WO2022033575A1 - Application of fluorine-containing heterocyclic derivative having macrocyclic structure - Google Patents

Abstract

The present invention relates to a use of a compound represented by formula (I), or a stereoisomer or pharmaceutically acceptable salt thereof in the preparation of a drug for the treatment of RET-related diseases.

Description

Application of Fluorinated Heterocyclic Derivatives with Macrocyclic Structure

This application is based on the CN application number 202010820363.5 and the filing date is August 14, 2020, and claims its priority. The disclosure of the CN application is hereby incorporated into this application as a whole.

technical field

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.

Background technique

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). The activation of RET is completed by the interaction between the four receptors of growth factor receptor α1/2/3/4 (GFRα1/2/3/4) and the four ligands of GDNFs, and GFRα 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-γ pathway, JAK-STAT pathway), etc. (Matti S. Airaksinen. Mol Cell Neurosci. 1999.13 (313-325)). Under normal conditions, 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. In other cancers, 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)). In preclinical and clinical studies, RET inhibitors have been shown to significantly inhibit the proliferation of tumor cells with RET gene fusions and mutations.

At present, 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. The FDA approved LOXO-292 (Selpercatinib) in May 2020 for the treatment of patients with RET fusion gene-positive non-small cell lung cancer, advanced or metastatic RET-mutated medullary thyroid cancer, and advanced or metastatic RET fusions It is the first RET inhibitor to be marketed in patients with gene-positive thyroid cancer. 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. In addition, there are other RET small molecule inhibitors in preclinical development.

As a proto-oncogene, positive mutations or fusions of RET play a key role in the proliferation and growth of tumors such as non-small cell lung cancer, medullary thyroid cancer, and thyroid cancer. More and more studies have shown that 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.

SUMMARY OF THE INVENTION

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. In certain embodiments, in the composition, 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. In certain embodiments, in the composition, 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.

In certain embodiments, the compound of formula (I) is a compound of formula (Ia).

In certain embodiments, the compound of formula (I) is a compound of formula (Ib).

In certain embodiments, the RET-related disease is a disease associated with dysregulation of the expression, activity or level of RET gene or RET kinase protein.

In certain embodiments, the RET-related disease is a disease associated with a mutation in a RET gene or a RET kinase protein. In certain embodiments, the RET gene or RET kinase protein mutation comprises a mutation at one or more sites.

In certain embodiments, the RET-related disease is a RET fusion gene-related disease.

In certain embodiments, 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 , 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 and any combination thereof.

In certain embodiments, 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 .

In certain embodiments, the RET fusion gene is selected from the group consisting of: RET(V804L)-KIF5B, RET(V804M)-KIF5B, and any combination thereof.

In certain embodiments, the RET fusion gene is RET(V804M)-KIF5B.

In certain embodiments, the RET fusion gene is RET(V804L)-KIF5B.

In certain embodiments, the RET fusion gene is RET-NCOA4 (PTC3).

In certain embodiments, the RET fusion gene is RET-CCDC6 (PTC1).

In certain embodiments, the RET fusion gene is RET-KIF5B (Kex15Rex14).

In certain embodiments, the RET fusion gene is RET-PRKAR1A (PTC2).

In certain embodiments, 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.

In certain embodiments, the RET gene is mutated to RET(S891A).

In certain embodiments, the RET gene is mutated to RET(L790F).

In certain embodiments, the RET gene is mutated to RET(R749T).

In certain embodiments, the RET gene is mutated to RET (S904A).

In certain embodiments, the RET gene is mutated to RET(R912P).

In certain embodiments, the RET gene is mutated to RET (Y791F).

In certain embodiments, the RET gene is mutated to RET(V778I).

In certain embodiments, the RET gene is mutated to RET(G691S).

In certain embodiments, the RET gene is mutated to RET(V804L).

In certain embodiments, the RET gene is mutated to RET(R813Q).

In certain embodiments, the RET gene is mutated to RET(E762Q).

In certain embodiments, the RET gene is mutated to RET(V804E).

In certain embodiments, the RET gene is mutated to RET(A883F).

In certain embodiments, the RET gene is mutated to RET(S904F).

In certain embodiments, the RET gene is mutated to RET(Y806H).

In certain embodiments, the RET gene is mutated to RET (M918T).

In certain embodiments, the RET-related disease is a cancer associated with dysregulation of RET gene or RET kinase protein expression, activity, or level.

In certain embodiments, the RET-related disease is a cancer associated with a mutation in a RET gene or a RET kinase protein. In certain embodiments, the RET gene or RET kinase protein mutation comprises a mutation at one or more sites.

In certain embodiments, 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.

In certain embodiments, the RET-related disease is RET gene mutation-related lung cancer.

In certain embodiments, the RET-related disease is medullary thyroid cancer associated with a RET gene mutation.

In certain embodiments, the RET-related disease is RET gene mutation-related colon cancer.

In certain embodiments, 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.

In certain embodiments, the RET-related disease is RET gene mutation-related small cell lung cancer.

In certain embodiments, the RET-related disease is RET gene mutation-related non-small cell lung cancer.

In certain embodiments, the RET-related disease is RET gene mutation-related bronchiolopulmonary cell carcinoma.

In certain embodiments, the RET-related disease is a RET gene mutation-related lung adenocarcinoma.

In certain embodiments, the RET-related disease is a RET fusion gene-related cancer.

In certain embodiments, 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.

In certain embodiments, the RET-related disease is a RET fusion gene-related lung cancer.

In certain embodiments, the RET-related disease is RET fusion gene-related medullary thyroid cancer.

In certain embodiments, the RET-related disease is RET fusion gene-related colon cancer.

In certain embodiments, the RET-related disease is RET-CCDC6-related colon cancer.

In certain embodiments, 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.

In certain embodiments, the RET-related disease is RET fusion gene-related small cell lung cancer.

