008
Figure PCTCN2020099916-appb-000009
Figure PCTCN2020099916-appb-000010
本发明提供制备通式(I)化合物的方法，它包括以下步骤：
Figure PCTCN2020099916-appb-000011
Figure PCTCN2020099916-appb-000012
其中，R 1、R 2、R 3、R 4、R 5、R 6和R 7与上述通式(I)中的含义相同。
以化合物(a)和(b)为起始原料，在碱作用下经取代反应得到中间体1，中间体1与中间体2经取代或偶联反应得到化合物(c)，化合物(c)经过亲核取代得到化合物(d),化合物(d)硝基被还原得到化合物(e),化合物(e)经酰化得到化合物(I)；或中间体1与中间体2’经取代或偶联反应直接得到化合物(I)。
在本发明制备通式(I)化合物的一个实施方案中，当中间体1为中间体1a时，其制备方法如下：
Figure PCTCN2020099916-appb-000013
在本发明制备通式(I)化合物的一个实施方案中，当中间体1为中间体1b时，其制备方法如下，
Figure PCTCN2020099916-appb-000014
在本发明制备通式(I)化合物的一个实施方案中，当中间体2、中间体2’的制备方法 包括以下步骤:
Figure PCTCN2020099916-appb-000015
其中，R 2、R 3和R 4与上述通式(I)中的含义相同。
以2,6-二氯-3-硝基吡啶为原料，经醚化反应得到化合物(g)，化合物(g)硝基被还原得到化合物(h)，化合物(h)经酰化得到化合物(i)，化合物(i)经硝化反应得到化合物(j)，再脱保护得到中间体2。
化合物(j)与R 3H经取代反应得到化合物(k)，化合物(k)经Boc保护得到化合物(l)，然后脱乙酰基保护得到化合物(m)，化合物(m)硝基被还原得到化合物(n)，化合物(n)经酰化得到化合物(o)，最后脱保护得到中间体2’。
在本发明所述中间体2’、中间体2’的制备方法中，醚化反应是在强碱作用下进行，所述强碱包括但不限于氢化钠、氢化钾、氢氧化钠、氢氧化钾、乙醇钠、甲醇钠；还原硝基的方法采用本领域中公知的常规还原剂，包括但不限于铁粉、锌粉、硫化钠、H 2/PtO 2；上保护基或脱保护基是采用本领域中公知的常规方法，再适当的酸性或碱性条件下进行。
本发明中“卤素”(或卤代基)是指氟、氯、溴或碘。
“C1-6烷基”是指含有1至6个碳原子的直链或支链烷基，优选为1至4个碳原子的直链或支链烷基。支链是指一个或多个1至4个碳原子的烷基如甲基、乙基或丙基等与直链烷基连接。优选的C1-6烷基包括但不限于甲基、乙基、正丙基、异丙基、正丁基、异丁基和叔丁基等。
“氘代烷基”是指烷基中的一个或多个氢原子被氘取代。例如，甲基中的三个氢原子都被氘取代，成为氘代甲基CD 3。
“C1-6卤代烷基”是指如上定义的C1-6烷基中含有一个或多个卤素原子取代基。
“C1-6杂烷基”是指如上定义的C1-6烷基中含有一个或多个选自以下基团中的取代基：O、S、N、-(S＝O)-、-(O＝S＝O)-等。
“C3-6环烷基”是指含有3至6个碳原子，优选3至6个碳原子的非芳族单环或多环基团。优选的单环C3-6环烷基包括但不限于环丙基、环戊基、环己基等。
“C1-6烷氧基”是指C1-6烷基-O-基团，通过氧与母体部分键接，其中C1-6烷基如上所述。优选的C1-6烷氧基包括但不限于甲氧基、乙氧基、正丙氧基、异丙氧基和正丁氧基。
本发明任何前述官能团可以是未经取代或被本发明所述取代基取代。术语“取代的”(或取代)是指将指定原子上的一个或多个氢原子替换为从指定基团中选择的基团，条件是不超出指定原子的正常价态，并且取代产生稳定的化合物。只有当所述组合形成稳定化合物时，所述取代基和/或变量的组合才是允许的。
本发明还包括通式(I)化合物在药学上可接受的盐。术语“药学上可接受的盐”是指相对无毒的本发明化合物的酸加成盐或碱加成盐。所述酸加成盐为本发明通式(I)化合物与合适的无机酸或者有机酸形成的盐，这些盐可在化合物最后的分离和提纯过程中制备，或者可使用纯化的通式(I)化合物以其游离碱形式与适宜的有机酸或无机酸进行反应来制备。代表性酸加成盐包括氢溴酸盐、盐酸盐、硫酸盐、硫酸氢盐、亚硫酸盐、乙酸盐、草酸盐、戊酸盐、油酸盐、棕榈酸盐、硬脂酸盐、月桂酸盐、硼酸盐、苯甲酸盐、乳酸盐、磷酸盐、磷酸氢盐、碳酸盐、碳酸氢盐、甲苯甲酸盐、柠檬酸盐、马来酸盐、富马酸盐、琥珀酸盐、酒石酸盐、苯甲酸盐、甲磺酸盐、对甲苯磺酸盐、葡萄糖酸盐、乳糖酸盐和月桂酸磺酸盐等。所述碱加成盐为本发明通式(I)化合物与合适的无机碱或者有机碱形成的盐，包括例如与碱金属、碱土金属、季铵阳离子形成的盐，例如钠盐、锂盐、钾盐、钙盐、镁盐、四甲基铵盐、四乙基铵盐等；胺盐，包括与氨(NH 3)、伯氨、仲氨、或叔胺形成的盐，如甲胺盐、二甲胺盐、三甲胺盐、三乙胺盐、乙胺盐等。
通过酶活实验证明本发明化合物对20号外显子插入性激活突变酶具有很好的活性； 通过细胞实验即对激活型突变即20号外显子插入型激活突变细胞、耐药性肿瘤细胞以及野生型EGFR人皮肤细胞的体外增殖抑制实验，证明本发明化合物对激活型突变或耐药性突变肿瘤细胞具有良好的增殖抑制作用，但对野生型EGFR癌细胞的增殖抑制作用较弱，具有良好的选择性。本发明化合物可用作治疗有EGFR激活型或耐药型突变体活性介导的疾病或病况、特别是肿瘤例如癌症的药物。所述癌症包括但不限于，例如肝细胞癌、肺癌、头颈癌、胰腺癌、乳腺癌、宫颈癌、子宫内膜癌、结肠直肠癌、胃癌、肺癌、鼻咽癌、卵巢癌、前列腺癌、白血病、淋巴瘤、非霍奇金淋巴瘤骨髓瘤、胶质瘤、胶质母细胞瘤、黑色素瘤、胃肠道基质瘤(GIST)、甲状腺癌、胆管癌、肾癌、间变性大细胞淋巴瘤、急性髓细胞白血病(AML)、多发性骨髓瘤、间皮瘤，尤其对对于表皮生长因子受体790位苏氨酸突变为蛋氨酸(EGFR T790M)和激活型突变即20号外显子插入型激活突变的肿瘤类型有更好的应用。
应理解，本发明的前述一般性描述和以下详细描述都是示例性和说明性的，旨在提供对所要求保护的本发明的进一步说明。
应理解，在不脱离本发明范围和精神的情况下，本领域技术人员可以对本发明进行各种改动或修改，这对于本领域技术人员是显而易见的，这些等价形式同样落于本申请所附权利要求书所限定的范围。
附图说明
图1为化合物1对PC9细胞脑原位裸小鼠存活率影响的结果图。
具体实施方式
以下将详细描述本发明的实施例。
实施例
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围，且本发明不限于这些实施例。本领域的技术人员将容易理解的是，可使用以下制备方法的条件和过程的已知变型来制备这些化合物。本发明中用到的起始反应物未经特别说明，均为商业购买。
缩写：室温(RT，rt)；水溶液(aq.)；石油醚(PE)；乙酸乙酯(EA)；二氯甲烷(DCM)；甲醇(MeOH)；乙醇(EtOH)；四氢呋喃(THF)；二甲基甲酰胺(DMF)；二甲基亚砜(DMSO)；三乙胺(TEA)；二异丙基乙胺(DI(P)EA)；4-二甲基氨基吡啶(DMAP)；钯碳(Pd/C)；当量 (eq.)；克/毫克(g/mg)；摩尔/毫摩尔(mol/mmol)；升/毫升(L/mL)；分钟(min(s))；小时(h,hr,hrs)；氮气(N 2)；核磁共振(NMR)；薄层色谱(TLC)。
