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WO2020228635A1 - Egfr kinase inhibitor and application thereof in preparing anti-cancer drug - Google Patents

Egfr kinase inhibitor and application thereof in preparing anti-cancer drug Download PDF

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WO2020228635A1
WO2020228635A1 PCT/CN2020/089390 CN2020089390W WO2020228635A1 WO 2020228635 A1 WO2020228635 A1 WO 2020228635A1 CN 2020089390 W CN2020089390 W CN 2020089390W WO 2020228635 A1 WO2020228635 A1 WO 2020228635A1
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substituted
alkyl
group
hydrogen
aromatic
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Chinese (zh)
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金洪传
冯利锋
应士龙
王娴
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Zhejiang University ZJU
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Zhejiang University ZJU
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/70—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings condensed with carbocyclic rings or ring systems
    • C07D239/72—Quinazolines; Hydrogenated quinazolines
    • C07D239/86—Quinazolines; Hydrogenated quinazolines with hetero atoms directly attached in position 4
    • C07D239/94—Nitrogen atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
    • C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
    • C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
    • C07D239/46—Two or more oxygen, sulphur or nitrogen atoms
    • C07D239/48—Two nitrogen atoms
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    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
    • C07D413/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links
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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
    • C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
    • C07D491/04—Ortho-condensed systems
    • C07D491/056—Ortho-condensed systems with two or more oxygen atoms as ring hetero atoms in the oxygen-containing ring
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    • C07D495/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms
    • C07D495/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
    • C07D495/04—Ortho-condensed systems

Definitions

  • the present invention relates to the field of medicine; specifically, the present invention relates to a novel EGFR kinase inhibitor and its application in the treatment of cancer.
  • Lung cancer is the malignant tumor with the highest incidence rate and the largest number of deaths in the world, and it poses a serious threat to human health.
  • Epidermal Growth Factor Receptor (Epidermal Growth Factor Receptor, EGFR, ErbB1) belongs to the ErbB family of receptor tyrosine kinases.
  • EGFR epidermal Growth Factor Receptor
  • L858R gene amplification or activation mutation
  • EGFR overexpression or activating mutations can cause excessive activation of downstream Ras/Raf/MAPK, PI3K/Akt and other cell proliferation and survival signaling pathways.
  • EGFR is considered to be a key oncogene that drives the occurrence and development of lung cancer, pancreatic cancer, breast cancer and other malignant tumors.
  • EGFR tyrosine kinase inhibitors (EGFR TKIs) have significant clinical benefits in the treatment of malignant tumors with abnormal activation of EGFR, and once brought great hope to people.
  • Gefitinib, Erlotinib, and other first-generation EGFR-TKIs inhibit the activation of tumor cell EGFR signaling pathways by reversibly binding to the ATP binding site of EGFR.
  • gefitinib can only maintain an effective remission period of six months to one year, and drug resistance soon develops.
  • the resistance mechanism of gefitinib mainly includes the following aspects: 1 Mutation of EGFR gene: the second mutation of specific EGFR exon 20 causes the threonine at position 790 to be replaced by the larger A Thionine substitution (T790M) prevents the binding of gefitinib and erlotinib to the ATP site; other secondary resistance mutations include L747S, D761Y and T854A; 2changes in EGFR signal transduction pathways, such as Ras activation Causes Raf-MEK-MAPK up-regulation, loss of coupling with EGFR, high expression of ERK and Met and inactivation of PTEN cause excessive activation of the AKT pathway, etc.; EGFR bypass effects: such as platelet-derived growth factor receptor (PDGFR), insulin Growth factor receptors such as growth factor receptor-1 (IGFR-1) receptors directly activate their downstream signaling pathways.
  • PDGFR platelet-derived growth factor receptor
  • IGFR-1 growth factor receptor-1
  • Autophagy is a biological process in which cells maintain their own homeostasis by degrading macromolecular substances and damaged organelles to maintain energy supply and remove harmful substances under stress.
  • a large number of studies have found that the progression of autophagy to malignant tumors is closely related to the development of EGFR TKI-acquired resistance.
  • Autophagy can not only prevent the malignant transformation of normal cells, but also help the cells that have been malignant to resist the unfavorable living environment and promote the development of drug resistance.
  • SQSTM1 Sequestosome-1, also known as p62
  • SQSTM1 As a key ligand protein SQSTM1 (Sequestosome-1, also known as p62) that mediates multiple signaling pathways such as autophagy, energy induction, and oxidative stress, it plays a very important role in tumor development and drug resistance. The role of. As a ligand protein, SQSTM1 plays an important role in regulating multiple cancer-promoting signal pathways. It not only mediates the activation of mTORC1 in the lysosome, but also dissociates the interaction with Nrf2 through direct action with keap1, thereby Inhibit keap1 mediates the ubiquitination and degradation of Nrf2.
  • SQSTM1 The increased protein level of SQSTM1 not only means that autophagy-related degradation pathways are weakened, but also can help tumor cells resist apoptosis. Studies have found that the expression of SQSTM1 in platinum drug-resistant cells is up-regulated. On the contrary, reducing the protein level of SQSTM1 can effectively improve the sensitivity of tumors to drugs. In addition, SQSTM1 can shuttle between plasma and nuclei to participate in the repair of DNA damage caused by radiotherapy and chemotherapy. It also proves that SQSTM1 is a key regulator that promotes tumor resistance. However, it is still unclear what role SQSTM1 plays in the development of EGFR TKI resistance.
  • the purpose of the present invention is to provide a new type of EGFR kinase inhibitor with strong anti-cancer effect without affecting the autophagy process and lysosomal function and its preparation, prevention and/or treatment related to receptor tyrosine protein kinase
  • the application of medicines for various diseases and conditions for example, cancer, especially non-small cell lung cancer.
  • the present invention provides a compound represented by formula I and a pharmaceutically acceptable salt thereof,
  • Ring A is a 5- to 18-membered ring, or, Ring A does not exist;
  • X is -O-, -S-, -N(R 4 )- or -(CHR 4 ) n -, n is 0 or 1;
  • R 1 is selected from: aryl, aralkyl, aromatic or non-aromatic heterocyclic group and heterocycloalkyl;
  • n is an integer of 0-3 (specifically 0, 1, 2, 3);
  • t 1, 2 or 3;
  • R 4 and R 5 are each independently selected from: hydrogen, alkyl, cycloalkyl, alkenyl, aryl, aromatic or non-aromatic heterocyclic group, alkoxy, aryloxy and halogen;
  • the structure of the compound represented by formula I contains at least one acidic substituent, and the acidic substituent contains at least one acidic functional group.
  • the acidic functional group is selected from one or more of phenolic hydroxyl group, carboxyl group, sulfonic acid group and phosphoric acid group.
  • the acidic substituent is selected from the group consisting of: hydroxyphenyl, carboxyphenyl, sulfophenyl, phosphate, hydroxybenzoyloxy substituted alkyl, sulfonate substituted alkyl , Carboxyl substituted alkyl, phosphate substituted alkyl, sulfonate phenyl substituted alkyl, carboxyphenyl substituted alkyl, phosphate phenyl substituted alkyl, hydroxybenzoyloxy substituted alkoxy, sulfonic acid group One or more of substituted alkoxy, carboxy substituted alkoxy, phosphate substituted alkoxy, sulfonate phenyl substituted alkoxy, carboxyphenyl substituted alkoxy, and phosphate phenyl substituted alkoxy .
  • the hydroxyl group in the hydroxyphenyl group, is located at least one of the 2-5 positions of the phenyl group.
  • the carboxy group in the carboxyphenyl group, is located at least one of the 2-5 positions of the phenyl group.
  • the sulfonic acid group is located at least one of the 2-5 positions of the phenyl group.
  • the phosphate group in the phosphate phenyl group, is located at least one of the 2-5 positions of the phenyl group.
  • the hydroxybenzoyloxy group is a dihydroxybenzoyloxy group (for example, 2,3-dihydroxybenzoyloxy, 2,4-dihydroxybenzoyloxy, 2,5-Dihydroxybenzoyloxy, 2,6-dihydroxybenzoyloxy, 3,4-dihydroxybenzoyloxy, 3,5-dihydroxybenzoyloxy, 3, 6-dihydroxybenzoyloxy, 4,5-dihydroxybenzoyloxy, 4,6-dihydroxybenzoyloxy or 5,6-dihydroxybenzoyloxy) or trihydroxybenzene
  • Formyloxy for example, 2,3,4-trihydroxybenzoyloxy, 2,3,5-trihydroxybenzoyloxy, 2,3,6-trihydroxybenzoyloxy, 2 ,4,5-trihydroxybenzoyloxy, 2,4,6-trihydroxybenzoyloxy, 2,5,6-trihydroxybenzoyloxy, 3,4,5-trihydroxybenzene Formyloxy, 3,4,6-tri-trihydroxybenzoy
  • the alkyl group is a C1-C6 alkyl group, preferably a C1-C3 alkyl group.
  • the alkoxy group is a C1-C6 alkoxy group, preferably a C1-C3 alkoxy group.
  • the structure of the compound represented by formula I contains at least one substituent with the following structure:
  • X is -O-.
  • X is -NH-.
  • X is -(CHR 4 ) n -, and n is 0, that is, X does not exist.
  • R 1 is a substituted aryl group, such as a substituted phenyl group.
  • R 1 is a substituted heterocyclic group, such as substituted indolyl (eg 3-indolyl).
  • n 2
  • n is zero.
  • the A ring is an aromatic ring, preferably a benzene ring, which forms a quinazoline ring structure with the connected pyrimidine ring.
  • ring A is a heteroaromatic ring, preferably a single heteroatom five-membered aromatic ring, for example, a pyrrole ring, a furan ring or a thiophene ring, which is fused with a pyrimidine ring to form a five-membered single heteroaromatic ring Ring [3,2-d] and pyrimidine ring structure.
  • a heteroaromatic ring preferably a single heteroatom five-membered aromatic ring, for example, a pyrrole ring, a furan ring or a thiophene ring, which is fused with a pyrimidine ring to form a five-membered single heteroaromatic ring Ring [3,2-d] and pyrimidine ring structure.
  • the A ring is absent.
  • ring A is an aromatic ring
  • R 1 is a substituted phenyl group
  • the compound has the structure shown in formula II:
  • X is -O-, -S-, -N(R 17 )- or -(CHR 17 ) n -, and n is 0 or 1;
  • t 1, 2 or 3;
  • R 17 and R 18 are independently selected from: hydrogen, alkyl, cycloalkyl, alkenyl, aryl, aromatic or non-aromatic heterocyclic group, alkoxy, aryloxy, halogen and amino;
  • R 19 is an alkylene group
  • the structure of the compound represented by formula II contains at least one acidic substituent, and the acidic substituent contains at least one acidic functional group.
  • the acidic functional group and acidic substituent have the above-mentioned definition of the present invention.
  • At least one of R 11 , R 12 , R 13 , R 14 , R 15 and R 16 contains an acidic functional group.
  • R 15 contains at least one acidic functional group.
  • R 15 is selected from the group consisting of: hydroxyphenyl, carboxyphenyl, sulfonate phenyl, phosphate, hydroxybenzoyloxy substituted alkyl, sulfonate substituted alkyl , Carboxyl substituted alkyl, phosphate substituted alkyl, sulfonate phenyl substituted alkyl, carboxyphenyl substituted alkyl, phosphate phenyl substituted alkyl, hydroxybenzoyloxy substituted alkoxy, sulfonic acid group Substituted alkoxy, carboxyl substituted alkoxy, phosphoric acid substituted alkoxy, sulfonic phenyl substituted alkoxy, carboxyphenyl substituted alkoxy, and phosphoric phenyl substituted alkoxy.
  • R 15 is
  • X is -NH-.
  • R 11 , R 12 and R 13 are independently selected from: hydrogen, halogen (such as F, Cl), -NO 2 , alkenyl, alkynyl, and -O(CH 2 ) x Ar , X is 0 or 1, and Ar is an aryl group (for example, a phenyl group) or a heterocyclic group (for example, a nitrogen-containing heterocyclic group).
  • R 11 is F
  • R 12 is Cl
  • R 13 is hydrogen
  • R 11 is hydrogen
  • R 12 is Cl
  • R 13 is F
  • R 11 is hydrogen
  • R 13 is hydrogen
  • R 12 is selected from halogen, vinyl and ethynyl.
  • R 14 is selected from: hydrogen, alkyl, -CN and halogen.
  • R 14 is hydrogen
  • R 16 is -OR 19 -R 18 , wherein R 19 is methylene, ethylene, propylene or butylene, and R 18 is selected from: aromatic or non-aromatic hetero Cyclic groups (such as morpholinyl, piperazinyl, piperidinyl, pyrrolidinyl, etc.), alkoxy (such as methoxy, ethoxy, propoxy), aryloxy (such as phenoxy) and Substituted amino (eg methylamino, dimethylamino).
  • aromatic or non-aromatic hetero Cyclic groups such as morpholinyl, piperazinyl, piperidinyl, pyrrolidinyl, etc.
  • alkoxy such as methoxy, ethoxy, propoxy
  • aryloxy such as phenoxy
  • Substituted amino eg methylamino, dimethylamino
  • R 16 is -OR 19 -R 18 , wherein R 19 is ethylene or propylene, and R 18 is selected from: morpholinyl, piperazinyl, dimethylamino, pyrrole Alkyl, piperidinyl, methoxy and ethoxy.
  • R 16 may be selected from: 3-(4-morpholinyl)propoxy, 3-(1-piperazinyl)propoxy, 3,3-dimethylaminopropoxy, 3-(1 -Pyrrolidinyl)propoxy, 3-(1-piperidinyl)propoxy, 3-(4-piperidinyl)propoxy, methoxyethoxy and ethoxyethoxy.
  • R 16 is
  • R 16 is -NHC(O)R 18 , wherein R 18 is selected from: substituted or unsubstituted alkenyl (such as vinyl, 1-propenyl, 1-butenyl),
  • the substituents on the alkenyl group can be selected from: aromatic or non-aromatic heterocyclic groups (such as pyrrolidinyl, piperidinyl), alkoxy (such as methoxy, ethoxy) and substituted amino (such as Methylamino, dimethylamino).
  • R 16 is selected from: 4-methoxy-2-butenamido, 4-pyrrolidin-2-butenamido, 4-dimethylamino-2-butene Enamido and 4-piperidinyl-2-butenamido.
  • the compound has the following structure:
  • ring A is a single heteroatom five-membered aromatic ring
  • R 1 is a substituted phenyl group
  • the compound has the structure shown in formula III:
  • X is -O-, -S-, -N(R 29 )- or -(CHR 29 ) n -, n is 0 or 1;
  • Y is -O-, -S- or -N(R 29 )-;
  • Z is -O-, -S- or -N(R 29 )-;
  • t 1, 2 or 3;
  • R 29 and R 210 are independently selected from: hydrogen, alkyl, cycloalkyl, alkenyl, aryl, aromatic or non-aromatic heterocyclic group, alkoxy, aryloxy, halogen and amino;
  • the structure of the compound represented by formula III contains at least one acidic substituent, and the acidic substituent contains at least one acidic functional group.
  • the acidic functional group and acidic substituent have the above-mentioned definition of the present invention.
  • At least one of R 21 , R 22 , R 23 , R 24 , R 25 , R 26 , R 27 and R 28 contains an acidic functional group.
  • R 26 contains at least one acidic functional group.
  • R 26 is selected from the group consisting of: hydroxyphenyl, carboxyphenyl, sulfonate phenyl, phosphate phenyl, hydroxybenzoyloxy substituted alkyl, sulfonate substituted alkyl , Carboxyl substituted alkyl, phosphate substituted alkyl, sulfonate phenyl substituted alkyl, carboxyphenyl substituted alkyl, phosphate phenyl substituted alkyl, hydroxybenzoyloxy substituted alkoxy, sulfonic acid group Substituted alkoxy, carboxyl substituted alkoxy, phosphoric acid substituted alkoxy, sulfonic phenyl substituted alkoxy, carboxyphenyl substituted alkoxy, and phosphoric phenyl substituted alkoxy.
  • R 26 is
  • X is -NH-.
  • X is -O-.
  • Y is -S-.
  • Z is -NH-.
  • R 21 and R 23 are independently selected from: hydrogen, alkyl and halogen.
  • R 21 is hydrogen
  • R 23 is hydrogen
  • R 22 is -NHC(O)R 210 , wherein R 210 is a substituted or unsubstituted alkenyl group, such as vinyl and 3-propenyl. Specifically, R 22 can be
  • R 24 is a heterocyclic group, such as a substituted or unsubstituted nitrogen-containing non-aromatic heterocyclic group (specifically, piperazinyl, piperidinyl), and the substituents on it are selected from: alkane Group (such as methyl, ethyl), acyl (such as acetyl, propionyl), heterocyclic group (such as pyrrolidinyl, piperidinyl, piperazinyl, etc.), the substitution site is a nitrogen-containing non-aromatic hetero The nitrogen position of the ring group.
  • alkane Group such as methyl, ethyl
  • acyl such as acetyl, propionyl
  • heterocyclic group such as pyrrolidinyl, piperidinyl, piperazinyl, etc.
  • R 24 is selected from: 4-(1-acetyl)piperazinyl, 4-(1-methyl)piperazinyl, 4-(1-pyrrolidinyl)piperazine Group, 4-(1-piperidinyl)piperazinyl and 4-methylpiperazinyl-1-piperidinyl.
  • R 24 is
  • R 24 is -NR 27 R 28 , R 27 and R 28 are independently selected from: hydrogen and substituted or unsubstituted alkyl (such as methyl, 3-(methylamino)propyl base).
  • R 24 is N,N-dimethylamino or N 1 ,N 1 ,N 2 -trimethylethylenediamino.
  • R 25 is hydrogen
  • R 27 and R 28 are independently selected from: hydrogen, alkyl, and halogen.
  • R 27 is hydrogen
  • R 28 is hydrogen
  • the compound has the following structure:
  • X is -O-, -S-, -N(R 39 )- or -(CHR 39 ) n -, n is 0 or 1;
  • Z is -O-, -S- or -N(R 39 )-;
  • R 31 is hydrogen, substituted or unsubstituted aryl or heterocyclic group
  • t 1, 2 or 3;
  • R 39 and R 310 are independently selected from: hydrogen, alkyl, cycloalkyl, alkenyl, aryl, aromatic or non-aromatic heterocyclic group, alkoxy, aryloxy, halogen and amino;
  • the structure of the compound represented by formula IV contains at least one acidic substituent, and the acidic substituent contains at least one acidic functional group.
  • the acidic functional group and acidic substituent have the above-mentioned definition of the present invention.
  • At least one of R 31 , R 32 , R 33 , R 34 , R 35 , R 36 , R 37 and R 38 contains an acidic functional group.
  • R 36 contains at least one acidic functional group.
  • R 36 is selected from: hydroxyphenyl, carboxyphenyl, sulfonate phenyl, phosphate phenyl, hydroxybenzoyloxy substituted alkyl, sulfonate substituted alkyl , Carboxyl substituted alkyl, phosphate substituted alkyl, sulfonate phenyl substituted alkyl, carboxyphenyl substituted alkyl, phosphate phenyl substituted alkyl, hydroxybenzoyloxy substituted alkoxy, sulfonic acid group Substituted alkoxy, carboxyl substituted alkoxy, phosphoric acid substituted alkoxy, sulfonic phenyl substituted alkoxy, carboxyphenyl substituted alkoxy, and phosphoric phenyl substituted alkoxy.
  • R 36 is
  • X is -(CHR 39 ) n -, and n is 0, that is, X does not exist.
  • Z is -NH-.
  • R 31 is a substituted aryl group, such as a substituted phenyl group, such as Among them, R 310 is a substituted or unsubstituted alkenyl group, such as vinyl, 3-propenyl, and the substituent on the alkenyl group is selected from: alkoxy (such as methoxy, ethoxy), heterocyclic group (such as Piperidinyl, pyrrolidinyl) and substituted amino (such as methylamino, dimethylamino).
  • alkoxy such as methoxy, ethoxy
  • heterocyclic group such as Piperidinyl, pyrrolidinyl
  • substituted amino such as methylamino, dimethylamino
  • R 31 is selected from: 3-acrylamidophenyl, 3-(4-methoxy-2-butenamido)phenyl, 3-(4-piperidinyl -2-butenamido)phenyl, 3-(4-pyrrolidinyl-2-butenamido)phenyl, 3-(4-dimethylamino-2-butenamido)phenyl.
  • R 31 is a substituted or unsubstituted heterocyclic group, such as pyrazole [1,5-a] pyridyl or substituted indolyl, and the substituent of indolyl is selected from : Hydrogen, alkyl and acyl.
  • R 31 is
  • R 32 is hydrogen
  • R 33 is Wherein X 3 is -NH- or -O-, R 39 is substituted or unsubstituted alkenyl, such as vinyl, 3-propenyl, and the substituents on the alkenyl are selected from: alkoxy (such as methoxy, Ethoxy), heterocyclic groups (such as piperidinyl, pyrrolidinyl), and substituted amino groups (such as methylamino, dimethylamino).
  • alkoxy such as methoxy, Ethoxy
  • heterocyclic groups such as piperidinyl, pyrrolidinyl
  • substituted amino groups such as methylamino, dimethylamino
  • R 33 is selected from: acryloxy group, acrylamido group, 4-methoxy-2-butenoyloxy group, 4-methoxy-2-butenamido group , 4-piperidinyl-2-butenoyloxy, 4-piperidinyl-2-butenamido, 4-pyrrolidinyl-2-butenoyloxy, 4-pyrrolidinyl-2- Butenamido, 4-dimethylamino-2-butenoyloxy and 4-dimethylamino-2-butenamido.
  • R 33 is
  • R 35 is hydrogen
  • R 34 is a substituted or unsubstituted heterocyclic group, such as a substituted nitrogen-containing non-aromatic heterocyclic group (specifically, piperazinyl, piperidinyl), and the substituents on it are selected from : Alkyl (such as methyl, ethyl), acyl (such as acetyl, propionyl), heterocyclic group (such as pyrrolidinyl, piperidinyl, piperazinyl, etc.), the substitution site is a nitrogen-containing non The nitrogen position of the aromatic heterocyclic group.
  • a substituted nitrogen-containing non-aromatic heterocyclic group specifically, piperazinyl, piperidinyl
  • the substituents on it are selected from : Alkyl (such as methyl, ethyl), acyl (such as acetyl, propionyl), heterocyclic group (such as pyrrolidinyl, piperidinyl, piperazinyl, etc.)
  • R 34 is selected from: 4-(1-acetyl)piperazinyl, 4-(1-methyl)piperazinyl, 4-(1-pyrrolidinyl)piperazine Group, 4-(1-piperidinyl)piperazinyl and 4-methylpiperazinyl-1-piperidinyl.
  • R 34 is -NR 27 R 28 , R 27 and R 28 are independently selected from: hydrogen and substituted or unsubstituted alkyl (such as methyl, 3-(methylamino)propyl) base).
  • R 34 is N,N-dimethylamino or N 1 ,N 1 ,N 2 -trimethylethylenediamino.
  • R 34 is
  • R 37 and R 38 are independently selected from: hydrogen, halogen, nitro, cyano and trifluoromethyl.
  • R 37 is hydrogen
  • R 38 is hydrogen
  • the compound has the following structure:
  • the present invention also provides a method for preparing the above compound, which includes the step of introducing at least one acidic functional group into the structure of a compound having EGFR inhibitory activity (for example, a known EGFR TKI compound) by reaction.
  • a compound having EGFR inhibitory activity for example, a known EGFR TKI compound
  • the acidic functional group is selected from one or more of phenolic hydroxyl group, carboxyl group, sulfonic acid group and phosphoric acid group.
  • the above preparation method includes introducing one or more groups selected from the group consisting of: hydroxyphenyl, carboxyphenyl, sulfophenyl, phosphorophenyl, hydroxybenzoyl Oxygen substituted alkyl, sulfonic acid substituted alkyl, carboxy substituted alkyl, phosphate substituted alkyl, sulfonic phenyl substituted alkyl, carboxyphenyl substituted alkyl, phosphate phenyl substituted alkyl, hydroxybenzene Formyloxy substituted alkoxy, sulfonic acid substituted alkoxy, carboxy substituted alkoxy, phosphoric acid substituted alkoxy, sulfonic phenyl substituted alkoxy, carboxyphenyl substituted alkoxy and phosphoric acid Phenyl replaces alkoxy.
  • the EGFR TKI compound in the above preparation method, is gefitinib.
  • the above preparation method includes the following reaction steps:
  • Ra is a halogen atom
  • Rp is a hydroxyl protecting group
  • the second step is the hydroxyl deprotection reaction.
  • the above preparation method includes the following reaction steps:
  • X, Y, Z, R 21 , R 22 , R 23 , R 24 , R 25 , R 27 , R 28 have the above definitions of the present invention
  • Ra is a halogen atom
  • Rp is a hydroxyl protecting group
  • the second step is the hydroxyl deprotection reaction.
  • the above preparation method includes the following reaction steps:
  • Ra is a halogen atom
  • Rp is a hydroxyl protecting group
  • the second step is the hydroxyl deprotection reaction.
  • the hydroxyl protecting group and the hydroxyl deprotection reaction can be selected from the conventional hydroxyl protecting group and hydroxyl deprotection reaction in the art, which is not specifically limited in the present invention.
  • the present invention also provides a method for transforming EGFR TKI, which includes the step of reducing the basicity of EGFR TKI, for example, the step of introducing at least one acidic functional group into the structure of the EGFR TKI.
  • the acidic functional group has the above definition of the present invention.
  • the present invention also provides stereoisomers, geometric isomers, tautomers, racemates, solvates, hydrates, metabolic precursors, and prodrugs of the above-mentioned compound or a pharmaceutically acceptable salt thereof.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the above compound or a pharmaceutically acceptable salt, stereoisomer, geometric isomer, tautomer, racemate, solvate, hydrate, metabolite Precursors or prodrugs, and one or more pharmaceutically acceptable excipients.
  • the pharmaceutically acceptable carrier refers to a conventional drug carrier in the pharmaceutical field.
  • diluents excipients such as water, fillers such as starch, sucrose, etc.; binders such as cellulose derivatives, alginate, gelatin and polyvinylpyrrolidone, etc.; wetting agents such as glycerin, etc.; disintegrating agents such as agar , Calcium carbonate and sodium bicarbonate, etc.; absorption enhancers such as quaternary ammonium compounds, etc.; surfactants such as cetyl alcohol, etc.; adsorption carriers such as kaolin and bentonite, etc.; lubricants such as talc, calcium and magnesium stearate, Polyethylene glycol and so on.
  • other adjuvants such as flavoring agents and sweetening agents can also be added to the composition.
  • the pharmaceutical composition of the present invention can be tablets (including sugar-coated tablets, film-coated tablets, sublingual tablets, orally disintegrating tablets, buccal tablets, etc.), pills, powders, granules, capsules (Including soft capsules, microcapsules), lozenges, syrups, liquids, emulsions, suspensions, controlled release formulations (e.g., instant release formulations, sustained release formulations, sustained release microcapsules), aerosols, films ( For example, oral disintegrating film, oral mucosa-adhesive film), injections (for example, subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection), intravenous drip, transdermal absorption preparation, ointment, lotion Preparations, adhesive preparations, suppositories (for example, rectal suppositories, vaginal suppositories), pellets, nasal preparations, lung preparations (inhalants), eye drops, etc., oral or parenteral preparations (for example, intrave
  • compositions of the present invention can be prepared according to conventional production methods in the pharmaceutical field.
  • the active ingredient is mixed with one or more excipients and then made into the desired dosage form.
  • the pharmaceutical composition of the present invention preferably contains the active ingredient in a weight ratio of 0.1-99.5 %, and more preferably contains the active ingredient in a weight ratio of 0.5-95%.
  • the dosage of the compound of the present invention may vary according to the route of administration, the age, weight of the patient, the type and severity of the disease to be treated, etc., and may be administered one or more times.
  • the present invention also provides a compound or a pharmaceutically acceptable salt, stereoisomer, geometric isomer, tautomer, racemate, solvate, hydrate, metabolic precursor and prodrug thereof Application in preparing medicines for preventing and/or treating diseases and disorders related to receptor tyrosine protein kinases.
  • the present invention also provides a method for preventing and/or treating diseases and disorders related to receptor tyrosine protein kinase, which comprises combining the compound of the present invention or a pharmaceutically acceptable salt, stereoisomer, geometric Isomers, tautomers, racemates, solvates, hydrates, metabolic precursors and prodrugs, or steps of administering the pharmaceutical composition to an individual in need thereof.
  • the diseases and disorders related to receptor tyrosine protein kinases are tumors, which include, but are not limited to: lung cancer, pancreatic cancer, breast cancer or colon cancer, particularly lung cancer, and more particularly non-small cell lung cancer.
  • the inventors of the present invention firstly discovered that the anti-tumor effect of EGFR TKIs is closely related to its own acidity and alkalinity.
  • the EGFR TKIs currently used in clinical practice can cause tumor cell lysis
  • the acidification process of the body is weakened, which in turn causes the accumulation of the oncoprotein SQSTM1, and ultimately leads to the generation of tumor-acquired drug resistance.
  • the inventors developed a series of EGFR TKI compounds by reducing the basicity of EGFR TKI, which can effectively improve the acquired drug resistance of EGFR TKI caused by the accumulation of SQSTM1, which is of great significance for improving the clinical treatment benefits of EGFR TKI.
  • Figure 1 shows the measurement results of SQSTM1 expression in A549, HCC827 and H460 cells treated with gefitinib in Example 1.
  • Figure 2 shows the measurement results of SQSTM1 expression in A549, HCC827 and H460 cells treated with AZD9291 in Example 1.
  • Figure 3 shows the measurement results of LC3 and SQSTM1 expression in A549 and H460 cells in Example 2.
  • Fig. 4 shows the detection result of Example 3;
  • Fig. 4A is a photo of GFP-LC3 and RFP-LC3 spots detected by immunofluorescence microscope.
  • the spots in the first column are all green fluorescent spots, and the spots in the second column are They are all red fluorescent spots, and the spots in the third column are all yellow fluorescent spots;
  • Figure 4B shows the results of LC3 yellow spots/red spots (%) in each experimental group.
  • Figure 5 shows the test results of Example 4; among them, the first column shows the transient transduction of the SQSTM1-Flag plasmid in A549 cells, followed by screening with antibiotic G418 to obtain stable transfected monoclonal cell clusters, using different concentrations Gefitinib and AZD9291 were treated for 48 hours, and the cell viability was detected by MTS method.
  • the second column of graphs shows the results of cell viability detected by MTS method after 48 hours of knockdown of SQSTM1 in H460 cells, and treatment with different concentrations of Gefitinib and AZD9291 for 24 hours.
  • Figure 6 shows the detection results of Example 5;
  • Figure 6A shows the results of immunofluorescence detection of LC3 protein expression changes after treating A549 with gefitinib and AZD9291 for different times;
  • Figure 6B shows the statistics of the results in Figure 6A In analysis, 10 random fields of view were counted, the scale is 10 ⁇ m, and the asterisk (*) represents P ⁇ 0.05, which means there is a statistical difference.
  • Figure 7 shows the detection results of Example 6; among them, Figure 7A is the immunofluorescence detection photos of LAMP1 and LC3, and Figure 7B shows the statistical results of the colocalization coefficients of each experimental group.
  • Figure 8 shows the results of determination of CTSB expression in mature body CTSB in A549 and HCC827 cells after treatment with gefitinib, AZD9291, CQ and CQ in combination with Baf A1 in Example 7.
  • Figure 9 shows the detection results of Example 8; among them, Figure 9A is a fluorescence microscope examination photo, in which the scale bar is 10 ⁇ M, the spots in the first and third rows are all red fluorescent spots, and in the second and fourth rows The spots are all green fluorescent spots, and Figure 9B shows the detection results of AO-red spots in each experimental group.
  • Figure 10 shows the synthesis route of compound II-1 (Gefi-2OH).
  • pKa basic
  • pKa 1 acidic
  • pKa 2 acidic
  • Figure 11 shows the synthetic route of compound III-1.
  • Figure 12 shows the synthetic route of compound IV-1 (AZD-2OH).
  • Figure 13 shows the test results of Example 13.
  • Figure 13A shows the results of determination of the expression levels of SQSTM1 and mature CTSB after treatment with Gefitinib and compound Gefi-2OH in H460 cells.
  • Figure 13B shows the results of EGFR treatment in A549 and HCC827 cells after treatment with Gefitinib and compound Gefi-2OH. And its downstream Akt, Erk1/2 protein phosphorylation modification degree and the detection results of the protein itself.
  • Figure 14 also shows the test results of Example 13. Among them, Figure 14A shows the results of determination of the expression levels of SQSTM1 and mature CTSB after treatment with AZD9291 and compound AZD-2OH in H460 cells, and Figure 14B shows EGFR and its downstream Akt and Erk1 after treatment with AZD9291 and compound AZD-2OH in HCC827 cells. /2 The degree of phosphorylation modification of the protein and the detection result of its protein expression.
  • Figure 15 shows the test results of Example 14.
  • the left side is the fluorescence microscope inspection photo, where the scale bar is 10 ⁇ M
  • the spots in the picture are all blue fluorescent spots formed by Lysotracker-blue
  • the right side shows the acidic vesicle spots in each experimental group (the number of blue spots) The test results.
  • Figure 16 shows the test results of Example 15, which are the test results of the proliferation inhibitory effects of Gefitinib and the compound Gefi-2OH on A549 (left) and H460 cells (right), respectively.
  • Figure 17 shows the test result of Example 15, which is the test result of the inhibitory effect of AZD9291 and compound AZD-2OH on the proliferation of H460 cells.
  • Figure 18 shows the detection results of Example 16; wherein, the left side is a flow cytometry detection result graph, and the right side shows the results of the apoptosis rate of each experimental group.
  • Halogen means fluorine, chlorine, bromine or iodine, preferably fluorine or chlorine.
  • Alkyl refers to straight or branched hydrocarbon chain radicals without unsaturated bonds. Typical alkyl groups contain 1-12, 1-8 or 1-6 carbon atoms, such as methyl, Ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, n-pentyl, etc. If the alkyl group is substituted with an aryl group, then the corresponding "aralkyl” radical, such as benzyl or phenethyl. If the alkyl group is substituted by a heterocyclic group, then it corresponds to a "heterocycloalkyl" radical.
  • alkenyl refers to a straight or branched hydrocarbon chain radical containing at least two carbon atoms and at least one unsaturated bond, and the hydrocarbon chain radical is connected to other parts of the molecule by a single bond.
  • Typical alkenyl groups contain 2-12, 2-8, or 2-6 carbon atoms, for example, vinyl, 1-propenyl or butenyl.
  • Alkynyl refers to a straight or branched hydrocarbon chain radical containing at least two carbon atoms and at least one carbon-carbon triple bond, and the hydrocarbon chain radical is connected to other parts of the molecule by a single bond.
  • Typical alkynyl groups contain 2-12, 2-8, or 2-6 carbon atoms, for example, ethynyl, propynyl (eg 1-propynyl, 2-propynyl).
  • Cycloalkyl refers to an alicyclic hydrocarbon.
  • a typical cycloalkyl group contains 1 to 3 monocyclic and/or fused rings, 3-18 carbon atoms, preferably 3-10 carbon atoms, such as cyclopropyl, cyclohexyl or adamantyl.
  • the cycloalkyl group contains 3-6 carbon atoms.
  • Aryl refers to monocyclic or polycyclic radicals, including polycyclic radicals containing monoaryl groups and/or condensed aryl groups. Typical aryl groups contain 1-3 monocyclic or fused rings and 6-18 carbon ring atoms, such as phenyl, naphthyl (such as 2-naphthyl) radicals.
  • Heterocyclyl refers to a stable, typically 3- to 18-membered cyclic radical consisting of carbon atoms and one to five heteroatoms selected from nitrogen, oxygen and sulfur, preferably with one or more A 4- to 8-membered ring with three heteroatoms, more preferably a 5- or 6-membered ring with one or more heteroatoms, which may be aromatic or non-aromatic.
  • the heterocyclic ring may be a monocyclic, bicyclic or tricyclic ring system, which may include a fused ring system; and the nitrogen, carbon or sulfur atoms in the heterocyclic radical may be optionally oxidized; the nitrogen atom may be any It is optionally quaternized; and the heterocyclic radical can be partially saturated or fully saturated or aromatic.
  • heterocyclic groups include, but are not limited to: benzimidazole, benzothiazole, furan, isothiazole, imidazole, indole, piperidine, piperazine, purine, quinoline, thiadiazole, tetrahydrofuran, coumarin, pyridine Pyroline, pyrrole, pyrazole, oxazole, isoxazole, triazole, imidazole, etc.
  • Alkoxy refers to a radical of formula -OR a, wherein R a is an alkyl radical as defined above having one or more (e.g. 2, 3 or 4) oxygen bond, and generally contain from 1 -12, 1-8 or 1-6 carbon atoms, such as methoxy, ethoxy, propoxy, etc.
  • Aryloxy refers to a radical of the formula -O-aryl, where aryl is as defined above. Some examples of aryloxy compounds are -O-phenyl, -O-p-tolyl, -O-m-tolyl, -O-o-tolyl, or -O-naphthyl.
  • A549 wild-type EGFR
  • HCC827 exon 19 deleted EGFR
  • NCI-H460 H460, wild-type EGFR
  • RPMI1640 medium, DMEM medium and fetal bovine serum (FBS) were all purchased from Hangzhou Sijiqing Co., Ltd. (Hangzhou, China);
  • Rapamycin purchased from Beyotime (Shanghai, China);
  • CQ Chloroquine
  • AO Acridine Orange
  • BCA protein determination kit purchased from Bio-Rad Laboratories (California, USA);
  • Horseradish peroxidase conjugated secondary antibody purchased from Jackson ImmunoResearch (Shanghai, China);
  • Chemiluminescence detection kit purchased from Biological Industries (USA);
  • Amersham Imager 600 system purchased from the Life Science Department of General Electric Medical Group (Shanghai, China);
  • siRNA transfection reagent Lipofectamine TM RNAiMAX (Invivogen, USA);
  • Plasmid transfection reagent X-tremeGENE HP DNA Transfection Reagent (Roche, Switzerland);
  • BD Pharmingen FITC Annexin V cell apoptosis detection kit purchased from BD Biosciences (Shanghai, China);
  • BD FACSCaliburTM flow cytometer purchased from BD Biosciences (Shanghai, China).
  • the inventor conducted the following experiments in the early stage:
  • treatment refers to the compound to be tested is diluted with a cell culture solution to a specific concentration, and then added to a culture dish and incubated with the cells for a specific time.
  • NSCLC non-small cell lung cancer cells
  • Figure 1 Treat non-small cell lung cancer cells (NSCLC) with the specified concentration (as shown in Figure 1) for 24 hours (for A549 and H460 cells) or 36 hours (for HCC827 cells) with Gefitinib, and pass the Western blotting method (Western Blot). ) Determine the expression of SQSTM1. Actin is used as an internal reference protein. The experimental results are shown in Figure 1.
  • A549, HCC827, and H460 cells were treated with AZD9291 at the specified concentration (as shown in Figure 2) for 24 hours, and the expression of SQSTM1 was determined by Western Blot. Actin is used as an internal reference protein. The experimental results are shown in Figure 2.
  • Cells were lysed with a lysis buffer containing 2% SDS, 0.1% bromophenol blue, 10% glycerol, 1.5% DTT (dithiothreitol), and 0.1M tris-HCl (pH 6.8).
  • the protein concentration in cell lysate was determined by BCA protein quantification kit.
  • the boiled and denatured lysate was separated by SDS-PAGE, and then transferred to a polyvinylidene fluoride membrane and blocked with 5% fat-free milk (dissolved in TBST).
  • the membrane was incubated with the designated primary antibody overnight at 4°C, washed with TBST (TBS containing 0.1% Tween-20) for 3 times, and the secondary antibody conjugated with horseradish peroxidase (HRP) at room temperature Incubate for 2 hours. Wash 3 times with TBST.
  • HRP horseradish peroxidase
  • the A549 and H460 cells were treated with DMSO, rapamycin, and gefitinib for 7 hours, and then the above cells were administered with Baf A1 (Bafilomycin A1) or not, and cultured for 5 hours.
  • Baf A1 Bafilomycin A1
  • the expression of LC3 and SQSTM1 was detected by Western Blot (refer to the Western Blot step in Example 1). The experimental results are shown in Figure 3.
  • A549 cells were transfected with the GFP-RFP-LC3 plasmid for 48 hours, and then the cells were treated with EBSS, CQ, gefitinib or AZD9291.
  • the spots of GFP-LC3 and RFP-LC3 were detected by immunofluorescence microscopy, and the experimental results are shown in Figure 4A.
  • High expression of SQSTM-1 gene Plant the cells in the logarithmic growth phase into the corresponding well plate, and cultivate overnight to make the cells adhere to the wall. Before transfection, replace the culture medium with the corresponding medium containing 10% fetal bovine serum without anti-antibody, take appropriate volume of Opti-MEM medium into the EP tube, add appropriate amount of plasmid and transfection reagent X-treme GENE HD, pipette and mix well Then let it stand at room temperature for 20-25 min. Add the mixture evenly to the cell culture medium in the form of drops, and gently shake to mix. Transfection for 48-72h according to experimental needs. And the Western blot method was used to evaluate the overexpression effect.
  • Knockdown of SQSTM-1 gene Plant the cells in the logarithmic growth phase into the corresponding well plate and culture overnight to make the cells adhere to the wall (different cells to be transfected require different coverage). Before transfection, replace the culture medium with the corresponding medium containing 10% fetal bovine serum, take an appropriate amount of Opti-MEM medium into the EP tube, add an appropriate amount of Lipofectamine RNAiMAX transfection reagent according to the experimental instructions, mix, and then take, etc. Add an appropriate amount of siRNA to a volume of Opti-MEM medium, and mix well (siRNA and negative control siRNA are synthesized by Shanghai Jima Pharmaceutical Technology Co., Ltd.).
  • EGFR TKIs such as gefitinib and AZD9291 inhibit the autophagic degradation of cells, leading to the accumulation of SQSTM1, thereby promoting the cells to develop acquired drug resistance to EGFR TKIs.
  • Immunofluorescence experiment procedure Place the sterilized cover glass in the well plate, inoculate the cell density to 1/5 to 1/3 of the conventional one, incubate overnight, add the medicine for a specific time, and take it out of the incubator. Wash once with 1 ⁇ PBS, then wash once with anhydrous methanol, and fix at room temperature for 10 minutes. Wash 3 times with PBS. Then it was permeabilized with 0.25% Triton X-100 for 25 minutes. Wash 3 times with PBS. Add 3% BSA (1 ⁇ PBS) and block for 30 minutes at room temperature. Add the diluted primary antibody (diluted with 3% BSA) and place it in a humidified box at 4°C overnight.
  • autophagy can be divided into three main processes, namely the formation of autophagosomes, the fusion of autophagosomes and lysosomes, and the degradation of autophagolysosomes.
  • Colocalization coefficient is expressed as the ratio of the colocalization points of LC3 and LAMP1 to the total LC3 points.
  • gefitinib does not affect the fusion of autophagosomes and lysosomes.
  • CTSB is a characteristic hydrolase of lysosome, which needs to be sheared into mature body in the acidic environment of lysosome before it can play the role of protein hydrolysis. Therefore, many studies use the expression level of mature CTSB as an indicator of the strength of lysosomal function.
  • the A549 and HCC827 cells were treated with Gefitinib, AZD9291, CQ, or Baf A1 for 24 hours, and actin was used as a control to evaluate the expression of CTSB mature body by Western Blot (refer to the Western Blot step in Example 1).
  • the experimental results are shown in Figure 8.
  • gefitinib and AZD9291 can significantly down-regulate the expression of CTSB mature body, which shows that these two compounds do have the effect of weakening lysosomal function.
  • Acridine orange dye is a protic fluorescent dye. Its aprotonated monomer can emit green fluorescence, and it enters the lysosome and is protonated to form a polymer and emit red fluorescence.
  • the A549 and HCC827 cells were treated with DMSO, Gefitinib, AZD9291, CQ or BafA1 for 12 hours, and then the A549 and HCC827 cells were incubated with AO for 15 minutes. Detect the number of AO-red fluorescent spots by fluorescence microscope. The experimental results are shown in Figure 9A.
  • EGFR TKIs such as gefitinib and AZD9291 inhibit lysosomal acidification, leading to the weakening of autophagic lysosomes and hindering the degradation of the encapsulated substrates.
  • Representative EGFR TKI chemical structure acid-base analysis selected from the first generation to the third generation of EGFR TKI, the eight representative drugs most commonly used in clinical practice, through the free and open source drug database (https://www .drugbank.ca/) Retrieve its physical and chemical properties.
  • the eight representative EGFR TKI chemical structures have three or more nitrogen atoms, but there is no Lewis acidic acid group that lacks electrons, and they all belong to Lewis base basic drugs.
  • Gefitinib, Afatinib, Dacomtinib, Ositinib, Olmutinib, etc. all have aliphatic amine groups with strong basicity, and their basic pKa values are as follows Shown.
  • the inventors infer that the root cause of EGFR TKIs such as gefitinib and AZD9291 inhibiting lysosomal acidification, which in turn leads to the hindered autophagy lysosomal degradation may be due to the fact that EGFR TKIs such as gefitinib and AZD9291 are in The chemical structure has a strong basic functional group, which makes it easy to enter the lysosome in the acidic microenvironment. By reducing the acidification of the lysosome, it causes the functional blockade of the autophagy lysosome and promotes cancer.
  • EGFR TKIs such as gefitinib and AZD9291.
  • the inventors carried out alkaline neutralization modification of EGFR TKI with strong basic groups in the structure to obtain a series of EGFR TKI alkaline neutralization analogs, such as the compounds synthesized in the following examples 10-12 .
  • Step I Preparation of 3-bromopropyl-3,5-dimethoxybenzoate (Intermediate 1-3): Weigh 3,5-dimethoxybenzoyl chloride (Intermediate 1-1 ) (2g, 10mmol) and 3-bromo-1-propanol (Intermediate 1-2) (1.07mL, 5mmol), dissolve it in 20mL of dichloromethane, stir until dissolved, and then slowly add triethylamine dropwise (1.5mL, 15mmol), stirred at room temperature, TLC monitored the reaction process, and the reaction was complete after about 5 hours.
  • Step II Preparation of 3-bromopropyl-3,5-dihydroxybenzoate (Intermediate 1-4): Weigh Intermediate 1-3 (604mg, 2mmol) and dissolve it in 20mL of dichloromethane, While stirring, aluminum trichloride (1333 mg, 10 mmol, three times) and sodium iodide (150 mg, 1 mmol) were added sequentially, and the reaction was carried out overnight at room temperature. When the reaction is complete, add 20 mL of saturated ammonium chloride solution to neutralize the reaction, then add 20 mL of dichloromethane for extraction, wash the dichloromethane layer with saturated NaCl solution 3 times, and dry with anhydrous MgSO 4 .
  • Step III Preparation of 3-bromopropyl-3,5-ditetrahydropyranylbenzoate (Intermediate 1-6): Weigh Intermediate 1-4 (550mg, 2mmol), dihydropyran (Intermediate 1-5) (841.2 mg, 10 mmol) and pyridinium p-toluenesulfonate (502 mg, 0.2 mmol) were dissolved in 20 mL of dichloromethane and reacted at room temperature for 6 hours. After the reaction is complete, add 20 mL of water and 20 mL of dichloromethane, extract and spin dry the dichloromethane.
  • Step IV Preparation of 4-((3-chloro-4-fluorophenyl)amino)-6-(3-morpholinopropoxy)quinazolin-7-ol (Intermediate 1-8): Weigh The crude drug gefitinib (Intermediate 1-7) (89.4mg, 0.2mmol) and L-methionine (49.23mg, 0.33mmol) were dissolved in 5mL of methanesulfonic acid, and the reaction was refluxed at 165°C for approximately 24 hours. After the reaction was completed, 5 mL of ultrapure water was added to quench the reaction, and the reaction was cooled at room temperature.
  • Step V-VI 3-((4-((3-chloro-4-fluorophenyl)amino)-6-(3-morpholinopropoxy)quinazolin-7-yl)oxy)propyl -3,5-Dihydroxybenzoic acid ester (general formula compound II-1, Gefi-2OH): Weigh intermediate 1-8 (43.2mg, 0.1mmol) and intermediate 1-6 (88.4mg, 0.2mmol) was dissolved in 5mL N,N-dimethylformamide, potassium carbonate (55.2mg, 0.4mmol) and sodium iodide (15mg, 0.1mmol) were added, and reacted at 80°C overnight.
  • Step I Preparation of 4-fluoro-2-((tetrahydro-2H-pyran-2-yl)oxy)aniline (Intermediate 2-2): The 2-amino-5-fluorophenol (Intermediate 2 -1) (1.27g, 10mmol), dihydropyran (2.52g, 30mmol) and pyridinium p-toluenesulfonate (502mg, 1mmol), dissolved in 20mL of dichloromethane, and reacted at room temperature for 6 hours. After the reaction is complete, add 20 mL of water and 20 mL of dichloromethane, extract and spin dry the dichloromethane.
  • Step II Preparation of 4-methylpiperazinyl-2-((tetrahydro-2H-pyran-2-yl)oxy)aniline (Intermediate 2-3): Weigh Intermediate 2-2 (1.05 g, 5mmol) and N-methylpiperazine (1.36mL, 15mmol) were dissolved in 20mL 2-pentanol and reacted at 120°C for 3 hours. After the reaction is complete, the solvent is spin-dried, ethyl acetate is added for extraction, concentrated, and separated and purified by silica gel column chromatography to obtain intermediate 2-3 as a red powder. The yield is 71%. MS(ESI)m/z:292.30(M+1) + ,calculated for C 16 H 25 N 3 O 2 :291.19.
  • Step IV 2-((4-(4-methylpiperazin-1-yl)-2-((tetrahydro-2H-pyran-2-yl)oxy)phenyl)amino)thiophene [3, 2-d]
  • Preparation of pyrimidine-4(3H)-one (Intermediate 2-6): Weigh Intermediate 2-5 (930mg, 5mmol) and dissolve in 50mL ethanol, add Intermediate 2-3 (1.75g, 6mmol) and 5mL acetic acid, reflux for 6 hours.
  • Step V 4-Chloro-N-(4-(4-methylpiperazin-1-yl)-2-((tetrahydro-2H-pyran-2-yl)oxy)phenyl)thiophene
  • Intermediate 2-7 Weigh Intermediate 2-6 (1.76g, 4mmol) and dissolve it in 5mL acetonitrile, and raise the reaction temperature to 75°C. Add 1 mL of a mixed solution of phosphorus oxychloride and 1 mL of acetonitrile, and react at 75°C for 1 hour.
  • Step VI N-(3-((2-((4-(4-methylpiperazin-1-yl)-2-((tetrahydro-2H-pyran-2-yl)oxy)phenyl ) Preparation of amino)thieno[3,2-d]pyrimidin-4-yl)oxy)phenyl)acrylamide (Intermediate 2-8): Weigh Intermediate 2-7 (1.76g, 4mmol), N-(3-hydroxyphenyl)acrylamide (815 mg, 5 mmol) and potassium carbonate (1.66 g, 12 mmol) were dissolved in 25 mL of an acetonitrile/water mixed solvent with a volume ratio of 8:2, and the reaction was refluxed for 6 hours.
  • Step VII-VIII N-(3-((2-((4-(4-methylpiperazin-1-yl)-2-((tetrahydro-2H-pyran-2-yl)oxy) (Phenyl) amino) thieno [3,2-d] pyrimidin-4-yl) oxy) phenyl) acrylamide (general formula compound III-1) preparation: refer to the step V and step VI in Example 10 Method: Intermediate 2-8 and Intermediate 1-6 are reacted to obtain target compound III-1 as a white solid powder, yield: 52%. MS(ESI)m/z:697.30(M+1) + , calculated for C 36 H 36 N 6 O 7 S: 696.24.
  • Step 1 Preparation of 3-(2-chloropyrimidin-4-yl)-1-methyl-1H-indole (Intermediate 3-3): Add 2,4-Dichloropyrimidine (Intermediate 3-2) (1.49g, 10mmol) and aluminum chloride (1.33g, 10mmol) were suspended in 20mL of N,N-dimethylacetamide (DME). After stirring for 5 minutes at room temperature, 1-methyl was added to the reaction system. Indole (Intermediate 3-1) (1.25 mL, 10 mmol) was reacted at 80°C for 2 hours, and the reaction process was monitored by TLC.
  • 2,4-Dichloropyrimidine (Intermediate 3-2) (1.49g, 10mmol)
  • aluminum chloride (1.33g, 10mmol)
  • 1-methyl was added to the reaction system.
  • Indole (Intermediate 3-1) (1.25 mL, 10 mmol) was reacted at 80°C for 2 hours, and the reaction process was monitored by TLC
  • Step II Preparation of 2-amino-4-nitro-5-fluorophenol (Intermediate 3-5): Weigh 4-fluoro-2-methoxy-5-nitroaniline (Intermediate 3-4) (372mg, 2mmol) was dissolved in 20mL of dichloromethane, aluminum trichloride (1333mg, 10mmol, three times) and sodium iodide (150mg, 1mmol) were added in sequence while stirring, and reacted overnight at room temperature. When the reaction is complete, add 20 mL of saturated ammonium chloride solution to neutralize the reaction, then add 20 mL of dichloromethane for extraction, wash the dichloromethane layer with saturated NaCl solution 3 times, and dry with anhydrous MgSO 4 .
  • Step III Preparation of 4-fluoro-5-nitro-2-((tetrahydro-2H-pyran-2-yl)oxy)aniline (Intermediate 3-6): Weigh Intermediate 3-5 ( 860 mg, 5 mmol), dihydropyran (1680 mg, 20 mmol) and pyridinium p-toluenesulfonate (125.5 mg, 0.5 mmol) were dissolved in 20 mL of dichloromethane and reacted at room temperature for 6 hours. After the reaction is complete, add 20 mL of water and 20 mL of dichloromethane, extract and spin dry the dichloromethane.
  • Step IV N-(4-Fluoro-5-nitro-2-((tetrahydro-2H-pyran-2-yl)oxy)phenyl)-4-(1-methyl-1H-indole
  • Intermediate 3-7 Weigh Intermediate 3-6 (1280mg, 5mmol) and Intermediate 3-3 (1215mg, 5mmol) and dissolve them in 20mL 2-pentanol , P-toluenesulfonic acid monohydrate (1140mg, 6mmol) was added and reacted at 105°C for 3 hours.
  • Step VI N-(2-fluoro-5-((4-(1-methyl-1H-indol-3-yl)pyrimidin-2-yl)amino)-4-((tetrahydro-2H- Preparation of pyran-2-yl)oxy)phenyl)acrylamide
  • Intermediate 3-9 Weigh Intermediate 3-8 (867mg, 2mmol) and N,N-diisopropylethylamine (310mg , 2.4 mmol), dissolved in 10 mL of dichloromethane, acryloyl chloride (181 mg, 2 mmol) was slowly added dropwise into the reaction solution, and reacted in an ice-water bath for about 2 hours.
  • Step VII-VIII N-(2-fluoro-5-((4-(1-methyl-1H-indol-3-yl)pyrimidin-2-yl)amino)-4-((tetrahydro- Preparation of 2H-pyran-2-yl)oxy)phenyl)acrylamide (Intermediate 3-10): According to the method of step V and step VI in Example 10, the intermediate 3-9 and intermediate 1 -6 was reacted to obtain white powder intermediate 3-10, yield: 63%. MS(ESI)m/z:598.30(M+1) + , calculated for C 32 H 28 FN 5 O 6 :597.20.
  • Step IX 3-(4-acrylamide-5-((2-(dimethylamino)ethyl)(methyl)amino)-2-((4-(1-methyl-1H-indole- Preparation of 3-yl)pyrimidin-2-yl)amino)phenoxy)propyl-3,5-dihydroxybenzoate (general formula compound IV-1, AZD-2OH): Weigh intermediate 3- 10 (597.6 mg, 1 mmol) and N,N-diisopropylethylamine (306 mg, 3 mmol) were dissolved in 10 mL of 2-pentanol and reacted at 120°C for about 3 hours.
  • A549 and H460 cells were seeded in a 6-well plate at a density of 1 ⁇ 10 5 /well and 0.8 ⁇ 10 5 /well, respectively. After overnight culture, gefitinib and the compound Gefi-2OH were diluted with complete medium to make specific Concentrations were added to a 6-well plate. After 24 hours of action, lysis buffer was added to lyse the cells. After the cell lysate was boiled and denatured, the Western Blot experiment was performed (see the Western Blot method in Example 1 for the steps). 13 shown.
  • Lysotracker is a weakly basic probe labeled with a chemical group that emits blue fluorescence.
  • the weakly basic part can provide protons to maintain the pH at neutral. It can be selectively retained in acidic lysosomes to achieve specific fluorescent labeling of lysosomes.
  • the specific steps are as follows: place the cover glass in the well plate, inoculate the cell density to 1/5 to 1/3 of the usual, and perform the corresponding treatment after sticking.
  • the Lysotracker dye and the culture medium were diluted 1:1000, then added to the cells, and incubated for 2 hours. Pour out the culture medium and wash 3 times with 1 ⁇ PBS for 5 minutes each time. Use a mixture of 20% glycerol and PBS as the mounting solution to directly mount the live cells and observe the fluorescence under a microscope.
  • Example 15 Compound Gefi-2OH inhibits proliferation of non-small cell lung cancer
  • the logarithmic growth phase A549 and H460 cells were seeded in a 96-well plate at a density of 5000/well and 6000/well respectively, and each experimental group was set up with 5 parallel replicate wells; after 24 hours of culture, the formulated drug concentration was added Gradient culture medium, continue to incubate for 48h; aspirate the medium in each well before testing, and add 100 ⁇ l of 1640 medium containing 10% MTS that has been prepared, and incubate in a 37°C incubator in the dark for 20-60 minutes. After shaking on the enzyme-linked immunoassay instrument for 1 minute, read the absorbance value at 490nm wavelength. When the net OD value reaches the range of 0.5-0.8, stop the detection, calculate the relative cell proliferation rate, and use Graphpad prism 6 to draw the proliferation curve. The result is as follows Shown in Figure 16.
  • the basic and neutralized gefitinib derivative Gefi-2OH has a significantly better proliferation inhibitory effect on non-small cell lung cancer cells A549 and H460 than gefitinib, and its half inhibitory concentration (IC 50 ) Gefitinib was increased by approximately 1.8 times (in A549 cells) and 3 times (in H460 cells), respectively.
  • the inhibitory effect of the compound AZD-2OH of the present invention on the proliferation of H460 cells was also tested, and the proliferation curve is shown in FIG. 17. From the results of FIG. 17, the compounds of the present invention AZD-2OH inhibited the proliferation of H460 cells was significantly better than the same compound AZD9291 before transformation, 50% inhibitory concentrations (IC 50) less than half the AZD9291.
  • the A549 and H460 cell supernatants were collected in a centrifuge tube, washed once with pre-cooled PBS, and also collected in the above centrifuge tube. Add an appropriate amount of 0.25% trypsin without EDTA to the cells, after a certain period of time, neutralize with 2 times the volume of trypsin in 1640 culture medium containing 10% FBS, and collect the cell suspension into the previous centrifuge tube. Centrifuge at 1500rpm for 5min, pour out the supernatant, resuspend the cell pellet in PBS, centrifuge again at 1500rpm for 5min, and discard the supernatant.
  • the modified compounds of general formula II, III, and IV all showed good lung cancer cell proliferation inhibitory activity.
  • the modified product of osimertinib (formula IV-1-formula IV-8) had the best activity, and its IC 50 The value is between 1.21-5.13 ⁇ M.
  • the acidity of the R substituent plays a decisive role in the activity level. The stronger the acidity, the better the overall anti-tumor activity of the compound.
  • the specific performance is that different substituents affect the compound.
  • the present invention is the first to carry out alkaline neutralization modification for EGFR TKIs, which has achieved unexpected good results and explored a new way to improve its activity.