In certain embodiments, the RET-related disease is RET fusion gene-related non-small cell lung cancer.

In certain embodiments, the RET-related disease is RET fusion gene-related bronchiolopulmonary cell carcinoma.

In certain embodiments, the RET-related disease is a RET fusion gene-related lung adenocarcinoma.

In certain embodiments, the RET-related disease is a human RET-related disease, and 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.

definition:

In this application, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. For a better understanding of the present application, definitions and explanations of related terms are provided below.

The term "stereoisomer" used in this application 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.

The term "RET (Rearranged during Transfection) gene" used in this application is a proto-oncogene, full name: 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.

The term "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.

The term "RET fusion gene" used in this application 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. In this application, "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. For example, RET-CCDC6 and CCDC6-RET have the same meaning, and they both mean the fusion gene of RET and CCDC6.

The term "RET gene mutation" used in this application 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.

The term "pharmaceutically acceptable salts" as used herein 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.

When referring to the compound of the present application in the present application, it includes the compound represented by formula (I), its stereoisomer or its pharmaceutically acceptable salt.

As used herein, 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.

As used herein, 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.

The term "treatment" as used herein 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.

In addition, it should be noted that 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.

For the compounds described in this application, if the name and the structural formula of the same compound are inconsistent, the structural formula of the compound shall prevail.

Description of drawings

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.

In Figure 1 and Figure 2: 1. The data are expressed as "mean ± standard error"; 2. G3-00442# mice were found dead on the 30th day after grouping; G3-00443# mice were found on the 33rd day after grouping Death; 3. BID means twice a day dosing; QD means once a day dosing.

detailed description

The substantive content of the present application will be further described below with reference to the specific embodiments of the present application. It should be understood that the following embodiments are only used to illustrate the present application, but are not intended to limit the protection scope of the present application. If the specific conditions are not indicated in the following examples, it is carried out according to the conventional conditions or the manufacturer's suggestion. The raw materials used without specifying the manufacturer are all conventional products that can be obtained from the market.

While many of the materials and methods of operation used in the following examples are known in the art, the present application is described in as much detail as possible herein. It is clear to those skilled in the art that the materials and operating methods used in the following examples are well known in the art unless otherwise specified.

The compounds used as RET inhibitors provided in the present application and their preparation methods and uses will be described in detail below with reference to the examples.

The following abbreviations have the meanings shown below:

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 means sodium hydroxide;

DIPEA or DIEA means N,N-diisopropylethylamine;

H ₂ O represents water;

HCl/Dioxane means hydrogen chloride dioxane solution;

rt means the reaction temperature is room temperature;

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;

EtOH means ethanol;

OTs represents p-methylbenzenesulfonyloxy,

ee indicates enantiomeric excess.

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 (R749T) 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) means that the 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 (M918T) represents the mutation of methionine at position 918 of the RET gene to threonine.

RET-CCDC6 (PTC1) 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 (PTC2) 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 (PTC3) 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).

Preparation Example 1: Preparation of ethyl 5-chloro-6-fluoropyrazolo[1,5-a]pyrimidine-3-carboxylate

Step 1: Preparation of 2-fluoromalonic acid

At room temperature, weigh diethyl 2-fluoromalonate (5.0 g) and sodium hydroxide (17.3 g) into a mixed solution of ethanol/water (100/100 mL), react overnight, and LCMS shows that the reaction is complete. The reaction solution was concentrated to remove ethanol, water (50 mL) was added, the pH was adjusted to about 1 with concentrated hydrochloric acid, extracted four times with methyl tert-butyl ether, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 3.7 g of the title compound, It was used in the next reaction without purification.

MS(ESI) m/z(MH) ⁺ = 121.1.

Step 2: Preparation of ethyl 5,7-dichloro-6-fluoropyrazolo[1,5-a]pyrimidine-3-carboxylate

At room temperature, 2-fluoromalonic acid (2.0 g) and ethyl 5-amino-1H-pyrazole-4-carboxylate (1.7 g) were weighed into phosphorus oxychloride (20 mL), and N,N - Dimethylformamide (2 mL) and N,N-diethylaniline (4.9 g) were added, and the temperature was raised to 110° C. to react for 3 hours. LCMS showed that the reaction was complete. The 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.

MS(ESI) m/z(M+H) ⁺ = 278.0.

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.80 (s, 1H), 4.33 (q, J=7.2 Hz, 2H), 1.32 (t, J=7.0 Hz, 3H).

Step 3: Preparation of ethyl 5-chloro-6-fluoropyrazolo[1,5-a]pyrimidine-3-carboxylate

Weigh 5,7-dichloro-6-fluoropyrazolo[1,5-a]pyrimidine-3-carboxylic acid ethyl ester (1.14g) and ammonium chloride (800mg) in ethanol/tetrahydrofuran/water (30/ 10/20mL) in the mixed solution, add zinc powder (1.3g) during stirring, filter the zinc powder after 5 minutes of reaction, wash the filter cake with ethyl acetate, collect the filtrate, dry over anhydrous sodium sulfate, filter, and concentrate, the resulting The crude product was purified by column chromatography to obtain 800 mg of the title compound.

MS(ESI) m/z(M+H) ⁺ = 244.0.

¹ H NMR (400MHz, DMSO-d ₆ ) δ 9.93 (d, J=4.4Hz, 1H), 8.68 (s, 1H), 4.31 (q, J=7.2Hz, 2H), 1.31 (t, J= 7.2Hz, 3H).

