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WO2022033575A1 - 具有大环结构的含氟并杂环衍生物的应用 - Google Patents

具有大环结构的含氟并杂环衍生物的应用 Download PDF

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WO2022033575A1
WO2022033575A1 PCT/CN2021/112413 CN2021112413W WO2022033575A1 WO 2022033575 A1 WO2022033575 A1 WO 2022033575A1 CN 2021112413 W CN2021112413 W CN 2021112413W WO 2022033575 A1 WO2022033575 A1 WO 2022033575A1
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Prior art keywords
ret
gene
compound
cancer
pharmaceutically acceptable
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French (fr)
Inventor
李志勇
张晓东
张德伟
阳安乐
黄浩喜
何权鸿
陈垌珲
唐军
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Scinnohub Pharmaceutical Co Ltd
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Scinnohub Pharmaceutical Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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

Definitions

  • the present application relates to the application of fluoro-heterocyclic derivatives with macrocyclic structure in the treatment of diseases, in particular to the preparation of compounds of formula (I), stereoisomers or pharmaceutically acceptable salts thereof for the treatment of RET-related Use in medicines for diseases, application in the treatment of RET-related diseases.
  • the proto-oncogene RET (Rearranged during Transfection) is responsible for encoding the receptor tyrosine kinase RET protein.
  • the kinase is a transmembrane protein, consisting of an extracellular domain, a transmembrane domain and an intracellular tyrosine kinase active domain.
  • the physiological ligands of RET belong to the family of glial cell-derived neurotrophic factors (GDNFs).
  • RET RET activation is completed by the interaction between the four receptors of growth factor receptor ⁇ 1/2/3/4 (GFR ⁇ 1/2/3/4) and the four ligands of GDNFs, and GFR ⁇ can specifically bind to GDNFs Family members, promote the phosphorylation of RET protein receptors and make RET enter an activated state, thereby activating downstream signaling pathways related to cell proliferation, migration and differentiation, mainly including Ras/Raf/MEK/ERK-MAPK pathway and PI3K/Akt/ mTOR pathway and other pathways (PLC- ⁇ pathway, JAK-STAT pathway), etc. (Matti S. Airaksinen. Mol Cell Neurosci. 1999.13 (313-325)).
  • receptor tyrosine kinases play a maintenance role in normal organs and adult tissues, but when the RET gene is mutated, its receptor tyrosine kinase activity is abnormally activated, thereby driving tumorigenesis and maintaining Tumor proliferation and survival.
  • RET gene variants have been observed in a variety of tumors, and aberrant activation of RET is a key driver of tumor growth and proliferation in a large number of solid tumors.
  • About 1% to 2% of patients with non-small cell lung cancer, 65% of patients with medullary thyroid cancer, and 10% to 20% of patients with papillary thyroid cancer have RET mutations or fusions.
  • the overall average incidence of RET mutations is less than 1. %.
  • Oncogenic RET-positive variants accounted for approximately 71.6% of the total RET variants.
  • Common RET variants included mutation (38.6%), fusion (30.7%), amplification (25%), rearrangement (3.4%), copy number gain (1.1%) and copy number loss (1.1%) (Shumei Kato. Clin Cancer Res. 2018.23(8)).
  • RET inhibitors have been shown to significantly inhibit the proliferation of tumor cells with RET gene fusions and mutations.
  • RET inhibitors are mainly divided into two categories, one is targeted RET small molecule inhibitors, including LOXO-292 and BLU-667, which are the current research focus of RET inhibitors; the other is multi-kinase inhibitors, which Class inhibitors have RET inhibitory activity.
  • LOXO-292 Selpercatinib
  • BLU-667 Another RET inhibitor, BLU-667, currently has an NDA application with the FDA for the treatment of patients with locally advanced or metastatic RET fusion-positive non-small cell lung cancer.
  • RET small molecule inhibitors in preclinical development.
  • RET inhibitors can effectively inhibit the proliferation of malignant tumors with RET fusion or mutation in vivo, and have a high disease remission rate in clinical practice. They are effective therapeutic targets regardless of cancer type.
  • the application provides the use of a compound represented by formula (I), a stereoisomer thereof or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment of RET-related diseases,
  • the present application also provides a compound represented by formula (I), a stereoisomer thereof or a pharmaceutically acceptable salt thereof, which is used for the treatment of RET-related diseases.
  • the present application also provides a method for treating RET-related diseases, comprising administering an effective amount of the compound represented by formula (I), a stereoisomer thereof or a pharmaceutically acceptable salt thereof to a subject in need thereof.
  • the application also provides a composition for treating RET-related diseases, which comprises a compound represented by formula (I), a stereoisomer or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable carrier and/or excipients.
  • a composition for treating RET-related diseases which comprises a compound represented by formula (I), a stereoisomer or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable carrier and/or excipients.
  • the compound of formula (I), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof is present in an amount effective for treating RET-related diseases.
  • the application also provides a medicament for the treatment of RET-related diseases, which comprises a compound represented by formula (I), a stereoisomer thereof or a pharmaceutically acceptable salt thereof, optionally a pharmaceutically acceptable carrier and / or excipients.
  • a medicament for the treatment of RET-related diseases which comprises a compound represented by formula (I), a stereoisomer thereof or a pharmaceutically acceptable salt thereof, optionally a pharmaceutically acceptable carrier and / or excipients.
  • the compound of formula (I), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof is present in an amount effective for treating RET-related diseases.
  • the compound of formula (I) is a compound of formula (Ia).
  • the compound of formula (I) is a compound of formula (Ib).
  • the RET-related disease is a disease associated with dysregulation of the expression, activity or level of RET gene or RET kinase protein.
  • the RET-related disease is a disease associated with a mutation in a RET gene or a RET kinase protein.
  • the RET gene or RET kinase protein mutation comprises a mutation at one or more sites.
  • the RET-related disease is a RET fusion gene-related disease.
  • the RET fusion gene is selected from the group consisting of: BCR-RET, CLIP1-RET, KIF5B-RET, CCDC6-RET, NCOA4-RET, TRIM33-RET, ERC1-RET, FGFR1OP-RET, RET-MBD1 , RET-RAB6IP2, RET-PRKAR1A, RET-TRIM24, RET-GOLGA5, HOOK3-RET, KTN1-RET, TRIM27-RET, AKAP13-RET, FKBP15-RET, SPECC1L-RET, TBL1XR1-RET, CEP55-RET, CUX1 -RET, KIAA1468-RET, RFG8-RET, ACBD5-RET, PTC1ex9-RET, MYH13-RET, PIBF1-RET, KIAA1217-RET, MPRIP-RET, HRH4-RET, Ria-RET, RET-PTC4, FRMD4A-RET, MYH13
  • the RET fusion gene is selected from the group consisting of: RET-CCDC6 (PTC1), RET-KIF5B (Kex15Rex14), RET-PRKAR1A (PTC2), RET-BCR, RET-NCOA4 (PTC3), and any combination thereof .
  • the RET fusion gene is selected from the group consisting of: RET(V804L)-KIF5B, RET(V804M)-KIF5B, and any combination thereof.
  • the RET fusion gene is RET(V804M)-KIF5B.
  • the RET fusion gene is RET(V804L)-KIF5B.
  • the RET fusion gene is RET-NCOA4 (PTC3).
  • the RET fusion gene is RET-CCDC6 (PTC1).
  • the RET fusion gene is RET-KIF5B (Kex15Rex14).
  • the RET fusion gene is RET-PRKAR1A (PTC2).
  • the RET gene mutation is selected from the group consisting of: RET(Y791F), RET(V778I), RET(G691S), RET(V804L), RET(R813Q), RET(E762Q), RET(V804E), RET(V804L)-KIF5B, RET(A883F), RET(S904F), RET(V804M), RET(V804M)-KIF5B, RET(Y806H), RET(M918T) and any combination thereof.
  • the RET gene is mutated to RET(S891A).
  • the RET gene is mutated to RET(L790F).
  • the RET gene is mutated to RET(R749T).
  • the RET gene is mutated to RET (S904A).
  • the RET gene is mutated to RET(R912P).
  • the RET gene is mutated to RET (Y791F).
  • the RET gene is mutated to RET(V778I).
  • the RET gene is mutated to RET(G691S).
  • the RET gene is mutated to RET(V804L).
  • the RET gene is mutated to RET(R813Q).
  • the RET gene is mutated to RET(E762Q).
  • the RET gene is mutated to RET(V804E).
  • the RET gene is mutated to RET(A883F).
  • the RET gene is mutated to RET(S904F).
  • the RET gene is mutated to RET(Y806H).
  • the RET gene is mutated to RET (M918T).
  • the RET-related disease is a cancer associated with dysregulation of RET gene or RET kinase protein expression, activity, or level.
  • the RET-related disease is a cancer associated with a mutation in a RET gene or a RET kinase protein.
  • the RET gene or RET kinase protein mutation comprises a mutation at one or more sites.
  • the RET mutation-related cancer is selected from the group consisting of: RET mutation-related lung cancer, papillary thyroid cancer, medullary thyroid cancer, differentiated thyroid cancer, recurrent thyroid cancer, refractory differentiated type thyroid cancer, multiple endocrine neoplasia type 2A or 2B (MEN2A or MEN2B, respectively), pheochromocytoma, parathyroid hyperplasia, breast cancer, colon cancer, colorectal cancer, papillary renal cell carcinoma, gastrointestinal mucosa One or more of gangliomas and cervical cancer.
  • RET mutation-related lung cancer papillary thyroid cancer
  • medullary thyroid cancer differentiated thyroid cancer
  • differentiated thyroid cancer differentiated thyroid cancer
  • recurrent thyroid cancer recurrent thyroid cancer
  • refractory differentiated type thyroid cancer multiple endocrine neoplasia type 2A or 2B (MEN2A or MEN2B, respectively)
  • pheochromocytoma parathyroid hyperplasia
  • the RET-related disease is RET gene mutation-related lung cancer.
  • the RET-related disease is medullary thyroid cancer associated with a RET gene mutation.
  • the RET-related disease is RET gene mutation-related colon cancer.
  • the RET-related disease is selected from RET gene mutation-related small cell lung cancer, RET gene mutation-related non-small cell lung cancer, RET gene mutation-related bronchiopulmonary cell carcinoma, or RET gene mutation-related One or more of lung adenocarcinomas.
  • the RET-related disease is RET gene mutation-related small cell lung cancer.
  • the RET-related disease is RET gene mutation-related non-small cell lung cancer.
  • the RET-related disease is RET gene mutation-related bronchiolopulmonary cell carcinoma.
  • the RET-related disease is a RET gene mutation-related lung adenocarcinoma.
  • the RET-related disease is a RET fusion gene-related cancer.
  • the RET fusion gene-related cancer is selected from the group consisting of: RET fusion gene-related lung cancer, papillary thyroid cancer, medullary thyroid cancer, differentiated thyroid cancer, recurrent thyroid cancer, refractory differentiated type thyroid cancer, multiple endocrine neoplasia type 2A or 2B (MEN2A or MEN2B, respectively), pheochromocytoma, parathyroid hyperplasia, breast cancer, colon cancer, colorectal cancer, papillary renal cell carcinoma, gastrointestinal mucosa One or more of gangliomas and cervical cancer.
  • RET fusion gene-related lung cancer papillary thyroid cancer, medullary thyroid cancer, differentiated thyroid cancer, recurrent thyroid cancer, refractory differentiated type thyroid cancer, multiple endocrine neoplasia type 2A or 2B (MEN2A or MEN2B, respectively), pheochromocytoma, parathyroid hyperplasia, breast cancer, colon cancer, colorectal cancer, pa
  • the RET-related disease is a RET fusion gene-related lung cancer.
  • the RET-related disease is RET fusion gene-related medullary thyroid cancer.
  • the RET-related disease is RET fusion gene-related colon cancer.
  • the RET-related disease is RET-CCDC6-related colon cancer.
  • the RET-related disease is selected from RET fusion gene-related small cell lung cancer, RET fusion gene-related non-small cell lung cancer, RET fusion gene-related bronchiopulmonary cell carcinoma, or RET fusion gene-related One or more of lung adenocarcinomas.
  • the RET-related disease is RET fusion gene-related small cell lung cancer.
  • the RET-related disease is RET fusion gene-related non-small cell lung cancer.
  • the RET-related disease is RET fusion gene-related bronchiolopulmonary cell carcinoma.
  • the RET-related disease is a RET fusion gene-related lung adenocarcinoma.
  • the RET-related disease is a human RET-related disease
  • the administration dose of the compound represented by formula (I), its stereoisomer or a pharmaceutically acceptable salt thereof is 0.5-4 mg/kg /day.
  • stereoisomer refers to the isomers produced by the different arrangements of atoms in the molecule in space, which can be divided into cis-trans isomers and enantiomers. It can also be divided into two categories: enantiomers and diastereomers. Isomers in which atoms or groups of atoms in a molecule are connected to each other in the same order but in different spatial arrangements are called stereoisomers, and there are two types of stereoisomers. Stereoisomers due to bond lengths, bond angles, double bonds in the molecule, rings, etc. are called configuration stereo-isomers. In general, configurational isomers cannot or are difficult to convert to each other.
  • Stereoisomers caused only by the rotation of a single bond are called conformational stereo-isomers, and are sometimes called rotamers.
  • Configuration isomers are divided into two categories. Among them, isomers caused by double bonds or single bonds forming ring carbon atoms cannot rotate freely are called geometric isomers, also known as cis-trans isomers.
  • Stereoisomers with different optical properties due to the absence of anti-axial symmetry are called optical isomers.
  • RET Rearranged during Transfection gene
  • RET proto-oncogene located on the long arm of autosomal chromosome 10 (10q11.2), full-length 53kb, including 20 penetrants child, encoding a tyrosine kinase receptor of 1114 amino acids.
  • RET kinase used in this application is a receptor tyrosine kinase, encoded by the RET gene, which is involved in signal transduction in processes such as cell proliferation, migration, differentiation, and survival of neural crest cells, formation of kidney organs, and spermatogenesis. guide related.
  • RET fusion gene refers to the rearrangement of the RET gene with other gene sequences, resulting in fusion after protein expression, resulting in abnormal expression or activity of RET at the gene level or protein level.
  • RET fusion gene is represented by "A-B” or “B-A”, wherein A and B respectively represent the RET gene and other genes fused with the RET gene.
  • A-B” or “B-A” have the same meaning and represent a fusion gene of A gene and B gene.
