WO2020038433A1 - 一种egfr激酶抑制剂及其制备方法和应用 - Google Patents

一种egfr激酶抑制剂及其制备方法和应用 Download PDF

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WO2020038433A1
WO2020038433A1 PCT/CN2019/101969 CN2019101969W WO2020038433A1 WO 2020038433 A1 WO2020038433 A1 WO 2020038433A1 CN 2019101969 W CN2019101969 W CN 2019101969W WO 2020038433 A1 WO2020038433 A1 WO 2020038433A1
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compound
pharmaceutically acceptable
acceptable salt
alkyl
formula
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PCT/CN2019/101969
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English (en)
French (fr)
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罗会兵
李庆
周华勇
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上海艾力斯医药科技有限公司
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Priority to CN201980055090.5A priority Critical patent/CN112638897B/zh
Publication of WO2020038433A1 publication Critical patent/WO2020038433A1/zh

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    • 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
    • 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
    • C07D401/14—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 three or more hetero rings

Definitions

  • the invention relates to an epidermal growth factor receptor (EGFR) kinase inhibitor that selectively inhibits a mutant form and a preparation method thereof, comprising the kinase inhibitor and a pharmaceutical composition thereof, and the treatment of the kinase inhibitor by certain Mutated morphology of EGFR-mediated diseases and use in the preparation of a medicament for the treatment of a disease mediated by certain morphology of EGFR.
  • EGFR epidermal growth factor receptor
  • Epidermal growth factor receptor belongs to the protein tyrosine kinase (PTK) family. It consists of EGFR (Erb-B1), Erb-B2 (HER-2 / neu), Erb-B3, and Erb-B4, and has been identified as It is a vital driving factor in the process of cell growth and appreciation. EGFR overexpression and mutations have been clearly confirmed to cause uncontrolled cell growth, which is related to most cancer disease processes, such as lung cancer, colon cancer, breast cancer, etc.
  • PTK protein tyrosine kinase
  • the first representative dermal growth factor receptor tyrosine kinase inhibitors include Gefitinib, Erlotinib, and Lapatinib.
  • Type mutations such as exon 19 deletion activation mutation or L858R activation mutation
  • NSCLC non-small cell lung cancer
  • breast cancer breast cancer
  • the second-generation EGFR-TKIs such as Afatinib and dacomitinib have very prominent advantages. They can irreversibly interact with EGFR through the Michael addition reaction. Covalent binding of the upper cysteine residue (Cys797) expands the EGFR inhibitor and ATP binding site, thereby overcoming the resistance caused by the T790M mutation to a certain extent (Li et D, Oncogene, 27: 4702-4711 , 2008).
  • Cys797 is present in all forms of EGFR, so the second-generation EGFR-TKI is not only active against EGFR with activating mutations and drug-resistant mutations, but also with wild-type EGFR, resulting in rash, diarrhea, and nausea. , Anorexia, weakness, and other toxic side effects (Curr. Med. Chem. 2006, 13, 3483-3492), ultimately limiting its clinical dose and effective blood concentration, failing to achieve impressive results in overcoming T790M resistance mutations Eye-catching effects.
  • Third generation EGFR-TKIs such as: AZD9291, CO-1686 and HM61713, they are a class of EGFR tyrosine kinase inhibitors with specific selectivity. Compared with the first and second generation EGFR-TKIs, they It has strong inhibitory activity on EGFR of activating mutations and drug-resistant mutations, but weaker inhibitory effect on wild-type EGFR. They are highly effective in T790M-positive tumors, but they still have some toxicity, such as still causing clinical side effects such as diarrhea, rash, nausea and even high blood sugar ((J Clin Oncol 2014; 32: abstr 8009; J Clin Oncol 2014; 32 : abstr 8010). Obviously, a compound with higher activity and lower toxicity will bring greater benefits.
  • AstraZeneca AZD9291 is an oral small molecule third representative dermal growth factor tyrosine kinase inhibitor.
  • AZD9291 has strong specific binding ability against drug-resistant mutations T790M and sensitive mutation sites (19Del and L858R). Patients with resistance to non-small cell lung cancer after first-line TKI treatment have a good therapeutic effect, but because it also inhibits EGFR wild type to a certain extent, it will clinically cause side effects such as diarrhea and rash.
  • the EGFR kinase inhibitor with weak inhibitory activity against wild-type EGFR has low toxic and side effects and good safety. It is expected that such inhibitors will have good curative effects, and it is expected to overcome the problems of drug resistance and toxic side effects, and have good development prospects.
  • the invention provides a compound of formula (I), or a pharmaceutically acceptable salt thereof:
  • Ring A is heteroaryl
  • R 1 is selected from hydrogen, halogen, C 1 -C 4 alkyl or halo C 1 -C 4 alkyl;
  • R 2 is a halogenated C 1 -C 4 alkyl
  • R 3 is selected from hydrogen, C 1 -C 4 alkyl or halo C 1 -C 4 alkyl
  • R 4 is selected from hydrogen, C 1 -C 4 alkyl or halo C 1 -C 4 alkyl
  • R 5 is selected from
  • Each R 6 is independently selected from hydrogen, halogen, -CN, C 1 -C 4 alkyl or halo C 1 -C 4 alkyl;
  • R 7 is selected from hydrogen, C 1 -C 4 alkyl or halo C 1 -C 4 alkyl
  • n 1, 2 or 3.
  • the present invention provides compounds of formula (I), which are capable of inhibiting one or more EGFR-activated or drug-resistant mutations, such as L858R-activated mutants, exon 19 deletion-activated mutants, and T790M-resistant mutants.
  • EGFR-activated or drug-resistant mutations such as L858R-activated mutants, exon 19 deletion-activated mutants, and T790M-resistant mutants.
  • such compounds are useful in the treatment of cancer in patients who have developed a degree of resistance to existing therapies based on EGFR inhibitors.
  • the present invention provides compounds of formula (I), which show higher inhibition of EGFR in the form of activated or drug-resistant mutants than wild-type EGFR. Due to the reduced toxicity associated with wild-type EGFR inhibition, this compound is expected to be more suitable as a therapeutic agent, especially for the treatment of cancer.
  • the invention also provides a method for preparing a compound of formula (I).
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the compound of formula (I) of the present invention or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, excipient or diluent.
  • the invention also provides the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof for the treatment of diseases, particularly cancer, mediated by EGFR-activated or resistant mutants.
  • the present invention also provides the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating a disease, particularly cancer, mediated by an EGFR-activated or resistant mutant.
  • the invention also provides a method of treating a disease, particularly cancer, mediated by an EGFR-activated or resistant mutant, said method comprising administering to a patient a compound of formula (I) or a pharmaceutically acceptable salt thereof, or A patient is administered a pharmaceutical composition containing a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.
  • the present invention also provides a method for treating mammals, especially humans, mediated by EGFR-activated or resistant mutants, particularly cancer, the method comprising administering to a patient a compound of formula (I) or a pharmaceutically acceptable compound thereof.
  • An acceptable salt, or a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (I) and a pharmaceutically acceptable carrier, excipient or diluent.
  • the invention also provides a method for selectively inhibiting EGFR-activated or drug-resistant mutations compared to wild-type EGFR (WTEGFR), the method comprising contacting a biological sample or administering a compound of formula (I) or a patient to the patient A pharmaceutically acceptable salt or a pharmaceutical composition thereof.
  • WTEGFR wild-type EGFR
  • the cancers mentioned in the present invention include lung cancer, ovarian cancer, cervical cancer, breast cancer, gastric cancer, colorectal cancer, pancreatic cancer, glioma, glioblastoma, melanoma, prostate cancer, leukemia, lymphoma, non-cancer Hodgkin's lymphoma, hepatocellular carcinoma, gastrointestinal stromal tumor (GIST), thyroid cancer, bile duct cancer, endometrial cancer, kidney cancer, anaplastic large cell lymphoma, acute myeloid leukemia (AML), multiple Myeloma, mesothelioma.
  • lung cancer ovarian cancer, cervical cancer, breast cancer, gastric cancer, colorectal cancer, pancreatic cancer, glioma, glioblastoma, melanoma, prostate cancer, leukemia, lymphoma, non-cancer Hodgkin's lymphoma, hepatocellular carcinoma, gastrointestinal stromal tumor (GIST), thyroid
  • ring A is indolyl, indazolyl, pyrrole [2,3-c] pyridyl, pyrrole [3, 2-c] pyridyl, pyrrolyl [2,3-b] pyridyl, pyrrolyl [3,2-b] pyridyl, pyrrolyl [2,3-b] pyridazinyl, indoline-2 -Keto, pyridyl, pyrazolyl or pyrimidinyl.
  • ring A is indolyl, pyrrole [2,3-c] pyridyl, pyrrole [3,2-c ] Pyridyl, pyrrolyl [2,3-b] pyridyl, pyrrolyl [3,2-b] pyridyl, pyrazolyl, or pyrimidinyl.
  • ring A is indolyl
  • R 1 is hydrogen or halogen.
  • R 1 is hydrogen
  • R 2 is trifluoromethyl, monofluoromethyl, difluoromethyl or 2,2,2-trifluoroethyl base.
  • R 2 is 2,2,2-trifluoroethyl.
  • R 3 is C 1 -C 4 alkyl.
  • R 3 is methyl
  • R 4 is a C 1 -C 4 group.
  • R 4 is methyl
  • R 5 is
  • R 5 is
  • each R 6 is independently halogen or C 1 -C 4 alkyl, and m is 1, 2 or 3.
  • each R 6 is independently a C 1 -C 4 alkyl group, and m is 1, 2 or 3.
  • R 6 is methyl and m is 1.
  • a particularly preferred compound of formula (I) or a pharmaceutically acceptable salt thereof is N- (2- (methyl (2- (methylamino) ethyl) amino) -6- (2 , 2,2-trifluoroethoxy) -5-((4- (1-methyl-1H-indol-3-yl) pyrimidin-2-yl) amino) pyridin-3-yl) acrylamide
  • the structure is as follows:
  • a particularly preferred pharmaceutically acceptable salt of the compound of formula (I) is N- (2- (methyl (2- (methylamino) ethyl) amino) -6- (2,2 , 2-trifluoroethoxy) -5-((4- (1-methyl-1H-indol-3-yl) pyrimidin-2-yl) amino) pyridin-3-yl) acrylamide methanesulfonate Acid salt.
  • the invention also provides a method for preparing a compound of formula (I), which comprises the following steps:
  • ring A, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 and m have the same meanings as in the above formula (I);
  • X and Y are halogens, respectively, specifically including fluorine, chlorine, and bromine , Iodine, preferably chlorine or bromine.
  • a compound of formula a is reacted with an alcohol R 2 OH to obtain a compound of formula b; a compound of formula b is reduced by a nitro group to obtain a compound of formula c; in the presence of a base, a compound of formula c is subjected to acylation to obtain a compound of formula d; Compound d undergoes a nitration reaction to obtain a compound of formula e; in the presence of an acid, a compound of formula e undergoes a hydrolysis reaction to obtain intermediate 1; in the presence of an acid, intermediate 1 and a compound of formula f are substituted to obtain a compound of formula g or a salt thereof; in the presence of a base A compound of formula g and a compound of formula h are substituted to obtain a compound of formula i; a compound of formula i is protected by Boc in the presence of a catalyst to obtain a compound of formula j; a compound of formula j is reduced to a compound
  • the base in the etherification reaction is an inorganic strong base including, but not limited to, NaH, LiHMDS, NaHMDS, or KHMDS; a compound of formula b 2.
  • the reducing agent is a conventional reducing agent, including but not limited to insurance powder, palladium carbon / hydrogen or ferric chloride / hydrazine hydrate.
  • An acid may be further added during the reduction reaction, and the acid is organic.