一般合成方法：
除非另有说明，否则所有反应均在惰性气体(如氩气或氮气)环境下进行，使用的市售试剂和无水溶剂无需进行进一步处理。
质谱用液相色谱-质谱联用仪(LC-MS)记录(安捷伦6120B型单四级杆液相色谱-质谱联用仪)。核磁共振谱(如氢谱( 1H)、碳谱( 13C)、磷谱( 31P)和氟谱( 19F)等)用BrukerAMX-400型、Gemini-300型或AMX–600型核磁共振仪记录，在氘代氯仿、氘代甲醇、氘代水或氘代二甲亚砜等氘代溶剂中记录并以氘代溶剂峰为参考标准。化学位移δ的单位为ppm，耦合常数(J)的单位为赫兹(Hz，Hertz)，核磁谱中耦合裂分峰表示为：宽单峰(brs)、单峰(s)、二重峰(d)、双二重峰(dd)、三重峰(t)、四重峰(q)和多重峰(m)。
具体实施方式
Ⅰ.本发明化合物制备实施例
中间体1a:1-(2-氯嘧啶-4-基)-8-氟-5,6-二氢-4H-咪唑[4,5,1-ij]喹啉-2(1H)-酮的合成
Figure PCTCN2020099916-appb-000016
步骤1：3-氯-N-(4-氟苯基)丙酰胺的合成
Figure PCTCN2020099916-appb-000017
3-氯丙酰氯(653g,1eq)溶于6.5L二氯甲烷中，干冰乙醇浴下滴加原料(783.6g,1.05eq)有大量固体析出，维持内温0～10度。滴加完毕搅拌0.5h，分批加入咪唑(405g，1.01eq)(升温明显)，维持内温0～10度。搅拌1h反应完全，反应液倒入到稀盐酸中，分液，有机相浓缩至大量固体析出，加入PE/EA(5/1)800ml搅拌过夜，过滤，PE/EA(5/1)洗涤得950g白色固体，MS(ESI):m/z＝202[M+H] +，HNMR: 1H NMR(400MHz,DMSO-d 6)δ10.16(s,1H),7.71–7.60(m,2H),7.23–7.13(m,2H),3.91(t,J＝6.3Hz,2H),2.84(t,J＝6.3Hz,2H).
步骤2：6-氟-3,4-二氢喹啉-2(1H)-酮的合成
Figure PCTCN2020099916-appb-000018
5L三口瓶中，加入3-氯-N-(4-氟苯基)丙酰胺(820g，1eq)，搅拌下加入无水三氯化铝(1640g,3eq)，氮气置换三次，设置外温60度，搅拌至熔融(内温升至70度)，待内温有所下降，升温至100度(内温97度)，搅拌4h，LCMS显示反应58％，补加500g三氯化铝，搅拌4h，LCMS显示反应73％，补加200g三氯化铝搅拌4h，TLC检测少量原料残留。待体系冷却至40度，往体系加入DCM(2L)，DCM溶液滴加到THF(6L)中，剧烈放热，完毕，体系粘稠，补加EA(3L)，继续加水,体系析出大量固体，分出有机相，有机相浓缩，水相过滤，合并产物分别用EA，水打浆得湿品600g白色固体，MS(ESI):m/z＝166[M+H] +，HNMR: 1H NMR(400MHz,DMSO-d 6)δ10.16(s,1H),7.71–7.61(m,2H),7.23–7.13(m,2H),3.92(t,J＝6.3Hz,2H),2.85(t,J＝6.3Hz,2H).
步骤3：6-氟-8-硝基-3,4-二氢喹啉-2(1H)-酮的合成
Figure PCTCN2020099916-appb-000019
5L三口瓶中，加入6-氟-3,4-二氢喹啉-2(1H)-酮(700g，1eq)，加入乙酸酐3.5L，控制内温15-20度，缓慢滴加浓硝酸(485g，1.2eq),滴加完毕，体系澄清，25度搅拌30min,有大量固体析出，将反应液倒入水(20L)中，搅拌至水解完全，过滤，滤饼用水洗至洗液无色，烘干得700g黄色固体，MS(ESI):m/z＝211[M+H] +，HNMR: 1H NMR(400MHz,DMSO-d 6)δ9.84(s,1H),7.91(dd,J＝8.9,2.9Hz,1H),7.70(dd,J＝8.2,2.8Hz,1H),3.15–3.04(m,2H),2.63(dd,J＝8.3,6.7Hz,2H).
步骤4：6-氟-8-氨基-1,2,3,4-四氢喹啉的合成
Figure PCTCN2020099916-appb-000020
LiAlH 4(48g,1.27mol)溶于THF(1L)，分批加入6-氟-8-硝基-3,4-二氢喹啉-2(1H)-酮(89g,0.42mol)的THF(100mL)悬浊液,保持内温5-10度。滴加完毕后自然恢复至12度下搅拌0.5h。体系冷却至低于0度后，水(48mL),15％NaOH(48mL)和水(144mL)依次淬灭，保持内温低于5度，加入硅藻土(90g)，低于5度下搅拌30min后，垫硅藻土过滤，THF洗涤，滤饼再次THF打浆，过滤，有机相浓缩，粗品柱层析(流动相PE/EA依次比例：1/10,1/4,2/3含0.1％TEA)分离得酒红色油状液体57g，MS(ESI):m/z＝167[M+H] +。
步骤5：8-氟-5,6-二氢-4H-咪唑[4,5,1-ij]喹啉-2(1H)-酮的合成
Figure PCTCN2020099916-appb-000021
6-氟-8-氨基-1,2,3,4-四氢喹啉(166g,1mol)溶于THF(1L)，滴加三光气(118g,0.4mol)的THF(300mL)悬浊液,保持内温5-10度。滴加完毕后继续搅拌0.5h，滴加咪唑(160g,20mol)，保持内温10-20度,恢复至室温后继续搅拌15min，LCMS监测，原料反应完全，加13％NaCl溶液1L，补加THF(1L)，分液，水相THF(2L*2)萃取，干燥浓缩，粗品EA打浆过夜，过滤得目标产物淡棕色固体168g，MS(ESI):m/z＝193[M+H] +。
步骤6：1-(2-氯嘧啶-4-基)-8-氟-5,6-二氢-4H-咪唑[4,5,1-ij]喹啉-2(1H)-酮的合成
Figure PCTCN2020099916-appb-000022
8-氟-5,6-二氢-4H-咪唑[4,5,1-ij]喹啉-2(1H)-酮(36g,0.19mol)和2,4-二氯嘧啶(34g,0.23mol)溶于DMF(400mL)，加入碳酸铯(122g,0.37mol)，室温下搅拌4h，LCMS监测原料反应完全，加水(250mL)稀释体系，固体过滤，拌样，柱层析(DCM/EA,100/1)分离，体系浓缩至50mL左右，加入PE(200mL)打浆，过滤得白色固体45g，MS(ESI):m/z＝305[M+H] +，HNMR: 1H NMR(400MHz,DMSO-d 6)δ8.81(d,J＝5.7Hz,1H),8.42(d,J＝5.8Hz,1H),7.75(d,J＝9.7Hz,1H),7.00(d,J＝9.6Hz,1H),3.82(t,J＝5.5Hz,2H),2.85(t,J＝5.6Hz,2H),2.15–2.01(m,2H).
中间体1b:1-(2-氯嘧啶-4-基)-5,6-二氢-4H-咪唑[4,5,1-ij]喹啉-2(1H)-酮的合成
Figure PCTCN2020099916-appb-000023
步骤1：N-甲氧基-3,4-二氢喹啉-1(2H)-甲酰胺的合成
Figure PCTCN2020099916-appb-000024
