CN114073704A

CN114073704A - Use of fluoro-heterocyclic derivatives having macrocyclic structure

Info

Publication number: CN114073704A
Application number: CN202110928936.0A
Authority: CN
Inventors: 李志勇; 张晓东; 张德伟; 阳安乐; 黄浩喜; 何权鸿; 陈垌珲; 唐军
Original assignee: Scinnohub Pharmaceutical Co Ltd
Current assignee: Scinnohub Pharmaceutical Co Ltd
Priority date: 2020-08-14
Filing date: 2021-08-13
Publication date: 2022-02-22
Anticipated expiration: 2041-08-13
Also published as: CN114073704B; WO2022033575A1

Abstract

The invention relates to the use of fluoro-heterocyclic derivatives having a macrocyclic structure. The present application relates to the use of a compound of formula (I), a stereoisomer or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of RET related diseases.

Description

Use of fluoro-heterocyclic derivatives having macrocyclic structure

This application is based on and claims priority from application No. 202010820363.5 filed on even 14/8/2020, the disclosure of which is hereby incorporated by reference in its entirety.

Technical Field

The application relates to application of a fluorine-containing heterocyclic derivative with a macrocyclic structure in treating diseases, in particular to application of a compound shown as a formula (I), a stereoisomer thereof or a pharmaceutically acceptable salt thereof in preparing a medicament for treating RET related diseases and application in treating RET related diseases.

Background

The protooncogene RET (Rearranged reduced transduction) is responsible for coding the receptor tyrosine kinase RET protein. The kinase belongs to transmembrane protein and consists of an extracellular region, a transmembrane region and an intracellular tyrosine kinase active region. The physiological ligands of RET belong to the glial cell line-derived neurotrophic factor (gdnf) family. Activation of RET is accomplished by the interaction between the four receptors for the growth factor receptor α 1/2/3/4(GFR α 1/2/3/4) and the four ligands for gdnf, GFR α specifically binds to gdnf family members, promotes phosphorylation of RET protein receptors and brings RET into an activated state, thereby activating downstream signaling pathways associated with Cell proliferation, migration and differentiation, including primarily the Ras/Raf/MEK/ERK-MAPK pathway and the PI3K/Akt/mTOR pathway, as well as other pathways (PLC- γ pathway, JAK-STAT pathway), among others (Matti. Under normal conditions, receptor tyrosine kinases play a maintenance role in normal organs and adult tissues, but when the RET gene is altered, the receptor tyrosine kinase activity is abnormally activated, thereby driving the occurrence of tumors and maintaining the proliferation and survival of the tumors.

RET gene variation is 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 carcinoma, and 10% to 20% of patients with papillary thyroid carcinoma develop RET mutations or fusions, with an overall average incidence of RET mutations of less than 1% in other cancers. The oncogenic RET positive variation accounts for approximately 71.6% of the total variation in RET. Common RET variations include mutations (38.6%), fusions (30.7%), amplifications (25%), rearrangements (3.4%), copy number increases (1.1%) and copy number decreases (1.1%) (Shumei kato. clin Cancer res.2018.23 (8)). It has been shown in preclinical and clinical studies that RET inhibitors can significantly inhibit the proliferation of RET gene-fused and mutated tumor cells.

At present, RET inhibitors are mainly divided into two classes, one class is targeted RET small molecule inhibitors, including LOXO-292, BLU-667 and the like, and is the research focus of the current RET inhibitors; another class are multi-kinase inhibitors, which have RET inhibitory activity. The FDA approved LOXO-292(Selpercatinib) for marketing at 5 months 2020, for the treatment of RET fusion gene positive non-small cell lung cancer patients, advanced or metastatic RET mutated medullary thyroid cancer patients, and advanced or metastatic RET fusion gene positive thyroid cancer patients, was the first RET inhibitor to market. Another RET inhibitor, BLU-667, has now been filed with NDA by the FDA for the treatment of locally advanced or metastatic RET fusion gene positive non-small cell lung cancer patients. In addition, there are other small molecule inhibitors of RET that are under clinical investigation.

As protooncogenes, 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 researches show that the RET inhibitor can effectively inhibit the in-vivo proliferation of RET fused or mutated malignant tumors, has higher disease remission rate clinically, and is an effective treatment target for cancer species.

Disclosure of Invention

The application provides an application of a compound shown as a formula (I), a stereoisomer thereof or a pharmaceutically acceptable salt thereof in preparing a medicament for treating 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 useful for treating RET-related diseases.