  • RET-CCDC6 and CCDC6-RET have the same meaning, and they both mean the fusion gene of RET and CCDC6.
  • RET gene mutation refers to a point mutation caused by a base change in the RET gene, or a deletion, duplication or insertion of one or more bases, resulting in corresponding changes in the protein.
  • pharmaceutically acceptable salts includes conventional salts formed with pharmaceutically acceptable inorganic or organic acids, or inorganic or organic bases. Methods for preparing pharmaceutically acceptable salts of the compounds of the present application are known to those skilled in the art.
  • the term "pharmaceutical composition” includes a product comprising a therapeutically effective amount of a compound of the present application, as well as any product that results, directly or indirectly, from a combination of the compounds of the present application.
  • the pharmaceutical composition can be administered, for example, orally or parenterally.
  • the pharmaceutical composition of the present application can be prepared into various dosage forms according to conventional methods in the art, including but not limited to tablets, capsules, solutions, suspensions, granules or injections, etc., and can be administered orally or parenterally.
  • the term "effective amount” refers to an amount sufficient to achieve the desired therapeutic effect, eg, an amount to achieve relief of symptoms associated with the disease to be treated.
  • treatment is intended to alleviate or eliminate the disease state or disorder targeted. If a subject receives a therapeutically effective amount of a compound, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof according to the methods described herein, the subject has one or more of the signs and symptoms A subject has been successfully "treated” if it exhibits an observable and/or detectable reduction or improvement. It should also be understood that the treatment of the disease state or disorder includes not only complete treatment, but also incomplete treatment, but the achievement of some biologically or medically relevant result.
  • the dosage and method of use of the compounds of the present application depend on many factors, including the patient's age, weight, sex, natural health, nutritional status, the active strength of the compound, the time of administration, the rate of metabolism, the severity of the disorder and Subjective judgment of the treating physician.
  • the preferred dosage used is between 0.001 and 1000 mg/kg body weight/day.
  • Figure 1 shows Growth curve of the average tumor volume of mice in each group in colon cancer CR2518 model
  • Figure 2 shows Body weight change curve of mice in each group with treatment time in colon cancer CR2518 model.
  • HATU stands for 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethylurea hexafluorophosphate
  • Cs 2 CO 3 represents cesium carbonate
  • THF stands for tetrahydrofuran
  • POCl 3 represents phosphorus oxychloride
  • DMF stands for N,N-dimethylformamide
  • NaOH sodium hydroxide
  • DIPEA or DIEA means N,N-diisopropylethylamine
  • H 2 O represents water
  • HCl/Dioxane means hydrogen chloride dioxane solution
  • N,N-Diethylaniline means N,N-diethylaniline
  • CH 3 CN or ACN means acetonitrile
  • Zn means zinc powder
  • NH 4 Cl means ammonium chloride
  • CH 3 MgBr represents methyl magnesium bromide
  • K 2 CO 3 represents potassium carbonate
  • OTs represents p-methylbenzenesulfonyloxy
  • RET (S891A) means that the serine at position 891 of the RET gene is mutated to alanine
  • RET (L790F) means that the leucine at position 790 of the RET gene is mutated to phenylalanine
  • RET V804M means that the valine at position 804 of the RET gene is mutated to methionine
  • RET means that the arginine at position 749 of the RET gene is mutated to threonine
  • RET (S904A) means that the serine at position 904 of the RET gene is mutated to alanine
  • RET(R912P) means that the arginine at position 912 of the RET gene is mutated to proline
  • RET (Y791F) means that the tyrosine at position 791 of the RET gene is mutated to phenylalanine
  • RET V778I
  • V778I valine at position 778 of the RET gene is mutated to isoleucine
  • RET (G691S) means that the glycine at position 691 of the RET gene is mutated to serine;
  • RET (V804L) means that the valine at position 804 of the RET gene is mutated to leucine
  • RET(R813Q) means that the arginine at position 813 of the RET gene is mutated to glutamine
  • RET (E762Q) means that the glutamic acid at position 762 of the RET gene is mutated to glutamine;
  • RET V804E means that the valine at position 804 of the RET gene is mutated to glutamic acid
  • RET (A883F) means that the alanine at position 883 of the RET gene is mutated to phenylalanine
  • RET (S904F) means that the serine at position 904 of the RET gene is mutated to phenylalanine
  • RET (Y806H) means that the tyrosine at position 806 of the RET gene is mutated to histidine;
  • RET represents the mutation of methionine at position 918 of the RET gene to threonine.
  • RET-CCDC6 is a fusion gene of RET and CCDC6 (alias PTC1, coiled-coil domain containing 6, coiled-coil domain protein 6, Gene ID (NCBI): 8030);
  • RET-KIF5B (Kex15Rex14) is a fusion gene of RET and KIF5B (the kinesin family 5B gene, kinesin family 5B, Gene ID (NCBI): 3799), and the fusion position starts at exon 15 of KIF5B gene (amino acids 1-575). ), ends at exons 14-21 (amino acid 713) of RET;
  • RET-PRKAR1A is a fusion gene of RET and PRKAR1A (alias PTC2, protein kinase cAMP-dependent type I regulatory subunit alpha, protein kinase c-AMP-dependent I regulatory subunit alpha, Gene ID (NCBI): 5573);
  • RET(V804L)-KIF5B is a fusion gene of RET(V804L) and KIF5B (the kinesin family 5B gene, kinesin family 5B, Gene ID (NCBI): 3799);
  • RET-BCR RET and BCR (alias BCR1; BCR activator of RhoGEF and GTPase, RhoGEF and GTO enzyme BCR activator, Gene ID (NCBI): 613) fusion gene;
  • RET(V804M)-KIF5B is a fusion gene of RET(V804M) and KIF5B (the kinesin family 5B gene, kinesin family 5B, Gene ID (NCBI): 3799);
  • RET-NCOA4 is a fusion gene of RET and NCOA4 (alias PTC3, nuclear receptor coactivator 4, Gene ID (NCBI): 8031);
  • CLIP1-RET is a fusion gene of RET and CLIP1 (CAP-Gly domain containing linker protein 1, Gene ID (NCBI): 6249);
  • CCDC6-RET is a fusion gene of RET and CCDC6 (coiled-coil domain containing 6, coiled-coil domain protein 6, Gene ID (NCBI): 8030);
  • TRIM33-RET is a fusion gene of RET and TRIM33 (tripartite motif containing 33, tripartite motif protein 33, Gene ID (NCBI): 51592);
  • ERC1-RET is a fusion gene of RET and ERC1 (ELKS/RAB6-interacting/CAST family member 1, ELKS/Rab6-interacting/CAST protein family member 1, Gene ID (NCBI): 23085);
  • FGFR1OP-RET is a fusion gene of RET and FGFR1OP (FGFR1 oncogene partner, FGFR1 oncogene partner, Gene ID (NCBI): 11116);
  • RET-MBD1 is a fusion gene of RET and MBD1 (methyl-CpG binding domain protein 1, methylphosphocytidylguanosine binding protein, Gene ID (NCBI): 4152);
  • RET-RAB6IP2 is a fusion gene of RET and RAB6IP2 (Rab6Interacting Protein 2A, alias ERC-1, Gene ID (NCBI): 23085);
  • RET-TRIM24 is a fusion gene of RET and TRIM24 (tripartite motif containing 24, tripartite motif protein 24, Gene ID (NCBI): 8805);
  • RET-GOLGA5 is a fusion gene of RET and GOLGA5 (golgin A5, Golgi protein 5, Gene ID (NCBI): 9950);
  • HOOK3-RET is a fusion gene of RET and HOOK3 (hook microtubule tethering protein 3, hook microtubule tethering protein 3, Gene ID (NCBI): 84376);
  • KTN1-RET is the fusion gene of RET and KTN1 (kinectin 1, kinesin 1, Gene ID (NCBI): 3895);
  • TRIM27-RET is a fusion gene of RET and TRIM27 (tripartite motif containing 27, motif protein 27, Gene ID (NCBI): 5987);
  • AKAP13-RET is a fusion gene of RET and AKAP13 (A-kinase anchoring protein 13, Gene ID (NCBI): 11214);
  • FKBP15-RET is a fusion gene of RET and FKBP15 (FKBP prolyl isomerase family member 15, Gene ID (NCBI): 23307);
  • SPECC1L-RET is a fusion gene of RET and SPECC1L (sperm antigen with calponin homology and coiled-coil domains 1like, calmodulin homology and coiled domain 1-like kd glycoprotein, Gene ID (NCBI): 23384);
  • TBL1XR1-RET is a fusion gene of RET and TBL1XR1 (TBL1X receptor 1, TBL1X receptor 1, Gene ID (NCBI): 79718);
  • CEP55-RET is the fusion gene of RET and CEP55 (centrosomal protein 55, centrosomal protein 55, Gene ID (NCBI): 55165);
  • CUX1-RET is a fusion gene of RET and CUX1 (cut like homeobox 1, homeobox cleavage protein 1, Gene ID (NCBI): 1523);
  • KIAA1468-RET is the fusion gene of RET and KIAA1468 (alias RAB11binding and LisH domain, coiled-coil and HEAT repeat containing, Gene ID(NCBI):57614)
  • RFG8-RET is the fusion gene of RET and RFG8 (RET-fused gene 8, RET fusion gene 8);
  • ACBD5-RET is a fusion gene of RET and ACBD5 (acyl-CoA binding domain containing 5, acyl-CoA binding domain protein 5, Gene ID (NCBI): 91452);
  • PTC1ex9-RET is a variant of PTC-RET fusion, which is the penetrance of RET extracellular domain exon 9 and CCDC6 (coiled-coil domain containing 6, coiled-coil domain 6, Gene ID (NCBI): 8030) The fusion gene of son 1;
  • MYH13-RET is a fusion gene of RET and MYH13 (myosin heavy chain 13, myosin heavy chain 13, Gene ID (NCBI): 8735);
  • PIBF1-RET is a fusion gene of RET and PIBF1 (progesterone immunomodulatory binding factor 1, progesterone immunomodulatory binding factor 1, Gene ID (NCBI): 10464);
  • KIAA1217-RET is the fusion gene of RET and KIAA1217 (Gene ID (NCBI): 56243);
  • MPRIP-RET is a fusion gene of RET and MPRIP (myosin phosphatase Rho interacting protein, myosin phosphatase-Rho interacting protein, Gene ID (NCBI): 23164);
  • HRH4-RET is the fusion gene of RET and HRH4 (histamine receptor H4, histamine H4 receptor, Gene ID (NCBI): 59340);
  • Ria-RET is a fusion gene of RET and RIA (the RIA regulatory subunit of the c-AMP dependent protein kinase A, the regulatory subunit RIA of protein kinase A,);
  • RET-PTC4 is a fusion gene of RET and PTC4 (type 2C protein phosphatase, type 2C protein phosphatase, Gene ID (NCBI): 5108);
  • FRMD4A-RET is a fusion gene of RET and FRMD4A (FERM domain containing 4A, FERM domain protein 4A, Gene ID (NCBI): 55691);
  • SQSTM1-RET is a fusion gene of RET and SQSTM1 (sequestosome 1, autophagy adaptor protein 1, Gene ID (NCBI): 8878);
  • AFAP1L2-RET is a fusion gene of RET and AFAP1L2 (actin filament associated protein 1like 2, Gene ID (NCBI): 84632);
  • PPFIBP2-RET is a fusion gene of RET and PPFIBP2 (PPFIA binding protein 2, PPFIA binding protein 2, Gene ID (NCBI): 8495);
  • EML4-RET is the fusion gene of RET and EML4 (EMAP like 4, EMAP like protein 4, Gene ID (NCBI): 27436);
  • PARD3-RET is a fusion gene of RET and PARD3 (par-3 family cell polarity regulator, Par-3 family cell polarity regulator, Gene ID (NCBI): 56288);
  • MYH10-RET is a fusion gene of RET and MYH10 (myosin heavy chain 10, myosin heavy chain 10, Gene ID (NCBI): 4628);
  • HTIF1-RET is the fusion gene of RET and HTIF1 (alias tripartite motif containing 24, triple motif protein 24, Gene ID (NCBI): 8805);
  • AFAP1-RET is a fusion gene of RET and AFAP1 (actin filament associated protein 1, actin filament associated protein 1, Gene ID (NCBI): 60312);
  • RASGEF1A-RET is a fusion gene of RET and RASGEF1A (RasGEF domain family member 1A, RasGEF domain family member 1A, Gene ID (NCBI): 221002);
  • TEL-RET is the fusion gene of RET and TEL (alias EVT6, ETS variant transcription factor 6Telomere elongation, (NCBI): 2120);
  • RUFY1-RET is a fusion gene of RET and RUFY1 (RUN and FYVE domain containing 1, RUN and FYVE domain containing protein 1, Gene ID (NCBI): 80230);
  • UEVLD-RET is a fusion gene of RET and UEVLD (UEV and lactate/malate dehyrogenase domains, UEV and lactate/malate dehydrogenase domains, Gene ID (NCBI): 55293);
  • DLG5-RET is a fusion gene of RET and DLG5 (discs large MAGUK scaffold protein 5, MAGUK scaffold protein 5, Gene ID (NCBI): 9231);
  • FOXP4-RET is a fusion gene of RET and FOXP4 (forkhead box P4, forkhead box protein P4, Gene ID (NCBI): 116113);
  • OLFM4-RET is the fusion gene of RET and OLFM4 (Olfactomedin-4, olfactory protein 4, Gene ID (NCBI): 418826);
  • RRBP1-RET is a fusion gene of RET and RRBP1 (ribosome binding protein 1, ribosome binding protein 1 Gene ID (NCBI): 6238).
  • Step 2 Preparation of ethyl 5,7-dichloro-6-fluoropyrazolo[1,5-a]pyrimidine-3-carboxylate
  • reaction solution was concentrated to remove phosphorus oxychloride, then poured into saturated sodium bicarbonate solution (100 mL) to keep the solution basic, extracted with ethyl acetate three times, dried over anhydrous sodium sulfate, filtered and concentrated, and the obtained crude product was filtered through a column. After purification by chromatography, the obtained solid was washed with petroleum ether and dried to obtain 1.7 g of the title compound.
  • Step 3 Preparation of ethyl 5-chloro-6-fluoropyrazolo[1,5-a]pyrimidine-3-carboxylate
  • Step 1 Preparation of (R)-N-(((5-fluoro-2-methoxypyridin-3-yl)methylene)-2-methylpropane-2-sulfinamide
  • Step 2 Preparation of (R)-N-((R)-1-(5-fluoro-2-methoxypyridin-3-yl)ethyl)-2-methylpropane-2-sulfinamide
  • Example 1 ( 31S ,33S, 63E ,64E,8R) -15,66 - difluoro- 8 -methyl-2-oxa-4,7-diazepine -6(3,5)-Pyrazolo[1,5-a]pyrimidine-1(2,3)-pyridine 3-3,(1,3)-cyclobutan-5-one (Compound Ib ) preparation
  • Step 1 (R)-6-Fluoro-5-((1-(5-Fluoro-2-methoxypyridin-3-yl)ethyl)amino)pyrazolo[1,5-a]pyrimidine- Preparation of ethyl 3-carboxylate
  • reaction solution was poured into water (50 mL), extracted with dichloromethane, the organic phases were combined, backwashed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered and concentrated.
  • the obtained crude product was purified by column chromatography to obtain 1.1 g of the title compound.
  • Step 2 (R)-6-Fluoro-5-(((1-(5-fluoro-2-methoxypyridin-3-yl)ethyl)amino)pyrazolo[1,5-a]pyrimidine - Preparation of 3-carboxylic acid
  • reaction solution was concentrated to remove ethanol, the residue was poured into water (20 mL), the pH was adjusted to about 5 with hydrogen chloride solution (2M), extracted with dichloromethane, the organic phases were combined, backwashed with saturated sodium chloride solution, anhydrous sodium sulfate Dry, filter, and concentrate to obtain 800 mg of crude product, which can be used directly in the next step without purification.
  • Step 3 (1R,3r)-3-(6-Fluoro-5-(((R)-1-(5-fluoro-2-methoxypyridin-3-yl)ethyl)amino)pyrazolo
  • reaction solution was poured into water (30 mL), extracted with ethyl acetate, the organic phases were combined, backwashed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated. The obtained crude product was filtered through the column layer. 800 mg of the title compound was obtained by analytical purification.
  • Step 4 (1R,3r)-3-(6-Fluoro-5-(((R)-1-(5-fluoro-2-hydroxypyridin-3-yl)ethyl)amino)pyrazolo[1 Preparation of ,5-a]pyrimidine-3-carboxamido)-4-methylbenzenesulfonate cyclobutyl hydrochloride
  • Step 5 ( 31S ,33S, 63E ,64E,8R) -15,66 - difluoro- 8 -methyl-2-oxa-4,7-diaza- Preparation of 6(3,5)-pyrazolo[1,5-a]pyrimidine-1(2,3)-pyridine 3-3(1,3)-cyclobutaneoctatan-5-one
