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WO2023061434A1 - 一种三环化合物的用途 - Google Patents

一种三环化合物的用途 Download PDF

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Publication number
WO2023061434A1
WO2023061434A1 PCT/CN2022/125057 CN2022125057W WO2023061434A1 WO 2023061434 A1 WO2023061434 A1 WO 2023061434A1 CN 2022125057 W CN2022125057 W CN 2022125057W WO 2023061434 A1 WO2023061434 A1 WO 2023061434A1
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Prior art keywords
mutation
egfr
compound
pharmaceutically acceptable
acceptable salt
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PCT/CN2022/125057
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English (en)
French (fr)
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郑善松
郑庆梅
王梅
邓伟
杨莹莹
张臣伟
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齐鲁制药有限公司
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Priority to CN202280066992.0A priority Critical patent/CN118043329A/zh
Publication of WO2023061434A1 publication Critical patent/WO2023061434A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D519/00—Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00

Definitions

  • the present invention provides a use of a tricyclic compound or a pharmaceutically acceptable salt thereof in treating cancer mediated by EGFR, FGFR2, KIT, ALK and/or ROS1 mutations.
  • EGFR the epidermal growth factor receptor
  • the EGFR signaling pathway plays an important role in physiological processes such as cell growth, proliferation and differentiation.
  • EGFR mutation is also the most common type of mutation in NSCLC patients, especially in Asian populations, accounting for 40% to 50%. Therefore, EGFR has always been one of the hottest targets in the field of drug development.
  • the EGFR inhibitors on the market are divided into the first, second and third generations.
  • the first generation is reversible targeted drugs, targeting L858R mutation and Del19 mutation, such as gefitinib, erlotinib, and icotinib.
  • the second generation is irreversible targeted drugs, such as afatinib and dacomitinib.
  • the first and second generation targeted drugs are effective, most patients will develop drug resistance after 1-2 years of drug use.
  • 50% of drug resistance is related to T790M mutation.
  • the third-generation EGFR-targeted drug osimertinib can overcome the tumor resistance caused by the T790M mutation and bring better survival benefits to more lung cancer patients.
  • the third-generation targeted drugs will inevitably produce drug resistance, and the reasons for drug resistance include further C797S mutation, G724S mutation, L792H mutation, E709K mutation, and EGFR amplification.
  • drug resistance include further C797S mutation, G724S mutation, L792H mutation, E709K mutation, and EGFR amplification.
  • Fibroblast growth factor and its receptor drive important developmental signaling pathways affecting cell proliferation, migration and survival. Aberrant FGF signaling plays a role in many cancers.
  • the FGFR family consists of FGFR1, FGFR2, FGFR3 and FGFR4.
  • FGFRs are tyrosine kinases that are activated in a subset of tumors by gene amplification, mutation, or chromosomal translocations or rearrangements. Amplification of FGFR1 occurs in squamous cell lung cancer and estrogen receptor-positive breast cancer.
  • FGFR2 is also amplified in gastric and breast cancers.
  • FGFR mutations have been observed in endometrial cancer and FGFR3 mutations in bladder cancer.
  • the encoded product of c-KIT is a transmembrane receptor protein with tyrosine kinase activity and a molecular weight of 145 kilodaltons. It has five immunoglobulin G-like domains in the extracellular region, so it belongs to Member of the type III tyrosine kinase superfamily. Under physiological conditions, a small amount of c-KIT is expressed in mast cells, stem cells, sperm cells and intestinal Cajal cells.
  • stem cell factor a ligand of c-KIT
  • the c-KIT molecule undergoes homodimerization, resulting in the Y568 and Y570 tyrosine Autophosphorylation of acid residues, which in turn leads to phosphorylation of tyrosine residues in many substrate proteins in cells, and activation of multiple signal transduction pathways related to cell proliferation, including Jak-Stat3/Stat5 pathway, Src kinase, Ras-MEK-Erk1/2 and PI3K-AKT pathways, thereby enabling cell proliferation.
  • GIST gastrointestinal stromal tumor
  • Gene fusion is a chimeric gene formed by connecting the coding regions of two or more genes end to end and placed under the control of the same set of regulatory sequences (including promoters, enhancers, ribosome binding sequences, terminators, etc.).
  • a fusion of the echinoderm microtubule-binding protein 4 (EML4) gene and the anaplastic lymphoma kinase (ALK) gene has been found in NSCLC.
  • EML4-ALK fusion gene is a cancer-promoting gene mutation that occurs in non-small cell lung cancer, accounting for 4-5% of the incidence of non-small cell lung cancer.
  • EML4-ALK leads to abnormal expression of tyrosine kinases, causing malignant transformation of cells.
  • the incidence of SLC34A2-ROS1 fusion gene in NSCLC is about 1.0%-3.4%, and the incidence in EGFR/KRAS/ALK negative population can reach 5.7%.
  • the pathological type is mainly adenocarcinoma.
  • SLC34A2-ROS1 gene fusion occurs, the extracellular region is lost, and the transmembrane and intracellular tyrosine kinase regions are retained.
  • the fusion sites mainly occur in exons 32, 34, 35, and 36 of the ROS1 gene.
  • ROS1 receptor tyrosine kinase is involved in the activation of multiple downstream signal transduction pathways, including RAS-MAPK/ERK, PI3K/AKT/mTOR, JAK/STAT3, PLC/IP3 and SHP2/VAV3 pathways, thereby regulating the growth and proliferation of tumor cells , cell cycle, differentiation, metastasis and migration.
  • ROS1 gene and ALK gene are homology between ROS1 gene and ALK gene in the sequence of tyrosine kinase region, and the homology between them is as high as 77% in the ATP binding site of the kinase catalytic region.
  • ROS1 fusion gene provides a new method for individualized treatment of lung cancer It is of great significance for clinical practice to clarify the positive rate of ROS1 fusion gene in lung adenocarcinoma.
  • the Del19/T790M/C797S mutation has good kinase inhibitory activity and cell anti-proliferation activity, and the molecule has good anti-tumor activity and tolerance in mouse models. In order to improve the clinical value of this compound, it is of great significance to develop more uses of it.
  • the invention provides a use of a tricyclic compound or a pharmaceutically acceptable salt thereof in preparing a medicine for treating cancer mediated by EGFR, FGFR2, KIT, ALK and/or ROS1 mutations.
  • the present invention provides the use of the compound of formula (A) or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating cancer mediated by EGFR mutation, and the type of EGFR mutation is Del19 mutation.
  • the present invention also provides the use of the compound of formula (A) or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating cancer mediated by EGFR mutation, and the type of EGFR mutation is L858R mutation.
  • the present invention also provides the use of the compound of formula (A) or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating cancer mediated by EGFR mutation, and the type of EGFR mutation is T790M mutation without C797S mutation.
  • the T790M mutation without the C797S mutation described in the above uses is selected from one or a combination of the following: L858R/T790M double mutation, Del19/G724S/T790M triple mutation, L858R/T790M/L792H triple mutation Mutation, E709K/T790M/L858R triple mutation.
  • the present invention also provides the use of the compound of formula (A) or a pharmaceutically acceptable salt thereof in preparing a medicine for treating cancer mediated by EGFR mutation, and the type of EGFR mutation is Del19/C797S double mutation.
  • the present invention also provides the use of the compound of formula (A) or a pharmaceutically acceptable salt thereof in preparing a medicine for treating cancer mediated by EGFR mutation, and the type of EGFR mutation is L858R/C797S double mutation.
  • the present invention also provides the use of the compound of formula (A) or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating cancer mediated by EGFR amplification.
  • the above-mentioned EGFR amplification is the amplification of Del19/T790M/C797S triple mutation, L858R/T790M/D537H triple mutation and V674L/E746_A750del/T790M triple mutation EGFR amplification.
  • the above-mentioned EGFR amplification is EGFR amplification accompanied by Del19/T790M/C797S triple mutation, EGFR amplification accompanied by L858R/T790M/D537H triple mutation or accompanied by V674L/E746_A750del/T790M triple mutation EGFR amplification.
  • the present invention also provides the use of the compound of formula (A) or a pharmaceutically acceptable salt thereof in preparing a medicine for treating cancer mediated by EGFR mutation, and the type of EGFR mutation is exon 20 insertion mutation.
  • the present invention also provides the use of the compound of formula (A) or a pharmaceutically acceptable salt thereof in the preparation of a medicine for treating cancer mediated by EGFR mutation or amplification, and the type of EGFR mutation is selected from one or any combination of the following: Del19 mutation, L858R mutation, L858R/T790M double mutation, Del19/G724S/T790M triple mutation, L858R/T790M/L792H triple mutation, E709K/T790M/L858R triple mutation, Del19/C797S double mutation, L858R/C797S double mutation, 20 outside Exon mutation; EGFR amplification selected from Del19/T790M/C797S triple mutation, L858R/T790M/D537H triple mutation, V674L/E746_A750del/T790M triple mutation EGFR amplification.
  • the present invention also provides the use of a compound of formula (A) or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating cancer mediated by EGFR mutation or amplification, wherein the type of EGFR mutation is selected from one or any combination of the following : Del19 mutation, L858R mutation, L858R/T790M double mutation, Del19/G724S/T790M triple mutation, L858R/T790M/L792H triple mutation, E709K/T790M/L858R triple mutation, Del19/C797S double mutation, L858R/C797S double mutation, 20 Exon mutation; the EGFR amplification is selected from one or any combination of the following: EGFR amplification accompanied by Del19/T790M/C797S triple mutation, L858R/T790M/D537H triple mutation or V674L/E746_A750del/T790M triple mutation .
  • the present invention also provides the use of the compound of formula (A) or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating cancer with high expression of FGFR2.
  • the present invention also provides the use of the compound of formula (A) or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating cancer with C-KIT mutation, and the type of C-KIT mutation is V560G mutation and/or D816Y mutation and/or D816H mutation and/or V559 and V560 amino acid deletion mutation and/or D816V mutation.
  • the present invention also provides the use of the compound of formula (A) or a pharmaceutically acceptable salt thereof in preparing a medicine for treating cancer mediated by EML4-ALK fusion protein.
  • the present invention also provides the use of the compound of formula (A) or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating cancer mediated by EML4-ALK fusion protein L1196M mutation and/or F1174L mutation and/or L1196M/L1198F double mutation.
  • the present invention also provides the use of the compound of formula (A) or a pharmaceutically acceptable salt thereof in preparing a medicine for treating cancer mediated by SLC34A2-ROS1 fusion protein.
  • the present invention also provides the use of the compound of formula (A) or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating cancer mediated by the D2033N mutation of the SLC34A2-ROS1 fusion protein.
  • the pharmaceutically acceptable salt of the compound (A) in any of the above uses is hydrochloride.
  • the pharmaceutically acceptable salt of the compound (A) in any of the above uses is monohydrochloride.
  • the present invention also provides the use of the compound of formula (A) or a pharmaceutically acceptable salt thereof in the treatment of cancer mediated by EGFR mutation, and the type of EGFR mutation is Del19 mutation.
  • the present invention also provides the use of the compound of formula (A) or a pharmaceutically acceptable salt thereof in the treatment of cancer mediated by EGFR mutation, and the type of EGFR mutation is L858R mutation.
  • the present invention also provides the use of the compound of formula (A) or a pharmaceutically acceptable salt thereof in the treatment of cancer mediated by EGFR mutation, and the type of EGFR mutation is T790M mutation without C797S mutation.
  • the T790M mutation without the C797S mutation described in the above uses is selected from one or a combination of the following: L858R/T790M double mutation, Del19/G724S/T790M triple mutation, L858R/T790M/L792H triple mutation Mutation, E709K/T790M/L858R triple mutation.
  • the present invention also provides the use of the compound of formula (A) or a pharmaceutically acceptable salt thereof in the treatment of cancer mediated by EGFR mutation, and the type of EGFR mutation is Del19/C797S double mutation.
  • the present invention also provides the use of the compound of formula (A) or a pharmaceutically acceptable salt thereof in the treatment of cancer mediated by EGFR mutation, and the type of EGFR mutation is L858R/C797S double mutation.
  • the present invention also provides the use of the compound of formula (A) or a pharmaceutically acceptable salt thereof in the treatment of cancer mediated by EGFR amplification.
  • the EGFR amplification described in the above uses is the amplification of Del19/T790M/C797S triple mutation, L858R/T790M/D537H triple mutation and V674L/E746_A750del/T790M triple mutation EGFR amplification.
  • the EGFR amplification in the above use is EGFR amplification accompanied by Del19/T790M/C797S triple mutation, L858R/T790M/D537H triple mutation or V674L/E746_A750del/T790M triple mutation.
  • the present invention also provides the use of the compound of formula (A) or a pharmaceutically acceptable salt thereof in the treatment of cancer mediated by EGFR mutation, and the type of EGFR mutation is exon 20 insertion mutation.
  • the present invention also provides the use of the compound of formula (A) or a pharmaceutically acceptable salt thereof in the treatment of cancer mediated by EGFR mutation or amplification, and the type of EGFR mutation is selected from one or any combination of the following: Del19 mutation, L858R mutation, L858R/T790M double mutation, Del19/G724S/T790M triple mutation, L858R/T790M/L792H triple mutation, E709K/T790M/L858R triple mutation, Del19/C797S double mutation, L858R/C797S double mutation, exon 20 mutation ; EGFR amplification is selected from the amplification of Del19/T790M/C797S triple mutation, L858R/T790M/D537H triple mutation, V674L/E746_A750del/T790M triple mutation EGFR.
  • the present invention also provides the use of the compound of formula (A) or a pharmaceutically acceptable salt thereof in the treatment of cancer mediated by EGFR mutation or amplification, wherein the type of EGFR mutation is selected from one or any combination of the following: Del19 mutation , L858R mutation, L858R/T790M double mutation, Del19/G724S/T790M triple mutation, L858R/T790M/L792H triple mutation, E709K/T790M/L858R triple mutation, Del19/C797S double mutation, L858R/C797S double mutation, exon 20 Mutation; the EGFR amplification is selected from one or any combination of the following: EGFR amplification accompanied by Del19/T790M/C797S triple mutation, L858R/T790M/D537H triple mutation or V674L/E746_A750del/T790M triple mutation.