Preparation Example 2: Preparation of (R)-1-(5-fluoro-2-methoxypyridin-3-yl)ethan-1-amine hydrochloride

Step 1: Preparation of (R)-N-(((5-fluoro-2-methoxypyridin-3-yl)methylene)-2-methylpropane-2-sulfinamide

(R)-2-methylpropane-2-sulfinamide (12.9 g) was dissolved in tetrahydrofuran (100 mL), 5-fluoro-2-methoxynicotinaldehyde (15.0 g) and cesium carbonate ( 40.9g). The resulting mixture was reacted at room temperature for 2 hours, TLC showed complete consumption of starting material. Suction filtration, the filter cake was washed three times with tetrahydrofuran, the obtained filtrate was backwashed once with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered and concentrated. The obtained crude product was purified by column chromatography to obtain 23.0 g of the title compound.

MS(ESI) m/z(M+H) ⁺ = 259.1.

¹ H NMR (400MHz, DMSO-d ₆ ) δ 8.67 (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).

Step 2: Preparation of (R)-N-((R)-1-(5-fluoro-2-methoxypyridin-3-yl)ethyl)-2-methylpropane-2-sulfinamide

Weigh (R)-N-(((5-fluoro-2-methoxypyridin-3-yl)methylene)-2-methylpropane-2-sulfinamide (5.0g) and dissolve it in tetrahydrofuran ( 40mL), the obtained mixture was cooled to -78°C and slowly added dropwise methylmagnesium bromide (7.8mL, 3M), maintaining the temperature below -65°C. After dropping, the temperature was naturally returned to room temperature, and the reaction was continued for 1 hour. TLC It showed that the reaction was complete. The reaction system was poured into saturated aqueous ammonium chloride solution (1 L), 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 a column. Purification by chromatography gave 4.5 g of the title compound.

MS(ESI) m/z(M+H) ⁺ = 275.2.

¹ H NMR (400MHz, DMSO-d ₆ ) δ 8.04 (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).

Step 3: Preparation of (R)-1-(5-fluoro-2-methoxypyridin-3-yl)ethan-1-amine hydrochloride

At room temperature, (R)-N-((R)-1-(5-fluoro-2-methoxypyridin-3-yl)ethyl)-2-methylpropane-2-sulfinamide (4.5 g) Dissolved in hydrogen chloride-dioxane solution (30 mL), reacted overnight, LCMS showed that the starting material was completely consumed. The reaction solution was concentrated to obtain 3.1 g of crude product, greater than 95% ee, which was directly used in the next step without purification.

MS(ESI) m/z(M+H) ⁺ = 171.2.

¹ H NMR (400MHz, DMSO-d ₆ ) δ 8.80-8.66 (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).

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

(R)-1-(5-Fluoro-2-methoxypyridin-3-yl)ethan-1-amine hydrochloride (1.0 g) was dissolved in acetonitrile (20 mL), followed by N,N-di Isopropylethylamine (1.9g) and ethyl 5-chloro-6-fluoropyrazolo[1,5-a]pyrimidine-3-carboxylate (1.2g), the resulting mixture was reacted at 60°C for 3 hours, TLC showed the reaction was complete. The 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.

MS(ESI) m/z(M+H) ⁺ = 378.2.

¹ H 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).

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

(R)-6-Fluoro-5-((1-(5-fluoro-2-methoxypyridin-3-yl)ethyl)amino)pyrazolo[1,5-a]pyrimidine Ethyl-3-carboxylate (1.1 g) was dissolved in a mixed solution of ethanol/water (5/15 mL), sodium hydroxide (584 mg) was added, the resulting mixture was reacted at 50°C overnight, and TLC showed that the reaction was complete. The 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.

MS(ESI) m/z(M+H) ⁺ =350.1.

¹ H NMR (400MHz, DMSO-d ₆ ) δ 11.68 (s, 1H), 9.13 (d, J=6.0 Hz, 1H), 8.51 (d, J=7.6 Hz, 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=6.8 Hz, 3H).

Step 3: (1R,3r)-3-(6-Fluoro-5-(((R)-1-(5-fluoro-2-methoxypyridin-3-yl)ethyl)amino)pyrazolo Preparation of [1,5-a]pyrimidine-3-carboxamido)-4-methylbenzenesulfonic acid cyclobutyl ester

(R)-6-Fluoro-5-(((1-(5-fluoro-2-methoxypyridin-3-yl)ethyl)amino)pyrazolo[1,5-a]pyrimidine-3 -Carboxylic acid (800mg), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (1.0g) and N,N-diiso Propylethylamine (886mg) was dissolved in dry tetrahydrofuran (10mL), the resulting mixture was reacted at room temperature for 1 hour, and then tert-butyl (3-hydroxycyclobutyl)carbamate hydrochloride (953mg) was added, and the reaction was continued for 1 After 2 hours, TLC showed that the reaction was complete. The 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.

MS(ESI) m/z(M+H) ⁺ =573.2.

¹ H NMR (400MHz, DMSO-d ₆ ) δ 9.21 (d, J=6.0 Hz, 1H), 8.57 (d, J=7.6 Hz, 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).

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

(1R,3r)-3-(6-fluoro-5-(((R)-1-(5-fluoro-2-methoxypyridin-3-yl)ethyl)amino)pyrazolo[1 ,5-a]pyrimidine-3-carboxamido)-4-methylbenzenesulfonic acid cyclobutyl ester (800 mg) was dissolved in hydrogen chloride in dioxane (4M, 10 mL), and the resulting mixture was reacted at 55°C Overnight, TLC showed the reaction was complete. The reaction solution was directly concentrated to remove most of the dioxane to obtain 600 mg of crude product, which was directly used in the next step without purification.

MS(ESI) m/z(M+H) ⁺ =559.2.

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

(1R,3r)-3-(6-fluoro-5-(((R)-1-(5-fluoro-2-hydroxypyridin-3-yl)ethyl)amino)pyrazolo[1,5 -a]Pyrimidine-3-carboxamido)-4-methylbenzenesulfonate cyclobutyl hydrochloride (250mg) was dissolved in N,N-dimethylformamide (6mL), potassium carbonate (232mg) was added ), the resulting mixture was reacted at room temperature for 5 hours, and TLC showed that the reaction was complete. The 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%.