335g(1.13mol)三光气溶于3L二氯甲烷，在0℃到5℃温度区间下滴加300g(2.26mol)1,2,3,4-四氢喹啉和390g(3.86mmol)三乙胺的二氯甲烷(2L)溶液，耗时1.5小时。滴加 完毕后℃下搅拌反应1小时。TLC(PE：EA＝5：1)检测大部分1,2,3,4-四氢喹啉反应完。接着0℃下一次性加入800g(7.92mol)三乙胺和375g(4.52mol)甲氧胺盐酸盐，加料完毕后升温至室温(15℃)反应16小时，TLC(PE：EA＝5：1)检测少部分(约20％)原料未反应完。将反应升温至30℃(水浴)继续反应3小时,TLC(PE：EA＝5：1)检测反应完毕。反应液用2M盐酸(3L)洗，水相用1L二氯甲烷萃取。所有有机相合并，3L饱和碳酸氢钠溶液洗，2L饱和食盐水洗，无水硫酸钠干燥，过滤旋干，得到黄色固体580g。
步骤2：1-甲氧基-5,6-二氢-4H-咪唑[4,5,1-ij]喹啉-2(1H)-酮的合成
Figure PCTCN2020099916-appb-000025
580g(1.13mol)N-甲氧基-3,4-二氢喹啉-1(2H)-甲酰胺(粗品)溶于500ml二氯甲烷，在-3℃到2℃温度区间下滴加1250g(2.91mol)二(三氟乙酸)碘苯的二氯甲烷(1.2L)溶液，滴加完毕自然升至室温(15℃)搅拌反应1小时。TLC(PE：EA＝1：1)检测反应完毕。向反应液中加入8L饱和碳酸氢钠溶液，分出有机相，旋干。粗品经柱层析分离(PE：EA＝5：1至1：1)纯化得黄色固体205g，产率44.5％。
步骤3：5,6-二氢-4H-咪唑[4,5,1-ij]喹啉-2(1H)-酮的合成
Figure PCTCN2020099916-appb-000026
51.25g(251.22mmol)1-甲氧基-5,6-二氢-4H-咪唑[4,5,1-ij]喹啉-2(1H)-酮溶于500mL乙醇，室温(15℃)下加入20g雷尼镍，加料完毕升温至50℃，氢气球下搅拌16小时，TLC(PE：EA＝1：1)检测约30％原料未反应完。重新至于新的氢气球下50℃搅拌4小时，TLC(PE：EA＝1：1)检测仍有约20％原料未反应完。室温补加8g雷尼镍，重新至于新的氢气球下50℃搅拌16小时，TLC(PE：EA＝1：1)检测原料反应完。反应液冷却到室温，硅藻土过滤，3次150mL甲醇洗滤饼，滤液旋干。粗品(4个批次合并)用PE：EA＝1：1(800mL)打浆，过滤得灰白色固体155g，产率88.6％。
步骤4：1-(2-氯嘧啶-4-基)-5,6-二氢-4H-咪唑[4,5,1-ij]喹啉-2(1H)-酮的合成
Figure PCTCN2020099916-appb-000027
155g(890.80mmol)5,6-二氢-4H-咪唑[4,5,1-ij]喹啉-2(1H)-酮溶于1.5L DMF，室温 (10℃)下加入158g(1.06mol)2,4-二氯嘧啶和580g(1.78mol)碳酸铯，升温至30℃，搅拌反应16小时。TLC(DCM：MeOH＝20：1)检测反应完毕。向反应液中加入3L水，搅拌1小时。过滤，滤饼1L水洗。滤饼重新用PE：EA＝1：1(1.5L)打浆，过滤得灰白色固体230g，产率90.2％。
中间体2a:N-(5-氨基-2-((2-(二甲氨基)乙基)(甲基)氨基)-4-甲氧基苯基)丙烯酰胺的合成
Figure PCTCN2020099916-appb-000028
步骤1：N 1-(2-(二甲氨基)乙基)-5-甲氧基-N 1-甲基-2-硝基苯-1,4-二胺的合成
Figure PCTCN2020099916-appb-000029
4-氟-2-甲氧基-5-硝基苯胺(3g,16mmol)和N 1,N 1,N 2-三甲基乙烷-1,2-二胺(2.47g,24mmol)溶于DMF(30mL)，加入碳酸钾(4.5g,32mmol)，80度下搅拌2h，LCMS监测原料反应完全，冷却至室温后加水(60mL)稀释体系，固体过滤，粗品EtOH/H 2O(1/1)打浆,过滤，干燥得黄色固体3.1g,MS(ESI):m/z＝269,[M+H] +。
步骤2：叔丁基(4-((2-(二甲氨基)乙基)(甲基)氨基)-2-甲氧基-5-硝基苯基)氨基甲酸酯的合成
Figure PCTCN2020099916-appb-000030
N 1-(2-(二甲氨基)乙基)-5-甲氧基-N 1-甲基-2-硝基苯-1,4-二胺(3.1g,12mmol)溶于THF(40mL)，加入二碳酸二叔丁酯(3.8g,17mmol)，70度下搅拌6h后反应完全，浓缩，粗品EA/PE(1/5)打浆得淡黄色固体3.8g,MS(ESI):m/z＝369,[M+H] +。
步骤3：叔丁基(5-氨基-4-((2-(二甲氨基)乙基)(甲基)氨基)-2-甲氧基苯基)氨基甲酸酯的合成
Figure PCTCN2020099916-appb-000031
叔丁基(4-((2-(二甲氨基)乙基)(甲基)氨基)-2-甲氧基-5-硝基苯基)氨基甲酸酯(3.8g,10.3mmol)溶于MeOH(40mL)，置换氮气三次后，加入Pd/C(0.4g)，置换氢气三次后，室温下搅拌4h，反应完全，过滤浓缩，粗品直接下一步反应，MS(ESI):m/z＝339,[M+H] +。
步骤4：叔丁基(5-丙烯酰胺基-4-((2-(二甲氨基)乙基)(甲基)氨基)-2-甲氧基苯基)氨基甲酸酯的合成
Figure PCTCN2020099916-appb-000032
叔丁基(5-氨基-4-((2-(二甲氨基)乙基)(甲基)氨基)-2-甲氧基苯基)氨基甲酸酯(10.3mmol)溶于DCM(50mL)，冰浴下依次滴加丙烯酰氯(1.36g,15mmol)，自然恢复至室温后反应0.5h，加入饱和碳酸氢钠溶液调节pH至8，水相分液，DCM(50mL)萃取，干燥浓缩，粗品柱层析(MeOH/DCM＝1/70至1/20)得灰色固体1.4g，MS(ESI):m/z＝393,[M+H] +。
步骤5：N-(5-氨基-2-((2-(二甲氨基)乙基)(甲基)氨基)-4-甲氧基苯基)丙烯酰胺的合成
Figure PCTCN2020099916-appb-000033
叔丁基(5-丙烯酰胺基-4-((2-(二甲氨基)乙基)(甲基)氨基)-2-甲氧基苯基)氨基甲酸酯(392mg,1mmol)溶于DCM(5mL)，滴加TFA(1mL)，室温下搅拌1h后反应完全，冰浴下加入饱和碳酸氢钠溶液调节pH至8，水相分液，DCM(50mL)萃取，干燥浓缩，粗品柱层析(MeOH/DCM＝1/20至1/10)得褐色糖浆状固体200mg，MS(ESI):m/z＝293,[M+H] +。
中间体2b:N-(5-氨基-2-((2-(二甲氨基)乙基)(甲基)氨基)-6-甲氧基吡啶-3-基)丙烯酰胺的合成
Figure PCTCN2020099916-appb-000034
步骤1：6-氯-2-三氟乙氧基-3-硝基吡啶的合成
Figure PCTCN2020099916-appb-000035
将2,6-二氯-3-硝基吡啶(500g，2.6mol)溶于THF(1L)中，降温至-10℃以下，加入钠氢(104g，2.6mol)，控制温度-15℃滴加三氟乙醇(260g，2.6mol)，加完缓慢回至室温反应过夜，TLC(PE/EA＝5/1)反应完全，体系倾至1L冰水中，搅拌分液，有机相浓缩至少量溶剂残余，以EA萃取两次，合并有机相，饱和食盐水洗，干燥，旋干得黄色油固混合物720g，MS(ESI):m/z＝257(M+H) +。