  • reaction solution was poured into water (10 mL), extracted with ethyl acetate, the organic phases were combined, backwashed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered and concentrated.
  • the obtained crude product was purified by preparative thin layer chromatography and preparative high performance liquid chromatography to obtain 58.0 mg of the title compound, ee value>99.5%.
  • Test Examples 1-7 of this application include:
  • LOXO-195 a second-generation TRK inhibitor, has the following chemical structure:
  • Positive control 1 is the compound of Example 5 of patent WO2019210835, and its chemical structure is:
  • Positive control 2 is the compound of Example 6 of patent WO2019210835, and its chemical structure is: Compound 1, the structural formula is:
  • LOXO-195 and control compounds 1-2 are commercially available, and can also be synthesized and prepared according to conventional routes.
  • LOXO-195 is commercially available, and positive controls 1 and 2 can be synthesized according to the method described in WO2019210835.
  • Compounds 1, 2 and 4-6 can be synthesized and prepared according to conventional routes, and can also be synthesized with reference to the method described in WO2021115401A1.
  • Test Example 1 Inhibitory effect of Compound Ib prepared in Example 1 on RET kinase at a dose of 1 ⁇ M.
  • Buffer preparation 20 mM HEPES (pH 7.5), 10 mM MgCl 2 , 1 mM EGTA, 0.01% Brij35 0.02 mg/mL
  • test substance was used in a single-dose experiment (2 duplicate wells) at a concentration of 1 ⁇ M, and the initial concentration of the reference substance Staurosporine (Med Chem, MC-2104) was 20 ⁇ M or 100 ⁇ M, and serially diluted at a ratio of 1:4 to obtain 10 reaction concentrations.
  • the experiment will determine the single-concentration inhibition rate of the test substance and the IC 50 value of the control substance.
  • kinase activity was expressed as the percentage of kinase activity after reaction of the corresponding kinase with Compound Ib with vehicle group (DMSO). Inhibition efficiency is expressed as 1-% kinase activity. The results are shown in Table 1.
  • Test Example 2 Compound Ib in In vivo pharmacodynamic study of colon cancer CR2518 subcutaneous xenograft model in BALB/c nude mouse model
  • Test animals BALB/c Nude mice, female, 6-8 weeks (the age of mice at the time of tumor cell inoculation), body weight 21-25g, 25 mice, purchased from Beijing Ankai Yibo Biotechnology Co., Ltd., animal Certificate number: 110330201100091758. Breeding environment: SPF grade.
  • CR2518 was established from a colon tumor in a female patient Xenograft model. This model is a CCDC6-RET fusion mutation, which is prone to tumor rupture and may cause a slight weight loss in animals.
  • Animal vaccination and grouping from Colon cancer xenograft model CR2518 (R14P5) tumor-bearing mice were harvested with tumor tissue, cut into 2-3mm diameter tumor pieces and inoculated subcutaneously at the right anterior scapula of BALB/c nude mice. Group administration was started when the average tumor volume reached 151 mm 3 .
  • the test is divided into 4 groups, the first group is the vehicle control group, and the second group is the test drug low-dose group (the test drug is compound Ib, the single dose is 10 mg/kg, QD*14 days, and then BID*21 days ), the 3rd group is the test drug high-dose group (the test drug is compound Ib, the single dose is 20mg/kg, QD*14 days, then BID*21 days), the 4th group is the positive control group (positive control The drug was BLU-667, the single dose was 10 mg/kg, BID*34 days) group, 5 animals in each group, oral gavage administration, and the experiment ended on the 35th day after administration.
  • TGI tumor growth inhibition rate
  • TGI(%) [1-(average tumor volume at the end of administration of a certain treatment group-average tumor volume at the beginning of administration of this treatment group)/(average tumor volume at the end of treatment in the solvent control group- The average tumor volume at the start of treatment in the solvent control group)] ⁇ 100%.
  • T/C (%) T RTV /C RTV ⁇ 100% (T RTV : RTV in the treatment group; C RTV : RTV in the vehicle control group).
  • T weight and C weight represent the tumor weight of the administration group and the vehicle control group, respectively.
  • the results of the analysis included the mean and standard error (SEM) of tumor volume at each time point for each group.
  • the treatment group exhibited a significant tumor-suppressing effect on the 35th day after administration at the end of the trial, so statistical analysis was performed based on this data to evaluate the differences between groups.
  • the Bartlett test was first used to test the assumption of homogeneity of variance between all groups. When the p-value of Bartlett's test is not less than 0.05, one-way ANOVA will be used to test that all group means are equal. If the p-value for one-way ANOVA was less than 0.05, we used Tukey's HSD test for pairwise comparisons between all groups, or Dunnett's t-test for pairwise comparisons between each treatment and control group.
  • the Kruskal Wallis test When the p-value for Bartlett's test is less than 0.05, the Kruskal Wallis test will be used to test that the medians of all groups are equal. If the p-value of the Kruskal Wallis test is less than 0.05, we will use the Conover test for pairwise comparisons between all groups or between each treatment group and the control group with the corresponding p-value based on the number of groups in the multiple test Correction. All statistical analysis and graphing were performed in the R language environment (version 3.3.1). All tests were two-tailed and p-values less than 0.05 were considered statistically significant.
  • the test drug compound Ib had no animal death at a dose of 10 mg/kg, no obvious drug toxicity, and was well tolerated during treatment.
  • Compound Ib was administered once a day at a dose of 20 mg/kg for 14 consecutive days, and the mice did not experience weight loss and did not exhibit obvious drug toxicity. Subsequently, the compound Ib was adjusted to be administered twice a day at a dose of 20 mg/kg for 21 consecutive days. The weight of the mice decreased, and some animals died, showing certain drug toxicity. The test drug Compound Ib was effective at the doses used. Colon cancer CR2518 showed a significant inhibitory effect on tumor growth. Tumor-bearing mice tolerated low doses of Compound Ib well. High dose compound Ib has certain drug toxicity in tumor-bearing mice.
  • the doses of mice can be calculated, as shown in the following table.
  • test drug compound Ib and the positive control drug BLU-667 were significantly different at the doses used.
  • Colon cancer CR2518 has a significant inhibitory effect on tumor growth.
  • Compound Ib and BLU-667 were well tolerated by tumor-bearing mice at the tested and low doses.
  • TRKa The inhibitory activities of the compounds of the present application on three kinases TRKa, TRKA (G595R) and TRKC (G623R) were determined.
  • Test compounds were accurately weighed and dissolved in 100% DMSO to make up a 10 mM solution.
  • Inhibition % (Inhibition) (Conversion % Maximum - Conversion % Sample) / (Conversion % Maximum - Conversion % Minimum) ⁇ 100
  • conversion rate % sample is the conversion rate reading of the sample
  • conversion rate % minimum the mean value of negative control wells, representing the conversion rate reading of wells without enzymatic activity
  • conversion rate % maximum the average value of the ratio of positive control wells, representing the wells without compound inhibition Conversion rate readings.
  • Fitting the dose-response curve take the log value of the concentration as the X-axis, and the percentage inhibition rate on the Y-axis, using the analysis software GraphPad Prism 5 log (inhibitor) vs. response-variable slope (Variable slope) to fit the dose-response curve (Four-parameter model fitting) to derive the IC50 value of each compound for enzymatic activity.
  • Fetal bovine serum FBS (GBICO, Cat#10099-141); Luminescent Cell Viability Assay (CTG, Promega, Cat#G7573); 96-well clear flat bottom black wall plate ( Cat#165305); RPMI-1640 (Hyclone, Cat#SH30809.01)
  • the compounds to be tested were prepared with a 10-fold drug solution in a medium containing 1% DMSO, and the highest concentration was 10 ⁇ M, and were sequentially diluted 3-fold to obtain a total of 9 concentrations of drug solutions.
  • the final concentration of DMSO in the wells was 0.1%, and three replicate wells were set for each concentration of the drug.
  • Cells in medicated 96-well plates were incubated at 37° C., 5% CO 2 , and 95% humidity for an additional 72 hours before CTG analysis was performed.
  • Cell survival rate (%) (luminescence value of drug well to be tested - luminescence value of culture solution control well)/(luminescence value of cell control well - luminescence value of culture solution control well) ⁇ 100%.
  • Step 3.2 To each incubation system described in the aforementioned “Step 3.2", start the reaction by adding 2.5 ⁇ L of the positive control compound or the solution of the test compound of the present application (100 ⁇ M), the positive control is verapamil (purchased from Sigma). ), so that the final concentration of the test compound of the present application or the positive control compound is 1 ⁇ M. Incubation solutions after compound addition were batch-incubated in water at 37°C.
  • Peak areas were determined from extracted ion chromatograms.
  • the slope value k was determined by linear regression of the residual percentage of parent drug versus the natural logarithm of the incubation time curve.
  • the in vitro half-life (t 1/2 ) was calculated and determined according to the slope value, and the in vitro half-life average value was converted into the in vitro intrinsic clearance (CLint, expressed in ⁇ L/min/mg protein).
  • the stability of compounds 1 and 2 in human, rat and mouse liver microsomes was not significantly different from that of the corresponding positive compounds, or the stability in most species of liver microsomes became worse, but the compound Ib of the present application was in The stability in human, rat and mouse liver microsomes was significantly better than that of the positive control 2.
  • Test Example 6 In vivo pharmacokinetic study of test compounds administered intravenously and orally to SD rats
  • Species SD rat, SPF grade.
  • Source Animals were transferred from the experimental institution animal bank (999M-017), Shanghai Sipple-Bikai Laboratory Animal Co., Ltd. Quantity: 3 for each dosage form.
  • test product 2.1 Accurately weigh an appropriate amount of the test product, add the final volume of 5% DMSO, 10% polyethylene glycol-15 hydroxystearate, 85% normal saline, vortex or sonicate to fully mix to obtain 0.2 mg/mL dosing solution for intravenous administration.
  • test product 2.2 Accurately weigh an appropriate amount of the test product, add the final volume of 5% DMSO, 10% polyethylene glycol-15 hydroxystearate, 85% normal saline, vortex or sonicate to fully mix to obtain 0.5 mg/mL dosing solution for oral gavage administration.
  • Blood is collected through the jugular vein or other suitable methods, each sample is collected about 0.20mL, heparin sodium is anticoagulated, the blood samples are placed on ice after collection, and centrifuged within 2 hours to separate the plasma (centrifugation conditions: centrifugal force 6800g, 6 minutes, 2 -8°C).
  • the collected plasma samples were stored in a -80°C refrigerator before analysis, and the remaining plasma samples were kept in a -80°C refrigerator for temporary storage after analysis.
  • the BLQ was recorded as 0.
  • concentration before administration is calculated as 0; the BLQ (including "No peak") before Cmax is calculated as 0; the BLQ (including "No peak") that appears after Cmax is not involved in the calculation.
  • WinNonlin to calculate the pharmacokinetic parameters, such as AUC(0-t), T 1/2 , Cmax, etc., through the plasma concentration data at different time points. The results are shown in Table 7.
  • Test Example 7 Bidirectional Permeability Study of Test Compounds on MDCK-MDR1 Cell Line
  • MDCK-MDR1 cells were purchased from the Netherlands Cancer Institute and cells between passages 10 and 20 were used.
  • the cell suspension was added to the chamber of a 96-well Transwell plate at 50 ⁇ L per well.
  • the incubator was set at 37°C, 5% CO 2 , and maintained at 95% relative humidity for 4-8 days.
  • the medium was changed 48 hours after inoculation, and the medium was cultured for 4-8 days, and the medium was changed every other day.
  • the positive control compound was prepared as a stock solution with a concentration of 10 mM in DMSO.
  • the internal standard contained (100 nM alprazolam, 200 nM caffeine and 100 nM tosylbutyramide), and 50 ⁇ L of the above-transferred sample solution was vortexed at 1000 rpm for 10 min.
  • the apical to basal direction and the basal to apical direction should be performed simultaneously.
  • the MDCK-MDR1 Transwell culture was incubated at 37°C for 2 hours.
  • MRM Multiple Reaction Detection
  • Peak areas were calculated from ion chromatography results.
  • the apparent permeability coefficient (Papp, unit: cm/s ⁇ 10 -6 ) of the compound is calculated by the following formula:
  • VA is the volume of the receiving end solution ( Ap ⁇ Bl is 0.3mL, Bl ⁇ Ap is 0.1mL), Area is the membrane area of the Transwell-96-well plate (0.143cm 2 ); time is the incubation time (unit: s) ); [drug] is the drug concentration.
  • the compounds to be tested were dissolved in a 10 mM stock solution prepared in DMSO, and then diluted to 1 mM with DMSO.
  • the prepared 10 test compounds with a concentration gradient of 1000 ⁇ were diluted 50 times with complete medium to 20 ⁇ compounds, and stored in a 96-well plate (Beaver, Suzhou), with a total of 10 concentration gradients. Volume of DMSO solvent served as a negative control.
  • the logarithmic growth phase cell suspension was taken and seeded in a 96-well white cell culture plate (Corning 3917, NY, USA), and the volume of each well was 95 ⁇ l (about 2000 cells/well).
  • the culture plate was placed at room temperature for 10 minutes to stabilize the luminescence signal.
  • the luminescence signal was detected on the MD SpectraMax Paradigm plate reader.
  • Cell viability (%) (RLU Drug -RLU Min )/(RLU Max -RLU Min )*100%. Calculate the cell viability corresponding to different concentrations of compounds in EXCEL, and then use the GraphPad Prism software to draw the cell viability curve and calculate the relevant parameters, including the maximum and minimum cell viability, IC 50 value.
  • the compound Ib of the present application has a strong growth inhibitory effect on 6 BaF3 cell lines.
  • the inhibitory activity of compound 2 on 6 BaF3 cell lines was not significantly different from that of the corresponding positive compounds, but the inhibitory activity of compound Ib in the present application on 6 BaF3 cell lines was significantly higher than that of positive control 2.