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

The present application also provides a composition for treating RET-related diseases, comprising a compound represented by formula (I), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, optionally further comprising a pharmaceutically acceptable carrier and/or excipient. In certain embodiments, the compound of formula (I), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, is present in the composition in an amount effective to treat the RET-associated disease.

The application also provides a medicament for treating RET related diseases, which comprises the compound shown in the formula (I), the stereoisomer thereof or the pharmaceutically acceptable salt thereof, and optionally further comprises a pharmaceutically acceptable carrier and/or excipient. In certain embodiments, the compound of formula (I), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, is present in the composition in an amount effective to treat the RET-associated disease.

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

In certain embodiments, the RET-associated disease is a disease associated with a mutation in the RET gene or 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-associated disease is a RET fusion gene-associated disease.

In certain embodiments, the RET fusion gene is selected from the group consisting of: BCR-RET, CLIP-RET, KIF 5-RET, CCDC-RET, NCOA-RET, TRIM-RET, ERC-RET, FGFR 1-RET, RET-MBD, RET-RAB6IP, RET-PRKAR1, RET-TRIM, RET-GOLGA, HOOK-RET, KTN-RET, TRIM-RET, AKAP-RET, FKBP-RET, SPECC 1-RET, TBL1 XR-RET, CEP-RET, CUX-RET, KIAA1468-RET, RFG-RET, ACBD-RET, PTC1 ex-RET, MYH-RET, PIBF-RET, AFKIAA 1217-RET, MPRIP-RET, Ria-RET, RET-RET, FRAP 4-RET, SQMD-RET, SQIF 1-RET, SGEF-RET, SAF-RET, MAG-RET, MYAA 1217-RET, MPIP-RET, HRH-RET, RIA-RET, RET-RET, CRIF-RET, SMIF-RET, SMAP 1-RET, SMIF-RET, SMIF-RET, SMIF-RET, SMIF-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) -KIF 5B.

In certain embodiments, the RET fusion gene is RET (V804L) -KIF 5B.

In certain embodiments, the RET fusion gene is RET-NCOA4(PTC 3).

In certain embodiments, the RET fusion gene is RET-CCDC6(PTC 1).

In certain embodiments, the RET fusion gene is RET-KIF5B (Kex15Rex 14).

In certain embodiments, the RET fusion gene is RET-PRKAR1A (PTC 2).

In certain embodiments, the 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.

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 (a 883F).

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

In certain embodiments, the RET-associated disease is a cancer associated with a mutation in the RET gene or the 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 gene mutation associated cancer is selected from the group consisting of: RET gene mutation-associated one or more of lung cancer, papillary thyroid carcinoma, medullary thyroid carcinoma, differentiated thyroid carcinoma, recurrent thyroid carcinoma, refractory differentiated thyroid carcinoma, multiple endocrine neoplasia type 2A or 2B (MEN 2A or MEN2B, respectively), pheochromocytoma, parathyroid hyperplasia, breast cancer, colon cancer, colorectal cancer, papillary renal cell carcinoma, gastrointestinal mucosal ganglion tumor, and cervical cancer.

In certain embodiments, the RET-associated disease is lung cancer associated with mutations in the RET gene.

In certain embodiments, the RET-associated disease is medullary thyroid cancer associated with mutations in the RET gene.

In certain embodiments, the RET-associated disease is colon cancer associated with mutations in the RET gene.

In certain embodiments, the RET-associated disease is one or more selected from the group consisting of small cell lung cancer associated with RET gene mutations, non-small cell lung cancer associated with RET gene mutations, bronchiolar lung cell carcinoma associated with RET gene mutations, or lung adenocarcinoma associated with RET gene mutations.

In certain embodiments, the RET-associated disease is small cell lung cancer associated with mutations in the RET gene.

In certain embodiments, the RET-associated disease is non-small cell lung cancer associated with mutations in the RET gene.

In certain embodiments, the RET-associated disease is a bronchioloalveolar carcinoma associated with a mutation in the RET gene.

In certain embodiments, the RET-associated disease is lung adenocarcinoma associated with mutations in the RET gene.

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

In certain embodiments, the RET fusion gene-associated cancer is selected from: RET fusion gene associated lung cancer, papillary thyroid carcinoma, medullary thyroid carcinoma, differentiated thyroid carcinoma, recurrent thyroid carcinoma, refractory differentiated thyroid carcinoma, multiple endocrine neoplasia type 2A or 2B (MEN 2A or MEN2B, respectively), pheochromocytoma, parathyroid hyperplasia, breast cancer, colon cancer, colorectal cancer, papillary renal cell carcinoma, gastrointestinal mucosal ganglion tumor, and cervical cancer.