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Abstract

Disclosed are an EGFR kinase inhibitor and application thereof in preparing an anti-cancer drug; said EGFR kinase inhibitor reduces the alkalinity of EGFR TKI, can effectively improve the acquired drug resistance of EGFR TKI caused by the accumulation of SQSTM1, and is of great significance to improving the clinical treatment benefits of EGFR TKI.

Description

一种EGFR激酶抑制剂及其在制备抗癌药物方面的应用An EGFR kinase inhibitor and its application in preparing anticancer drugs 技术领域Technical field

本发明涉及药物领域；具体来说，本发明涉及一种新型EGFR激酶抑制剂及其在治疗癌症方面的应用。The present invention relates to the field of medicine; specifically, the present invention relates to a novel EGFR kinase inhibitor and its application in the treatment of cancer.

背景技术Background technique

肺癌是全球范围内发病率最高、导致死亡人数最多的恶性肿瘤，对人类健康造成了严重的威胁。然而，传统的化学治疗方法治疗晚期肺癌的疗效却十分有限。表皮生长因子受体(Epidermal Growth Factor Receptor,EGFR，ErbB1)属于受体酪氨酸激酶ErbB家族中的一员。现有的研究证据表明，在多种实体瘤内普遍存在EGFR高表达、基因扩增或激活型突变(L858R)的现象。EGFR过表达或者激活型突变，会引起下游Ras/Raf/MAPK、PI3K/Akt等细胞增殖、生存信号通路的过度活化。因此，EGFR被认为是一种驱动肺癌、胰腺癌、乳腺癌等恶性肿瘤发生发展的关键癌基因。EGFR酪氨酸激酶抑制剂(EGFR TKI)在治疗上述EGFR异常激活的恶性肿瘤方面，临床收益十分显著，曾一度为人们带来巨大的希望。Lung cancer is the malignant tumor with the highest incidence rate and the largest number of deaths in the world, and it poses a serious threat to human health. However, the efficacy of traditional chemotherapy in the treatment of advanced lung cancer is very limited. Epidermal Growth Factor Receptor (Epidermal Growth Factor Receptor, EGFR, ErbB1) belongs to the ErbB family of receptor tyrosine kinases. Existing research evidence shows that high expression of EGFR, gene amplification or activation mutation (L858R) is common in a variety of solid tumors. EGFR overexpression or activating mutations can cause excessive activation of downstream Ras/Raf/MAPK, PI3K/Akt and other cell proliferation and survival signaling pathways. Therefore, EGFR is considered to be a key oncogene that drives the occurrence and development of lung cancer, pancreatic cancer, breast cancer and other malignant tumors. EGFR tyrosine kinase inhibitors (EGFR TKIs) have significant clinical benefits in the treatment of malignant tumors with abnormal activation of EGFR, and once brought great hope to people.

吉非替尼(Gefitinib)、厄洛替尼(Erlotinib)等第一代EGFR-TKI通过可逆性地结合EGFR的ATP结合位点而抑制肿瘤细胞EGFR信号通路的激活。然而，临床实践发现，吉非替尼只能维持半年到一年的有效缓解期，很快便产生耐药性。目前，研究揭示吉非替尼的耐药机制主要有以下几个方面：①EGFR基因的突变：特异性EGFR外显子20的二次突变，引起第790位的苏氨酸被体积较大的甲硫氨酸取代(T790M)，阻止吉非替尼和厄洛替尼与ATP位点的结合；其他二次耐药突变包括L747S、D761Y以及T854A等；②EGFR信号转导路径的变化，如Ras活化引起Raf-MEK-MAPK的上调，与EGFR失去偶联，ERK、Met高表达以及PTEN失活引起AKT通路的过度活化等；③EGFR旁路效应：如血小板源性生长因子受体(PDGFR)、胰岛素样生长因子受体-1(IGFR-1)受体等生长因子受体直接活化其下游信号通路。针对EGFR的T790M突变开发的第二代EGFR-TKI阿法替尼(Afatinib，BIBW2992)、第三代EGFR-TKI奥西替尼(Osimertinib，AZD9291)已经开始应用于临床，但亦不可避免地产生了各种耐药性，其机制包括产生C797S突变、EGFR、HER2、c-Met基因扩增等。Gefitinib, Erlotinib, and other first-generation EGFR-TKIs inhibit the activation of tumor cell EGFR signaling pathways by reversibly binding to the ATP binding site of EGFR. However, clinical practice has found that gefitinib can only maintain an effective remission period of six months to one year, and drug resistance soon develops. At present, studies have revealed that the resistance mechanism of gefitinib mainly includes the following aspects: ① Mutation of EGFR gene: the second mutation of specific EGFR exon 20 causes the threonine at position 790 to be replaced by the larger A Thionine substitution (T790M) prevents the binding of gefitinib and erlotinib to the ATP site; other secondary resistance mutations include L747S, D761Y and T854A; ②changes in EGFR signal transduction pathways, such as Ras activation Causes Raf-MEK-MAPK up-regulation, loss of coupling with EGFR, high expression of ERK and Met and inactivation of PTEN cause excessive activation of the AKT pathway, etc.; EGFR bypass effects: such as platelet-derived growth factor receptor (PDGFR), insulin Growth factor receptors such as growth factor receptor-1 (IGFR-1) receptors directly activate their downstream signaling pathways. The second-generation EGFR-TKI Afatinib (BIBW2992) and the third-generation EGFR-TKI Osimertinib (Osimertinib, AZD9291) developed for the T790M mutation of EGFR have begun to be used in clinical practice, but they have inevitably produced The mechanisms of various drug resistance include the generation of C797S mutation, EGFR, HER2, and c-Met gene amplification.