  • the present invention also provides a method for treating cancer mediated by EGFR mutation or amplification, which comprises administering a compound of formula (A) or a pharmaceutically acceptable salt thereof to a patient, wherein the type of EGFR mutation is selected from one or any of the following Combination: Del19 mutation, L858R mutation, L858R/T790M double mutation, Del19/G724S/T790M triple mutation, L858R/T790M/L792H triple mutation, E709K/T790M/L858R triple mutation, Del19/C797S double mutation, L858R/C797S double mutation, Exon 20 mutation; the EGFR amplification is selected from one or any combination of the following: EGFR amplification accompanied by Del19/T790M/C797S triple mutation, L858R/T790M/D537H triple mutation or V674L/E746_A750del/T790M triple mutation increase.
  • the present invention also provides the compound of formula (A) for preparing the above-mentioned Del19 mutation, L858R mutation, L858R/T790M double mutation, Del19/G724S/T790M triple mutation, L858R/T790M/L792H triple mutation, E709K/T790M/L858R triple mutation, Del19 Application in EGFR mutation regulators of /C797S double mutation, L858R/C797S double mutation and exon 20 mutation.
  • the above-mentioned EGFR mutation regulator is used as an inhibitor of the above-mentioned mutation of EGFR.
  • the present invention also provides the use of the compound of formula (A) in preparing regulators for EGFR amplification of the above-mentioned Del19/T790M/C797S triple mutation, the above-mentioned L858R/T790M/D537H triple mutation or the above-mentioned V674L/E746_A750del/T790M triple mutation.
  • the cancer in any of the above uses is lung cancer.
  • the cancer in any of the above uses is non-small cell lung cancer.
  • the cancer in any of the above uses is treatment-naive non-small cell lung cancer.
  • the cancer in any of the above uses is non-small cell lung cancer that develops drug resistance after receiving EGFR inhibitor therapy in the past.
  • the aforementioned EGFR inhibitors include first-generation EGFR inhibitors, second-generation or third-generation EGFR inhibitors.
  • the above-mentioned first-generation EGFR inhibitors include gefitinib, icotinib, and erlotinib.
  • the above-mentioned second-generation EGFR inhibitors include afatinib and dacomitinib.
  • the aforementioned third-generation EGFR inhibitor includes osimertinib.
  • the present invention also provides the use of the compound of formula (A) or a pharmaceutically acceptable salt thereof in the treatment of cancers with high FGFR2 expression.
  • the present invention also provides the use of the compound of formula (A) or a pharmaceutically acceptable salt thereof in the treatment of cancer with C-KIT mutation, and the type of C-KIT mutation is V560G mutation and/or D816Y mutation and/or D816H mutation and /or 559 and 560 amino acid deletion mutations and/or D816V mutations.
  • the present invention also provides the use of the compound of formula (A) or a pharmaceutically acceptable salt thereof in the treatment of cancer mediated by EML-ALK fusion protein.
  • the present invention also provides the use of the compound of formula (A) or a pharmaceutically acceptable salt thereof in the treatment of cancers mediated by L1196M mutation and/or F1174L mutation and/or L1196M/L1198F double mutation of EML4-ALK fusion protein.
  • the present invention also provides the use of the compound of formula (A) or a pharmaceutically acceptable salt thereof in the treatment of cancer mediated by SLC34A2-ROS1 fusion protein.
  • the present invention also provides the use of the compound of formula (A) or a pharmaceutically acceptable salt thereof in the treatment of cancer mediated by the D2033N mutation of the SLC34A2-ROS1 fusion protein.
  • the present invention also provides a method for treating the above-mentioned cancer with high FGFR2 expression, the above-mentioned cancer with C-KIT mutation, the above-mentioned cancer mediated by EML-ALK fusion protein, and the above-mentioned cancer mediated by SLC34A2-ROS1 fusion protein, which comprises administering to patients A compound of formula (A) or a pharmaceutically acceptable salt thereof.
  • the present invention also provides the use of the compound of formula (A) in preparing regulators of the above-mentioned FGFR2, the above-mentioned C-KIT mutation, the above-mentioned EML-ALK fusion protein, and the above-mentioned SLC34A2-ROS1.
  • the aforementioned modulators are inhibitors.
  • the pharmaceutically acceptable salt of the compound of formula (A) in any of the above uses is hydrochloride.
  • the pharmaceutically acceptable salt of the compound of formula (A) in any of the above uses is monohydrochloride.
  • the compound of formula (A) of the present invention not only has good activity against L858R/T790M/C797S triple mutation and Del19/T790M/C797S triple mutation, but also has good activity against L858R or Del19 single mutation, exon 20 insertion mutation, L858R/T790M or L858R/C797S or Del19/C797S double mutation, Del19/G724S/T790M triple mutation, L858R/T790M/L792H triple mutation, E709K/T790M/L858R triple mutation and Del19/T790M/C797S triple mutation accompanied by EGFR amplification, L858R/ The T790M/D537H triple mutation and the V674L/E746_A750del/T790M triple mutation also have good in vitro kinase or cell anti-proliferation activities, and the compound has shown a strong anti-proliferative activity in the mouse models of
  • the compound of formula (A) of the present invention is effective against high expression of FGFR2, C-KIT V560G mutation, C-KIT D816Y mutation, C-KIT D816H mutation, C-KIT V559 and V560 amino acid deletion mutation, C-KIT D816V mutation , EML4-ALK fusion protein mutation, EML4-ALK fusion protein L1196M or F1174L mutation or L1196M/L1198F double mutation, SLC34A2-ROS1 fusion protein mutation, SLC34A2-ROS1 fusion protein D2033N mutation cell lines all have good anti-proliferation activity.
  • pharmaceutically acceptable salt refers to derivatives prepared from the compounds of the present invention with relatively non-toxic acids or bases. These salts can be prepared during compound synthesis, isolation, purification, or alone by reacting the free form of the purified compound with an appropriate acid or base.
  • the compound contains relatively acidic functional groups, it can react with alkali metal, alkaline earth metal hydroxide or organic amine to obtain base addition salts, including cations based on alkali metals and alkaline earth metals and non-toxic ammonium, quaternary ammonium and amine cations, Salts of amino acids and the like are also contemplated.
  • the compound contains a relatively basic functional group, it reacts with an organic acid or an inorganic acid to form an acid addition salt.
  • the EGFR mutation-mediated tumor or cancer refers to the cancer driver mutation (driver mutation) of EGFR that can be detected in these tumors or cancer patients, including but not limited to Del19 mutation, L858R mutation, T790M mutation , 20 exon insertion mutation (Exon 20ins), C797S and other mutations.
  • the Del19 mutation refers to the deletion of some bases in exon 19, resulting in a non-frameshift partial amino acid deletion
  • L858R refers to the change of amino acid 858 from L to R due to a missense mutation of the base
  • T790M refers to the change of amino acid 790 from T to M due to the missense mutation of the base in the gene
  • the exon 20 insertion (Exon 20ins) mutation refers to the in-frame duplication/ Insertion mutation
  • C797S mutation refers to the mutation of cysteine residue at position 797 to serine.
  • the EGFR mutations include not only the above-mentioned single mutants of EGFR, but also compound mutants of T790M, Del19, L858R, Exon 20ins, C797S and other sites freely combined, including but not limited to the L858R/T790M double mutation , Del19/G724S/T790M triple mutation, L858R/T790M/L792H triple mutation, E709K/T790M/L858R triple mutation, Del19/C797S double mutation, L858R/C797S double mutation, etc.
  • the EGFR amplification refers to the increase of the copy number of EGFR gene or the high-level expression of protein. It can occur on mutant cells as well as on EGFR receptor cells without the mutation (wild type).
  • Figure 1 is the animal tumor growth curve in the in vivo pharmacodynamic study of EGFR Del19/C797S mutation.
  • Fig. 2 is a graph of animal body weight in the in vivo pharmacodynamic study of EGFR Del19/C797S mutation.
  • Figure 3 is the animal tumor growth curve in the in vivo pharmacodynamic study of EGFR L858R mutation.
  • Fig. 4 is the animal body weight curve in the in vivo pharmacodynamic study of EGFR L858R mutation.
  • Figure 5 is a graph of animal tumor growth curves in the in vivo pharmacodynamic study of EGFR Del19 mutation.
  • Figure 6 is a graph of animal body weights in the in vivo pharmacodynamic study of EGFR Del19 mutation.
  • Figure 7 is the animal tumor growth curve in the PDX model study of Osimertinib-resistant human lung cancer.
  • Fig. 8 is a graph of animal body weight in the PDX model study of Osimertinib-resistant human lung cancer.
  • Figure 9 is a graph of animal tumor growth curves in the in vivo pharmacodynamic study of EGFR L858R/C797S mutation.
  • Fig. 10 is a curve diagram of animal body weight in the in vivo pharmacodynamic study of EGFR L858R/C797S mutation.
  • Figure 11 is the animal tumor growth curve in the in vivo pharmacodynamic study of EGFR L858R/T790M mutation.
  • Figure 12 is a graph of animal body weights in the in vivo pharmacodynamic study of the EGFR L858R/T790M mutation.
  • 6-Aminoquinoxaline (10 g, 68.89 mmol) was dissolved in concentrated sulfuric acid (20 mL). At 0° C., potassium nitrate (9.054 g, 89.55 mmol) was added in portions to the reaction solution and stirring was continued at this temperature for 30 minutes. After LCMS monitoring showed that the starting material disappeared, the reaction solution was poured into ice water (100 g). Its pH was adjusted to 8 with 1M aqueous sodium hydroxide solution. The mixture was extracted with ethyl acetate (200 mL x 2 times), and the organic phases were combined.
  • Embodiment 2 biological test evaluation:
  • TR-FRET fluorescence resonance energy transfer
  • EGFR, EGFR Del19, EGFR L858R, EGFR L858R/T790M, EGFR L858R/C797S, EGFR ex19del/C797S recombinases were purchased from Signalchem.
  • HTRF KinEASE-TK kit was purchased from Cisbio.
  • DTT, MnCl2, and MgCl2 were purchased from Sigma.
  • ATP was purchased from Promega.
  • a microplate reader detects the 615nm and 665nm fluorescence signal values of each well.
  • the CellTiter-Glo method was used to test the inhibitory effect of compound A on the proliferation of A431 cells, and the concentration IC 50 at which the compound inhibited half of the cell growth was obtained.
  • A431 cells were purchased from ATCC.
  • DMEM medium fetal bovine serum (FBS), and Penicillin-Streptomycin were purchased from GIBCO.
  • CellTiter-Glo reagent was purchased from Promega Company.
  • A431 cells were seeded in a 384-well culture plate at a density of 800 cells per well, 30 ⁇ l per well, and placed in a cell culture incubator for 24 hours (37° C., 5% CO 2 ).
  • Envision microplate reader detects chemiluminescent signal.
  • the CellTiter-Glo method was used to test the inhibitory effect of compound A on the proliferation of NCI-H3255 (EGFR L858R mutation) cells, and the concentration IC 50 of compound A inhibiting half of the cell growth was obtained.
  • NCI-H3255 cells were purchased from Nanjing Kebai Biotechnology Co., Ltd.
  • FBS fetal bovine serum
  • Penicillin-Streptomycin purchased from GIBCO.
  • CellTiter-Glo reagent was purchased from Promega Company.
  • Envision microplate reader detects chemiluminescent signal.
  • the CellTiter-Glo method was used to test the inhibitory effect of compound A on PC-9 (EGFR Del19 mutation) cell proliferation, and the concentration IC 50 of the compound inhibiting half of the cell growth was obtained.
  • PC-9 cells were purchased from European Collection of Authenticated Cell Cultures.
  • FBS fetal bovine serum
  • Penicillin-Streptomycin purchased from GIBCO.
  • CellTiter-Glo reagent was purchased from Promega Company.
  • Envision microplate reader detects chemiluminescent signal.
  • the CellTiter-Glo method was used to test the inhibitory effect of compound A on the proliferation of NCI-H1975 (EGFR L858R/T790M mutation) cells, and the concentration IC 50 of compound A inhibiting half of the cell growth was obtained.
  • NCI-H1975 cells were from ATCC.
  • FBS fetal bovine serum
  • Penicillin-Streptomycin purchased from GIBCO.
  • CellTiter-Glo reagent was purchased from Promega Company.
  • Envision microplate reader detects chemiluminescent signal.
  • the CellTiter-Glo method was used to test the inhibitory effect of compound A on the proliferation of HCC827 (EGFR Del mutation) cells, and the concentration IC 50 of compound A inhibiting half of the cell growth was obtained.
  • HCC827 cells were purchased from ATCC.
  • FBS fetal bovine serum
  • Penicillin-Streptomycin purchased from GIBCO.
  • CellTiter-Glo reagent was purchased from Promega Company.
  • Envision microplate reader detects chemiluminescent signal.
  • the CellTiter-Glo method was used to test the inhibitory effect of compound A on the proliferation of Ba/F3 EGFR-Del19/G724S/T790M and Ba/F3 EGFR-E709K/T790M/L858R and Ba/F3 EGFR-L858R/T790M/L792H cells. And the concentration IC 50 of compound A inhibiting half of the cell growth was obtained.
  • Ba/F3 EGFR-Del19/G724S/T790M cells were from Kangyuan Biotech (Beijing) Co., Ltd.
  • Ba/F3 EGFR-E709K/T790M/L858R cells were from Kangyuan Biotech (Beijing) Co., Ltd.
  • Ba/F3 EGFR-L858R/T790M/L792H cells were from Kangyuan Biotech (Beijing) Co., Ltd.
  • FBS fetal bovine serum
  • CellTiter-Glo reagent was purchased from Promega Company.
  • the CellTiter-Glo method was used to test the inhibitory effect of compound A on the proliferation of Ba/F3 (EGFR-Del19/C797S) and Ba/F3 (EGFR-L858R/C797S) cells, and the concentration of compound A that inhibited half of the cell growth was obtained IC50 .
  • Ba/F3 EGFR-Del19/C797S cells were from Kangyuan Bochuang Biotechnology (Beijing) Co., Ltd.
  • Ba/F3 EGFR-L858R/C797S cells were from Kangyuan Biotech (Beijing) Co., Ltd.