步骤2：6-氯-2-三氟乙氧基吡啶-3-胺的合成
Figure PCTCN2020099916-appb-000036
将6-氯-2-三氟乙氧基-3-硝基吡啶(150g，0.58mol)溶于乙醇/水(1.2/0.3L)混合溶剂中，加入氯化铵(160g，2.9mol)，升温至内温50℃，开始分批次缓慢加入铁粉(166g，2.9mol)，控制内温80℃反应1h，TLC(PE/EA＝5/1)反应完全。降温至内温40℃，加入碳酸钠160g，硅藻土160g，加完搅拌20min，抽滤，硅藻土助滤，滤饼以DCM打浆，乙醇-水母液浓缩至干，以滤饼打浆的DCM相萃取两次。合并有机相，饱和食盐水洗，干燥，旋干得黑色油状物122g，MS(ESI):m/z＝227(M+H) +。
步骤3：N-(6-氯-2-三氟乙氧基-吡啶-3-基)乙酰胺的合成
Figure PCTCN2020099916-appb-000037
将6-氯-2-三氟乙氧基吡啶-3-胺(570g，2.5mol)溶于DCM(4.5L)中，加入DIPEA(540mL，3.8mol)，降温至0℃，开始滴加乙酰氯(200mL，3mol)，控制温度10℃反应，滴加1h。加完30min，TLC(PE/EA＝5/1)显示反应完全。冰浴下加入水(2L)，分液，水相以DCM萃取。合并有机相，1M盐酸洗，饱和食盐水洗，干燥，旋干柱层析，PE/EA＝5/1洗脱得 黄色固液混合物480g，MS(ESI):m/z＝269(M+H) +。
步骤4：N-(6-氯-2-三氟乙氧基-5-硝基吡啶-3-基)乙酰胺的合成
Figure PCTCN2020099916-appb-000038
将N-(6-氯-2-三氟乙氧基-吡啶-3-基)乙酰胺(300g，1.1mol)悬浊于三氟乙酸酐(1.5L)中，降温至-5℃以下，滴加浓硝酸(125g，1.2mol)，1h加完，-5℃反应3h，TLC(PE/EA＝2/1)显示反应完全。搅拌下体系加至冰水混合物中，搅拌分散，抽滤，依次以水、PE淋洗，湿品185g以PE/EA混合溶剂400ml打浆过夜，抽滤，滤饼再以PE/EA＝5/1混合溶剂打浆得黄色固体220g，MS(ESI):m/z＝314(M+H) +。
步骤5：6-氯-2-三氟乙氧基-5-硝基吡啶-3-胺的合成
Figure PCTCN2020099916-appb-000039
将N-(6-氯-2-三氟乙氧基-5-硝基吡啶-3-基)乙酰胺(220g，0.7mol)悬浊于甲醇/浓盐酸(900/220mL)混合溶剂中，升温至50℃反应4h，体系溶清，TLC监测反应完全，搅拌下体系加至水中，搅拌分散，抽滤，水洗，滤饼再以饱和碳酸氢钠溶液打浆，抽滤，依次以水、PE淋洗，抽干得黄色固体175g，MS(ESI):m/z＝272(M+H) +。
步骤6：N2-(2-(二甲氨基)乙基)-N2-甲基-3-硝基-6-(2,2,2-三氟乙氧基)-2,5-二胺的合成
Figure PCTCN2020099916-appb-000040
6-氯-2-三氟乙氧基-5-硝基吡啶-3-胺(950mg，3.5mmol)溶于乙腈(15mL)，室温下加入K 2CO 3(967mg，7mmol)，N，N，N’-三甲基乙二胺(643mg，6.3mmol)，油浴80度中搅拌过夜。反应液经滤纸抽滤，滤液旋干，硅胶柱层析，收集目标组分，旋干后得到1.16g红色油状物，MS＝338.2(M+H) +。
步骤7：N2-(2-(二甲氨基)乙基)-N2-甲基-3-硝基-5-二叔丁氧羰基氨基-6-(2,2,2-三氟乙氧基)-2-胺的合成
Figure PCTCN2020099916-appb-000041
N2-(2-(二甲氨基)乙基)-N2-甲基-3-硝基-6-(2,2,2-三氟乙氧基)-2,5-二胺(1.01g， 3.5mmol)和DMAP(110mg，0.9mmol)溶于1，4-二氧六环(30mL)，加入二碳酸二叔丁酯(1.96g，10.5mmol)，油浴100度中搅拌8h，浓缩，柱层析分离得黄色油状物680mg，MS＝538(M+H) +。
步骤8：N2-(2-(二甲氨基)乙基)-N2-甲基-5-二叔丁氧羰基氨基-6-(2,2,2-三氟乙氧基)-2,3-二胺的合成
Figure PCTCN2020099916-appb-000042
N2-(2-(二甲氨基)乙基)-N2-甲基-3-硝基-5-二叔丁氧羰基氨基-6-(2,2,2-三氟乙氧基)-2-胺(680mg，1.3mmol)溶于MeOH(30mL)，加入10％Pd-C(136mg)，置换氢气三次后，室温下搅拌1h，反应结束后经硅藻土抽滤，滤液浓缩柱层析分离得棕色油状物415mg，MS＝508.3(M+H) +。
步骤9：N-(5-二叔丁氧羰基氨基-2-((2-(二甲氨基)乙基)(甲基)氨基)-6-(2,2,2-三氟乙氧基)吡啶-3-基)丙烯酰胺的合成
Figure PCTCN2020099916-appb-000043
N2-(2-(二甲氨基)乙基)-N2-甲基-5-二叔丁氧羰基氨基-6-(2,2,2-三氟乙氧基)-2,3-二胺(415mg，0.8mmol)溶于DCM(15mL)，加入三乙胺(248mg，2.4mmol)，冰水浴下搅拌，滴加丙烯酰氯(148mg，1.6mmol)，自然恢复至室温后继续搅拌10min，加水淬灭体系，DCM(15mLx3)萃取，合并的有机相，干燥，浓缩柱层析得棕色油状物318mg，MS＝562.3(M+H) +。
步骤10：N-(5-氨基-2-((2-(二甲氨基)乙基)(甲基)氨基)-6-(2,2,2-三氟乙氧基)吡啶-3-基)丙烯酰胺的合成
Figure PCTCN2020099916-appb-000044
N-(5-二叔丁氧羰基氨基-2-((2-(二甲氨基)乙基)(甲基)氨基)-6-(2,2,2-三氟乙氧基)吡 啶-3-基)丙烯酰胺(318mg，0.57mmol)溶于DCM(20mL)，冰水浴下滴加甲磺酸(1.63g,5.7mmol)，自然恢复至室温后继续搅拌2.5h。冰水浴下缓慢滴加饱和碳酸氢钠溶液调节pH至8，DCM(25mLx3)萃取，合并有机相，干燥，浓缩，柱层析分离得浅棕绿色固体176mg，MS＝362.2(M+H) +。
实施例1：N-(2-((2-(二甲氨基)乙基)(甲基)氨基)-5-((4-(8-氟-2-氧代-5,6-二氢-4H-咪唑[4,5,1-ij]喹啉-1(2H)-基)嘧啶-2-基)氨基)-4-甲氧基苯基)丙烯酰胺
Figure PCTCN2020099916-appb-000045
中间体1a(152mg,0.2mmol)，中间体2a(200mg,0.68mmol)，醋酸钯(45mg,0.2mmol)，Xanphos(116mg,0.2mmol)和碳酸铯(130mg,0.4mmol)加入到1,4-二氧六环(5mL)中，90度下搅拌10h，反应完全，垫硅藻土过滤，浓缩，柱层析分离(MeOH/DCM 1/10)得淡棕色固体41mg。
用相同方法合成的化合物如下表：
表2
Figure PCTCN2020099916-appb-000046
Figure PCTCN2020099916-appb-000047
Figure PCTCN2020099916-appb-000048
Figure PCTCN2020099916-appb-000049
Figure PCTCN2020099916-appb-000050