自噬是在应激状态下，细胞通过降解大分子物质和损伤的细胞器来维持能量供应及清除有害物质，从而维持自身稳态的一种生物学过程。近年来，大量研究发现自噬对于恶性肿瘤的进展和EGFR TKI获得性耐药的产生密切相关。自噬既可以阻止正常细胞恶变，又可以帮助已经恶变的细胞抵抗不利的生存环境，促进耐药性的产生。作为介导细胞自噬、能量感应、氧化应激等多个信号通路的关键配体蛋白SQSTM1(Sequestosome-1，又称为p62)，其在肿瘤的发生发展及耐药中均发挥了非常重要的作用。作为配体蛋白，SQSTM1对多个促癌信号通路有重要调控作用，其不仅介导mTORC1在溶酶体中的活化，也可以通过与keap1的直接作用使其与Nrf2的相互作用解离，从而抑制keap1介导Nrf2的泛素化降解。SQSTM1的蛋白水平升高，不仅意味着细胞自噬相关降解途径被削弱，同时也可以帮助肿瘤细胞抵抗凋亡。有研究发现铂类药物耐药性细胞内SQSTM1的表达出现上调，相反地，降低SQSTM1的蛋白水平可有效提高肿瘤对药物的敏感性。此外，SQSTM1可发生浆核穿梭，从而参与对放疗、化疗等引起的DNA损伤的修复，同样地证明SQSTM1是促进肿瘤耐药性产生的关键调节因子。然而，对于SQSTM1在EGFR TKI耐药性产生过程中扮演着怎样的角色，目前仍未可知。通过阐明SQSTM1在EGFR TKI耐药性产生过程的重要作用，并通过针对SQSTM1调控EGFR TKI耐药性的作用机制，进行EGFR TKI的合理改造，有望克服临床上EGFR TKI获得性耐药性这一难题，对提高EGFR TKI的临床治疗收益具有重要意义。Autophagy is a biological process in which cells maintain their own homeostasis by degrading macromolecular substances and damaged organelles to maintain energy supply and remove harmful substances under stress. In recent years, a large number of studies have found that the progression of autophagy to malignant tumors is closely related to the development of EGFR TKI-acquired resistance. Autophagy can not only prevent the malignant transformation of normal cells, but also help the cells that have been malignant to resist the unfavorable living environment and promote the development of drug resistance. As a key ligand protein SQSTM1 (Sequestosome-1, also known as p62) that mediates multiple signaling pathways such as autophagy, energy induction, and oxidative stress, it plays a very important role in tumor development and drug resistance. The role of. As a ligand protein, SQSTM1 plays an important role in regulating multiple cancer-promoting signal pathways. It not only mediates the activation of mTORC1 in the lysosome, but also dissociates the interaction with Nrf2 through direct action with keap1, thereby Inhibit keap1 mediates the ubiquitination and degradation of Nrf2. The increased protein level of SQSTM1 not only means that autophagy-related degradation pathways are weakened, but also can help tumor cells resist apoptosis. Studies have found that the expression of SQSTM1 in platinum drug-resistant cells is up-regulated. On the contrary, reducing the protein level of SQSTM1 can effectively improve the sensitivity of tumors to drugs. In addition, SQSTM1 can shuttle between plasma and nuclei to participate in the repair of DNA damage caused by radiotherapy and chemotherapy. It also proves that SQSTM1 is a key regulator that promotes tumor resistance. However, it is still unclear what role SQSTM1 plays in the development of EGFR TKI resistance. By clarifying the important role of SQSTM1 in the process of EGFR TKI resistance, and by targeting the mechanism of SQSTM1 regulating EGFR TKI resistance, the rational transformation of EGFR TKI is expected to overcome the clinical problem of EGFR TKI acquired drug resistance. , It is of great significance to improve the clinical treatment benefits of EGFR TKI.

发明内容Summary of the invention

本发明目的在于提供一种抗癌作用强，同时又不会影响细胞自噬进程和溶酶体功能的新型EGFR激酶抑制剂及其在制备预防和/或治疗与受体酪氨酸蛋白激酶有关的疾病和病症(例如，癌症，特别是非小细胞肺癌)的药物中的应用。The purpose of the present invention is to provide a new type of EGFR kinase inhibitor with strong anti-cancer effect without affecting the autophagy process and lysosomal function and its preparation, prevention and/or treatment related to receptor tyrosine protein kinase The application of medicines for various diseases and conditions (for example, cancer, especially non-small cell lung cancer).

本发明提供一种如式Ⅰ所示的化合物及其药学上可接受的盐，The present invention provides a compound represented by formula I and a pharmaceutically acceptable salt thereof,

Figure PCTCN2020089390-appb-000001
Figure PCTCN2020089390-appb-000001

式Ⅰ中，In formula I,

A环为5至18元环，或，A环不存在；Ring A is a 5- to 18-membered ring, or, Ring A does not exist;

X为-O-、-S-、-N(R 4)-或-(CHR 4) n-，n为0或1； X is -O-, -S-, -N(R 4 )- or -(CHR 4 ) n -, n is 0 or 1;

R 1选自：芳基、芳烷基、芳香族或非芳香族杂环基和杂环烷基； R 1 is selected from: aryl, aralkyl, aromatic or non-aromatic heterocyclic group and heterocycloalkyl;

R 2选自：氢、烷基、环烷基、烯基、炔基、芳基、芳烷基、芳香族或非芳香族杂环基、杂环烷基、-COR 4、-C(O)OR 4、-C(O)NR 4R 5、-CH＝NR 4、-CN、-OR 4、-OC(O)R 4、-S(O) t-R 4、-NR 4R 5、-NR 4C(O)R 5、-NO 2、-N＝CR 4R 5和卤素； R 2 is selected from: hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, aromatic or non-aromatic heterocyclic group, heterocycloalkyl, -COR 4 , -C(O )OR 4 , -C(O)NR 4 R 5 , -CH=NR 4 , -CN, -OR 4 , -OC(O)R 4 , -S(O) t -R 4 , -NR 4 R 5 , -NR 4 C(O)R 5 , -NO 2 , -N=CR 4 R 5 and halogen;

R 3每次出现时独立地选自：氢、烷基、环烷基、烯基、炔基、芳基、芳烷基、芳香族或非芳香族杂环基、杂环烷基、-COR 4、-C(O)OR 4、-C(O)NR 4R 5、-CH＝NR 4、-CN、-OR 4、-OC(O)R 4、-S(O) t-R 4、-NR 4R 5、-NR 4C(O)R 5、-NO 2、-N＝CR 4R 5和卤素； Each occurrence of R 3 is independently selected from: hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, aromatic or non-aromatic heterocyclic group, heterocycloalkyl, -COR 4 , -C(O)OR 4 , -C(O)NR 4 R 5 , -CH=NR 4 , -CN, -OR 4 , -OC(O)R 4 , -S(O) t -R 4 , -NR 4 R 5 , -NR 4 C(O)R 5 , -NO 2 , -N=CR 4 R 5 and halogen;

m为0-3的整数(具体如0、1、2、3)；m is an integer of 0-3 (specifically 0, 1, 2, 3);

t为1、2或3；t is 1, 2 or 3;

R 4和R 5分别独立地选自：氢、烷基、环烷基、烯基、芳基、芳香族或非芳香族杂环基、烷氧基、芳氧基和卤素； R 4 and R 5 are each independently selected from: hydrogen, alkyl, cycloalkyl, alkenyl, aryl, aromatic or non-aromatic heterocyclic group, alkoxy, aryloxy and halogen;

并且，式Ⅰ所示的化合物结构中含有至少一个酸性取代基，该酸性取代基含有至少一个酸性官能团。In addition, the structure of the compound represented by formula I contains at least one acidic substituent, and the acidic substituent contains at least one acidic functional group.

在本发明的一个实施方式中，酸性官能团选自：酚羟基、羧基、磺酸基和磷酸基中的一种或多种。In an embodiment of the present invention, the acidic functional group is selected from one or more of phenolic hydroxyl group, carboxyl group, sulfonic acid group and phosphoric acid group.

在本发明的一个实施方式中，酸性取代基选自：羟基苯基、羧基苯基、磺酸基苯基、磷酸基苯基、羟基苯甲酰氧基取代烷基、磺酸基取代烷基、羧基取代烷基、磷酸基取代烷基、磺酸基苯基取代烷基、羧基苯基取代烷基、磷酸基苯基取代烷基、羟基苯甲酰氧基取代烷氧基、磺酸基取代烷氧基、羧基取代烷氧基、磷酸基取代烷氧基、磺酸基苯基取代烷氧基、羧基苯基取代烷氧基和磷酸基苯基取代烷氧基中的一个或多种。In one embodiment of the present invention, the acidic substituent is selected from the group consisting of: hydroxyphenyl, carboxyphenyl, sulfophenyl, phosphate, hydroxybenzoyloxy substituted alkyl, sulfonate substituted alkyl , Carboxyl substituted alkyl, phosphate substituted alkyl, sulfonate phenyl substituted alkyl, carboxyphenyl substituted alkyl, phosphate phenyl substituted alkyl, hydroxybenzoyloxy substituted alkoxy, sulfonic acid group One or more of substituted alkoxy, carboxy substituted alkoxy, phosphate substituted alkoxy, sulfonate phenyl substituted alkoxy, carboxyphenyl substituted alkoxy, and phosphate phenyl substituted alkoxy .

在本发明的一个实施例中，羟基苯基中，羟基位于苯基的2-5位中的至少一个。In one embodiment of the present invention, in the hydroxyphenyl group, the hydroxyl group is located at least one of the 2-5 positions of the phenyl group.

在本发明的一个实施例中，羧基苯基中，羧基位于苯基的2-5位中的至少一个。In an embodiment of the present invention, in the carboxyphenyl group, the carboxy group is located at least one of the 2-5 positions of the phenyl group.

在本发明的一个实施例中，磺酸基苯基中，磺酸基位于苯基的2-5位中的至少一个。In an embodiment of the present invention, in the sulfonic phenyl group, the sulfonic acid group is located at least one of the 2-5 positions of the phenyl group.

在本发明的一个实施例中，磷酸基苯基中，磷酸基位于苯基的2-5位中的至少一个。In an embodiment of the present invention, in the phosphate phenyl group, the phosphate group is located at least one of the 2-5 positions of the phenyl group.

在本发明的一个实施例中，羟基苯甲酰氧基为二羟基苯甲酰氧基(例如，2,3-二羟基苯甲酰氧基、2,4-二羟基苯甲酰氧基、2,5-二羟基苯甲酰氧基、2,6-二羟基苯甲酰氧基、3,4-二羟基苯甲酰氧基、3,5-二羟基苯甲酰氧基、3,6-二羟基苯甲酰氧基、4,5-二羟基苯甲酰氧基、4,6-二羟基苯甲酰氧基或5,6-二羟基苯甲酰氧基)或三羟基苯甲酰氧基(例如，2,3,4-三羟基苯甲酰氧基、2,3,5-三羟基苯甲酰氧基、2,3,6-三羟基苯甲酰氧基、2,4,5-三羟基苯甲酰氧基、2,4,6-三羟基苯甲酰氧基、2,5,6-三羟基苯甲酰氧基、3,4,5-三羟基苯甲酰氧基、3,4,6-三羟基苯甲酰氧基或4,5,6-三羟基苯甲酰氧基)。In an embodiment of the present invention, the hydroxybenzoyloxy group is a dihydroxybenzoyloxy group (for example, 2,3-dihydroxybenzoyloxy, 2,4-dihydroxybenzoyloxy, 2,5-Dihydroxybenzoyloxy, 2,6-dihydroxybenzoyloxy, 3,4-dihydroxybenzoyloxy, 3,5-dihydroxybenzoyloxy, 3, 6-dihydroxybenzoyloxy, 4,5-dihydroxybenzoyloxy, 4,6-dihydroxybenzoyloxy or 5,6-dihydroxybenzoyloxy) or trihydroxybenzene Formyloxy (for example, 2,3,4-trihydroxybenzoyloxy, 2,3,5-trihydroxybenzoyloxy, 2,3,6-trihydroxybenzoyloxy, 2 ,4,5-trihydroxybenzoyloxy, 2,4,6-trihydroxybenzoyloxy, 2,5,6-trihydroxybenzoyloxy, 3,4,5-trihydroxybenzene Formyloxy, 3,4,6-trihydroxybenzoyloxy or 4,5,6-trihydroxybenzoyloxy).

在本发明的一个实施方式，烷基为C1-C6烷基，优选为C1-C3烷基。In one embodiment of the present invention, the alkyl group is a C1-C6 alkyl group, preferably a C1-C3 alkyl group.

在本发明的一个实施方式，烷氧基为C1-C6烷氧基，优选为C1-C3烷氧基。In one embodiment of the present invention, the alkoxy group is a C1-C6 alkoxy group, preferably a C1-C3 alkoxy group.

在本发明的一个实施例中，式Ⅰ所示的化合物结构中含有至少一个如下结构的取代基：In an embodiment of the present invention, the structure of the compound represented by formula I contains at least one substituent with the following structure:

Figure PCTCN2020089390-appb-000002
Figure PCTCN2020089390-appb-000002

在本发明的一个实施例中，X为-O-。In one embodiment of the present invention, X is -O-.

在本发明的另一个实施例中，X为-NH-。In another embodiment of the present invention, X is -NH-.

在本发明的另一个实施例中，X为-(CHR 4) n-，n为0，即X不存在。 In another embodiment of the present invention, X is -(CHR 4 ) n -, and n is 0, that is, X does not exist.

在本发明的一个实施方式中，R 1为取代的芳基，如取代的苯基。 In one embodiment of the present invention, R 1 is a substituted aryl group, such as a substituted phenyl group.

在本发明的另一个实施方式中，R 1为取代的杂环基，如取代的吲哚基(如3-吲哚基)。 In another embodiment of the present invention, R 1 is a substituted heterocyclic group, such as substituted indolyl (eg 3-indolyl).

在本发明的一个实施例中，m为2。In an embodiment of the present invention, m is 2.

在本发明的另一个实施例中，m为0。In another embodiment of the present invention, m is zero.

在本发明的一个实施方式中，A环为芳环，优选为苯环，其与相连的嘧啶环形成喹唑啉环结构。In one embodiment of the present invention, the A ring is an aromatic ring, preferably a benzene ring, which forms a quinazoline ring structure with the connected pyrimidine ring.

在本发明的另一个实施方式中，A环为杂芳环，优选为单杂原子五元芳环，例如，吡咯环、呋喃环或噻吩环，其与嘧啶环稠合形成五元单杂芳环[3,2-d]并嘧啶环结构。In another embodiment of the present invention, ring A is a heteroaromatic ring, preferably a single heteroatom five-membered aromatic ring, for example, a pyrrole ring, a furan ring or a thiophene ring, which is fused with a pyrimidine ring to form a five-membered single heteroaromatic ring Ring [3,2-d] and pyrimidine ring structure.

在本发明的另一个实施方式中，A环不存在。In another embodiment of the invention, the A ring is absent.

在本发明的一个具体实施方式中，式Ⅰ中，A环为芳环，R 1为取代苯基，所述化合物具有通式Ⅱ所示结构： In a specific embodiment of the present invention, in formula I, ring A is an aromatic ring, R 1 is a substituted phenyl group, and the compound has the structure shown in formula II:

Figure PCTCN2020089390-appb-000003
Figure PCTCN2020089390-appb-000003

式Ⅱ中，X为-O-、-S-、-N(R 17)-或-(CHR 17) n-，n为0或1； In formula II, X is -O-, -S-, -N(R 17 )- or -(CHR 17 ) n -, and n is 0 or 1;

R 11、R 12和R 13独立地选自：氢、烷基、环烷基、烯基、炔基、芳基、芳烷基、芳香族或非芳香族杂环基、杂环烷基、-COR 17、-C(O)OR 17、-C(O)NR 17R 18、-CH＝NR 17、-CN、-OR 17、-OC(O)R 17、-S(O) t-R 17、-NR 17R 18、-NR 17C(O)R 18、-NO 2、-N＝CR 17R 18和卤素； R 11 , R 12 and R 13 are independently selected from: hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, aromatic or non-aromatic heterocyclic group, heterocycloalkyl, -COR 17 , -C(O)OR 17 , -C(O)NR 17 R 18 , -CH=NR 17 , -CN, -OR 17 , -OC(O)R 17 , -S(O) t- R 17 , -NR 17 R 18 , -NR 17 C(O)R 18 , -NO 2 , -N=CR 17 R 18 and halogen;

R 14选自：氢、烷基、环烷基、烯基、炔基、芳基、芳烷基、芳香族或非芳香族杂环基、杂环烷基、-COR 17、-C(O)OR 17、-C(O)NR 17R 18、-CH＝NR 17、-CN、-OR 17、-OC(O)R 17、-S(O) t-R 17、-NR 17R 18、-NR 17C(O)R 18、-NO 2、-N＝CR 17R 18和卤素； R 14 is selected from: hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, aromatic or non-aromatic heterocyclic group, heterocycloalkyl, -COR 17 , -C(O )OR 17 , -C(O)NR 17 R 18 , -CH=NR 17 , -CN, -OR 17 , -OC(O)R 17 , -S(O) t -R 17 , -NR 17 R 18 , -NR 17 C(O)R 18 , -NO 2 , -N=CR 17 R 18 and halogen;

R 15和R 16独立地选自：氢、烷基、环烷基、烯基、炔基、芳基、芳烷基、芳香族或非芳香族杂环基、杂环烷基、-COR 17、-C(O)OR 17、-C(O)NR 17R 18、-CH＝NR 17、-CN、-OR 17、-OR 19-R 18、-OC(O)R 17、-S(O) t-R 17、-NR 17R 18、-NR 17C(O)R 18、-NO 2、-N＝CR 17R 18和卤素；或R 15和R 16与其所连接的苯环形成任意取代的稠环体系； R 15 and R 16 are independently selected from: hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, aromatic or non-aromatic heterocyclic group, heterocycloalkyl, -COR 17 , -C(O)OR 17 , -C(O)NR 17 R 18 , -CH=NR 17 , -CN, -OR 17 , -OR 19 -R 18 , -OC(O)R 17 , -S( O) t -R 17 , -NR 17 R 18 , -NR 17 C(O)R 18 , -NO 2 , -N=CR 17 R 18 and halogen; or R 15 and R 16 are formed with the benzene ring to which they are connected Arbitrarily substituted fused ring system;

t为1、2或3；t is 1, 2 or 3;

R 17和R 18独立地选自：氢、烷基、环烷基、烯基、芳基、芳香族或非芳香族杂环基、烷氧基、芳氧基、卤素和氨基； R 17 and R 18 are independently selected from: hydrogen, alkyl, cycloalkyl, alkenyl, aryl, aromatic or non-aromatic heterocyclic group, alkoxy, aryloxy, halogen and amino;

R 19为亚烷基； R 19 is an alkylene group;

并且，式Ⅱ所示的化合物结构中含有至少一个酸性取代基，该酸性取代基含有至少一个酸性官能团。所述酸性官能团和酸性取代基具有本发明上述定义。In addition, the structure of the compound represented by formula II contains at least one acidic substituent, and the acidic substituent contains at least one acidic functional group. The acidic functional group and acidic substituent have the above-mentioned definition of the present invention.

在本发明的一个实施方式中，R 11、R 12、R 13、R 14、R 15和R 16中至少一个含有酸性官能团。 In one embodiment of the present invention, at least one of R 11 , R 12 , R 13 , R 14 , R 15 and R 16 contains an acidic functional group.

在本发明的一个具体实施方式中，R 15含有至少一个酸性官能团。 In a specific embodiment of the present invention, R 15 contains at least one acidic functional group.

在本发明的一个具体实施方式中，R 15选自：羟基苯基、羧基苯基、磺酸基苯基、磷酸基苯基、羟基苯甲酰氧基取代烷基、磺酸基取代烷基、羧基取代烷基、磷酸基取代烷基、磺酸基苯基取代烷基、羧基苯基取代烷基、磷酸基苯基取代烷基、羟基苯甲酰氧基取代烷氧基、磺酸基取代烷氧基、羧基取代烷氧 基、磷酸基取代烷氧基、磺酸基苯基取代烷氧基、羧基苯基取代烷氧基和磷酸基苯基取代烷氧基。 In a specific embodiment of the present invention, R 15 is selected from the group consisting of: hydroxyphenyl, carboxyphenyl, sulfonate phenyl, phosphate, hydroxybenzoyloxy substituted alkyl, sulfonate substituted alkyl , Carboxyl substituted alkyl, phosphate substituted alkyl, sulfonate phenyl substituted alkyl, carboxyphenyl substituted alkyl, phosphate phenyl substituted alkyl, hydroxybenzoyloxy substituted alkoxy, sulfonic acid group Substituted alkoxy, carboxyl substituted alkoxy, phosphoric acid substituted alkoxy, sulfonic phenyl substituted alkoxy, carboxyphenyl substituted alkoxy, and phosphoric phenyl substituted alkoxy.

在本发明的一个实施例中，R 15为

Figure PCTCN2020089390-appb-000004
In an embodiment of the present invention, R 15 is
Figure PCTCN2020089390-appb-000004

在本发明的一个实施例中，X为-NH-。In one embodiment of the present invention, X is -NH-.

在本发明的一个实施方式中，R 11、R 12和R 13独立地选自：氢、卤素(如F、Cl)、-NO 2、烯基、炔基和-O(CH 2) xAr，x为0或1，Ar为芳基(例如苯基)或杂环基(例如，含氮杂环基)。 In one embodiment of the present invention, R 11 , R 12 and R 13 are independently selected from: hydrogen, halogen (such as F, Cl), -NO 2 , alkenyl, alkynyl, and -O(CH 2 ) x Ar , X is 0 or 1, and Ar is an aryl group (for example, a phenyl group) or a heterocyclic group (for example, a nitrogen-containing heterocyclic group).

在本发明的一个实施例中，R 11为F，R 12为Cl，R 13为氢。 In an embodiment of the present invention, R 11 is F, R 12 is Cl, and R 13 is hydrogen.

在本发明的另一个实施例中，R 11为氢，R 12为Cl，R 13为F。 In another embodiment of the present invention, R 11 is hydrogen, R 12 is Cl, and R 13 is F.

在本发明的一个实施方式中，R 11为氢，R 13为氢，R 12选自：卤素、乙烯基和乙炔基。 In one embodiment of the present invention, R 11 is hydrogen, R 13 is hydrogen, and R 12 is selected from halogen, vinyl and ethynyl.

在本发明的一个实施方式中，R 14选自：氢、烷基、-CN和卤素。 In one embodiment of the present invention, R 14 is selected from: hydrogen, alkyl, -CN and halogen.

在本发明的一个实施例中，R 14为氢。 In one embodiment of the present invention, R 14 is hydrogen.

在本发明的一个实施方式中，R 16为-OR 19-R 18，其中R 19为亚甲基、亚乙基、亚丙基或亚丁基，R 18选自：芳香族或非芳香族杂环基(如吗啉基、哌嗪基、哌啶基、吡咯烷基等)、烷氧基(如甲氧基、乙氧基、丙氧基)、芳氧基(如苯氧基)和取代的氨基(如甲氨基、二甲氨基)。 In one embodiment of the present invention, R 16 is -OR 19 -R 18 , wherein R 19 is methylene, ethylene, propylene or butylene, and R 18 is selected from: aromatic or non-aromatic hetero Cyclic groups (such as morpholinyl, piperazinyl, piperidinyl, pyrrolidinyl, etc.), alkoxy (such as methoxy, ethoxy, propoxy), aryloxy (such as phenoxy) and Substituted amino (eg methylamino, dimethylamino).

在本发明的一个具体实施方式中，R 16为-OR 19-R 18，其中R 19为亚乙基或亚丙基，R 18选自：吗啉基、哌嗪基、二甲氨基、吡咯烷基、哌啶基、甲氧基和乙氧基。 In a specific embodiment of the present invention, R 16 is -OR 19 -R 18 , wherein R 19 is ethylene or propylene, and R 18 is selected from: morpholinyl, piperazinyl, dimethylamino, pyrrole Alkyl, piperidinyl, methoxy and ethoxy.

具体地，R 16可选自：3-(4-吗啉基)丙氧基、3-(1-哌嗪基)丙氧基、3,3-二甲氨基丙氧基、3-(1-吡咯烷基)丙氧基、3-(1-哌啶基)丙氧基、3-(4-哌啶基)丙氧基、甲氧乙氧基和乙氧乙氧基。 Specifically, R 16 may be selected from: 3-(4-morpholinyl)propoxy, 3-(1-piperazinyl)propoxy, 3,3-dimethylaminopropoxy, 3-(1 -Pyrrolidinyl)propoxy, 3-(1-piperidinyl)propoxy, 3-(4-piperidinyl)propoxy, methoxyethoxy and ethoxyethoxy.

在本发明的一个实施例中，R 16为

Figure PCTCN2020089390-appb-000005
In an embodiment of the present invention, R 16 is
Figure PCTCN2020089390-appb-000005

在本发明的一个实施方式中，R 16为-NHC(O)R 18，其中R 18选自：取代或未取代的烯基(如乙烯基、1-丙烯基、1-丁烯基)，该烯基上的取代基可选自：芳香族或非芳香族杂环基(如吡咯烷基、哌啶基)、烷氧基(如甲氧基、乙氧基)和取代的氨基(如甲氨基、二甲氨基)。 In one embodiment of the present invention, R 16 is -NHC(O)R 18 , wherein R 18 is selected from: substituted or unsubstituted alkenyl (such as vinyl, 1-propenyl, 1-butenyl), The substituents on the alkenyl group can be selected from: aromatic or non-aromatic heterocyclic groups (such as pyrrolidinyl, piperidinyl), alkoxy (such as methoxy, ethoxy) and substituted amino (such as Methylamino, dimethylamino).

在本发明的一个具体实施方式中，R 16选自：4-甲氧基-2-丁烯酰胺基、4-吡咯烷基-2-丁烯酰胺基、4-二甲氨基-2-丁烯酰胺基和4-哌啶基-2-丁烯酰胺基。 In a specific embodiment of the present invention, R 16 is selected from: 4-methoxy-2-butenamido, 4-pyrrolidin-2-butenamido, 4-dimethylamino-2-butene Enamido and 4-piperidinyl-2-butenamido.

在本发明的一个实施例中，所述化合物具有如下结构：In an embodiment of the present invention, the compound has the following structure:

Figure PCTCN2020089390-appb-000006
Figure PCTCN2020089390-appb-000006

在本发明的另一个实施方式中，式Ⅰ中，A环为单杂原子五元芳环，R 1为取代苯基，所述化合物具有通式Ⅲ所示结构： In another embodiment of the present invention, in formula I, ring A is a single heteroatom five-membered aromatic ring, R 1 is a substituted phenyl group, and the compound has the structure shown in formula III:

Figure PCTCN2020089390-appb-000007
Figure PCTCN2020089390-appb-000007

式Ⅲ中，In formula Ⅲ,

X为-O-、-S-、-N(R 29)-或-(CHR 29) n-，n为0或1； X is -O-, -S-, -N(R 29 )- or -(CHR 29 ) n -, n is 0 or 1;

Y为-O-、-S-或-N(R 29)-； Y is -O-, -S- or -N(R 29 )-;

Z为-O-、-S-或-N(R 29)-； Z is -O-, -S- or -N(R 29 )-;

R 21、R 22和R 23独立地选自：氢、烷基、环烷基、烯基、炔基、芳基、芳烷基、芳香族或非芳香族杂环基、杂环烷基、-COR 29、-C(O)OR 29、-C(O)NR 29R 28、-CH＝NR 29、-CN、-OR 29、-OC(O)R 29、-S(O) t-R 29、-NR 29R 210、-NR 29C(O)R 210、-NO 2、-N＝CR 29R 210和卤素； R 21 , R 22 and R 23 are independently selected from: hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, aromatic or non-aromatic heterocyclic group, heterocycloalkyl, -COR 29 , -C(O)OR 29 , -C(O)NR 29 R 28 , -CH=NR 29 , -CN, -OR 29 , -OC(O)R 29 , -S(O) t- R 29 , -NR 29 R 210 , -NR 29 C(O)R 210 , -NO 2 , -N=CR 29 R 210 and halogen;

R 24、R 25和R 26独立地选自：氢、烷基、环烷基、烯基、炔基、芳基、芳烷基、芳香族或非芳香族杂环基、杂环烷基、-COR 29、-C(O)OR 29、-C(O)NR 29R 28、-CH＝NR 29、-CN、-OR 29、-OC(O)R 29、-S(O) t-R 29、-NR 29R 210、-NR 29C(O)R 210、-NO 2、-N＝CR 29R 210和卤素； R 24 , R 25 and R 26 are independently selected from: hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, aromatic or non-aromatic heterocyclic group, heterocycloalkyl, -COR 29 , -C(O)OR 29 , -C(O)NR 29 R 28 , -CH=NR 29 , -CN, -OR 29 , -OC(O)R 29 , -S(O) t- R 29 , -NR 29 R 210 , -NR 29 C(O)R 210 , -NO 2 , -N=CR 29 R 210 and halogen;

R 27和R 28独立地选自：氢、烷基、环烷基、烯基、炔基、芳基、芳烷基、芳香族或非芳香族杂环基、杂环烷基、-COR 29、-C(O)OR 29、-C(O)NR 29R 28、-CH＝NR 29、-CN、-OR 29、-OC(O)R 29、-S(O) t-R 29、-NR 29R 210、-NR 29C(O)R 210、-NO 2、-N＝CR 29R 210和卤素；R 27和R 28与其所连接的杂环形成任意取代的稠环体系； R 27 and R 28 are independently selected from: hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, aromatic or non-aromatic heterocyclic group, heterocycloalkyl, -COR 29 , -C(O)OR 29 , -C(O)NR 29 R 28 , -CH=NR 29 , -CN, -OR 29 , -OC(O)R 29 , -S(O) t -R 29 , -NR 29 R 210 , -NR 29 C(O)R 210 , -NO 2 , -N=CR 29 R 210 and halogen; R 27 and R 28 and the heterocyclic ring to which they are connected form an optionally substituted fused ring system;

t为1、2或3；t is 1, 2 or 3;

R 29和R 210独立地选自：氢、烷基、环烷基、烯基、芳基、芳香族或非芳香族杂环基、烷氧基、芳氧基、卤素和氨基； R 29 and R 210 are independently selected from: hydrogen, alkyl, cycloalkyl, alkenyl, aryl, aromatic or non-aromatic heterocyclic group, alkoxy, aryloxy, halogen and amino;

并且，式Ⅲ所示的化合物结构中含有至少一个酸性取代基，该酸性取代基含有至少一个酸性官能团。所述酸性官能团和酸性取代基具有本发明上述定义。In addition, the structure of the compound represented by formula III contains at least one acidic substituent, and the acidic substituent contains at least one acidic functional group. The acidic functional group and acidic substituent have the above-mentioned definition of the present invention.

在本发明的一个实施方式中，R 21、R 22、R 23、R 24、R 25、R 26、R 27和R 28中至少一个含有酸性官能团。 In one embodiment of the present invention, at least one of R 21 , R 22 , R 23 , R 24 , R 25 , R 26 , R 27 and R 28 contains an acidic functional group.

在本发明的一个具体实施方式中，R 26含有至少一个酸性官能团。 In a specific embodiment of the present invention, R 26 contains at least one acidic functional group.

在本发明的一个具体实施方式中，R 26选自：羟基苯基、羧基苯基、磺酸基苯基、磷酸基苯基、羟基苯甲酰氧基取代烷基、磺酸基取代烷基、羧基取代烷基、磷酸基取代烷基、磺酸基苯基取代烷基、羧基苯基取代烷基、磷酸基苯基取代烷基、羟基苯甲酰氧基取代烷氧基、磺酸基取代烷氧基、羧基取代烷氧基、磷酸基取代烷氧基、磺酸基苯基取代烷氧基、羧基苯基取代烷氧基和磷酸基苯基取代烷氧基。 In a specific embodiment of the present invention, R 26 is selected from the group consisting of: hydroxyphenyl, carboxyphenyl, sulfonate phenyl, phosphate phenyl, hydroxybenzoyloxy substituted alkyl, sulfonate substituted alkyl , Carboxyl substituted alkyl, phosphate substituted alkyl, sulfonate phenyl substituted alkyl, carboxyphenyl substituted alkyl, phosphate phenyl substituted alkyl, hydroxybenzoyloxy substituted alkoxy, sulfonic acid group Substituted alkoxy, carboxyl substituted alkoxy, phosphoric acid substituted alkoxy, sulfonic phenyl substituted alkoxy, carboxyphenyl substituted alkoxy, and phosphoric phenyl substituted alkoxy.