  • Acids or inorganic acids include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid or acetic acid;
  • the base in the amidation reaction of the compound of formula c is an organic base or an inorganic base Including but not limited to triethylamine, N, N-diisopropylethylamine, sodium bicarbonate, potassium carbonate, cesium carbonate, potassium tert-butoxide or sodium tert-butoxide;
  • the acylating agents include, but are not limited to, trifluoroamine Acetic anhydride or trifluoroacetyl chloride;
  • the nitrating agent is a conventional nitrating agent including but not limited to HNO 3 / H 2 SO 4 , KNO 3 / H 2 SO 4 or fuming
  • halogen refers to fluorine, chlorine, bromine, iodine, and the like, and fluorine, chlorine, and bromine are preferred.
  • C 1 -C 4 alkyl refers to a straight or branched chain alkyl group containing 1 to 4 carbon atoms
  • alkyl refers to a saturated aliphatic hydrocarbon group, which specifically includes, but is not limited to, methyl and ethyl.
  • halogenated C 1 -C 4 alkyl refers to a C 1 -C 4 alkyl group, as defined herein, substituted with one or more halogens, preferably one to five halogen atoms, and specifically includes, but is not limited to, trifluoro Methyl, monofluoromethyl, difluoromethyl, 2,2,2-trifluoroethyl, 2-fluoroethyl or 1-chloro-2-fluoroethyl, preferably trifluoromethyl, monofluoromethyl , Difluoromethyl or 2,2,2-trifluoroethyl, more preferably 2,2,2-trifluoroethyl.
  • a heteroaryl group refers to a 5- to 6-membered monocyclic heteroaryl group containing 1 to 4 heteroatoms selected from N, S, or O or a fused with a benzene ring, a pyridine ring, or a pyrrole ring.
  • Bicyclic heteroaryl which may be partially saturated.
  • the heteroaryl group includes, but is not limited to, furyl, thienyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, triazolyl, tetrazolyl, thiazyl Diazolyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, benzofuranyl, benzothienyl, benzothiadiazolyl, benzothiazolyl, benzimidazolyl, indolyl, isopropyl Indolyl, indazolyl, quinolinyl, isoquinolinyl, quinazolinyl, 1,2,3,4-tetrahydroisoquinolinyl, pyrrole [2,3-c] pyridyl, pyrrole [3,2-c] pyridyl, pyrroly
  • the present invention also includes a pharmaceutically acceptable salt of a compound of formula (I).
  • pharmaceutically acceptable salt refers to an acid or base addition salt of a compound of the invention that is relatively non-toxic.
  • the acid addition salt is a salt formed by the compound of formula (I) of the present invention with a suitable inorganic or organic acid, and these salts can be prepared during the final isolation and purification of the compound, or the purified formula (I) can be used.
  • the compounds are prepared by reacting their free base form with a suitable organic or inorganic acid.
  • Representative acid addition salts include hydrobromide, hydrochloride, sulfate, bisulfate, sulfite, acetate, oxalate, valerate, oleate, palmitate, stearic acid Salt, moon silicate, borate, benzoate, lactate, phosphate, hydrogen phosphate, carbonate, bicarbonate, toluate, citrate, maleate, rich Maleate, succinate, tartrate, benzoate, mesylate, p-toluenesulfonate, gluconate, lactate, and laurylsulfonate.
  • the base addition salt is a salt formed by a compound of formula (I) with a suitable inorganic or organic base, and includes, for example, a salt formed with an alkali metal, alkaline earth metal, quaternary ammonium cation, such as sodium salt, lithium salt, potassium salt, Calcium, magnesium, tetramethyl quaternary ammonium, tetraethyl quaternary ammonium, etc .; amine salts, including salts with ammonia (NH 3 ), primary, secondary or tertiary amines, such as methylamine, diamine Methylamine salt, trimethylamine salt, triethylamine salt, ethylamine salt, and the like.
  • a salt formed with an alkali metal, alkaline earth metal, quaternary ammonium cation such as sodium salt, lithium salt, potassium salt, Calcium, magnesium, tetramethyl quaternary ammonium, tetraethyl quaternary ammonium,
  • the compounds of the present invention or a pharmaceutically acceptable salt thereof can be administered to mammals, including humans, and can be administered orally, rectally, parenterally (intravenously, intramuscularly or subcutaneously), topically (powders, ointments or drops) , Or intratumoral administration.
  • the compound of the present invention may be administered at a dose of about 0.3-30 mg / kg body weight / day, such as 0.5-20 mg / kg body weight / day, 10 mg / kg body weight / day.
  • the application amount is calculated based on the parent compound.
  • the compound of the present invention or a pharmaceutically acceptable salt thereof may be formulated into a solid dosage form for oral administration, including, but not limited to, capsules, tablets, pills, powders, granules, and the like.
  • the compound of formula (I) according to the invention is mixed as an active ingredient with at least one conventional inert excipient (or carrier), for example with sodium citrate or dicalcium phosphate, or with the following ingredients: (1 ) Fillers or compatibilizers, such as starch, lactose, sucrose, glucose, mannitol and silicic acid, etc .; (2) binders, such as hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and Gum arabic, etc .; (3) humectants, such as glycerol, etc .; (4) disintegrating agents, such as agar, calcium carbonate, potato starch or cassava starch, alginic acid, some complex silicates and sodium carbon
  • the solid dosage forms such as tablets, dragees, capsules, pills and granules can be coated or microencapsulated with coating and shell materials such as enteric coatings and other materials known in the art. They may contain opaque agents and the release of the active ingredient in such a composition may be released in a certain part of the digestive tract in a delayed manner. Examples of embedding components that can be used are polymeric substances and waxes. If necessary, the active ingredient may also be microencapsulated with one or more of the above-mentioned excipients.
  • liquid dosage forms for oral administration, including, but not limited to, pharmaceutically acceptable emulsions, solutions, suspensions, syrups, elixirs and the like.
  • liquid dosage forms may contain inert diluents such as water and other solvents, solubilizers and emulsifiers, such as ethanol, Propanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butanediol, dimethylformamide and oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, etc.
  • liquid dosage forms of the present invention may also contain conventional auxiliaries such as wetting agents, emulsifying and suspending agents, sweeteners, flavoring agents, flavorings and the like.
  • the suspending agent includes, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide, agar and the like or a mixture of these materials.
  • the compound of the present invention or a pharmaceutically acceptable salt thereof may be formulated into a dosage form for parenteral injection, including, but not limited to, a physiologically acceptable sterile aqueous or anhydrous solution, dispersion, suspension or emulsion, and for Redissolve a sterile powder into a sterile injectable solution or dispersion.
  • Suitable carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.
  • the compound of the present invention or a pharmaceutically acceptable salt thereof can also be formulated into dosage forms for topical administration, including, for example, ointments, powders, suppositories, drops, sprays, inhalants and the like.
  • the compound of formula (I) of the present invention or a pharmaceutically acceptable salt thereof as an active ingredient is, under sterile conditions, together with a physiologically acceptable carrier and optional preservatives, buffers, or propellants that may be required if necessary mixing.
  • the present invention also provides a pharmaceutical composition containing the compound of formula (I) of the present invention or a pharmaceutically acceptable salt thereof as an active ingredient, and a pharmaceutically acceptable carrier, excipient or diluent.
  • a pharmaceutically acceptable carrier excipient or diluent.
  • composition of the present invention can be formulated into a conventional pharmaceutical preparation according to a conventional preparation method.
  • a conventional preparation method for example, tablets, pills, capsules, powders, granules, emulsions, suspensions, dispersions, solutions, syrups, elixirs, ointments, drops, suppositories, inhalants, propellants and the like.
  • the compound of the present invention or a pharmaceutically acceptable salt thereof can be administered alone or in combination with other pharmaceutically acceptable therapeutic agents, especially in combination with other antitumor drugs.
  • the therapeutic agents include, but are not limited to, drugs that act on the chemical structure of DNA, such as cisplatin, and antitumor drugs that affect nucleotide synthesis, such as methotrexate (MTX), 5-fluorouracil (5FU), and so on.
  • MTX methotrexate
  • 5FU 5-fluorouracil
  • Nucleic acid-transcribed anti-tumor drugs such as doxorubicin, epirubicin, clarithromycin, radiomycin, etc., anti-tumor drugs acting on tubulin synthesis such as paclitaxel, vinorelbine, etc., aromatase inhibitors such Aminutamide, Lantron, Letrozole, Reynolds, etc.
  • Cell signal pathway inhibitors such as epidermal growth factor receptor inhibitor Imatinib, Gefitinib, Erlotinib (Erlotinib) and others.
  • the ingredients to be combined can be administered simultaneously or sequentially, in the form of a single preparation or in the form of different preparations.
  • the combination includes not only a combination of a compound of the present invention and one other active agent, but also a combination of a compound of the present invention and two or more other active agents.
  • the compounds of the invention are useful as medicaments for the treatment of diseases or conditions mediated by the activity of EGFR sensitive or resistant mutants, particularly tumors such as cancer.
  • the cancer includes, but is not limited to, for example, lung cancer, ovarian cancer, cervical cancer, breast cancer, gastric cancer, colorectal cancer, pancreatic cancer, glioma, glioblastoma, melanoma, prostate cancer, leukemia, lymphoma, Non-Hodgkin's lymphoma, hepatocellular carcinoma, gastrointestinal stromal tumor (GIST), thyroid cancer, bile duct cancer, endometrial cancer, kidney cancer, anaplastic large cell lymphoma, acute myeloid leukemia (AML), multiple Myeloma, mesothelioma, especially for the tumor type of the epidermal growth factor receptor 790 threonine mutation to methionine (EGFR T790M) has a better application.
  • AML acute myeloid leukemia
  • the compounds of the present invention are useful as and as a medicament for the treatment of non-small cell lung cancer (EGFR T790M). It can be used to overcome the resistance problem caused by EGFR T790M after clinical application of gefitinib and erlotinib. And due to the reduced toxicity associated with wild-type EGFR inhibition, it is expected that the compounds of the present invention will have relatively small toxic and side effects during their application in the treatment of cancer.
  • EGFR T790M non-small cell lung cancer
  • the compound kinase activity inhibition experiment of the present invention can be measured by conventional methods.
  • One preferred evaluation method is enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • the inhibition of the kinase by the drug is calculated by measuring the change in the light absorption value produced by the drug acting on the kinase. rate,
  • the drug efficacy of the compound of the present invention in inhibiting the proliferation of cancer cells can be determined by conventional methods.
  • One preferred evaluation method is the Sulforhodamin B (SRB) protein staining method, which measures the light absorption produced by drugs acting on cancer cells. Value change to calculate the inhibition rate of the cancer cell proliferation by the drug,
  • the OD value of the negative control group was the OD value of the wells of normal growing cells without adding any compound (containing 0.5% DMSO).
  • the OD value of the administration group was the OD value after adding the test compound (containing 0.5% DMSO).
  • the half inhibitor concentration (IC50) value was calculated using GraphPad's PrIsm software version 5.0, a four-parameter fitting method. Each experiment was repeated 3 times, and the average IC50 value of the 3 experiments was obtained as the final index of the inhibition ability.
  • a preferred evaluation method is the inhibition of the growth of human squamous cell carcinoma A431 nude mice subcutaneously transplanted tumors; another preferred evaluation method is Growth inhibitory effect on human non-small cell lung cancer cell HCC827 subcutaneously transplanted tumors in nude mice; another preferred method of evaluation is growth inhibitory effect on human lung adenocarcinoma H1975 nude mice.
  • Test methods Human squamous cell carcinoma A431, human non-small cell lung cancer cells HCC827, and human lung adenocarcinoma H1975 were inoculated subcutaneously in the right front back of BALB / cNude mice, respectively, and the tumors were grown to an average suitable size according to tumor size and mice The body weight was randomly divided into groups, and the test compound was administered orally by a certain dose. The solvent control group was orally administered with the same amount of solvent once a day for 21 consecutive days. During the entire experiment, the mouse weight and tumor size were measured twice a week. Observe whether there is a toxic reaction.