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Abstract

涉及式(I)化合物、其立体异构体或药学上可接受的盐在制备用于治疗RET相关疾病的药物中的用途。

Description

具有大环结构的含氟并杂环衍生物的应用
本申请是以CN申请号为202010820363.5，申请日为2020年8月14日的申请为基础，并主张其优先权，该CN申请的公开内容在此作为整体引入本申请中。
技术领域
本申请涉及具有大环结构的含氟并杂环衍生物在治疗疾病中的应用，具体涉及式(I)化合物、其立体异构体或其药学上可接受的盐在制备用于治疗RET相关疾病的药物中的用途、在治疗RET相关疾病中的应用。
背景技术
原癌基因RET(Rearranged during Transfection)负责编码受体酪氨酸激酶RET蛋白。该激酶属于跨膜蛋白，由胞外区、跨膜区和胞内酪氨酸激酶活性区三部分组成。RET的生理配体属于神经胶质细胞源性神经营养因子(GDNFs)家族。RET的激活是由生长因子受体α1/2/3/4(GFRα1/2/3/4)四个受体和GDNFs四个配体之间的相互作用来完成，GFRα能特异性地结合GDNFs家族成员，促进RET蛋白受体的磷酸化并使RET进入激活状态，从而激活与细胞增殖，迁移和分化有关的下游信号通路，主要包括Ras/Raf/MEK/ERK-MAPK通路和PI3K/Akt/mTOR通路以及其他通路(PLC-γ通路、JAK-STAT通路)等(Matti S.Airaksinen.Mol Cell Neurosci.1999.13(313–325))。在正常情况下，受体酪氨酸激酶在正常器官和成年组织中起维护作用，但当RET基因出现变异时，其受体酪氨酸激酶活性会异常激活，从而驱动肿瘤的发生，并维持肿瘤的增殖和生存。
在多种肿瘤中观察到RET基因变异，RET的异常激活是大量实体瘤中肿瘤生长和增殖的关键驱动因素。约有1％～2％的非小细胞肺癌患者、65％髓样甲状腺癌、10％～20％乳头状甲状腺癌患者发生RET突变或融合，在其它癌症中，RET变异总体平均发生率小于1％。致癌性的RET阳性变异约占RET总体变异的71.6％。常见的RET变异包括突变(38.6％)、融合(30.7％)、扩增(25％)、重排(3.4％)、拷贝数增加(1.1％)和拷贝数减少(1.1％)(Shumei Kato.Clin Cancer Res.2018.23(8))。在临床前和临床研究中表明，RET抑制剂能显著抑制RET基因融合和突变的肿瘤细胞增殖。
目前RET抑制剂主要分为两类，一类是靶向RET小分子抑制剂，包括LOXO-292和BLU-667等，是目前RET抑制剂的研究重点；另一类是多激酶抑制剂，这类抑制 剂具有RET抑制活性。FDA于2020年5月批准LOXO-292(Selpercatinib)上市，用于治疗RET融合基因阳性的非小细胞肺癌患者、晚期或转移性的RET突变的甲状腺髓样癌患者以及晚期或转移性的RET融合基因阳性的甲状腺癌患者，是第一个上市的RET抑制剂。另一个RET抑制剂BLU-667目前已向FDA进行了NDA申请，用于治疗局部晚期或转移性RET融合基因阳性的非小细胞肺癌患者。此外，还有其他一些RET小分子抑制剂临床前在研。
作为原癌基因，RET的阳性突变或融合在非小细胞肺癌、甲状腺髓样癌、甲状腺癌等肿瘤的增殖及生长中发挥关键作用。越来越多的研究表明，RET抑制剂可有效抑制RET融合或突变的恶性肿瘤体内增殖，并且在临床上有较高的疾病缓解率，是不分癌种的有效治疗靶点。
发明内容
本申请提供式(I)所示化合物、其立体异构体或其药学上可接受的盐在制备用于治疗RET相关疾病的药物中的用途，
Figure PCTCN2021112413-appb-000001
本申请还提供式(I)所示化合物、其立体异构体或其药学上可接受的盐，其用于治疗RET相关疾病。
本申请还提供一种治疗RET相关疾病的方法，包括给有需要的受试者施用有效量的式(I)所示化合物、其立体异构体或其药学上可接受的盐。
本申请还提供一种用于治疗RET相关疾病的组合物，其中包含式(I)所示化合物、其立体异构体或其药学上可接受的盐，任选地还包含药学可接受的载体和/或赋形剂。在某些实施方案中，所述的组合物中，式(I)所示化合物、其立体异构体或其药学上可接受的盐以治疗RET相关疾病的有效量存在。
本申请还提供一种用于治疗RET相关疾病的药物，其中包含式(I)所示化合物、其立体异构体或其药学上可接受的盐，任选地还包含药学可接受的载体和/或赋形剂。在某些实施方案中，所述的组合物中，式(I)所示化合物、其立体异构体或其药学上可接受的盐以治疗RET相关疾病的有效量存在。
在某些实施方案中，所述式(I)化合物为式(Ia)所示的化合物。
Figure PCTCN2021112413-appb-000002
在某些实施方案中，所述式(I)化合物为式(Ib)所示的化合物。
Figure PCTCN2021112413-appb-000003
在某些实施方案中，所述RET相关疾病是RET基因或RET激酶蛋白的表达、活性或水平调节异常相关的疾病。
在某些实施方案中，所述RET相关疾病是RET基因或RET激酶蛋白突变相关的疾病。在某些实施方案中，所述RET基因或RET激酶蛋白突变包含一个或多个位点上的突变。
在某些实施方案中，所述RET相关疾病是RET融合基因相关的疾病。
在某些实施方案中，所述RET融合基因选自：BCR-RET、CLIP1-RET、KIF5B-RET、CCDC6-RET、NCOA4-RET、TRIM33-RET、ERC1-RET、FGFR1OP-RET、RET-MBD1、RET-RAB6IP2、RET-PRKAR1A、RET-TRIM24、RET-GOLGA5、HOOK3-RET、KTN1-RET、TRIM27-RET、AKAP13-RET、FKBP15-RET、SPECC1L-RET、TBL1XR1-RET、CEP55-RET、CUX1-RET、KIAA1468-RET、RFG8-RET、ACBD5-RET、PTC1ex9-RET、MYH13-RET、PIBF1-RET、KIAA1217-RET、MPRIP-RET、HRH4-RET、Ria-RET、RET-PTC4、FRMD4A-RET、SQSTM1-RET、AFAP1L2-RET、PPFIBP2-RET、EML4-RET、PARD3-RET、MYH10-RET、HTIF1-RET、AFAP1-RET、RASGEF1A-RET、TEL-RET、RUFY1-RET、UEVLD-RET、DLG5-RET、FOXP4-RET、OFLM4-RET、RRBP1-RET及其任意组合。
在某些实施方案中，所述RET融合基因选自：RET-CCDC6(PTC1)、RET-KIF5B(Kex15Rex14)、RET-PRKAR1A(PTC2)、RET-BCR、RET-NCOA4(PTC3)及其任意组合。
在某些实施方案中，所述RET融合基因选自：RET(V804L)-KIF5B、RET(V804M)-KIF5B及其任意组合。
在某些实施方案中，所述RET融合基因为RET(V804M)-KIF5B。
在某些实施方案中，所述RET融合基因为RET(V804L)-KIF5B。
在某些实施方案中，所述RET融合基因为RET-NCOA4(PTC3)。
在某些实施方案中，所述RET融合基因为RET-CCDC6(PTC1)。
在某些实施方案中，所述RET融合基因为RET-KIF5B(Kex15Rex14)。
在某些实施方案中，所述RET融合基因为RET-PRKAR1A(PTC2)。
在某些实施方案中，所述RET基因突变选自：RET(Y791F)、RET(V778I)、RET(G691S)、RET(V804L)、RET(R813Q)、RET(E762Q)、RET(V804E)、RET(V804L)-KIF5B、RET(A883F)、RET(S904F)、RET(V804M)、RET(V804M)-KIF5B、RET(Y806H)、RET(M918T)及其任意组合。
某些实施方案中，所述RET基因突变为RET(S891A)。
某些实施方案中，所述RET基因突变为RET(L790F)。
某些实施方案中，所述RET基因突变为RET(R749T)。
某些实施方案中，所述RET基因突变为RET(S904A)。
某些实施方案中，所述RET基因突变为RET(R912P)。
某些实施方案中，所述RET基因突变为RET(Y791F)。
某些实施方案中，所述RET基因突变为RET(V778I)。
某些实施方案中，所述RET基因突变为RET(G691S)。
某些实施方案中，所述RET基因突变为RET(V804L)。
某些实施方案中，所述RET基因突变为RET(R813Q)。
某些实施方案中，所述RET基因突变为RET(E762Q)。
某些实施方案中，所述RET基因突变为RET(V804E)。
某些实施方案中，所述RET基因突变为RET(A883F)。
某些实施方案中，所述RET基因突变为RET(S904F)。
某些实施方案中，所述RET基因突变为RET(Y806H)。
某些实施方案中，所述RET基因突变为RET(M918T)。
在某些实施方案中，所述RET相关疾病为RET基因或RET激酶蛋白的表达、活性或水平调节异常相关的癌症。
在某些实施方案中，所述RET相关疾病为RET基因或RET激酶蛋白突变相关的癌症。在某些实施方案中，所述RET基因或RET激酶蛋白突变包含一个或多个位点上的突变。
在某些实施方案中，所述RET基因突变相关的癌症是选自：RET基因突变相关的肺癌、乳头状甲状腺癌、甲状腺髓样癌、分化型甲状腺癌、复发性甲状腺癌、难治性分化型甲状腺癌、多发性内分泌瘤形成2A或2B型(分别是MEN2A或MEN2B)、嗜铬细胞瘤、甲状旁腺增生、乳腺癌、结肠癌、结肠直肠癌、乳头状肾细胞癌、胃肠粘膜神经节瘤和宫颈癌中的一种或多种。
在某些实施方案中，所述RET相关疾病是RET基因突变相关的肺癌。
在某些实施方案中，所述RET相关疾病是RET基因突变相关的甲状腺髓样癌。
在某些实施方案中，所述RET相关疾病是RET基因突变相关的结肠癌。
在某些实施方案中，所述RET相关疾病是选自RET基因突变相关的小细胞肺癌、RET基因突变相关的非小细胞肺癌、RET基因突变相关的细支气管肺细胞癌或RET基因突变相关的肺腺癌中的一种或多种。
在某些实施方案中，所述RET相关疾病是RET基因突变相关的小细胞肺癌。
在某些实施方案中，所述RET相关疾病是RET基因突变相关的非小细胞肺癌。
在某些实施方案中，所述RET相关疾病是RET基因突变相关的细支气管肺细胞癌。
在某些实施方案中，所述RET相关疾病是RET基因突变相关的肺腺癌。
在某些实施方案中，所述RET相关疾病是RET融合基因相关的癌症。
在某些实施方案中，所述RET融合基因相关的癌症是选自：RET融合基因相关的肺癌、乳头状甲状腺癌、甲状腺髓样癌、分化型甲状腺癌、复发性甲状腺癌、难治性分化型甲状腺癌、多发性内分泌瘤形成2A或2B型(分别是MEN2A或MEN2B)、嗜铬细胞瘤、甲状旁腺增生、乳腺癌、结肠癌、结肠直肠癌、乳头状肾细胞癌、胃肠粘膜神经节瘤和宫颈癌中的一种或多种。
在某些实施方案中，所述RET相关疾病是RET融合基因相关的肺癌。
在某些实施方案中，所述RET相关疾病是RET融合基因相关的甲状腺髓样癌。
在某些实施方案中，所述RET相关疾病是RET融合基因相关的结肠癌。
在某些实施方案中，所述RET相关疾病是RET-CCDC6相关的结肠癌。
在某些实施方案中，所述RET相关疾病是选自RET融合基因相关的小细胞肺癌、RET融合基因相关的非小细胞肺癌、RET融合基因相关的细支气管肺细胞癌或RET融合基因 相关的肺腺癌中的一种或多种。
在某些实施方案中，所述RET相关疾病是RET融合基因相关的小细胞肺癌。
在某些实施方案中，所述RET相关疾病是RET融合基因相关的非小细胞肺癌。
在某些实施方案中，所述RET相关疾病是RET融合基因相关的细支气管肺细胞癌。
在某些实施方案中，所述RET相关疾病是RET融合基因相关的肺腺癌。
在某些实施方案中，所述RET相关疾病是人的RET相关疾病，式(I)所示化合物、其立体异构体或其药学上可接受的盐的给药剂量为0.5-4mg/kg/day。
定义：
在本申请中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。为了更好地理解本申请，下面提供相关术语的定义和解释。
本申请中所用术语“立体异构体(stereoisomer)”指由分子中原子在空间上排列方式不同所产生的异构体，它可分为顺反异构体、对映异构体两种，也可分为对映异构体和非对映异构体两大类。分子中原子或原子团互相连接次序相同，但空间排列不同而引起的异构体称为立体异构体，有两类立体异构体。因键长、键角、分子内有双键、有环等原因引起的立体异构体称为构型异构体(configuration stereo-isomer)。一般来讲，构型异构体之间不能或很难互相转换。仅由于单键的旋转而引起的立体异构体称为构象异构体(conformational stereo-isomer)，有时也称为旋转异构体(rotamer)。构型异构体又分为两类。其中因双键或成环碳原子的单键不能自由旋转而引起的异构体称为几何异构体(geometric isomer)，也称为顺反异构体(cis-trans isomer)，将因分子中没有反轴对称性而引起的具有不同旋光性能的立体异构体称为旋光异构体(optical isomer)。
本申请中所用术语“RET(Rearranged during Transfection)基因”是一个原癌基因，全名：RET proto-oncogene：位于10号常染色体长臂(10q11.2)，全长53kb，包含20个外显子，编码1114个氨基酸的酪氨酸激酶受体。
本申请中所用术语“RET激酶”是一种受体酪氨酸激酶，由RET基因编码，与细胞增殖、迁移、分化及神经嵴细胞的生存、肾脏器官的形成、精子发生等过程的信号转导有关。
本申请中所用术语“RET融合基因”指RET基因与其它基因序列发生重排，导致蛋白表达后发生融合，使RET的基因水平或蛋白水平的表达或活性异常。本申请中“RET融合基因”用“A-B”或“B-A”表示，其中A、B分别代表RET基因和与RET基因融合的其他基因。“A-B”或“B-A”的含义相同，表示A基因和B基因的融合基因。例如，RET-CCDC6和CCDC6-RET的含义相同，它们含义均是RET与CCDC6的融合基因。
本申请中所用术语“RET基因突变”是指RET基因的碱基改变所引起的点突变，或一个或多个碱基的缺失、重复或插入，造成蛋白发生相应变化。
本申请所用术语“药学上可接受的盐”包括与药学上可以接受的无机酸或者有机酸、或者无机碱或有机碱形成的常规盐。用于制备本申请的化合物的可药用的盐的方法为本领域技术人员已知的。
本申请中涉及到本申请化合物时，包括式(I)所示化合物、其立体异构体或其药学上可接受的盐。
本申请所用术语“药物组合物”包括包含治疗有效量的本申请的化合物的产品，以及直接地或间接地由本申请化合物的组合产生的任何产品。该药物组合物可通过例如口服或非肠道等途径给药。本申请的药物组合物可按本领域常规方法制备成各种剂型，包括但不限于片剂、胶囊、溶液、悬浮液、颗粒剂或注射剂等，经口服或非肠道等途径给药。
本申请所用术语“有效量”是指足以实现所需治疗效果的量，例如，实现减轻与待治疗疾病相关的症状的量。
本申请所用的术语“治疗”目的是减轻或消除所针对的疾病状态或病症。如果受试者按照本申请所述方法接受了治疗有效量的化合物、其立体异构体、其药学上可接受的盐或其药物组合物，该受试者一种或多种指征和症状表现出可观察到的和/或可检测出的降低或改善，则受试者被成功地“治疗”了。还应当理解，所述的疾病状态或病症的治疗的不仅包括完全地治疗，还包括未达到完全地治疗，但实现了一些生物学或医学相关的结果。
另外需要指出，本申请化合物的使用剂量和使用方法取决于诸多因素，包括患者的年龄、体重、性别、自然健康状况、营养状况、化合物的活性强度、服用时间、代谢速率、病症的严重程度以及诊治医师的主观判断。优选的使用剂量介于0.001－1000mg/kg体重/天。
本申请中所述的化合物，对于同一化合物而言，若名称与结构式不一致，以化合物结构式为准。
附图说明
图1显示了
Figure PCTCN2021112413-appb-000004
结肠癌CR2518模型中各组小鼠平均肿瘤体积的生长曲线；
图2显示了
Figure PCTCN2021112413-appb-000005
结肠癌CR2518模型中各组小鼠随治疗时间的体重变化曲线。
图1和图2中：1.数据以“平均值±标准误差”表示；2.G3-00442#小鼠在分组后第30天发现死亡；G3-00443#小鼠在分组后第33天发现死亡；3.BID代表一天给药两次；QD代表一天给药一次。
具体实施方式
下面结合本申请的具体实施例来进一步说明本申请的实质性内容，应理解，以下实施例仅用于说明本申请，但并不以此来限定本申请的保护范围。下面实施例中未注明具体条件者，按照常规条件或制造商建议的进行。所用原料未注明生产厂商者，均为可以通过市购获得的常规产品。
虽然以下实施例中所使用的许多材料和操作方法是本领域公知的，但是本申请仍然在此作尽可能详细描述。本领域技术人员清楚，如果未特别说明，下面实施例中所用的材料和操作方法是本领域公知的。
下面结合实施例对本申请提供的用作RET抑制剂的化合物及其制备方法和用途进行详细描述。
下面的缩写具有如下所示的意义：
HATU表示2-(7-氧化苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸盐；
Cs 2CO 3表示碳酸铯；
THF表示四氢呋喃；
POCl 3表示三氯氧磷；
DMF表示N,N-二甲基甲酰胺；
NaOH表示氢氧化钠；
DIPEA或DIEA表示N,N-二异丙基乙胺；
H 2O表示水；
HCl/Dioxane表示氯化氢二氧六环溶液；
rt表示反应温度为室温；
N,N-Diethylaniline表示N,N-二乙基苯胺；
CH 3CN或ACN表示乙腈；
Zn表示锌粉；
NH 4Cl表示氯化铵；
CH 3MgBr表示甲基溴化镁；
K 2CO 3表示碳酸钾；
EtOH表示乙醇；
OTs表示对甲基苯磺酰基氧基，
Figure PCTCN2021112413-appb-000006
ee表示对映异构体过量。
RET(S891A)表示RET基因在891位的丝氨酸突变为丙氨酸；
RET(L790F)表示RET基因在790位的亮氨酸突变为苯丙氨酸；
RET(V804M)表示RET基因在804位的缬氨酸突变为甲硫氨酸；
RET(R749T)表示RET基因在749位的精氨酸突变为苏氨酸；
RET(S904A)表示RET基因在904位的丝氨酸突变为丙氨酸；
RET(R912P)表示RET基因在912位的精氨酸突变为脯氨酸；
RET(Y791F)表示RET基因在791位的酪氨酸突变为苯丙氨酸；