在本发明的一个实施例中，R 26为

Figure PCTCN2020089390-appb-000008
In one embodiment of the present invention, R 26 is
Figure PCTCN2020089390-appb-000008

在本发明的一个实施例中，X为-NH-。In one embodiment of the present invention, X is -NH-.

在本发明的另一个实施例中，X为-O-。In another embodiment of the present invention, X is -O-.

在本发明的一个实施例中，Y为-S-。In one embodiment of the present invention, Y is -S-.

在本发明的一个实施例中，Z为-NH-。In one embodiment of the present invention, Z is -NH-.

在本发明的一个实施方式中，R 21和R 23独立地选自：氢、烷基和卤素。 In one embodiment of the present invention, R 21 and R 23 are independently selected from: hydrogen, alkyl and halogen.

在本发明的一个实施例中，R 21为氢。 In one embodiment of the present invention, R 21 is hydrogen.

在本发明的一个实施例中，R 23为氢。 In one embodiment of the present invention, R 23 is hydrogen.

在本发明的一个实施方式中，R 22为-NHC(O)R 210、其中，R 210为取代或未取代的烯基，如乙烯基、3-丙烯基。具体地，R 22可为

Figure PCTCN2020089390-appb-000009
In one embodiment of the present invention, R 22 is -NHC(O)R 210 , wherein R 210 is a substituted or unsubstituted alkenyl group, such as vinyl and 3-propenyl. Specifically, R 22 can be
Figure PCTCN2020089390-appb-000009

在本发明的一个实施方式中，R 24为杂环基，如取代或未取代的含氮非芳香杂环基(具体如，哌嗪基、哌啶基)，其上取代基选自：烷基(如甲基、乙基)、酰基(如乙酰基、丙酰基)、杂环基(如吡咯烷基、哌啶基、哌嗪基等)，所述取代位点为含氮非芳香杂环基的氮位。 In one embodiment of the present invention, R 24 is a heterocyclic group, such as a substituted or unsubstituted nitrogen-containing non-aromatic heterocyclic group (specifically, piperazinyl, piperidinyl), and the substituents on it are selected from: alkane Group (such as methyl, ethyl), acyl (such as acetyl, propionyl), heterocyclic group (such as pyrrolidinyl, piperidinyl, piperazinyl, etc.), the substitution site is a nitrogen-containing non-aromatic hetero The nitrogen position of the ring group.

在本发明的一个具体实施方式中，R 24选自：4-(1-乙酰基)哌嗪基、4-(1-甲基)哌嗪基、4-(1-吡咯烷基)哌嗪基、4-(1-哌啶基)哌嗪基和4-甲基哌嗪基-1-哌啶基。 In a specific embodiment of the present invention, R 24 is selected from: 4-(1-acetyl)piperazinyl, 4-(1-methyl)piperazinyl, 4-(1-pyrrolidinyl)piperazine Group, 4-(1-piperidinyl)piperazinyl and 4-methylpiperazinyl-1-piperidinyl.

在本发明的一个实施例中，R 24为

Figure PCTCN2020089390-appb-000010
In one embodiment of the present invention, R 24 is
Figure PCTCN2020089390-appb-000010

在本发明的另一个实施方式中，R 24为-NR 27R 28，R 27和R 28独立地选自：氢和取代或未取代的烷基(如甲基、3-(甲氨基)丙基)。 In another embodiment of the present invention, R 24 is -NR 27 R 28 , R 27 and R 28 are independently selected from: hydrogen and substituted or unsubstituted alkyl (such as methyl, 3-(methylamino)propyl base).

在本发明的一个实施例中，R 24为N,N-二甲氨基或N 1,N 1,N 2-三甲基乙二胺基。 In an embodiment of the present invention, R 24 is N,N-dimethylamino or N 1 ,N 1 ,N 2 -trimethylethylenediamino.

在本发明的一个实施例中，R 25为氢。 In one embodiment of the present invention, R 25 is hydrogen.

在本发明的一个实施方式中，R 27和R 28独立地选自：氢、烷基和卤素。 In one embodiment of the present invention, R 27 and R 28 are independently selected from: hydrogen, alkyl, and halogen.

在本发明的一个实施例中，R 27为氢。 In one embodiment of the present invention, R 27 is hydrogen.

在本发明的一个实施例中，R 28为氢。 In one embodiment of the present invention, R 28 is hydrogen.

在本发明的一个实施例中，所述化合物具有如下结构：In an embodiment of the present invention, the compound has the following structure:

Figure PCTCN2020089390-appb-000011
Figure PCTCN2020089390-appb-000011

在本发明的另一个实施方式中，式Ⅰ中，A环不存在，R 1为取代苯基，所述化合物具有通式Ⅳ所示结构： In another embodiment of the present invention, in formula I, ring A does not exist, R 1 is substituted phenyl, and the compound has the structure shown in formula IV:

Figure PCTCN2020089390-appb-000012
Figure PCTCN2020089390-appb-000012

式Ⅳ中，In formula IV,

X为-O-、-S-、-N(R 39)-或-(CHR 39) n-，n为0或1； X is -O-, -S-, -N(R 39 )- or -(CHR 39 ) n -, n is 0 or 1;

Z为-O-、-S-或-N(R 39)-； Z is -O-, -S- or -N(R 39 )-;

R 31为氢、取代或未取代的芳基或杂环基； R 31 is hydrogen, substituted or unsubstituted aryl or heterocyclic group;

R 32、R 33、R 34、R 35和R 36独立地选自：氢、烷基、环烷基、烯基、炔基、芳基、芳烷基、芳香族或非芳香族杂环基、杂环烷基、-COR 39、-C(O)OR 39、-C(O)NR 39R 310、-CH＝NR 39、-CN、-OR 39、-OC(O)R 39、-S(O) t-R 39、-NR 39R 310、-NR 39C(O)R 310、-NO 2、-N＝CR 39R 310和卤素； R 32 , R 33 , R 34 , R 35 and R 36 are independently selected from: hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, aromatic or non-aromatic heterocyclic group , Heterocycloalkyl, -COR 39 , -C(O)OR 39 , -C(O)NR 39 R 310 , -CH=NR 39 , -CN, -OR 39 , -OC(O)R 39 ,- S(O) t -R 39 , -NR 39 R 310 , -NR 39 C(O)R 310 , -NO 2 , -N=CR 39 R 310 and halogen;

R 37和R 38独立地选自：氢、烷基、环烷基、烯基、炔基、芳基、芳烷基、芳香族或非芳香族杂环基、杂环烷基、-COR 39、-C(O)OR 39、-C(O)NR 39R 310、-CH＝NR 39、-CN、-OR 39、-OC(O)R 39、-S(O) t-R 39、-NR 39R 310、-NR 39C(O)R 310、-NO 2、-N＝CR 39R 310和卤素； R 37 and R 38 are independently selected from: hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, aromatic or non-aromatic heterocyclic group, heterocycloalkyl, -COR 39 , -C(O)OR 39 , -C(O)NR 39 R 310 , -CH=NR 39 , -CN, -OR 39 , -OC(O)R 39 , -S(O) t -R 39 , -NR 39 R 310 , -NR 39 C(O)R 310 , -NO 2 , -N=CR 39 R 310 and halogen;

t为1、2或3；t is 1, 2 or 3;

R 39和R 310独立地选自：氢、烷基、环烷基、烯基、芳基、芳香族或非芳香族杂环基、烷氧基、芳氧基、卤素和氨基； R 39 and R 310 are independently selected from: hydrogen, alkyl, cycloalkyl, alkenyl, aryl, aromatic or non-aromatic heterocyclic group, alkoxy, aryloxy, halogen and amino;

并且，式Ⅳ所示的化合物结构中含有至少一个酸性取代基，该酸性取代基含有至少一个酸性官能团。所述酸性官能团和酸性取代基具有本发明上述定义。In addition, the structure of the compound represented by formula IV contains at least one acidic substituent, and the acidic substituent contains at least one acidic functional group. The acidic functional group and acidic substituent have the above-mentioned definition of the present invention.

在本发明的一个实施方式中，R 31、R 32、R 33、R 34、R 35、R 36、R 37和R 38中至少一个含有酸性官能团。 In one embodiment of the present invention, at least one of R 31 , R 32 , R 33 , R 34 , R 35 , R 36 , R 37 and R 38 contains an acidic functional group.

在本发明的一个具体实施方式中，R 36含有至少一个酸性官能团。 In a specific embodiment of the present invention, R 36 contains at least one acidic functional group.

在本发明的一个具体实施方式中，R 36选自：羟基苯基、羧基苯基、磺酸基苯基、磷酸基苯基、羟基苯甲酰氧基取代烷基、磺酸基取代烷基、羧基取代烷基、磷酸基取代烷基、磺酸基苯基取代烷基、羧基 苯基取代烷基、磷酸基苯基取代烷基、羟基苯甲酰氧基取代烷氧基、磺酸基取代烷氧基、羧基取代烷氧基、磷酸基取代烷氧基、磺酸基苯基取代烷氧基、羧基苯基取代烷氧基和磷酸基苯基取代烷氧基。 In a specific embodiment of the present invention, R 36 is selected from: hydroxyphenyl, carboxyphenyl, sulfonate phenyl, phosphate phenyl, hydroxybenzoyloxy substituted alkyl, sulfonate substituted alkyl , Carboxyl substituted alkyl, phosphate substituted alkyl, sulfonate phenyl substituted alkyl, carboxyphenyl substituted alkyl, phosphate phenyl substituted alkyl, hydroxybenzoyloxy substituted alkoxy, sulfonic acid group Substituted alkoxy, carboxyl substituted alkoxy, phosphoric acid substituted alkoxy, sulfonic phenyl substituted alkoxy, carboxyphenyl substituted alkoxy, and phosphoric phenyl substituted alkoxy.

在本发明的一个实施例中，R 36为

Figure PCTCN2020089390-appb-000013
In one embodiment of the present invention, R 36 is
Figure PCTCN2020089390-appb-000013

在本发明的一个实施例中，X为-(CHR 39) n-，n为0，即X不存在。 In an embodiment of the present invention, X is -(CHR 39 ) n -, and n is 0, that is, X does not exist.

在本发明的一个实施例中，Z为-NH-。In one embodiment of the present invention, Z is -NH-.

在本发明的一个实施方式中，R 31为取代的芳基，如取代的苯基，具体如

Figure PCTCN2020089390-appb-000014
其中，R 310为取代或未取代的烯基，如乙烯基、3-丙烯基，烯基上的取代基选自：烷氧基(如甲氧基、乙氧基)、杂环基(如哌啶基、吡咯烷基)和取代的氨基(如甲氨基、二甲氨基)。 In one embodiment of the present invention, R 31 is a substituted aryl group, such as a substituted phenyl group, such as
Figure PCTCN2020089390-appb-000014
Among them, R 310 is a substituted or unsubstituted alkenyl group, such as vinyl, 3-propenyl, and the substituent on the alkenyl group is selected from: alkoxy (such as methoxy, ethoxy), heterocyclic group (such as Piperidinyl, pyrrolidinyl) and substituted amino (such as methylamino, dimethylamino).

在本发明的一个具体实施方式中，R 31选自：3-丙烯酰胺基苯基、3-(4-甲氧基-2-丁烯酰胺基)苯基、3-(4-哌啶基-2-丁烯酰胺基)苯基、3-(4-吡咯烷基-2-丁烯酰胺基)苯基、3-(4-二甲胺基-2-丁烯酰胺基)苯基。 In a specific embodiment of the present invention, R 31 is selected from: 3-acrylamidophenyl, 3-(4-methoxy-2-butenamido)phenyl, 3-(4-piperidinyl -2-butenamido)phenyl, 3-(4-pyrrolidinyl-2-butenamido)phenyl, 3-(4-dimethylamino-2-butenamido)phenyl.

在本发明的另一个实施方式中，R 31为取代或未取代的杂环基，如吡唑[1,5-a]并吡啶基或取代的吲哚基，吲哚基的取代基选自：氢、烷基和酰基。 In another embodiment of the present invention, R 31 is a substituted or unsubstituted heterocyclic group, such as pyrazole [1,5-a] pyridyl or substituted indolyl, and the substituent of indolyl is selected from : Hydrogen, alkyl and acyl.

在本发明的一个实施例中，R 31为

Figure PCTCN2020089390-appb-000015
In an embodiment of the present invention, R 31 is
Figure PCTCN2020089390-appb-000015

在本发明的一个实施例中，R 32为氢。 In one embodiment of the present invention, R 32 is hydrogen.

在本发明的一个实施方式中，R 33为

Figure PCTCN2020089390-appb-000016
其中X 3为-NH-或-O-，R 39为取代或未取代的烯基，如乙烯基、3-丙烯基，烯基上的取代基选自：烷氧基(如甲氧基、乙氧基)、杂环基(如哌啶基、吡咯烷基)和取代的氨基(如甲氨基、二甲氨基)。 In one embodiment of the present invention, R 33 is
Figure PCTCN2020089390-appb-000016
Wherein X 3 is -NH- or -O-, R 39 is substituted or unsubstituted alkenyl, such as vinyl, 3-propenyl, and the substituents on the alkenyl are selected from: alkoxy (such as methoxy, Ethoxy), heterocyclic groups (such as piperidinyl, pyrrolidinyl), and substituted amino groups (such as methylamino, dimethylamino).

在本发明的一个具体实施方式中，R 33选自：丙烯酰氧基、丙烯酰胺基、4-甲氧基-2-丁烯酰氧基、4-甲氧基-2-丁烯酰胺基、4-哌啶基-2-丁烯酰氧基、4-哌啶基-2-丁烯酰胺基、4-吡咯烷基-2-丁烯酰氧基、4-吡咯烷基-2-丁烯酰胺基、4-二甲胺基-2-丁烯酰氧基和4-二甲胺基-2-丁烯酰胺基。 In a specific embodiment of the present invention, R 33 is selected from: acryloxy group, acrylamido group, 4-methoxy-2-butenoyloxy group, 4-methoxy-2-butenamido group , 4-piperidinyl-2-butenoyloxy, 4-piperidinyl-2-butenamido, 4-pyrrolidinyl-2-butenoyloxy, 4-pyrrolidinyl-2- Butenamido, 4-dimethylamino-2-butenoyloxy and 4-dimethylamino-2-butenamido.

在本发明的一个实施例中，R 33为

Figure PCTCN2020089390-appb-000017
In an embodiment of the present invention, R 33 is
Figure PCTCN2020089390-appb-000017

在本发明的一个实施例中，R 35为氢。 In one embodiment of the present invention, R 35 is hydrogen.

在本发明的一个实施方式中，R 34为取代或未取代的杂环基，如取代的含氮非芳香杂环基(具体如，哌嗪基、哌啶基)，其上取代基选自：烷基(如甲基、乙基)、酰基(如乙酰基、丙酰基)、杂环基(如吡咯烷基、哌啶基、哌嗪基等)，所述取代位点为含氮非芳香杂环基的氮位。 In one embodiment of the present invention, R 34 is a substituted or unsubstituted heterocyclic group, such as a substituted nitrogen-containing non-aromatic heterocyclic group (specifically, piperazinyl, piperidinyl), and the substituents on it are selected from : Alkyl (such as methyl, ethyl), acyl (such as acetyl, propionyl), heterocyclic group (such as pyrrolidinyl, piperidinyl, piperazinyl, etc.), the substitution site is a nitrogen-containing non The nitrogen position of the aromatic heterocyclic group.

在本发明的一个具体实施方式中，R 34选自：4-(1-乙酰基)哌嗪基、4-(1-甲基)哌嗪基、4-(1-吡咯烷基)哌嗪基、4-(1-哌啶基)哌嗪基和4-甲基哌嗪基-1-哌啶基。 In a specific embodiment of the present invention, R 34 is selected from: 4-(1-acetyl)piperazinyl, 4-(1-methyl)piperazinyl, 4-(1-pyrrolidinyl)piperazine Group, 4-(1-piperidinyl)piperazinyl and 4-methylpiperazinyl-1-piperidinyl.

在本发明的另一个实施方式中，R 34为-NR 27R 28，R 27和R 28独立地选自：氢和取代或未取代的烷基(如甲基、3-(甲氨基)丙基)。 In another embodiment of the present invention, R 34 is -NR 27 R 28 , R 27 and R 28 are independently selected from: hydrogen and substituted or unsubstituted alkyl (such as methyl, 3-(methylamino)propyl) base).

在本发明的一个具体实施方式中，R 34为N,N-二甲氨基或N 1,N 1,N 2-三甲基乙二胺基。 In a specific embodiment of the present invention, R 34 is N,N-dimethylamino or N 1 ,N 1 ,N 2 -trimethylethylenediamino.

在本发明的一个实施例中，R 34为

Figure PCTCN2020089390-appb-000018
In an embodiment of the present invention, R 34 is
Figure PCTCN2020089390-appb-000018

在本发明的一个实施方式中，R 37和R 38独立地选自：氢、卤素、硝基、氰基和三氟甲基。 In one embodiment of the present invention, R 37 and R 38 are independently selected from: hydrogen, halogen, nitro, cyano and trifluoromethyl.

在本发明的一个实施例中，R 37为氢。 In one embodiment of the invention, R 37 is hydrogen.

在本发明的一个实施例中，R 38为氢。 In one embodiment of the present invention, R 38 is hydrogen.

在本发明的一个实施例中，所述化合物具有如下结构：In an embodiment of the present invention, the compound has the following structure:

Figure PCTCN2020089390-appb-000019
Figure PCTCN2020089390-appb-000019

本发明还提供一种上述化合物的制备方法，其包括向具有EGFR抑制活性的化合物(例如，已知EGFR TKI化合物)结构中通过反应引入至少一个酸性官能团的步骤。The present invention also provides a method for preparing the above compound, which includes the step of introducing at least one acidic functional group into the structure of a compound having EGFR inhibitory activity (for example, a known EGFR TKI compound) by reaction.

在本发明的一个实施方式中，酸性官能团选自：酚羟基、羧基、磺酸基和磷酸基中的一种或多种。In an embodiment of the present invention, the acidic functional group is selected from one or more of phenolic hydroxyl group, carboxyl group, sulfonic acid group and phosphoric acid group.

在本发明的一个实施方式中，上述制备方法包括通过反应引入一个或多个选自以下的基团：羟基苯基、羧基苯基、磺酸基苯基、磷酸基苯基、羟基苯甲酰氧基取代烷基、磺酸基取代烷基、羧基取代烷基、磷酸基取代烷基、磺酸基苯基取代烷基、羧基苯基取代烷基、磷酸基苯基取代烷基、羟基苯甲酰氧基取代烷氧基、磺酸基取代烷氧基、羧基取代烷氧基、磷酸基取代烷氧基、磺酸基苯基取代烷氧基、羧基苯基取代烷氧基和磷酸基苯基取代烷氧基。In one embodiment of the present invention, the above preparation method includes introducing one or more groups selected from the group consisting of: hydroxyphenyl, carboxyphenyl, sulfophenyl, phosphorophenyl, hydroxybenzoyl Oxygen substituted alkyl, sulfonic acid substituted alkyl, carboxy substituted alkyl, phosphate substituted alkyl, sulfonic phenyl substituted alkyl, carboxyphenyl substituted alkyl, phosphate phenyl substituted alkyl, hydroxybenzene Formyloxy substituted alkoxy, sulfonic acid substituted alkoxy, carboxy substituted alkoxy, phosphoric acid substituted alkoxy, sulfonic phenyl substituted alkoxy, carboxyphenyl substituted alkoxy and phosphoric acid Phenyl replaces alkoxy.

在本发明的一个实施方式中，上述制备方法中，EGFR TKI化合物为吉非替尼。In one embodiment of the present invention, in the above preparation method, the EGFR TKI compound is gefitinib.

在本发明的一个实施例中，上述制备方法包括如下反应步骤：In an embodiment of the present invention, the above preparation method includes the following reaction steps:

Figure PCTCN2020089390-appb-000020
Figure PCTCN2020089390-appb-000020

其中，X、R 11、R 12、R 13、R 14、R 16具有本发明上述定义， Wherein, X, R 11 , R 12 , R 13 , R 14 , and R 16 have the above definitions of the present invention,

Ra为卤原子，Ra is a halogen atom,

Rp为羟基保护基团；Rp is a hydroxyl protecting group;

第2步反应为羟基脱保护反应。The second step is the hydroxyl deprotection reaction.

在本发明的另一个实施例中，上述制备方法包括如下反应步骤：In another embodiment of the present invention, the above preparation method includes the following reaction steps:

Figure PCTCN2020089390-appb-000021
Figure PCTCN2020089390-appb-000021

其中，X、Y、Z、R 21、R 22、R 23、R 24、R 25、R 27、R 28具有本发明上述定义， Among them, X, Y, Z, R 21 , R 22 , R 23 , R 24 , R 25 , R 27 , R 28 have the above definitions of the present invention,

Ra为卤原子，Ra is a halogen atom,

Rp为羟基保护基团；Rp is a hydroxyl protecting group;

第2步反应为羟基脱保护反应。The second step is the hydroxyl deprotection reaction.

在本发明的另一个实施例中，上述制备方法包括如下反应步骤：In another embodiment of the present invention, the above preparation method includes the following reaction steps:

Figure PCTCN2020089390-appb-000022
Figure PCTCN2020089390-appb-000022

其中，X、Z、R 31、R 32、R 33、R 34、R 35、R 37、R 38具有本发明上述定义， Wherein, X, Z, R 31 , R 32 , R 33 , R 34 , R 35 , R 37 , R 38 have the above definition of the present invention,

Ra为卤原子，Ra is a halogen atom,

Rp为羟基保护基团；Rp is a hydroxyl protecting group;

第2步反应为羟基脱保护反应。The second step is the hydroxyl deprotection reaction.

为实现羟基保护和脱保护的目的，所述羟基保护基团和羟基脱保护反应可选择本领域常规的羟基保护基团和羟基脱保护反应，本发明对此不作具体限定。In order to achieve the purpose of protecting and deprotecting the hydroxyl group, the hydroxyl protecting group and the hydroxyl deprotection reaction can be selected from the conventional hydroxyl protecting group and hydroxyl deprotection reaction in the art, which is not specifically limited in the present invention.

本发明还提供一种改造EGFR TKI的方法，其包括减弱EGFR TKI的碱性的步骤，例如，在该EGFR TKI的结构中引入至少一个酸性官能团的步骤。The present invention also provides a method for transforming EGFR TKI, which includes the step of reducing the basicity of EGFR TKI, for example, the step of introducing at least one acidic functional group into the structure of the EGFR TKI.

所述酸性官能团具有本发明上述定义。The acidic functional group has the above definition of the present invention.

本发明还提供一种上述化合物或其药学上可接受的盐的立体异构体、几何异构体、互变异构体、消旋体、溶剂化物、水合物、代谢前体和前药。The present invention also provides stereoisomers, geometric isomers, tautomers, racemates, solvates, hydrates, metabolic precursors, and prodrugs of the above-mentioned compound or a pharmaceutically acceptable salt thereof.

本发明还提供一种药物组合物，其包含上述化合物或其药学上可接受的盐、立体异构体、几何异构体、互变异构体、消旋体、溶剂化物、水合物、代谢前体或前药，以及一种或多种药学上可接受的辅料。The present invention also provides a pharmaceutical composition comprising the above compound or a pharmaceutically acceptable salt, stereoisomer, geometric isomer, tautomer, racemate, solvate, hydrate, metabolite Precursors or prodrugs, and one or more pharmaceutically acceptable excipients.

所述药学上可接受的载体是指药学领域常规的药物载体。例如：稀释剂、赋形剂如水等，填充剂如淀粉、蔗糖等；粘合剂如纤维素衍生物、藻酸盐、明胶和聚乙烯吡咯烷酮等；湿润剂如甘油等；崩解剂如琼脂、碳酸钙和碳酸氢钠等；吸收促进剂如季铵化合物等；表面活性剂如十六烷醇等；吸附载体如高岭土和皂粘土等；润滑剂如滑石粉、硬脂酸钙和镁、聚乙二醇等。另外，还可以在组合物中加入其它辅剂如香味剂、甜味剂等。The pharmaceutically acceptable carrier refers to a conventional drug carrier in the pharmaceutical field. For example: diluents, excipients such as water, fillers such as starch, sucrose, etc.; binders such as cellulose derivatives, alginate, gelatin and polyvinylpyrrolidone, etc.; wetting agents such as glycerin, etc.; disintegrating agents such as agar , Calcium carbonate and sodium bicarbonate, etc.; absorption enhancers such as quaternary ammonium compounds, etc.; surfactants such as cetyl alcohol, etc.; adsorption carriers such as kaolin and bentonite, etc.; lubricants such as talc, calcium and magnesium stearate, Polyethylene glycol and so on. In addition, other adjuvants such as flavoring agents and sweetening agents can also be added to the composition.

本发明所述的药物组合物可以为片剂(包括糖衣片剂、膜包衣片剂、舌下片剂、口腔崩解片、口腔片剂等等)、丸剂、粉剂、颗粒剂、胶囊剂(包括软胶囊、微胶囊)、锭剂、糖浆剂、液体、乳剂、混悬剂、控制释放制剂(例如，瞬时释放制剂、缓释制剂、缓释微囊)、气雾剂、膜剂(例如，口服崩解膜剂、口腔粘膜-粘附膜剂)、注射剂(例如，皮下注射、静脉注射、肌内注射、腹膜内注射)、静脉滴注剂、透皮吸收制剂、软膏剂、洗剂、粘附制剂、栓剂(例如，直肠栓剂、阴道栓剂)、小药丸、鼻制剂、肺制剂(吸入剂)、眼睛滴剂等等、口服或胃肠外制剂(例如，通过静脉内、肌内、皮下、器官内、鼻内、皮内、滴注、脑内、直肠内等给药形式，给药至肿瘤的附近和直接给药至病变处)。在本发明的一个实施方式中，所述的药物组合物为注射剂。The pharmaceutical composition of the present invention can be tablets (including sugar-coated tablets, film-coated tablets, sublingual tablets, orally disintegrating tablets, buccal tablets, etc.), pills, powders, granules, capsules (Including soft capsules, microcapsules), lozenges, syrups, liquids, emulsions, suspensions, controlled release formulations (e.g., instant release formulations, sustained release formulations, sustained release microcapsules), aerosols, films ( For example, oral disintegrating film, oral mucosa-adhesive film), injections (for example, subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection), intravenous drip, transdermal absorption preparation, ointment, lotion Preparations, adhesive preparations, suppositories (for example, rectal suppositories, vaginal suppositories), pellets, nasal preparations, lung preparations (inhalants), eye drops, etc., oral or parenteral preparations (for example, intravenous, intramuscular Intradermal, subcutaneous, intra-organ, intranasal, intradermal, drip, intracerebral, intrarectal and other forms of administration, administered to the vicinity of the tumor and directly to the lesion). In one embodiment of the present invention, the pharmaceutical composition is an injection.

本发明的药物组合物的各种剂型可以按照药学领域的常规生产方法制备。例如使活性成分与一种或多种辅料混合，然后将其制成所需的剂型。Various dosage forms of the pharmaceutical composition of the present invention can be prepared according to conventional production methods in the pharmaceutical field. For example, the active ingredient is mixed with one or more excipients and then made into the desired dosage form.

本发明的药物组合物优选含有重量比为0.1-99.5％的活性成分，更优选含有重量比为0.5-95％的活性成分。The pharmaceutical composition of the present invention preferably contains the active ingredient in a weight ratio of 0.1-99.5 %, and more preferably contains the active ingredient in a weight ratio of 0.5-95%.

本发明化合物的施用量可根据用药途径、患者的年龄、体重、所治疗的疾病的类型和严重程度等变化，可以一次或多次施用。The dosage of the compound of the present invention may vary according to the route of administration, the age, weight of the patient, the type and severity of the disease to be treated, etc., and may be administered one or more times.

本发明还提供一种上述化合物或其药学上可接受的盐、立体异构体、几何异构体、互变异构体、消旋体、溶剂化物、水合物、代谢前体和前药在制备预防和/或治疗与受体酪氨酸蛋白激酶有关的疾病和病症的药物中的应用。The present invention also provides a compound or a pharmaceutically acceptable salt, stereoisomer, geometric isomer, tautomer, racemate, solvate, hydrate, metabolic precursor and prodrug thereof Application in preparing medicines for preventing and/or treating diseases and disorders related to receptor tyrosine protein kinases.

本发明还提供一种预防和/或治疗与受体酪氨酸蛋白激酶有关的疾病和病症的方法，其包括将本发明所述化合物或其药学上可接受的盐、立体异构体、几何异构体、互变异构体、消旋体、溶剂化物、水合物、代谢前体和前药、或所述药物组合物施用至由此需求的个体的步骤。The present invention also provides a method for preventing and/or treating diseases and disorders related to receptor tyrosine protein kinase, which comprises combining the compound of the present invention or a pharmaceutically acceptable salt, stereoisomer, geometric Isomers, tautomers, racemates, solvates, hydrates, metabolic precursors and prodrugs, or steps of administering the pharmaceutical composition to an individual in need thereof.

所述与受体酪氨酸蛋白激酶有关的疾病和病症为肿瘤，其包括但不限于：肺癌、胰腺癌、乳腺癌或结肠癌，特别是肺癌，更特别是非小细胞肺癌。The diseases and disorders related to receptor tyrosine protein kinases are tumors, which include, but are not limited to: lung cancer, pancreatic cancer, breast cancer or colon cancer, particularly lung cancer, and more particularly non-small cell lung cancer.

本发明的发明人经过大量实验首创性地发现EGFR TKIs的抗肿瘤效果和其自身的酸碱性密切相关，目前临床上使用的EGFR TKI(如吉非替尼和AZD9291)会使肿瘤细胞溶酶体酸化过程减弱，进而造成促癌蛋白SQSTM1的积累，最终导致肿瘤获得性耐药性的产生。发明人通过减弱EGFR TKI的碱性的方式，开发了一系列EGFR TKI化合物，其可有效改善由SQSTM1积聚引起的EGFR TKI获得性耐药性，对提高EGFR TKI的临床治疗收益具有重要意义。After a large number of experiments, the inventors of the present invention firstly discovered that the anti-tumor effect of EGFR TKIs is closely related to its own acidity and alkalinity. The EGFR TKIs currently used in clinical practice (such as gefitinib and AZD9291) can cause tumor cell lysis The acidification process of the body is weakened, which in turn causes the accumulation of the oncoprotein SQSTM1, and ultimately leads to the generation of tumor-acquired drug resistance. The inventors developed a series of EGFR TKI compounds by reducing the basicity of EGFR TKI, which can effectively improve the acquired drug resistance of EGFR TKI caused by the accumulation of SQSTM1, which is of great significance for improving the clinical treatment benefits of EGFR TKI.

附图说明Description of the drawings

图1所示为实施例1中用吉非替尼处理后的A549、HCC827和H460细胞中SQSTM1表达量的测定结果。Figure 1 shows the measurement results of SQSTM1 expression in A549, HCC827 and H460 cells treated with gefitinib in Example 1.

图2所示为实施例1中用AZD9291处理后的A549、HCC827和H460细胞中SQSTM1表达量的测定结果。Figure 2 shows the measurement results of SQSTM1 expression in A549, HCC827 and H460 cells treated with AZD9291 in Example 1.

图3所示为实施例2中A549和H460细胞中LC3和SQSTM1表达量的测定结果。Figure 3 shows the measurement results of LC3 and SQSTM1 expression in A549 and H460 cells in Example 2.

图4所示为实施例3的检测结果；图4A为免疫荧光显微镜检测GFP-LC3和RFP-LC3的斑点照片，其中，第一列图中斑点均为绿色荧光斑点，第二列图中斑点均为红色荧光斑点，第三列图中斑点均为黄色荧光斑点；图4B所示为各实验组LC3黄色斑点/红色斑点(％)的结果。Fig. 4 shows the detection result of Example 3; Fig. 4A is a photo of GFP-LC3 and RFP-LC3 spots detected by immunofluorescence microscope. The spots in the first column are all green fluorescent spots, and the spots in the second column are They are all red fluorescent spots, and the spots in the third column are all yellow fluorescent spots; Figure 4B shows the results of LC3 yellow spots/red spots (%) in each experimental group.

图5所示为实施例4的检测结果；其中，第一列图表示在A549细胞中瞬转SQSTM1-Flag的质粒，接着用抗生素G418筛选得到稳定转染的单克隆细胞团后，用不同浓度的Gefitinib和AZD9291处理48小时，MTS法检测细胞活力的结果。第二列图表示在H460细胞中敲减SQSTM1 48小时后，用不同浓度的Gefitinib和AZD9291处理24小时，MTS法检测细胞活力的结果。Figure 5 shows the test results of Example 4; among them, the first column shows the transient transduction of the SQSTM1-Flag plasmid in A549 cells, followed by screening with antibiotic G418 to obtain stable transfected monoclonal cell clusters, using different concentrations Gefitinib and AZD9291 were treated for 48 hours, and the cell viability was detected by MTS method. The second column of graphs shows the results of cell viability detected by MTS method after 48 hours of knockdown of SQSTM1 in H460 cells, and treatment with different concentrations of Gefitinib and AZD9291 for 24 hours.