  • FIG. 1 is a body weight change curve of the human squamous cell carcinoma A431 nude mouse at a dose of 10 mg / kg of the compound salt of Example 2 and AZD9291.
  • FIG. 2 is a TGI% curve of tumor growth inhibition rate of a subcutaneous xenograft of human squamous cell carcinoma A431 nude mice at a dose of 10 mg / kg of the compound salt of Example 2 and AZD9291.
  • FIG. 3 is a curve of body weight of a human non-small cell lung cancer cell HCC827 nude mouse at different doses of the compound salt of Example 2.
  • FIG. 4 is a TGI% curve of the tumor growth inhibition rate of subcutaneous xenografts of human non-small cell lung cancer cell HCC827 nude mice at different dosages of the compound salt of Example 2.
  • FIG. 5 is a curve of body weight of a human lung adenocarcinoma cell H1975 nude mouse at different doses of the compound salt of Example 2.
  • FIG. 6 is a TGI% curve of the tumor growth inhibition rate of subcutaneous xenografts of human lung adenocarcinoma cells H1975 nude mice at different doses of the compound salt of Example 2.
  • Step 1 Synthesis of 6-chloro-3-nitro-2- (2,2,2-trifluoroethoxy) pyridine
  • Step 2 Synthesis of 6-chloro-3-amino-2- (2,2,2-trifluoroethoxy) pyridine
  • Step 3 Synthesis of 6-chloro-3- (2,2,2-trifluoroacetamido) -2- (2,2,2-trifluoroethoxy) pyridine
  • Step 4 Synthesis of 6-chloro-5-nitro-3- (2,2,2-trifluoroacetamido) -2- (2,2,2-trifluoroethoxy) pyridine
  • Step 5 Synthesis of 6-chloro-5-nitro-3-amino-2- (2,2,2-trifluoroethoxy) pyridine
  • the total yield of the five-step reaction from step 1 to step 5 is 34.9%.
  • Example 1 N- (2- (methyl (2- (methylamino) ethyl) amino) -6- (2,2,2-trifluoroethoxy) -5-((4- (1-methyl-1H-indole-3-yl) pyrimidin-2-yl) amino) pyridin-3-yl) acrylamide
  • Toluene (7.43L) was added to a 20L reactor, and the intermediate 1a compound 6-chloro-5-nitro-3-amino-2- (2,2,2-trifluoroethoxy) pyridine (743.0 g, 2.74 mol), compound 3- (2-chloropyrimidin-4-yl) -1-methyl-1H-indole (866.7 g, 3.56 mol) (prepared from CN105315259A intermediate 2a), p-toluenesulfonic acid-1 Hydrate (780.7 g, 4.10 mol), stirred, and heated the reaction mixture to 110-115 ° C for 36 hours.
  • Step 2 N 2 - methyl -N 2 - (2- (methylamino) ethyl) -6- (2,2,2-trifluoroethoxy) -N 5 - (4- (1- methyl Of phenyl-1H-3-yl) pyrimidin-2-yl) -3-nitropyridine-2,5-diamine
  • Step 3 N 2 - methyl -N 2 - (2- (methyl-t-butoxycarbonyl amino) ethyl) -6- (2,2,2-trifluoroethoxy) -N 5 - (4 Synthesis of-(1-methyl-1H-3-yl) pyrimidin-2-yl) -3-nitropyridine-2,5-diamine
  • Step 4 N 2 - methyl -N 2 - (2- (methyl-t-butoxycarbonyl amino) ethyl) -6- (2,2,2-trifluoroethoxy) -N 5 - (4 Synthesis of-(1-methyl-1H-3-yl) pyrimidin-2-yl) pyridine-2,3,5-, triamine
  • Step 5 N- (2- (methyl (2- (methyl-tert-butoxycarbonylamino) ethyl) amino) -6- (2,2,2-trifluoroethoxy) -5- ( Synthesis of (4- (1-methyl-1H-indole-3-yl) pyrimidin-2-yl) amino) pyridin-3-yl) acrylamide
  • Step 6 N- (2- (methyl (2- (methylamino) ethyl) amino) -6- (2,2,2-trifluoroethoxy) -5-((4- ( Synthesis of 1-methyl-1H-indole-3-yl) pyrimidin-2-yl) amino) pyridin-3-yl) acrylamide
  • reaction solution was concentrated under reduced pressure, ethyl acetate (100 mL) was added to the residue, and the organic phase was washed with a saturated NaHCO 3 solution and a saturated NaCl solution successively.
  • the organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a yellow solid (535 mg, recovered Rate 81%).
  • Test Example 1 EGFR tyrosine kinase inhibitor activity test
  • ELISA method enzyme reaction substrate polyglutamic acid: tyrosine (4: 1) coated 96-well microtiter plate, T-PBS (phosphate buffer solution containing 0.1% Tween-20) was washed 5 times The plate was dried in an oven at 37 ° C for 2 hours. Add 80 ⁇ l of adenosine triphosphate solution diluted in reaction buffer to each well, then add 10 ⁇ l of the test compound (compound well) with a different concentration gradient or a certain concentration of dimethyl sulfoxide solution (negative control well), and finally add dilution with reaction buffer 10 ⁇ l of the protein tyrosine kinase solution was used to start the reaction or 10 ⁇ l of the reaction buffer was used as an enzyme-free control well.
  • the reaction was carried out at 37 ° C with a shaker for 1 hour, and the plate was washed 5 times with T-PBS.
  • 100 ⁇ l / well of a monoclonal antibody (PY99) against phosphorylated tyrosine was added, the reaction was performed at 25 ° C. for 1 hour, and the plate was washed 5 times with T-PBS.
  • 100 ⁇ l / well of horseradish peroxidase-labeled goat anti-mouse IgG shake at 25 ° C for 1 hour, and wash the plate 5 times with T-PBS.
  • Add 2mg / ml o-phenylenediamine OPD color development solution and react at 25 ° C in the dark for 10min.
  • the reaction was stopped by adding 50 ⁇ l / well of 2M sulfuric acid solution, and the reading was read with Synergy H4, a microplate reader with adjustable wavelength, at a wavelength of 490 nm.
  • the data were collated to calculate the inhibition rate. According to the inhibition rate of each concentration, the IC 50 values of each test compound for EGFR L858R , EGFR L858R / T790M and HER2 were calculated by non-linear regression method.
  • AZD9291 was purchased from Shanghai Yanyan Chemical Co., Ltd.
  • test results show that the salt of the compound of Example 2 has good inhibitory activity on HER2, EGFR mutations (such as EGFR L858R , EGFR L858R / T790M, etc.).
  • Test Example 2 Inhibition of EGFR-sensitive mutations in human lung cancer cells PC-9 (EGFR ex19del), HCC827 (EGFR E746_A750del), and resistant mutant human lung cancer cells H1975 (EGFR L858R / T790M)
  • Sulfonhodamine B protein staining method (SRB method): take cells in log phase growth and inoculate them in 96-well plates (cell concentration: 5000 cells / well; cell suspension: 180 ⁇ l / well), 37 ° C, 5% CO 24H 2 cultured adherent cells.
  • the test drug was dissolved in dimethyl sulfoxide to prepare a 10 mM storage solution, which was diluted to 10 times the target concentration when tested, and then 20 ⁇ l / well of the compound was added to a 96-well plate inoculated with cells to reach the target concentration. Set three replicates for each concentration and set a blank control. The culture was continued for 72H at 37 ° C, 5% CO 2 .
  • Terminate the culture add 50 ⁇ l of pre-cooled (4 ° C) 50% trichloroacetic acid, TCA (final concentration 10%), place at 4 ° C for 1H, wash with purified water at least 5 times, and dry naturally in the air or 60 ° C Oven drying.
  • TCA trichloroacetic acid
  • test results show that the salt of the compound of Example 2 has a strong inhibitory effect on tumor cells with EGFR sensitive mutations and drug resistance mutations.
  • Test Example 3 Growth inhibition of human squamous cell carcinoma A431, human non-small cell lung cancer cells HCC827, and human lung adenocarcinoma H1975 in nude mice subcutaneously transplanted tumors.
  • Example 2 of the present invention Observe the inhibitory effect of the compound salt of Example 2 of the present invention and AZD9291 on human squamous cell carcinoma A431, human lung adenocarcinoma HCC827, and human lung adenocarcinoma H1975 subcutaneously transplanted tumors in nude mice and their corresponding safety conditions.
  • mice weight change rate body weight on the day / D0 body weight of the animal ⁇ 100%.
  • Test Example 3.1 Growth inhibition of subcutaneous xenografts of human squamous cell carcinoma A431 nude mice.
  • Cell culture After cell recovery, use DMEM medium containing 10% fetal bovine serum and culture in a constant temperature incubator at 37 ° C and 5% CO 2. When the cell fusion degree is about 80% -90% (logarithmic growth) Phase). Passage expansion and culture to 5 T175cm 2 culture flasks. Cells in logarithmic growth phase are collected and counted for seeding.
  • mice BALB / c Nude nude mice, 12 females, 4-5 weeks, purchased from Shanghai Xipuer-Bikai Experimental Animal Co., Ltd.
  • mice Human squamous cell carcinoma cell A431 was inoculated with 3 * 10 6 /0.2mL subcutaneously in the right front back of BALB / c Nude mice, and the tumor growth was regularly observed. When the tumor grew to an average of about 150-200mm 3 , according to the tumor size And mouse weights were randomly grouped. Three experimental groups were set up: a 0.5% methyl cellulose vehicle control group, an AZD929 110 mg / kg group, and a compound salt of Example 2 at a 10 mg / kg group. After grouping, each group was administered according to the corresponding administration dose, the administration volume was 10 ⁇ L / g, once a day for 21 consecutive days. During the entire experiment, the body weight and tumor size of the mice were measured twice a week to observe whether a toxic reaction occurred.
  • the rate of change in body weight of the three experimental groups is shown in FIG. 1, and the tumor growth inhibition rate TGI% is shown in FIG. 2.
  • the results show that the salt of the compound of Example 2 has no effect on the body weight of the animal, and has a weak inhibitory effect on the growth of human squamous cell carcinoma A431 nude mice subcutaneous xenografts, that is, the compound of the present invention has better safety.
  • Test Example 3.2 Growth inhibition of subcutaneous xenografts of human non-small cell lung cancer HCC827 nude mice.
  • Cell culture After cell recovery, use RPMI-1640 medium containing 10% fetal bovine serum and culture in a constant temperature incubator at 37 ° C and 5% CO 2. When the cell fusion degree is about 80% -90% (for (Growth phase)) Passage expansion and culture to 24 T175cm 2 culture flasks, the cells in the log phase are collected and counted for seeding.
  • mice BALB / c Nude nude mice, 32, female, 4-5 weeks, purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.
  • mice Human non-small cell lung cancer cells HCC827 were inoculated 5 * 10 6 /0.1mL subcutaneously into the right front dorsal surface of BALB / c Nude mice, and the tumor growth was regularly observed. When the tumor grew to an average of about 150-200 mm 3 , according to the tumor size And mouse weights were randomly grouped. Set up 4 experimental groups, respectively: 20% polyethylene glycol 400 + 80% physiological saline solvent control group, the compound of Example 2 salt 1mg / kg group, 3mg / kg group and 10mg / kg group. After grouping, each group was administered according to the corresponding administration dose, the administration volume was 10 ⁇ L / g, once a day for 21 consecutive days. During the entire experiment, the body weight and tumor size of the mice were measured twice a week to observe whether a toxic reaction occurred.
  • the rate of change in body weight of the four experimental groups of mice is shown in Fig. 3, and the tumor growth inhibition rate TGI% is shown in Fig. 4.
  • the experimental results show that the salt of the compound of Example 2 has a good inhibitory effect on the growth of human non-small cell lung cancer cell HCC827 subcutaneously transplanted tumors in nude mice, has no significant effect on the body weight of animals, and shows good safety.