Figure PCTCN2020099916-appb-000051
Figure PCTCN2020099916-appb-000052
Figure PCTCN2020099916-appb-000053
Figure PCTCN2020099916-appb-000054
Figure PCTCN2020099916-appb-000055
参考化合物1的合成，可以得到下表所示化合物：
表3
Figure PCTCN2020099916-appb-000056
Figure PCTCN2020099916-appb-000057
Figure PCTCN2020099916-appb-000058
Figure PCTCN2020099916-appb-000059
Figure PCTCN2020099916-appb-000060
本发明化合物活性测试实施例
测试实施例1：对野生型EGFR、HER2以及HER4和突变型EGFR激酶活性的抑制作用
用Echo 550向反应板(784075,Greiner)每孔转移10nL稀释好的化合物，用封板膜封住反应板，1000g离心1分钟；用1X的激酶反应缓冲液配制准备2X Enzyme，向反应板中每孔加入5μL 2X Enzyme，用封板膜封住板子1000g离心30秒，室温放置10分钟；用1X的激酶反应缓冲液配制2X TK-substrate-biotin和ATP混合液，向反应板中加入5μL TK-substrate-biotin/ATP混合液，用封板膜封住板子1000g离心30秒，室温反应40分钟；用HTRF检测缓冲液配制2X Sa-XL 665和TK-antibody-Cryptate混合液，每孔加入10μL Sa-XL 665/TK-antibody-Cryptate混合液，1000g离心30秒，室温反应1小时；用Envision 2104读取615nm(Cryptate)和665nm(XL665)的荧光信号，计算各组665nm和615nm的比值，与对照组对比计算抑制百分率，进而计算药物抑制酶活的IC 50。
表4
Figure PCTCN2020099916-appb-000061
Figure PCTCN2020099916-appb-000062
测试结果表明：上述化合物与AZD9291相比，对具有20号外显子插入突变或者点突变的EGFR激酶具有很高的活性，本发明的其他化合物也具有很好的活性。
测试实施例2：对人皮肤癌细胞(A431，野生型EGFR)、人肺癌细胞(H1975，EGFR T790M耐药型突变)、Ba/F 3(过表达EGFR D770_N771insSVD，20号外显子插入突变型EGFR的pro-B细胞)、Ba/F 3(过表达V769_D770insASV 20号外显子插入突变型EGFR的pro-B细胞)增殖抑制作用
取处于对数生长期的细胞接种在96孔板中(A431细胞浓度为5000个/孔，细胞悬液180μL/孔；H1975细胞浓度为3000个/孔，细胞悬液180μL/孔；Ba/F 3细胞浓度为10000个/孔，细胞悬液180μL/孔)，5％CO 2，37℃孵育过夜使细胞贴壁，加入药液20μL/孔，药物最高浓度为30μM，3倍稀释(药物浓度梯度为30μM，10μM，3μM，1μM，0.3μM，0.1μM，0.03μM，0.01μM)，每个药物浓度设3个复孔。另外设对照组(加培液和细胞，无药物)和空白组(仅加培液，无细胞和药物)。继续在5％CO 2，37℃中培养72小时，期间倒置显微镜下观察细胞增殖和药物析出情况。每孔吸出80μL的新鲜培液，再每孔加入10μL/孔MTT溶液(5mg/ml，即0.5％MTT)，继续培养4h后加入50μL/孔的三联液(10％SDS-5％异丁醇-0.01mol/L HCl)，于CO 2培养箱中培养过夜。用酶标仪测OD570值，与对照组对比计算抑制百分率，进而计算药物抑制细胞增殖的IC 50。结果见表5：
表5
Figure PCTCN2020099916-appb-000063
测试结果表明：上述化合物对Ba/F 3(表达并依赖EGFR 20号外显子插入突变包括D770_N771insSVD，V769_D770insASV生长的proB细胞)、人肺癌细胞(H1975，EGFR T790M耐药型突变)具有很强的增殖抑制作用，对人皮肤癌细胞(A431，野生型EGFR)的增殖抑制作用较弱，即本发明化合物与AZD9291相比，在保持了T790M耐药型突变活性的同时，大大的提高了依赖EGFR 20号外显子插入突变活性增殖的Ba/F3细胞的活性，同时相对于野生型EGFR具有良好的选择性。本发明的其他化合物也具有很好的活性和选择性。
测试实施例3对小鼠皮下PDX和CDX模型中肿瘤生长的作用
裸小鼠皮下左侧背部接种携带EGFR外显子20插入突变的LU-01-0493,LU-01-0426和LU-0387的肿瘤块或者携带EGFR T790M的H1975细胞。待肿瘤生长至100-150mm 3，分组，每周一次口服灌胃给药化合物1或者poziotinib。一周两次，以及给药终点第28天(LU-01-0493,LU-01-0426和LU-0387)或者21天(H1975)测量肿瘤体积和小鼠体重。按照肿瘤生长抑制率(TGI)＝1-(给药组第28天肿瘤体积-给药组第一天肿瘤体积)/(对照组第28天给药体积-对照组第一天肿瘤体积)，评价化合物抑制肿瘤生长能力。根据小 鼠体重评价化合物的毒性。
表6
Figure PCTCN2020099916-appb-000064
NA：未评价该剂量组或者不适用
如上表所示，与poziotinib相比，化合物1更加安全有效，其具有更强的抑制携带EGFR外显子20插入突变或EGFR T790M突变的体内肿瘤生长的活性，有效剂量下，对小鼠体重影响较小。
测试实施例4对小鼠脑原位PC9模型的作用
裸小鼠脑部原位注射用luciferase标记的3*10 5携带EGFR Del 19的PC9细胞，注射后三天后，按照荧光强度和体重随机分组，给药。每天给药化合物1评价小鼠的生存率，除了自然死亡小鼠外，体重降低20％以上提示小鼠濒死，按照动物福利，小鼠将被安乐死，计入小鼠死亡。
由表7和图1结果可见，对照组小鼠在给药后28天内全部死亡，而给药化合物1后，小鼠全部存活至28天，表明化合物1可以抑制脑内肿瘤诱发的小鼠死亡，提示化合物1可以入脑，抑制脑内肿瘤生长。
表7化合物1对原位脑癌模型PC9小鼠存活率的影响
Figure PCTCN2020099916-appb-000065
从上述测试实施例1-4的结果可以看出，本发明化合物在酶水平对20号外显子插入突变或者点突变的激酶具有很高的抑制活性，细胞水平上大大的提高了抑制依赖EGFR20号外显子插入突变活性增殖的Ba/F3细胞的活性，同时相对于野生型EGFR具有良好的选择性。对多种小鼠PDX模型，化合物1均显示具有很强的抑制肿瘤活性，和poziotinib相比，化合物1更加安全有效，小鼠脑原位PC9模型也显示化合物1可以入脑，抑制脑内肿瘤生长。本发明其它化合物也具有很强的抑制体内肿瘤生长的能力。
虽然以上描述了本发明的具体实施方式，但是本领域的技术人员应当理解，这些仅是举例说明，在不背离本发明的原理和实质的前提下，可以对这些实施方式做出多种变更或修改。因此，本发明的保护范围由所附权利要求书限定。