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

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

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

In certain embodiments, the RET-associated disease is RET-CCDC 6-associated colon cancer.

In certain embodiments, the RET-associated disease is one or more selected from RET fusion gene-associated small cell lung cancer, RET fusion gene-associated non-small cell lung cancer, RET fusion gene-associated bronchiolar lung cell carcinoma, or RET fusion gene-associated lung adenocarcinoma.

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

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

In certain embodiments, the RET-associated disease is RET fusion gene-associated bronchogenic carcinoma of the lung.

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

In certain embodiments, the RET associated disease is human RET associated disease and the compound of formula (I), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, is administered at a dose of 0.5 to 4 mg/kg/day.

Defining:

in the present application, unless otherwise indicated, scientific and technical terms used herein have the meanings that are commonly understood by those of skill in the art. For a better understanding of the present application, the following provides definitions and explanations of relevant terms.

The term "stereoisomer" as used herein refers to an isomer produced by the different arrangement of atoms in a molecule, and can be divided into two types, cis-trans isomers, enantiomers, and enantiomers and diastereomers. Isomers resulting from the fact that atoms or groups of atoms in a molecule are bonded to each other in the same order but in different spatial arrangements are called stereoisomers, and there are two types of stereoisomers. Stereoisomers caused by bond length, bond angle, double bonds in the molecule, rings and the like are called configurational isomers (configurational stereoisomers). Generally, configurational isomers cannot or are difficult to interconvert. Stereoisomers resulting from rotation of single bonds alone are called conformational isomers (conformational stereo-isomers) and sometimes also called rotamers. Configurational isomers fall into two categories. Among them, isomers caused by the fact that a double bond or a single bond of a ring-forming carbon atom cannot rotate freely are called geometrical isomers (cis-trans isomers), and also called cis-trans isomers (cis-trans isomers), and stereoisomers having different optical rotation properties caused by the fact that there is no axial symmetry in the molecule are called optical isomers (optical isomers).

The term "ret (rearranged during transformation) gene" as used in this application is a protooncogene, full name: RET proto-oncogene: located on the long arm of 10 autosomal chromosome (10q11.2), 53kb in length, contains 20 exons and encodes the 1114 amino acid tyrosine kinase receptor.

The term "RET kinase" as used in this application is a receptor tyrosine kinase, encoded by the RET gene, involved in signal transduction during cell proliferation, migration, differentiation and survival of neural crest cells, kidney organ formation, spermatogenesis and the like.

The term "RET fusion gene" as used herein refers to a RET gene that is rearranged with other gene sequences to cause fusion after protein expression, resulting in abnormal expression or activity at the gene level or protein level of RET. The "RET fusion gene" is referred to as "A-B" or "B-A" in the present application, wherein A, B represents the RET gene and other genes fused to the RET gene, respectively. "A-B" or "B-A" have the same meaning and refer to a fusion gene of the A gene and the B gene. For example, RET-CCDC6 and CCDC6-RET have the same meaning, and they are fusion genes of RET and CCDC 6.

The term "RET gene mutation" as used herein refers to a point mutation caused by a base change of the RET gene, or a deletion, repetition or insertion of one or more bases, resulting in a corresponding change in a protein.

The term "pharmaceutically acceptable salt" as used herein includes conventional salts formed with pharmaceutically acceptable inorganic or organic acids or bases. Methods for preparing pharmaceutically acceptable salts of the compounds of the present application are known to those skilled in the art.

Reference herein to a compound of the present application includes a compound of formula (I), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

The term "pharmaceutical composition" as used herein includes products comprising a therapeutically effective amount of a compound of the present application, as well as any product which results, directly or indirectly, from combination of 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 including, but not limited to, tablets, capsules, solutions, suspensions, granules, injections, etc. according to conventional methods in the art, and can be administered orally or parenterally, etc.

The term "effective amount" as used herein refers to an amount sufficient to achieve a desired therapeutic effect, e.g., to achieve alleviation of symptoms associated with the disease being treated.

The term "treatment" as used herein is intended to reduce or eliminate the disease state or condition for which it is directed. A subject is successfully "treated" if the subject receives a therapeutically effective amount of a compound, stereoisomer thereof, pharmaceutically acceptable salt thereof, or pharmaceutical composition thereof according to the methods described herein, and the subject exhibits an observable and/or detectable decrease or improvement in one or more of the indications and symptoms of the subject. It is also understood that treatment of the disease state or condition described includes not only complete treatment, but also less than complete treatment, but achieves some biologically or medically relevant result.