  • Test Example 3.3 Growth inhibition of subcutaneous xenografts of human lung adenocarcinoma H1975 nude mice.
  • Cell culture After cell recovery, use RPMI-1640 medium containing 10% fetal bovine serum and culture in a constant temperature incubator at 37 ° C and 5% CO 2. When the cell fusion degree is about 80% -90% (for (Growth phase)) Passage expansion and culture to 27 T175cm 2 culture flasks, the cells in the log phase are collected and counted for seeding.
  • mice BALB / c Nude nude mice, 32, female, 4-5 weeks, purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.
  • the rate of change in body weight of the four experimental groups of mice is shown in FIG. 5, and the tumor growth inhibition rate TGI% is shown in FIG. 6.
  • the experimental results show that the salt of the compound of Example 2 has a good inhibitory effect on the growth of human lung adenocarcinoma H1975 nude mice subcutaneously transplanted tumors, has no significant effect on the weight of the animals, and shows good safety.
  • the salt of the compound of Example 2 has a good inhibitory effect on the growth of human non-small cell lung cancer HCC827 and human lung adenocarcinoma H1975 transplanted tumor, and it has a wild-type EGFR effect.
  • the inhibitory effect of human squamous cell carcinoma A431 transplantation tumor is relatively weak.
  • the compound salt of Example 2 of the present invention has good selectivity and good safety.

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Abstract

一种式(I)所示的EGFR激酶抑制剂化合物，以及所述激酶抑制剂化合物的盐、制备方法、药物组合物及其在治疗由EGFR介导的疾病特别是癌症方面的应用。

Description

一种EGFR激酶抑制剂及其制备方法和应用 技术领域
本发明涉及一种选择性抑制突变形态的表皮生长因子受体(EGFR)激酶抑制剂及其制备方法，包含所述激酶抑制剂及其药物组合物，以及所述激酶抑制剂在治疗由某些突变形态的EGFR介导的疾病以及制备用于治疗由某些突变形态的EGFR介导的疾病的药物方面的应用。
背景技术
表皮生长因子受体(EGFR)属于蛋白酪氨酸激酶(PTK)家族，由EGFR(Erb-B1)、Erb-B2(HER-2/neu)、Erb-B3和Erb-B4组成，被确认为是在细胞生长和增值过程中至关重要的驱动因素。EGFR的过度表达和突变已被明确证实将导致不可控的细胞生长，与大部分癌症疾病进程有关，如肺癌、结肠癌、乳腺癌等。
作为潜在的抗癌治疗药物的特异性PTK抑制剂备受关注。第一代表皮生长因子受体酪氨酸激酶抑制剂(EGFR-TKI)包括吉非替尼(Gefitinib)、埃罗替尼(Erlotinib)和拉帕替尼(Lapatinib)，对EGFR野生型和激活型突变(例如19号外显子缺失激活突变、或L858R激活突变)均有抑制作用，分别用于非小细胞肺癌(NSCLC)和乳腺癌的治疗。然而，患者在接受治疗后产生耐药而导致肿瘤产生继发性生长，使得此类抑制剂在临床上的进一步应用受到限制。研究表明，50％的吉非替尼、埃罗替尼治疗后耐药性的产生与EGFR 790号位的苏氨酸置换为蛋氨酸(T790M)发生二次突变相关(Pao W.等，Plos Med.，2:1-11,2005)。
与第一代EGFR-TKI相比，第二代EGFR-TKI如阿法替尼(Afatinib)和达克替尼(dacomitinib)具有非常突出的优点，它们可以通过麦克尔加成反应不可逆的与EGFR上半胱氨酸残基(Cys797)共价结合，使EGFR抑制剂与ATP结合位点扩大，从而在一定程度上克服T790M突变引起的耐药性(Li D等，Oncogene，27:4702-4711,2008)。然而Cys797存在于所有形式的EGFR中，因此第二代EGFR-TKI，不仅对激活性突变和耐药型突变的EGFR有活性，而且对野生型的EGFR也具有活性，从而导致皮疹、腹泻、恶心、厌食，虚弱无力等毒副作用(Curr.Med.Chem.2006,13,3483-3492)，最终限制了其临床给药剂量及 有效血药浓度，在克服T790M耐药突变方面未能取得令人瞩目的效果。
第三代EGFR-TKI如：AZD9291,CO-1686和HM61713,它们是一类具有特异选择性的EGFR酪氨酸激酶抑制剂，相较于第一代、第二代EGFR-TKI而言，它们对激活性突变和耐药型突变的EGFR有较强抑制活性，而对野生型EGFR抑制作用较弱。它们在T790M阳性肿瘤中有高效，但他们仍然存在一定的毒性，像依然会产生腹泻，皮疹，恶心甚至高血糖等临床副作用((J Clin Oncol 2014；32:abstr 8009；J Clin Oncol 2014；32:abstr 8010)。很显然，一个具有更高活性和低毒性的化合物会带来更大好处。
阿斯利康AZD9291是一种口服的小分子第三代表皮生长因子络氨酸激酶抑制剂，AZD9291针对耐药突变T790M和敏感突变位点(19Del和L858R)的特异性结合能力较强，因此对于一线TKI治疗后耐药的非小细胞肺癌患者具有很好的治疗效果，但由于其对EGFR野生型也有一定的抑制，因此临床上会产生腹泻、皮疹等副作用，
Figure PCTCN2019101969-appb-000001
为克服临床中常见的EGFR耐药性突变(例如T790M突变)以及现有EGFR抑制剂的毒副作用问题，即开发更多的对某些激活突变体和耐药型突变体形式的EGFR显示较高的抑制同时对野生型EGFR显示相对较低的抑制的小分子抑制剂已经是当前抗肿瘤领域的迫切需要。本发明人在研究EGFR抑制剂的过程中，惊喜地发现了一种对EGFR激活型突变(如19号外显子缺失激活突变、或L858R激活突变)和T790M耐药型突变具有很好的抑制活性，而对野生型EGFR(WT EGFR)的抑制活性较弱的EGFR激酶抑制剂，且毒副作用较低，安全性好。预期此类抑制剂将会有好的疗效，有望克服耐药性问题及毒副作用问题，具有良好的开发前景。
发明内容
本发明提供如下式(I)化合物，或其药学上可接受的盐：
Figure PCTCN2019101969-appb-000002
式中：
环A为杂芳基；
R 1选自氢、卤素、C 1-C 4烷基或卤代C 1-C 4烷基；
R 2为卤代C 1-C 4烷基；
R 3选自氢、C 1-C 4烷基或卤代C 1-C 4烷基；
R 4选自氢、C 1-C 4烷基或卤代C 1-C 4烷基；
R 5选自
Figure PCTCN2019101969-appb-000003
每个R 6独立的选自氢、卤素、-CN、C 1-C 4烷基或卤代C 1-C 4烷基；
R 7选自氢、C 1-C 4烷基或卤代C 1-C 4烷基；
m为1、2或3。
本发明提供式(I)化合物，其能够抑制一种或多种EGFR激活型或耐药型突变，例如L858R激活突变体、19号外显子缺失激活突变体、T790M耐药型突变体。有利地，这种化合物可用于对基于EGFR抑制剂的现有疗法已产生一定程度的耐药性患者的癌症治疗。
本发明提供式(I)化合物，其对激活或耐药型突变体形式的EGFR显示比野生型EGFR更高的抑制。由于与野生型EGFR抑制相关的毒性降低，因而预期这种化合物更适于用作治疗剂，尤其适用于癌症的治疗。
本发明还提供了式(I)化合物的制备方法。
本发明还提供药物组合物，它含有上述本发明式(I)化合物或其药学上可接受的盐，以及药学上可以接受的载体、赋形剂或稀释剂。
本发明还提供式(I)化合物或其药学上可接受的盐在治疗由EGFR激活型或耐药型突变体介导的疾病、特别是癌症方面的应用。
本发明还提供本发明式(I)化合物或其药学上可接受的盐在制备 治疗由EGFR激活型或耐药型突变体介导的疾病、特别是癌症的药物中的应用。
本发明还提供一种治疗由EGFR激活型或耐药型突变体介导的疾病、特别是癌症的方法，所述方法包括对患者施用式(I)化合物或其药学上可接受的盐，或对患者施用含有治疗有效量的式(I)化合物或其药学上可接受的盐的药物组合物。
本发明还提供一种治疗哺乳动物尤其人类由EGFR激活型或耐药型突变体介导的疾病、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载体、赋形剂或稀释剂的药物组合物。
本发明还提供一种相比于野生型EGFR(WT EGFR)选择性地抑制EGFR激活型或耐药型突变的方法，所述方法包括使生物样品接触或向患者投与式(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，包括肺癌、卵巢癌、宫颈癌、乳腺癌、胃癌、结肠直肠癌、胰腺癌、胶质瘤、胶质母细胞瘤、黑色素瘤、前列腺癌、白血病、淋巴瘤、非霍奇金淋巴瘤、肝细胞癌、胃肠道基质瘤(GIST)、甲状腺癌、胆管癌、子宫内膜癌、肾癌、间变性大细胞淋巴瘤、急性髓细胞白血病(AML)、多发性骨髓瘤、间皮瘤。
在本发明式(I)化合物或其药学上可接受的盐的一个优选实施方案中，环A为吲哚基、吲唑基、吡咯[2，3-c]骈吡啶基、吡咯[3，2-c]骈吡啶基、吡咯[2，3-b]骈吡啶基、吡咯[3，2-b]骈吡啶基、吡咯[2，3-b]骈吡嗪基、吲哚啉-2-酮基、吡啶基、吡唑基或嘧啶基。
在本发明式(I)化合物或其药学上可接受的盐的一个更优选实施方案中，环A为吲哚基、吡咯[2，3-c]骈吡啶基、吡咯[3，2-c]骈吡啶基、吡咯[2，3-b]骈吡啶基、吡咯[3，2-b]骈吡啶基、吡唑基或嘧啶基。
在本发明式(I)化合物或其药学上可接受的盐的一个最优选实施方案中，环A为吲哚基。
在本发明式(I)化合物或其药学上可接受的盐的一个优选实施方案中，R 1为氢或卤素。