RET(V778I)表示RET基因在778位的缬氨酸突变为异亮氨酸；
RET(G691S)表示RET基因在691位的甘氨酸突变为丝氨酸；
RET(V804L)表示RET基因在804位的缬氨酸突变为亮氨酸；
RET(R813Q)表示RET基因在813位的精氨酸突变为谷氨酰胺；
RET(E762Q)表示RET基因在762位的谷氨酸突变为谷氨酰胺；
RET(V804E)表示RET基因在804位的缬氨酸突变为谷氨酸；
RET(A883F)表示RET基因在883位的丙氨酸突变为苯丙氨酸；
RET(S904F)表示RET基因在904位的丝氨酸突变为苯丙氨酸；
RET(Y806H)表示RET基因在806位的酪氨酸突变为组氨酸；
RET(M918T)表示RET基因在918位的甲硫氨酸突变为苏氨酸。
RET-CCDC6(PTC1)为RET与CCDC6(别名PTC1，coiled-coil domain containing 6，卷曲螺旋结构域蛋白6，Gene ID(NCBI):8030)的融合基因；
RET-KIF5B(Kex15Rex14)为RET与KIF5B(the kinesin family 5B gene，驱动蛋白家族5B，Gene ID(NCBI):3799)的融合基因，融合位置在KIF5B基因的15外显子开始(氨基酸1-575)，在RET的14-21外显子(氨基酸713)结束；
RET-PRKAR1A(PTC2)为RET与PRKAR1A(别名PTC2，protein kinase cAMP-dependent type I regulatory subunit alpha，蛋白激酶c-AMP依赖型I调控亚基α，Gene ID(NCBI):5573)的融合基因；
RET(V804L)-KIF5B为RET(V804L)与KIF5B(the kinesin family 5B gene，驱动蛋 白家族5B，Gene ID(NCBI):3799)的融合基因；
RET-BCR：RET与BCR(别名BCR1；BCR activator of RhoGEF and GTPase，RhoGEF和GTO酶BCR激活子，Gene ID(NCBI):613)的融合基因；
RET(V804M)-KIF5B为RET(V804M)与KIF5B(the kinesin family 5B gene，驱动蛋白家族5B，Gene ID(NCBI):3799)的融合基因；
RET-NCOA4(PTC3)为RET与NCOA4(别名PTC3，nuclear receptor coactivator 4，Gene ID(NCBI):8031)的融合基因；
CLIP1-RET为RET与CLIP1(CAP-Gly domain containing linker protein 1，含CAP-Gly结构域接头蛋白1，Gene ID(NCBI):6249)的融合基因；
CCDC6-RET为RET与CCDC6(coiled-coil domain containing 6，卷曲螺旋结构域蛋白6，Gene ID(NCBI):8030)的融合基因；
TRIM33-RET为RET与TRIM33(tripartite motif containing 33，三基序蛋白33，Gene ID(NCBI):51592)的融合基因；
ERC1-RET为RET与ERC1(ELKS/RAB6-interacting/CAST family member 1，ELKS/Rab6相互作用/CAST蛋白家族成员1，Gene ID(NCBI):23085)的融合基因；
FGFR1OP-RET为RET与FGFR1OP(FGFR1oncogene partner，FGFR1致癌基因伴侣，Gene ID(NCBI):11116)的融合基因；
RET-MBD1为RET与MBD1(methyl-CpG binding domain protein 1，甲基磷酸胞苷酰鸟苷结合蛋白，Gene ID(NCBI):4152)的融合基因；
RET-RAB6IP2为RET与RAB6IP2(Rab6Interacting Protein 2A，别名ERC-1，Gene ID(NCBI):23085)的融合基因；
RET-TRIM24为RET与TRIM24(tripartite motif containing 24，三基序蛋白24，Gene ID(NCBI):8805)的融合基因；
RET-GOLGA5为RET与GOLGA5(golgin A5，高尔基体蛋白5，Gene ID(NCBI):9950)的融合基因；
HOOK3-RET为RET与HOOK3(hook microtubule tethering protein 3，钩状微管栓系蛋白3，Gene ID(NCBI):84376)的融合基因；
KTN1-RET为RET与KTN1(kinectin 1，驱动结合蛋白1，Gene ID(NCBI):3895)的融合基因；
TRIM27-RET为RET与TRIM27(tripartite motif containing 27，基序蛋白27，Gene  ID(NCBI):5987)的融合基因；
AKAP13-RET为RET与AKAP13(A-kinase anchoring protein 13，激酶锚定蛋白13，Gene ID(NCBI):11214)的融合基因；
FKBP15-RET为RET与FKBP15(FKBP prolyl isomerase family member 15，脯氨酸异构酶家族成员15，Gene ID(NCBI):23307)的融合基因；
SPECC1L-RET为RET与SPECC1L(sperm antigen with calponin homology and coiled-coil domains 1like，钙调节蛋白同源及卷曲结构域1样kd糖蛋白，Gene ID(NCBI):23384)的融合基因；
TBL1XR1-RET为RET与TBL1XR1(TBL1X receptor 1，TBL1X受体1，Gene ID(NCBI):79718)的融合基因；
CEP55-RET为RET与CEP55(centrosomal protein 55，中心体蛋白55，Gene ID(NCBI):55165)的融合基因；
CUX1-RET为RET与CUX1(cut like homeobox 1，同源框切割蛋白1，Gene ID(NCBI):1523)的融合基因；
KIAA1468-RET为RET与KIAA1468(别名RAB11binding and LisH domain,coiled-coil and HEAT repeat containing，Gene ID(NCBI):57614)的融合基因
RFG8-RET为RET与RFG8(RET-fused gene 8，RET融合基因8)的融合基因；
ACBD5-RET为RET与ACBD5(acyl-CoA binding domain containing 5，酰基辅酶A结合域蛋白5，Gene ID(NCBI):91452)的融合基因；
PTC1ex9-RET为PTC-RET融合的一种变体，为RET细胞外结构域外显子9与CCDC6(coiled-coil domain containing 6，卷曲螺旋结构域6，Gene ID(NCBI):8030)的外显子1的融合基因；
MYH13-RET为RET与MYH13(myosin heavy chain 13，肌球蛋白重链13，Gene ID(NCBI):8735)的融合基因；
PIBF1-RET为RET与PIBF1(progesterone immunomodulatory binding factor 1，孕酮免疫调节结合因子1，Gene ID(NCBI):10464)的融合基因；
KIAA1217-RET为RET与KIAA1217(Gene ID(NCBI):56243)的融合基因；
MPRIP-RET为RET与MPRIP(myosin phosphatase Rho interacting protein，肌球蛋白磷酸酶-Rho作用蛋白，Gene ID(NCBI):23164)的融合基因；
HRH4-RET为RET与HRH4(histamine receptor H4，组胺H4受体，Gene ID(NCBI): 59340)的融合基因；
Ria-RET为RET与RIA(the RIA regulatory subunit of the c-AMP dependent protein kinase A,蛋白激酶A的调控亚基RIA,)的融合基因；
RET-PTC4为RET与PTC4(type 2C protein phosphatase，2C型蛋白磷酸酶，Gene ID(NCBI):5108)的融合基因；
FRMD4A-RET为RET与FRMD4A(FERM domain containing 4A，FERM域蛋白4A，Gene ID(NCBI):55691)的融合基因；
SQSTM1-RET为RET与SQSTM1(sequestosome 1，自噬接头蛋白1，Gene ID(NCBI):8878)的融合基因；
AFAP1L2-RET为RET与AFAP1L2(actin filament associated protein 1like 2，肌动蛋白丝相关蛋白1样蛋白2，Gene ID(NCBI):84632)的融合基因；
PPFIBP2-RET为RET与PPFIBP2(PPFIA binding protein 2，PPFIA结合蛋白2，Gene ID(NCBI):8495)的融合基因；
EML4-RET为RET与EML4(EMAP like 4，EMAP样蛋白4，Gene ID(NCBI):27436)的融合基因；
PARD3-RET为RET与PARD3(par-3family cell polarity regulator，Par-3家族细胞极性调节器，Gene ID(NCBI):56288)的融合基因；
MYH10-RET为RET与MYH10(myosin heavy chain 10，肌球蛋白重链10，Gene ID(NCBI):4628)的融合基因；
HTIF1-RET为RET与HTIF1(别名tripartite motif containing 24，三基序蛋白24，Gene ID(NCBI):8805)的融合基因；
AFAP1-RET为RET与AFAP1(actin filament associated protein 1，肌动蛋白丝相关蛋白1，Gene ID(NCBI):60312)的融合基因；
RASGEF1A-RET为RET与RASGEF1A(RasGEF domain family member 1A，RasGEF域家族成员1A，Gene ID(NCBI):221002)的融合基因；
TEL-RET为RET与TEL(别名EVT6，ETS variant transcription factor 6Telomere elongation，(NCBI):2120)的融合基因；
RUFY1-RET为RET与RUFY1(RUN and FYVE domain containing 1，RUN和FYVE域包含蛋白1，Gene ID(NCBI):80230)的融合基因；
UEVLD-RET为RET与UEVLD(UEV and lactate/malate dehyrogenase domains， UEV和乳酸/苹果酸脱氢酶结构域，Gene ID(NCBI):55293)的融合基因；
DLG5-RET为RET与DLG5(discs large MAGUK scaffold protein 5，MAGUK支架蛋白5，Gene ID(NCBI):9231)的融合基因；
FOXP4-RET为RET与FOXP4(forkhead box P4，叉头框蛋白P4，Gene ID(NCBI):116113)的融合基因；
OLFM4-RET为RET与OLFM4(Olfactomedin-4，嗅质蛋白4，Gene ID(NCBI):418826)的融合基因；
RRBP1-RET为RET与RRBP1(ribosome binding protein 1，核糖体结合蛋白1Gene ID(NCBI):6238)的融合基因。
制备例1：5-氯-6-氟吡唑并[1,5-a]嘧啶-3-羧酸乙酯的制备
Figure PCTCN2021112413-appb-000007
步骤1：2-氟丙二酸的制备
Figure PCTCN2021112413-appb-000008
室温下，称取2-氟丙二酸二乙酯(5.0g)及氢氧化钠(17.3g)于乙醇/水(100/100mL)的混合溶液中，反应过夜，LCMS显示反应完全。将反应液浓缩除去乙醇，加水(50mL)，浓盐酸调节pH约为1，甲基叔丁基醚萃取四次，合并有机相，无水硫酸钠干燥，过滤，浓缩，得标题化合物3.7g，无需纯化即可用于下一步反应。
MS(ESI)m/z(M-H) +＝121.1.
步骤2：5,7-二氯-6-氟吡唑并[1,5-a]嘧啶-3-羧酸乙酯的制备
Figure PCTCN2021112413-appb-000009
室温下，称取2-氟丙二酸(2.0g)及5-氨基-1H-吡唑-4-羧酸乙酯(1.7g)于三氯氧磷(20mL)中，再将N,N-二甲基甲酰胺(2mL)和N,N-二乙基苯胺(4.9g)加入，升温至110℃下反 应3小时，LCMS显示反应完全。将反应液浓缩除去三氯氧磷，再倒入饱和碳酸氢钠溶液(100mL)中，使溶液保持为碱性，乙酸乙酯萃取三次，无水硫酸钠干燥，过滤，浓缩，所得粗品经柱层析纯化，得到的固体再用石油醚洗涤，干燥后得标题化合物1.7g。
MS(ESI)m/z(M+H) +＝278.0.
1H NMR(400MHz,DMSO-d 6)δ8.80(s,1H),4.33(q,J＝7.2Hz,2H),1.32(t,J＝7.0Hz,3H).
步骤3：5-氯-6-氟吡唑并[1,5-a]嘧啶-3-羧酸乙酯的制备
Figure PCTCN2021112413-appb-000010
称取5,7-二氯-6-氟吡唑并[1,5-a]嘧啶-3-羧酸乙酯(1.14g)及氯化铵(800mg)于乙醇/四氢呋喃/水(30/10/20mL)的混合溶液中，搅拌过程中加入锌粉(1.3g)，反应5分钟后将锌粉过滤，乙酸乙酯洗涤滤饼，收集滤液，无水硫酸钠干燥，过滤，浓缩，所得粗品经柱层析纯化得标题化合物800mg。
MS(ESI)m/z(M+H) +＝244.0.
1H NMR(400MHz,DMSO-d 6)δ9.93(d,J＝4.4Hz,1H),8.68(s,1H),4.31(q,J＝7.2Hz,2H),1.31(t,J＝7.2Hz,3H).
制备例2：(R)-1-(5-氟-2-甲氧基吡啶-3-基)乙-1-胺盐酸盐的制备
Figure PCTCN2021112413-appb-000011
步骤1：(R)-N-(((5-氟-2-甲氧基吡啶-3-基)亚甲基)-2-甲基丙烷-2-亚磺酰胺的制备
Figure PCTCN2021112413-appb-000012
将(R)-2-甲基丙烷-2-亚磺酰胺(12.9g)溶于四氢呋喃(100mL)中，依次加入5-氟-2-甲氧基烟碱醛(15.0g)和碳酸铯(40.9g)。所得混合物于室温反应2小时，TLC显示原料消耗完毕。抽滤，滤饼用四氢呋喃洗涤三次，所得滤液用饱和氯化钠溶液反洗一次，无水硫酸钠干燥，过滤，浓缩。所得粗品经柱层析纯化得标题化合物23.0g。
MS(ESI)m/z(M+H) +＝259.1.
1H NMR(400MHz,DMSO-d 6)δ8.67(d,J＝2.4Hz,1H),8.42(d,J＝3.2Hz,1H),8.14(dd,J＝8.4,3.2Hz,1H),3.98(s,3H),1.18(s,9H).
步骤2：(R)-N-((R)-1-(5-氟-2-甲氧基吡啶-3-基)乙基)-2-甲基丙烷-2-亚磺酰胺的制备
Figure PCTCN2021112413-appb-000013
称取(R)-N-(((5-氟-2-甲氧基吡啶-3-基)亚甲基)-2-甲基丙烷-2-亚磺酰胺(5.0g)溶于四氢呋喃(40mL)中，所得混合物冷却至-78℃后缓慢滴加甲基溴化镁(7.8mL,3M)，维持温度低于-65℃。滴毕，自然回温至室温，继续反应1小时，TLC显示反应完全。将反应体系倒入饱和氯化铵水溶液(1L)中，乙酸乙酯萃取，合并有机相，饱和氯化钠溶液反洗，无水硫酸钠干燥，过滤，浓缩。所得粗品经柱层析纯化得标题化合物4.5g。
MS(ESI)m/z(M+H) +＝275.2.
1H NMR(400MHz,DMSO-d 6)δ8.04(d,J＝2.8Hz,1H),7.74(dd,J＝9.2,3.2Hz,1H),5.80(d,J＝8.8Hz,1H),4.57-4.50(m,1H),3.88(s,3H),1.33(d,J＝6.8Hz,3H),1.11(s,9H).
步骤3：(R)-1-(5-氟-2-甲氧基吡啶-3-基)乙-1-胺盐酸盐的制备
Figure PCTCN2021112413-appb-000014
室温下，将(R)-N-((R)-1-(5-氟-2-甲氧基吡啶-3-基)乙基)-2-甲基丙烷-2-亚磺酰胺(4.5g)溶于氯化氢-二氧六环溶液(30mL)中，反应过夜，LCMS显示原料消耗完全。将反应液浓缩得粗品3.1g，大于95％ee，无需纯化即可直接用于下一步。
MS(ESI)m/z(M+H) +＝171.2.
1H NMR(400MHz,DMSO-d 6)δ8.80-8.66(m,3H),8.18(d,J＝2.8Hz,1H),8.04-8.00(m,1H),7.09–6.60(m,1H),4.51–4.45(m,1H),3.90(s,3H),1.49(d,J＝6.4Hz,3H).
实施例1：(3 1S,3 3S,6 3E,6 4E,8R)-1 5,6 6-二氟-8-甲基-2-氧杂-4,7-二氮杂-6(3,5)-吡唑并[1,5-a]嘧啶-1(2,3)-吡啶3-3,(1,3)-环丁环八烷-5-酮(化合物Ib)的制备
Figure PCTCN2021112413-appb-000015
步骤1：(R)-6-氟-5-((1-(5-氟-2-甲氧基吡啶-3-基)乙基)氨基)吡唑并[1,5-a]嘧啶-3-羧酸乙酯的制备
Figure PCTCN2021112413-appb-000016
将(R)-1-(5-氟-2-甲氧基吡啶-3-基)乙-1-胺盐酸盐(1.0g)溶于乙腈(20mL)中，依次加入N,N-二异丙基乙胺(1.9g)和5-氯-6-氟吡唑并[1,5-a]嘧啶-3-羧酸乙酯(1.2g)，所得混合物于60℃下反应3小时，TLC显示反应完全。将反应液倒入水(50mL)中，二氯甲烷萃取，合并有机相，饱和氯化钠溶液反洗，无水硫酸钠干燥，过滤，浓缩。所得粗品经柱层析纯化得标题化合物1.1g。
MS(ESI)m/z(M+H) +＝378.2.