It is further noted that the dosage and method of administration of the compounds of the present application will depend upon a variety of factors including the age, weight, sex, physical condition, nutritional status, the strength of the activity of the compound, the time of administration, the metabolic rate, the severity of the condition, and the subjective judgment of the treating physician. The preferred dosage is between 0.001-1000mg/kg body weight/day.

The compound described in the present application is based on the structural formula of the compound if the name and the structural formula are inconsistent for the same compound.

Drawings

FIG. 1 shows

Growth curves of mean tumor volume in groups of mice in colon cancer CR2518 model;

FIG. 2 shows

Body weight change curves over treatment time for various groups of mice in the colon cancer CR2518 model.

In fig. 1 and 2: 1. data are expressed as "mean ± standard error"; g3-00442# mice found death on day 30 after the cohort; g3-00443# mice found death on day 33 post-cohort; BID represents twice daily dosing; QD stands for once daily dosing.

Detailed Description

The following examples are presented to further illustrate the substance of the present application and should not be construed as limiting the scope of the present application. The following examples, which do not indicate specific conditions, were conducted according to conventional conditions or as recommended by the manufacturer. The raw materials are not indicated by manufacturers, and are all conventional products which can be obtained commercially.

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

The compounds provided herein as RET inhibitors, their preparation and use are described in detail below with reference to the examples.

The following abbreviations have the meanings indicated below:

HATU represents 2- (7-benzotriazol oxide) -N, N' -tetramethyluronium hexafluorophosphate;

Cs₂CO₃represents cesium carbonate;

THF represents tetrahydrofuran;

POCl₃represents phosphorus oxychloride;

DMF means N, N-dimethylformamide;

NaOH represents sodium hydroxide;

DIPEA or DIEA represents N, N-diisopropylethylamine;

H₂o represents water;

HCl/Dioxane stands for hydrogen chloride Dioxane solution;

rt represents the reaction temperature at room temperature;

n, N-Diethyllaniline represents N, N-Diethylaniline;

CH₃CN or ACN represents acetonitrile;

zn represents zinc powder;

NH₄cl represents ammonium chloride;

CH₃MgBr represents methyl bromideMagnesium;

K₂CO₃represents potassium carbonate;

EtOH stands for ethanol;

OTs represent a p-toluenesulfonyloxy group,

ee denotes enantiomeric excess.

RET (S891A) indicates that serine at position 891 of RET gene is mutated to alanine;

RET (L790F) indicated that leucine at position 790 of the RET gene was mutated to phenylalanine;

RET (V804M) indicates that valine at position 804 of RET gene is mutated into methionine;

RET (R749T) indicates that arginine at position 749 of the RET gene is mutated to threonine;

RET (S904A) indicates that serine at position 904 of the RET gene was mutated to alanine;

RET (R912P) indicates that arginine at position 912 of RET gene is mutated into proline;

RET (Y791F) indicates that tyrosine at position 791 of the RET gene was mutated to phenylalanine;

RET (V778I) indicates that valine at position 778 of RET gene is mutated into isoleucine;

RET (G691S) indicates that glycine at 691 position of RET gene is mutated into serine;

RET (V804L) indicates that valine at position 804 of RET gene is mutated into leucine;

RET (R813Q) indicates that arginine at position 813 of RET gene is mutated to glutamine;

RET (E762Q) indicates that glutamic acid at 762 position of RET gene is mutated into glutamine;

RET (V804E) indicates that valine at position 804 of RET gene is mutated into glutamic acid;

RET (a883F) indicates that alanine at position 883 of the RET gene was mutated to phenylalanine;

RET (S904F) indicates that serine at position 904 of the RET gene was mutated to phenylalanine;

RET (Y806H) indicates that tyrosine at position 806 of RET gene is mutated into histidine;

RET (M918T) indicates that the methionine at position 918 of the RET gene is mutated to threonine.

RET-CCDC6(PTC1) is a fusion Gene of RET and CCDC6 (alias PTC1, woven-coil domain conjugation 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), the fusion position starts at exon 15 of KIF5B Gene (amino acids 1-575) and ends at exon 14-21 of RET (amino acid 713);

RET-PRKAR1A (PTC2) is fusion Gene of RET and PRKAR1A (alias PTC2, protein kinase cAMP-dependent type I regulation subunit alpha, protein kinase c-AMP dependent type 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: fusion genes of RET and BCR (alias BCR 1; BCR activator of RhoGEF and GTPase, RhoGEF and GTO enzyme BCR activator, Gene ID (NCBI): 613);

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 activator 4, Gene ID (NCBI): 8031);

CLIP1-RET is fusion Gene of RET and CLIP1(CAP-Gly domain linking protein 1, CAP-Gly domain linker-containing protein 1, Gene ID (NCBI): 6249);