MS(ESI) m/z(M+H) ⁺ = 387.1.

¹ H 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).

The compounds used in Test Examples 1-7 of this application include:

Compound Ib, prepared from Example 1 of the present application;

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:

Compound 2, the structural formula is:

Compound 4, the structural formula is:

Compound 5, the structural formula is:

Compound 6, the structural formula is:

The above-mentioned LOXO-195 and control compounds 1-2 are commercially available, and can also be synthesized and prepared according to conventional routes. For example, 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 μM.

2.1 Preparation of the test product

2.2 Compound Dosage

Concentration of test substance: 1 μM

Compound incubation time: 20 minutes

ATP concentration: 10 μM

Response time: 2 hours

2.3: Detection conditions and plans

Detection conditions:

Buffer preparation: 20 mM HEPES (pH 7.5), 10 mM MgCl ₂ , 1 mM EGTA, 0.01% Brij35 0.02 mg/mL

BSA, 0.1 mM Na ₃ VO ₄ , 2 mM DTT, 1% DMSO

*The required coenzyme factor is added separately to the corresponding kinase reaction

reaction process:

The test substance was used in a single-dose experiment (2 duplicate wells) at a concentration of 1 μM, and the initial concentration of the reference substance Staurosporine (Med Chem, MC-2104) was 20 μM or 100 μM, 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.

1. Prepare the reaction buffer and prepare the corresponding substrate with the newly prepared reaction buffer;

2. Transfer the desired coenzyme factor into the above-mentioned substrate solution;

3. Add the corresponding kinase to the substrate solution and mix gently;

4. Use a sonic pipette (Echo550) to transfer the test substance dissolved in DMSO to the kinase reaction mixture;

5. Transfer 10 μM of ³³ P-ATP to the above solution, at which point the reaction begins;

6. Incubate for 120 minutes at room temperature;

7. Spot the above reaction solution on P81 ion exchange chromatography paper (Whatman#3698-915);

8. Rinse the chromatography paper with a large amount of 0.75% phosphoric acid solution;

9. Detect the radiophosphorylated substrate remaining on the ion exchange chromatography paper.

data analysis:

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.

Table 1. Kinase Activity

The results show that compound Ib has strong inhibitory activity against a variety of RET mutants or fusion kinases, with an inhibition rate of over 85% at 1 μM, and an inhibition rate of over 95% for some RET mutants and fusion kinases.

Test Example 2: Compound Ib in

In vivo pharmacodynamic study of colon cancer CR2518 subcutaneous xenograft model in BALB/c nude mouse model

1. Experimental materials and reagents

1.1 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.

1.2 Test compounds

1.3 Model Information

Colon cancer xenograft model CR2518 information

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.

2. Test method

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.

3. Tumor Measurements and Experimental Indicators

The experimental indicator is to examine whether tumor growth is inhibited, delayed or cured. Tumor diameters were measured with vernier calipers twice a week. The calculation formula of tumor volume is: V=0.5a×b ² , a and b represent the long and short diameters of the tumor, respectively.

The antitumor efficacy of the compounds was evaluated by TGI (%) or relative tumor proliferation rate T/C (%). TGI (%), reflecting tumor growth inhibition rate. Calculation of TGI(%): 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%.

Relative tumor proliferation rate T/C (%): the calculation formula is as follows: T/C (%) = T _RTV /C _RTV × 100% (T _RTV : RTV in the treatment group; C _RTV : RTV in the vehicle control group). The relative tumor volume (RTV) is calculated according to the results of tumor measurement, and the calculation formula is RTV=V _n /V ₀ , where V ₀ is the tumor volume measured during group administration (ie, d0), and V _n is a certain The tumor volume at one measurement, T _RTV and C _RTV were taken on the same day.

After the experiment, the tumor weight will be detected, and the percentage of T/C _weight will be calculated. T _weight and C _weight represent the tumor weight of the administration group and the vehicle control group, respectively.

4. Statistical analysis

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. 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.

5. Test results

5.1 Test drug compound Ib in

Results and discussion of antitumor effects in colon cancer CR2518 model

5.1.1 The growth curve of the average tumor volume of mice in each group and the analysis of tumor volume are shown in Figure 1 and Table 2.

Table 2. In

Tumor volume analysis table of each group in colon cancer CR2518 model

Note: 1. The data are expressed as "mean ± standard error"; 2. T/C%=T _RTV /C _RTV ×100%; TGI%=(1-T/C)×100%; 3. Use Conover test Comparisons between each treatment group and the control group were made.

5.2 Test Drug Compound Ib and BLU-667 in

Safety Study Results and Discussion in the Colon Cancer CR2518 Model

5.2.1 The average body weight change curve and body weight change analysis table of mice in each group are shown in Figure 2 and Table 3.

Table 3. In

Changes of body weight in each group in colon cancer CR2518 model

Note: 1. Data are expressed as "mean ± standard error"; 2. G3-00442# mice were found dead on the 30th day after grouping; G3-00443# mice were found dead on the 33rd day after grouping.

After 35 days of administration, the positive control group (BLU-667, 10 mg/kg) had a significant tumor-suppressing effect compared with the vehicle control group (T/C=0.0%, TGI=100%, p<0.001); group 2 (Compound Ib, 10 mg/kg) and group 3 (Compound Ib, 20 mg/kg) had T/C values of 29.3% and 9.5%, TGI values of 70.7% and 90.5%, respectively, p-values <0.05 and < 0.001, compared with the vehicle control group, there is a significant tumor inhibitory effect. 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.

According to the above-mentioned doses of mice, the doses of humans can be calculated, as shown in the following table.

6 Conclusion

The 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.

Test Example 3: Kinase inhibitory activity

This test case was entrusted to Sandia Medical Technology (Shanghai) Co., Ltd. to complete.