1H NMR(400MHz,DMSO-d 6)δ9.15(d,J＝6.4Hz,1H),8.49(d,J＝8.0Hz,1H),8.18(s,1H),8.02(d,J＝3.2Hz,1H),7.67(dd,J＝9.0,3.0Hz,1H),5.60-5.52(m,1H),4.18-4.10(m,2H),3.93(s,3H),1.50(d,J＝6.8Hz,3H),1.22(t,J＝7.0Hz,3H).
步骤2：(R)-6-氟-5-(((1-(5-氟-2-甲氧基吡啶-3-基)乙基)氨基)吡唑并[1,5-a]嘧啶-3-羧酸的制备
Figure PCTCN2021112413-appb-000017
室温下，将(R)-6-氟-5-((1-(5-氟-2-甲氧基吡啶-3-基)乙基)氨基)吡唑并[1,5-a]嘧啶-3-羧 酸乙酯(1.1g)溶于乙醇/水(5/15mL)的混合溶液中，加入氢氧化钠(584mg)，所得混合物于50℃反应过夜，TLC显示反应完全。将反应液浓缩除去乙醇，残余液倒入水(20mL)中，用氯化氢溶液(2M)调pH至5左右，二氯甲烷萃取，合并有机相，饱和氯化钠溶液反洗，无水硫酸钠干燥，过滤，浓缩得粗品800mg，无需纯化即可直接用于下一步。
MS(ESI)m/z(M+H) +＝350.1.
1H NMR(400MHz,DMSO-d 6)δ11.68(s,1H),9.13(d,J＝6.0Hz,1H),8.51(d,J＝7.6Hz,1H),8.14(s,1H),8.01(d,J＝2.8Hz,1H),7.72(dd,J＝9.0,3.0Hz,1H),5.59-5.52(m,1H),3.92(s,3H),1.50(d,J＝6.8Hz,3H).
步骤3：(1R,3r)-3-(6-氟-5-(((R)-1-(5-氟-2-甲氧基吡啶-3-基)乙基)氨基)吡唑并[1,5-a]嘧啶-3-甲酰胺基)-4-甲基苯磺酸环丁酯的制备
Figure PCTCN2021112413-appb-000018
将(R)-6-氟-5-(((1-(5-氟-2-甲氧基吡啶-3-基)乙基)氨基)吡唑并[1,5-a]嘧啶-3-羧酸(800mg)、2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(1.0g)及N,N-二异丙基乙胺(886mg)溶于干燥四氢呋喃(10mL)中，所得混合物在室温下反应1小时后再加入(3-羟基环丁基)氨基甲酸叔丁酯盐酸盐(953mg)，继续反应1小时，TLC显示反应完全。将反应液倒入水(30mL)中，乙酸乙酯萃取，合并有机相，饱和氯化钠溶液反洗，无水硫酸钠干燥，过滤，浓缩。所得粗品经柱层析纯化得标题化合物800mg。
MS(ESI)m/z(M+H) +＝573.2.
1H NMR(400MHz,DMSO-d 6)δ9.21(d,J＝6.0Hz,1H),8.57(d,J＝7.6Hz,1H),8.09(s,1H),8.05(d,J＝2.8Hz,1H),7.80–7.78(m,2H),7.66(dd,J＝8.8,2.8Hz,1H),7.60(d,J＝6.8Hz,1H),7.47(d,J＝8.0Hz,2H),5.44-5.37(m,1H),4.96–4.92(m,1H),4.33-4.28(m,1H),3.80(s,3H),2.47–2.38(m,5H),2.24-2.18(m,1H),2.12–2.08(m,1H),1.52(d,J＝6.8Hz,3H).
步骤4：(1R,3r)-3-(6-氟-5-(((R)-1-(5-氟-2-羟基吡啶-3-基)乙基)氨基)吡唑并[1,5-a]嘧啶-3-甲酰胺基)-4-甲基苯磺酸盐环丁基盐酸盐的制备
Figure PCTCN2021112413-appb-000019
将(1R,3r)-3-(6-氟-5-(((R)-1-(5-氟-2-甲氧基吡啶-3-基)乙基)氨基)吡唑并[1,5-a]嘧啶-3-甲酰胺基)-4-甲基苯磺酸环丁酯(800mg)溶于氯化氢的二氧六环(4M,10mL)中，所得混合物于55℃条件下反应过夜，TLC显示反应完全。将反应液直接浓缩以除去大部分二氧六环，得粗品600mg，无需纯化即可直接用于下一步。
MS(ESI)m/z(M+H) +＝559.2.
步骤5：(3 1S,3 3S,6 3E,6 4E,8R)-1 5,6 6-二氟-8-甲基-2-氧杂-4,7-二氮杂-6(3,5)-吡唑并[1,5-a]嘧啶-1(2,3)-吡啶3-3(1,3)-环丁环八烷-5-酮的制备
Figure PCTCN2021112413-appb-000020
将(1R,3r)-3-(6-氟-5-(((R)-1-(5-氟-2-羟基吡啶-3-基)乙基)氨基)吡唑并[1,5-a]嘧啶-3-甲酰胺基)-4-甲基苯磺酸盐环丁基盐酸盐(250mg)溶于N,N-二甲基甲酰胺(6mL)中，加入碳酸钾(232mg)，所得混合物室温反应5小时，TLC显示反应完全。将反应液倒入水(10mL)中，乙酸乙酯萃取，合并有机相，饱和氯化钠溶液反洗，无水硫酸钠干燥，过滤，浓缩。所得粗品经制备薄层色谱纯化和制备高效液相色谱纯化得标题化合物58.0mg，ee值>99.5％。
MS(ESI)m/z(M+H) +＝387.1.
1H NMR(400MHz,DMSO-d6)δ9.22–9.16(m,1H),9.15(d,J＝12.0Hz,1H),8.93(d,J＝8.0Hz,1H),8.10(s,1H),8.05(d,J＝4.0Hz,1H),7.87(dd,J＝8.8,3.0Hz,1H),5.79–5.67(m,1H),5.15–5.12(m,1H),4.70–4.63(m,1H),3.10–3.04(m,1H),2.92–2.86(m,1H),2.18–2.12(m,1H),1.69–1.63(m,1H),1.54(d,J＝8.0Hz,3H).
本申请测试例1-7中所用到的化合物包括：
化合物Ib，由本申请实施例1制备；
LOXO-195，为第二代TRK抑制剂，化学结构为：
Figure PCTCN2021112413-appb-000021
阳性对照1，为专利WO2019210835实施例5化合物，化学结构为：
Figure PCTCN2021112413-appb-000022
阳性对照2，为专利WO2019210835实施例6化合物，化学结构为：
Figure PCTCN2021112413-appb-000023
化合物1，结构式为：
Figure PCTCN2021112413-appb-000024
化合物2，结构式为：
Figure PCTCN2021112413-appb-000025
化合物4，结构式为：
Figure PCTCN2021112413-appb-000026
化合物5，结构式为：
Figure PCTCN2021112413-appb-000027
化合物6，结构式为：
Figure PCTCN2021112413-appb-000028
上述LOXO-195和对照化合物1-2可商购获得，也可依据常规路线合成制备获得，例如，LOXO-195可商购获得，阳性对照1和2可参照WO2019210835记载的方法合成。化合物1、2和4-6均可依据常规路线合成制备获得，也可参照WO2021115401A1记载的方法合成。
测试例1：实施例1制备的化合物Ib在1μM的剂量下对RET激酶的抑制作用。
2.1供试品制备
Figure PCTCN2021112413-appb-000029
2.2化合物剂量
供试品浓度：1μM
化合物孵育时间：20分钟
ATP浓度：10μM
反应时间：2小时
2.3：检测条件与方案
检测条件：
缓冲液配制：20mM HEPES(pH 7.5),10mM MgCl 2,1mM EGTA,0.01％Brij35 0.02mg/mL
BSA,0.1mM Na 3VO 4,2mM DTT,1％DMSO
*所需辅酶因子单独加入相应的激酶反应中
反应过程：
受试物以1μM的浓度进行单剂量实验(2个复孔)，对照品Staurosporine(Med Chem，MC-2104)初始浓度为20μM或者100μM，按1：4的比例连续稀释得到10个反应浓度。实验将测定受试物的单浓度抑制率和对照品的IC 50值。
1.配制反应缓冲液，用新配制的反应缓冲液制备相应底物；
2.将所需辅酶因子转移到上述底物溶液中；
3.将相应激酶加入到底物溶液中，并轻柔混匀；
4.使用声波移液器(Echo550)将溶解在DMSO中的供试品转移到激酶反应混合液中；
5.将10μM的 33P-ATP转移到上述溶液，此时反应开始；
6.在室温条件下，孵育120分钟；
7.将上述反应液点到P81离子交换层析纸(Whatman#3698-915)上；
8.用大量的0.75％的磷酸液冲洗层析纸；
9.检测离子交换层析纸上剩下的放射性磷酸化底物。
数据分析：
激酶活性表达为相应激酶与化合物Ib反应后激酶活性与溶媒组(DMSO)反应后激酶活性的百分比。抑制效率表达为1-％激酶活性。结果见表1。
表1.激酶活性
Figure PCTCN2021112413-appb-000030
Figure PCTCN2021112413-appb-000031
结果显示化合物Ib对多种RET突变或融合激酶均有较强抑制活性，在1μM能达到85％以上的抑制率，对某些RET突变及融合激酶能达到95％以上的抑制效率。
测试例2：化合物Ib在
Figure PCTCN2021112413-appb-000032
结肠癌CR2518皮下异种移植模型BALB/c裸小鼠模型上的体内药效学研究
1.实验材料及试剂
1.1试验动物：BALB/c Nude小鼠，雌性，6-8周(肿瘤细胞接种时的小鼠周龄)，体重21-25g，25只，购自北京安凯毅博生物技术有限公司，动物合格证编号：110330201100091758。饲养环境：SPF级。
1.2试验化合物
Figure PCTCN2021112413-appb-000033
1.3模型信息
Figure PCTCN2021112413-appb-000034
结肠癌异体移植模型CR2518信息
CR2518是源于一名女性患者的结肠肿瘤建立的
Figure PCTCN2021112413-appb-000035
异种移植模型。该模型为CCDC6-RET融合突变，具有肿瘤破溃倾向，可能会引起动物轻微的体重降低。
Figure PCTCN2021112413-appb-000036
2.试验方法
动物接种及分组：从
Figure PCTCN2021112413-appb-000037
结肠癌异种移植模型CR2518(R14P5)荷瘤小鼠收取肿瘤组织，切成直径为2-3mm的瘤块接种于BALB/c裸小鼠右前肩胛处皮下。肿瘤平均体积达到151mm 3时开始分组给药。试验分为4组，第1组为溶媒对照组，第2组为测试药物低剂量组(测试药物为化合物Ib，单次给药剂量为10mg/kg，QD*14天，之后BID*21天)、第3组为测试药物高剂量组(测试药物为化合物Ib，单次给药剂量为20mg/kg， QD*14天，之后BID*21天)、第4组为阳性对照组(阳性对照药为BLU-667，单次给药剂量为10mg/kg，BID*34天)组，每组5只，口服灌胃给药，给药后第35天结束实验。
3.肿瘤测量和实验指标
实验指标是考察肿瘤生长是否被抑制、延缓或治愈。每周两次用游标卡尺测量肿瘤直径。肿瘤体积的计算公式为：V＝0.5a×b 2，a和b分别表示肿瘤的长径和短径。
化合物的抑瘤疗效用TGI(％)或相对肿瘤增殖率T/C(％)评价。TGI(％)，反映肿瘤生长抑制率。TGI(％)的计算：TGI(％)＝[1-(某处理组给药结束时平均瘤体积－该处理组开始给药时平均瘤体积)/(溶剂对照组治疗结束时平均瘤体积－溶剂对照组开始治疗时平均瘤体积)]×100％。
相对肿瘤增殖率T/C(％)：计算公式如下：T/C(％)＝T RTV/C RTV×100％(T RTV：治疗组RTV；C RTV：溶媒对照组RTV)。根据肿瘤测量的结果计算出相对肿瘤体积(relative tumor volume，RTV)，计算公式为RTV＝V n/V 0，其中V 0是分组给药时(即d0)测量所得肿瘤体积，V n为某一次测量时的肿瘤体积，T RTV与C RTV取同一天数据。
在实验结束后将检测肿瘤重量，并计算T/C weight百分比，T weight和C weight分别表示给药组和溶媒对照组的瘤重。
4.统计分析
分析结果包括每个组的每个时间点的肿瘤体积的平均值和标准误(SEM)。治疗组在试验结束时给药后第35天表现显著的抑瘤作用，因此基于此数据进行统计学分析评估组间差异。首先使用Bartlett检验来验证所有组间的方差齐性假设。当Bartlett检验的p值不小于0.05时，单因素方差分析将被用于检验所有组均值是否相等。若单因素方差分析的p值小于0.05，我们将用Tukey HSD检验进行所有组之间两两比较，或用Dunnett’s t检验进行每个治疗组和对照组之间的两两比较。当Bartlett检验的p值小于0.05时，Kruskal Wallis检验将被用于检验所有组的中位数是否相等。若Kruskal Wallis检验的p值小于0.05，我们将用Conover检验进行所有组之间两两比较或每个治疗组和对照组之间的两两比较，并根据多重检验的组数进行相应的p值校正。所有的统计分析和图形绘制都在R语言环境中进行(3.3.1版)。所有检验均为双尾检验，p值小于0.05时被认为具有统计显著性。
5.试验结果
5.1测试药化合物Ib在
Figure PCTCN2021112413-appb-000038
结肠癌CR2518模型中的抗肿瘤作用研究结果和讨论
5.1.1各组小鼠平均肿瘤体积的生长曲线及瘤体积分析表见图1和表2。
表2.在
Figure PCTCN2021112413-appb-000039
结肠癌CR2518模型中各组瘤体积分析表
Figure PCTCN2021112413-appb-000040
注：1.数据以“平均值±标准误差”表示；2.T/C％＝T RTV/C RTV×100％；TGI％＝(1-T/C)×100％；3.使用Conover检验进行每个治疗组和对照组之间的比较。
5.2测试药化合物Ib和BLU-667在
Figure PCTCN2021112413-appb-000041
结肠癌CR2518模型中的安全性研究结果和讨论
5.2.1各组小鼠平均体重变化曲线及体重变化分析表见图2和表3。
表3.在
Figure PCTCN2021112413-appb-000042
结肠癌CR2518模型中各组体重变化情况
Figure PCTCN2021112413-appb-000043
注：1.数据以“平均值±标准误差”表示；2.G3-00442#小鼠在分组后第30天发现死亡；G3-00443#小鼠在分组后第33天发现死亡。
给药35天后，阳性对照组(BLU-667，10mg/kg)与溶媒对照组相比具有显著的抑瘤作用(T/C＝0.0％，TGI＝100％，p<0.001)；第2组(化合物Ib，10mg/kg)和第3组(化合物Ib，20mg/kg)的T/C值分别为29.3％和9.5％，TGI值分别为70.7％和90.5％，p值分别<0.05和<0.001，与溶媒对照组相比存在具有显著的抑瘤作用。测试药化合物Ib在10mg/kg使用剂量下无动物死亡，没有明显的药物毒性，治疗期间耐受良好。化合物Ib在20mg/kg使用剂量下每天一次给药，连续给药14天，小鼠未出现体重降低，没有表 现明显的药物毒性。随后化合物Ib在20mg/kg使用剂量下调整为每天两次给药，连续给药21天，小鼠出现体重降低，有部分动物死亡，表现出一定的药物毒性。测试药化合物Ib在所使用剂量下对
Figure PCTCN2021112413-appb-000044
结肠癌CR2518表现出明显抑制肿瘤生长的作用。荷瘤鼠对低剂量化合物Ib耐受良好。高剂量化合物Ib在对荷瘤鼠有一定的药物毒性。
根据上述小鼠的用药剂量可以推算出人的用药剂量，如下表所示。
Figure PCTCN2021112413-appb-000045
6.结论
测试药化合物Ib和阳性对照药BLU-667在所使用剂量下对
Figure PCTCN2021112413-appb-000046
结肠癌CR2518均具有显著抑制肿瘤生长的作用。荷瘤小鼠对化合物Ib和BLU-667在测试剂量下及低剂量下耐受良好。
测试例3：激酶抑制活性
该测试例委托桑迪亚医药技术(上海)有限责任公司完成。
1.实验目的
测定本申请化合物对TRKa、TRKA(G595R)和TRKC(G623R)三种激酶的抑制活性。
2.实验材料
2.2.1试剂及耗材
试剂名称 供货商 货号 批号
TRKa Carna 08-186 13CBS-0565G
TRKA(G595R) signalchem N16-12BG-100 H2714-7
TRKC(G623R) signalchem N18-12CH-100 D2567-8
激酶底物22 GL 112393 P190329-SL112393
DMSO Sigma D8418-1L SHBG3288V
384孔板(白色) PerkinElmer 6007290 810712
2.2.2仪器
离心机(生产厂家：Eppendorf，型号：5430)；酶标仪(生产厂家：Perkin Elmer，型号：Caliper EZ ReaderII)；Echo 550(生产厂家：Labcyte，型号：Echo 550)
3.实验方法
3.1受试化合物精确称量并溶解在100％DMSO中，配制成10mM溶液。
3.2激酶反应过程
3.2.1配制1×激酶缓冲液。
3.2.2化合物浓度梯度的配制：受试化合物测试起始浓度为1000nM，在384板中稀释成100倍终浓度的100％DMSO溶液，然后3倍稀释，得到10个浓度的化合物的DMSO溶液。使用分液器Echo 550向反应板OptiPlate-384F转移250nL 100倍终浓度的化合物。
3.2.3用1×激酶缓冲液配制2.5倍终浓度的激酶溶液。
3.2.4在化合物孔和阳性对照孔分别加10μL的2.5倍终浓度的激酶溶液；在阴性对照孔中加10μL的1×激酶缓冲液。
3.2.5反应板振荡混匀后室温孵育10分钟。
3.2.6用1×激酶缓冲液配制
Figure PCTCN2021112413-appb-000047
倍终浓度的ATP和激酶底物22的混合溶液。
3.2.7加入15μL的
Figure PCTCN2021112413-appb-000048
倍终浓度的ATP和底物的混合溶液。
3.2.8将384孔板1000rpm离心30秒，振荡混匀后室温孵育相应的时间。
3.2.9终止激酶反应。
3.2.10用酶标仪Caliper EZ Reader读取转化率。
4.数据分析
计算公式
抑制率％(Inhibition)＝(转化率％最大—转化率％样品)/(转化率％最大—转化率％最小)×100
其中：转化率％样品是样品的转化率读数；转化率％最小：阴性对照孔均值，代表没有酶活孔的转化率读数；转化率％最大：阳性对照孔比值均值，代表没有化合物抑制孔的转化率读数。
拟合量效曲线：以浓度的log值作为X轴，百分比抑制率为Y轴，采用分析软件GraphPad Prism 5的log(抑制剂)vs.响应-可变斜率(Variable slope)拟合量效曲线(四参数模型拟合)，从而得出各个化合物对酶活性的IC 50值。