在本发明式(I)化合物或其药学上可接受的盐的一个更优选实施方案中，R 1为氢。
在本发明式(I)化合物或其药学上可接受的盐的一个优选实施方 案中，R 2为三氟甲基、一氟甲基、二氟甲基或2,2,2-三氟乙基。
在本发明式(I)化合物或其药学上可接受的盐的一个更优选实施方案中，R 2为2,2,2-三氟乙基。
在本发明式(I)化合物或其药学上可接受的盐的一个优选实施方案中，R 3为C 1-C 4烷基。
在本发明式(I)化合物或其药学上可接受的盐的一个更优选实施方案中，R 3为甲基。
在本发明式(I)化合物或其药学上可接受的盐的一个优选实施方案中，R 4为C 1-C 4基。
在本发明式(I)化合物或其药学上可接受的盐的一个更优选实施方案中，R 4为甲基。
在本发明式(I)化合物或其药学上可接受的盐的一个优选实施方案中，R 5为
Figure PCTCN2019101969-appb-000004
在本发明式(I)化合物或其药学上可接受的盐的一个更优选实施方案中，R 5为
Figure PCTCN2019101969-appb-000005
在本发明式(I)化合物或其药学上可接受的盐的一个优选实施方案中，每个R 6独立的为卤素或C 1-C 4烷基，m为1、2或3。
在本发明式(I)化合物或其药学上可接受的盐的一个更优选实施方案中，每个R 6独立的为C 1-C 4烷基，m为1、2或3。
在本发明式(I)化合物或其药学上可接受的盐的一个最优选实施方案中，R 6为甲基，m为1。
在本发明中，具体优选的式(Ⅰ)化合物或其药学上可接受的盐为N-(2-(甲基(2-(甲基胺基)乙基)胺基)-6-(2，2，2-三氟乙氧基)-5-((4-(1-甲基-1H-吲哚-3-基)嘧啶-2-基)胺基)吡啶-3-基)丙烯酰胺，结构如下所示：
Figure PCTCN2019101969-appb-000006
在本发明中，具体优选的式(Ⅰ)化合物药学上可接受的盐为N-(2-(甲基(2-(甲基胺基)乙基)胺基)-6-(2，2，2-三氟乙氧基)-5-((4-(1- 甲基-1H-吲哚-3-基)嘧啶-2-基)胺基)吡啶-3-基)丙烯酰胺甲磺酸盐。
本发明还提供制备式(I)化合物的方法，它包括以下步骤：
Figure PCTCN2019101969-appb-000007
其中，环A、R 1、R 2、R 3、R 4、R 5、R 6和m与上述式(I)中的含义相同；X、Y分别为卤素，具体的包括氟、氯、溴、碘，优选氯或溴。
碱存在下，式a化合物与醇R 2OH经醚化反应得到式b化合物；式b化合物经硝基还原得到式c化合物；碱存在下，式c化合物经酰化反应得到式d化合物；式d化合物经硝化反应得到式e化合物；酸存在下，式e化合物经水解反应得到中间体1；酸存在下，中间体1与式f化合物经取代反应得到式g化合物或其盐；碱存在下，式g化合物与式h化合物经取代反应得到式i化合物；式i化合物在催化剂存在下经Boc保护得到式j化合物；式j化合物经硝基还原得到式k化合物；碱存在下，式k化合物经酰胺化得到式m化合物；最后，酸存在下，式m化合物脱Boc得到式(Ⅰ)化合物。
在本发明通式(Ⅰ)化合物的制备方法中，均可按照常规方法使用常规试剂制备，其中醚化反应所述碱为无机强碱包括但不限于NaH、LiHMDS、NaHMDS或KHMDS；式b化合物、式j化合物硝基还原所述还原剂为常规还原剂，包括但不限于保险粉、钯碳/氢气或三氯化铁/水合肼，还原反应中可进一步的加入酸，所述酸为有机酸或无机酸包括但不限于盐酸、氢溴酸、硫酸、磷酸、甲磺酸、苯磺酸、对甲苯磺酸或乙酸；式c化合物酰胺化反应中所述碱为有机碱或是无机碱包括但不限于三乙胺、N,N-二异丙基乙胺、碳酸氢钠、碳酸钾、碳酸铯、叔丁醇钾或叔丁醇钠；所述酰化试剂包括但不限于三氟乙酸酐或三氟乙酰氯；式d化合物硝化反应中，所述硝化剂为常规硝化剂包括但不限于HNO 3/H 2SO 4、KNO 3/H 2SO 4或发烟硝酸；式e化合物水解反应中，所述酸为无机酸或是有机酸包括但不限于盐酸、三氟乙酸、TsOH.H 2O或TsOH；式f化合物与中间体1的取代反应中所述酸为有机酸包括但不限于一水对甲苯磺酸或对甲苯磺酸；式g化合物与胺的取代反应中，所述碱为无机碱或有机碱包括但不限于碳酸钾、碳酸氢钠、三乙胺、N,N-二异丙基乙胺；式i化合物Boc保护反应中所述催化剂为常规催化剂包括但不限于4-二甲氨基吡啶(DMAP)；式k化合物酰胺化反应所述碱为有机碱或无机碱包括但不限于三乙胺、N,N-二异丙基乙胺、碳酸氢钠、碳酸钾；式m化合物脱Boc保护反应中所述酸为无机酸或有机酸包括但不限于盐酸、三氟乙酸。
在本发明中，卤素是指氟、氯、溴、碘等，优选氟、氯、溴。
在本发明中，C 1-C 4烷基是指含有1～4个碳原子的直链或支链烷基，烷基指饱和的脂肪族烃基团，具体包括但不限于甲基、乙基、丙 基、异丙基、丁基、异丁基、仲丁基或叔丁基，优选甲基、乙基或异丙基，更优选甲基。
在本发明中，卤代C 1-C 4烷基是指被一个或多个卤素，优选一至五个卤原子取代的如本文定义的C 1-C 4烷基，具体包括但不限于三氟甲基、一氟甲基、二氟甲基、2,2,2-三氟乙基、2-氟乙基或1-氯-2-氟乙基，优选三氟甲基、一氟甲基、二氟甲基或2,2,2-三氟乙基，更优选2,2,2-三氟乙基。
在本发明中，杂芳基是指含有1至4个选自N、S或O的杂原子的5至6元单环杂芳基或其与苯环、吡啶环或吡咯环稠而成的双环式杂芳基，它可以是部分饱和的。所述杂芳基包括但不限于呋喃基、噻吩基、吡咯基、咪唑基、吡唑基、噻唑基、异噻唑基、噁唑基、异噁唑基、三唑基、四唑基、噻二唑基、吡啶基、嘧啶基、哒嗪基、吡嗪基、苯并呋喃基、苯并噻吩基、苯并噻二唑基、苯并噻唑基、苯并咪唑基、吲哚基、异吲哚基、吲唑基、喹啉基、异喹啉基、喹唑啉基、1，2，3，4-四氢异喹啉基、吡咯[2,3-c]骈吡啶基、吡咯[3,2-c]骈吡啶基、吡咯[2,3-b]骈吡啶基、吡咯[3,2-b]骈吡啶基、吡咯[2,3-b]骈吡嗪基、吲哚啉-2-酮基，优选吲哚基、吲唑基、吡咯[2,3-c]骈吡啶基、吡咯[3,2-c]骈吡啶基、吡咯[2,3-b]骈吡啶基、吡咯[3,2-b]骈吡啶基、吡咯[2,3-b]骈吡嗪基、吲哚啉-2-酮基、吡啶基、吡唑基或嘧啶基、咪唑基、吡嗪基、苯并咪唑基、吲哚基、异吲哚基或1，2，3，4-四氢异喹啉基，更优选吲哚基、吲唑基、吡咯[2,3-c]骈吡啶基、吡咯[3,2-c]骈吡啶基、吡咯[2,3-b]骈吡啶基、吡咯[3,2-b]骈吡啶基、吡咯[2,3-b]骈吡嗪基、吲哚啉-2-酮基、吡啶基、吡唑基或嘧啶基，最优选吲哚基、吡咯[2，3-c]骈吡啶基、吡咯[3，2-c]骈吡啶基、吡咯[2，3-b]骈吡啶基、吡咯[3，2-b]骈吡啶基、吡唑基或嘧啶基。
本发明还包含式(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，或者可用过使纯化的式(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、月硅酸盐、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、乳糖酸盐和月桂基磺酸盐等。所述碱加成盐为式(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、四甲基季铵盐、四乙基季铵盐等；胺盐，包括与氨(NH 3)、伯胺、仲胺或叔胺形成的盐，如甲胺盐、二甲胺盐、三甲胺盐、三乙胺盐、乙胺盐等。
本发明的化合物或其药学上可接受的盐可给药于哺乳动物包括人，可以口服、直肠、肠胃外(静脉内、肌肉内或皮下)、局部给药(粉剂、软膏剂或滴剂)、或瘤内给药。
本发明化合物的给药剂量可以大约为0.3-30mg/kg体重/天，例如0.5-20mg/kg体重/天，10mg/kg体重/天。当本发明化合物为盐、酯、前药等时，以母体化合物为基础计算施用量。
本发明化合物或其药学上可接受的盐可以配制为用于口服给药的固体剂型，包括，但不限于胶囊剂、片剂、丸剂、散剂和颗粒剂等。在这些固体剂型中，本发明式(I)化合物作为活性成分与至少一种常规惰性赋形剂(或载体)混合，例如与柠檬酸钠或磷酸二钙，或与下述成分混合：(1)填料或增容剂，例如，淀粉、乳糖、蔗糖、葡萄糖、甘露醇和硅酸等；(2)粘合剂，例如羟甲基纤维素、藻酸盐、明胶、聚乙烯基吡咯烷酮、蔗糖和阿拉伯胶等；(3)保湿剂，例如，甘油等；(4)崩解剂，例如琼脂、碳酸钙、马铃薯淀粉或木薯淀粉、藻酸、某些复合硅酸盐和碳酸钠等；(5)缓溶剂，例如石蜡等；(6)吸收加速剂，例如，季铵化合物等；(7)润湿剂，例如鲸蜡醇和单硬脂酸甘油酯等；(8)吸附剂，例如，高岭土等；和(9)润滑剂，例如，滑石、硬脂酸钙、硬脂酸镁、固体聚乙二醇、十二烷基硫酸钠等，或其混合物。胶囊剂、片剂和丸剂中也可包含缓冲剂。
所述固体剂型如片剂、糖丸、胶囊剂、丸剂和颗粒剂可采用包衣和壳材料例如肠溶衣和其他本领域公知的材料进行包衣或微囊化。它们可包含不透明剂，并且，这种组合物中活性成分的释放可以延迟的方式在消化道内的某一部分中释放。可采用的包埋组分的实例是聚合物质和蜡类物质。必要时，活性成分也可与上述赋形剂中的一种或多 种形成微胶囊形式。
本发明化合物或其药学上可接受的盐可以配制为用于口服给药的液体剂型，包括，但不限于药学上可接受的乳液、溶液、悬浮液、糖浆和酊剂等。除了作为活性成分的式(I)化合物或其药学上可接受的盐外，液体剂型可包含本领域中常规采用的惰性稀释剂，如水和其他溶剂，增溶剂和乳化剂，例如，乙醇、异丙醇、碳酸乙酯、乙酸乙酯、丙二醇、1,3-丁二醇、二甲基甲酰胺以及油类，特别是棉籽油、花生油、玉米胚油、橄榄油、蓖麻油和芝麻油等或这些物质的混合物等。除了这些惰性稀释剂外，本发明液体剂型也可包含常规助剂，如润湿剂、乳化剂和悬浮剂、甜味剂、矫味剂和香料等。
所述悬浮剂包括，例如，乙氧基化异十八烷醇、聚氧乙烯山梨醇和脱水山梨醇酯、微晶纤维素、甲醇铝和琼脂等或这些物质的混合物。
本发明化合物或其药学上可接受的盐可以配制为用于肠胃外注射的剂型，包括，但不限于生理上可接受的无菌含水或无水溶液、分散液、悬浮液或乳液，以及用于重新溶解成无菌的可注射溶液或分散液的无菌粉末。适宜的载体、稀释剂、溶剂或赋形剂包括水、乙醇、多元醇及其适宜的混合物。
本发明化合物或其药学上可接受的盐也可以配制为用于局部给药的剂型，包括如软膏剂、散剂、栓剂、滴剂、喷射剂和吸入剂等。作为活性成分的本发明式(I)化合物或其药学上可接受的盐在无菌条件下和生理上可接受的载体及任选的防腐剂、缓冲剂，或必要时可能需要的推进剂一起混合。
本发明还提供药物组合物，它含有本发明式(I)化合物或其药学上可接受的盐作为活性成分，以及药学上可接受载体、赋形剂或稀释剂。在制备药物组合物时，通常是将本发明式(I)化合物或其药学上可接受的盐与药学上可接受载体、赋形剂或稀释剂混合。
可以按常规制备方法将所述本发明组合物配制为常规药物制剂。例如片剂、丸剂、胶囊剂、散剂、颗粒剂、乳液剂、混浮剂、分散液、溶液剂、糖浆剂、酏剂、软膏剂、滴剂、栓剂、吸入剂、喷射剂等。
本发明所述的化合物或其药学上可接受的盐可以单独给药，或者与其他药学上可接受的治疗剂联合给药，特别是与其他抗肿瘤药物组合。所述治疗剂包括但不限于：作用于DNA化学结构的药物抗肿瘤药 如顺铂，影响核苷酸合成的抗肿瘤药物如甲氨蝶呤(MTX)、5-氟尿嘧啶(5FU)等，影响核酸转录的抗肿瘤药物如阿霉素、表阿霉素、阿克拉霉素、光辉霉素等，作用于微管蛋白合成的抗肿瘤药物如紫杉醇、长春瑞滨等，芳香化酶抑制剂如氨鲁米特、兰特隆、来曲唑、瑞宁德等，细胞信号通路抑制剂如表皮生长因子受体抑制剂伊马替尼(Imatinib)、吉非替尼(Gefitinib)、埃罗替尼(Erlotinib)等。待组合的各成分可同时或顺序的给予，以单一制剂形式或以不同制剂的形式给予。所述组合不仅包括本发明化合物和一种其他活性剂的组合，而且也包括本发明化合物和两种或更多种其他活性剂的组合。