1. Experimental purpose

The inhibitory activities of the compounds of the present application on three kinases TRKa, TRKA (G595R) and TRKC (G623R) were determined.

2. Experimental materials

2.2.1 Reagents and consumables

Reagent name	supplier	article number	batch number
TRKa	Carna	08-186	13CBS-0565G
TRKA(G595R)	signalchem	N16-12BG-100	H2714-7
TRKC(G623R)	signalchem	N18-12CH-100	D2567-8
Kinase substrate 22	GL	112393	P190329-SL112393
DMSO	Sigma	D8418-1L	SHBG3288V
384-well plate (white)	PerkinElmer	6007290	810712

2.2.2 Instruments

Centrifuge (manufacturer: Eppendorf, model: 5430); microplate reader (manufacturer: Perkin Elmer, model: Caliper EZ ReaderII); Echo 550 (manufacturer: Labcyte, model: Echo 550)

3. Experimental method

3.1 Test compounds were accurately weighed and dissolved in 100% DMSO to make up a 10 mM solution.

3.2 Kinase reaction process

3.2.1 Prepare 1× Kinase Buffer.

3.2.2 Preparation of compound concentration gradient: the initial concentration of the test compound was 1000 nM, diluted into 100-fold final concentration 100% DMSO solution in 384 plate, and then 3-fold diluted to obtain 10 concentrations of compound DMSO solution. Use a dispenser Echo 550 to transfer 250 nL of 100x final concentration of compound to the reaction plate OptiPlate-384F.

3.2.3 Prepare 2.5x final concentration of kinase solution with 1x kinase buffer.

3.2.4 Add 10 μL of 2.5 times final concentration of kinase solution to compound well and positive control well respectively; add 10 μL of 1× kinase buffer to negative control well.

3.2.5 The reaction plate was shaken and mixed and incubated at room temperature for 10 minutes.

3.2.6 Prepare with 1× Kinase Buffer

Mixed solution of ATP and kinase substrate 22 at the final concentration.

3.2.7 Add 15 μL of

times the final concentration of the mixed solution of ATP and substrate.

3.2.8 Centrifuge the 384-well plate at 1000 rpm for 30 seconds, shake and mix well, and incubate at room temperature for the corresponding time.

3.2.9 Terminate the kinase reaction.

3.2.10 Read the conversion rate with the microplate reader Caliper EZ Reader.

4. Data Analysis

Calculation formula

Inhibition % (Inhibition) = (Conversion % Maximum - Conversion % Sample) / (Conversion % Maximum - Conversion % Minimum) × 100

Among them: 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.

The results are shown in Table 4 below:

Table 4. IC ₅₀ values of the compounds of the present application for the inhibitory activities of three kinases

The results showed that compound Ib had high inhibitory activity on three kinases, TRKa, TRKA(G595R) and TRKC(G623R). Compared with compounds 4-6, the activity of compound Ib is significantly different, which indicates that the fluorine substitution at different positions has different effects on the inhibitory activities of the three kinases.

Test Example 4: In Vitro Cell Viability

This test case was entrusted to Hefei Zhongkepu Ruisheng Biomedical Technology Co., Ltd., and the NTRK mutant cells used in it were constructed by the company.

1. Experimental purpose

Determination of the inhibitory effect of the compounds of the present application on the growth of three NTRK mutant cells (Ba/F3 LMNA-NTRK1-G667C, Ba/F3 EVT6-NTRK3-G623R, Ba/F3 LMNA-NTRK1-G595R)

2. Reagents and Consumables

cell line:

Reagents:

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)

3. Experimental procedure

3.1 Cell culture and seeding:

Collect the cells in the logarithmic growth phase and count the cells with a platelet counter, adjust the cell concentration to 3-6×10 ⁴ cells/mL, add 90 μL of cell suspension to a 96-well plate, and place the cells in the 96-well plate in a 96-well plate. Incubate overnight at 37°C, 5% CO ₂ , and 95% humidity.

3.2 Drug dilution and dosing:

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 μM, and were sequentially diluted 3-fold to obtain a total of 9 concentrations of drug solutions. Add more than 10 μL of the prepared drug solution to each well of the 96-well plate inoculated with cells, and then add 90 μL of medium containing 1% DMSO to obtain a drug solution with a maximum concentration of 1 μM, which is diluted 3 times in sequence, with a total of 9 concentrations. 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.

3.3 End-point plate reading

Add an equal volume of CTG solution to each well, place the 96-well plate at room temperature for 20 minutes to stabilize the luminescence signal, and read the luminescence value.

4. Data processing

Data were analyzed using GraphPad Prism 7.0 software, and a dose-response curve was fitted to the data using nonlinear S-curve regression, from which _IC50 values were calculated.

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%.

The experimental results are shown in Table 5 below:

Table 5. IC ₅₀ values of the compounds of the present application for the inhibitory activity of the three cell lines

The results showed that compound Ib had a strong growth inhibitory effect on three NTRK mutant cells (Ba/F3LMNA-NTRK1-G667C, Ba/F3EVT6-NTRK3-G623R, Ba/F3LMNA-NTRK1-G595R). The inhibitory activities of compounds 1 and 2 on the three NTRK mutant cells were not significantly different from the corresponding positive compounds, but the inhibitory activity of compound Ib on the three NTRK mutant cells was significantly higher than the corresponding positive control 2.