  • FBS fetal bovine serum
  • CellTiter-Glo reagent was purchased from Promega Company.
  • Envision microplate reader detects chemiluminescent signal.
  • the CellTiter-Glo method was used to test the inhibitory effect of compound A on the proliferation of NCI-H716 (highly expressed FGFR2) cells, and the concentration IC 50 of the compound inhibiting half of the cell growth was obtained.
  • NCI-H716 cells were purchased from ATCC.
  • FBS fetal bovine serum
  • Penicillin-Streptomycin purchased from GIBCO.
  • CellTiter-Glo reagent was purchased from Promega Company.
  • Envision microplate reader detects chemiluminescent signal.
  • the method of CellTiter-Glo was used to test the effect of compound A on Ba/F3 C-KIT-V560G, Ba/F3 C-KIT-D816Y, Ba/F3 C-KIT-D816H, Ba/F3 C-KIT-Del(V559- V560), Ba/F3 C-KIT-D816V, NCI-H3122(EML4-ALK), Ba/F3-EML4-ALK-L1196M, Ba/F3 EML4-ALK-F1174L, Ba/F3-EML4-ALK-L1196M/ Inhibitory effect of L1198F, Ba/F3 SLC34A2/ROS1, Ba/F3 SLC34A2-ROS1-D2033N cell proliferation, and the concentration IC 50 of compound A inhibiting half of cell growth was obtained.
  • Ba/F3 C-KIT-V560G cells were from Kangyuan Bochuang Biotechnology (Beijing) Co., Ltd.
  • Ba/F3 C-KIT-D816Y cells were from Kangyuan Bochuang Biotechnology (Beijing) Co., Ltd.
  • Ba/F3 C-KIT-D816H cells were from Kangyuan Bochuang Biotechnology (Beijing) Co., Ltd.
  • Ba/F3 C-KIT-Del (V559V560) cells were from Kangyuan Biotech (Beijing) Co., Ltd.
  • Ba/F3 C-KIT-D816V cells were from Kangyuan Bochuang Biotechnology (Beijing) Co., Ltd.
  • NCI-H3122 (EML4-ALK) cells were from Kangyuan Bochuang Biotechnology (Beijing) Co., Ltd.
  • Ba/F3-EML4-ALK-L1196M cells were from Kangyuan Bochuang Biotechnology (Beijing) Co., Ltd.
  • Ba/F3 EML4-ALK-F1174L cells were from Kangyuan Bochuang Biotechnology (Beijing) Co., Ltd.
  • Ba/F3-EML4-ALK-L1196M/L1198F cells were from Kangyuan Biotech (Beijing) Co., Ltd.
  • Ba/F3 SLC34A2/ROS1 cells were from Kangyuan Bochuang Biotechnology (Beijing) Co., Ltd.
  • Ba/F3 SLC34A2-ROS1-D2033N cells were from Kangyuan Biotech (Beijing) Co., Ltd.
  • FBS fetal bovine serum
  • CellTiter-Glo reagent was purchased from Promega Company.
  • compound A of the present invention is effective against NCI-H3255 L858R EGFR mutation, PC9 Del19 EGFR mutation, HCC827 Del19 EGFR mutation, NCI-H1975 L858R/T790M EGFR mutation, Ba/F3 (Del19/G724S/ T790M) EGFR triple mutant cell line, Ba/F3 (L858R/T790M/L792H) EGFR triple mutant cell line, Ba/F3 (E709K/T790M/L858R) EGFR triple mutant cell line, Ba/F3 (Del19/C797S) EGFR double mutant cell line Mutant cell lines and Ba/F3 (L858R/C797S) EGFR double mutant cell lines, NCI-H716 (FGFR2), Ba/F3 C-KIT-V560G, Ba/F3 C-KIT-D816Y, Ba/F3 C-KIT-
  • the CellTiter-Glo method was used to test the inhibitory effect of compound A on the proliferation of osimertinib-resistant PDO (Patient Derived Tumor Organoids), and the concentration IC 50 of compound A inhibiting half of the cell growth was obtained.
  • CellTiter-Glo reagent was purchased from Promega Company.
  • NU/NU mice female, SPF grade, were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.
  • Ba/F3 EGFR Del19/C797S cells were purchased from Kangyuan Bochuang Biotechnology (Beijing) Co., Ltd.
  • Ba/F3 EGFR Del19/C797S cells were cultured in RPMI1640 medium containing 10% fetal bovine serum at 37°C in a 5% carbon dioxide incubator, and cells in the exponential growth phase were collected for inoculation.
  • mice with moderate tumor volume were selected and randomly divided into 4 groups according to the tumor volume: G1: vehicle control group, G2: compound A (15mg/kg), G3 : Compound A (30 mg/kg) and G4: Compound A (65 mg/kg), 8 rats/group.
  • the drug was administered, and the volume of the drug was 10 mL/kg, and the drug was administered orally (po); the drug was weighed once a day, and the drug was administered continuously for 14 days; the tumor diameter was measured twice a week.
  • TGI tumor growth inhibition rate
  • mice During the experiment, the living conditions of the mice were closely observed, including appearance signs, general behavioral activities, mental state, food intake, respiratory state, feces and urine properties, injection site and other toxic manifestations.
  • mice were euthanized, and the animal corpses were stored in a freezer and handed over to a qualified medical waste disposal unit for disposal.
  • NOD SCID mice female, SPF grade, were purchased from Beijing Huafukang Biotechnology Co., Ltd.
  • NCI-H3255 (L858R) cells were purchased from Nanjing Kebai Biotechnology Co., Ltd.
  • NCI-H3255 tumor cells were cultured in RPMI-1640 medium containing inactivated 10% fetal bovine serum in an incubator at 37°C and 5% CO 2 . Tumor cells in logarithmic growth phase were used for inoculation of tumors in vivo.
  • NCI-H3255 tumor cells resuspended in serum-free RPMI-1640 culture medium at a concentration of 1 ⁇ 10 7 /100 ⁇ L were inoculated subcutaneously on the right flank of experimental animals, and the day of inoculation was set as day 0.
  • mice with moderate tumor volume were selected and randomly divided into 3 groups according to the tumor volume: G1: vehicle control group, G2: compound A (15mg/kg) and G3: Compound A (60mg/kg), 8 rats/group.
  • the drug was administered, and the volume of the drug was 10 mL/kg, and the drug was administered orally (po); the drug was administered once a day by weight, and the drug was administered continuously for 22 days; the tumor diameter was measured twice a week.
  • TGI tumor growth inhibition rate
  • mice During the experiment, the living conditions of the mice were closely observed, including appearance signs, general behavioral activities, mental state, food intake, respiratory state, feces and urine properties, injection site and other toxic manifestations.
  • mice were euthanized, and the animal corpses were stored in a freezer and handed over to a qualified medical waste disposal unit for disposal.
  • CB-17 SCID mice female, SPF grade, were purchased from Beijing Huafukang Biotechnology Co., Ltd.
  • PC-9 (Del19) cells were purchased from European Collection of Authenticated Cell Cultures.
  • PC-9 (Del19) tumor cells resuspended in serum-free RPMI-1640 culture medium at a concentration of 5 ⁇ 10 6 /100 uL were inoculated subcutaneously on the right flank of the experimental animal, and the day of inoculation was set as day 0.
  • mice with moderate tumor volume were selected and randomly divided into 4 groups according to the tumor volume: G1: vehicle control group, G2: Gefitinib (gefitinib, 100 mg/kg ), G3: Compound A (15 mg/kg) and G4: Compound A (45/60 mg/kg), 5 rats/group.
  • Animals were grouped and started to be dosed with a volume of 10 mL/kg, administered orally (po); the dose was weighed once a day for 21 consecutive days; the tumor diameter was measured twice a week.
  • TGI tumor growth inhibition rate
  • mice During the experiment, the living conditions of the mice were closely observed, including appearance signs, general behavioral activities, mental state, food intake, respiratory state, feces and urine properties, injection site and other toxic manifestations.
  • mice were euthanized, and the animal corpses were stored in a freezer and handed over to a qualified medical waste disposal unit for disposal.
  • NU/NU mice female, SPF grade, were purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd.
  • LD1-0025-200717 human lung cancer tissue, 54-year-old male patient, clinical diagnosis: left upper lung primary bronchial lung cancer, adenocarcinoma; EGFR triple mutation, 19del&T790M&C797S; Osimertinib resistance; PDX pathological diagnosis: poorly-moderately differentiated adenocarcinoma cancer. Passed to FP2+5 generation for this efficacy test.
  • the LD1-0025-200717 tumor tissue was evenly cut into a tumor mass of about 3mm ⁇ 3mm ⁇ 3mm (about 50-90mg) and inoculated subcutaneously on the right side of NU/NU mice. Post-inoculation mice were then observed and tumor growth monitored.
  • Animals were grouped into groups and administered on the same day, with a volume of 10 mL/kg, administered orally (po); administered once a day by weight, for 21 consecutive days; tumor diameter was measured twice a week.
  • TGI tumor growth inhibition rate
  • mice During the experiment, the living conditions of the mice were closely observed, including appearance signs, general behavioral activities, mental state, food intake, respiratory state, feces and urine properties, injection site and other toxic manifestations.
  • mice were euthanized, and the animal corpses were stored in a freezer and handed over to a qualified medical waste disposal unit for disposal.
  • NU/NU mice female, SPF grade, were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.
  • Ba/F3 EGFR L858R/C797S cells were purchased from Kangyuan Bochuang Biotechnology (Beijing) Co., Ltd.
  • Ba/F3 EGFR L858R/C797S cells were inoculated subcutaneously in the right axilla of NU/NU mice at 2 ⁇ 10 6 cells/0.1 mL, and the day of inoculation was set as day 0.
  • G1 Vehicle
  • G2 Osimertinib (10mg/kg)
  • G3 Compound A (15mg/kg)
  • G4 Compound A (30mg /kg)
  • G5 Compound A (65mg/kg), 7 rats/group.
  • the drug was administered, and the volume of the drug was 10 mL/kg, and the drug was administered orally (po); the drug was weighed once a day, and the drug was administered continuously for 14 days; the tumor diameter was measured twice a week.
  • TGI tumor growth inhibition rate
  • mice During the experiment, the living conditions of the mice were closely observed, including appearance signs, general behavioral activities, mental state, food intake, respiratory state, feces and urine properties, injection site and other toxic manifestations.
  • mice were euthanized, and the animal corpses were stored in a freezer and handed over to a qualified medical waste disposal unit for disposal.
  • H1975 cells were purchased from ATCC.
  • PBS containing 5 ⁇ 106 H1975 cells (final volume: 100uL) was inoculated subcutaneously in the axilla of the right forelimb of each mouse.
  • group administration began.
  • the drug was administered, and the volume of the drug was 10 mL/kg, and the drug was administered orally (po); the drug was weighed once a day, and the drug was administered continuously for 21 days; the diameter of the tumor was measured twice a week.
  • TGI tumor growth inhibition rate
  • mice Closely observe the living conditions of the mice during the experiment, including appearance signs, general behavioral activities, mental state, feeding situation, respiratory state, feces and urine properties, injection site and other toxic manifestations.
  • mice were euthanized, and the animal corpses were stored in a freezer and handed over to a qualified medical waste disposal unit for disposal.