CCDC6-RET is a fusion Gene of RET and CCDC6 (ground-coil domain linking 6, coiled-coil domain protein 6, Gene ID (NCBI): 8030);

TRIM33-RET is a fusion Gene of RET and TRIM33(tripartite motif binding 33, Trimotif protein 33, Gene ID (NCBI): 51592);

ERC1-RET is fusion Gene of RET and ERC1(ELKS/RAB 6-interaction/CAST family member 1, ELKS/Rab6 interaction/CAST protein family member 1, Gene ID (NCBI): 23085);

FGFR1OP-RET is fusion Gene of RET and FGFR1OP (FGFR1 oncogene partner, FGFR1 oncogene partner, Gene ID (NCBI): 11116);

RET-MBD1 is fusion Gene of RET and MBD1(methyl-CpG binding domain protein 1, Cytidinylguanosine methylphosphonate binding protein, Gene ID (NCBI): 4152);

RET-RAB6IP2 is a fusion Gene of RET and RAB6IP2(Rab6 Interacting Protein 2A, alias ERC-1, Gene ID (NCBI): 23085);

RET-TRIM24 is a fusion Gene of RET and TRIM24(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 fusion Gene of RET and HOOK3(HOOK microtubular thermal protein 3, HOOK microtubular tetherin 3, Gene ID (NCBI): 84376);

KTN1-RET is a fusion Gene of RET and KTN1(kinectin 1, driver binding protein 1, Gene ID (NCBI): 3895);

TRIM27-RET is a fusion Gene of RET and TRIM27(tripartite motif protein 27, Gene ID (NCBI): 5987);

AKAP13-RET is 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 (sphere antisense with calponin homology and finished-coil domains 1like kd glycoprotein, Gene ID (NCBI): 23384);

TBL1XR1-RET is fusion Gene of RET and TBL1XR1(TBL1X receptor 1, TBL1X receptor 1, Gene ID (NCBI): 79718);

CEP55-RET is a fusion Gene of RET and CEP55(centrosomal protein 55, centrosome protein 55, Gene ID (NCBI): 55165);

CUX1-RET is a fusion Gene of RET and CUX1(cut like houseobox 1, homeobox cleavage protein 1, Gene ID (NCBI): 1523);

KIAA1468-RET is a fusion Gene of RET and KIAA1468 (alias RAB11 binding and LisH domain, bound-protein and Heat repeat binding, Gene ID (NCBI):57614)

RFG8-RET is 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 binding 5, acyl-CoA binding domain protein 5, Gene ID (NCBI): 91452);

PTC1ex9-RET is a variant of PTC-RET fusion, and is a fusion Gene of RET extracellular domain exon 9 and exon 1 of CCDC6 (ground-coil domain conjugation 6, coiled-coil domain 6, Gene ID (NCBI): 8030);

MYH13-RET is a fusion Gene of RET and MYH13 (myostatin latent chain 13, myosin heavy chain 13, Gene ID (NCBI): 8735);

PIBF1-RET is fusion Gene of RET and PIBF1 (progressisterone immunomodulating factor 1, progesterone immunomodulating binding factor 1, Gene ID (NCBI): 10464);

KIAA1217-RET is a 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, Gene ID (NCBI): 23164);

HRH4-RET is 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, RIA of protein kinase A);

RET-PTC4 is fusion Gene of RET and PTC4(type 2C protein phosphatase, Gene ID (NCBI): 5108);

FRMD4A-RET is fusion Gene of RET and FRMD4A (FERM domain containment 4A, FERM domain protein 4A, Gene ID (NCBI): 55691);

SQSTM1-RET is fusion Gene of RET and SQSTM1 (sequenstosome 1, autophagy linker protein 1, Gene ID (NCBI): 8878);

AFAP1L2-RET is a fusion Gene of RET and AFAP1L2(actin filament associated protein 1like 2, actin filament associated protein 1like protein 2, Gene ID (NCBI): 84632);

PPFIBP2-RET is fusion Gene of RET and PPFIBP2(PPFIA binding protein 2, Gene ID (NCBI): 8495);

EML4-RET is a 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-3family cell polarity regulator, Par-3family cell polarity regulator, Gene ID (NCBI): 56288);

MYH10-RET is a fusion Gene of RET and MYH10 (myostatin latent chain 10, myosin heavy chain 10, Gene ID (NCBI): 4628);

HTIF1-RET is a fusion Gene of RET and HTIF1 (also known as tripartite motif protein 24, Gene ID (NCBI): 8805);