结果如下表4显示：
表4.本申请化合物对三种激酶抑制活性的IC 50值
Figure PCTCN2021112413-appb-000049
Figure PCTCN2021112413-appb-000050
结果显示，化合物Ib对TRKa、TRKA(G595R)和TRKC(G623R)三种激酶具有高抑制活性。化合物Ib与化合物4-6相比，活性有显著差异，表明不同位置的氟取代对三种激酶抑制活性的影响区别较大。
测试例4：体外细胞活性
该测试例委托合肥中科普瑞昇生物医药科技有限公司完成，其中所用的NTRK突变细胞由该公司构建。
1.实验目的
测定本申请化合物对三种NTRK突变细胞(Ba/F3 LMNA-NTRK1-G667C，Ba/F3 EVT6-NTRK3-G623R，Ba/F3 LMNA-NTRK1-G595R)生长的抑制作用
2.试剂和耗材
细胞系：
Figure PCTCN2021112413-appb-000051
试剂：
胎牛血清FBS(GBICO,Cat#10099-141)；
Figure PCTCN2021112413-appb-000052
Luminescent Cell Viability Assay(CTG,Promega,Cat#G7573)；96孔透明平底黑壁板(
Figure PCTCN2021112413-appb-000053
Cat#165305)；RPMI-1640(Hyclone，Cat#SH30809.01)
3.实验过程
3.1细胞培养和接种：
收集处于对数生长期的细胞并采用血小板计数器进行细胞计数，调整细胞浓度至3-6×10 4cell/mL，添加90μL细胞悬液至96孔板中，将96孔板中的细胞置于37℃、5％CO 2、95％湿度条件下培养过夜。
3.2药物稀释和加药：
用含1％的DMSO的培养基将待测化合物配制10倍药物溶液，最高浓度为10μM，依次3倍稀释，共得到9个浓度的药物溶液。在接种细胞的96孔板中每孔加入10μL以上配制的药物溶液，再加入90μL含1％的DMSO的培养基，得到最高浓度为1μM的药物溶液，依次3倍稀释，共9个浓度，每孔中DMSO终浓度为0.1％，每个浓度的药物设置三个复孔。将已加药的96孔板中的细胞置于37℃、5％CO 2、95％湿度条件下继续培养72小时，之后进行CTG分析。
3.3终点读板
每孔加入等体积的CTG溶液，将96孔板放置于室温20分钟以稳定冷光信号，读取冷光值。
4.数据处理
使用GraphPad Prism 7.0软件分析数据，利用非线性S曲线回归来拟合数据得出剂量-效应曲线，并由此计算IC 50值。
细胞存活率(％)＝(待测药孔冷光值-培养液对照孔冷光值)/(细胞对照孔冷光值-培养液对照孔冷光值)×100％。
实验结果如下表5示：
表5.本申请化合物对三株细胞抑制活性的IC 50值
Figure PCTCN2021112413-appb-000054
结果显示，化合物Ib对三种NTRK突变细胞(Ba/F3LMNA-NTRK1-G667C，Ba/F3EVT6-NTRK3-G623R，Ba/F3LMNA-NTRK1-G595R)具有强的生长抑制作用。化合物1和2对三种NTRK突变细胞的抑制活性与对应的阳性化合物无明显差别，但化合物Ib对三种NTRK突变细胞的抑制活性明显高于对应的阳性对照2。
测试例5：肝微粒体稳定性试验
1.实验目的
测定本申请化合物在人、大鼠和小鼠肝微粒体中的稳定性
2.试验材料及仪器
试剂及耗材：
试剂名称 供应商 货号 批号
人肝微粒体 BiolVT X008070 SDL
大鼠肝微粒体 BiolVT M00001 TIQ
小鼠肝微粒体 Biopredic MIC255037 BQM
3.实验步骤
3.1缓冲液和肝微粒体按下表进行准备，制成孵化液：
试剂 浓度 体积
磷酸盐缓冲液 100mM 216.25μL
肝微粒体 20mg/mL 6.25μL
3.2分别进行了以下两个实验：a)添加辅酶因子NADPH的孵化体系：向孵化液(主要包含肝微粒体、磷酸盐缓冲液)中添加25μL NADPH(10mM)，使得肝微粒体和NADPH的终浓度分别为0.5mg/mL和1mM；b)不添加辅酶因子NADPH的孵化体系：向孵化液中添加25μL磷酸盐缓冲剂(100mM)，使得肝微粒体的最终浓度为0.5mg/mL。上述孵化体系分别在37℃预热10分钟。
3.3向前述“步骤3.2”中所述的各孵化体系中，分别通过添加2.5μL阳性对照化合物或本申请受试化合物溶液(100μM)开始反应，所述阳性对照为维拉帕米(购自Sigma)，使得本申请受试化合物或阳性对照化合物的最终浓度为1μM。将添加化合物后的孵化溶液在37℃的水中分批孵化。
3.4在0.5、5、15、30和45分钟时分别从反应溶液中取出30μL等分试样，通过加入5倍体积的含有200nM咖啡因和100nM甲苯磺丁脲的冷乙腈来终止反应。将等分试样在3220g重力加速度下离心40分钟，取100μL上清液与100μL超纯水混合，然后用于LC-MS/MS分析。
3.5数据分析
峰面积由提取的离子色谱图确定。斜率值k是通过母体药物的剩余百分比相对于孵育时间曲线的自然对数的线性回归来确定的。
根据斜率值分别计算确定体外半衰期(t 1/2)，通过体外半衰期平均值转换为体外固有清除率(CLint，以μL/min/mg蛋白表示)。
实验结果如下表6所示：
表6.本申请化合物在人、大鼠和小鼠肝微粒体中的稳定性数据
Figure PCTCN2021112413-appb-000055
结果显示，本申请化合物Ib在人、大鼠和小鼠肝微粒体中具有良好的稳定性。化合物1和2在人、大鼠和小鼠肝微粒体中的稳定性与对应的阳性化合物无明显差别，或在多数种属的肝微粒体中的稳定性变差，但本申请化合物Ib在人、大鼠和小鼠肝微粒体中的稳定性明显优于阳性对照2。
测试例6：SD大鼠静脉和口服给予受试化合物的体内药代动力学研究
1.试验动物
种属：SD大鼠，SPF级。来源：动物转移自实验机构动物储备库(999M-017)，上海西普尔-必凯实验动物有限公司。数量：每种剂型3只。
2.供试品配制
2.1准确称取适量的供试品，加入终体积5％DMSO、10％聚乙二醇-15羟基硬脂酸酯、85％生理盐水，涡旋或超声使充分混匀，得到0.2mg/mL的给药溶液，用于静脉注射给药。
2.2准确称取适量的供试品，加入终体积5％DMSO、10％聚乙二醇-15羟基硬脂酸酯、85％生理盐水，涡旋或超声使充分混匀，得到0.5mg/mL的给药溶液，用于口服灌胃给药。
3.实验设计
Figure PCTCN2021112413-appb-000056
Figure PCTCN2021112413-appb-000057
4.给药方式
给药前称重，根据体重，计算给药量。通过静脉或灌胃口服给药。
5.采血时间点
给药前及给药后0.083h，0.25h，0.5h，1h，2h，4h，6h，8h，24h。
6.样品采集和处置
经颈静脉或其它合适方式采血，每个样品采集约0.20mL，肝素钠抗凝，血液样本采集后放置于冰上，并于2小时内离心分离血浆(离心条件：离心力6800g，6分钟，2-8℃)。采集的血浆样本在分析前存放于-80℃冰箱内，分析后剩余血浆样本继续存放于-80℃冰箱暂存。
7.生物分析和数据处理
检测受试物血药浓度，进行血浆药物浓度-时间曲线绘制时，BLQ均记为0。进行药代参数计算时，给药前的浓度按照0计算；C max之前的BLQ(包括“No peak”)按照0计算；Cmax之后出现的BLQ(包括“No peak”)一律不参与计算。通过不同时间点的血药浓度数据，运用WinNonlin计算药代动力学参数，如AUC(0-t)，T 1/2，Cmax等。结果见表7。
表7.SD大鼠静脉和口服给予受试化合物的体内药代动力学研究数据
Figure PCTCN2021112413-appb-000058
Figure PCTCN2021112413-appb-000059
结果显示，化合物2在SD大鼠体内药代动力学参数(AUC/CL/F％)与阳性化合物相比变差，化合物Ib在SD大鼠体内药代动力学参数(AUC/CL/F％)与阳性对照2相比显著提高。
测试例7：受试化合物在MDCK-MDR1细胞系上的双向渗透性研究
1.1材料
MDCK-MDR1细胞购自荷兰癌症研究所，使用10到20代之间的细胞。
1.2试验设计
1.2.1细胞培养和种板
1)细胞接种前，向Transwell上室每孔中加入50μL细胞培养基，下层培养板内加入25mL细胞培养基。将培养板置于37℃,5％CO 2培养箱内孵育1小时后可用于接种细胞。
2)使用培养基重悬MDCK-MDR1细胞，使得终浓度为1.56×10 6cells/mL。将细胞悬液以50μL每孔加入到96孔Transwell培养板上室中。培养箱设置为37℃、5％CO 2、保证相对湿度95％培养4-8天。接种后48小时开始换液，培养4-8天，隔一天换一次培养基。
1.2.2细胞单层膜完整性的评价
1)将下层培养板中的原培养基移除并在上室加入新鲜预热的培养基。
2)用电阻仪(Millipore，USA)测量单层膜电阻，记录每孔电阻。
3)测定结束后，将Transwell培养板放回培养箱。
4)电阻值的计算：测定电阻值(ohms)×膜面积(cm 2)＝TEER值(ohm·cm 2)，若TEER值<42ohms·cm 2，则该孔不能用于穿透试验。
1.2.3溶液配制
1)使用DMSO将待测化合物配置成10mM的母液，
2)阳性对照化合物用DMSO配制为浓度10mM的母液。
1.2.4药物穿透试验
1)从培养箱中取出MDCK-MDR1Transwell培养板。使用预热的HBSS(25mM HEPES,pH 7.4)缓冲液润洗细胞单层膜两次，37℃条件下孵育30分钟。
2)对照化合物和试验化合物的原液在DMSO中稀释得到200μM溶液，然后用HBSS(10mM HEPES，pH7.4)稀释得到1μM工作溶液。孵育体系中DMSO的最终浓度为0.5％。
3)测定化合物由顶端到基底端的转运速率。向上层小室(顶端)加入125μL工作液，然后从下层小室(基底端)立刻转移50μL样本溶液加到装有200μL含内标的乙腈的96孔板作为0分钟给药样品(A-B)进行检测，并将235μL HBSS(25mM HEPES,pH 7.4)缓冲液加入到下层小室中。内标包含了(100nM阿普唑仑，200nM咖啡因和100nM甲苯磺丁酰胺)。将上述转移的50μL样本溶液以1000rpm速度涡旋10min。
4)测定化合物由基底端到顶端的转运速率。将285μL的工作溶液添加到下层小室(基底端)，然后立刻转移50μL至上层小室(顶端)样本溶液加到装有200μL含内标的乙腈的96孔板作为0分钟给药样品(B-A)进行检测，并将75μL HBSS(25mM HEPES,pH 7.4)缓冲液加入到上层小室中。内标包含了(100nM阿普唑仑，200nM咖啡因和100nM甲苯磺丁酰胺)，将上述转移的50μL样本溶液以1000rpm速度涡旋10min。由顶端到基底端方向与基底端到顶端的实验应同时进行。
5)下室和上室分别加入缓冲液后，将MDCK-MDR1 Transwell培养置于37℃条件下孵育2小时。
6)孵育结束后，分别从给药侧(上室：Ap→Bl flux，下室：Bl→Ap)与接收侧(下室：Ap→Bl flux，上室Bl→Ap)取50μL样本溶液至新的96孔板中，向孔板中加入4倍体积的含内标物的乙醇，内标物质包含(100nM阿普唑仑，200nM咖啡因和100nM甲苯磺丁酰胺)，涡旋10分钟后，于3220g离心40分钟。吸取100μL上清液与等体积超纯水混合后进行LC-MS/MS分析。
7)用荧光黄的渗漏评价孵育2小时后细胞单层膜的完整性。用HBSS(10mM HEPES,pH 7.4)稀释荧光黄储备液至最终浓度100μM，在上层小室(顶端)每个孔中加入荧光黄溶液100μL，下层小室(基底端)基底的每个孔中加300μL HBSS(25mM HEPES,pH 7.4)。37℃下孵育30分钟后，分别从每孔上下层吸出80μL溶液至一个新的96孔板中。使用酶标仪，激发波长485nm和发射波长530nm条件下进行荧光测定。
1.2.5分析条件
LC system:Shimadzu
MS analysis:Triple Quad 5500 instrument from AB Inc with an ESI interface
1)LC参数
柱温:40℃
色谱柱:Waters XSelect HSS T3 C18,2.5μM,2.1 x 50mm
流动相:0.1％甲酸溶于水(A)和0.1％甲酸溶于乙腈(B)
进样体积:5μL
洗脱速率:0.6mL/min
Time(min) 0 0.2 0.7 1.2 1.25 1.5
％B 5 5 95 95 5 5
2)MS参数
离子源:Turbo spray
电离模型:ESI
扫描类型:多反应检测(MRM)
帘气:35L/min
碰撞气:9L/min
载气:50L/min
辅助气体:50L/min
温度:500℃
离子喷雾电压:+5500V(positive)
1.3数据分析
峰面积由离子色谱结果计算得出。化合物的表观渗透系数(Papp，单位：cm/s×10 -6)用以下公式计算得出：
Figure PCTCN2021112413-appb-000060
公式中：V A为接收端溶液的体积(Ap→Bl是0.3mL，Bl→Ap是0.1mL)，Area为Transwell-96孔板膜面积(0.143cm 2)；time为孵育时间(单位：s)；[drug]为药物浓度。
实验结果如下表8所示：
表8.受试化合物在同批次MDCK-MDR1细胞系上的双向渗透性研究数据
Figure PCTCN2021112413-appb-000061
*外排比＝P app(B-A)/P app(A-B)
结果显示，化合物Ib的渗透性(P app(A-B))优于阳性对照2，表明本申请化合物Ib更容易吸收进入细胞，同时，本申请化合物Ib具有较低的外排比，表明本申请化合物Ib不易被外排，从而在细胞中能维持较高的药物浓度，产生更好的药效。因此，结合SD大鼠体内药代动力学研究，更加充分说明本申请化合物Ib在体内药代动力学参数(AUC/CL/F％)与阳性对照2相比具有显著提高。
测试例8：体外细胞活性评估
该测试例委托合肥中科普瑞昇生物医药科技有限公司完成，其中所用的细胞系由该公司构建。
1.实验目的
测定本申请化合物对6株BaF3细胞系的体外抗增殖作用。
2.试剂和耗材
细胞系：
细胞系 细胞类型 细胞数量/孔 培养基
Ba/F3-LMNA-NTRK1 悬浮 2000 RPMI 1640+10％FBS+1％PS
Ba/F3-LMNA-NTRK1-V573I 悬浮 2000 RPMI 1640+10％FBS+1％PS
Ba/F3-LMNA-NTRK1-F589L 悬浮 2000 RPMI 1640+10％FBS+1％PS
Ba/F3-LMNA-NTRK1-G667S 悬浮 2000 RPMI 1640+10％FBS+1％PS
Ba/F3-TEL-NTRK2 悬浮 2000 RPMI 1640+10％FBS+1％PS
Ba/F3-TEL-NTRK3 悬浮 2000 RPMI 1640+10％FBS+1％PS
材料：
Figure PCTCN2021112413-appb-000062
Figure PCTCN2021112413-appb-000063
3.实验过程
细胞处理及给药
细胞培养条件
6株Ba/F3细胞系采用RPMI 1640(Biological Industries，Israel)+10％胎牛血清(Biological Industries，Israel)+1％双抗(Penicillin Streptomycin solution，Coring，USA)进行培养，细胞复苏后培养两代，待测试。
1000×化合物的制备
待测化合物溶于DMSO中配成的10mM的母液，再用DMSO稀释至1mM。按照3倍稀释制备成1.0000mM、0.3333mM、0.1111mM、0.03704mM、0.01235mM、0.00412mM、0.00137mM、0.00046mM、0.00015mM、0.00005mM储存于96孔药板中(Beaver,Suzhou)，共10个浓度梯度，同时采用同等体积的DMSO溶剂作为阴性对照。
20×化合物制备
将制备好的10个浓度梯度1000×的待测化合物，分别用完全培养基稀释50倍至20×化合物，储存于96孔药板中(Beaver,Suzhou)，共10个浓度梯度，同时采用同等体积的DMSO溶剂作为阴性对照。
铺板
取对数生长期细胞悬液，接种于96孔白色细胞培养板(Corning 3917，NY，USA)，每孔体积为95μl(约2000个细胞/孔)。
取5μl 20×待测化合物分别加入上述含有95μl细胞悬液的培养板中，混匀，每个浓度梯度2个孔。待测化合物最终检测浓度分别为1.0000μM、0.3333μM、0.1111μM、0.03704μM、0.01235μM、0.00412μM、0.00137μM、0.00046μM、0.00015μM、0.00005μM。
将培养板37℃、5％CO 2培养箱中孵育72小时。
读板
以下步骤按照Promega CellTiter-Glo发光法细胞活性检测试剂盒(Promega-G7573)的说明书来进行。
(1).将CellTiter-Glo缓冲液融化并放置至室温。
(2).将CellTiter-Glo底物放置至室温。
(3).在一瓶CellTiter-Glo底物中加入CellTiter-Glo缓冲液以溶解底物，从而配制CellTiter-Glo工作液。
(4).缓慢涡旋震荡使充分溶解。
(5).取出细胞培养板放置10分钟使其平衡至室温。
(6).在每孔中加入50μl的CellTiter-Glo工作液。
(7).将培养板在轨道摇床上振摇2分钟以诱导细胞裂解。
(8).培养板在室温放置10分钟以稳定发光信号。
(9).在MD SpectraMax Paradigm读板器上检测发光信号。
4.数据分析
SpectraMax Paradigm读数得出对应的每孔荧光值RLU。细胞活力(Cell viability)数据采用下列公式来处理：
Cell viability(％)＝(RLU Drug-RLU Min)/(RLU Max-RLU Min)*100％。在EXCEL中计算不同浓度化合物对应的细胞活力，然后用GraphPad Prism软件作细胞活力曲线图并计算相关参数，参数包括细胞最大和最小活力，IC 50值。
实验结果如下表9示：
表9.本申请化合物对同批次6株BaF3细胞系抑制活性的IC 50值
Figure PCTCN2021112413-appb-000064
从具体实施例化合物生物活性数据来看，本申请化合物Ib对6株BaF3细胞系具有强的生长抑制作用。化合物2对6株BaF3细胞系的抑制活性与对应的阳性化合物无明显差别，但本申请化合物Ib对6株BaF3细胞系的抑制活性明显高于阳性对照2。