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Abstract

本发明提供一种式(A)化合物或其可药用的盐在制备治疗EGFR、FGFR2、KIT、ALK和/或ROS1突变介导的癌症的药物中的用途。该化合物对该些突变类型介导的癌症具有明显的抑制活性。

Description

一种三环化合物的用途
本申请要求申请日为2021年10月14日的中国专利申请202111200131.0、申请日为2022年3月31日的中国专利申请202210330403.7的优先权，2022年10月8日递交的中国专利申请202211221824.2的优先权，本申请引用上述中国专利申请的全文。
技术领域
本发明提供了一种三环化合物或其可药用的盐在治疗EGFR、FGFR2、KIT、ALK和/或ROS1突变介导的癌症中的用途。
背景技术
EGFR，即表皮生长因子受体(epidermal growth factor receptor)，广泛分布于哺乳动物上皮细胞、成纤维细胞、胶质细胞等细胞表面。EGFR信号通路对细胞的生长、增殖和分化等生理过程发挥着重要的作用。EGFR突变也是NSCLC患者中最常见的一种突变类型，尤其是在亚洲人群中可以占到40％～50％。因此EGFR一直是药物研发领域的最热门靶点之一。
目前，上市的EGFR抑制剂分为第一、二、三代。第一代为可逆的靶向药物，针对L858R突变以及Del19突变，例如吉非替尼、厄洛替尼、埃克替尼。第二代为不可逆的靶向药物，例如阿法替尼以及达克替尼。第一、二代靶向药物虽然疗效显著，但多数患者都会在使用药物1-2年后出现耐药性。EGFR抑制剂耐药的患者中，有50％的耐药与T790M突变有关。第三代EGFR靶向药物奥希替尼能克服由于T790M突变引起的肿瘤耐药，给更多的肺癌患者带来了更好的生存获益。然而第三代靶向药也不可避免的产生耐药，其耐药原因包括进一步的C797S突变，G724S突变、L792H突变、E709K突变以及EGFR扩增等。目前临床上对于奥希替尼耐药的应对尚无成熟的治疗手段，临床需求迫在眉睫。
成纤维细胞生长因子及其受体(FGFR)驱动影响细胞增殖、迁移和存活的重要发育信号传导途径。异常的FGF信号传导在许多癌症中起作用。FGFR家族由FGFR1、FGFR2、FGFR3和FGFR4组成。FGFR是在一部分肿瘤中通过基因扩增、突变或染色体易位或重排激活的酪氨酸激酶。FGFR1的扩增发生在鳞状细胞肺癌和雌激素受体阳性乳腺癌中。FGFR2还在胃癌和乳腺癌中扩增。在子宫内膜癌中观察到FGFR突变，并在膀胱癌中观察到FGFR3突变。
c-KIT的编码产物是一种分子量为145千道尔顿的具有酪氨酸激酶活性的跨膜受体蛋白，它在细胞外区域有5个免疫球蛋白G样结构域，因此，它属于III型酪氨酸激酶超家族成员。生理情况下，c-KIT少量表达于肥大细胞、干细胞、精细胞及肠Cajal细胞等。生理情况下，当干细胞因子(c-KIT的配体)结合c-KIT的免疫球蛋白G样结构域时，c-KIT分子发生同源二聚化，使得临膜域的Y568和Y570酪氨酸残基发生自动磷酸化，进而导致细胞内许多底物蛋白的酪氨酸残基发生磷酸化，引起细胞增殖有关的多条信号转导通路激活，包括Jak-Stat3/Stat5通路、Src激酶、Ras-MEK-Erk1/2及PI3K-AKT通路，从而使细胞增殖。c-KIT酪氨酸激酶域的功能获得性点突变可引起配体非依赖性持续激活，进而导致细胞的失控性生长和对凋亡的抵抗。已经明确c-KIT突变是造成胃肠间质瘤(GIST)、系统性肥大细胞增多症的原因，与小细胞肺癌有密切关系。
基因融合是由两个或多个基因的编码区首尾相连，置于同一套调控序列(包括启动子、增强子、核糖体结合序列、终止子等)控制之下，构成的嵌合基因。在NSCLC中已发现棘皮动物微管结合蛋白4(EML4)基因和间变性淋巴瘤激酶(ALK)基因的融合。EML4-ALK融合基因是发生于非小细胞肺癌中的促癌的基因突变，占非小细胞肺癌发生率的4-5％。EML4-ALK导致酪氨酸激酶异常表达，引起细胞的恶性转化。SLC34A2-ROS1融合基因在NSCLC中的发生率约为1.0％-3.4％，在EGFR/KRAS/ALK均阴性的人群中的发生率则可至5.7％，病理类型主要是腺癌。SLC34A2-ROS1基因发生融合时丢失细胞外区域，保留跨膜和细胞内酪氨酸激酶区域，融合位点主要发生在ROS1基因的第32、34、35、36号外显子。ROS1受体酪氨酸激酶参与激活多条下游信号转导通路，包括RAS-MAPK/ERK、PI3K/AKT/mTOR、JAK/STAT3、PLC/IP3和SHP2/VAV3途径，进而调控肿瘤细胞的生长增殖、细胞周期、分化、转移和迁移。ROS1基因和ALK基因在酪氨酸激酶区域序列存在49％同源性，而在激酶催化区的ATP结合位点二者同源性高达77％，ROS1融合基因为肺癌的个体化治疗提供新的方案，明确ROS1融合基因在肺腺癌中的阳性率，对临床实践具有重要的意义。
本申请人在专利PCT/CN2021/086941中公开了一种针对C797S突变的小分子EGFR抑制剂，其结构如式(A)所示，该小分子抑制剂针对EGFR L858R/T790M/C797S突变以及EGFR Del19/T790M/C797S突变具有良好的激酶抑制活性和细胞抗增殖活性，同时该分子在小鼠模型上体现了较好的抗肿瘤活性及耐受性。为了提高该化合物的临 床价值，开发其更多的用途具有重要的意义。
Figure PCTCN2022125057-appb-000001
发明内容
本发明提供了一种三环化合物或其可药用的盐在制备治疗EGFR、FGFR2、KIT、ALK和/或ROS1突变介导的癌症的药物中的用途。
具体的，
本发明提供式(A)化合物或其可药用的盐在制备治疗EGFR突变介导的癌症的药物中的用途，所述的EGFR突变类型为Del19突变。
Figure PCTCN2022125057-appb-000002
本发明还提供式(A)化合物或其可药用的盐在制备治疗EGFR突变介导的癌症的药物中的用途，所述的EGFR突变类型为L858R突变。
Figure PCTCN2022125057-appb-000003
本发明还提供式(A)化合物或其可药用的盐在制备治疗EGFR突变介导的癌症的药物中的用途，所述的EGFR突变类型为不伴随C797S突变的T790M突变。
Figure PCTCN2022125057-appb-000004
在本发明的一些方案中，上述用途中所述的不伴随C797S突变的T790M突变选自以下的一种或组合：L858R/T790M双突变、Del19/G724S/T790M三突变、L858R/T790M/L792H三突变、E709K/T790M/L858R三突变。
本发明还提供式(A)化合物或其可药用的盐在制备治疗EGFR突变介导的癌症的药物中的用途，所述的EGFR突变类型为Del19/C797S双突变。
Figure PCTCN2022125057-appb-000005
本发明还提供式(A)化合物或其可药用的盐在制备治疗EGFR突变介导的癌症的药物中的用途，所述的EGFR突变类型为L858R/C797S双突变。
Figure PCTCN2022125057-appb-000006
本发明还提供式(A)化合物或其可药用的盐在制备治疗EGFR扩增介导的癌症的药物中的用途。
Figure PCTCN2022125057-appb-000007
在本发明的一些方案中，上述EGFR扩增为Del19/T790M/C797S三突变、L858R/T790M/D537H三突变以及V674L/E746_A750del/T790M三突变EGFR的扩增。
在本发明的一些方案中，上述EGFR扩增为伴随着Del19/T790M/C797S三突变的EGFR扩增、伴随着L858R/T790M/D537H三突变的EGFR扩增或伴随着V674L/E746_A750del/T790M三突变的EGFR扩增。
本发明还提供式(A)化合物或其可药用的盐在制备治疗EGFR突变介导的癌症的药物中的用途，所述的EGFR突变类型为20外显子插入突变。
Figure PCTCN2022125057-appb-000008
本发明还提供式(A)化合物或其可药用的盐在制备治疗EGFR突变或扩增介导的癌症的药物中的用途，所述的EGFR突变类型选自以下的一种或任意组合：Del19突变、L858R突变、L858R/T790M双突变、Del19/G724S/T790M三突变、L858R/T790M/L792H三突变、E709K/T790M/L858R三突变、Del19/C797S双突变、L858R/C797S双突变、20外显子突变；EGFR扩增选自Del19/T790M/C797S三突变、L858R/T790M/D537H三突变、V674L/E746_A750del/T790M三突变EGFR的扩增。
本发明还提供式(A)化合物或其可药用的盐在制备治疗EGFR突变或扩增介导的癌症的药物中的用途，其中所述的EGFR突变类型选自以下的一种或任意组合：Del19突变、L858R突变、L858R/T790M双突变、Del19/G724S/T790M三突变、L858R/T790M/L792H三突变、E709K/T790M/L858R三突变、Del19/C797S双突变、L858R/C797S双突变、20外显子突变；所述的EGFR扩增选自以下的一种或任意组合：伴随着Del19/T790M/C797S 三突变、L858R/T790M/D537H三突变或V674L/E746_A750del/T790M三突变的EGFR扩增。
Figure PCTCN2022125057-appb-000009
本发明还提供式(A)化合物或其可药用的盐在制备治疗FGFR2高表达的癌症的药物中的用途。
本发明还提供式(A)化合物或其可药用的盐在制备治疗C-KIT突变的癌症的药物中的用途，所述的C-KIT突变类型为V560G突变和/或D816Y突变和/或D816H突变和/或V559和V560氨基酸缺失突变和/或D816V突变。
本发明还提供式(A)化合物或其可药用的盐在制备治疗EML4-ALK融合蛋白介导的癌症的药物中的用途。
在本发明还提供式(A)化合物或其可药用的盐在制备治疗EML4-ALK融合蛋白L1196M突变和/或F1174L突变和/或L1196M/L1198F双突变介导的癌症的药物中的用途。
本发明还提供式(A)化合物或其可药用的盐在制备治疗SLC34A2-ROS1融合蛋白介导的癌症的药物中的用途。
本发明还提供式(A)化合物或其可药用的盐在制备治疗SLC34A2-ROS1融合蛋白D2033N突变介导的癌症的药物中的用途。
在本发明的一些方案中，上述任一用途中的(A)化合物的可药用盐为盐酸盐。
在本发明的一些方案中，上述任一用途中的(A)化合物的可药用盐为一盐酸盐。
本发明还提供式(A)化合物或其可药用的盐在治疗EGFR突变介导的癌症中的用途，所述的EGFR突变类型为Del19突变。
Figure PCTCN2022125057-appb-000010
本发明还提供式(A)化合物或其可药用的盐在治疗EGFR突变介导的癌症中的用途，所述的EGFR突变类型为L858R突变。
Figure PCTCN2022125057-appb-000011
本发明还提供式(A)化合物或其可药用的盐在治疗EGFR突变介导的癌症中的用途，所述的EGFR突变类型为不伴随C797S突变的T790M突变。
Figure PCTCN2022125057-appb-000012
在本发明的一些方案中，上述用途中所述的不伴随C797S突变的T790M突变选自以下的一种或组合：L858R/T790M双突变、Del19/G724S/T790M三突变、L858R/T790M/L792H三突变、E709K/T790M/L858R三突变。
本发明还提供式(A)化合物或其可药用的盐在治疗EGFR突变介导的癌症中的用途，所述的EGFR突变类型为Del19/C797S双突变。
Figure PCTCN2022125057-appb-000013
本发明还提供式(A)化合物或其可药用的盐在治疗EGFR突变介导的癌症中的用途，所述的EGFR突变类型为L858R/C797S双突变。
Figure PCTCN2022125057-appb-000014
本发明还提供式(A)化合物或其可药用的盐在治疗EGFR扩增介导的癌症中的用途。
Figure PCTCN2022125057-appb-000015
在本发明的一些方案中，上述用途中所述的EGFR扩增为Del19/T790M/C797S三突变、L858R/T790M/D537H三突变以及V674L/E746_A750del/T790M三突变EGFR的扩增。
在本发明的一些方案中，上述用途中的EGFR扩增为伴随着Del19/T790M/C797S三突变、L858R/T790M/D537H三突变或V674L/E746_A750del/T790M三突变的EGFR扩增。
本发明还提供式(A)化合物或其可药用的盐在治疗EGFR突变介导的癌症中的用途，所述的EGFR突变类型为20外显子插入突变。
Figure PCTCN2022125057-appb-000016
本发明还提供式(A)化合物或其可药用的盐在治疗EGFR突变或扩增介导的癌症中的用途，所述的EGFR突变类型选自以下的一种或任意组合：Del19突变、L858R突变、L858R/T790M双突变、Del19/G724S/T790M三突变、L858R/T790M/L792H三突变、E709K/T790M/L858R三突变、Del19/C797S双突变、L858R/C797S双突变、20外显 子突变；EGFR扩增选自Del19/T790M/C797S三突变、L858R/T790M/D537H三突变、V674L/E746_A750del/T790M三突变EGFR的扩增。
本发明还提供式(A)化合物或其可药用的盐在治疗EGFR突变或扩增介导的癌症中的用途，其中所述的EGFR突变类型选自以下的一种或任意组合：Del19突变、L858R突变、L858R/T790M双突变、Del19/G724S/T790M三突变、L858R/T790M/L792H三突变、E709K/T790M/L858R三突变、Del19/C797S双突变、L858R/C797S双突变、20外显子突变；所述的EGFR扩增选自以下的一种或任意组合：伴随着Del19/T790M/C797S三突变、L858R/T790M/D537H三突变或V674L/E746_A750del/T790M三突变的EGFR扩增。