Claims (3)

  1. 一种通式(I)所示化合物或其药学上可接受的盐：
    Figure PCTCN2020099916-appb-100001
    其中：
    X选自N和CH；
    R 1选自氢，卤素，C1-6烷基，C3-6环烷基，-C(O)OR 8，和-CN；
    R 2选自C1-6烷基，C3-6环烷基，氘代C1-6烷基，和C1-6卤代烷基；
    R 3选自-NR 9(CH 2) 2NR 9’R 9”，
    Figure PCTCN2020099916-appb-100002
    Figure PCTCN2020099916-appb-100003
    R 4为
    Figure PCTCN2020099916-appb-100004
    R 5，R 6，和R 7独立地选自氢，卤素，C1-6烷基，C1-6卤代烷基，C1-6烷氧基，和-CN；
    R 8选自氢，C1-6烷基，和C1-6卤代烷基；
    R 9选自氢，C1-6烷基，氘代C1-6烷基，和C1-6卤代烷基；
    R 9’和R 9”独立地选自氢，C1-6烷基，C3-6环烷基，氘代C1-6烷基，和C1-6卤代烷基，或R 9’和R 9”与所相连的氮原子一起环合形成未取代或被1-3个选自卤素，C1-6烷基，C1-3烷氧基，甲巯基，甲砜基，和C1-6卤代烷基取代的杂环烷基；
    R 10选自氢,卤素，C1-6烷基,和-CH 2NR 12’R 12”；
    R 11选自氢,卤素，和C1-6烷基；和
    R 12’和R 12”独立地选自氢，C1-6烷基，C1-6卤代烷基，或R 12’和R 12”与所相连的氮原子一起环合形成未取代或被1-3个选自卤素，C1-6烷基，和C1-6卤代烷基取代的杂环烷基。
  2. 如权利要求1所述的通式(I)所示化合物或其药学上可接受的盐，其中：
    R 1选自氢,卤素，C1-6烷基，-C(O)OR 8，和-CN；和
    R 5，R 6，和R 7独立地选自氢和卤素。
  3. 如权利要求2所述的通式(I)所示化合物或其药学上可接受的盐，其中化合物为以下化合物：
    Figure PCTCN2020099916-appb-100005
    Figure PCTCN2020099916-appb-100006
    Figure PCTCN2020099916-appb-100007
    Figure PCTCN2020099916-appb-100008
PCT/CN2020/099916 2019-07-04 2020-07-02 一类抑制egfr激酶的化合物及其制备方法和用途 WO2021000912A1 (zh)