经激酶抑制实验(酶联免疫吸附法)，证明本发明化合物对EGFR突变(如EGFR L858R、EGFR L858R/T790M等)具有很好的抑制活性，尤其是对EGFR L858R/T790M。
此外，通过细胞实验即对敏感型突变肿瘤细胞如HCC827(EGFR E746_A750del)细胞、PC-9(EGFR ex19del)细胞，耐药型突变肿瘤细胞如H1975(EGFR L858R/T790M)体外增值抑制实验，证明本发明化合物对敏感型突变或耐药型突变肿瘤细胞具有良好的增值抑制作用；通过动物实验即对人鳞状细胞癌A431、人非小细胞肺癌HCC827、人肺腺癌H1975裸小鼠皮下移植瘤的生长抑制实验，证明本发明化合物对敏感型突变和耐药型突变肿瘤如人非小细胞肺癌HCC827、人肺腺癌H1975移植瘤的生长具有良好的抑制作用，而对野生型EGFR人鳞状细胞癌A431的抑制作用相对较弱，说明本发明化合物具有良好的特异选择性抑制作用，并且具有较好的安全性。本发明化合物可用作治疗由EGFR敏感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，例如肺癌、卵巢癌、宫颈癌、乳腺癌、胃癌、结肠直肠癌、胰腺癌、胶质瘤、胶质母细胞瘤、黑色素瘤、前列腺癌、白血病、淋巴瘤、非霍奇金淋巴瘤、肝细胞癌、胃肠道基质瘤(GIST)、甲状腺癌、胆管癌、子宫内膜癌、肾癌、间变性大细胞淋巴瘤、急性髓细胞白血病(AML)、多发性骨髓瘤、间皮瘤，尤其对于表皮生长因子受体790位苏氨酸突变为蛋氨酸(EGFR T790M)的肿瘤类型有更好的应用。举例来说，本发明化合物可作为和用于治疗非小细胞肺癌(EGFR T790M)的药物。可用于克服临床上应用吉非替尼、埃罗替尼后由EGFR T790M引起的耐药性问题。且因 与野生型EGFR抑制相关的毒性降低，预期本发明化合物在应用于治疗癌症的过程当中产生的毒副作用相对较小。
本发明化合物激酶活性抑制实验可用常规方法测定，一种优选的评价方法为酶联免疫吸附法(ELISA)，通过测定药物作用于激酶后所产生的光吸收值的变化来计算药物对激酶的抑制率，
Figure PCTCN2019101969-appb-000008
根据各浓度抑制率，采用非线性回归方法计算出各测试化合物的半数抑制浓度IC50。
本发明化合物抑制癌细胞增殖的药效可用常规方法测定，一种优选的评价方法为磺酰罗丹明B(SulforhodamIne B,SRB)蛋白染色法，通过测定药物作用于癌细胞后所产生的光吸收值的变化来计算药物对癌细胞增殖的抑制率，
Figure PCTCN2019101969-appb-000009
阴性对照组OD值为不加入任何化合物(含0.5％的DMSO)的正常生长的细胞孔的OD值。
给药组OD值为加入待测化合物(含0.5％的DMSO)作用后的OD值。
半数抑制剂浓度(IC50)值采用GraphPad公司PrIsm软件5.0版本，四参数拟合方法计算。每个实验重复3次，求出3次实验的平均IC50值为抑制能力的最终指标。
本发明化合物抑制动物移植瘤生长的药效可用常规方法测定，一种优选的评价方法为对对人鳞状细胞癌A431裸小鼠皮下移植瘤的生长抑制作用；另一种优选的评价方法为对人非小细胞肺癌细胞HCC827裸小鼠皮下移植瘤的生长抑制作用；另一种优选的评价方法为对人肺腺癌H1975裸小鼠皮下移植瘤的生长抑制作用。试验方法：人鳞状细胞癌A431、人非小细胞肺癌细胞HCC827、人肺腺癌H1975分别接种于BALB/c Nude小鼠右前背部皮下，待肿瘤生长至平均合适大小时根据肿瘤大小和小鼠体重随机分组，测试化合物按一定剂量灌胃给药， 溶剂对照组灌胃给予等量溶剂，每天一次，连续21天，整个实验过程中，每周测量两次小鼠的体重和肿瘤的大小，观察是否出现毒性反应。
肿瘤大小计算公式：肿瘤体积(mm 3)＝0.5×(肿瘤长径×肿瘤短径 2)。
附图说明
附图1是实施例2化合物盐和AZD9291在10mg/kg给药剂量下人鳞状细胞癌A431裸小鼠的体重变化曲线。
附图2是实施例2化合物盐和AZD9291在10mg/kg给药剂量下人鳞状细胞癌A431裸小鼠皮下移植瘤的肿瘤生长抑制率TGI％曲线。
附图3是实施例2化合物盐在不同给药剂量下人非小细胞肺癌细胞HCC827裸小鼠的体重变化曲线。
附图4是实施例2化合物盐在不同给药剂量下人非小细胞肺癌细胞HCC827裸小鼠皮下移植瘤的肿瘤生长抑制率TGI％曲线。
附图5是实施例2化合物盐在不同给药剂量下人肺腺癌细胞H1975裸小鼠的体重变化曲线。
附图6是实施例2化合物盐在不同给药剂量下人肺腺癌细胞H1975裸小鼠皮下移植瘤的肿瘤生长抑制率TGI％曲线。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于举例说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。除非另外说明，否则份数和百分比分别为重量份和重量百分比。
具体实施方式
I.本发明化合物制备实施例
中间体1a：6-氯-5-硝基-3-氨基-2-(2,2,2-三氟乙氧基)吡啶
Figure PCTCN2019101969-appb-000010
步骤1：6-氯-3-硝基-2-(2,2,2-三氟乙氧基)吡啶的合成
Figure PCTCN2019101969-appb-000011
加入甲苯(24.0L)至反应釜中，再加入2,6-二氯-3-硝基吡啶(3000g， 15.54mol)，调整内温在-20℃至-10℃之间，分批加入钠氢(933g，23.33mol)。滴加2,2,2-三氟乙醇(1586g，16.00mol)甲苯(6.0L)溶液。反应2h，TLC及HPLC监控反应终点。反应完毕，滴加入10％氯化铵溶液(6.0L)。静置，分层。有机相使用水(6.0L)洗涤，减压浓缩。加入乙酸乙酯(0.3L)，升温至40～50℃，滴加正庚烷(2.7L)，滴完降温到-15至-5℃下继续析晶3小时，抽滤。得到产物固体3017g，收率75.65％。
1H NMR(500MHz,DMSO-d6)δ8.60(d,J＝8.0Hz,1H),7.50(d,J＝8.5Hz,1H),5.13(q,J＝9.0Hz,2H)；
13C NMR(126MHz,DMSO-d6)δ153.20,151.09,139.34,132.67,123.38(q,J＝277.2Hz),119.14,63.34(q,J＝36Hz)；
MS m/z：256.99[M+1]。
步骤2：6-氯-3-氨基-2-(2,2,2-三氟乙氧基)吡啶的合成
Figure PCTCN2019101969-appb-000012
室温下，加入乙腈(21.0L)和水(21.0L)至反应釜中，开启搅拌，加入实施例1得到的6-氯-3-硝基-2-(2,2,2-三氟乙氧基)吡啶(3017.0g，11.76mol)，加入保险粉(15.1Kg，70.54mol)。控制温度27～33℃条件下反应2小时。滴加入36％的浓盐酸(11.9Kg，117.60mol)，继续反应1.5小时。加入碳酸氢钠固体(12.8Kg，12.96mol)。过滤，母液分层，有机相使用饱和食盐水(21.0L)洗涤，减压浓缩，得到油状物质理论计算投下步反应。
1H NMR(500MHz,DMSO-d6)δ7.03(d,J＝8.0Hz,1H),6.90(d,J＝8.0Hz,1H),5.21(s,2H),4.93(q,J＝9.0Hz,2H)；
13C NMR(126MHz,DMSO-d6)δ148.16,131.72,130.55,123.93(q,J＝278.5Hz),121.02,118.42,61.72(q,J＝34.0Hz)；
MS m/z：227.01[M+1]。
步骤3：6-氯-3-(2,2,2-三氟乙酰胺基)-2-(2,2,2-三氟乙氧基)吡啶的合成
Figure PCTCN2019101969-appb-000013
室温下，反应釜中加入二氯甲烷(10.4L)，开启搅拌，加入实施例2得到的6-氯-3-氨基-2-(2,2,2-三氟乙氧基)吡啶(2664g，11.76mol)，加入二异丙基乙基胺(2279g，17.64mol)，控温-15至-10℃，滴加三氟乙酸酐(2963g，14.11mol)的二氯甲烷(5.2L)溶液，滴完继续搅拌20分钟。滴加水(13.0L)，分层，有机相减压浓缩，理论计算投下步反应。
1H NMR(400MHz,DMSO-d6)δ11.23(s,7H),7.95(d,J＝8.0Hz,1H),7.34(d,J＝8.0Hz,1H),5.03(q,J＝8.9Hz,2H)；
13C NMR(101MHz,DMSO-d6)δ155.74(q,J＝46.6Hz),155.60,145.37,140.24,124.01(q,J＝278.8Hz),119.07,118.30,116.19(q,J＝289.9Hz),62.99(q,J＝35.4Hz)；
MS m/z：322.99[M+1]。
步骤4：6-氯-5-硝基-3-(2,2,2-三氟乙酰胺基)-2-(2,2,2-三氟乙氧基)吡啶的合成
Figure PCTCN2019101969-appb-000014
室温下，反应釜中加入浓硫酸(11.7L)，开启搅拌，加入实施例3得到的6-氯-3-(2,2,2-三氟乙酰胺基)-2-(2,2,2-三氟乙氧基)吡啶(3.9Kg，11.76mol)，分批加入硝酸钾固体(1783.4g，17.64mol)，加完继续搅拌约40分钟，监测反应完毕后，降温，控制内温在10～25℃,开始滴加二氯甲烷(27.3L)，搅拌45分钟，分层，取有机相，用水(11.7L)洗涤一次。有机相减压浓缩，理论计算投下步反应。
1H NMR(500MHz,DMSO-d6)δ11.58(s,1H),8.78(s,1H),5.17(q,J＝8.7Hz,2H)；
13C NMR(126MHz,DMSO-d6)δ155.89,155.43(q,J＝37.8Hz),138.84,138.57,135.05,123.22(q,J＝273.4Hz),118.47,115.51(q,J＝ 278.5Hz),63.65(q,J＝35.3Hz)；
MS m/z：367.98[M+1]。
步骤5：6-氯-5-硝基-3-氨基-2-(2,2,2-三氟乙氧基)吡啶的合成
Figure PCTCN2019101969-appb-000015
室温下，反应釜中加入甲醇(13.0L)，加入实施例4得到的6-氯-5-硝基-3-(2,2,2-三氟乙酰胺基)-2-(2,2,2-三氟乙氧基)吡啶(4322g,11.76mol)，加入对甲苯磺酸一水合物(3355g，17.64mol)，控制温度60～65℃反应15小时，减压除去甲醇。加入甲基叔丁基醚(13.0L)和水(6.5L)，用碳酸钾调pH 7-8。分层，有机相用水(8.6L)洗一次，分液，减压浓缩。加入正庚烷(21.5L)，控温60～65℃搅拌1小时，降至室温，抽滤，滤饼50℃鼓风干燥18小时，得到产品1475g。
步骤1至步骤5五步反应合计收率34.9％。
1H NMR(500MHz,DMSO-d6)δ7.62(s,1H),5.92(s,2H),5.05(q,J＝8.9Hz,2H).