本发明还提供了治疗EGFR突变或扩增介导的癌症的方法，其包括向患者施用式(A)化合物或其可药用的盐,所述的EGFR突变类型选自以下的一种或任意组合：Del19突变、L858R突变、L858R/T790M双突变、Del19/G724S/T790M三突变、L858R/T790M/L792H三突变、E709K/T790M/L858R三突变、Del19/C797S双突变、L858R/C797S双突变、20外显子突变；所述的EGFR扩增选自以下的一种或任意组合：伴随着Del19/T790M/C797S三突变、L858R/T790M/D537H三突变或V674L/E746_A750del/T790M三突变的EGFR扩增。
本发明还提供了式(A)化合物在制备上述Del19突变、L858R突变、L858R/T790M双突变、Del19/G724S/T790M三突变、L858R/T790M/L792H三突变、E709K/T790M/L858R三突变、Del19/C797S双突变、L858R/C797S双突变、20外显子突变的EGFR突变调节剂中的用途。
在本发明的一些方案中，上述EGFR突变调节剂为EGFR上述突变的抑制剂中的用途。
本发明还提供了式(A)化合物在制备上述Del19/T790M/C797S三突变、上述L858R/T790M/D537H三突变或上述V674L/E746_A750del/T790M三突变的EGFR扩增的调节剂中的用途。
在本发明的一些方案中，上述任一用途中的癌症为肺癌。
在本发明的一些方案中，上述任一用途中的癌症为非小细胞肺癌。
在本发明的一些方案中，上述任一用途中的癌症为未曾接受过治疗的非小细胞肺癌。
在本发明的一些方案中，上述任一用途中的癌症为既往接受过EGFR抑制剂治疗后，产生耐药的非小细胞肺癌。
在本发明的一些方案中，上述的EGFR抑制剂包括第一代EGFR抑制剂、第二代或第三代EGFR抑制剂。
在本发明的一些方案中，上述第一代EGFR抑制剂包括吉非替尼、埃克替尼、厄洛替尼。
在本发明的一些方案中，上述第二代EGFR抑制剂包括阿法替尼、达克替尼。
在本发明的一些方案中，上述第三代EGFR抑制剂包括奥希替尼。
Figure PCTCN2022125057-appb-000017
本发明还提供式(A)化合物或其可药用的盐在治疗FGFR2高表达的癌症中的用途。
本发明还提供式(A)化合物或其可药用的盐在治疗C-KIT突变的癌症中的用途，所述的C-KIT突变类型为V560G突变和/或D816Y突变和/或D816H突变和/或559和560氨基酸缺失突变和/或D816V突变。
本发明还提供式(A)化合物或其可药用的盐在治疗EML-ALK融合蛋白介导的癌症中的用途。
在本发明还提供式(A)化合物或其可药用的盐在治疗EML4-ALK融合蛋白L1196M突变和/或F1174L突变和/或L1196M/L1198F双突变介导的癌症中的用途。
本发明还提供式(A)化合物或其可药用的盐在治疗SLC34A2-ROS1融合蛋白介导的癌症中的用途。
本发明还提供式(A)化合物或其可药用的盐在治疗SLC34A2-ROS1融合蛋白D2033N突变介导的癌症中的用途。
本发明还提供了治疗上述FGFR2高表达的癌症、上述C-KIT突变的癌症、上述EML-ALK融合蛋白介导的癌症、上述SLC34A2-ROS1融合蛋白介导的癌症的方法，其包括向患者施用式(A)化合物或其可药用的盐。
本发明还提供了式(A)化合物在制备上述FGFR2、上述C-KIT突变、上述EML-ALK融合蛋白、上述SLC34A2-ROS1的调节剂中的用途。
在本发明的一些方案中，上述调节剂为抑制剂。
在本发明的一些方案中，上述任一用途中的式(A)化合物的可药用盐为盐酸盐。
在本发明的一些方案中，上述任一用途中的式(A)化合物的可药用盐为一盐酸盐。
技术效果
本发明式(A)化合物不仅对L858R/T790M/C797S三突变以及Del19/T790M/C797S三突变具有较好的活性，同时对L858R或者Del19的单突变、20外显子插入突变、L858R/T790M或L858R/C797S或Del19/C797S的双突变、Del19/G724S/T790M三突变、L858R/T790M/L792H三突变、E709K/T790M/L858R三突变以及伴随EGFR扩增的Del19/T790M/C797S三突变、L858R/T790M/D537H三突变、V674L/E746_A750del/T790M三突变也有着较好的体外激酶或细胞抗增殖活性，并且该化合物在Del19单突变、L858R单突变以及Del19/C797S双突变的小鼠模型体现出了较好的抗肿瘤活性，且小鼠耐受性良好。
除此之外，本发明式(A)化合物对FGFR2高表达、C-KIT V560G突变、C-KIT D816Y突变、C-KIT D816H突变、C-KIT V559和V560氨基酸缺失突变、C-KIT D816V突变、EML4-ALK融合蛋白突变、EML4-ALK融合蛋白L1196M或F1174L突变或L1196M/L1198F双突变、SLC34A2-ROS1融合蛋白突变、SLC34A2-ROS1融合蛋白D2033N突变的细胞系均有着较好的抗增殖活性。
定义和说明
除非另有说明，本文所用的下列术语和短语旨在具有下列含义。一个特定的术语或短语在没有特别定义的情况下不应该被认为是不确定的或不清楚的，而应该按照普通的含义去理解。
术语“药学上可接受的盐”是指本发明化合物与相对无毒的酸或碱制备得到的衍生物。这些盐可以在化合物合成、分离、纯化期间就被制备，或者单独使用经过纯化的化合物的游离形式与适合的酸或碱反应。当化合物中含有相对酸性的官能团时，与碱金属、碱土金属氢氧化物或有机胺反应得到碱加成盐，包括基于碱金属与碱土金属的阳离子以及无毒的铵、季铵和胺阳离子，还涵盖氨基酸的盐等。当化合物中含有相对碱性的官能团时，与有机酸或无机酸反应得到酸加成盐。在本发明中，所述EGFR突变介导的肿瘤或癌症指的是在这些肿瘤或癌症患者中可检测出EGFR的癌症驱动突变(driver mutation)，包括但不限于Del19突变、L858R突变、T790M突变，20外显子插入突变(Exon 20ins)，C797S等突变。其中，Del19突变是指第19号外显子内部分碱基的缺失造成了非移码性部分氨基酸缺失；L858R指的是由于碱基的错义突变造成了858号氨基酸由L变成了R；T790M指的是由于基因中碱基的错义突变造成了790号氨基酸由T变成了M；20外显子插入(Exon 20ins)突变是指发生于EGFR的20外显子的框内重复/插入的突变；C797S突变是指797位的半胱氨酸残基突变成丝氨酸。在本发明中，所述EGFR突变不但包括上述EGFR的单突变型，还包括T790M、Del19、L858R、Exon 20ins、C797S以及其他位点自由组合的复合突变型，包括但不限于L858R/T790M双突变、Del19/G724S/T790M三突变、L858R/T790M/L792H三突变、E709K/T790M/L858R三突变、Del19/C797S双突变、L858R/C797S双突变等。
在本发明中，所述EGFR扩增是指EGFR基因拷贝数的增加或蛋白的高水平表达。它可以发生在突变细胞上，也可以发生在没有突变(野生型)的EGFR受体细胞上。
附图说明
图1为EGFR Del19/C797S突变的体内药效研究中的动物肿瘤生长曲线图。
图2为EGFR Del19/C797S突变的体内药效研究中的动物体重曲线图。
图3为EGFR L858R突变的体内药效研究中的动物肿瘤生长曲线图。
图4为EGFR L858R突变的体内药效研究中的动物体重曲线图。
图5为EGFR Del19突变的体内药效研究中的动物肿瘤生长曲线图。
图6为EGFR Del19突变的体内药效研究中的动物体重曲线图。
图7为Osimertinib耐药人源肺癌PDX模型研究中的动物肿瘤生长曲线图。
图8为Osimertinib耐药人源肺癌PDX模型研究中的动物体重曲线图。
图9为EGFR L858R/C797S突变的体内药效研究中的动物肿瘤生长曲线图。
图10为EGFR L858R/C797S突变的体内药效研究中的动物体重曲线图。
图11为EGFR L858R/T790M突变的体内药效研究中的动物肿瘤生长曲线图。
图12为EGFR L858R/T790M突变的体内药效研究中的动物体重曲线图。
具体实施方式
下面通过实施例对本发明进行详细描述，但并不意味着对本发明任何不利限制。本发明的化合物可以通过本领域技术人员所熟知的多种合成方法来制备，包括下面列举的具体实施方式、其与其他化学合成方法的结合所形成的实施方式以及本领域技术上人员所熟知的等同替换方式，优选的实施方式包括但不限于本发明的实施例。对本领域的技术人员而言，在不脱离本发明精神和范围的情况下针对本发明具体实施方式进行各种变化和改进将是显而易见的。
实施例1，式(A)化合物的制备
1.1中间体6A的制备
Figure PCTCN2022125057-appb-000018
化合物6A-1：
将化合物1C-4(3.5g,15.5mmol)溶于乙腈(40mL)，在0℃条件下，加入N-碘代丁二酰亚胺(4.9g,21.7mmol)。室温反应搅拌5小时，LCMS监控显示原料消失后，减压浓缩，加入水(30mL)，用二氯甲烷(45mL×3次)萃取，合并有机相，有机相先用饱和食盐水(60mL×2次)洗涤，然后用无水硫酸钠干燥，过滤，减压浓缩。所得残余物用硅胶柱层析纯化(洗脱剂：乙酸乙酯/石油醚＝1/1)得到3.57g化合物6A-1。
MS(ESI,m/z):352.0[M+H] +。
化合物6A-2：
将化合物6A-1(3.4g,9.7mmol)和1A(3.7g,12.5mmol)溶于1,4-二氧六环(30mL)和水(6mL)中，向上述反应液中加入碳酸钾(2.7g,19.4mmol)和[1,1'-双(二苯基膦)二茂铁]二氯化钯二氯甲烷络合物(790mg,1.0mmol)。在氮气保护下，将反应体系加热至80℃并继续搅拌2小时。LCMS监控显示原料消失后，将反应液冷却至室温，减压浓缩，所得残余物用硅胶柱层析纯化(洗脱剂：乙酸乙酯/石油醚＝2/1)得到2.8g化合物6A-2。
MS(ESI,m/z):395.3[M+H] +。
化合物6A-3：
将化合物6A-2(2.7g,6.8mmol)溶于N,N-二甲基甲酰胺(28mL)中。随后，向上述反应液中加入碳酸钾(1.9g，13.5mmol)。将反应体系加热至100℃并继续搅拌24小时。LCMS监控显示原料消失后，将反应液冷却至室温并加入水(50mL)淬灭。混合液用乙酸乙酯(60mL×4次)萃取，合并有机相，有机相先用饱和食盐水(50mL×3次)洗涤，然后用无水硫酸钠干燥，过滤，最后减压浓缩。所得残余物用硅胶柱层析纯化(洗脱剂：二氯甲烷/乙酸乙酯＝15/1)得到1.2g化合物6A-3。
MS(ESI,m/z):375.2[M+H] +。
化合物6A-4：
将化合物6A-3(1.2g,3.3mmol)溶于氯化氢的1,4-二氧六环溶液(4M,15mL)中。在30℃条件下搅拌6小时，LCMS监控显示原料消失后，将反应液浓缩，加入水(40mL)，用饱和碳酸氢钠水溶液调节其pH到9。混合液用氯仿/异丙醇＝3/1(50mL×3次)萃取，合并有机相，然后用无水硫酸钠干燥，过滤，最后减压浓缩。所得残余物用硅胶柱层析纯化(洗脱剂：二氯甲烷/甲醇＝30/1)得到842mg化合物6A-4。
MS(ESI,m/z):275.0[M+H] +。
化合物6A-5：
将化合物6A-4(300mg，1.1mmol)和碳酸铯(1.07g，3.3mmol)溶于N,N-二甲基甲酰胺(6mL)中。随后， 向上述反应液中加入碘代异丙烷(1.86g，10.9mmol)。将反应体系加热至80℃并继续搅拌16小时。LCMS监控显示原料消失后，将反应液冷却至室温并加入水(30mL)淬灭。混合液用乙酸乙酯(50mL×3次)萃取，合并有机相，有机相先用饱和食盐水(50mL×3次)洗涤，然后用无水硫酸钠干燥，过滤，最后减压浓缩。所得残余物用硅胶柱层析纯化(洗脱剂：二氯甲烷/甲醇＝10/1)得到75mg化合物6A-5。
MS(ESI,m/z):317.2[M+H] +。
中间体6A：
将化合物6A-5(75mg，0.2mmol)溶于乙醇(8mL)和水(1.6mL)中。随后，向上述反应液中加入氯化铵(50.7mg，0.9mmol)和还原铁粉(132.4mg，2.4mmol)。将反应体系加热至80℃并继续搅拌5小时。LCMS监控显示原料消失后，将反应液冷却至室温并减压浓缩。所得残余物用硅胶柱层析纯化(洗脱剂：二氯甲烷/甲醇＝10/1)得到48mg化合物6A。
MS(ESI,m/z):287.2[M+H] +。
中间体35A的制备
Figure PCTCN2022125057-appb-000019
化 合物35A-l，
将6-氨基喹喔啉(10g,68.89mmol)溶于浓硫酸(20mL)中。在0℃条件下，向反应液中分批加入硝酸钾(9.054g,89.55mmol)并在该温度下继续搅拌30分钟。LCMS监控显示原料消失后，将反应液倒入冰水(100g)中。用1M氢氧化钠水溶液调节其pH到8。混合液用乙酸乙酯(200mL×2次)萃取，合并有机相，有机相先用饱和食盐水(100mL×3次)洗涤，然后用无水硫酸钠干燥，过滤，减压浓缩。所得残余物用硅胶柱层析纯化(洗脱剂：二氯甲烷/甲醇＝10/1)得到2g化合物35A-1。
MS(ESI)M/Z:191.2[M+H] +。
1.2中间体35A：
将化合物35A-1(2g,10.5mmol)溶于N,N-二甲基甲酰胺(20mL)中。将反应液降至0℃，氮气保护下，分批加入氢化钠(60wt,1.3g,31.5mmol)并继续搅拌20分钟。随后，向上述反应液中加入2,4-二氯-5-溴嘧啶(4.8g,21.0mmol)，将反应升至室温并继续搅拌1小时。LCMS监控显示原料消失后，将反应液降温至0℃并加入饱和氯化铵水溶液(80mL)淬灭。混合液用乙酸乙酯(100mL×3次)萃取，合并有机相，有机相先用饱和食盐水(80mL×3次)洗涤，然后用无水硫酸钠干燥，过滤，减压浓缩。所得残余物用硅胶柱层析纯化(洗脱剂：乙酸乙酯/石油醚＝1/2)得到2.7g化合物35A。
MS(ESI,m/z):381.0,383.0[M+H] +。
Figure PCTCN2022125057-appb-000020
化合物53A：
将化合物6A(2.7g,9.43mmol)和35A(3.6g,9.43mmol)溶于N-甲基吡咯烷酮(30mL)中。随后，向上述反应液中加入甲烷磺酸(2.72g,28.28mmol)。将反应体系加热至95℃并继续搅拌3小时。LCMS监控显示原料消失后，将反应液冷却至室温并经反相C18柱纯化。纯化条件：色谱柱330g C18反相柱；流动相水(含0.1％甲酸)和乙腈；流速70mL/分钟；梯度在20分钟内，乙腈从10％升到50％；检测波长254nm。收集产品，减压浓缩，得到3.4g化合物53A。