AFAP1-RET is a fusion Gene of RET and AFAP1(actin filament associated protein 1, Gene ID (NCBI): 60312);

RASGEF1A-RET is a fusion Gene of RET and RASGEF1A (RasGEF domain family member 1A, Gene ID (NCBI): 221002);

TEL-RET is a fusion gene of RET and TEL (alias EVT6, ETS variable transcription factor 6Telomere interaction, (NCBI): 2120);

RUFY1-RET is a fusion Gene of RET and RUFY1(RUN and FYVE domain containing protein 1, Gene ID (NCBI): 80230);

UEVLD-RET is fusion Gene of RET and UEVLD (UEV and lactate/malate dehydrogenase domains, Gene ID (NCBI): 55293);

DLG5-RET is fusion Gene of RET and DLG5(disc large 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 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, 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-fluoro malonic acid

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

MS(ESI)m/z(M-H)⁺＝121.1.

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

2-Fluoromalonic acid (2.0g) and ethyl 5-amino-1H-pyrazole-4-carboxylate (1.7g) were weighed into phosphorus oxychloride (20mL) at room temperature, N-dimethylformamide (2mL) and N, N-diethylaniline (4.9g) were added, the temperature was raised to 110 ℃ for 3 hours, and LCMS indicated completion of the reaction. The reaction solution was concentrated to remove phosphorus oxychloride, and then poured into saturated sodium bicarbonate solution (100mL) to keep the solution alkaline, extracted with ethyl acetate three times, dried over anhydrous sodium sulfate, filtered, concentrated, the crude product obtained was purified by column chromatography, the solid obtained was washed with petroleum ether and dried to give the title compound 1.7 g.

MS(ESI)m/z(M+H)⁺＝278.0.

¹H NMR(400MHz,DMSO-d₆)δ8.80(s,1H),4.33(q,J＝7.2Hz,2H),1.32(t,J＝7.0Hz,3H).

And step 3: preparation of ethyl 5-chloro-6-fluoropyrazolo [1,5-a ] pyrimidine-3-carboxylate

Weighing ethyl 5, 7-dichloro-6-fluoropyrazolo [1,5-a ] pyrimidine-3-carboxylate (1.14g) and ammonium chloride (800mg) into a mixed solution of ethanol/tetrahydrofuran/water (30/10/20mL), adding zinc powder (1.3g) during stirring, filtering the zinc powder after 5 minutes of reaction, washing a filter cake with ethyl acetate, collecting a filtrate, drying with anhydrous sodium sulfate, filtering, concentrating, and purifying an obtained crude product by column chromatography to obtain the title compound 800 mg.

MS(ESI)m/z(M+H)⁺＝244.0.

¹H NMR(400MHz,DMSO-d₆)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).

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 (12.9g) was dissolved in tetrahydrofuran (100mL), and 5-fluoro-2-methoxynicotinaldehyde (15.0g) and cesium carbonate (40.9g) were added in this order. The resulting mixture was allowed to react at room temperature for 2 hours and TLC showed complete consumption of starting material. Suction filtering, washing filter cake with tetrahydrofuran three times, backwashing the obtained filtrate once with saturated sodium chloride solution, drying with anhydrous sodium sulfate, filtering and concentrating. The resulting crude product was purified by column chromatography to give the title compound 23.0 g.

MS(ESI)m/z(M+H)⁺＝259.1.

¹H NMR(400MHz,DMSO-d₆)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).

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

Weighing (R) -N- (((5-fluoro-2-methoxypyridin-3-yl) methylene) -2-methylpropane-2-sulfinamide (5.0g) and dissolving in tetrahydrofuran (40mL), cooling the obtained mixture to-78 ℃, slowly adding methyl magnesium bromide (7.8mL,3M) dropwise, maintaining the temperature below-65 ℃, after dripping, naturally returning to room temperature, continuing to react for 1 hour, TLC (thin layer chromatography) shows that the reaction is complete, pouring the reaction system into saturated ammonium chloride aqueous solution (1L), extracting with ethyl acetate, combining organic phases, backwashing with saturated sodium chloride solution, drying with anhydrous sodium sulfate, filtering, concentrating, and purifying the obtained crude product by column chromatography to obtain 4.5g of a title compound.

MS(ESI)m/z(M+H)⁺＝275.2.

¹H NMR(400MHz,DMSO-d₆)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).