Test Example 5: Liver Microsome Stability Test

1. Experimental purpose

Determination of the stability of the compounds of the present application in human, rat and mouse liver microsomes

2. Test materials and instruments

Reagents and consumables:

Reagent name	supplier	article number	batch number
human liver microsomes	BiolVT	X008070	SDL
Rat liver microsomes	BiolVT	M00001	TIQ
mouse liver microsomes	Biopredic	MIC255037	BQM

3. Experimental steps

3.1 Buffer and liver microsomes were prepared according to the following table to make incubation solution:

reagent	concentration	volume
Phosphate buffer	100mM	216.25μL
liver microsomes	20mg/mL	6.25μL

3.2 The following two experiments were carried out: a) Incubation system with coenzyme factor NADPH: 25 μL of NADPH (10 mM) was added to the incubation solution (mainly including liver microsomes and phosphate buffer) to make the final concentration of liver microsomes and NADPH. The concentrations were 0.5 mg/mL and 1 mM, respectively; b) Incubation system without coenzyme factor NADPH: 25 μL of phosphate buffer (100 mM) was added to the incubation solution to make the final concentration of liver microsomes 0.5 mg/mL. The above incubation systems were preheated at 37°C for 10 minutes.

3.3 To each incubation system described in the aforementioned "Step 3.2", start the reaction by adding 2.5 μL of the positive control compound or the solution of the test compound of the present application (100 μM), 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 μM. Incubation solutions after compound addition were batch-incubated in water at 37°C.

3.4 Take 30 μL aliquots from the reaction solution at 0.5, 5, 15, 30 and 45 minutes, respectively, and stop the reaction by adding 5 volumes of cold acetonitrile containing 200 nM caffeine and 100 nM tolbutamide. An aliquot was centrifuged at 3220 g for 40 min, and 100 μL of the supernatant was mixed with 100 μL of ultrapure water and used for LC-MS/MS analysis.

3.5 Data Analysis

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 μL/min/mg protein).

The experimental results are shown in Table 6 below:

Table 6. Stability data of compounds of the present application in human, rat and mouse liver microsomes

The results show that the compound Ib of the present application has good stability in human, rat and mouse liver microsomes. 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

1. Experimental animals

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.

2. Preparation of the 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.

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.

3. Experimental Design

4. Mode of administration

Weigh before administration, and calculate the dosage according to body weight. It is administered orally by intravenous or gavage.

5. Blood collection time point

Before administration and 0.083h, 0.25h, 0.5h, 1h, 2h, 4h, 6h, 8h, 24h after administration.

6. Sample Collection and Disposal

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.

7. Bioanalysis and Data Processing

When the blood concentration of the test substance was detected and the plasma drug concentration-time curve was drawn, the BLQ was recorded as 0. When calculating the pharmacokinetic parameters, the 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. Using 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.

Table 7. Data from in vivo pharmacokinetic studies of test compounds administered intravenously and orally to SD rats

The results showed that the pharmacokinetic parameters (AUC/CL/F%) of compound 2 in SD rats were worse than that of the positive compound, and the pharmacokinetic parameters (AUC/CL/F%) of compound Ib in SD rats were worse. ) was significantly increased compared to the positive control 2.

Test Example 7: Bidirectional Permeability Study of Test Compounds on MDCK-MDR1 Cell Line

1.1 Materials

MDCK-MDR1 cells were purchased from the Netherlands Cancer Institute and cells between passages 10 and 20 were used.

1.2 Experimental Design

1.2.1 Cell culture and seeding

1) Before cell seeding, add 50 μL of cell culture medium to each well of the upper chamber of the Transwell, and add 25 mL of cell culture medium to the lower plate. Plates can be used to seed cells after 1 hour of incubation in a 37°C, 5% CO ₂ incubator.

2) Resuspend the MDCK-MDR1 cells in the medium so that the final concentration is 1.56×10 ⁶ cells/mL. The cell suspension was added to the chamber of a 96-well Transwell plate at 50 μL 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.

1.2.2 Evaluation of cell monolayer integrity

1) Remove the original medium in the lower plate and add fresh pre-warmed medium in the upper chamber.

2) The single-layer membrane resistance was measured with a resistance meter (Millipore, USA), and the resistance of each pore was recorded.

3) After the measurement, put the Transwell plate back into the incubator.

4) Calculation of resistance value: measure resistance value (ohms)×film area (cm ² )=TEER value (ohm·cm ² ), if TEER value<42ohms·cm ² , the hole cannot be used for penetration test.

1.2.3 Solution Preparation

1) Use DMSO to prepare the test compound as a 10 mM stock solution,

2) The positive control compound was prepared as a stock solution with a concentration of 10 mM in DMSO.

1.2.4 Drug Penetration Test

1) Remove the MDCK-MDR1 Transwell plate from the incubator. Cell monolayers were rinsed twice with pre-warmed HBSS (25mM HEPES, pH 7.4) buffer and incubated at 37°C for 30 minutes.

2) Stock solutions of control and test compounds were diluted in DMSO to obtain a 200 μM solution, and then diluted with HBSS (10 mM HEPES, pH 7.4) to obtain a 1 μM working solution. The final concentration of DMSO in the incubation system was 0.5%.

3) Measure the transport rate of the compound from the apical to the basal end. Add 125 μL of working solution to the upper chamber (top), then transfer 50 μL of sample solution from the lower chamber (basal end) immediately to a 96-well plate containing 200 μL of acetonitrile containing internal standard as a 0-minute dosing sample (AB) for detection, and 235 μL of HBSS (25 mM HEPES, pH 7.4) buffer was added to the lower chamber. Internal standards were included (100 nM alprazolam, 200 nM caffeine and 100 nM tosylbutyramide). The 50 μL sample solution transferred above was vortexed at 1000 rpm for 10 min.

4) Determination of the rate of transport of the compound from the basolateral to the apical. Add 285 μL of the working solution to the lower chamber (basal side), then immediately transfer 50 μL to the upper chamber (apical) Sample solution was added to a 96-well plate containing 200 μL of acetonitrile containing internal standard as a 0-minute dosing sample (BA) for detection , and 75 μL of HBSS (25 mM HEPES, pH 7.4) buffer was added to the upper chamber. The internal standard contained (100 nM alprazolam, 200 nM caffeine and 100 nM tosylbutyramide), and 50 μL 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.