MS(ESI,m/z):631.2,633.2[M+H] +。
化合物53B:
将化合物53A(3.4g,5.38mmol)溶于乙醇(40mL)和水(8mL)的混合溶剂中。随后，向上述反应液中加入铁粉(1.50g,26.92mmol)和氯化铵(0.86g,16.15mmol)并将反应体系加热至80℃并继续搅拌2小时。LCMS监控显示原料消失后，将反应液冷却至室温并减压浓缩。所得残余物用硅胶柱层析纯化(洗脱剂：二氯甲烷/甲醇＝10/1)，得到2.8g化合物53B。
MS(ESI,m/z):601.2,603.2[M+H] +。
1.3化合物A：
将化合物53B(5g,8.31mmol)溶于吡啶(50mL)中。随后向反应液中滴加甲基磺酰氯(1.9g,16.62mmol)。将反应体系升温至50℃并继续搅拌2小时。LCMS监控显示原料消失后，将反应液冷却至室温并减压浓缩。残余物溶于甲醇/四氢呋喃(1/1，50mL)的混合溶剂中，在0℃条件下，向反应液中加入氢氧化钠(2M，5mL)的水溶液。将反应体系升温至室温并继续搅拌1小时后，减压浓缩。所得残余物用硅胶柱层析纯化(洗脱剂：二氯甲烷/甲醇＝10/1)，粗产品经过二氯甲烷/甲醇(20/1，30mL)打浆后，用乙腈/水(50mL)冻干，得到3g化合物A。
MS(ESI,m/z):679.0,681.0[M+H] +。
1H NMR(400MHz,DMSO-d 6)δ9.88(br s,1H),8.94(d,J＝2.0Hz,1H),8.85(d,J＝2.0Hz,1H),8.76(s,1H),8.67(br s,1H),8.35(s,1H),8.27(s,1H),7.73(s,1H),7.49(s,1H),7.38(s,1H),6.58(s,1H),3.99-3.91(m,1H),3.76(s,3H),3.71(s,3H),3.21(t,J＝5.6Hz,2H),3.00(s,3H),2.94(t,J＝5.6Hz,2H),1.29(d,J＝6.4Hz,6H)。
1.4化合物A盐酸盐：
将化合物A(67g,98.59mmol)溶于二氯甲烷(880mL)和甲醇(440mL)的混合溶剂中并在室温下继续搅拌1小时。随后在室温条件下，向反应液中滴加氯化氢的甲醇溶液(4M,24.65mL,98.59mmol)。该反应体系继续在室温搅拌4小时后，将反应液减压浓缩至70mL。向上述混合物中加入甲基叔丁基醚(880mL)并继续在室温搅拌2小时。析出的固体经过过滤，乙腈/水(500mL)冻干，得到60.2g化合物A盐酸盐。
MS(ESI)M/Z:679.0,681.0[M+H] +。
1H NMR(300MHz,DMSO-d 6)δ9.97(s,1H),9.30-9.16(m,2H),8.99(s,1H),8.91(s,1H),8.43(s,2H),7.75-7.30(m,3H),6.57(s,1H),3.95-3.86(m,1H),3.79(s,3H),3.69(s,3H),3.28-3.14(m,2H),3.03(s,3H),2.98-2.88(m,2H),1.27(brs,6H)。
实施例2：生物学测试评价:
(一)体外酶学实验
本实验采用荧光共振能量转移(TR-FRET)的方法测试化合物A对EGFR WT、EGFR Del19、EGFR L858R、EGFR L858R/T790M、EGFR L858R/C797S和EGFR ex19del/C797S激酶活性的抑制作用，并得出化合物A对EGFR激酶活性的半数抑制浓度IC 50。
2.实验材料
EGFR、EGFR Del19、EGFR L858R、EGFR L858R/T790M、EGFR L858R/C797S、EGFR ex19del/C797S重组酶购自Signalchem公司。
HTRF KinEASE-TK kit试剂盒，购自Cisbio公司。
DTT，MnCl2，MgCl2购自Sigma公司。
ATP购自Promega公司。
3.实验方法
1)准备1×工作液：5mM MgCl 2；1mM DTT；1mM MnCl 2和1×激酶缓冲液(试剂盒中)，其中EGFR L858R/T790M的缓冲液中加入了SEB。
2)使用Echo 550(Labcyte)转移10nL(或1μL)梯度稀释的化合物到384孔实验板中。
3)加入5μL(或2μL)2×重组酶溶液到384孔实验板中，室温孵育10分钟。
4)加入5μL(或2μL)2×TK-substrate-biotin底物溶液(包含ATP)到384孔实验板中，室温孵育40分钟(或1h)。
5)加入5μL含有Sa-XL 665HTRF检测液，以及5μL TK-antibody-Cryptate，室温孵育1小时。
6)酶标仪检测各孔的615nm和665nm荧光信号值。
7)计算每孔荧光信号665nm/615nm的比值。
8)使用GraphPad Prism软件进行数据分析，得出化合物A的IC 50。
激酶活性抑制结果见表1。
4.实验结果及结论
从表1中我们可以看出，化合物A对Del19单突变、L858R单突变激酶均有很好的抑制作用。
表1.酶学抑制结果
Figure PCTCN2022125057-appb-000021
(二)细胞增殖抑制实验
I.A431细胞增殖抑制实验
本实验采用CellTiter-Glo的方法测试化合物A对A431细胞增殖的抑制作用，并得出化合物抑制细胞生长半数的浓度IC 50。
1.实验材料
A431细胞购自ATCC。
DMEM培养基，胎牛血清(FBS)，Penicillin-Streptomycin购自GIBCO。
Brigatinib购自Selleck公司。
CellTiter-Glo试剂，购自Promega公司。
2.实验方法
1)按照每孔800个细胞的密度将A431细胞接种于384孔培养板，每孔30μl，置于细胞培养箱中培养24小时(37℃，5％CO 2)。
2)Day 0：使用Echo向培养板细胞中加入30nL梯度稀释的待测化合物，DMSO终浓度为0.1％，将培养板置于细胞培养箱中孵育72小时(37℃，5％CO 2)。空白对照加入每孔30nL的DMSO。
3)Day 3：每孔加入30μL Cell Titer-Glo试剂，室温避光30分钟。
4)Envision酶标仪(PerkinElmer)检测化学发光信号。
5)使用GraphPad Prism软件进行数据分析，得出化合物A的IC 50。
II.NCI-H3255细胞增殖抑制实验
1.实验目的
本实验采用CellTiter-Glo的方法测试化合物A对NCI-H3255(EGFR L858R突变)细胞增殖的抑制作用，并得出化合物A抑制细胞生长半数的浓度IC 50。
2.实验材料
NCI-H3255细胞，购自南京科佰生物科技有限公司。
1640培养基，胎牛血清(FBS)，Penicillin-Streptomycin，购自GIBCO。
CellTiter-Glo试剂，购自Promega公司。
3.实验方法
1)将NCI-H3255细胞接种于384孔培养板，每孔30μL。
2)Day0：使用Echo向培养板细胞中加入30nL梯度稀释的待测化合物，DMSO终浓度为0.1％，将培养板置于细胞培养箱中孵育72小时(37℃，5％CO 2)。空白对照加入每孔30nL的DMSO。
3)Day3：每孔加入30μL Cell Titer-Glo试剂，室温避光30分钟。
4)Envision酶标仪(PerkinElmer)检测化学发光信号。
5)使用GraphPad Prism软件进行数据分析，得出化合物A的IC 50。
细胞活性抑制结果见表2。
III.PC-9细胞增殖抑制实验
1.实验目的
本实验采用CellTiter-Glo的方法测试化合物A对PC-9(EGFR Del19突变)细胞增殖的抑制作用，并得出化合物抑制细胞生长半数的浓度IC 50。
2.实验材料
PC-9细胞，购自European Collection of Authenticated Cell Cultures。
1640培养基，胎牛血清(FBS)，Penicillin-Streptomycin，购自GIBCO。
CellTiter-Glo试剂，购自Promega公司。
3.实验方法
1)将PC-9细胞接种于384孔培养板，每孔30μL。
2)Day0：使用Echo向培养板细胞中加入30nL梯度稀释的待测化合物，DMSO终浓度为0.1％，将培养板置于细胞培养箱中孵育72小时(37℃，5％CO 2)。空白对照加入每孔30nL的DMSO。
3)Day3：每孔加入30μL Cell Titer-Glo试剂，室温避光30分钟。
4)Envision酶标仪(PerkinElmer)检测化学发光信号。
5)使用GraphPad Prism软件进行数据分析，得出化合物A的IC 50。
细胞活性抑制结果见表2。
IV.NCI-H1975细胞增殖抑制实验
1.实验目的
本实验采用CellTiter-Glo的方法测试化合物A对NCI-H1975(EGFR L858R/T790M突变)细胞增殖的抑制作用，并得出化合物A抑制细胞生长半数的浓度IC 50。
2.实验材料
NCI-H1975细胞来自ATCC。
1640培养基，胎牛血清(FBS)，Penicillin-Streptomycin购自GIBCO。
CellTiter-Glo试剂，购自Promega公司。
3.实验方法
1)将NCI-H1975细胞接种于384孔培养板，每孔30μL。
2)Day0：使用Echo向培养板细胞中加入30nL梯度稀释的待测化合物，DMSO终浓度为0.1％，将培养板置于细胞培养箱中孵育72小时(37℃，5％CO 2)。空白对照加入每孔30nL的DMSO。
3)Day3：每孔加入30μL Cell Titer-Glo试剂，室温避光30分钟
4)Envision酶标仪(PerkinElmer)检测化学发光信号。
5)使用GraphPad Prism软件进行数据分析，得出化合物A的IC 50。
细胞活性抑制结果见表2。
V.HCC827细胞增殖抑制实验
1.实验目的
本实验采用CellTiter-Glo的方法测试化合物A对HCC827(EGFR Del突变)细胞增殖的抑制作用，并得出化合物A抑制细胞生长半数的浓度IC 50。
2.实验材料
HCC827细胞，购自ATCC。
1640培养基，胎牛血清(FBS)，Penicillin-Streptomycin，购自GIBCO。
CellTiter-Glo试剂，购自Promega公司。
3.实验方法
1)将HCC827细胞接种于384孔培养板，每孔30μL。
2)Day0：使用Echo向培养板细胞中加入30nL梯度稀释的待测化合物，DMSO终浓度为0.1％，将培养板置于细胞培养箱中孵育72小时(37℃，5％CO 2)。空白对照加入每孔30nL的DMSO。
3)Day3：每孔加入30μL Cell Titer-Glo试剂，室温避光30分钟。
4)Envision酶标仪(PerkinElmer)检测化学发光信号。
5)使用GraphPad Prism软件进行数据分析，得出化合物A的IC 50。
细胞活性抑制结果见表2。
VI.Ba/F3 EGFR-Del19/G724S/T790M、Ba/F3 EGFR-E709K/T790M/L858R、Ba/F3 EGFR- L858R/T790M/L792H细胞增殖抑制实验
1.实验目的
本实验采用CellTiter-Glo的方法测试化合物A对Ba/F3 EGFR-Del19/G724S/T790M和Ba/F3 EGFR-E709K/T790M/L858R和Ba/F3 EGFR-L858R/T790M/L792H细胞增殖的抑制作用，并得出化合物A抑制细胞生长半数的浓度IC 50。
2.实验材料
Ba/F3 EGFR-Del19/G724S/T790M细胞来自康源博创生物科技(北京)有限公司。
Ba/F3 EGFR-E709K/T790M/L858R细胞来自康源博创生物科技(北京)有限公司。
Ba/F3 EGFR-L858R/T790M/L792H细胞来自康源博创生物科技(北京)有限公司。
1640培养基，胎牛血清(FBS)购自Hyclone和GIBCO。
CellTiter-Glo试剂，购自Promega公司。
3.实验方法
1)收获处于对数生长期的细胞进行细胞计数。用台盼蓝排斥法检测细胞活力，确保细胞活力在90％以上。
2)使用完全培养基调整细胞密度，随后接种于96孔细胞培养板，每孔接种90μL，共3000个细胞。
3)将96孔板中的细胞置于37℃、5％CO2条件下培养。
4)配制10倍药物溶液，转移连续稀释化合物各10μL至96孔细胞板的相应实验孔中，使得化合物检测浓度为1μM起，9个浓度，3倍稀释，然后，每个药物浓度设置三个复孔。
5)将已加药的96孔板置于37℃、5％CO2条件下继续培养72小时，之后进行CTG分析。
6)融化CTG试剂并平衡细胞板至室温30分钟。
7)每孔加入等体积的CTG溶液。
8)在定轨摇床上振动5分钟使细胞裂解。
9)将细胞板放置于室温20分钟以稳定冷光信号。
10)读取冷光值，收集数据。
11)使用GraphPad Prism软件进行数据分析，得出化合物A的IC 50。
细胞活性抑制结果见表2。
VII.Ba/F3(EGFR-Del19/C797S)、Ba/F3(EGFR-L858R/C797S)细胞增殖抑制实验
1.实验目的
本实验采用CellTiter-Glo的方法测试化合物A对Ba/F3(EGFR-Del19/C797S)和Ba/F3(EGFR-L858R/C797S)细胞增殖的抑制作用，并得出化合物A抑制细胞生长半数的浓度IC 50。
2.实验材料
Ba/F3 EGFR-Del19/C797S细胞来自康源博创生物科技(北京)有限公司。
Ba/F3 EGFR-L858R/C797S细胞来自康源博创生物科技(北京)有限公司。
1640培养基，胎牛血清(FBS)购自GIBCO。
CellTiter-Glo试剂，购自Promega公司。
3.实验方法
1)按照每孔3000个细胞的密度将Ba/F3(EGFR Del19/C797S)和Ba/F3(EGFR L858R/C797S)细胞分别接种于96孔培养板，每孔90μL。
2)Day 0：向培养板细胞中加入10μL梯度稀释的待测化合物，DMSO终浓度为0.2％，将培养板置于细胞培养箱中孵育72小时(37℃，5％CO 2)。空白对照加入每孔10μL的DMSO。
3)Day3：每孔加入100μL CellTiter-Glo试剂，室温避光振荡10分钟