Priority Applications (9)

Application Number Priority Date Filing Date Title
EP20835453.0A EP3998268A4 (en) 2019-07-04 2020-07-02 COMPOUND FOR INHIBITING EGFR KINASE AND METHODS OF MAKING AND USE THEREOF
KR1020217036806A KR20220027817A (ko) 2019-07-04 2020-07-02 Egfr 키나아제를 억제하기 위한 화합물, 이의 제조 방법 및 용도
CN202080005303.6A CN114026090B (zh) 2019-07-04 2020-07-02 一类抑制egfr激酶的化合物及其制备方法和用途
BR112021024139A BR112021024139A2 (pt) 2019-07-04 2020-07-02 Composto de fórmula geral (i) ou um sal farmaceuticamente aceitável do mesmo
CA3142071A CA3142071A1 (en) 2019-07-04 2020-07-02 Compound for inhibiting egfr kinase and preparation method and uses thereof
AU2020299659A AU2020299659A1 (en) 2019-07-04 2020-07-02 Compound for inhibiting EGFR kinase and preparation method and use thereof
JP2021578076A JP2022539224A (ja) 2019-07-04 2020-07-02 Ｅｇｆｒキナーゼを阻害する化合物、調製方法、及びその使用
MX2021013982A MX2021013982A (es) 2019-07-04 2020-07-02 Compuestos para inhibir la quinasa del egfr, metodos de preparacion y usos de los mismos.
US17/200,856 US20210230161A1 (en) 2019-07-04 2021-03-14 Compounds for inhibiting egfr kinase, preparation methods and uses thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201910600229.1A CN112174961A (zh) 2019-07-04 2019-07-04 一类抑制egfr激酶的化合物及其制备方法和用途
CN201910600229.1 2019-07-04