13C NMR(126MHz,DMSO-d6)δ149.30,139.53,132.84,123.46,123.44(q,J＝278.5Hz),116.25,62.52(q,J＝35.3Hz)；
MS m/z：272.00[M+1]。
实施例1：N-(2-(甲基(2-(甲基胺基)乙基)胺基)-6-(2，2，2-三氟乙氧基)-5-((4-(1-甲基-1H-吲哚-3-基)嘧啶-2-基)胺基)吡啶-3-基)丙烯酰胺
Figure PCTCN2019101969-appb-000016
步骤1：2-氯-3-硝基-5-[4-(1-甲基-1H-吲哚-3-基)嘧啶-2-胺基]-6-(2,2,2-三氟乙氧基)吡啶对甲苯磺酸盐的合成
Figure PCTCN2019101969-appb-000017
将甲苯(7.43L)加入到20L反应釜中，依次加入中间体1a化合物6-氯-5-硝基-3-氨基-2-(2,2,2-三氟乙氧基)吡啶(743.0g，2.74mol)、化合物3-(2-氯嘧啶-4-基)-1-甲基-1H-吲哚(866.7g，3.56mol)(参照CN105315259A中间体2a制备)、对甲苯磺酸一水合物(780.7g，4.10mol)，搅拌，将反应混合物升温至110-115℃，反应36小时。控制温度15～30℃，加入四氢呋喃(3.72L)搅拌30分钟。抽滤，将滤饼转移至50L反应釜中，加入四氢呋喃(4.46L)，加热回流3小时。降至15～25℃，抽滤，滤饼50℃鼓风干燥17小时，得2-氯-3-硝基-5-[4-(1-甲基-1H-吲哚-3-基)嘧啶-2-胺基]-6-(2,2,2-三氟乙氧基)吡啶对甲苯磺酸盐(1719g，85.96HPLC area％purity)。按照HPLC纯度折算，投下步反应。
熔点：216-218℃；
1H NMR(500MHz,DMSO-d6)δ9.70(s,1H),9.21(s,1H),8.62(s,1H),8.40(d,J＝6.2Hz,1H),8.24(d,J＝7.8Hz,1H),7.59(d,J＝8.3Hz,1H),7.50(d,J＝6.5Hz,1H),7.49(d,J＝8.3Hz,2H),7.32(t,J＝7.6Hz,1H),7.18(t,J＝7.5Hz,1H),7.12(d,J＝7.9Hz,2H),5.17(q,J＝8.8Hz,2H),3.91(s,3H),2.29(d,J＝5.2Hz,3H)；
13C NMR(126MHz,DMSO-d6)δ166.66,157.35,155.72,147.40,140.87,139.90,139.72,138.59,135.83,130.09,129.99,129.98,129.97,127.39,127.38,127.37,127.15,125.22(q,J＝278.5Hz),124.97,123.85,123.69,113.63,112.97,110.27,63.58(q,J＝35.3Hz),35.57,22.81。
步骤2：N 2-甲基-N 2-(2-(甲胺基)乙基)-6-(2，2，2-三氟乙氧基)-N 5-(4-(1-甲基-1H-3-基)嘧啶-2-基)-3-硝基吡啶-2，5-二胺的合成
Figure PCTCN2019101969-appb-000018
500mL单口瓶中加入2-氯-3-硝基-5-[4-(1-甲基-1H-吲哚-3-基)嘧啶-2-胺基]-6-(2,2,2-三氟乙氧基)吡啶对甲苯磺酸盐(15.0g，31.3mmol)、碳酸钾(10.4g，75mmol)、N,N'-二甲基乙二胺(3.3g，37.6mmol)及DMF(75mL)。反应物置于80℃油浴中搅拌反应1小时。向反应物中滴加400mL水，抽滤收集析出的固体，80℃烘干，得棕黄色固体(7.6g，收率46％)。
MS(ESI)m/z 531[M+1] +。
步骤3：N 2-甲基-N 2-(2-(甲基叔丁氧羰基胺基)乙基)-6-(2，2，2-三氟乙氧基)-N 5-(4-(1-甲基-1H-3-基)嘧啶-2-基)-3-硝基吡啶-2，5-二胺的合成
Figure PCTCN2019101969-appb-000019
250mL单口瓶中加入N 2-甲基-N 2-(2-(甲胺基)乙基)-6-(2，2，2-三氟乙氧基)-N 5-(4-(1-甲基-1H-3-基)嘧啶-2-基)-3-硝基吡啶-2，5-二胺(7.0g，13.2mmol)、二碳酸二叔丁酯(4.3mL，18.5mmol)、DMAP(0.16g，1.32mmol)及乙腈(100mL)。反应物置于80℃油浴中搅拌反应4小时。反应液减压蒸干，固体残留物用乙酸乙酯(80mL)打浆处理，抽滤得棕黄色固体(5.5g，收率66％)。
MS(ESI)m/z 631[M+1] +。
步骤4：N 2-甲基-N 2-(2-(甲基叔丁氧羰基胺基)乙基)-6-(2，2，2-三氟乙氧基)-N 5-(4-(1-甲基-1H-3-基)嘧啶-2-基)吡啶-2，3，5-,三胺的合成
Figure PCTCN2019101969-appb-000020
1L单口瓶中加入N 2-甲基-N 2-(2-(甲基叔丁氧羰基胺基)乙基)-6-(2，2，2-三氟乙氧基)-N 5-(4-(1-甲基-1H-3-基)嘧啶-2-基)-3-硝基吡啶-2，5-二胺(5.5g，8.7mmol)、10％钯碳(0.5g)及甲醇(100mL)。反应物室温下以氢气球加压氢化反应2小时。反应液通过硅藻土过滤，滤液减压蒸干得棕褐色固体(3.8g，收率72％)，直接用于下一步反应。
MS(ESI)m/z 601[M+1] +。
步骤5：N-(2-(甲基(2-(甲基叔丁氧羰基胺基)乙基)胺基)-6-(2，2，2-三氟乙氧基)-5-((4-(1-甲基-1H-吲哚-3-基)嘧啶-2-基)氨基)吡啶-3-基)丙烯酰胺的合成
Figure PCTCN2019101969-appb-000021
将N 2-甲基-N 2-(2-(甲基叔丁氧羰基胺基)乙基)-6-(2，2，2-三氟乙氧基)-N 5-(4-(1-甲基-1H-3-基)嘧啶-2-基)吡啶-2，3，5-,三胺(3.8g，6.3mmol)及三乙胺(1.76mL，12.6mmol)溶于二氯甲烷(120mL)。反应物以冰水浴冷却，加入丙烯酰氯(0.56mL，6.9mmol)，于冰水浴中搅拌反应1小时。再加入丙烯酰氯(0.11mL，1.2mmol)并继续反应1小时。反应物依次以饱和NaHCO 3溶液、饱和NaCl溶液洗涤，有机相以无水硫酸钠干燥，减压浓缩，残留物以硅胶柱层析(石油醚/乙酸乙酯＝4/1洗脱)，得黄色固体(1.2g，收率29％)。
MS(ESI)m/z 655[M+1] +；
1H NMR(400MHz,DMSO-d 6)δ9.52(s,1H),8.38(s,1H),8.26(d,J＝5.4Hz,2H),8.10(s,1H),7.51(d,J＝8.2Hz,1H),7.20(m,3H),6.58(dd,J＝16.9,10.1Hz,1H),6.25(dd,J＝17.0,2.4Hz,1H),5.75(dd,J＝ 10.1,2.4Hz,1H),4.97(m,2H),3.88(s,3H),3.36(m,4H),2.92(s,3H),2.76(m,3H),1.37(s,9H)。
步骤6：N-(2-(甲基(2-(甲基胺基)乙基)胺基)-6-(2，2，2-三氟乙氧基)-5-((4-(1-甲基-1H-吲哚-3-基)嘧啶-2-基)胺基)吡啶-3-基)丙烯酰胺的合成
Figure PCTCN2019101969-appb-000022
将N-(2-(甲基(2-(甲基叔丁氧羰基胺基)乙基)胺基)-6-(2，2，2-三氟乙氧基)-5-((4-(1-甲基-1H-吲哚-3-基)嘧啶-2-基)氨基)吡啶-3-基)丙烯酰胺(780mg，1.2mmol)溶于二氯甲烷(20mL)。反应物以冰水浴冷却，加入三氟乙酸(10mL)，然后于室温搅拌反应3小时。反应液减压浓缩，残留物中加入乙酸乙酯(100mL)，再依次以饱和NaHCO 3溶液、饱和NaCl溶液洗涤，有机相以无水硫酸钠干燥，减压浓缩，得黄色固体(535mg，收率81％)。
MS(ESI)m/z 555[M+1] +；
1H NMR(400MHz,DMSO-d 6)δ10.15(s,1H),8.90(s,2H),8.64(s,1H),8.44(s,1H),8.29(t,J＝6.1Hz,2H),8.18(s,1H),7.52(d,J＝8.1Hz,1H),7.22(m,2H),6.91(dd,J＝17.0,10.2Hz,1H),6.29(m,1H),5.78(dd,J＝10.1,2.0Hz,1H),5.00(q,J＝9.1Hz,2H),3.90(s,3H),3.58(t,J＝5.5Hz,2H),3.13(m,2H),2.79(s,3H),2.60(t,J＝5.0Hz,3H)。
实施例2：N-(2-(甲基(2-(甲基胺基)乙基)胺基)-6-(2，2，2-三氟乙氧基)-5-((4-(1-甲基-1H-吲哚-3-基)嘧啶-2-基)胺基)吡啶-3-基)丙烯酰胺甲磺酸盐的合成
Figure PCTCN2019101969-appb-000023
游离碱产物N-(2-(甲基(2-(甲基胺基)乙基)胺基)-6-(2，2，2-三氟乙氧基)-5-((4-(1-甲基-1H-吲哚-3-基)嘧啶-2-基)胺基)吡啶-3-基)丙烯酰胺(50mg，0.09mmol)加入到丙酮/水混合溶剂(2mL，v/v＝20/1)中，再加入甲磺酸(8μL)，室温下搅拌0.5小时后抽滤收集固体，干燥得黄色固体(48mg，收率83％)。
1H NMR(400MHz,DMSO-d 6)δ10.19(s,1H),8.86(brs,2H),8.75(s,1H),8.21(brs,2H),7.60(d,J＝8.0Hz,1H),7.41(d,J＝6.6Hz,1H),7.28(m,2H),6.81(dd,J＝17.0,10.3Hz,1H),6.27(d,J＝16.4Hz,1H),5.78(d,J＝11.4Hz,1H),4.96(q,J＝9.0Hz,2H),3.93(s,3H),3.68(m,2H),3.16(m,2H),2.88(s,3H),2.60(t,J＝5.2Hz,3H),2.34(s,3H)。
II.本发明化合物活性测试实施例
测试实施例1：EGFR酪氨酸激酶抑制剂活性检测