图6所示为实施例5的检测结果；图6A为采用吉非替尼和AZD9291处理A549不同时间后，免疫荧光法检测LC3蛋白表达变化的结果；图6B所示为图6A中结果的统计分析，分别统计10个随机视野，标尺为10μm，星号(*)代表P<0.05，为有统计差异。Figure 6 shows the detection results of Example 5; Figure 6A shows the results of immunofluorescence detection of LC3 protein expression changes after treating A549 with gefitinib and AZD9291 for different times; Figure 6B shows the statistics of the results in Figure 6A In analysis, 10 random fields of view were counted, the scale is 10μm, and the asterisk (*) represents P<0.05, which means there is a statistical difference.

图7所示为实施例6的检测结果；其中，图7A为LAMP1和LC3的免疫荧光检测照片，图7B所示为各实验组的共定位系数的统计结果。Figure 7 shows the detection results of Example 6; among them, Figure 7A is the immunofluorescence detection photos of LAMP1 and LC3, and Figure 7B shows the statistical results of the colocalization coefficients of each experimental group.

图8所示为实施例7中吉非替尼、AZD9291、CQ和CQ联用Baf A1处理后A549和HCC827细胞中成熟体CTSB表达量的测定结果。Figure 8 shows the results of determination of CTSB expression in mature body CTSB in A549 and HCC827 cells after treatment with gefitinib, AZD9291, CQ and CQ in combination with Baf A1 in Example 7.

图9所示为实施例8的检测结果；其中，图9A为荧光显微镜检测照片，其中，比例尺为10μM，第一、三行图中的斑点均为红色荧光斑点，第二、四行图片中的斑点均为绿色荧光斑点，图9B所示为各实验组AO-红色斑点的检测结果。Figure 9 shows the detection results of Example 8; among them, Figure 9A is a fluorescence microscope examination photo, in which the scale bar is 10 μM, the spots in the first and third rows are all red fluorescent spots, and in the second and fourth rows The spots are all green fluorescent spots, and Figure 9B shows the detection results of AO-red spots in each experimental group.

图10所示为化合物Ⅱ-1(Gefi-2OH)的合成路径。其中，pKa(碱性)表示化合物中碱性最强的吗啡啉基团的酸度系数，而pKa 1(酸性)、pKa 2(酸性)则分别表示化合物中呈酸性的两个酚羟基的酸度系数。 Figure 10 shows the synthesis route of compound II-1 (Gefi-2OH). Among them, pKa (basic) represents the acidity coefficient of the most basic morpholine group in the compound, while pKa 1 (acidic) and pKa 2 (acidic) respectively represent the acidity coefficients of the two acidic phenolic hydroxyl groups in the compound. .

图11所示为化合物Ⅲ-1的合成路径。Figure 11 shows the synthetic route of compound III-1.

图12所示为化合物Ⅳ-1(AZD-2OH)的合成路径。Figure 12 shows the synthetic route of compound IV-1 (AZD-2OH).

图13所示为实施例13的检测结果。其中，图13A为H460细胞中吉非替尼和化合物Gefi-2OH处理后SQSTM1和成熟体CTSB的表达量的测定结果，图13B为A549和HCC827细胞中吉非替尼和化合物Gefi-2OH处理后，EGFR及其下游Akt、Erk1/2蛋白的磷酸化修饰程度以及其蛋白本身表达量的检测结果。Figure 13 shows the test results of Example 13. Among them, Figure 13A shows the results of determination of the expression levels of SQSTM1 and mature CTSB after treatment with Gefitinib and compound Gefi-2OH in H460 cells. Figure 13B shows the results of EGFR treatment in A549 and HCC827 cells after treatment with Gefitinib and compound Gefi-2OH. And its downstream Akt, Erk1/2 protein phosphorylation modification degree and the detection results of the protein itself.

图14所示同样为实施例13的检测结果。其中，图14A为H460细胞中AZD9291和化合物AZD-2OH 处理后SQSTM1和成熟体CTSB的表达量的测定结果，图14B为HCC827细胞中AZD9291和化合物AZD-2OH处理后，EGFR及其下游Akt、Erk1/2蛋白的磷酸化修饰程度以及其蛋白本身表达量的检测结果。Figure 14 also shows the test results of Example 13. Among them, Figure 14A shows the results of determination of the expression levels of SQSTM1 and mature CTSB after treatment with AZD9291 and compound AZD-2OH in H460 cells, and Figure 14B shows EGFR and its downstream Akt and Erk1 after treatment with AZD9291 and compound AZD-2OH in HCC827 cells. /2 The degree of phosphorylation modification of the protein and the detection result of its protein expression.

图15所示为实施例14的检测结果。其中，左侧为荧光显微镜检测照片，其中，比例尺为10μM，图片中的斑点均为Lysotracker-blue形成的蓝色荧光斑点，右侧所示为各实验组酸性囊泡斑点(蓝色斑点数量)的检测结果。Figure 15 shows the test results of Example 14. Among them, the left side is the fluorescence microscope inspection photo, where the scale bar is 10μM, the spots in the picture are all blue fluorescent spots formed by Lysotracker-blue, and the right side shows the acidic vesicle spots in each experimental group (the number of blue spots) The test results.

图16所示为实施例15的检测结果，其分别为吉非替尼和化合物Gefi-2OH对A549(左侧)和H460细胞(右侧)的增殖抑制作用的检测结果。Figure 16 shows the test results of Example 15, which are the test results of the proliferation inhibitory effects of Gefitinib and the compound Gefi-2OH on A549 (left) and H460 cells (right), respectively.

图17所示同样为实施例15的检测结果，为AZD9291和化合物AZD-2OH对H460细胞的增殖抑制作用的检测结果。Figure 17 shows the test result of Example 15, which is the test result of the inhibitory effect of AZD9291 and compound AZD-2OH on the proliferation of H460 cells.

图18所示为实施例16的检测结果；其中，左侧为流式细胞术检测结果图，右侧所示为各实验组的细胞凋亡率的结果。Figure 18 shows the detection results of Example 16; wherein, the left side is a flow cytometry detection result graph, and the right side shows the results of the apoptosis rate of each experimental group.

具体实施方式Detailed ways

除非另有定义，本文使用的所有的技术和科学术语具有与本发明涉及领域的技术人员通常理解的相同的含义。在描述和要求保护本发明中，将使用以下术语。Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art related to the present invention. In describing and claiming the present invention, the following terms will be used.

“卤素”表示氟、氯、溴或碘，优选为氟或氯。"Halogen" means fluorine, chlorine, bromine or iodine, preferably fluorine or chlorine.

“烷基”是指直链或支链的且不含不饱和键的烃链自由基，典型的烷基含有1-12个、1-8个或1-6个碳原子，如甲基、乙基、正丙基、异丙基、正丁基、叔丁基、正戊基等。如果烷基被芳基取代，那么其相应为“芳烷基”自由基，如苯甲基或苯乙基。如果烷基被杂环基取代，那么其相应为“杂环烷基”自由基。"Alkyl" refers to straight or branched hydrocarbon chain radicals without unsaturated bonds. Typical alkyl groups contain 1-12, 1-8 or 1-6 carbon atoms, such as methyl, Ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, n-pentyl, etc. If the alkyl group is substituted with an aryl group, then the corresponding "aralkyl" radical, such as benzyl or phenethyl. If the alkyl group is substituted by a heterocyclic group, then it corresponds to a "heterocycloalkyl" radical.

“烯基”指的是至少含两个碳原子、至少一个不饱和键的直链或支链的烃链自由基，且该烃链自由基以单键与分子其它部分连接。典型的烯基基团含有2-12个、2-8个或者2-6个碳原子，例如，乙烯基、1-丙烯基或丁烯基。"Alkenyl" refers to a straight or branched hydrocarbon chain radical containing at least two carbon atoms and at least one unsaturated bond, and the hydrocarbon chain radical is connected to other parts of the molecule by a single bond. Typical alkenyl groups contain 2-12, 2-8, or 2-6 carbon atoms, for example, vinyl, 1-propenyl or butenyl.

“炔基”指的是含至少两个碳原子、至少一个碳碳三键的直链或支链的烃链自由基，且该烃链自由基以单键与分子其它部分连接。典型的炔基基团含有2-12个、2-8个或者2-6个碳原子，例如，乙炔基、丙炔基(例如1-丙炔基、2-丙炔基)。"Alkynyl" refers to a straight or branched hydrocarbon chain radical containing at least two carbon atoms and at least one carbon-carbon triple bond, and the hydrocarbon chain radical is connected to other parts of the molecule by a single bond. Typical alkynyl groups contain 2-12, 2-8, or 2-6 carbon atoms, for example, ethynyl, propynyl (eg 1-propynyl, 2-propynyl).

“环烷基”指的是脂环烃。典型的环烷基含1至3个单环和/或稠环、含3-18个碳原子，优选3-10个碳原子，如环丙基、环己基或金刚烷基。在具体实施方式中，环烷基含3-6个碳原子。"Cycloalkyl" refers to an alicyclic hydrocarbon. A typical cycloalkyl group contains 1 to 3 monocyclic and/or fused rings, 3-18 carbon atoms, preferably 3-10 carbon atoms, such as cyclopropyl, cyclohexyl or adamantyl. In a specific embodiment, the cycloalkyl group contains 3-6 carbon atoms.

“芳基”指的是单环或多环自由基，包括含单芳基基团和/或稠芳基基团的多环自由基。典型的芳基基团包含1-3个单环或稠环及6-18个碳环原子，如苯基、萘基(如2-萘基)自由基。"Aryl" refers to monocyclic or polycyclic radicals, including polycyclic radicals containing monoaryl groups and/or condensed aryl groups. Typical aryl groups contain 1-3 monocyclic or fused rings and 6-18 carbon ring atoms, such as phenyl, naphthyl (such as 2-naphthyl) radicals.

“杂环基”指的是稳定的、典型地为3元至18元的环自由基，其由碳原子和一至五个选自氮、氧和硫的杂原子构成，优选为具有一个或多个杂原子的4元至8元环，更优选为具有一个或多个杂原子的5元或6元环，其可以是芳香族的或者非芳香族的。对于本发明的目的，杂环可以是单环、双环或三环体系，其可以包括稠环体系；且杂环自由基中的氮、碳或硫原子可以任选地被氧化；氮原子可以任选地被季胺化；且杂环自由基可以是部分饱和或完全饱和的或为芳族的。杂环基的例子包括但不限于：苯并咪唑、苯并噻唑、呋喃、异噻唑、咪唑、吲哚、哌啶、哌嗪、嘌呤、喹啉、噻二唑、四氢呋喃、香豆素、吗啉、吡咯、吡唑、恶唑、异恶唑、三唑、咪唑等。"Heterocyclyl" refers to a stable, typically 3- to 18-membered cyclic radical consisting of carbon atoms and one to five heteroatoms selected from nitrogen, oxygen and sulfur, preferably with one or more A 4- to 8-membered ring with three heteroatoms, more preferably a 5- or 6-membered ring with one or more heteroatoms, which may be aromatic or non-aromatic. For the purpose of the present invention, the heterocyclic ring may be a monocyclic, bicyclic or tricyclic ring system, which may include a fused ring system; and the nitrogen, carbon or sulfur atoms in the heterocyclic radical may be optionally oxidized; the nitrogen atom may be any It is optionally quaternized; and the heterocyclic radical can be partially saturated or fully saturated or aromatic. Examples of heterocyclic groups include, but are not limited to: benzimidazole, benzothiazole, furan, isothiazole, imidazole, indole, piperidine, piperazine, purine, quinoline, thiadiazole, tetrahydrofuran, coumarin, pyridine Pyroline, pyrrole, pyrazole, oxazole, isoxazole, triazole, imidazole, etc.

“烷氧基”是指式为-OR a的自由基，其中R a为如上定义的烷基自由基，具有一个或多个(如1、2、3或4)氧键，且通常含有1-12个、1-8个或者1-6个碳原子，如甲氧基、乙氧基、丙氧基等。 "Alkoxy" refers to a radical of formula -OR a, wherein R a is an alkyl radical as defined above having one or more (e.g. 2, 3 or 4) oxygen bond, and generally contain from 1 -12, 1-8 or 1-6 carbon atoms, such as methoxy, ethoxy, propoxy, etc.

“芳氧基”是指式为-O-芳基的自由基，其中芳基为如上所定义的。芳氧基化合物的一些例子为-O-苯基、-O-对甲苯基、-O-间甲苯基、-O-邻甲苯基或-O-萘基。"Aryloxy" refers to a radical of the formula -O-aryl, where aryl is as defined above. Some examples of aryloxy compounds are -O-phenyl, -O-p-tolyl, -O-m-tolyl, -O-o-tolyl, or -O-naphthyl.

除非在本发明中特别说明，否则，如果适用的话，所有的基团均可被任选地取代。Unless specifically stated in the present invention, all groups can be optionally substituted if applicable.

下面将结合实施例及说明书附图详细说明本发明。The present invention will be described in detail below in conjunction with the embodiments and the accompanying drawings of the specification.

这里所列举的化合物只是为了更好地说明本发明的化合物类别和结构形式，并非限制本发明。The compounds listed here are only to better illustrate the compound categories and structural forms of the present invention, and do not limit the present invention.

本发明在以下的实施例中进一步说明。这些实施例只是为了说明的目的，而不是用来限制本发明的范围。由于通式(Ⅱ)-(Ⅳ)中每个通式所包含化合物的合成采用了完全相同的方法，只在反应原料上有差别，因此，实施例10-12中仅选用了通式化合物Ⅱ-1、Ⅲ-1、Ⅳ-1为代表，对通式(Ⅱ)-(Ⅳ)化合物的合成进行概述。The invention is further illustrated in the following examples. These examples are for illustrative purposes only, and are not used to limit the scope of the present invention. Since the synthesis of the compounds contained in each of the general formulas (II)-(IV) uses exactly the same method, only the reaction materials are different, therefore, only the general formula II is used in Examples 10-12 -1, Ⅲ-1, Ⅳ-1 as representatives, the synthesis of compounds of general formula (II)-(IV) is summarized.

实验中所用部分实验材料和设备信息如下：Some experimental materials and equipment information used in the experiment are as follows:

A549(野生型EGFR)、HCC827(外显子19缺失型EGFR)和NCI-H460(H460，野生型EGFR)，购自中国科学院细胞库(中国上海)；A549 (wild-type EGFR), HCC827 (exon 19 deleted EGFR) and NCI-H460 (H460, wild-type EGFR), purchased from the Chinese Academy of Sciences Cell Bank (Shanghai, China);

RPMI1640培养基、DMEM培养基和胎牛血清(FBS)，均购自杭州四季青有限公司(中国杭州)；RPMI1640 medium, DMEM medium and fetal bovine serum (FBS) were all purchased from Hangzhou Sijiqing Co., Ltd. (Hangzhou, China);

PBS缓冲液、EBSS缓冲液、含0.02％EDTA的0.25％胰酶和Opti-MEM培养基(吉诺生物医药技术有限公司，中国)；PBS buffer, EBSS buffer, 0.25% pancreatin containing 0.02% EDTA and Opti-MEM medium (Jinuo Biomedical Technology Co., Ltd., China);

二甲基亚砜DMSO(AMRESCO，美国)；Dimethyl sulfoxide DMSO (AMRESCO, USA);

细胞培养皿及培养板(Corning Costar，美国)；Cell culture dishes and culture plates (Corning Costar, USA);

吉非替尼、奥西替尼(AZD9291)、巴弗洛霉素A1(Baf A1)，购自Selleck(中国上海)；Gefitinib, Ositinib (AZD9291), Bafilomycin A1 (Baf A1), purchased from Selleck (Shanghai, China);

雷帕霉素(Rapamycin)，购自Beyotime(中国上海)；Rapamycin, purchased from Beyotime (Shanghai, China);

氯喹(CQ)、吖啶橙染料(AO)，购自Sigma Aldrich(中国上海)；Chloroquine (CQ) and Acridine Orange (AO), purchased from Sigma Aldrich (Shanghai, China);

BCA蛋白质测定试剂盒，购自Bio-Rad Laboratories(美国加州)；BCA protein determination kit, purchased from Bio-Rad Laboratories (California, USA);

辣根过氧化物酶偶联二抗，购自Jackson ImmunoResearch(中国上海)；Horseradish peroxidase conjugated secondary antibody, purchased from Jackson ImmunoResearch (Shanghai, China);

化学发光检测试剂盒，购自Biological Industries(美国)；Chemiluminescence detection kit, purchased from Biological Industries (USA);

Amersham Imager 600系统，购自通用电气医疗集团生命科学部(中国上海)；Amersham Imager 600 system, purchased from the Life Science Department of General Electric Medical Group (Shanghai, China);

siRNA转染试剂：Lipofectamine TM RNAiMAX(Invivogen，美国)； siRNA transfection reagent: Lipofectamine TM RNAiMAX (Invivogen, USA);

质粒转染试剂：X-tremeGENE HP DNA Transfection Reagent(Roche，瑞士)；Plasmid transfection reagent: X-tremeGENE HP DNA Transfection Reagent (Roche, Switzerland);

CellTiter

Figure PCTCN2020089390-appb-000023
AQueous非放射性细胞增殖检测试剂盒，购自Promega(中国北京)； CellTiter
Figure PCTCN2020089390-appb-000023
AQueous non-radioactive cell proliferation detection kit, purchased from Promega (Beijing, China);

BD Pharmingen FITC Annexin V细胞凋亡检测试剂盒，购自BD Biosciences(中国上海)；BD Pharmingen FITC Annexin V cell apoptosis detection kit, purchased from BD Biosciences (Shanghai, China);

BD FACSCaliburTM流式细胞仪，购自BD Biosciences(中国上海)。BD FACSCaliburTM flow cytometer, purchased from BD Biosciences (Shanghai, China).

发明人前期进行了如下实验：The inventor conducted the following experiments in the early stage:

在下述的细胞生物学实验步骤中，“处理”是指将待试化合物用细胞培养液稀释到特定浓度后，加入培养皿中与细胞孵育特定时间。In the following cell biology experiment procedures, "treatment" refers to the compound to be tested is diluted with a cell culture solution to a specific concentration, and then added to a culture dish and incubated with the cells for a specific time.

实施例1Example 1

用指定浓度(如图1所示)的吉非替尼处理非小细胞肺癌细胞(NSCLC)24小时(对A549和H460细胞)或36小时(对HCC827细胞)，并通过蛋白质印迹法(Western Blot)测定SQSTM1的表达。肌动蛋白用作内部参照蛋白。实验结果如图1所示。Treat non-small cell lung cancer cells (NSCLC) with the specified concentration (as shown in Figure 1) for 24 hours (for A549 and H460 cells) or 36 hours (for HCC827 cells) with Gefitinib, and pass the Western blotting method (Western Blot). ) Determine the expression of SQSTM1. Actin is used as an internal reference protein. The experimental results are shown in Figure 1.

用指定浓度(如图2所示)的AZD9291处理A549、HCC827和H460细胞24小时，并通过Western Blot测定SQSTM1的表达。肌动蛋白用作内部参照蛋白。实验结果如图2所示。A549, HCC827, and H460 cells were treated with AZD9291 at the specified concentration (as shown in Figure 2) for 24 hours, and the expression of SQSTM1 was determined by Western Blot. Actin is used as an internal reference protein. The experimental results are shown in Figure 2.

Western Blot操作步骤：Western Blot operation steps:

用含有2％SDS、0.1％溴酚蓝、10％甘油、1.5％DTT(二硫苏糖醇)和0.1M tris-HCl(pH6.8)的裂解缓冲液裂解细胞。通过BCA法蛋白定量试剂盒测定细胞裂解物中蛋白浓度。通过SDS-PAGE分离煮沸变性的裂解物，再将其转移至聚偏二氟乙烯膜上，用5％无脂牛奶(溶解在TBST中)封闭。然后，将该膜与指定的一抗在4℃孵育过夜，用TBST(含0.1％Tween-20的TBS)洗涤3次后，与辣根过氧化物酶(HRP)偶联的二抗室温下孵育2小时。TBST洗涤3次。最后，在Amersham Imager 600系统中，通过化学发光试剂盒检测膜上HRP的水平，以此表征相应抗原的含量。Cells were lysed with a lysis buffer containing 2% SDS, 0.1% bromophenol blue, 10% glycerol, 1.5% DTT (dithiothreitol), and 0.1M tris-HCl (pH 6.8). The protein concentration in cell lysate was determined by BCA protein quantification kit. The boiled and denatured lysate was separated by SDS-PAGE, and then transferred to a polyvinylidene fluoride membrane and blocked with 5% fat-free milk (dissolved in TBST). Then, the membrane was incubated with the designated primary antibody overnight at 4°C, washed with TBST (TBS containing 0.1% Tween-20) for 3 times, and the secondary antibody conjugated with horseradish peroxidase (HRP) at room temperature Incubate for 2 hours. Wash 3 times with TBST. Finally, in the Amersham Imager 600 system, the level of HRP on the membrane was detected by a chemiluminescence kit to characterize the content of the corresponding antigen.

由图1和2中结果可知，吉非替尼、AZD9291在三种具有不同遗传背景的NSCLC细胞(A549、HCC827和H460)中，均以剂量依赖性的方式上调SQSTM1蛋白水平。From the results in Figures 1 and 2, it can be seen that gefitinib and AZD9291 up-regulated SQSTM1 protein levels in three NSCLC cells (A549, HCC827 and H460) with different genetic backgrounds in a dose-dependent manner.

实施例2Example 2

用DMSO、雷帕霉素、吉非替尼处理A549和H460细胞7小时，然后对上述细胞给药Baf A1(巴弗 洛霉素A1)或不给药，培养5小时。通过Western Blot(参照实施例1中的Western Blot步骤)检测LC3和SQSTM1表达。实验结果如图3所示。The A549 and H460 cells were treated with DMSO, rapamycin, and gefitinib for 7 hours, and then the above cells were administered with Baf A1 (Bafilomycin A1) or not, and cultured for 5 hours. The expression of LC3 and SQSTM1 was detected by Western Blot (refer to the Western Blot step in Example 1). The experimental results are shown in Figure 3.

由图3结果可知，单用吉非替尼的作用和自噬抑制剂Baf A1类似，而和自噬诱导剂雷帕霉素相反；雷帕霉素和BafA1联用后，LC3-II和SQSTM1的蛋白水平均明显上升，而吉非替尼和BafA1联合后却没有表现出明显的增强效果。以上实验结果表明吉非替尼很可能通过抑制细胞自噬而阻断SQSTM1自噬性降解，导致其在细胞内积聚。From the results in Figure 3, it can be seen that the effect of gefitinib alone is similar to the autophagy inhibitor Baf A1, but opposite to the autophagy inducer rapamycin; after rapamycin and BafA1 are used in combination, LC3-II and SQSTM1 The protein level of gefitinib increased significantly, but the combination of gefitinib and BafA1 did not show a significant enhancement effect. The above experimental results indicate that gefitinib is likely to block the autophagic degradation of SQSTM1 by inhibiting autophagy, leading to its accumulation in cells.

实施例3Example 3

用GFP-RFP-LC3质粒转染A549细胞48小时，然后分别用EBSS、CQ、吉非替尼或AZD9291处理该细胞。通过免疫荧光显微镜检测GFP-LC3和RFP-LC3的斑点，实验结果如图4A所示。A549 cells were transfected with the GFP-RFP-LC3 plasmid for 48 hours, and then the cells were treated with EBSS, CQ, gefitinib or AZD9291. The spots of GFP-LC3 and RFP-LC3 were detected by immunofluorescence microscopy, and the experimental results are shown in Figure 4A.

LC3黄色斑点/红色斑点(％)的定量表示为3个独立实验的平均值±SD，n＝10个细胞。统计结果如图4B所示，其中，星号(*)表示统计学差异(学生t检验，P<0.05)。The quantification of LC3 yellow spots/red spots (%) is expressed as the mean±SD of 3 independent experiments, n=10 cells. The statistical results are shown in Figure 4B, where asterisks (*) indicate statistical differences (Student's t-test, P<0.05).

由实施例2和3的结果可知，吉非替尼、AZD9291对自噬的抑制作用与已知的自噬抑制剂巴佛洛霉素A1(Baf A1)和氯喹(CQ)类似，虽然能上调LC3Ⅱ的表达(如图3所示)，但却负向调控细胞的自噬流进程(如图4所示)。From the results of Examples 2 and 3, it can be seen that the inhibitory effects of gefitinib and AZD9291 on autophagy are similar to those of the known autophagy inhibitors Bafilomycin A1 (Baf A1) and chloroquine (CQ), although they can increase The expression of LC3Ⅱ (as shown in Figure 3), but negatively regulates the process of cell autophagy (as shown in Figure 4).

实施例4Example 4

SQSTM-1基因的高表达：将处于对数生长期的细胞种到相应的孔板内，培养过夜使细胞贴壁。转染前将培养液更换为无抗含10％胎牛血清的相应培养基，取适量体积的Opti-MEM培养基于EP管中，加入适量质粒和转染试剂X-treme GENE HD，吹打混匀后室温静置20-25min。将混合物以滴的形式均匀加到细胞培养基中，轻轻晃动使其混匀。根据实验需要转染48-72h。并采用Western blot法评估过表达效果。High expression of SQSTM-1 gene: Plant the cells in the logarithmic growth phase into the corresponding well plate, and cultivate overnight to make the cells adhere to the wall. Before transfection, replace the culture medium with the corresponding medium containing 10% fetal bovine serum without anti-antibody, take appropriate volume of Opti-MEM medium into the EP tube, add appropriate amount of plasmid and transfection reagent X-treme GENE HD, pipette and mix well Then let it stand at room temperature for 20-25 min. Add the mixture evenly to the cell culture medium in the form of drops, and gently shake to mix. Transfection for 48-72h according to experimental needs. And the Western blot method was used to evaluate the overexpression effect.

SQSTM-1基因的敲低：将处于对数生长期的细胞种到相应的孔板内，培养过夜使细胞贴壁(不同的待转染细胞需要不同的覆盖率)。转染前将培养液更换为无抗含10％胎牛血清的相应培养基，取适量的Opti-MEM培养基于EP管中，根据实验说明书加入适量的Lipofectamine RNAiMAX transfection reagent，混匀，再取等体积的Opti-MEM培养基加入适量siRNA，混匀(siRNA和阴性对照negative control siRNA由上海吉玛制药技术有限公司合成)。再将两管液体混合，吹打混匀，室温静置约15min。将混合物以滴的形式均匀加到细胞培养基中，轻轻晃动使其混匀。根据实验需要转染48-72h。并采用Western blot法评估敲低效果。Knockdown of SQSTM-1 gene: Plant the cells in the logarithmic growth phase into the corresponding well plate and culture overnight to make the cells adhere to the wall (different cells to be transfected require different coverage). Before transfection, replace the culture medium with the corresponding medium containing 10% fetal bovine serum, take an appropriate amount of Opti-MEM medium into the EP tube, add an appropriate amount of Lipofectamine RNAiMAX transfection reagent according to the experimental instructions, mix, and then take, etc. Add an appropriate amount of siRNA to a volume of Opti-MEM medium, and mix well (siRNA and negative control siRNA are synthesized by Shanghai Jima Pharmaceutical Technology Co., Ltd.). Then mix the two tubes of liquid, mix by pipetting, and let stand at room temperature for about 15 minutes. Add the mixture evenly to the cell culture medium in the form of drops, and gently shake to mix. Transfection for 48-72h according to experimental needs. The Western blot method was used to evaluate the knockdown effect.

实验结果如图5所示。由图5中结果证明高表达SQSTM1导致细胞对吉非替尼、AZD9291的耐受性，而敲低SQSTM1可增加细胞对二者的敏感性。The experimental results are shown in Figure 5. The results in Figure 5 prove that high expression of SQSTM1 leads to cell tolerance to gefitinib and AZD9291, while knocking down SQSTM1 can increase the sensitivity of cells to both.

根据实施例1-4的实验结果，发明人推断，吉非替尼、AZD9291等EGFR TKI通过抑制细胞的自噬性降解，导致SQSTM1的积聚从而促进细胞产生对EGFR TKI的获得性耐药性。Based on the experimental results of Examples 1-4, the inventors infer that EGFR TKIs such as gefitinib and AZD9291 inhibit the autophagic degradation of cells, leading to the accumulation of SQSTM1, thereby promoting the cells to develop acquired drug resistance to EGFR TKIs.

实施例5Example 5

免疫荧光实验步骤：将灭菌过的盖玻片置于孔板中，细胞密度接种为常规的1/5到1/3，过夜培养，加入药物处理特定时间后，从孵箱中取出。1×PBS洗1次，再用无水甲醇洗1次，室温固定10分钟。PBS洗3次。然后用0.25％Triton X-100透化25分钟。PBS再洗3次。加入3％的BSA(1×PBS配制)室温封闭30分钟。加稀释好的一抗(用3％BSA稀释)，放在湿盒内，4℃过夜。次日，PBS洗3次后，加稀释好的荧光二抗(用3％BSA稀释，1:200稀释)室温孵育1小时，避光。PBS再洗3次。使用混有DAPI的封片剂封片，封片后室温静置1小时，荧光显微镜下检测DAPI和LC3荧光的强度。实验结果如图6所示。Immunofluorescence experiment procedure: Place the sterilized cover glass in the well plate, inoculate the cell density to 1/5 to 1/3 of the conventional one, incubate overnight, add the medicine for a specific time, and take it out of the incubator. Wash once with 1×PBS, then wash once with anhydrous methanol, and fix at room temperature for 10 minutes. Wash 3 times with PBS. Then it was permeabilized with 0.25% Triton X-100 for 25 minutes. Wash 3 times with PBS. Add 3% BSA (1×PBS) and block for 30 minutes at room temperature. Add the diluted primary antibody (diluted with 3% BSA) and place it in a humidified box at 4°C overnight. The next day, after washing 3 times with PBS, add the diluted fluorescent secondary antibody (diluted with 3% BSA, 1:200 dilution) and incubate for 1 hour at room temperature, protected from light. Wash 3 times with PBS. Mount the slide with a mounting tablet mixed with DAPI, and then let it stand at room temperature for 1 hour after mounting, and detect the fluorescence intensity of DAPI and LC3 under a fluorescent microscope. The experimental results are shown in Figure 6.

已知，自噬可分成3个主要的过程，即自噬体的形成、自噬体和溶酶体的融合、自噬溶酶体的降解。It is known that autophagy can be divided into three main processes, namely the formation of autophagosomes, the fusion of autophagosomes and lysosomes, and the degradation of autophagolysosomes.

由图6中结果可知，以细胞内表达的LC3Ⅱ产生的荧光为检测信号，吉非替尼、AZD9291对自噬体的形成不仅没有抑制作用，反而起到促进作用。It can be seen from the results in Figure 6 that with the fluorescence produced by LC3II expressed in the cell as the detection signal, gefitinib and AZD9291 not only have no inhibitory effect on the formation of autophagosomes, but promote the formation of autophagosomes.

实施例6Example 6

以溶酶体膜蛋白LAMP1和LC3Ⅱ的共定位为评价指标，用雷帕霉素或吉非替尼处理A549细胞24小时，通过免疫荧光检测LAMP1和LC3的共定位，实验结果如图7A所示。Taking the co-localization of lysosomal membrane proteins LAMP1 and LC3Ⅱ as the evaluation index, A549 cells were treated with rapamycin or gefitinib for 24 hours, and the co-localization of LAMP1 and LC3 was detected by immunofluorescence. The experimental results are shown in Figure 7A. .

共定位系数(Colocalization coefficient)表示为LC3和LAMP1的共定位点与总LC3点的比率。共定位系数的定量表示为3个独立实验的平均值±SD，n＝10个细胞。统计结果如图7B所示，其中，NS表示 无显著意义(学生t检验，P>0.05)。Colocalization coefficient (Colocalization coefficient) is expressed as the ratio of the colocalization points of LC3 and LAMP1 to the total LC3 points. The quantification of the colocalization coefficient is expressed as the mean±SD of 3 independent experiments, n=10 cells. The statistical results are shown in Figure 7B, where NS means insignificant (Student's t-test, P>0.05).

由图7中结果可知，吉非替尼并不影响自噬体与溶酶体的融合。It can be seen from the results in Figure 7 that gefitinib does not affect the fusion of autophagosomes and lysosomes.