And step 3: preparation of (R) -1- (5-fluoro-2-methoxypyridin-3-yl) ethan-1-amine hydrochloride

(R) -N- ((R) -1- (5-fluoro-2-methoxypyridin-3-yl) ethyl) -2-methylpropane-2-sulfinamide (4.5g) was dissolved in hydrogen chloride-dioxane solution (30mL) at room temperature and reacted overnight with LCMS to show complete consumption of starting material. The reaction was concentrated to give a crude product of 3.1g, greater than 95% ee, which was used directly in the next step without purification.

MS(ESI)m/z(M+H)⁺＝171.2.

¹H NMR(400MHz,DMSO-d₆)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).

Example 1: (3¹S,3³S,6³E,6⁴E,8R)-1⁵,6⁶-difluoro-8-methyl-2-oxa-4, 7-diaza-6 (3,5) -pyrazolo [1,5-a]Preparation of pyrimidine-1 (2,3) -pyridine 3-3, (1,3) -cyclobutyloctan-5-one (Compound Ib)

Step 1: preparation of ethyl (R) -6-fluoro-5- ((1- (5-fluoro-2-methoxypyridin-3-yl) ethyl) amino) pyrazolo [1,5-a ] pyrimidine-3-carboxylate

(R) -1- (5-fluoro-2-methoxypyridin-3-yl) ethan-1-amine hydrochloride (1.0g) was dissolved in acetonitrile (20mL), N-diisopropylethylamine (1.9g) and ethyl 5-chloro-6-fluoropyrazolo [1,5-a ] pyrimidine-3-carboxylate (1.2g) were added in this order, and the resulting mixture was reacted at 60 ℃ for 3 hours with TLC showing completion of the reaction. The reaction was poured into water (50mL), extracted with dichloromethane, the organic phases combined, back-washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered and concentrated. The resulting crude product was purified by column chromatography to give the title compound 1.1 g.

MS(ESI)m/z(M+H)⁺＝378.2.

¹H NMR(400MHz,DMSO-d₆)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: preparation of (R) -6-fluoro-5- (((1- (5-fluoro-2-methoxypyridin-3-yl) ethyl) amino) pyrazolo [1,5-a ] pyrimidine-3-carboxylic acid

Ethyl (R) -6-fluoro-5- ((1- (5-fluoro-2-methoxypyridin-3-yl) ethyl) amino) pyrazolo [1,5-a ] pyrimidine-3-carboxylate (1.1g) was dissolved in a mixed solution of ethanol/water (5/15mL) at room temperature, sodium hydroxide (584mg) was added, and the resulting mixture was reacted at 50 ℃ overnight, as indicated by TLC completion of the reaction. The reaction solution was concentrated to remove ethanol, the residue was poured into water (20mL), the pH was adjusted to about 5 with hydrogen chloride solution (2M), dichloromethane was extracted, the organic phases were combined, back washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, concentrated to obtain 800mg of crude product, which was used directly in the next step without purification.

MS(ESI)m/z(M+H)⁺＝350.1.

¹H NMR(400MHz,DMSO-d₆)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).

And step 3: preparation of Cyclobutyl (1R,3R) -3- (6-fluoro-5- (((R) -1- (5-fluoro-2-methoxypyridin-3-yl) ethyl) amino) pyrazolo [1,5-a ] pyrimidine-3-carboxamido) -4-methylbenzenesulfonate

(R) -6-fluoro-5- (((1- (5-fluoro-2-methoxypyridin-3-yl) ethyl) amino) pyrazolo [1,5-a ] pyrimidine-3-carboxylic acid (800mg), 2- (7-azobenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate (1.0g) and N, N-diisopropylethylamine (886mg) were dissolved in dry tetrahydrofuran (10mL), the resulting mixture was reacted at room temperature for 1 hour, t-butyl (3-hydroxycyclobutyl) carbamate hydrochloride (953mg) was added thereto, the reaction was continued for 1 hour, TLC showed completion, the reaction mixture was poured into water (30mL), extracted with ethyl acetate, the organic phase was combined, and back-washed with a saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting crude product was purified by column chromatography to give the title compound 800 mg.

MS(ESI)m/z(M+H)⁺＝573.2.

¹H NMR(400MHz,DMSO-d₆)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).

And 4, step 4: preparation of (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

(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 (800mg) was dissolved in hydrogen chloride dioxane (4M,10mL), and the resulting mixture was reacted at 55 ℃ overnight, with TLC showing completion of the reaction. The reaction solution was directly concentrated to remove most of dioxane to give 600mg of crude product, which was used in the next step without purification.

MS(ESI)m/z(M+H)⁺＝559.2.