ELISA法：酶反应底物多聚谷氨酸:酪氨酸(4:1)包被96孔酶标板，T-PBS(含0.1％吐温-20的磷酸盐缓冲液)洗板5次，于37℃烘箱中干燥酶标版2小时。每孔先加入用反应缓冲液稀释的三磷酸腺苷溶液80μl，然后加入不同浓度梯度的待测化合物(化合物孔)或一定浓度的二甲亚砜溶液(阴性对照孔)10μl，最后加入用反应缓冲液稀释的蛋白酪氨酸激酶溶液10μl以启动反应或10μl的反应缓冲液作为无酶对照孔，置37℃摇床反应1小时，T-PBS洗板5次。加入抗磷酸化酪氨酸的单抗(PY99)100μl/孔，25℃摇床反应1小时，T-PBS洗板5次。加入辣根过氧化物酶标记的羊抗鼠IgG 100μl/孔，25℃摇床反应1小时，T-PBS洗板5次。加入2mg/ml的邻苯二胺OPD显色液，25℃避光反应10min。加入2M硫酸溶液50μl/孔中止反应，用可调波长式微孔板酶标仪Synergy H4读数，波长490nm。并进行数据整理计算抑制率，根据各浓度抑制率，通过非线性回归方法计算出各测试化合物对EGFR L858R、EGFR L858R/T790M和HER2的IC 50值。
Figure PCTCN2019101969-appb-000024
注：AZD9291购自上海岱岩化工有限公司
测试结果表明：实施例2化合物盐对HER2、EGFR突变(如EGFR L858R、EGFR L858R/T790M等)具有良好的抑制活性。
测试实施例2：对EGFR敏感突变的人肺癌细胞PC-9(EGFR ex19del)、HCC827(EGFR E746_A750del)以及耐药突变的人肺癌细胞H1975(EGFR L858R/T790M)的增值抑制作用
磺酰罗丹明B蛋白染色法(SRB法)：取处于对数生长期的细胞接种在96孔板中(细胞浓度为5000个/孔；细胞悬液180μl/孔)，37℃、5％CO 2培养24H使细胞贴壁。将受试药物事先溶解在二甲亚砜中配制成10mM储存液，当检测时稀释到目的浓度的10倍，然后在接种细胞的96孔板中加入化合物20μl/孔，即到达目的浓度。每个浓度设3个复孔，并设空白对照。继续在37℃、5％CO 2中继续培养72H。终止培养，每孔加入50μl预冷(4℃)的50％三氯乙酸即TCA(终浓度10％)，放置在4℃固定1H，用纯化水洗涤至少5次，空气中自然干燥或60℃烘箱干燥。用含1％冰乙酸的纯化水配制4mg/ml的磺酰罗丹明B即SRB，每孔加入100μl，室温染色1H，弃上清，用1％冰乙酸洗涤至少5次除去非特异结合，干燥待用。每孔加入150μl的10mM的三羟甲基氨基甲烷—盐酸缓冲溶液溶解，在510nm波长处测OD值，并进行数据整理计算抑制率，根据各浓度抑制率，计算半数抑制浓度IC50。
Figure PCTCN2019101969-appb-000025
测试结果表明：实施例2化合物盐对EGFR敏感突变及耐药突变的肿瘤细胞具有很强的增殖抑制作用。
测试实施例3：对人鳞状细胞癌A431、人非小细胞肺癌细胞 HCC827、人肺腺癌H1975裸小鼠皮下移植瘤的生长抑制作用。
观察本发明实施例2化合物盐和AZD9291对人鳞状细胞癌A431、人肺腺癌HCC827、人肺腺癌H1975裸小鼠皮下移植瘤的抑制作用及其相应的安全情况。
肿瘤大小计算公式：肿瘤体积(mm 3)＝0.5×(肿瘤长径×肿瘤短径 2)。
小鼠体重变化率(％)公式：体重变化率＝当天体重/该动物D0体重×100％。
肿瘤生长抑制率TGI％：若肿瘤体积≥D0体积，公式为TGI％＝[1-(肿瘤体积–该肿瘤D0体积)/(当天对照组肿瘤体积–对照组D0肿瘤体积)]×100％；若肿瘤体积<D0体积，公式为TGI％＝(1-(肿瘤体积–该肿瘤D0体积)/该肿瘤D0体积)×100％。
测试实施例3.1：对人鳞状细胞癌A431裸小鼠皮下移植瘤的生长抑制作用。
细胞培养：细胞复苏后，使用含有10％胎牛血清的DMEM培养基，并置于37℃、5％CO 2的恒温培养箱中培养，细胞融合度约80％-90％时(对数生长期)传代扩增，培养至5个T175cm 2培养瓶，收取对数生长期的细胞并计数，以供接种。
实验动物：BALB/c Nude裸鼠，12只，雌性，4-5周，购自上海西普尔－必凯实验动物有限公司。
实验方法：人鳞状细胞癌细胞A431以3*10 6/0.2mL接种于BALB/c Nude小鼠右前背部皮下，定期观察肿瘤生长情况，待肿瘤生长至平均约150-200mm 3时根据肿瘤大小和小鼠体重随机分组。设3个实验组，分别为：0.5％甲基纤维素溶媒对照组，AZD929110mg/kg组，实施例2化合物盐10mg/kg组。分组后各组按照对应给药剂量给药，给药体积为10μL/g，每天一次，连续21天。整个实验过程中，每周测量两次小鼠的体重和肿瘤的大小，观察是否出现毒性反应。
3个实验组小鼠体重变化率见附图1，肿瘤生长抑制率TGI％见附图2。结果表明实施例2化合物盐对动物体重无影响，对人鳞状细胞癌A431裸小鼠皮下移植瘤的生长抑制作用较弱，即本发明化合物具有较好的安全性。
测试实施例3.2：对人非小细胞肺癌HCC827裸小鼠皮下移植瘤的生长抑制作用。
细胞培养：细胞复苏后，使用含有10％胎牛血清的RPMI-1640培养基，并置于37℃、5％CO 2的恒温培养箱中培养，细胞融合度约80％-90％时(对数生长期)传代扩增，培养至24个T175cm 2培养瓶，收取对数生长期的细胞并计数，以供接种。
实验动物：BALB/c Nude裸鼠，32只，雌性，4-5周，购自北京维通利华实验动物技术有限公司。
实验方法：人非小细胞肺癌细胞HCC827以5*10 6/0.1mL接种于BALB/c Nude小鼠右前背部皮下，定期观察肿瘤生长情况，待肿瘤生长至平均约150-200mm 3时根据肿瘤大小和小鼠体重随机分组。设4个实验组，分别为：20％聚乙二醇400+80％生理盐水溶媒对照组，实施例2化合物盐1mg/kg组、3mg/kg组和10mg/kg组。分组后各组按照对应给药剂量给药，给药体积为10μL/g，每天一次，连续21天。整个实验过程中，每周测量两次小鼠的体重和肿瘤的大小，观察是否出现毒性反应。
4组实验组小鼠体重变化率见附图3，肿瘤生长抑制率TGI％见附图4。实验结果表明实施例2化合物盐对人非小细胞肺癌细胞HCC827裸小鼠皮下移植瘤的生长具有较好的抑制作用，对动物体重无明显影响，显示出较好的安全性。
测试实施例3.3：对人肺腺癌H1975裸小鼠皮下移植瘤的生长抑制作用。
细胞培养：细胞复苏后，使用含有10％胎牛血清的RPMI-1640培养基，并置于37℃、5％CO 2的恒温培养箱中培养，细胞融合度约80％-90％时(对数生长期)传代扩增，培养至27个T175cm 2培养瓶，收取对数生长期的细胞并计数，以供接种。
实验动物：BALB/c Nude裸鼠，32只，雌性，4-5周，购自北京维通利华实验动物技术有限公司。
实验方法：人肺腺癌细胞H1975以5*10 6/0.1mL接种于BALB/c Nude小鼠右前背部皮下，定期观察肿瘤生长情况，待肿瘤生长至平均约150-200mm 3时根据肿瘤大小和小鼠体重随机分组。设4个实验组，分别为：20％聚乙二醇400+80％生理盐水溶媒对照组，实施例2化合物盐3mg/kg组、10mg/kg组和30mg/kg组。分组后各组按照对应给药剂量给药，给药体积为10μL/g，每天一次，连续21天。整个实验过 程中，每周测量两次小鼠的体重和肿瘤的大小，观察是否出现毒性反应。
4组实验组小鼠体重变化率见附图5，肿瘤生长抑制率TGI％见附图6。实验结果表明实施例2化合物盐对人肺腺癌H1975裸小鼠皮下移植瘤的生长具有较好的抑制作用，对动物体重无明显影响，显示出较好的安全性。
结合测试实施例3.1、测试实施例3.2、测试实施例3.3，表明实施例2化合物盐对人非小细胞肺癌HCC827、人肺腺癌H1975移植瘤的生长具有良好的抑制作用，而对野生型EGFR人鳞状细胞癌A431移植瘤的抑制作用相对较弱，本发明实施例2化合物盐具有良好的选择性，且具有较好安全性。
在本文中提及的所有文献均通过引用被并入本申请中。此外还应指明的是，在阅读了本申请的上述公开内容之后，本领域技术人员可以无需背离本发明的精神和范围，对本发明作出各种修饰、改动或修改，但这些变化形式同样都应落于本申请所附权利要求书所记载的范围。

Claims (11)

  1. 式(I)所示的化合物，或其药学上可接受的盐，
    Figure PCTCN2019101969-appb-100001
    式中：
    环A为杂芳基；
    R 1选自氢、卤素、C 1-C 4烷基或卤代C 1-C 4烷基；
    R 2为卤代C 1-C 4烷基；
    R 3选自氢、C 1-C 4烷基或卤代C 1-C 4烷基；
    R 4选自氢、C 1-C 4烷基或卤代C 1-C 4烷基；
    R 5选自
    Figure PCTCN2019101969-appb-100002
    每个R 6独立的选自氢、卤素、-CN、C 1-C 4烷基或卤代C 1-C 4烷基；
    R 7选自氢、C 1-C 4烷基或卤代C 1-C 4烷基；
    m为1、2或3。
  2. 如权利要求1所述的化合物或其药学上可接受的盐，其特征在于环A为吲哚基、吡咯[2，3-c]骈吡啶基、吡咯[3，2-c]骈吡啶基、吡咯[2，3-b]骈吡啶基、吡咯[3，2-b]骈吡啶基、吡唑基或嘧啶基。
  3. 如权利要求1所述的化合物或其药学上可接受的盐，其特征在于R 1为氢。
  4. 如权利要求1所述的化合物或其药学上可接受的盐，其特征在于R 2为三氟甲基、一氟甲基、二氟甲基或2,2,2-三氟乙基。
  5. 如权利要求1所述的化合物或其药学上可接受的盐，其特征在于R 3为C 1-C 4烷基，R 4为C 1-C 4烷基。
  6. 如权利要求1所述的化合物或其药学上可接受的盐，其特征在于R 5为
    Figure PCTCN2019101969-appb-100003
    R 7为氢。
  7. 如权利要求1-6之任一项所述的化合物或其药学上可接受的盐， 其特征在于每个R 6独立的为卤素或C 1-C 4烷基，m为1、2或3。
  8. 如权利要求1所述的化合物或其药学上可接受的盐，其特征在于所述化合物为N-(2-(甲基(2-(甲基胺基)乙基)胺基)-6-(2，2，2-三氟乙氧基)-5-((4-(1-甲基-1H-吲哚-3-基)嘧啶-2-基)胺基)吡啶-3-基)丙烯酰胺，其结构式如下所示，
    Figure PCTCN2019101969-appb-100004
  9. 如权利要求1所述的化合物或其药学上可接受的盐，其特征在于所述化合物药学上可接受的盐为N-(2-(甲基(2-(甲基胺基)乙基)胺基)-6-(2，2，2-三氟乙氧基)-5-((4-(1-甲基-1H-吲哚-3-基)嘧啶-2-基)胺基)吡啶-3-基)丙烯酰胺甲磺酸盐。
  10. 一种药物组合物，包括权利要求1～9任一项所述的化合物或其药学上可接受的盐，以及药学上可以接受的载体、赋形剂或稀释剂。
  11. 权利要求1～9任一项所述的化合物或其药学上可接受的盐在制备治疗由EGFR激活型或耐药型突变体介导的疾病、特别是癌症的药物中的应用。
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