由实施例5和6，发明人认为，吉非替尼、AZD9291等EGFR-TKI并不影响自噬体形成(反而起到促进作用，以LC3Ⅱ的表达量为标记物，如图6所示)和自噬体与溶酶体的融合(以溶酶体膜蛋白LAMP1和LC3Ⅱ的共定位为评价指标，如图7所示)，而可能是通过抑制溶酶体的功能。From Examples 5 and 6, the inventor believes that EGFR-TKIs such as gefitinib and AZD9291 do not affect the formation of autophagosomes (instead, they play a role in promoting, using the expression of LC3II as a marker, as shown in Figure 6) And the fusion of autophagosomes and lysosomes (using the co-localization of lysosomal membrane proteins LAMP1 and LC3Ⅱ as the evaluation index, as shown in Figure 7), and may be by inhibiting the function of lysosomes.

实施例7Example 7

CTSB是溶酶体的一个特征性水解酶，其需要在溶酶体的酸性环境中被剪切为成熟体后才能发挥水解蛋白的作用。因此，很多研究将CTSB成熟体的表达量作为溶酶体功能强弱的指标。CTSB is a characteristic hydrolase of lysosome, which needs to be sheared into mature body in the acidic environment of lysosome before it can play the role of protein hydrolysis. Therefore, many studies use the expression level of mature CTSB as an indicator of the strength of lysosomal function.

分别用吉非替尼、AZD9291、CQ或Baf A1处理A549和HCC827细胞24小时，以肌动蛋白作为对照，通过Western Blot(参照实施例1中的Western Blot步骤)评估CTSB成熟体的表达。实验结果如图8所示。The A549 and HCC827 cells were treated with Gefitinib, AZD9291, CQ, or Baf A1 for 24 hours, and actin was used as a control to evaluate the expression of CTSB mature body by Western Blot (refer to the Western Blot step in Example 1). The experimental results are shown in Figure 8.

由图8结果可知，吉非替尼、AZD9291可显著地下调CTSB成熟体的表达，由此说明了这两个化合物确实存在削弱溶酶体功能的作用。It can be seen from the results in Fig. 8 that gefitinib and AZD9291 can significantly down-regulate the expression of CTSB mature body, which shows that these two compounds do have the effect of weakening lysosomal function.

实施例8Example 8

吖啶橙染料(AO)是一种亲质子的荧光染料，其非质子化单体能发出绿色荧光，而进入溶酶体被质子化后形成聚合体并发出红色荧光。Acridine orange dye (AO) is a protic fluorescent dye. Its aprotonated monomer can emit green fluorescence, and it enters the lysosome and is protonated to form a polymer and emit red fluorescence.

分别用DMSO、吉非替尼、AZD9291、CQ或BafA1处理A549和HCC827细胞12小时，然后将A549和HCC827细胞与AO孵育15分钟。通过荧光显微镜检测AO-红色荧光斑点的数量。实验结果如图9A所示。The A549 and HCC827 cells were treated with DMSO, Gefitinib, AZD9291, CQ or BafA1 for 12 hours, and then the A549 and HCC827 cells were incubated with AO for 15 minutes. Detect the number of AO-red fluorescent spots by fluorescence microscope. The experimental results are shown in Figure 9A.

每个细胞的AO-红色荧光斑点的定量表示为3个独立实验的平均值±SD，n＝10个细胞。统计结果如图9B所示，其中，星号(*)表示统计学差异(学生t检验，P<0.05)，ND表示未检测到。The quantification of the AO-red fluorescent spot of each cell is expressed as the mean ± SD of 3 independent experiments, n=10 cells. The statistical results are shown in Figure 9B, where an asterisk (*) indicates a statistical difference (Student's t test, P<0.05), and ND indicates not detected.

由图9结果可知，吉非替尼、AZD9291均抑制溶酶体的酸化过程。It can be seen from the results in Figure 9 that both gefitinib and AZD9291 inhibit the acidification process of lysosomes.

根据实施例4-8的实验结果，发明人推测，吉非替尼、AZD9291等EGFR TKI通过抑制溶酶体酸化，导致自噬溶酶体的功能减弱及其所包裹的底物的降解受阻。According to the experimental results of Examples 4-8, the inventors speculate that EGFR TKIs such as gefitinib and AZD9291 inhibit lysosomal acidification, leading to the weakening of autophagic lysosomes and hindering the degradation of the encapsulated substrates.

实施例9Example 9

代表性EGFR TKI化学结构酸碱性分析：从第一代到第三代EGFR TKI中挑选了目前临床上最常用的8个代表性药物，通过在免费开源的药物数据库中(https://www.drugbank.ca/)检索其理化性质。8个代表性EGFR TKI化学结构中的氮原子个数都在三个或三个以上，而没有缺电子的路易斯酸酸性基团，均属于路易斯碱碱性药物。其中，吉非替尼、阿法替尼、达克替尼(Dacomtinib)、奥西替尼、奥姆替尼(Olmutinib)等都具有碱性较强的脂肪胺基，其碱性pKa值如下表所示。Representative EGFR TKI chemical structure acid-base analysis: selected from the first generation to the third generation of EGFR TKI, the eight representative drugs most commonly used in clinical practice, through the free and open source drug database (https://www .drugbank.ca/) Retrieve its physical and chemical properties. The eight representative EGFR TKI chemical structures have three or more nitrogen atoms, but there is no Lewis acidic acid group that lacks electrons, and they all belong to Lewis base basic drugs. Among them, Gefitinib, Afatinib, Dacomtinib, Ositinib, Olmutinib, etc. all have aliphatic amine groups with strong basicity, and their basic pKa values are as follows Shown.

表1代表性EGFR TKI的碱性pKa值Table 1 Basic pKa value of representative EGFR TKI

Figure PCTCN2020089390-appb-000024
Figure PCTCN2020089390-appb-000024

Figure PCTCN2020089390-appb-000025
Figure PCTCN2020089390-appb-000025

根据以上实验结果，发明人推断，造成吉非替尼、AZD9291等EGFR TKI抑制溶酶体酸化，进而导致自噬溶酶体降解受阻的根本原因可能是由于吉非替尼、AZD9291等EGFR TKI在化学结构上存在碱性较强的官能团，使其容易进入到酸性微环境的溶酶体中，通过减少溶酶体的酸化，进而造成了对自噬溶酶体的功能性阻断和促癌蛋白SQSTM1的积累，最终导致肿瘤细胞对吉非替尼、AZD9291等EGFR TKI产生耐药性。在此基础上，发明人对结构上含有强碱性基团的EGFR TKI进行碱性中和改造而得到一系列EGFR TKI碱性中和类似物，例如以下实施例10-12中所合成的化合物。Based on the above experimental results, the inventors infer that the root cause of EGFR TKIs such as gefitinib and AZD9291 inhibiting lysosomal acidification, which in turn leads to the hindered autophagy lysosomal degradation may be due to the fact that EGFR TKIs such as gefitinib and AZD9291 are in The chemical structure has a strong basic functional group, which makes it easy to enter the lysosome in the acidic microenvironment. By reducing the acidification of the lysosome, it causes the functional blockade of the autophagy lysosome and promotes cancer. The accumulation of protein SQSTM1 eventually leads to tumor cells becoming resistant to EGFR TKIs such as gefitinib and AZD9291. On this basis, the inventors carried out alkaline neutralization modification of EGFR TKI with strong basic groups in the structure to obtain a series of EGFR TKI alkaline neutralization analogs, such as the compounds synthesized in the following examples 10-12 .

实施例10通式化合物Ⅱ-1的合成(合成路线如图10所示)Example 10 Synthesis of compound II-1 of general formula (the synthetic route is shown in Figure 10)

步骤Ⅰ、3-溴丙基-3,5-二甲氧基苯甲酸酯(中间体1-3)的制备：称取3,5-二甲氧基苯甲酰氯(中间体1-1)(2g，10mmol)和3-溴-1-丙醇(中间体1-2)(1.07mL，5mmol)，将其溶解在20mL二氯甲烷中，搅拌至溶解，再缓慢滴加三乙胺(1.5mL，15mmol)，在室温下搅拌，TLC监测反应过程，约5小时后反应完毕。加入20mL超纯水猝灭反应后，再加入20mL二氯甲烷进行萃取，饱和NaCl溶液洗涤3次，二氯甲烷层用无水MgSO 4干燥，过滤后，旋转蒸发除去后浓缩得粗产物，硅胶柱层析纯化得到无色油状液体中间体1-3，产率：80％。 Step I. Preparation of 3-bromopropyl-3,5-dimethoxybenzoate (Intermediate 1-3): Weigh 3,5-dimethoxybenzoyl chloride (Intermediate 1-1 ) (2g, 10mmol) and 3-bromo-1-propanol (Intermediate 1-2) (1.07mL, 5mmol), dissolve it in 20mL of dichloromethane, stir until dissolved, and then slowly add triethylamine dropwise (1.5mL, 15mmol), stirred at room temperature, TLC monitored the reaction process, and the reaction was complete after about 5 hours. After adding 20 mL of ultrapure water to quench the reaction, adding 20 mL of dichloromethane for extraction, washing with saturated NaCl solution 3 times, drying the dichloromethane layer with anhydrous MgSO 4 , filtering, removing by rotary evaporation, and concentrating to obtain a crude product, silica gel Purification by column chromatography to obtain colorless oily liquid intermediate 1-3, yield: 80%.

步骤Ⅱ、3-溴丙基-3,5-二羟基苯甲酸酯(中间体1-4)的制备：称取中间体1-3(604mg，2mmol)溶于 20mL的二氯甲烷中，边搅拌边将三氯化铝(1333mg，10mmol，分三次)，碘化钠(150mg，1mmol)依次加入，室温下反应过夜。待反应完全，加入20mL饱和氯化铵溶液中和反应，再加入20mL二氯甲烷进行萃取，用饱和NaCl溶液洗涤二氯甲烷层3次，并用无水MgSO 4干燥。滤液浓缩后，通过快速硅胶柱层析纯化得到淡黄色油状液体中间体1-4，产率：30％。 1H-NMR(CDCl 3,600MHz)δ:7.087(d,J＝2.4Hz,2H,H-2,H-6),6.572(t,J＝2.4Hz,1H,H-4),5.486(s,2H,-OH×2),4.449(t,J＝6.0Hz,2H,-OC H 2CH 2CH 2Br),3.676(t,J＝6.6Hz,2H,-OCH 2CH 2C H 2Br),2.212(t,J＝6.0Hz,2H,-OCH 2C H 2CH 2Br). Step II. Preparation of 3-bromopropyl-3,5-dihydroxybenzoate (Intermediate 1-4): Weigh Intermediate 1-3 (604mg, 2mmol) and dissolve it in 20mL of dichloromethane, While stirring, aluminum trichloride (1333 mg, 10 mmol, three times) and sodium iodide (150 mg, 1 mmol) were added sequentially, and the reaction was carried out overnight at room temperature. When the reaction is complete, add 20 mL of saturated ammonium chloride solution to neutralize the reaction, then add 20 mL of dichloromethane for extraction, wash the dichloromethane layer with saturated NaCl solution 3 times, and dry with anhydrous MgSO 4 . After the filtrate was concentrated, it was purified by flash silica gel column chromatography to obtain the pale yellow oily liquid intermediate 1-4, the yield: 30%. 1 H-NMR(CDCl 3 ,600MHz)δ:7.087(d,J=2.4Hz,2H,H-2,H-6),6.572(t,J=2.4Hz,1H,H-4),5.486( s, 2H, -OH×2), 4.449 (t, J = 6.0Hz, 2H, -OC H 2 CH 2 CH 2 Br), 3.676 (t, J = 6.6 Hz, 2H, -OCH 2 CH 2 C H 2 Br),2.212(t,J=6.0Hz,2H,-OCH 2 C H 2 CH 2 Br).

步骤Ⅲ、3-溴丙基-3,5-二四氢吡喃基苯甲酸酯(中间体1-6)的制备：称取中间体1-4(550mg，2mmol)、二氢吡喃(中间体1-5)(841.2mg，10mmol)和对甲苯磺酸吡啶盐(502mg，0.2mmol)，溶于20mL二氯甲烷中，室温下反应6小时。反应完全后，加入20mL水和20mL二氯甲烷，萃取并旋干二氯甲烷。硅胶柱层析法分离纯化后得到淡黄色油状物中间体1-6，产率：63％。 1H-NMR(CDCl 3,500MHz)δ:7.348(t,J＝2.5Hz,2H,H-2,H-6),6.984(q,J＝2.5Hz,1H,H-4),5.435-5.463(m,2H,-O-pyranyl-H-2×2),4.447(t,J＝6.0Hz,2H,-OC H 2CH 2CH 2Br),3.886(td,J 1＝2.5Hz,J 2＝10.5Hz,2H,-O-pyranyl-H-6×2),3.688(t,J＝6.5Hz,2H,-OCH 2CH 2C H 2Br),3.596-3.632(m,2H,-O-pyranyl-H-6×2),2.196-2.246(m,2H,-OCH 2C H 2CH 2Br),1.958-2.016(m,2H,-O-pyranyl-H-3×2),1.844-1.894(m,4H,-O-pyranyl-H-3×2,-O-pyranyl-H-4×2),1.644-1.712(m,4H,-O-pyranyl-H-4×2,-O-pyranyl-H-5×2),1.584-1.613(m,2H,-O-pyranyl-H-5×2). Step III. Preparation of 3-bromopropyl-3,5-ditetrahydropyranylbenzoate (Intermediate 1-6): Weigh Intermediate 1-4 (550mg, 2mmol), dihydropyran (Intermediate 1-5) (841.2 mg, 10 mmol) and pyridinium p-toluenesulfonate (502 mg, 0.2 mmol) were dissolved in 20 mL of dichloromethane and reacted at room temperature for 6 hours. After the reaction is complete, add 20 mL of water and 20 mL of dichloromethane, extract and spin dry the dichloromethane. After separation and purification by silica gel column chromatography, intermediate 1-6 was obtained as a pale yellow oily substance, with a yield of 63%. 1 H-NMR (CDCl 3 , 500MHz) δ: 7.348 (t, J = 2.5 Hz, 2H, H-2, H-6), 6.984 (q, J = 2.5 Hz, 1H, H-4), 5.435- 5.463(m,2H,-O-pyranyl-H-2×2), 4.447(t,J=6.0Hz,2H,-OC H 2 CH 2 CH 2 Br), 3.886(td,J 1 ＝2.5Hz, J 2 = 10.5Hz, 2H, -O-pyranyl-H-6×2), 3.688 (t, J = 6.5Hz, 2H, -OCH 2 CH 2 C H 2 Br), 3.596-3.632 (m, 2H, -O-pyranyl-H-6×2), 2.196-2.246(m,2H,-OCH 2 C H 2 CH 2 Br), 1.958-2.016(m,2H,-O-pyranyl-H-3×2) ,1.844-1.894(m,4H,-O-pyranyl-H-3×2,-O-pyranyl-H-4×2), 1.644-1.712(m,4H,-O-pyranyl-H-4×2 ,-O-pyranyl-H-5×2), 1.584-1.613(m,2H,-O-pyranyl-H-5×2).

步骤Ⅳ、4-((3-氯-4-氟苯基)氨基)-6-(3-吗啡啉丙氧基)喹唑啉-7-醇(中间体1-8)的制备：称取原料药吉非替尼(中间体1-7)(89.4mg，0.2mmol)和L-甲硫氨酸(49.23mg，0.33mmol)，溶解于5mL的甲磺酸中，165℃下回流反应约24小时。反应完全后，加入5mL的超纯水猝灭反应，并置于室温下冷却。再逐渐滴加40％NaOH水溶液至体系pH接近7左右后，加入20mL乙酸乙酯萃取并旋干溶剂，硅胶柱层析法分离纯化后得到白色固体粉末中间体1-8，产率：52％。 1H-NMR(CD 3OD,400MHz)δ:8.367(s,1H,H-2),7.964(dd,J 1＝2.8Hz,J 2＝6.8Hz,1H,H-8),7.762(s,1H,H-5),7.616-7.656(m,1H,H-2’),7.230(t,J＝8.8Hz,1H,H-6’),7.033(s,1H,H-5’),4.267(t,J＝6.0Hz,2H,-NCH 2CH 2C H 2O-),3.783(t,J＝4.8Hz,4H,-OCH 2×2),2.871(t,J＝7.2Hz,2H,-NC H 2CH 2CH 2O-),2.784(s,4H,-NCH 2×2),2.145-2.208(m,2H,-NCH 2C H 2CH 2O-).MS(ESI)m/z:432.97(M+1) +,calculated for C 21H 22ClFN 4O 3:432.14. Step IV. Preparation of 4-((3-chloro-4-fluorophenyl)amino)-6-(3-morpholinopropoxy)quinazolin-7-ol (Intermediate 1-8): Weigh The crude drug gefitinib (Intermediate 1-7) (89.4mg, 0.2mmol) and L-methionine (49.23mg, 0.33mmol) were dissolved in 5mL of methanesulfonic acid, and the reaction was refluxed at 165℃ for approximately 24 hours. After the reaction was completed, 5 mL of ultrapure water was added to quench the reaction, and the reaction was cooled at room temperature. Then gradually add 40% NaOH aqueous solution until the pH of the system is close to about 7, add 20mL ethyl acetate for extraction and spin-dry the solvent. After separation and purification by silica gel column chromatography, a white solid powder intermediate 1-8 is obtained. Yield: 52% . 1 H-NMR(CD 3 OD,400MHz)δ:8.367(s,1H,H-2),7.964(dd,J 1 =2.8Hz,J 2 =6.8Hz,1H,H-8),7.762(s ,1H,H-5),7.616-7.656(m,1H,H-2'),7.230(t,J=8.8Hz,1H,H-6'),7.033(s,1H,H-5') ,4.267(t,J=6.0Hz,2H,-NCH 2 CH 2 C H 2 O-),3.783(t,J=4.8Hz,4H,-OCH 2 ×2),2.871(t,J=7.2Hz ,2H,-NC H 2 CH 2 CH 2 O-),2.784(s,4H,-NCH 2 ×2),2.145-2.208(m,2H,-NCH 2 C H 2 CH 2 O-).MS( ESI)m/z:432.97(M+1) + ,calculated for C 21 H 22 ClFN 4 O 3 :432.14.

步骤Ⅴ-Ⅵ、3-((4-((3-氯-4-氟苯基)氨基)-6-(3-吗啡啉丙氧基)喹唑啉-7-基)氧基)丙基-3,5-二羟基苯甲酸酯(通式化合物Ⅱ-1，Gefi-2OH)的制备：称取中间体1-8(43.2mg，0.1mmol)和中间体1-6(88.4mg，0.2mmol)，溶于5mL N,N-二甲基甲酰胺中，加入碳酸钾(55.2mg，0.4mmol)和碘化钠(15mg，0.1mmol)后，80℃反应过夜。反应完全后，加入5mL的超纯水和20mL的乙酸乙酯进行萃取，然后旋干溶剂。经柱层析色谱纯化，得到无色油状液体。将此油状物重新溶解在5mL的无水乙醇中，加入1mL 10％HCl溶液，室温下搅拌反应2h。旋干体系中的无水乙醇后，加入4mL的超纯水和10mL乙酸乙酯进行萃取。旋干乙酸乙酯后，通过快速柱层析法分离纯化得到白色粉末状的目标产物Gefi-2OH，产率：30％。 1H-NMR(DMSO-d 6,600MHz)δ:8.471(s,1H,H-2),8.116(d,J＝4.8Hz,1H,H-8),7.849(s,1H,H-5),7.785(s,1H,H-2’),7.410(t,J＝9.0Hz,1H,H-6’),7.222(s,1H,H-5’),6.803(d,J＝2.4Hz,2H,H-2”,H-6”),6.410(t,J＝2.4Hz,1H,H-4”),4.400(t,J＝6.0Hz,2H,O＝COC H 2),4.280(t,J＝6.0Hz,2H,O＝COCH 2CH 2C H 2),4.177(s,2H,-NCH 2CH 2C H 2O-),3.556(s,4H,-OCH 2×2),2.475(t,J＝1.8Hz,2H,-NC H 2CH 2CH 2O-),2.359(s,4H,-NCH 2×2),2.218(m,J＝6.0Hz,2H,O＝COCH 2C H 2CH 2),1.983(m,J＝7.8Hz,2H,-NCH 2C H 2CH 2O-).MALDI-TOF MS m/z:627.475(M+1) +,calculated for C 31H 32ClFN 4O 7:626.19. Step V-VI, 3-((4-((3-chloro-4-fluorophenyl)amino)-6-(3-morpholinopropoxy)quinazolin-7-yl)oxy)propyl -3,5-Dihydroxybenzoic acid ester (general formula compound II-1, Gefi-2OH): Weigh intermediate 1-8 (43.2mg, 0.1mmol) and intermediate 1-6 (88.4mg, 0.2mmol) was dissolved in 5mL N,N-dimethylformamide, potassium carbonate (55.2mg, 0.4mmol) and sodium iodide (15mg, 0.1mmol) were added, and reacted at 80°C overnight. After the reaction was complete, 5 mL of ultrapure water and 20 mL of ethyl acetate were added for extraction, and then the solvent was spin-dried. Purified by column chromatography to obtain a colorless oily liquid. The oil was re-dissolved in 5 mL of absolute ethanol, 1 mL of 10% HCl solution was added, and the reaction was stirred at room temperature for 2 h. After the absolute ethanol in the system was spin-dried, 4 mL of ultrapure water and 10 mL of ethyl acetate were added for extraction. After the ethyl acetate was spin-dried, the target product Gefi-2OH was obtained as a white powder by separation and purification by flash column chromatography. The yield was 30%. 1 H-NMR(DMSO-d 6 ,600MHz)δ: 8.471(s,1H,H-2),8.116(d,J=4.8Hz,1H,H-8),7.849(s,1H,H-5 ),7.785(s,1H,H-2'),7.410(t,J=9.0Hz,1H,H-6'),7.222(s,1H,H-5'),6.803(d,J=2.4 Hz,2H,H-2”,H-6”),6.410(t,J=2.4Hz,1H,H-4”),4.400(t,J=6.0Hz,2H,O=COC H 2 ), 4.280 (t, J = 6.0 Hz, 2H, O = COCH 2 CH 2 C H 2 ), 4.177 (s, 2H, -NCH 2 CH 2 C H 2 O-), 3.556 (s, 4H, -OCH 2 × 2), 2.475 (t, J = 1.8 Hz, 2H, -NC H 2 CH 2 CH 2 O-), 2.359 (s, 4H, -NCH 2 × 2), 2.218 (m, J = 6.0 Hz, 2H, O=COCH 2 C H 2 CH 2 ),1.983(m,J=7.8Hz,2H,-NCH 2 C H 2 CH 2 O-).MALDI-TOF MS m/z:627.475(M+1) + , calculated for C 31 H 32 ClFN 4 O 7 :626.19.

实施例11通式化合物Ⅲ-1的合成(合成路线如图11所示)Example 11 Synthesis of compound III-1 of general formula (the synthetic route is shown in Figure 11)

步骤Ⅰ、4-氟-2-((四氢-2H-吡喃-2-基)氧基)苯胺(中间体2-2)的制备：将2-氨基-5-氟苯酚(中间体2-1)(1.27g，10mmol)、二氢吡喃(2.52g，30mmol)和对甲苯磺酸吡啶盐(502mg，1mmol)，溶于20mL二氯甲烷中，室温下反应6小时。反应完全后，加入20mL水和20mL二氯甲烷，萃取并旋干二氯甲烷。硅胶柱层析法分离纯化后得到无色油状物中间体2-2，产率：77％。MS(ESI)m/z:212.10(M+1) +,calculated for C 11H 14FNO 2:211.10. Step I. Preparation of 4-fluoro-2-((tetrahydro-2H-pyran-2-yl)oxy)aniline (Intermediate 2-2): The 2-amino-5-fluorophenol (Intermediate 2 -1) (1.27g, 10mmol), dihydropyran (2.52g, 30mmol) and pyridinium p-toluenesulfonate (502mg, 1mmol), dissolved in 20mL of dichloromethane, and reacted at room temperature for 6 hours. After the reaction is complete, add 20 mL of water and 20 mL of dichloromethane, extract and spin dry the dichloromethane. After separation and purification by silica gel column chromatography, intermediate 2-2 was obtained as a colorless oily substance, and the yield was 77%. MS(ESI)m/z:212.10(M+1) + ,calculated for C 11 H 14 FNO 2 : 211.10.

步骤Ⅱ、4-甲基哌嗪基-2-((四氢-2H-吡喃-2-基)氧基)苯胺(中间体2-3)的制备：称取中间体2-2(1.05g，5mmol)和N-甲基哌嗪(1.36mL，15mmol)溶于20mL 2-戊醇中，120℃下反应3小时。反应完全后，旋干溶剂，加入乙酸乙酯萃取，浓缩后经硅胶柱层析法分离纯化后得到红色粉末状中间体2-3，产率：71％。 MS(ESI)m/z:292.30(M+1) +,calculated for C 16H 25N 3O 2:291.19. Step II. Preparation of 4-methylpiperazinyl-2-((tetrahydro-2H-pyran-2-yl)oxy)aniline (Intermediate 2-3): Weigh Intermediate 2-2 (1.05 g, 5mmol) and N-methylpiperazine (1.36mL, 15mmol) were dissolved in 20mL 2-pentanol and reacted at 120°C for 3 hours. After the reaction is complete, the solvent is spin-dried, ethyl acetate is added for extraction, concentrated, and separated and purified by silica gel column chromatography to obtain intermediate 2-3 as a red powder. The yield is 71%. MS(ESI)m/z:292.30(M+1) + ,calculated for C 16 H 25 N 3 O 2 :291.19.

步骤Ⅲ、2-氯噻吩并[3,2-d]嘧啶-4(3H)-酮(中间体2-5)的制备：称取2,4-氯噻吩并[3,2-d]嘧啶(中间体2-4)(1.025g，5mmol)溶于10mL乙醇/水＝4:1的混合溶剂中，再称取500mg氢氧化钠溶于3mL蒸馏水中，并将此溶液滴加进入反应体系，40℃下反应4小时。反应完全后，加入1mL乙酸，在35℃下继续反应2小时，将所得沉淀抽滤，蒸馏水洗涤后，得到白色粉末状中间体2-5，产率：89％。MS(ESI)m/z:187.96(M+2) +,calculated for C 6H 3ClN 2OS:185.97. Step III. Preparation of 2-chlorothieno[3,2-d]pyrimidine-4(3H)-one (Intermediate 2-5): Weigh 2,4-chlorothieno[3,2-d]pyrimidine (Intermediate 2-4) (1.025g, 5mmol) was dissolved in a mixed solvent of 10mL ethanol/water = 4:1, then 500mg sodium hydroxide was weighed and dissolved in 3mL distilled water, and this solution was added dropwise into the reaction system , React at 40°C for 4 hours. After the reaction was completed, 1 mL of acetic acid was added, and the reaction was continued at 35° C. for 2 hours. The resulting precipitate was suction filtered and washed with distilled water to obtain a white powdery intermediate 2-5. The yield: 89%. MS(ESI)m/z:187.96(M+2) + , calculated for C 6 H 3 ClN 2 OS: 185.97.

步骤Ⅳ、2-((4-(4-甲基哌嗪-1-基)-2-((四氢-2H-吡喃-2-基)氧基)苯基)氨基)噻吩[3,2-d]嘧啶-4(3H)-酮(中间体2-6)的制备：称取中间体2-5(930mg，5mmol)溶于50mL乙醇中，加入中间体2-3(1.75g，6mmol)和5mL乙酸，回流反应6小时。待反应完全后将反应物冷却至室温，将6mL三乙胺缓慢滴加进反应物，然后将抽滤，并用20mL乙醇洗涤2次，真空干燥得到黄色固体粉末状中间体2-6，产率：69％。MS(ESI)m/z:442.30(M+1) +,calculated for C 22H 27N 5O 3S:441.18. Step IV, 2-((4-(4-methylpiperazin-1-yl)-2-((tetrahydro-2H-pyran-2-yl)oxy)phenyl)amino)thiophene [3, 2-d] Preparation of pyrimidine-4(3H)-one (Intermediate 2-6): Weigh Intermediate 2-5 (930mg, 5mmol) and dissolve in 50mL ethanol, add Intermediate 2-3 (1.75g, 6mmol) and 5mL acetic acid, reflux for 6 hours. After the reaction is complete, the reactant is cooled to room temperature, 6 mL of triethylamine is slowly added dropwise to the reactant, and then filtered with suction, washed twice with 20 mL of ethanol, and dried in vacuo to obtain a yellow solid powdery intermediate 2-6, yield : 69%. MS(ESI)m/z: 442.30(M+1) + , calculated for C 22 H 27 N 5 O 3 S: 441.18.

步骤Ⅴ、4-氯-N-(4-(4-甲基哌嗪-1-基)-2-((四氢-2H-吡喃-2-基)氧基)苯基)噻吩[3,2-d]嘧啶-2-胺(中间体2-7)的制备：称取中间体2-6(1.76g，4mmol)溶于5mL乙腈中，将反应温度升至75℃。加入1mL三氯氧磷和1mL乙腈的混合溶液，75℃下反应1小时。反应完全后，分别加入10mL冰水和40％氢氧化钠溶液，在室温下搅拌1小时后，抽滤后，用20mL蒸馏水洗2次，烘干得到粗产品。然后，将此粗产品重新溶于20mL体积比为3:2的二氯甲烷/甲醇混合溶剂中，室温下搅拌1小时后，旋干溶剂。再将产物重新溶于10mL体积比为8:2的乙腈/水混合溶剂中，室温下搅拌2小时后，减压抽滤，得到白色固体粉末状中间体2-7，产率：77％。MS(ESI)m/z:461.20(M+2) +,calculated for C 22H 26ClN 5O 2S:459.15. Step V. 4-Chloro-N-(4-(4-methylpiperazin-1-yl)-2-((tetrahydro-2H-pyran-2-yl)oxy)phenyl)thiophene [3 Preparation of ,2-d]pyrimidin-2-amine (Intermediate 2-7): Weigh Intermediate 2-6 (1.76g, 4mmol) and dissolve it in 5mL acetonitrile, and raise the reaction temperature to 75°C. Add 1 mL of a mixed solution of phosphorus oxychloride and 1 mL of acetonitrile, and react at 75°C for 1 hour. After the reaction is complete, add 10 mL of ice water and 40% sodium hydroxide solution respectively, stir at room temperature for 1 hour, and after suction filtration, wash with 20 mL of distilled water twice, and dry to obtain a crude product. Then, the crude product was re-dissolved in 20 mL of a dichloromethane/methanol mixed solvent with a volume ratio of 3:2, stirred at room temperature for 1 hour, and the solvent was spin-dried. The product was re-dissolved in 10 mL of an acetonitrile/water mixed solvent with a volume ratio of 8:2, stirred at room temperature for 2 hours, and filtered under reduced pressure to obtain a white solid powder intermediate 2-7, with a yield of 77%. MS(ESI)m/z:461.20(M+2) + , calculated for C 22 H 26 ClN 5 O 2 S: 459.15.

步骤Ⅵ、N-(3-((2-((4-(4-甲基哌嗪-1-基)-2-((四氢-2H-吡喃-2-基)氧基)苯基)氨基)噻吩并[3,2-d]嘧啶-4-基)氧基)苯基)丙烯酰胺(中间体2-8)的制备：称取中间体2-7(1.76g，4mmol)、N-(3-羟苯基)丙烯酰胺(815mg，5mmol)和碳酸钾(1.66g，12mmol)溶于25mL体积比为8:2的乙腈/水混合溶剂中，回流反应6小时。反应完全后，将反应物降至室温，加入50mL蒸馏水，在室温下反应过夜。将反应液抽滤，用10mL体积比为1:2的乙腈/水混合溶剂洗涤1次，得到白色固体粉末中间体2-8，产率：84％。MS(ESI)m/z:587.30(M+1) +,calculated for C 31H 34N 6O 4S:586.24. Step VI, N-(3-((2-((4-(4-methylpiperazin-1-yl)-2-((tetrahydro-2H-pyran-2-yl)oxy)phenyl ) Preparation of amino)thieno[3,2-d]pyrimidin-4-yl)oxy)phenyl)acrylamide (Intermediate 2-8): Weigh Intermediate 2-7 (1.76g, 4mmol), N-(3-hydroxyphenyl)acrylamide (815 mg, 5 mmol) and potassium carbonate (1.66 g, 12 mmol) were dissolved in 25 mL of an acetonitrile/water mixed solvent with a volume ratio of 8:2, and the reaction was refluxed for 6 hours. After the reaction was completed, the reactant was cooled to room temperature, 50 mL of distilled water was added, and the reaction was carried out at room temperature overnight. The reaction solution was suction filtered and washed once with 10 mL of a 1:2 volume ratio of acetonitrile/water mixed solvent to obtain a white solid powder intermediate 2-8, yield: 84%. MS(ESI)m/z:587.30(M+1) + ,calculated for C 31 H 34 N 6 O 4 S:586.24.