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US17/200,856 Continuation US20210230161A1 (en) 2019-07-04 2021-03-14 Compounds for inhibiting egfr kinase, preparation methods and uses thereof

Publications (1)

Publication Number Publication Date
WO2021000912A1 true WO2021000912A1 (zh) 2021-01-07

Family

ID=73914611

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/099916 WO2021000912A1 (zh) 2019-07-04 2020-07-02 一类抑制egfr激酶的化合物及其制备方法和用途

Country Status (10)

Country Link
US (1) US20210230161A1 (zh)
EP (1) EP3998268A4 (zh)
JP (1) JP2022539224A (zh)
KR (1) KR20220027817A (zh)
CN (2) CN112174961A (zh)
AU (1) AU2020299659A1 (zh)
BR (1) BR112021024139A2 (zh)
CA (1) CA3142071A1 (zh)
MX (1) MX2021013982A (zh)
WO (1) WO2021000912A1 (zh)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017008761A1 (zh) * 2015-07-16 2017-01-19 正大天晴药业集团股份有限公司 苯胺嘧啶衍生物及其用途
WO2018019204A1 (zh) * 2016-07-26 2018-02-01 深圳市塔吉瑞生物医药有限公司 用于抑制蛋白酪氨酸激酶活性的氨基嘧啶类化合物
CN109328059A (zh) * 2016-01-07 2019-02-12 Cs制药技术有限公司 Egfr酪氨酸激酶的临床重要突变体的选择性抑制剂

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015188747A1 (zh) * 2014-06-12 2015-12-17 南京圣和药业股份有限公司 作为egfr抑制剂的苯基取代的三嗪类化合物及其应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017008761A1 (zh) * 2015-07-16 2017-01-19 正大天晴药业集团股份有限公司 苯胺嘧啶衍生物及其用途
CN109328059A (zh) * 2016-01-07 2019-02-12 Cs制药技术有限公司 Egfr酪氨酸激酶的临床重要突变体的选择性抑制剂
WO2018019204A1 (zh) * 2016-07-26 2018-02-01 深圳市塔吉瑞生物医药有限公司 用于抑制蛋白酪氨酸激酶活性的氨基嘧啶类化合物

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP3998268A4

Also Published As

Publication number Publication date
MX2021013982A (es) 2022-01-04
CA3142071A1 (en) 2021-01-07
KR20220027817A (ko) 2022-03-08
CN114026090B (zh) 2023-01-06
US20210230161A1 (en) 2021-07-29
EP3998268A4 (en) 2023-06-21
AU2020299659A1 (en) 2022-01-27
EP3998268A1 (en) 2022-05-18
CN114026090A (zh) 2022-02-08
BR112021024139A2 (pt) 2022-01-11
CN112174961A (zh) 2021-01-05
JP2022539224A (ja) 2022-09-07

Similar Documents

Publication Publication Date Title
JP7181558B2 (ja) Ｅｇｆｒモジュレーターとしての置換２－アミノピリミジン誘導体
JP6418622B2 (ja) ２−（２，４，５−置換アニリン）ピリミジン誘導体、その薬物組成物及びその用途
CN106928150B (zh) 丙烯酰胺苯胺衍生物及其药学上的应用
TWI662026B (zh) 吡啶酮類衍生物、其製備方法及其在醫藥上的應用
WO2015158310A1 (zh) 一种酪氨酸激酶抑制剂及其用途
CN104736155B (zh) 双环化合物作为激酶的抑制剂
CN108349981A (zh) 新型的吡唑并[3，4－d]嘧啶化合物或其盐
TW201512176A (zh) 酪氨酸蛋白激酶調節劑及其應用方法
CN105315285A (zh) 2,4-二取代7H-吡咯并[2,3-d]嘧啶衍生物、其制法与医药上的用途
KR20140025484A (ko) 브로모도메인 억제제로서 유용한 테트라하이드로퀴놀린 유도체
WO2008106635A1 (en) Pyrimidine-2,4-diamine derivatives and their use as jak2 kinase inhibitors
CA3050239A1 (en) Pyrimidine compound and pharmaceutical use thereof
US20150266878A1 (en) Novel bi-ring phenyl-pyridines/pyrazines for the treatment of cancer
WO2020233669A1 (zh) 含吲哚类衍生物抑制剂、其制备方法和应用
JP2015515995A (ja) キナーゼ阻害剤として有用な置換アミノキナゾリン
TW201934546A (zh) 嘧啶類化合物、其製備方法及其醫藥用途
TWI448461B (zh) 4-aniline-6-butenamide-7-alkyl ether quinazoline derivatives, methods and uses thereof
JP2021501215A (ja) アミノ置換窒素含有縮合環化合物、その調製方法及び使用
JP6945963B2 (ja) 新規な化合物、それらの調製及びそれらの使用
CN111196814B (zh) 芳环连二噁烷并喹唑啉或喹啉类化合物、组合物及其应用
WO2022017408A1 (zh) 芳胺类衍生物及其制备方法和医药用途
TWI691500B (zh) 作為tyro3、axl和mertk(tam)家族受體酪胺酸激酶抑制劑之雜環化合物
WO2021000912A1 (zh) 一类抑制egfr激酶的化合物及其制备方法和用途
WO2022007841A1 (zh) 一种egfr抑制剂、其制备方法和在药学上的应用
TWI820414B (zh) 喹唑啉類化合物、製備方法及其應用

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: 20835453

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 3142071

Country of ref document: CA

REG Reference to national code

Ref country code: BR

Ref legal event code: B01A

Ref document number: 112021024139

Country of ref document: BR

ENP Entry into the national phase

Ref document number: 2021578076

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 112021024139

Country of ref document: BR

Kind code of ref document: A2

Effective date: 20211130

ENP Entry into the national phase

Ref document number: 2020299659

Country of ref document: AU

Date of ref document: 20200702

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 2020835453

Country of ref document: EP

Effective date: 20220204