Claims (17)

  1. 式(I)所示化合物、其立体异构体或其药学上可接受的盐在制备用于治疗RET相关疾病的药物中的用途，
    Figure PCTCN2021112413-appb-100001
  2. 式(I)所示化合物、其立体异构体或其药学上可接受的盐，其用于治疗RET相关疾病，
    Figure PCTCN2021112413-appb-100002
  3. 一种治疗RET相关疾病的方法，包括给有需要的受试者施用有效量的式(I)所示化合物、其立体异构体或其药学上可接受的盐，
    Figure PCTCN2021112413-appb-100003
  4. 一种用于治疗RET相关疾病的组合物或药物，其中包含式(I)所示化合物、其立体异构体或其药学上可接受的盐，任选地还包含药学可接受的载体和/或赋形剂，
    Figure PCTCN2021112413-appb-100004
    优选地，所述的组合物中，式(I)所示化合物、其立体异构体或其药学上可接受的盐以治疗RET相关疾病的有效量存在。
  5. 权利要求1的用途、权利要求2的化合物、其立体异构体或其药学上可接受的盐、权利要求3的方法或权利要求4的组合物或药物，其中所述式(I)化合物为式(Ia)所示的化合物，
    Figure PCTCN2021112413-appb-100005
  6. 权利要求1的用途、权利要求2的化合物、其立体异构体或其药学上可接受的盐、权利要求3的方法或权利要求4的组合物或药物，其中所述式(I)化合物为式(Ib)所示的化合物，
    Figure PCTCN2021112413-appb-100006
  7. 权利要求1的用途、权利要求2的化合物、其立体异构体或其药学上可接受的盐、权利要求3的方法或权利要求4的组合物或药物，其中所述RET相关疾病是RET基因或RET激酶蛋白的表达、活性或水平调节异常相关的疾病。
  8. 权利要求1的用途、权利要求2的化合物、其立体异构体或其药学上可接受的盐、权利要求3的方法或权利要求4的组合物或药物，其中所述RET相关疾病是RET基因或RET激酶蛋白突变相关的疾病或RET融合基因相关的疾病，
    优选地，所述RET基因或RET激酶蛋白突变包含一个或多个位点上的突变。
  9. 权利要求8的用途、化合物、其立体异构体或其药学上可接受的盐、方法、组合物或药物，其中所述RET融合基因选自：BCR-RET、CLIP1-RET、KIF5B-RET、CCDC6-RET、NCOA4-RET、TRIM33-RET、ERC1-RET、FGFR1OP-RET、RET-MBD1、RET-RAB6IP2、RET-PRKAR1A、RET-TRIM24、RET-GOLGA5、HOOK3-RET、 KTN1-RET、TRIM27-RET、AKAP13-RET、FKBP15-RET、SPECC1L-RET、TBL1XR1-RET、CEP55-RET、CUX1-RET、KIAA1468-RET、RFG8-RET、ACBD5-RET、PTC1ex9-RET、MYH13-RET、PIBF1-RET、KIAA1217-RET、MPRIP-RET、HRH4-RET、Ria-RET、RET-PTC4、FRMD4A-RET、SQSTM1-RET、AFAP1L2-RET、PPFIBP2-RET、EML4-RET、PARD3-RET、MYH10-RET、HTIF1-RET、AFAP1-RET、RASGEF1A-RET、TEL-RET、RUFY1-RET、UEVLD-RET、DLG5-RET、FOXP4-RET、OFLM4-RET、RRBP1-RET及其任意组合。
  10. 权利要求8的用途、化合物、其立体异构体或其药学上可接受的盐、方法、组合物或药物，其中所述RET融合基因选自：RET-CCDC6(PTC1)、RET-KIF5B(Kex15Rex14)、RET-PRKAR1A(PTC2)、RET-BCR、RET-NCOA4(PTC3)及其任意组合。
  11. 权利要求8的用途、化合物、其立体异构体或其药学上可接受的盐、方法、组合物或药物，其中所述RET融合基因选自：RET(V804L)-KIF5B、RET(V804M)-KIF5B及其任意组合。
  12. 权利要求8的用途、化合物、其立体异构体或其药学上可接受的盐、方法、组合物或药物，其中所述RET基因突变选自：RET(Y791F)、RET(V778I)、RET(G691S)、RET(V804L)、RET(R813Q)、RET(E762Q)、RET(V804E)、RET(V804L)-KIF5B、RET(A883F)、RET(S904F)、RET(V804M)、RET(V804M)-KIF5B、RET(Y806H)、RET(M918T)及其任意组合。
  13. 权利要求1的用途、权利要求2的化合物、其立体异构体或其药学上可接受的盐、权利要求3的方法或权利要求4的组合物或药物，其中所述RET相关疾病为RET基因或RET激酶蛋白的表达、活性或水平调节异常相关的癌症。
  14. 权利要求13的用途、化合物、其立体异构体或其药学上可接受的盐、方法、组合物或药物，其中所述RET相关疾病为RET基因或RET激酶蛋白突变相关的癌症，或RET融合基因相关的癌症，
    优选地，所述RET基因或RET激酶蛋白突变包含一个或多个位点上的突变。
  15. 权利要求14的用途、化合物、其立体异构体或其药学上可接受的盐、方法、组合物或药物，其中：
    所述RET基因突变相关的癌症是选自：RET基因突变相关的肺癌、乳头状甲状腺癌、甲状腺髓样癌、分化型甲状腺癌、复发性甲状腺癌、难治性分化型甲状腺癌、多发性内分泌瘤形成2A或2B型(分别是MEN2A或MEN2B)、嗜铬细胞瘤、甲状旁腺增生、乳腺癌、 结肠癌、结肠直肠癌、乳头状肾细胞癌、胃肠粘膜神经节瘤和宫颈癌中的一种或多种，
    所述RET融合基因相关的癌症是选自：RET融合基因相关的肺癌、乳头状甲状腺癌、甲状腺髓样癌、分化型甲状腺癌、复发性甲状腺癌、难治性分化型甲状腺癌、多发性内分泌瘤形成2A或2B型(分别是MEN2A或MEN2B)、嗜铬细胞瘤、甲状旁腺增生、乳腺癌、结肠癌、结肠直肠癌、乳头状肾细胞癌、胃肠粘膜神经节瘤和宫颈癌中的一种或多种。
  16. 权利要求15的用途、化合物、其立体异构体或其药学上可接受的盐、方法、组合物或药物，其中：所述RET相关疾病是RET基因突变相关的肺癌、RET基因突变相关的甲状腺髓样癌或RET基因突变相关的结肠癌，或者所述RET相关疾病是RET融合基因相关的肺癌、RET融合基因相关的甲状腺髓样癌或RET融合基因相关的结肠癌，
    优选地，所述RET相关疾病是RET-CCDC6相关的结肠癌，
    优选地，所述RET相关疾病是选自RET基因突变相关的小细胞肺癌、RET基因突变相关的非小细胞肺癌、RET基因突变相关的细支气管肺细胞癌或RET基因突变相关的肺腺癌中的一种或多种，
    优选地，所述RET相关疾病是选自RET融合基因相关的小细胞肺癌、RET融合基因相关的非小细胞肺癌、RET融合基因相关的细支气管肺细胞癌或RET融合基因相关的肺腺癌中的一种或多种。
  17. 权利要求1的用途、权利要求2的化合物、其立体异构体或其药学上可接受的盐、权利要求3的方法或权利要求4的组合物或药物，其中RET相关疾病是人的RET相关疾病，式(I)所示化合物、其立体异构体或其药学上可接受的盐的给药剂量为0.5-4mg/kg/day。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022218276A1 (zh) * 2021-04-12 2022-10-20 成都倍特药业股份有限公司 含氟大环结构化合物的固体形态、制备方法和应用

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115813930A (zh) * 2021-08-17 2023-03-21 赛诺哈勃药业（成都）有限公司 大环化合物与mek抑制剂联用在用于治疗疾病药物中的用途

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102971322A (zh) * 2010-05-20 2013-03-13 阵列生物制药公司 作为trk激酶抑制剂的大环化合物
CN107735399A (zh) * 2015-07-02 2018-02-23 Tp生物医药公司 作为蛋白质激酶的调节剂的手性二芳基大环
WO2019210835A1 (zh) * 2018-05-04 2019-11-07 正大天晴药业集团股份有限公司 作为蛋白激酶调节剂的二芳基大环化合物
WO2021115401A1 (zh) * 2019-12-13 2021-06-17 成都倍特药业股份有限公司 具有大环结构的含氟并杂环衍生物及其用途

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20210131432A (ko) * 2010-12-30 2021-11-02 파운데이션 메디신 인코포레이티드 종양 샘플의 다유전자 분석의 최적화
EP3571203B1 (en) * 2017-01-18 2023-06-07 Array BioPharma Inc. Substituted pyrazolo[1,5-a]pyrazine compounds as ret kinase inhibitors

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102971322A (zh) * 2010-05-20 2013-03-13 阵列生物制药公司 作为trk激酶抑制剂的大环化合物
CN107735399A (zh) * 2015-07-02 2018-02-23 Tp生物医药公司 作为蛋白质激酶的调节剂的手性二芳基大环
WO2019210835A1 (zh) * 2018-05-04 2019-11-07 正大天晴药业集团股份有限公司 作为蛋白激酶调节剂的二芳基大环化合物
WO2021115401A1 (zh) * 2019-12-13 2021-06-17 成都倍特药业股份有限公司 具有大环结构的含氟并杂环衍生物及其用途

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022218276A1 (zh) * 2021-04-12 2022-10-20 成都倍特药业股份有限公司 含氟大环结构化合物的固体形态、制备方法和应用

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