步骤Ⅶ-Ⅷ、N-(3-((2-((4-(4-甲基哌嗪-1-基)-2-((四氢-2H-吡喃-2-基)氧基)苯基)氨基)噻吩并[3,2-d]嘧啶-4-基)氧基)苯基)丙烯酰胺(通式化合物Ⅲ-1)的制备：参照实施例10中步骤Ⅴ和步骤Ⅵ的方法，将中间体2-8和中间体1-6进行反应，得到白色固体粉末目标化合物Ⅲ-1，产率：52％。MS(ESI)m/z:697.30(M+1) +,calculated for C 36H 36N 6O 7S:696.24. Step Ⅶ-Ⅷ, N-(3-((2-((4-(4-methylpiperazin-1-yl)-2-((tetrahydro-2H-pyran-2-yl)oxy) (Phenyl) amino) thieno [3,2-d] pyrimidin-4-yl) oxy) phenyl) acrylamide (general formula compound III-1) preparation: refer to the step V and step VI in Example 10 Method: Intermediate 2-8 and Intermediate 1-6 are reacted to obtain target compound III-1 as a white solid powder, yield: 52%. MS(ESI)m/z:697.30(M+1) + , calculated for C 36 H 36 N 6 O 7 S: 696.24.

实施例12通式化合物Ⅳ-1的合成(合成路线如图12所示)Example 12 Synthesis of general formula compound IV-1 (the synthetic route is shown in Figure 12)

步骤Ⅰ、3-(2-氯嘧啶-4-基)-1-甲基-1H-吲哚(中间体3-3)的制备：将2,4-二氯嘧啶(中间体3-2)(1.49g，10mmol)和氯化铝(1.33g，10mmol)悬浮在20mL的N,N-二甲基乙酰胺(DME)中，室温下搅拌5分钟后，向反应体系中加入1-甲基吲哚(中间体3-1)(1.25mL，10mmol)，80℃反应2小时，TLC监测反应过程。在5分钟内，将50mL超纯水缓慢滴入反应体系中猝灭反应，搅拌30分钟，将所得固体减压过滤后，快速硅胶柱层析纯化得到白色固体中间体3-3，产率93％。MS(ESI)m/z:244.10(M+1) +,calculated for C 13H 10ClN 3:243.06. Step 1. Preparation of 3-(2-chloropyrimidin-4-yl)-1-methyl-1H-indole (Intermediate 3-3): Add 2,4-Dichloropyrimidine (Intermediate 3-2) (1.49g, 10mmol) and aluminum chloride (1.33g, 10mmol) were suspended in 20mL of N,N-dimethylacetamide (DME). After stirring for 5 minutes at room temperature, 1-methyl was added to the reaction system. Indole (Intermediate 3-1) (1.25 mL, 10 mmol) was reacted at 80°C for 2 hours, and the reaction process was monitored by TLC. Within 5 minutes, 50 mL of ultrapure water was slowly dropped into the reaction system to quench the reaction, stirred for 30 minutes, the obtained solid was filtered under reduced pressure, and purified by flash silica gel column chromatography to obtain a white solid intermediate 3-3 with a yield of 93 %. MS(ESI) m/z: 244.10(M+1) + , calculated for C 13 H 10 ClN 3 : 243.06.

步骤Ⅱ、2-氨基-4-硝基-5-氟苯酚(中间体3-5)的制备：称取4-氟-2-甲氧基-5-硝基苯胺(中间体3-4)(372mg，2mmol)溶于20mL的二氯甲烷中，边搅拌边将三氯化铝(1333mg，10mmol，分三次)、碘化钠(150mg，1mmol)依次加入，室温下反应过夜。待反应完全，加入20mL饱和氯化铵溶液中和反应，再加入20mL二氯甲烷进行萃取，用饱和NaCl溶液洗涤二氯甲烷层3次，并用无水MgSO 4干燥。滤液浓缩后，通过快速硅胶柱层析纯化得到淡黄色油状液体中间体3-5，产率：37％。MS(ESI)m/z:173.10(M+1) +,calculated for C 6H 5FN 2O 3:172.03. Step II. Preparation of 2-amino-4-nitro-5-fluorophenol (Intermediate 3-5): Weigh 4-fluoro-2-methoxy-5-nitroaniline (Intermediate 3-4) (372mg, 2mmol) was dissolved in 20mL of dichloromethane, aluminum trichloride (1333mg, 10mmol, three times) and sodium iodide (150mg, 1mmol) were added in sequence while stirring, and reacted overnight at room temperature. When the reaction is complete, add 20 mL of saturated ammonium chloride solution to neutralize the reaction, then add 20 mL of dichloromethane for extraction, wash the dichloromethane layer with saturated NaCl solution 3 times, and dry with anhydrous MgSO 4 . After the filtrate was concentrated, it was purified by flash silica gel column chromatography to obtain the pale yellow oily liquid intermediate 3-5, the yield: 37%. MS(ESI)m/z:173.10(M+1) + ,calculated for C 6 H 5 FN 2 O 3 :172.03.

步骤Ⅲ、4-氟-5-硝基-2-((四氢-2H-吡喃-2-基)氧基)苯胺(中间体3-6)的制备：称取中间体3-5(860mg，5mmol)、二氢吡喃(1680mg，20mmol)和对甲苯磺酸吡啶盐(125.5mg，0.5mmol)，溶于20mL二氯甲烷中，室温下反应6小时。反应完全后，加入20mL水和20mL二氯甲烷，萃取并旋干二氯甲烷。硅胶柱层析法分离纯化后得到无色油状物中间体3-6，产率：70％。MS(ESI)m/z:257.13(M+1) +,calculated for C 11H 13FN 2O 4:256.09. Step III. Preparation of 4-fluoro-5-nitro-2-((tetrahydro-2H-pyran-2-yl)oxy)aniline (Intermediate 3-6): Weigh Intermediate 3-5 ( 860 mg, 5 mmol), dihydropyran (1680 mg, 20 mmol) and pyridinium p-toluenesulfonate (125.5 mg, 0.5 mmol) were dissolved in 20 mL of dichloromethane and reacted at room temperature for 6 hours. After the reaction is complete, add 20 mL of water and 20 mL of dichloromethane, extract and spin dry the dichloromethane. After separation and purification by silica gel column chromatography, intermediate 3-6 was obtained as a colorless oily substance with a yield of 70%. MS(ESI)m/z:257.13(M+1) + ,calculated for C 11 H 13 FN 2 O 4 :256.09.

步骤Ⅳ、N-(4-氟-5-硝基-2-((四氢-2H-吡喃-2-基)氧基)苯基)-4-(1-甲基-1H-吲哚-3-基)嘧啶-2-胺(中间体3-7)的制备：称取中间体3-6(1280mg，5mmol)和中间体3-3(1215mg，5mmol)溶于20mL2-戊醇中，加入对甲基苯磺酸一水合物(1140mg，6mmol)，105℃反应3小时。待反应完全后将反应物冷却至室温，抽滤，并用10mL2-戊醇洗涤2次。再用5mL乙腈洗涤1次，真空干燥得到黄色固体粉末中间体3-7，产率：95％。MS(ESI)m/z:464.30(M+1) +,calculated for C 24H 22FN 5O 4:463.17. Step IV, N-(4-Fluoro-5-nitro-2-((tetrahydro-2H-pyran-2-yl)oxy)phenyl)-4-(1-methyl-1H-indole Preparation of -3-yl)pyrimidin-2-amine (Intermediate 3-7): Weigh Intermediate 3-6 (1280mg, 5mmol) and Intermediate 3-3 (1215mg, 5mmol) and dissolve them in 20mL 2-pentanol , P-toluenesulfonic acid monohydrate (1140mg, 6mmol) was added and reacted at 105°C for 3 hours. After the reaction is complete, the reactant is cooled to room temperature, filtered with suction, and washed twice with 10 mL of 2-pentanol. It was washed with 5 mL of acetonitrile once, and dried in vacuum to obtain a yellow solid powder intermediate 3-7, yield: 95%. MS(ESI)m/z:464.30(M+1) + , calculated for C 24 H 22 FN 5 O 4 :463.17.

步骤Ⅴ、4-氟-N 1-(4-(1-甲基-1H-吲哚-3-基)嘧啶-2-基)-6-((四氢-2H-吡喃-2-基)氧基)苯基-1,3-二胺(中间体3-8)的制备：称取中间体3-7(927mg，2mmol)、铁粉(560mg，10mmol)和氯化铵(80.25mg，1.5mmol)，溶解于16mL的乙醇/水＝3:1的混合溶剂中，78℃回流反应2小时。反应完全后，通过磺酸基离子交换柱将溶液初步提纯，再通过快速硅胶柱层析法，分离纯化后得到浅褐色油状物中间体3-8，产率：82％。MS(ESI)m/z:434.30(M+1) +,calculated for C 24H 24FN 5O 2:433.19. Step V. 4-Fluoro-N 1 -(4-(1-methyl-1H-indol-3-yl)pyrimidin-2-yl)-6-((tetrahydro-2H-pyran-2-yl ) Oxy) phenyl-1, 3-diamine (Intermediate 3-8) preparation: Weigh intermediate 3-7 (927mg, 2mmol), iron powder (560mg, 10mmol) and ammonium chloride (80.25mg , 1.5mmol), dissolved in 16mL ethanol/water=3:1 mixed solvent, refluxed at 78°C for 2 hours. After the reaction is completed, the solution is preliminarily purified through a sulfonic acid group ion exchange column, and then through flash silica gel column chromatography to obtain a light brown oily intermediate 3-8 after separation and purification. The yield: 82%. MS(ESI)m/z:434.30(M+1) + ,calculated for C 24 H 24 FN 5 O 2 : 433.19.

步骤Ⅵ、N-(2-氟-5-((4-(1-甲基-1H-吲哚-3-基)嘧啶-2-基)胺基)-4-((四氢-2H-吡喃-2-基)氧基)苯基)丙烯酰胺(中间体3-9)的制备：称取中间体3-8(867mg，2mmol)和N,N-二异丙基乙胺(310mg，2.4mmol)，溶解于10mL的二氯甲烷中，将丙烯酰氯(181mg，2mmol)缓慢滴加进入反应液，在冰水浴中反应约2小时。反应完全后，分别加入50mL二氯甲烷和50mL饱和碳酸氢钠，萃取3次，硅胶柱层析法分离纯化后得到白色粉末中间体3-9，产率：32％。MS(ESI)m/z:488.30(M+1) +,calculated for C 27H 26FN 5O 3:487.20. Step VI, N-(2-fluoro-5-((4-(1-methyl-1H-indol-3-yl)pyrimidin-2-yl)amino)-4-((tetrahydro-2H- Preparation of pyran-2-yl)oxy)phenyl)acrylamide (Intermediate 3-9): Weigh Intermediate 3-8 (867mg, 2mmol) and N,N-diisopropylethylamine (310mg , 2.4 mmol), dissolved in 10 mL of dichloromethane, acryloyl chloride (181 mg, 2 mmol) was slowly added dropwise into the reaction solution, and reacted in an ice-water bath for about 2 hours. After the completion of the reaction, 50 mL of dichloromethane and 50 mL of saturated sodium bicarbonate were added respectively, and the extraction was carried out 3 times, and the white powder intermediate 3-9 was obtained after separation and purification by silica gel column chromatography. The yield: 32%. MS(ESI)m/z:488.30(M+1) + , calculated for C 27 H 26 FN 5 O 3 :487.20.

步骤Ⅶ-Ⅷ、N-(2-氟-5-((4-(1-甲基-1H-吲哚-3-基)嘧啶-2-基)胺基)-4-((四氢-2H-吡喃-2-基)氧基)苯基)丙烯酰胺(中间体3-10)的制备：参照实施例10中步骤Ⅴ和步骤Ⅵ的方法，将中间体3-9和中间体1-6进行反应，得到白色粉末状中间体3-10，产率：63％。MS(ESI)m/z:598.30(M+1) +,calculated for C 32H 28FN 5O 6:597.20. Step Ⅶ-Ⅷ, N-(2-fluoro-5-((4-(1-methyl-1H-indol-3-yl)pyrimidin-2-yl)amino)-4-((tetrahydro- Preparation of 2H-pyran-2-yl)oxy)phenyl)acrylamide (Intermediate 3-10): According to the method of step V and step VI in Example 10, the intermediate 3-9 and intermediate 1 -6 was reacted to obtain white powder intermediate 3-10, yield: 63%. MS(ESI)m/z:598.30(M+1) + , calculated for C 32 H 28 FN 5 O 6 :597.20.

步骤Ⅸ、3-(4-丙烯酰胺-5-((2-(二甲基氨基)乙基)(甲基)氨基)-2-((4-(1-甲基-1H-吲哚-3-基)嘧啶-2-基)氨基)苯氧基)丙基-3,5-二羟基苯甲酸酯(通式化合物Ⅳ-1，AZD-2OH)的制备：称取中间体3-10(597.6mg，1mmol)和N,N-二异丙基乙胺(306mg，3mmol)，溶解于10mL的2-戊醇中，在120℃中反应约3小时。反应完全后，旋干溶剂，分别加入10mL乙酸乙酯和10mL饱和硫酸钠，萃取3次，快速硅胶柱层析法分离得到白色固体粉末目标化合物Ⅳ-1，产率：72％。MS(ESI)m/z:680.30(M+1) +,calculated for C 37H 41N 7O 6:679.31. Step IX, 3-(4-acrylamide-5-((2-(dimethylamino)ethyl)(methyl)amino)-2-((4-(1-methyl-1H-indole- Preparation of 3-yl)pyrimidin-2-yl)amino)phenoxy)propyl-3,5-dihydroxybenzoate (general formula compound IV-1, AZD-2OH): Weigh intermediate 3- 10 (597.6 mg, 1 mmol) and N,N-diisopropylethylamine (306 mg, 3 mmol) were dissolved in 10 mL of 2-pentanol and reacted at 120°C for about 3 hours. After the reaction is complete, the solvent is spin-dried, 10 mL of ethyl acetate and 10 mL of saturated sodium sulfate are added respectively, and extraction is performed 3 times. The target compound IV-1 is obtained as a white solid powder by rapid silica gel column chromatography. The yield is 72%. MS(ESI)m/z:680.30(M+1) + , calculated for C 37 H 41 N 7 O 6 : 679.31.

实施例13免疫印迹法检测化合物Gefi-2OH对细胞中p62、CTSB等蛋白表达量的影响Example 13 Detection of the effect of compound Gefi-2OH on the expression of p62, CTSB and other proteins in cells by immunoblotting

将A549、H460细胞分别以1×10 5/孔和0.8×10 5/孔的密度接种于6孔板中，过夜培养后，将吉非替尼和化合物Gefi-2OH用完全培养基稀释成特定浓度，分别加入6孔板中，作用24小时后，加入裂解缓冲液裂解细胞，将此细胞裂解液煮沸变性后，进行Western Blot实验(步骤参照实施例1中的Western Blot方法)，结果如图13所示。 A549 and H460 cells were seeded in a 6-well plate at a density of 1×10 5 /well and 0.8×10 5 /well, respectively. After overnight culture, gefitinib and the compound Gefi-2OH were diluted with complete medium to make specific Concentrations were added to a 6-well plate. After 24 hours of action, lysis buffer was added to lyse the cells. After the cell lysate was boiled and denatured, the Western Blot experiment was performed (see the Western Blot method in Example 1 for the steps). 13 shown.

实验结果如图13A所示，改造后的碱性中和类似物Gefi-2OH(实施例10制备所得)对SQSTM1的积聚效果以及对溶酶体功能的影响(CTSB的表达量)较吉非替尼均大大减弱，提示碱性中和的改造策略有效地削弱了吉非替尼对溶酶体功能的阻断。如图13B所示，相同浓度的Gefi-2OH和吉非替尼均可以有效抑制EGFR以及其下游Akt、Erk1/2的磷酸化程度，表示此类改造并不会影响吉非替尼等EGFR TKI原有的EGFR激酶抑制活性。此外，我们同样检测了改造后的类似物AZD-2OH对SQSTM1和CTSB表达量的影响以及其对EGFR信号通路的抑制作用。实验结果如图14A和14B所示，以AZD9291为对照，AZD-2OH对SQSTM1的积聚效果以及对溶酶体功能的影响同样明显减弱，并对EGFR信号通路产生与AZD9291相同的抑制作用。The experimental results are shown in Figure 13A. The modified alkaline neutralizing analogue Gefi-2OH (prepared in Example 10) has a greater effect on SQSTM1 accumulation and on lysosomal function (CTSB expression) than Gefi-2OH Nijun was greatly weakened, suggesting that the alkaline neutralization strategy effectively weakened the blockade of gefitinib on lysosomal function. As shown in Figure 13B, both Gefi-2OH and Gefitinib at the same concentration can effectively inhibit the phosphorylation of EGFR and its downstream Akt and Erk1/2, indicating that such modifications will not affect EGFR TKIs such as Gefitinib. The original EGFR kinase inhibitory activity. In addition, we also tested the effect of the modified analogue AZD-2OH on the expression of SQSTM1 and CTSB and its inhibitory effect on the EGFR signaling pathway. The experimental results are shown in Figures 14A and 14B. With AZD9291 as a control, the accumulation effect of AZD-2OH on SQSTM1 and the effect on lysosome function are also significantly weakened, and the EGFR signaling pathway has the same inhibitory effect as AZD9291.

实施例14采用溶酶体示踪荧光探针(Lysotracker)检测化合物Gefi-2OH对细胞中溶酶体功能的影响Example 14 Using Lysotracker to Detect the Effect of Compound Gefi-2OH on Lysosome Function in Cells

荧光染料Lysotracker的荧光实验检测原理及具体步骤：Lysotracker是用能发出蓝色荧光的化学基团标记的带有弱碱性的探针，其中弱碱部分可提供质子，以维持pH在中性,可以选择性地滞留在偏酸性的溶酶体中，从而实现对于溶酶体的特异性荧光标记。其具体步骤如下：将盖玻片置于孔板中，细胞密度接种为平时的1/5到1/3，贴壁后进行相应的处理。将Lysotracker染料与培养液进行1:1000稀释后加入细胞内，孵育2小时。倒掉培养液，用1×PBS洗3次，每次5分钟。使用20％的甘油与PBS的混合液作为 封片液，直接将活细胞封片并在显微镜下观察荧光。The fluorescence experiment detection principle and specific steps of the fluorescent dye Lysotracker: Lysotracker is a weakly basic probe labeled with a chemical group that emits blue fluorescence. The weakly basic part can provide protons to maintain the pH at neutral. It can be selectively retained in acidic lysosomes to achieve specific fluorescent labeling of lysosomes. The specific steps are as follows: place the cover glass in the well plate, inoculate the cell density to 1/5 to 1/3 of the usual, and perform the corresponding treatment after sticking. The Lysotracker dye and the culture medium were diluted 1:1000, then added to the cells, and incubated for 2 hours. Pour out the culture medium and wash 3 times with 1×PBS for 5 minutes each time. Use a mixture of 20% glycerol and PBS as the mounting solution to directly mount the live cells and observe the fluorescence under a microscope.

前面的研究中，发明人观察到吉非替尼和AZD9291等化合物通过影响溶酶体的功能而抑制自噬溶酶体的降解。因此，针对改造后的类似物Gefi-2OH，发明人同样检测了其对溶酶体功能的影响。通过将不同药物处理组的细胞用溶酶体的特异性探针Lysotracker进行示踪，并进行荧光检测，结果如图15所示。实验结果显示，吉非替尼使溶酶体的体积明显增加，数量明显减少，表明吉非替尼显著削弱细胞内溶酶体的功能；而相较于吉非替尼组，其碱性中和类似物Gefi-2OH对溶酶体的体积影响较微弱，对溶酶体的数量改变也弱于吉非替尼组。基于上述结果，揭示了经过碱性中和改造的类似物Gefi-2OH对溶酶体功能的阻断比吉非替尼减弱，并且这种作用有望使Gefi-2OH对肿瘤细胞的杀伤作用提升。In previous studies, the inventors observed that compounds such as gefitinib and AZD9291 inhibit autophagolysosome degradation by affecting lysosome functions. Therefore, for the modified analogue Gefi-2OH, the inventors also tested its effect on lysosome function. The cells in different drug treatment groups were traced with Lysotracker, a specific probe of lysosome, and fluorescence detection was performed. The results are shown in Figure 15. Experimental results showed that gefitinib significantly increased the volume of lysosomes and decreased the number, indicating that gefitinib significantly weakened the function of intracellular lysosomes; compared with gefitinib group, its alkaline neutralization The analogue Gefi-2OH has a weaker effect on the volume of lysosomes, and the change in the number of lysosomes is also weaker than that of the gefitinib group. Based on the above results, it is revealed that the alkali-neutralized modified analogue Gefi-2OH is less effective than gefitinib in blocking lysosomal function, and this effect is expected to enhance the killing effect of Gefi-2OH on tumor cells.

实施例15化合物Gefi-2OH对非小细胞肺癌的增殖抑制作用Example 15 Compound Gefi-2OH inhibits proliferation of non-small cell lung cancer

取对数生长期细胞A549和H460细胞分别以5000个/孔和6000/孔的密度接种于96孔板内，每个实验组设置5个平行重复孔；培养24h后，加入配制好的药物浓度梯度的培养液，继续培养48h；检测前吸尽每孔内的培养基，并加入已经配制好的含10％MTS的1640培养基100μl，在37℃孵箱中避光孵育20-60分钟，酶联免疫检测仪上震荡1分钟后用490nm波长读取吸光度值，当净OD值达到0.5-0.8范围时停止检测，计算细胞的相对增殖率，并使用Graphpad prism 6画出增殖曲线，结果如图16所示。The logarithmic growth phase A549 and H460 cells were seeded in a 96-well plate at a density of 5000/well and 6000/well respectively, and each experimental group was set up with 5 parallel replicate wells; after 24 hours of culture, the formulated drug concentration was added Gradient culture medium, continue to incubate for 48h; aspirate the medium in each well before testing, and add 100μl of 1640 medium containing 10% MTS that has been prepared, and incubate in a 37℃ incubator in the dark for 20-60 minutes. After shaking on the enzyme-linked immunoassay instrument for 1 minute, read the absorbance value at 490nm wavelength. When the net OD value reaches the range of 0.5-0.8, stop the detection, calculate the relative cell proliferation rate, and use Graphpad prism 6 to draw the proliferation curve. The result is as follows Shown in Figure 16.

由图16结果可知，碱性中和的吉非替尼衍生物Gefi-2OH对非小细胞肺癌细胞A549、H460的增殖抑制作用要明显优于吉非替尼，其半数抑制浓度(IC 50)较吉非替尼分别提高了约1.8倍(A549细胞中)和3倍(H460细胞中)。此外，按照上述方法亦检测了本发明的化合物AZD-2OH对H460细胞的增殖抑制作用，其增殖曲线如图17所示。由图17结果可知，本发明的化合物AZD-2OH对H460细胞的增殖抑制作用同样明显优于改造前化合物AZD9291，其半数抑制浓度(IC 50)仅为AZD9291的一半不到。 From the results in Figure 16, it can be seen that the basic and neutralized gefitinib derivative Gefi-2OH has a significantly better proliferation inhibitory effect on non-small cell lung cancer cells A549 and H460 than gefitinib, and its half inhibitory concentration (IC 50 ) Gefitinib was increased by approximately 1.8 times (in A549 cells) and 3 times (in H460 cells), respectively. In addition, according to the above method, the inhibitory effect of the compound AZD-2OH of the present invention on the proliferation of H460 cells was also tested, and the proliferation curve is shown in FIG. 17. From the results of FIG. 17, the compounds of the present invention AZD-2OH inhibited the proliferation of H460 cells was significantly better than the same compound AZD9291 before transformation, 50% inhibitory concentrations (IC 50) less than half the AZD9291.

实施例16流式细胞术检测化合物Gefi-2OH对NSCLC细胞凋亡的影响Example 16 Detection of the effect of compound Gefi-2OH on NSCLC cell apoptosis by flow cytometry

分别收集A549和H460细胞上清于离心管中，用预冷的PBS洗涤一次，同样收集到上述离心管中。在细胞中加入适量不含EDTA的0.25％的胰酶，一定时间后，用2倍胰酶体积的含10％FBS的1640培养液中和，收集细胞悬液至之前的离心管中。离心1500rpm，5min，倒去上清，PBS重悬细胞沉淀，再次1500rpm离心5min，弃上清。加入100μl 1×Binding Buffer重悬细胞，将细胞的密度调整为5-10×10 5/mL。各加入5μl的Annexin V-FITC和5μl的PI Staining Solution，轻柔地混匀细胞悬液。避光、室温孵育15min。加入400μl 1×Binding Buffer稀释，轻轻混匀，1小时内上机检测。每个样采集10000events。结果如图18所示。 The A549 and H460 cell supernatants were collected in a centrifuge tube, washed once with pre-cooled PBS, and also collected in the above centrifuge tube. Add an appropriate amount of 0.25% trypsin without EDTA to the cells, after a certain period of time, neutralize with 2 times the volume of trypsin in 1640 culture medium containing 10% FBS, and collect the cell suspension into the previous centrifuge tube. Centrifuge at 1500rpm for 5min, pour out the supernatant, resuspend the cell pellet in PBS, centrifuge again at 1500rpm for 5min, and discard the supernatant. Add 100μl 1×Binding Buffer to resuspend the cells and adjust the cell density to 5-10×10 5 /mL. Add 5μl of Annexin V-FITC and 5μl of PI Staining Solution to each, and gently mix the cell suspension. Incubate at room temperature for 15min in the dark. Add 400μl 1×Binding Buffer to dilute, mix gently, and test on the machine within 1 hour. 10000 events are collected for each sample. The result is shown in Figure 18.

由图18结果可知，Gefi-2OH显著诱导A549、H460细胞凋亡，且在相同浓度作用下，Gefi-2OH诱导凋亡的效果要明显优于吉非替尼。From the results in Figure 18, it can be seen that Gefi-2OH significantly induces apoptosis of A549 and H460 cells, and at the same concentration, the effect of Gefi-2OH in inducing apoptosis is significantly better than that of gefitinib.

实施例17不同通式化合物的H460细胞增殖抑制活性筛选Example 17 H460 cell proliferation inhibitory activity screening of compounds of different general formulas

为了说明本发明的通式化合物对非小细胞肺癌细胞的增殖抑制作用，以下合成了典型的通式(Ⅰ)化合物，并进行了细胞增殖实验检测，化合物具体合成方法同实施例10-12，检测方法同实施例15，其检测结果表1-3所示；其中表1、2、3结果分别表示通式化合物Ⅱ、Ⅲ、Ⅳ对H460细胞的增殖活性的影响。由表1-3结果可知，对吉非替尼、奥姆替尼以及奥西替尼进行碱性中和改造，极大地提升了其对H460细胞的杀伤作用。改造所得的通式化合物Ⅱ、Ⅲ、Ⅳ均表现出良好的肺癌细胞增殖抑制活性，其中，奥西替尼的改造物(通式Ⅳ-1-通式Ⅳ-8)活性最优，其IC 50值在1.21-5.13μM之间。此外，通过对三类化合物的构效关系分析，发现R取代基的酸性对活性的高低起决定性作用，其酸性越强，化合物整体的抗肿瘤活性也越好，具体表现为不同取代基对化合物增殖抑制活性提升的贡献排名：烷基磺酸>烷基羧酸>苯磺酸>苯甲酸>三元酚>二元酚，由此进一步说明了EGFR TKIs分子结构所带碱性的确是导致肿瘤细胞对其敏感性降低、耐药性产生的重要原因，而本发明首次针对EGFR TKIs进行碱性中和改造，取得了意想不到的良好效果，探索出了一条提高其活性的新途径。 In order to illustrate the inhibitory effect of the compound of the present invention on the proliferation of non-small cell lung cancer cells, a typical compound of the general formula (I) was synthesized below, and the cell proliferation test was carried out. The specific synthesis method of the compound is the same as in Examples 10-12 The detection method is the same as in Example 15, and the detection results are shown in Tables 1-3; the results of Tables 1, 2, and 3 respectively indicate the effects of compounds II, III, and IV on the proliferation activity of H460 cells. It can be seen from the results in Table 1-3 that the alkaline neutralization modification of gefitinib, omitinib and osimertinib greatly enhances its killing effect on H460 cells. The modified compounds of general formula II, III, and IV all showed good lung cancer cell proliferation inhibitory activity. Among them, the modified product of osimertinib (formula IV-1-formula IV-8) had the best activity, and its IC 50 The value is between 1.21-5.13μM. In addition, through the analysis of the structure-activity relationship of the three types of compounds, it is found that the acidity of the R substituent plays a decisive role in the activity level. The stronger the acidity, the better the overall anti-tumor activity of the compound. The specific performance is that different substituents affect the compound. The ranking of the contribution to the enhancement of proliferation inhibitory activity: alkyl sulfonic acid> alkyl carboxylic acid> benzene sulfonic acid> benzoic acid> trihydric phenol> dihydric phenol, which further illustrates that the basicity of the molecular structure of EGFR TKIs is indeed responsible for tumors It is an important reason for cells to reduce their sensitivity and drug resistance. The present invention is the first to carry out alkaline neutralization modification for EGFR TKIs, which has achieved unexpected good results and explored a new way to improve its activity.