5) After adding buffer to the lower chamber and the upper chamber respectively, the MDCK-MDR1 Transwell culture was incubated at 37°C for 2 hours.

6) After the incubation, take 50 μL of sample solution from the administration side (upper chamber: Ap→Bl flux, lower chamber: Bl→Ap) and the receiving side (lower chamber: Ap→Bl flux, upper chamber Bl→Ap) respectively. In a new 96-well plate, add 4 volumes of ethanol containing the internal standard (100 nM alprazolam, 200 nM caffeine, and 100 nM tosylbutyramide) to the well plate, and vortex for 10 minutes. , and centrifuged at 3220g for 40 minutes. 100 μL of the supernatant was mixed with an equal volume of ultrapure water for LC-MS/MS analysis.

7) Leakage of fluorescent yellow was used to evaluate the integrity of the cell monolayer after 2 hours of incubation. Dilute the Lucifer Yellow stock solution with HBSS (10 mM HEPES, pH 7.4) to a final concentration of 100 μM, add 100 μL of Lucifer Yellow solution to each well of the upper chamber (apex), and add 300 μL of HBSS to each well of the base of the lower chamber (basal side). (25mM HEPES, pH 7.4). After 30 minutes of incubation at 37°C, aspirate 80 μL of the solution from the upper and lower layers of each well into a new 96-well plate. Using a microplate reader, fluorescence measurement was performed under the conditions of excitation wavelength 485 nm and emission wavelength 530 nm.

1.2.5 Analysis Conditions

LC system: Shimadzu

MS analysis:Triple Quad 5500 instrument from AB Inc with an ESI interface

1) LC parameters

Column temperature: 40℃

Column: Waters XSelect HSS T3 C18, 2.5μM, 2.1 x 50mm

Mobile phase: 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B)

Injection volume: 5 μL

Elution rate: 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 parameters

Ion source: Turbo spray

Ionization Model: ESI

Scan Type: Multiple Reaction Detection (MRM)

Curtain air: 35L/min

Collision gas: 9L/min

Carrier gas: 50L/min

Auxiliary gas: 50L/min

Temperature: 500℃

Ion spray voltage: +5500V (positive)

1.3 Data Analysis

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:

In the 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 experimental results are shown in Table 8 below:

Table 8. Bidirectional permeability study data of test compounds on the same batch of MDCK-MDR1 cell line

*efflux ratio = P _app(BA) /P _app(AB)

The results show that the permeability (P _app(AB) ) of compound Ib is better than that of positive control 2, indicating that compound Ib of the present application is more easily absorbed into cells, and at the same time, the compound Ib of the present application has a lower efflux ratio, indicating that the compound of the present application Ib It is not easy to be effluxed, so that a higher drug concentration can be maintained in the cells, resulting in better drug efficacy. Therefore, combined with the in vivo pharmacokinetic study in SD rats, it is more fully demonstrated that the in vivo pharmacokinetic parameters (AUC/CL/F%) of the compound Ib of the present application are significantly improved compared with the positive control 2.

Test Example 8: In Vitro Cell Viability Assessment

This test case was entrusted to Hefei Zhongkepu Ruisheng Biomedical Technology Co., Ltd. to complete, and the cell line used in it was constructed by the company.

1. Experimental purpose

The in vitro anti-proliferative effects of the compounds of the present application on 6 BaF3 cell lines were determined.

2. Reagents and Consumables

cell line:

cell line	cell type	Number of cells/well	culture medium
Ba/F3-LMNA-NTRK1	Suspended	2000	RPMI 1640+10%FBS+1%PS
Ba/F3-LMNA-NTRK1-V573I	Suspended	2000	RPMI 1640+10%FBS+1%PS
Ba/F3-LMNA-NTRK1-F589L	Suspended	2000	RPMI 1640+10%FBS+1%PS
Ba/F3-LMNA-NTRK1-G667S	Suspended	2000	RPMI 1640+10%FBS+1%PS
Ba/F3-TEL-NTRK2	Suspended	2000	RPMI 1640+10%FBS+1%PS
Ba/F3-TEL-NTRK3	Suspended	2000	RPMI 1640+10%FBS+1%PS

Material:

3. Experimental procedure

Cell processing and drug delivery

cell culture conditions

Six Ba/F3 cell lines were cultured with RPMI 1640 (Biological Industries, Israel) + 10% fetal bovine serum (Biological Industries, Israel) + 1% double antibody (Penicillin Streptomycin solution, Coring, USA), and the cells were recovered and cultured for two days. generation, to be tested.

1000× compound preparation

The compounds to be tested were dissolved in a 10 mM stock solution prepared in DMSO, and then diluted to 1 mM with DMSO. Prepare 1.0000 mM, 0.3333 mM, 0.1111 mM, 0.03704 mM, 0.01235 mM, 0.00412 mM, 0.00137 mM, 0.00046 mM, 0.00015 mM, 0.00005 mM according to 3-fold dilution and store in 96-well plates (Beaver, Suzhou), a total of 10 A concentration gradient was used, and the same volume of DMSO solvent was used as a negative control.

20× compound preparation

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.

plank

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 μl (about 2000 cells/well).

Take 5 μl of 20× test compounds and add them to the above-mentioned culture plate containing 95 μl of cell suspension respectively, mix well, and have 2 wells for each concentration gradient. The final detection concentrations of the compounds to be tested were 1.0000 μM, 0.3333 μM, 0.1111 μM, 0.03704 μM, 0.01235 μM, 0.00412 μM, 0.00137 μM, 0.00046 μM, 0.00015 μM, and 0.00005 μM, respectively.

Incubate the plates in a 37°C, 5% _CO2 incubator for 72 hours.

board reading

The following steps were carried out according to the instructions of Promega CellTiter-Glo Luminescence Cell Viability Detection Kit (Promega-G7573).