4)Envision酶标仪(PerkinElmer)检测化学发光信号。
5)使用GraphPad Prism软件进行数据分析，得出化合物的IC 50。
细胞活性抑制结果见表2。
VIII.NCI-H716细胞增殖抑制实验
1.实验目的
本实验采用CellTiter-Glo的方法测试化合物A对NCI-H716(FGFR2高表达)细胞增殖的抑制作用，并得出化合物抑制细胞生长半数的浓度IC 50。
2.实验材料
NCI-H716细胞，购自ATCC。
1640培养基，胎牛血清(FBS)，Penicillin-Streptomycin，购自GIBCO。
CellTiter-Glo试剂，购自Promega公司。
3.实验方法
1)将NCI-H716细胞接种于96孔培养板，每孔100μL。
2)Day0：向培养板细胞中加入92μL培养基和8μL梯度稀释的待测化合物，DMSO终浓度为0.1％，将培养板置于细胞培养箱中孵育72小时(37℃，5％CO 2)。空白对照加入每孔92μL培养基和8μL的DMSO。
3)Day3：每孔加入100μL Cell Titer-Glo试剂，室温避光30分钟。
4)Envision酶标仪(PerkinElmer)检测化学发光信号。
5)使用GraphPad Prism软件进行数据分析，得出化合物A的IC 50。
细胞活性抑制结果见表2。
IX.Ba/F3 C-KIT-V560G、Ba/F3 C-KIT-D816Y、Ba/F3 C-KIT-D816H、Ba/F3 C-KIT-Del(V559V560)、Ba/F3 C-KIT-D816V、NCI-H3122(EML4-ALK)、Ba/F3-EML4-ALK-L1196M、Ba/F3 EML4-ALK-F1174L、Ba/F3-EML4-ALK-L1196M/L1198F、Ba/F3 SLC34A2/ROS1、BaF3 SLC34A2-ROS1-D2033N细胞增殖抑制实验
1.实验目的
本实验采用CellTiter-Glo的方法测试化合物A对Ba/F3 C-KIT-V560G、Ba/F3 C-KIT-D816Y、Ba/F3 C-KIT-D816H、Ba/F3 C-KIT-Del(V559-V560)、Ba/F3 C-KIT-D816V、NCI-H3122(EML4-ALK)、Ba/F3-EML4-ALK-L1196M、Ba/F3 EML4-ALK-F1174L、Ba/F3-EML4-ALK-L1196M/L1198F、Ba/F3 SLC34A2/ROS1、Ba/F3 SLC34A2-ROS1-D2033N细胞增殖的抑制作用，并得出化合物A抑制细胞生长半数的浓度IC 50。
2.实验材料
Ba/F3 C-KIT-V560G细胞来自康源博创生物科技(北京)有限公司。
Ba/F3 C-KIT-D816Y细胞来自康源博创生物科技(北京)有限公司。
Ba/F3 C-KIT-D816H细胞来自康源博创生物科技(北京)有限公司。
Ba/F3 C-KIT-Del(V559V560)细胞来自康源博创生物科技(北京)有限公司。
Ba/F3 C-KIT-D816V细胞来自康源博创生物科技(北京)有限公司。
NCI-H3122(EML4-ALK)细胞来自康源博创生物科技(北京)有限公司。
Ba/F3-EML4-ALK-L1196M细胞来自康源博创生物科技(北京)有限公司。
Ba/F3 EML4-ALK-F1174L细胞来自康源博创生物科技(北京)有限公司。
Ba/F3-EML4-ALK-L1196M/L1198F细胞来自康源博创生物科技(北京)有限公司。
Ba/F3 SLC34A2/ROS1细胞来自康源博创生物科技(北京)有限公司。
Ba/F3 SLC34A2-ROS1-D2033N细胞来自康源博创生物科技(北京)有限公司。
1640培养基，胎牛血清(FBS)购自Hyclone和GIBCO。
CellTiter-Glo试剂，购自Promega公司。
3.实验方法
1)收获处于对数生长期的细胞进行细胞计数。用台盼蓝排斥法检测细胞活力，确保细胞活力在90％以上。
2)使用完全培养基调整细胞密度，随后接种于96孔细胞培养板，每孔接种90μL，共3000个细胞。
3)将96孔板中的细胞置于37℃、5％CO2条件下培养。
4)配制10倍药物溶液，转移连续稀释化合物各10μL至96孔细胞板的相应实验孔中，每个药物浓度设置三个复孔。
5)将已加药的96孔板置于37℃、5％CO2条件下继续培养72小时，之后进行CTG分析。
6)融化CTG试剂并平衡细胞板至室温30分钟。
7)每孔加入等体积的CTG溶液。
8)在定轨摇床上振动5分钟使细胞裂解。
9)将细胞板放置于室温20分钟以稳定冷光信号。
10)读取冷光值，收集数据。
11)使用GraphPad Prism软件进行数据分析，得出化合物A的IC 50。
细胞活性抑制结果见表2。
4.实验结果及结论
从表2中的实验结果可以看出，本发明化合物A对NCI-H3255 L858R EGFR突变、PC9 Del19 EGFR突变、HCC827 Del19 EGFR突变、NCI-H1975 L858R/T790M EGFR突变、Ba/F3(Del19/G724S/T790M)EGFR三突变细胞株、Ba/F3(L858R/T790M/L792H)EGFR三突变细胞株、Ba/F3(E709K/T790M/L858R)EGFR三突变细胞株、Ba/F3(Del19/C797S)EGFR双突变细胞株和Ba/F3(L858R/C797S)EGFR双突变细胞株、NCI-H716(FGFR2)、Ba/F3 C-KIT-V560G、Ba/F3 C-KIT-D816Y、Ba/F3 C-KIT-D816H、Ba/F3 C-KIT-Del(V559V560)、Ba/F3 C-KIT-D816V、NCI-H3122(EML4-ALK)、Ba/F3-EML4-ALK-L1196M、Ba/F3 EML4-ALK-F1174L、Ba/F3-EML4-ALK-L1196M/L1198F、Ba/F3 SLC34A2/ROS1、Ba/F3 SLC34A2-ROS1-D2033N的细胞增殖有较好的抑制作用；对EGFR野生型细胞系A431的抑制作用较弱，有较好的选择性。
表2.细胞增殖抑制试验数据结果
Figure PCTCN2022125057-appb-000022
(三)PDO增殖抑制试验
1.实验目的
本实验采用CellTiter-Glo的方法测试化合物A对奥西替尼耐药的PDO(Patient Derived Tumor Organoids)增殖的抑制作用，并得出化合物A抑制细胞生长半数的浓度IC 50。
2.实验材料
PDO，来自北京科途医学科技有限公司。
Accutase，购自Sigma。
K2 Oncology，来自北京科途医学科技有限公司。
CellTiter-Glo试剂，购自Promega公司。
3.实验方法
1)PDO在6孔细胞培养板中生长到直径200μm后，用细胞消化液在37℃消化10-30分钟，用头部灼烧钝化的巴适管吹打几次，直到细胞呈单细胞或者寡细胞团的状态。
2)细胞计数后用预冷的完全培养基调整浓度到100-160个细胞/μL，加入等体积的20％的基质胶，完全混合后，按照50μL/孔，加入低吸附细胞培养板底部。
3)在37℃培养箱孵育30分钟后，上层加入50μL/孔完全培养基，在37℃，5％二氧化碳培养箱中继续培养2天。
4)向培养板细胞中加入梯度稀释的待测化合物，DMSO终浓度为0.2％，将培养板置于细胞培养箱中孵育5天(37℃，5％CO 2)。
5)加入70μL化学发光细胞裂解液，震荡5分钟后，轻轻吹打至细胞完全裂解，从细胞培养板中转移100μL到白色低透酶标检测板中。
6)在化学发光酶标仪(FLUOstar Omega)中检测化学发光信号。
7)使用GraphPad Prism软件进行数据分析，得出化合物A的IC 50。
4.实验结果及结论
从表3结果来看，化合物A对奥西替尼耐药的PDO增殖有较好的抑制作用。
表3.PDO增殖抑制试验数据结果
Figure PCTCN2022125057-appb-000023
(四)EGFR Dellg/C797S突变的体内药效研究
1.实验目的
评价化合物A(本实验使用的为化合物A的盐酸盐)连续14天口服给药，对Ba/F3 EGFR Del19/C797S的抗肿瘤活性及毒副作用。
2.实验材料
NU/NU小鼠，雌性，SPF级，购自北京维通利华实验动物技术有限公司。
Ba/F3 EGFR Del19/C797S细胞，购自康源博创生物科技(北京)有限公司。
3.实验步骤
3.1细胞培养
Ba/F3 EGFR Del19/C797S细胞用含有10％的胎牛血清的RPMI1640培养基在37℃，5％二氧化碳培养箱中培养，收集指数生长期的细胞进行接种。
3.2细胞接种
在无菌条件下，取体外培养的Ba/F3 EGFR Del19/C797S细胞悬液，离心后调整细胞浓度至3×10 7个/mL，接种于小鼠右侧腋窝皮下(0.1mL/只)，接种当天设为第0天。
3.3肿瘤分组、给药及测量
a，当平均肿瘤体积100-200mm 3时，挑选32只肿瘤体积适中小鼠入组，按照肿瘤体积大小随机分为4组：G1：溶媒对照组、G2：化合物A(15mg/kg)、G3：化合物A(30mg/kg)和G4：化合物A(65mg/kg)，8只/组。
b，动物分组后开始给药，给药体积均为10mL/kg，口服给药(po)；每天称重给药1次，连续给药14天；每周测量瘤径2次。
c，肿瘤体积(Tumor volume,TV)：每周测量2次肿瘤体积，以观察瘤块体积变化和生长速度。肿瘤体积V＝1/2×a×b 2，其中a、b分别表示肿瘤长径和短径。化合物对肿瘤组织的生长抑制作用采用肿瘤生长抑制率TGI(％)评价。TGI(％)＝[1-(某给药组的平均肿瘤体积-该给药组分组当天的平均肿瘤体积)/(阴性对照组的平均肿瘤体积-阴性对照组分组当天的平均肿瘤体积)]×100％。给药组和阴性对照组取同一天数据。
d，在试验过程中密切观察小鼠生活状态，包括外观体征、一般行为活动、精神状态、摄食情况、呼吸状态、粪便和尿液性状、注射局部及其它毒性表现。
e，试验达到终点后，将小鼠实施安乐死，动物尸体冻存至冰柜，移交至有资质的医疗废弃物处理单位进行处置。
4.实验结果
表4.实验数据
Figure PCTCN2022125057-appb-000024
a，平均值±标准误；
b，P值肿瘤体积进行统计分析，与G1组比较，*P﹤0.05；**P﹤0.01。
5.实验结论
从上数结果可以看出，化合物A给药剂量15、30、65mg/kg均能显著抑制肿瘤生长(图1)，且呈明显的量效关系，小鼠耐受性良好(图2)。
(五)L858R突变的体内药效研究
1.实验目的
评价化合物A连续22天口服给药，对NCI-H3255(L858R)的抗肿瘤活性及毒副作用。
2.实验材料
NOD SCID小鼠，雌性，SPF级，购自北京华阜康生物科技股份有限公司。
NCI-H3255(L858R)细胞，购自南京科佰生物科技有限公司。
3.实验步骤
3.1细胞培养
用含有灭活的10％胎牛血清的RPMI-1640培养基在37℃、5％CO 2的培养箱中培养NCI-H3255肿瘤细胞，待细胞长满后分瓶传代。将处于对数生长期的肿瘤细胞用于体内肿瘤的接种。
3.2细胞接种
将无血清RPMI-1640培养液重悬的NCI-H3255肿瘤细胞1×10 7/100μL浓度接种于实验动物的右侧胁肋部皮下，接种当天设为第0天。
3.3肿瘤分组、给药及测量
a，当平均肿瘤体积约236mm 3时，挑选24只肿瘤体积适中小鼠入组，按照肿瘤体积大小随机分为3组：G1：溶媒对照组、G2：化合物A(15mg/kg)和G3：化合物A(60mg/kg)，8只/组。
b，动物分组后开始给药，给药体积均为10mL/kg，口服给药(po)；每天称重给药1次，连续给药22天；每周测量瘤径2次。
c，肿瘤体积(Tumor volume,TV)：每周测量2次肿瘤体积，以观察瘤块体积变化和生长速度。肿瘤体积V＝1/2×a×b 2，其中a、b分别表示肿瘤长径和短径。化合物对肿瘤组织的生长抑制作用采用肿瘤生长抑制率TGI(％)评价。TGI(％)＝[1-(某给药组的平均肿瘤体积-该给药组分组当天的平均肿瘤体积)/(阴性对照组的平均肿瘤体积-阴性对照组分组当天的平均肿瘤体积)]×100％。给药组和阴性对照组取同一天数据。
d，在试验过程中密切观察小鼠生活状态，包括外观体征、一般行为活动、精神状态、摄食情况、呼吸状态、粪便和尿液性状、注射局部及其它毒性表现。
e，试验达到终点后，将小鼠实施安乐死，动物尸体冻存至冰柜，移交至有资质的医疗废弃物处理单位进行处置。
4.实验结果
表5.实验数据
Figure PCTCN2022125057-appb-000025
a，平均值±标准误；
b，P值肿瘤体积进行统计分析，与G1组比较，*P﹤0.05；**P﹤0.01。
5.实验结论
从上数结果可以看出，化合物A给药剂量15、60mg/kg均能显著抑制肿瘤生长(图3)，且呈一定的量效关系。 小鼠耐受性良好(图4)。
(六)Del19突变的体内药效研究
1.实验目的
评价化合物A连续21天口服给药，对PC-9(Del19)的抗肿瘤活性及毒副作用。
2.实验材料
CB-17 SCID小鼠，雌性，SPF级，购自北京华阜康生物科技股份有限公司。
PC-9(Del19)细胞，购自European Collection of Authenticated Cell Cultures。
3.实验步骤
3.1细胞培养
用含有灭活的10％胎牛血清，100U/mL的青霉素和100μg/mL的链霉素的RPMI-1640培养基在37℃、5％CO2的培养箱中培养PC-9(Del19)肿瘤细胞，待细胞长满后分瓶传代。将处于对数生长期的肿瘤细胞用于体内肿瘤的接种。
3.2细胞接种
将无血清RPMI-1640培养液重悬的PC-9(Del19)肿瘤细胞5×10 6/100uL浓度接种于实验动物的右侧胁肋部皮下，接种当天设为第0天。
3.3肿瘤分组、给药及测量
a，当平均肿瘤体积约181mm 3时，挑选20只肿瘤体积适中小鼠入组，按照肿瘤体积大小随机分为4组：G1：溶媒对照组、G2：Gefitinib(吉非替尼，100mg/kg)、G3：化合物A(15mg/kg)和G4：化合物A(45/60mg/kg)，5只/组。
b，动物分组后开始给药，给药体积均为10mL/kg，口服给药(po)；每天称重给药1次，连续给药21天；每周测量瘤径2次。