表1Table 1

Figure PCTCN2020089390-appb-000026
Figure PCTCN2020089390-appb-000026

化合物Ⅱ-1-化合物Ⅱ-8Compound Ⅱ-1-Compound Ⅱ-8

Figure PCTCN2020089390-appb-000027
Figure PCTCN2020089390-appb-000027

表2Table 2

Figure PCTCN2020089390-appb-000028
Figure PCTCN2020089390-appb-000028

化合物Ⅲ-1-化合物Ⅲ-8Compound Ⅲ-1-Compound Ⅲ-8

Figure PCTCN2020089390-appb-000029
Figure PCTCN2020089390-appb-000029

表3table 3

Figure PCTCN2020089390-appb-000030
Figure PCTCN2020089390-appb-000030

化合物Ⅳ-1-化合物Ⅳ-8Compound IV-1-Compound IV-8

Figure PCTCN2020089390-appb-000031
Figure PCTCN2020089390-appb-000031

Figure PCTCN2020089390-appb-000032
Figure PCTCN2020089390-appb-000032

Claims (16)

一种如通式Ⅰ所示的化合物或其药学上可接受的盐，A compound represented by formula I or a pharmaceutically acceptable salt thereof,
Figure PCTCN2020089390-appb-100001
Figure PCTCN2020089390-appb-100001
 式Ⅰ中，In formula I, A环为5至18元环，或，A环不存在；Ring A is a 5- to 18-membered ring, or, Ring A does not exist; X为-O-、-S-、-N(R 4)-或-(CHR 4) n-，n为0或1； X is -O-, -S-, -N(R 4 )- or -(CHR 4 ) n -, n is 0 or 1; R 1选自：芳基、芳烷基、芳香族或非芳香族杂环基和杂环烷基； R 1 is selected from: aryl, aralkyl, aromatic or non-aromatic heterocyclic group and heterocycloalkyl; R 2选自：氢、烷基、环烷基、烯基、炔基、芳基、芳烷基、芳香族或非芳香族杂环基、杂环烷基、-COR 4、-C(O)OR 4、-C(O)NR 4R 5、-CH＝NR 4、-CN、-OR 4、-OC(O)R 4、-S(O) t-R 4、-NR 4R 5、-NR 4C(O)R 5、-NO 2、-N＝CR 4R 5和卤素； R 2 is selected from: hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, aromatic or non-aromatic heterocyclic group, heterocycloalkyl, -COR 4 , -C(O )OR 4 , -C(O)NR 4 R 5 , -CH=NR 4 , -CN, -OR 4 , -OC(O)R 4 , -S(O) t -R 4 , -NR 4 R 5 , -NR 4 C(O)R 5 , -NO 2 , -N=CR 4 R 5 and halogen; R 3每次出现时独立地选自：氢、烷基、环烷基、烯基、炔基、芳基、芳烷基、芳香族或非芳香族杂环基、杂环烷基、-COR 4、-C(O)OR 4、-C(O)NR 4R 5、-CH＝NR 4、-CN、-OR 4、-OC(O)R 4、-S(O) t-R 4、-NR 4R 5、-NR 4C(O)R 5、-NO 2、-N＝CR 4R 5和卤素； Each occurrence of R 3 is independently selected from: hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, aromatic or non-aromatic heterocyclic group, heterocycloalkyl, -COR 4 , -C(O)OR 4 , -C(O)NR 4 R 5 , -CH=NR 4 , -CN, -OR 4 , -OC(O)R 4 , -S(O) t -R 4 , -NR 4 R 5 , -NR 4 C(O)R 5 , -NO 2 , -N=CR 4 R 5 and halogen; m为0-3的整数；m is an integer of 0-3; t为1、2或3；t is 1, 2 or 3; R 4和R 5分别独立地选自：氢、烷基、环烷基、烯基、芳基、芳香族或非芳香族杂环基、烷氧基、芳氧基和卤素； R 4 and R 5 are each independently selected from: hydrogen, alkyl, cycloalkyl, alkenyl, aryl, aromatic or non-aromatic heterocyclic group, alkoxy, aryloxy and halogen; 并且，通式Ⅰ所示的化合物结构中含有至少一个酸性取代基，所述酸性取代基含有至少一个酸性官能团；In addition, the structure of the compound represented by Formula I contains at least one acidic substituent, and the acidic substituent contains at least one acidic functional group; 优选地，所述酸性官能团选自：酚羟基、羧基、磺酸基和磷酸基中的一种或多种；Preferably, the acidic functional group is selected from one or more of phenolic hydroxyl group, carboxyl group, sulfonic acid group and phosphoric acid group; 更优选地，所述酸性取代基选自：羟基苯基、羧基苯基、磺酸基苯基、磷酸基苯基、羟基苯甲酰氧基取代烷基、磺酸基取代烷基、羧基取代烷基、磷酸基取代烷基、磺酸基苯基取代烷基、羧基苯基取代烷基、磷酸基苯基取代烷基、羟基苯甲酰氧基取代烷氧基、磺酸基取代烷氧基、羧基取代烷氧基、磷酸基取代烷氧基、磺酸基苯基取代烷氧基、羧基苯基取代烷氧基和磷酸基苯基取代烷氧基中的一种或多种；More preferably, the acidic substituent is selected from: hydroxyphenyl, carboxyphenyl, sulfophenyl, phosphorophenyl, hydroxybenzoyloxy substituted alkyl, sulfonic acid substituted alkyl, carboxyl substituted Alkyl, phosphate substituted alkyl, sulfonic phenyl substituted alkyl, carboxyphenyl substituted alkyl, phosphate phenyl substituted alkyl, hydroxybenzoyloxy substituted alkoxy, sulfonic acid substituted alkoxy One or more of the group, carboxyl substituted alkoxy, phosphoric acid substituted alkoxy, sulfonic phenyl substituted alkoxy, carboxyphenyl substituted alkoxy, and phosphoric phenyl substituted alkoxy; 更进一步优选地，所述化合物的结构中含有至少一个如下结构的取代基：More preferably, the structure of the compound contains at least one substituent of the following structure:
Figure PCTCN2020089390-appb-100002
Figure PCTCN2020089390-appb-100002
 如权利要求1所述的化合物，其特征在于，所述A环为芳环，优选为苯环，其与相连的嘧啶环形成喹唑啉环结构；或，The compound of claim 1, wherein the A ring is an aromatic ring, preferably a benzene ring, which forms a quinazoline ring structure with the connected pyrimidine ring; or, 所述A环为杂芳环，优选为单杂原子五元芳环，更优选为吡咯环、呋喃环或噻吩环，其与嘧啶环稠合形成五元单杂芳环[3,2-d]并嘧啶环结构；或，The A ring is a heteroaromatic ring, preferably a single heteroatom five-membered aromatic ring, more preferably a pyrrole ring, a furan ring or a thiophene ring, which is fused with a pyrimidine ring to form a five-membered single heteroaromatic ring [3,2-d ] Pyrimidine ring structure; or, 所述A环不存在。The A ring does not exist. 如权利要求1所述的化合物，其特征在于，所述化合物具有通式Ⅱ所示结构：The compound of claim 1, wherein the compound has a structure represented by Formula II:
Figure PCTCN2020089390-appb-100003
Figure PCTCN2020089390-appb-100003
 式Ⅱ中，X为-O-、-S-、-N(R 17)-或-(CHR 17) n-，n为0或1； In formula II, X is -O-, -S-, -N(R 17 )- or -(CHR 17 ) n -, and n is 0 or 1; R 11、R 12和R 13独立地选自：氢、烷基、环烷基、烯基、炔基、芳基、芳烷基、芳香族或非芳香族杂环基、杂环烷基、-COR 17、-C(O)OR 17、-C(O)NR 17R 18、-CH＝NR 17、-CN、-OR 17、-OC(O)R 17、-S(O) t-R 17、-NR 17R 18、-NR 17C(O)R 18、-NO 2、-N＝CR 17R 18和卤素； R 11 , R 12 and R 13 are independently selected from: hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, aromatic or non-aromatic heterocyclic group, heterocycloalkyl, -COR 17 , -C(O)OR 17 , -C(O)NR 17 R 18 , -CH=NR 17 , -CN, -OR 17 , -OC(O)R 17 , -S(O) t- R 17 , -NR 17 R 18 , -NR 17 C(O)R 18 , -NO 2 , -N=CR 17 R 18 and halogen; R 14选自：氢、烷基、环烷基、烯基、炔基、芳基、芳烷基、芳香族或非芳香族杂环基、杂环烷基、-COR 17、-C(O)OR 17、-C(O)NR 17R 18、-CH＝NR 17、-CN、-OR 17、-OC(O)R 17、-S(O) t-R 17、-NR 17R 18、-NR 17C(O)R 18、-NO 2、-N＝CR 17R 18和卤素； R 14 is selected from: hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, aromatic or non-aromatic heterocyclic group, heterocycloalkyl, -COR 17 , -C(O )OR 17 , -C(O)NR 17 R 18 , -CH=NR 17 , -CN, -OR 17 , -OC(O)R 17 , -S(O) t -R 17 , -NR 17 R 18 , -NR 17 C(O)R 18 , -NO 2 , -N=CR 17 R 18 and halogen; R 15和R 16独立地选自：氢、烷基、环烷基、烯基、炔基、芳基、芳烷基、芳香族或非芳香族杂环基、杂环烷基、-COR 17、-C(O)OR 17、-C(O)NR 17R 18、-CH＝NR 17、-CN、-OR 17、-OR 19-R 18、-OC(O)R 17、-S(O) t-R 17、-NR 17R 18、-NR 17C(O)R 18、-NO 2、-N＝CR 17R 18和卤素；或R 15和R 16与其所连接的苯环形成任意取代的稠环体系； R 15 and R 16 are independently selected from: hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, aromatic or non-aromatic heterocyclic group, heterocycloalkyl, -COR 17 , -C(O)OR 17 , -C(O)NR 17 R 18 , -CH=NR 17 , -CN, -OR 17 , -OR 19 -R 18 , -OC(O)R 17 , -S( O) t -R 17 , -NR 17 R 18 , -NR 17 C(O)R 18 , -NO 2 , -N=CR 17 R 18 and halogen; or R 15 and R 16 are formed with the benzene ring to which they are connected Arbitrarily substituted fused ring system; t为1、2或3；t is 1, 2 or 3; R 17和R 18独立地选自：氢、烷基、环烷基、烯基、芳基、芳香族或非芳香族杂环基、烷氧基、芳氧基、卤素和氨基； R 17 and R 18 are independently selected from: hydrogen, alkyl, cycloalkyl, alkenyl, aryl, aromatic or non-aromatic heterocyclic group, alkoxy, aryloxy, halogen and amino; R 19为亚烷基； R 19 is an alkylene group; 并且，R 11、R 12、R 13、R 14、R 15和R 16中至少一个含有酸性官能团。 In addition, at least one of R 11 , R 12 , R 13 , R 14 , R 15 and R 16 contains an acidic functional group. 如权利要求3所述的化合物，其特征在于，所述R 15含有至少一个酸性官能团； The compound of claim 3, wherein said R 15 contains at least one acidic functional group; 优选地，所述R 15选自：羟基苯基、羧基苯基、磺酸基苯基、磷酸基苯基、羟基苯甲酰氧基取代烷基、磺酸基取代烷基、羧基取代烷基、磷酸基取代烷基、磺酸基苯基取代烷基、羧基苯基取代烷基、磷酸基苯基取代烷基、羟基苯甲酰氧基取代烷氧基、磺酸基取代烷氧基、羧基取代烷氧基、磷酸基取代烷氧基、磺酸基苯基取代烷氧基、羧基苯基取代烷氧基和磷酸基苯基取代烷氧基； Preferably, the R 15 is selected from: hydroxyphenyl, carboxyphenyl, sulfophenyl, phosphorophenyl, hydroxybenzoyloxy substituted alkyl, sulfonic acid substituted alkyl, carboxyl substituted alkyl , Phosphate substituted alkyl, sulfonic phenyl substituted alkyl, carboxyphenyl substituted alkyl, phosphate phenyl substituted alkyl, hydroxybenzoyloxy substituted alkoxy, sulfonic acid substituted alkoxy, Carboxyl substituted alkoxy, phosphoric acid substituted alkoxy, sulfonic phenyl substituted alkoxy, carboxyphenyl substituted alkoxy and phosphoric phenyl substituted alkoxy; 更优选地，所述R 15为
Figure PCTCN2020089390-appb-100004
More preferably, the R 15 is
Figure PCTCN2020089390-appb-100004
 如权利要求3或4所述的化合物，其特征在于，所述X为-NH-；The compound of claim 3 or 4, wherein said X is -NH-; 所述R 11、R 12和R 13独立地选自：氢、卤素、-NO 2、烯基、炔基、-O(CH 2) xAr，x为0或1，Ar为芳基或杂环基；优选地，所述R 11为F，R 12为Cl，R 13为氢； The R 11 , R 12 and R 13 are independently selected from: hydrogen, halogen, -NO 2 , alkenyl, alkynyl, -O(CH 2 ) x Ar, x is 0 or 1, Ar is aryl or hetero Cyclic group; preferably, said R 11 is F, R 12 is Cl, and R 13 is hydrogen; 所述R 14选自：氢、烷基、-CN和卤素；优选地，所述R 14为氢； Said R 14 is selected from: hydrogen, alkyl, -CN and halogen; preferably, said R 14 is hydrogen; 所述R 16为-OR 19-R 18，其中R 19为亚甲基、亚乙基、亚丙基或亚丁基，R 18选自：芳香族或非芳香族杂环基、烷氧基、芳氧基和取代的氨基；优选地，所述R 16选自：3-(4-吗啉基)丙氧基、3-(1-哌嗪基)丙氧基、3,3-二甲氨基丙氧基、3-(1-吡咯烷基)丙氧基、3-(1-哌啶基)丙氧基、3-(4-哌啶基)丙氧基、甲氧乙氧基和乙氧乙氧基，更优选为
Figure PCTCN2020089390-appb-100005
或，所述R 16为-NHC(O)R 18，其中R 18选自：取代或未取代的烯基，所述烯基上的取代基选自：芳香族或非芳香族杂环基、烷氧基和取代的氨基，优选地，所述R 16选自：4-甲氧基-2-丁烯酰胺基、4-吡咯烷基-2-丁烯酰胺基、4-二甲氨基-2-丁烯酰胺基和4-哌啶基-2-丁烯酰胺基。 The R 16 is -OR 19 -R 18 , wherein R 19 is methylene, ethylene, propylene or butylene, and R 18 is selected from: aromatic or non-aromatic heterocyclic groups, alkoxy groups, Aryloxy and substituted amino; preferably, the R 16 is selected from: 3-(4-morpholinyl)propoxy, 3-(1-piperazinyl)propoxy, 3,3-dimethyl Aminopropoxy, 3-(1-pyrrolidinyl)propoxy, 3-(1-piperidinyl)propoxy, 3-(4-piperidinyl)propoxy, methoxyethoxy and Ethoxyethoxy, more preferably
Figure PCTCN2020089390-appb-100005
Or, the R 16 is -NHC(O)R 18 , wherein R 18 is selected from a substituted or unsubstituted alkenyl group, and the substituent on the alkenyl group is selected from an aromatic or non-aromatic heterocyclic group, Alkoxy and substituted amino, preferably, said R 16 is selected from: 4-methoxy-2-butenamido, 4-pyrrolidin-2-butenamido, 4-dimethylamino- 2-Butenamido and 4-piperidinyl-2-butenamido. 如权利要求1所述的化合物，其特征在于，所述化合物具有通式Ⅲ所示结构：The compound of claim 1, wherein the compound has a structure represented by Formula III:
Figure PCTCN2020089390-appb-100006
Figure PCTCN2020089390-appb-100006
Figure PCTCN2020089390-appb-100007
Figure PCTCN2020089390-appb-100007
 式Ⅲ中，In formula Ⅲ, X为-O-、-S-、-N(R 29)-或-(CHR 29) n-，n为0或1； X is -O-, -S-, -N(R 29 )- or -(CHR 29 ) n -, n is 0 or 1; Y为-O-、-S-或-N(R 29)-； Y is -O-, -S- or -N(R 29 )-; Z为-O-、-S-或-N(R 29)-； Z is -O-, -S- or -N(R 29 )-; R 21、R 22和R 23独立地选自：氢、烷基、环烷基、烯基、炔基、芳基、芳烷基、芳香族或非芳香族杂环基、杂环烷基、-COR 29、-C(O)OR 29、-C(O)NR 29R 28、-CH＝NR 29、-CN、-OR 29、-OC(O)R 29、-S(O) t-R 29、-NR 29R 210、-NR 29C(O)R 210、-NO 2、-N＝CR 29R 210和卤素； R 21 , R 22 and R 23 are independently selected from: hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, aromatic or non-aromatic heterocyclic group, heterocycloalkyl, -COR 29 , -C(O)OR 29 , -C(O)NR 29 R 28 , -CH=NR 29 , -CN, -OR 29 , -OC(O)R 29 , -S(O) t- R 29 , -NR 29 R 210 , -NR 29 C(O)R 210 , -NO 2 , -N=CR 29 R 210 and halogen; R 24、R 25和R 26独立地选自：氢、烷基、环烷基、烯基、炔基、芳基、芳烷基、芳香族或非芳香族杂环基、杂环烷基、-COR 29、-C(O)OR 29、-C(O)NR 29R 28、-CH＝NR 29、-CN、-OR 29、-OC(O)R 29、-S(O) t-R 29、-NR 29R 210、-NR 29C(O)R 210、-NO 2、-N＝CR 29R 210和卤素； R 24 , R 25 and R 26 are independently selected from: hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, aromatic or non-aromatic heterocyclic group, heterocycloalkyl, -COR 29 , -C(O)OR 29 , -C(O)NR 29 R 28 , -CH=NR 29 , -CN, -OR 29 , -OC(O)R 29 , -S(O) t- R 29 , -NR 29 R 210 , -NR 29 C(O)R 210 , -NO 2 , -N=CR 29 R 210 and halogen; R 27和R 28独立地选自：氢、烷基、环烷基、烯基、炔基、芳基、芳烷基、芳香族或非芳香族杂环基、杂环烷基、-COR 29、-C(O)OR 29、-C(O)NR 29R 28、-CH＝NR 29、-CN、-OR 29、-OC(O)R 29、-S(O) t-R 29、-NR 29R 210、-NR 29C(O)R 210、-NO 2、-N＝CR 29R 210和卤素；R 27和R 28与其所连接的杂环形成任意取代的稠环体系； R 27 and R 28 are independently selected from: hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, aromatic or non-aromatic heterocyclic group, heterocycloalkyl, -COR 29 , -C(O)OR 29 , -C(O)NR 29 R 28 , -CH=NR 29 , -CN, -OR 29 , -OC(O)R 29 , -S(O) t -R 29 , -NR 29 R 210 , -NR 29 C(O)R 210 , -NO 2 , -N=CR 29 R 210 and halogen; R 27 and R 28 and the heterocyclic ring to which they are connected form an optionally substituted fused ring system; t为1、2或3；t is 1, 2 or 3; R 29和R 210独立地选自：氢、烷基、环烷基、烯基、芳基、芳香族或非芳香族杂环基、烷氧基、芳氧基、卤素和氨基； R 29 and R 210 are independently selected from: hydrogen, alkyl, cycloalkyl, alkenyl, aryl, aromatic or non-aromatic heterocyclic group, alkoxy, aryloxy, halogen and amino; 并且，R 21、R 22、R 23、R 24、R 25、R 26、R 27和R 28中至少一个含有酸性官能团。 In addition, at least one of R 21 , R 22 , R 23 , R 24 , R 25 , R 26 , R 27 and R 28 contains an acidic functional group. 如权利要求6所述的化合物，其特征在于，所述R 26含有至少一个酸性官能团； The compound of claim 6, wherein the R 26 contains at least one acidic functional group; 优选地，所述R 26选自：羟基苯基、羧基苯基、磺酸基苯基、磷酸基苯基、羟基苯甲酰氧基取代烷基、磺酸基取代烷基、羧基取代烷基、磷酸基取代烷基、磺酸基苯基取代烷基、羧基苯基取代烷基、磷酸基苯基取代烷基、羟基苯甲酰氧基取代烷氧基、磺酸基取代烷氧基、羧基取代烷氧基、磷酸基取代烷氧基、磺酸基苯基取代烷氧基、羧基苯基取代烷氧基和磷酸基苯基取代烷氧基； Preferably, the R 26 is selected from: hydroxyphenyl, carboxyphenyl, sulfophenyl, phosphorophenyl, hydroxybenzoyloxy substituted alkyl, sulfonic acid substituted alkyl, carboxy substituted alkyl , Phosphate substituted alkyl, sulfonic phenyl substituted alkyl, carboxyphenyl substituted alkyl, phosphate phenyl substituted alkyl, hydroxybenzoyloxy substituted alkoxy, sulfonic acid substituted alkoxy, Carboxyl substituted alkoxy, phosphoric acid substituted alkoxy, sulfonic phenyl substituted alkoxy, carboxyphenyl substituted alkoxy and phosphoric phenyl substituted alkoxy; 更优选地，所述R 26为
Figure PCTCN2020089390-appb-100008
More preferably, the R 26 is
Figure PCTCN2020089390-appb-100008
 如权利要求6或7所述的化合物，其特征在于，所述X为-NH-为-O-；The compound of claim 6 or 7, wherein said X is -NH- is -O-; 所述Y为-S-；The Y is -S-; 所述Z为-NH-；The Z is -NH-; 所述R 21和R 23独立地选自：氢、烷基和卤素；优选地，所述R 21为氢，R 23为氢； Said R 21 and R 23 are independently selected from: hydrogen, alkyl and halogen; preferably, said R 21 is hydrogen and R 23 is hydrogen; 所述R 22为-NHC(O)R 210、其中，R 210为取代或未取代的烯基；优选地，所述R 22为
Figure PCTCN2020089390-appb-100009
The R 22 is -NHC(O)R 210 , wherein R 210 is a substituted or unsubstituted alkenyl group; preferably, the R 22 is
Figure PCTCN2020089390-appb-100009
 所述R 24为杂环基，优选为取代的含氮非芳香杂环基，其上取代基选自：烷基、酰基和杂环基，优选地，所述R 24选自：4-(1-乙酰基)哌嗪基、4-(1-甲基)哌嗪基、4-(1-吡咯烷基)哌嗪基、4-(1-哌啶基)哌嗪基和4-甲基哌嗪基-1-哌啶基，最优选为
Figure PCTCN2020089390-appb-100010
或，所述R 24为-NR 27R 28，R 27和R 28独立地选自：氢和取代或未取代的烷基，优选为N,N-二甲氨基或N 1,N 1,N 2-三甲基乙二胺基； Said R 24 is a heterocyclic group, preferably a substituted nitrogen-containing non-aromatic heterocyclic group, the substituents on which are selected from: alkyl, acyl and heterocyclic group, preferably, said R 24 is selected from: 4-( 1-acetyl)piperazinyl, 4-(1-methyl)piperazinyl, 4-(1-pyrrolidinyl)piperazinyl, 4-(1-piperidinyl)piperazinyl and 4-methyl Piperazinyl-1-piperidinyl, most preferably
Figure PCTCN2020089390-appb-100010
Or, the R 24 is -NR 27 R 28 , R 27 and R 28 are independently selected from: hydrogen and substituted or unsubstituted alkyl, preferably N,N-dimethylamino or N 1 ,N 1 ,N 2 -Trimethylethylenediamine group; 所述R 25为氢； The R 25 is hydrogen; 所述R 27和R 28独立地选自：氢、烷基和卤素；优选地，所述R 27为氢，R 28为氢。 Said R 27 and R 28 are independently selected from: hydrogen, alkyl and halogen; preferably, said R 27 is hydrogen and R 28 is hydrogen. 如权利要求1所述的化合物，其特征在于，所述化合物具有通式Ⅳ所示结构：The compound of claim 1, wherein the compound has a structure represented by Formula IV:
Figure PCTCN2020089390-appb-100011
Figure PCTCN2020089390-appb-100011
 式Ⅳ中，In formula IV, X为-O-、-S-、-N(R 39)-或-(CHR 39) n-，n为0或1； X is -O-, -S-, -N(R 39 )- or -(CHR 39 ) n -, n is 0 or 1; Z为-O-、-S-或-N(R 39)-； Z is -O-, -S- or -N(R 39 )-; R 31为氢、取代或未取代的芳基或杂环基； R 31 is hydrogen, substituted or unsubstituted aryl or heterocyclic group; R 32、R 33、R 34、R 35和R 36独立地选自：氢、烷基、环烷基、烯基、炔基、芳基、芳烷基、芳香族或非芳香族杂环基、杂环烷基、-COR 39、-C(O)OR 39、-C(O)NR 39R 310、-CH＝NR 39、-CN、-OR 39、-OC(O)R 39、-S(O) t-R 39、-NR 39R 310、-NR 39C(O)R 310、-NO 2、-N＝CR 39R 310和卤素； R 32 , R 33 , R 34 , R 35 and R 36 are independently selected from: hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, aromatic or non-aromatic heterocyclic group , Heterocycloalkyl, -COR 39 , -C(O)OR 39 , -C(O)NR 39 R 310 , -CH=NR 39 , -CN, -OR 39 , -OC(O)R 39 ,- S(O) t -R 39 , -NR 39 R 310 , -NR 39 C(O)R 310 , -NO 2 , -N=CR 39 R 310 and halogen; R 37和R 38独立地选自：氢、烷基、环烷基、烯基、炔基、芳基、芳烷基、芳香族或 非芳香族杂环基、杂环烷基、-COR 39、-C(O)OR 39、-C(O)NR 39R 310、-CH＝NR 39、-CN、-OR 39、-OC(O)R 39、-S(O) t-R 39、-NR 39R 310、-NR 39C(O)R 310、-NO 2、-N＝CR 39R 310和卤素； R 37 and R 38 are independently selected from: hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, aromatic or non-aromatic heterocyclic group, heterocycloalkyl, -COR 39 , -C(O)OR 39 , -C(O)NR 39 R 310 , -CH=NR 39 , -CN, -OR 39 , -OC(O)R 39 , -S(O) t -R 39 , -NR 39 R 310 , -NR 39 C(O)R 310 , -NO 2 , -N=CR 39 R 310 and halogen; t为1、2或3；t is 1, 2 or 3; R 39和R 310独立地选自：氢、烷基、环烷基、烯基、芳基、芳香族或非芳香族杂环基、烷氧基、芳氧基、卤素和氨基； R 39 and R 310 are independently selected from: hydrogen, alkyl, cycloalkyl, alkenyl, aryl, aromatic or non-aromatic heterocyclic group, alkoxy, aryloxy, halogen and amino; 并且，R 31、R 32、R 33、R 34、R 35、R 36、R 37和R 38中至少一个含有酸性官能团； In addition, at least one of R 31 , R 32 , R 33 , R 34 , R 35 , R 36 , R 37 and R 38 contains an acidic functional group; 优选地，所述酸性官能团选自：酚羟基、羧基、磺酸基和磷酸基中的一种或多种。Preferably, the acidic functional group is selected from one or more of phenolic hydroxyl group, carboxyl group, sulfonic acid group and phosphoric acid group. 如权利要求9所述的化合物，其特征在于，所述R 36含有至少一个酸性官能团； The compound of claim 9, wherein said R 36 contains at least one acidic functional group; 优选地，所述R 36选自：羟基苯基、羧基苯基、磺酸基苯基、磷酸基苯基、羟基苯甲酰氧基取代烷基、磺酸基取代烷基、羧基取代烷基、磷酸基取代烷基、磺酸基苯基取代烷基、羧基苯基取代烷基、磷酸基苯基取代烷基、羟基苯甲酰氧基取代烷氧基、磺酸基取代烷氧基、羧基取代烷氧基、磷酸基取代烷氧基、磺酸基苯基取代烷氧基、羧基苯基取代烷氧基和磷酸基苯基取代烷氧基； Preferably, the R 36 is selected from: hydroxyphenyl, carboxyphenyl, sulfophenyl, phosphorophenyl, hydroxybenzoyloxy substituted alkyl, sulfonic acid substituted alkyl, carboxyl substituted alkyl , Phosphate substituted alkyl, sulfonic phenyl substituted alkyl, carboxyphenyl substituted alkyl, phosphate phenyl substituted alkyl, hydroxybenzoyloxy substituted alkoxy, sulfonic acid substituted alkoxy, Carboxyl substituted alkoxy, phosphoric acid substituted alkoxy, sulfonic phenyl substituted alkoxy, carboxyphenyl substituted alkoxy and phosphoric phenyl substituted alkoxy; 更优选地，所述R 36为
Figure PCTCN2020089390-appb-100012
More preferably, the R 36 is
Figure PCTCN2020089390-appb-100012
 如权利要求9或10所述的化合物，其特征在于，所述X为-(CHR 39) n-，n为0； The compound of claim 9 or 10, wherein the X is -(CHR 39 ) n -, and n is 0; 所述Z为-NH-；The Z is -NH-; 所述R 31为取代的芳基，优选为
Figure PCTCN2020089390-appb-100013
其中，R 310为取代或未取代的烯基，所述烯基上的取代基选自：烷氧基、杂环基和取代的氨基，优选地，所述R 31选自：3-丙烯酰胺基苯基、3-(4-甲氧基-2-丁烯酰胺基)苯基、3-(4-哌啶基-2-丁烯酰胺基)苯基、3-(4-吡咯烷基-2-丁烯酰胺基)苯基、3-(4-二甲胺基-2-丁烯酰胺基)苯基；或，所述R 31为取代或未取代的杂环基，优选为吡唑[1,5-a]并吡啶基或取代的吲哚基，所述吲哚基上的取代基选自：氢、烷基和酰基，更优选地，所述R 31为
Figure PCTCN2020089390-appb-100014
The R 31 is a substituted aryl group, preferably
Figure PCTCN2020089390-appb-100013
Wherein, R 310 is a substituted or unsubstituted alkenyl group, and the substituent on the alkenyl group is selected from: alkoxy, heterocyclic group and substituted amino group, preferably, said R 31 is selected from: 3-acrylamide Phenyl, 3-(4-methoxy-2-butenamido)phenyl, 3-(4-piperidinyl-2-butenamido)phenyl, 3-(4-pyrrolidinyl -2-butenamido)phenyl, 3-(4-dimethylamino-2-butenamido)phenyl; or, said R 31 is a substituted or unsubstituted heterocyclic group, preferably pyridine Azole [1,5-a] pyridyl or substituted indolyl, the substituent on the indolyl is selected from the group consisting of hydrogen, alkyl and acyl, more preferably, said R 31 is
Figure PCTCN2020089390-appb-100014
 所述R 32为氢； The R 32 is hydrogen; 所述R 33为
Figure PCTCN2020089390-appb-100015
其中X 3为-NH-或-O-，R 39为取代或未取代的烯基，所述烯基上的取代基选自：烷氧基、杂环基和取代的氨基，优选地，所述R 33选自：丙烯酰氧基、丙烯酰胺基、4-甲氧基-2-丁烯酰氧基、4-甲氧基-2-丁烯酰胺基、4-哌啶基-2-丁烯酰氧基、4-哌啶基-2-丁烯酰胺基、4-吡咯烷基-2-丁烯酰氧基、4-吡咯烷基-2-丁烯酰胺基、4-二甲胺基-2-丁烯酰氧基和4-二甲胺基-2-丁烯酰胺基，更优选地，所述R 33为
Figure PCTCN2020089390-appb-100016
The R 33 is
Figure PCTCN2020089390-appb-100015
Wherein X 3 is -NH- or -O-, R 39 is a substituted or unsubstituted alkenyl group, and the substituent on the alkenyl group is selected from: alkoxy, heterocyclic group and substituted amino group, preferably, The R 33 is selected from: acryloyloxy, acrylamido, 4-methoxy-2-butenoyloxy, 4-methoxy-2-butenamido, 4-piperidinyl-2- Butenoyloxy, 4-piperidinyl-2-butenamido, 4-pyrrolidinyl-2-butenoyloxy, 4-pyrrolidin-2-butenamido, 4-dimethyl Amino-2-butenoyloxy and 4-dimethylamino-2-butenamido, more preferably, the R 33 is
Figure PCTCN2020089390-appb-100016
 所述R 35为氢； The R 35 is hydrogen; 所述R 34为取代或未取代的杂环基，优选为取代的含氮非芳香杂环基，其上取代基选自：烷基、酰基和杂环基，优选地，所述R 34选自：4-(1-乙酰基)哌嗪基、4-(1-甲基)哌嗪基、4-(1-吡咯烷基)哌嗪基、4-(1-哌啶基)哌嗪基和4-甲基哌嗪基-1-哌啶基；或，所述R 34为-NR 27R 28，其中R 27和R 28独立地选自：氢和取代或未取代的烷基，优选为N,N-二甲氨基或N 1,N 1,N 2-三甲基乙二胺基； Said R 34 is a substituted or unsubstituted heterocyclic group, preferably a substituted nitrogen-containing non-aromatic heterocyclic group, the substituents on which are selected from: alkyl, acyl and heterocyclic group, preferably, said R 34 is selected From: 4-(1-acetyl)piperazinyl, 4-(1-methyl)piperazinyl, 4-(1-pyrrolidinyl)piperazinyl, 4-(1-piperidinyl)piperazine Or 4-methylpiperazinyl-1-piperidinyl; or, said R 34 is -NR 27 R 28 , wherein R 27 and R 28 are independently selected from: hydrogen and substituted or unsubstituted alkyl, It is preferably N,N-dimethylamino or N 1 ,N 1 ,N 2 -trimethylethylenediamino; 所述R 37和R 38独立地选自：氢、卤素、硝基、氰基和三氟甲基；优选地，所述R 37为氢，R 38为氢。 Said R 37 and R 38 are independently selected from: hydrogen, halogen, nitro, cyano and trifluoromethyl; preferably, said R 37 is hydrogen and R 38 is hydrogen. 如权利要求1所述的化合物，其特征在于，所述化合物选自如下结构：The compound of claim 1, wherein the compound is selected from the following structures:
Figure PCTCN2020089390-appb-100017
Figure PCTCN2020089390-appb-100017
Figure PCTCN2020089390-appb-100018
Figure PCTCN2020089390-appb-100018
 一种权利要求1-12任一项所述化合物的制备方法，其包括向具有EGFR抑制活性的化合物结构中引入至少一个酸性官能团的步骤。A method for preparing the compound according to any one of claims 1-12, which comprises the step of introducing at least one acidic functional group into the structure of the compound having EGFR inhibitory activity. 一种权利要求1-12任一项所述化合物或其药学上可接受的盐的立体异构体、几何异构体、互变异构体、消旋体、溶剂化物、水合物、代谢前体和前药。A stereoisomer, geometric isomer, tautomer, racemate, solvate, hydrate, pre-metabolism of the compound according to any one of claims 1-12 or a pharmaceutically acceptable salt thereof Body and prodrug. 一种药物组合物，其包含权利要求1-12任一项所述的化合物或其药学上可接受的盐、或权利要求14所述的立体异构体、几何异构体、互变异构体、消旋体、溶剂化物、水合物、代谢前体或前药，以及一种或多种药学上可接受的辅料。A pharmaceutical composition comprising the compound of any one of claims 1-12 or a pharmaceutically acceptable salt thereof, or the stereoisomer, geometric isomer, or tautomer of claim 14 Forms, racemates, solvates, hydrates, metabolic precursors or prodrugs, and one or more pharmaceutically acceptable excipients. 一种权利要求1-12任一项所述的化合物或其药学上可接受的盐、或权利要求14所述的立体异构体、几何异构体、互变异构体、消旋体、溶剂化物、水合物、代谢前体或前药，或权利要求15所述的药物组合物在制备预防和/或治疗与受体酪氨酸蛋白激酶有关的疾病和病症的药物中的应用；A compound of any one of claims 1-12 or a pharmaceutically acceptable salt thereof, or a stereoisomer, geometric isomer, tautomer, racemate, Use of solvates, hydrates, metabolic precursors or prodrugs, or the pharmaceutical composition of claim 15 in the preparation of drugs for the prevention and/or treatment of diseases and disorders related to receptor tyrosine protein kinases; 优选地，所述与受体酪氨酸蛋白激酶有关的疾病和病症为肿瘤，优选自：肺癌、胰腺癌、乳腺癌或结肠癌，更优选为肺癌，最优选为非小细胞肺癌。Preferably, the diseases and disorders related to receptor tyrosine protein kinases are tumors, preferably from lung cancer, pancreatic cancer, breast cancer or colon cancer, more preferably lung cancer, most preferably non-small cell lung cancer.
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