(1). Thaw the CellTiter-Glo buffer and let it come to room temperature.

(2). Bring the CellTiter-Glo substrate to room temperature.

(3). Add CellTiter-Glo buffer to a bottle of CellTiter-Glo substrate to dissolve the substrate, thereby preparing CellTiter-Glo working solution.

(4). Slowly vortex and shake to fully dissolve.

(5). Take out the cell culture plate and let it equilibrate to room temperature for 10 minutes.

(6). Add 50 μl of CellTiter-Glo working solution to each well.

(7). Shake the plate on an orbital shaker for 2 minutes to induce cell lysis.

(8). The culture plate was placed at room temperature for 10 minutes to stabilize the luminescence signal.

(9). The luminescence signal was detected on the MD SpectraMax Paradigm plate reader.

4. Data Analysis

SpectraMax Paradigm readings yield the corresponding RLU per well fluorescence value. Cell viability data were processed using the following formula:

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 experimental results are shown in Table 9 below:

Table 9. IC ₅₀ values of the compounds of this application for the inhibitory activity of the same batch of 6 BaF3 cell lines

Judging from the biological activity data of the compounds in the specific examples, 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.

Claims (17)

1. 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,

   
2. 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,

   
3. A method for treating RET-related diseases, comprising administering to a subject in need an effective amount of a compound represented by formula (I), a stereoisomer thereof or a pharmaceutically acceptable salt thereof,

   
4. A composition or medicine for the treatment of RET-related diseases, comprising a compound shown in formula (I), a stereoisomer thereof or a pharmaceutically acceptable salt thereof, optionally also comprising a pharmaceutically acceptable carrier and/ or excipients,

   

   Preferably, in the composition, the compound represented by formula (I), its stereoisomer or a pharmaceutically acceptable salt thereof is present in an effective amount for treating RET-related diseases.
5. The use of claim 1, the compound of claim 2, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, the method of claim 3 or the composition or medicament of claim 4, wherein the compound of formula (I) is The compound represented by formula (Ia),

   
6. The use of claim 1, the compound of claim 2, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, the method of claim 3 or the composition or medicament of claim 4, wherein the compound of formula (I) is A compound represented by formula (Ib),

   
7. The use of claim 1, the compound of claim 2, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, the method of claim 3 or the composition or medicament of claim 4, wherein the RET-related disease is the RET gene Or diseases related to abnormal regulation of expression, activity or level of RET kinase protein.
8. The use of claim 1, the compound of claim 2, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, the method of claim 3 or the composition or medicament of claim 4, wherein the RET-related disease is the RET gene or RET kinase protein mutation-related disease or RET fusion gene-related disease,

   Preferably, the RET gene or RET kinase protein mutation comprises a mutation at one or more sites.
9. The purposes, compound, its stereoisomer or its pharmaceutically acceptable salt, method, composition or medicine of claim 8, wherein said RET fusion gene is selected from: 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 and any combination thereof.
10. The purposes, compound, its stereoisomer or its pharmaceutically acceptable salt, method, composition or medicine of claim 8, wherein said RET fusion gene is selected from: RET-CCDC6 (PTC1), RET-KIF5B (Kex15Rex14 ), RET-PRKAR1A (PTC2), RET-BCR, RET-NCOA4 (PTC3), and any combination thereof.
11. The purposes, compound, its stereoisomer or its pharmaceutically acceptable salt, method, composition or medicine of claim 8, wherein said RET fusion gene is selected from: RET(V804L)-KIF5B, RET(V804M)- KIF5B and any combination thereof.
12. The purposes, compound, its stereoisomer or its pharmaceutically acceptable salt, method, composition or medicine of claim 8, wherein said RET gene mutation is selected from: 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.
13. The use of claim 1, the compound of claim 2, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, the method of claim 3 or the composition or medicine of claim 4, wherein the RET-related disease is RET gene Cancers associated with dysregulation of expression, activity or level of RET kinase proteins.
14. The purposes, compounds, stereoisomers or pharmaceutically acceptable salts thereof, methods, compositions or medicaments of claim 13, wherein the RET-related disease is a cancer related to RET gene or RET kinase protein mutation, or RET fusion genetically related cancers,

    Preferably, the RET gene or RET kinase protein mutation comprises a mutation at one or more sites.
15. The use, compound, stereoisomer thereof or pharmaceutically acceptable salt, method, composition or medicament of claim 14, wherein:

    The RET gene mutation-related cancer is selected from the group consisting of: RET gene mutation-related lung cancer, papillary thyroid cancer, medullary thyroid cancer, differentiated thyroid cancer, recurrent thyroid cancer, refractory differentiated thyroid cancer, and 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 mucosal ganglionoma, and cervical cancer one or more of

    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 thyroid cancer, and 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 mucosal gangliomas, and cervical cancer one or more of.
16. The use, compound, stereoisomer or pharmaceutically acceptable salt thereof, method, composition or medicine of claim 15, wherein: said RET-related disease is RET gene mutation-related lung cancer, RET gene mutation-related thyroid gland Medullary cancer or RET gene mutation-related colon cancer, or the RET-related disease is RET fusion gene-related lung cancer, RET fusion gene-related medullary thyroid cancer, or RET fusion gene-related colon cancer,

    Preferably, the RET-related disease is RET-CCDC6-related colon cancer,

    Preferably, 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 lung adenocarcinoma one or more of

    Preferably, 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 lung adenocarcinoma one or more of.
17. The use of claim 1, the compound of claim 2, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, the method of claim 3 or the composition or medicament of claim 4, wherein the RET-related disease is a human RET-related disease Diseases, the compound represented by formula (I), its stereoisomer or its pharmaceutically acceptable salt is administered at a dose of 0.5-4 mg/kg/day.

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