c，肿瘤体积(Tumor volume,TV)：每周测量2次肿瘤体积，以观察瘤块体积变化和生长速度。肿瘤体积V＝1/2×a×b 2，其中a、b分别表示肿瘤长径和短径。化合物对肿瘤组织的生长抑制作用采用肿瘤生长抑制率TGI(％)评价。TGI(％)＝[1-(某给药组的平均肿瘤体积-该给药组分组当天的平均肿瘤体积)/(阴性对照组的平均肿瘤体积-阴性对照组分组当天的平均肿瘤体积)]×100％。给药组和阴性对照组取同一天数据。
d，在试验过程中密切观察小鼠生活状态，包括外观体征、一般行为活动、精神状态、摄食情况、呼吸状态、粪便和尿液性状、注射局部及其它毒性表现。
e，试验达到终点后，将小鼠实施安乐死，动物尸体冻存至冰柜，移交至有资质的医疗废弃物处理单位进行处置。
4.实验结果
表6.实验数据
Figure PCTCN2022125057-appb-000026
a，平均值±标准误；
b，P值肿瘤体积进行统计分析，与G1组比较，*P﹤0.05；**P﹤0.01。
5.实验结论
从上数结果可以看出，化合物A剂量15、45/60mg/kg和Gefitinib剂量100mg/kg均能显著抑制肿瘤生长，且化合物A呈明显的量效关系(图5)。化合物A 45/60mg/kg与Gefitinib 100mg/kg疗效相当无统计差异。小鼠耐受性良好(图6)。
(七)Osimertinib(奥希替尼)耐药人源肺癌PDX模型研究
1.实验目的
评价化合物A连续21天口服给药，对Osimertinib耐药人源肺癌PDX模型LD1-0025-200717的抗肿瘤活性及毒副作用。
2.实验材料
NU/NU小鼠，雌性，SPF级，购自浙江维通利华实验动物技术有限公司。
LD1-0025-200717，人源肺癌肿瘤组织，54岁男性患者，临床诊断：左上肺原发性支气管肺癌，腺癌；EGFR三突变，19del&T790M&C797S；Osimertinib耐药；PDX病理诊断：低-中分化腺癌。传至FP2+5代用于本次药效试验。
3.实验步骤
3.1人源肺癌移植瘤体内接种
将LD1-0025-200717肿瘤组织均匀切成大小约为3mm×3mm×3mm(约50～90mg)肿瘤块并接种于NU/NU小鼠右侧皮下。随后观察接种后小鼠并监测肿瘤的生长。
3.2肿瘤分组、给药及测量
a，当肿瘤平均体积达到143.53mm 3时，根据瘤体积大小随机分为3组：G1：溶媒对照组、G2：化合物A(15mg/kg)和G3：化合物A(60mg/kg)，每组8只。分组当天为第0天。
b，动物分组当天开始给药，给药体积均为10mL/kg，口服给药(po)；每天称重给药1次，连续给药21天；每周测量瘤径2次。
c，肿瘤体积(Tumor volume,TV)：每周测量2次肿瘤体积，以观察瘤块体积变化和生长速度。肿瘤体积V＝1/2×a×b 2，其中a、b分别表示肿瘤长径和短径。化合物对肿瘤组织的生长抑制作用采用肿瘤生长抑制率TGI(％)评价。TGI(％)＝[1-(某给药组的平均肿瘤体积-该给药组分组当天的平均肿瘤体积)/(阴性对照组的平均肿瘤体积-阴性对照组分组当天的平均肿瘤体积)]×100％。给药组和阴性对照组取同一天数据。
d，在试验过程中密切观察小鼠生活状态，包括外观体征、一般行为活动、精神状态、摄食情况、呼吸状态、粪便和尿液性状、注射局部及其它毒性表现。
e，试验达到终点后，将小鼠实施安乐死，动物尸体冻存至冰柜，移交至有资质的医疗废弃物处理单位进行处置。
4.实验结果
表7.实验数据
Figure PCTCN2022125057-appb-000027
a，平均值±标准误；
b，P值肿瘤体积进行统计分析，与G1组比较，*P﹤0.05；**P﹤0.01。
5.实验结论
从以上结果可以看出，在Osimertinib耐药人源肺癌PDX模型LD1-0025-200717中，化合物A给药剂量15、60mg/kg均能显著抑制肿瘤生长，且呈明显的量效关系(图7)。小鼠耐受性良好(图8)。
(八)L858R/C797S突变的体内药效研究
1.实验目的
评价化合物A连续14天口服给药，对Ba/F3 EGFR L858R/C797S的抗肿瘤活性及毒副作用。
2.实验材料
NU/NU小鼠，雌性，SPF级，购自北京维通利华实验动物技术有限公司。
Ba/F3 EGFR L858R/C797S细胞，购自康源博创生物科技(北京)有限公司。
3.实验步骤
3.1细胞培养
用含有10％的胎牛血清的RPMI1640培养基在37℃，5％二氧化碳培养箱中培养。细胞培养起始浓度5×10 6个，隔2-3天待细胞长满后分瓶传代。将处于对数生长期的肿瘤细胞用于体内肿瘤的接种。
3.2细胞接种
将Ba/F3 EGFR L858R/C797S细胞以2×10 6个/0.1mL接种于NU/NU小鼠的右侧腋窝皮下，接种当天设为第0天。
3.3肿瘤分组、给药及测量
a，当平均肿瘤体积约120mm 3时，按照肿瘤体积大小随机分为5组：G1：Vehicle、G2：Osimertinib(10mg/kg)、 G3：化合物A(15mg/kg)、G4：化合物A(30mg/kg)和G5：化合物A(65mg/kg)，7只/组。
b，动物分组后开始给药，给药体积均为10mL/kg，口服给药(po)；每天称重给药1次，连续给药14天；每周测量瘤径2次。
c，肿瘤体积(Tumor volume,TV)：每周测量2次肿瘤体积，以观察瘤块体积变化和生长速度。肿瘤体积V＝1/2×a×b 2，其中a、b分别表示肿瘤长径和短径。化合物对肿瘤组织的生长抑制作用采用肿瘤生长抑制率TGI(％)评价。TGI(％)＝[1-(某给药组的平均肿瘤体积-该给药组分组当天的平均肿瘤体积)/(阴性对照组的平均肿瘤体积-阴性对照组分组当天的平均肿瘤体积)]×100％。给药组和阴性对照组取同一天数据。
d，在试验过程中密切观察小鼠生活状态，包括外观体征、一般行为活动、精神状态、摄食情况、呼吸状态、粪便和尿液性状、注射局部及其它毒性表现。
e，试验达到终点后，将小鼠实施安乐死，动物尸体冻存至冰柜，移交至有资质的医疗废弃物处理单位进行处置。
4.实验结果
表8.实验数据
Figure PCTCN2022125057-appb-000028
a，平均值±标准误；
b，P值肿瘤体积进行统计分析，与G1组比较，*P﹤0.05；**P﹤0.01。
5.实验结论
从上数结果可以看出，化合物A给药剂量15、30、65mg/kg均能显著抑制肿瘤生长，且呈明显的量效关系，Osimertinib对L858R/C797S双突变模型几乎无抑制作用(图9)，证实其耐药性。小鼠耐受性良好(图10)。
(九)L858R/T790M突变的体内药效研究
1.实验目的
评价化合物A连续21天口服给药，对H1975(L858R/T790M)的抗肿瘤活性及毒副作用。
2.实验材料
BALB/c裸小鼠，雌性，SPF级，江苏集萃药康生物科技股份有限公司。
H1975细胞，购自ATCC。
3.实验步骤
3.1细胞培养
用含有RPMI1640培养基中加10％胎牛血清，1％双抗(青霉素/链霉素溶液)，37℃5％CO 2培养。一周两次0.25％胰酶常规离心处理传代。当细胞饱和度为80％-90％，数量到达要求时，收取细胞，计数，接种。
3.2细胞接种
将含有5×10 6个H1975细胞的PBS(终体积为100uL)皮下接种于每只小鼠的右前肢腋窝皮下，入组动物肿瘤平均体积达到127mm 3时开始分组给药。
3.3肿瘤分组、给药及测量
a，当平均肿瘤体积达到127mm 3时，按照肿瘤体积大小随机分为4组：G1：溶媒对照组、G2：化合物A(15mg/kg)、G3：化合物A(30mg/kg)和G4：化合物A(65mg/kg)，8只/组。分组当天为第0天。
b，动物分组后开始给药，给药体积均为10mL/kg，口服给药(po)；每天称重给药1次，连续给药21天；每周测量瘤径2次。
c，肿瘤体积(Tumor volume,TV)：每周测量2次肿瘤体积，以观察瘤块体积变化和生长速度。肿瘤体积V＝1/2×a×b 2，其中a、b分别表示肿瘤长径和短径。化合物对肿瘤组织的生长抑制作用采用肿瘤生长抑制率TGI(％)评价。TGI(％)＝[1-(某给药组的平均肿瘤体积-该给药组分组当天的平均肿瘤体积)/(阴性对照组的平均肿瘤体积-阴性对照组分组当天的平均肿瘤体积)]×100％。给药组和阴性对照组取同一天数据。
d，在试验过程中密切观察小鼠生活状态，包括外观体征、一般行为活动、精神状态、摄食情况、呼吸状态、粪 便和尿液性状、注射局部及其它毒性表现。
e，试验达到终点后，将小鼠实施安乐死，动物尸体冻存至冰柜，移交至有资质的医疗废弃物处理单位进行处置。
4.实验结果
表9.实验数据
Figure PCTCN2022125057-appb-000029
a，平均值±标准误；
b，P值肿瘤体积进行统计分析，与G1组比较，*P﹤0.05；**P﹤0.01。
5.实验结论
从上数结果可以看出，化合物A剂量15、30、65mg/kg均能显著抑制肿瘤生长，且化合物A呈一定的量效关系(图11)。小鼠耐受性良好(图12)。

Claims (17)

  1. 式(A)化合物或其可药用的盐在制备治疗EGFR突变介导的癌症的药物中的用途，
    Figure PCTCN2022125057-appb-100001
    其特征在于，所述的EGFR突变类型为Del19突变。
  2. 式(A)化合物或其可药用的盐在制备治疗EGFR突变介导的癌症的药物中的用途，
    Figure PCTCN2022125057-appb-100002
    其特征在于，所述的EGFR突变类型为L858R突变。
  3. 式(A)化合物或其可药用的盐在制备治疗EGFR突变介导的癌症的药物中的用途，
    Figure PCTCN2022125057-appb-100003
    其特征在于，所述的EGFR突变类型为不伴随C797S突变的T790M突变；
    优选地，所述的不伴随C797S突变的T790M突变选自以下的一种L858R/T790M双突变、Del19/G724S/T790M三突变、L858R/T790M/L792H三突变、E709K/T790M/L858R三突变。
  4. 式(A)化合物或其可药用的盐在制备治疗EGFR突变介导的癌症的药物中的用途，
    Figure PCTCN2022125057-appb-100004
    其特征在于，所述的EGFR突变类型为Del19/C797S双突变。
  5. 式(A)化合物或其可药用的盐在制备治疗EGFR突变介导的癌症的药物中的用途，
    Figure PCTCN2022125057-appb-100005
    其特征在于，所述的EGFR突变类型为L858R/C797S双突变。
  6. 式(A)化合物或其可药用的盐在制备治疗EGFR扩增介导的癌症的药物中的用途，
    Figure PCTCN2022125057-appb-100006
    优选地，所述的EGFR扩增为伴随着Del19/T790M/C797S三突变、L858R/T790M/D537H三突变或V674L/E746_A750del/T790M三突变的EGFR扩增。
  7. 式(A)化合物或其可药用的盐在制备治疗EGFR突变介导的癌症的药物中的用途，
    Figure PCTCN2022125057-appb-100007
    其特征在于，所述的EGFR突变类型为20外显子插入突变；
    优选地，所述的20外显子插入突变为伴随L747_P753突变的插入突变。
  8. 式(A)化合物或其可药用的盐在制备治疗EGFR突变或扩增介导的癌症的药物中的用途，
    Figure PCTCN2022125057-appb-100008
    其特征在于，所述的EGFR突变类型选自以下的一种或任意组合：Del19突变、L858R突变、L858R/T790M双突变、Del19/G724S/T790M三突变、L858R/T790M/L792H三突变、E709K/T790M/L858R三突变、Del19/C797S双突变、L858R/C797S双突变、20外显子突变；EGFR扩增可选自以下一种或任意组合：伴随着Del19/T790M/C797S三突变、L858R/T790M/D537H三突变或V674L/E746_A750del/T790M三突变的EGFR扩增。
  9. 如权利要求1-8任一项所述的用途，其特征在于，所述癌症为肺癌；
    优选地，所述癌症为非小细胞肺癌；
    优选地，所述癌症为未曾接受过治疗的非小细胞肺癌；
    优选地，所述癌症为既往接受过EGFR抑制剂治疗后，产生耐药的非小细胞肺癌。
  10. 如权利要求9所述的用途，其特征在于，所述的EGFR抑制剂包括第一代EGFR抑制剂、第二代EGFR抑制剂或第三代EGFR抑制剂；
    优选地，所述的第一代EGFR抑制剂包括吉非替尼、埃克替尼、厄洛替尼；
    优选地，所述的第二代EGFR抑制剂包括阿法替尼、达克替尼；
    优选地，所述的第三代EGFR抑制剂包括奥希替尼、阿美替尼、伏美替尼、贝福替尼。
  11. 式(A)化合物或其可药用的盐在制备治疗FGFR2高表达的癌症的药物中的用途，
    Figure PCTCN2022125057-appb-100009
  12. 式(A)化合物或其可药用的盐在制备治疗C-KIT突变的癌症的药物中的用途，
    Figure PCTCN2022125057-appb-100010
    其特征在于，所述的C-KIT突变类型为V560G突变和/或D816Y突变和/或D816H突变和/或V559和V560氨基酸缺失突变和/或D816V突变。
  13. 式(A)化合物或其可药用的盐在制备治疗EML4-ALK融合蛋白介导的癌症的药物中的用途，
    Figure PCTCN2022125057-appb-100011
  14. 式(A)化合物或其可药用的盐在制备治疗EML4-ALK融合蛋白L1196M突变和/或F1174L突变和/或L1196M/L1198F双突变介导的癌症的药物中的用途，
    Figure PCTCN2022125057-appb-100012
  15. 式(A)化合物或其可药用的盐在制备治疗SLC34A2-ROS1融合蛋白介导的癌症的药物中的用途，
    Figure PCTCN2022125057-appb-100013
  16. 式(A)化合物或其可药用的盐在制备治疗SLC34A2-ROS1融合蛋白D2033N突变介导的癌症的药物中的用途，
    Figure PCTCN2022125057-appb-100014
  17. 如权利要求1-16任一项所述的用途，其特征在于，(A)化合物的可药用盐为盐酸盐。
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