And 5: (3¹S,3³S,6³E,6⁴E,8R)-1⁵,6⁶-difluoro-8-methyl-2-oxa-4, 7-diaza-6 (3,5) -pyrazolo [1,5-a]Preparation of pyrimidin-1 (2,3) -pyridine 3-3(1,3) -cyclooctan-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, and the resulting mixture was reacted at room temperature for 5 hours, and TLC showed completion of the reaction. The reaction was poured into water (10mL), extracted with ethyl acetate, the organic phases combined, back-washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered and concentrated. The crude product was purified by preparative thin layer chromatography and preparative high performance liquid chromatography to give 58.0mg of the title compound with an ee value of > 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).

The compounds used in test examples 1 to 7 of the present application include:

compound Ib, prepared from example 1 herein;

LOXO-195, a second generation TRK inhibitor, having the chemical structure:

positive control 1, a compound of example 5 of patent WO2019210835, has the chemical structure:

positive control 2, isPatent WO2019210835 example 6 compound, the chemical structure is:

compound 1, structural formula:

compound 2, structural formula:

compound 4, structural formula:

compound 5, structural formula:

compound 6, structural formula:

the LOXO-195 and control compounds 1-2 described above are commercially available or can be prepared synthetically according to conventional routes, for example LOXO-195 is commercially available and positive controls 1 and 2 can be synthesized according to the methods described in WO 2019210835. Compounds 1, 2 and 4-6 can be prepared by conventional synthetic routes, and can also be synthesized by the methods described in WO2021115401A 1.

Test example 1: the compound Ib prepared in example 1 has an inhibitory effect on RET kinase at a dose of 1. mu.M.

2.1 preparation of test articles

2.2 Compound dosages

The concentration of the test sample: 1 μ M

Compound incubation time: 20 minutes

ATP concentration: 10 μ M

Reaction time: 2 hours

2.3: detection conditions and protocols

Detection conditions are as follows:

preparing a buffer solution: 20mM HEPES (pH 7.5),10mM MgCl₂,1mM EGTA,0.01％Brij35 0.02mg/mL

BSA,0.1mM Na₃VO₄,2mM DTT,1％DMSO

Coenzyme factors required for the growth of the tumor separately into the corresponding kinase reactions

The reaction process is as follows:

the test subjects were subjected to a single dose experiment (2 replicates) at a concentration of 1 μ M, and the control Staurosporine (Med Chem, MC-2104) was initially at a concentration of 20 μ M or 100 μ M, as 1: serial dilutions at a ratio of 4 gave 10 reaction concentrations. The experiment will determine the single concentration inhibition of the test substance and the IC of the control₅₀The value is obtained.

1. Preparing reaction buffer solution, and preparing corresponding substrates by using newly prepared reaction buffer solution;

2. transferring the needed coenzyme factor into the substrate solution;

3. adding the stress enzyme into the substrate solution, and gently and uniformly mixing;

4. transferring the test article dissolved in DMSO into the kinase reaction mixture using a sonic pipette (Echo 550);

5. mixing 10 mu M of³³P-ATP is transferred to the above solution, at which point the reaction begins;

6. incubating for 120 minutes at room temperature;

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

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

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

And (3) data analysis:

kinase activity is expressed as the percentage of kinase activity after reaction of the corresponding kinase with compound Ib and after reaction with vehicle group (DMSO). The inhibition efficiency was expressed as 1-% kinase activity. The results are shown in Table 1.

TABLE 1 kinase Activity

The result shows that the compound Ib has stronger inhibitory activity on various RET mutations or fusion kinases, the inhibition rate can reach more than 85% at 1 mu M, and the inhibition efficiency can reach more than 95% on certain RET mutations and fusion kinases.

Test example 2: compound Ib in

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

1. Test materials and reagents

1.1 test animals: BALB/c Nude mice, female, 6-8 weeks (week old mice when tumor cells were inoculated), 21-25g in weight, 25, purchased from Beijing Ankayibo Biotech, Inc., animal certification number: 110330201100091758. a breeding environment: SPF grade.

1.2 test Compounds

1.3 model information

Colon cancer allograft model CR2518 information

CR2518 is established from a colon tumor derived from a female patient

A xenograft model. The model is a CCDC6-RET fusion mutation, has a tumor ulceration tendency, and may cause slight weight loss of animals.

Model ID

Cancer type

Subtype of cell

Race of a ethnic group

Sex

Age (age)

Pathological diagnosis

Characteristics of

CR2518

Cancer of colon

ADC

Asian descendent

Woman

82

Is provided with

RET-CCDC6 fusion

2. Test method

Animal inoculation and grouping: from

Colon cancer xenograft model CR2518(R14P5) tumor-bearing mice harvested tumor tissue, cut into tumor masses of 2-3mm in diameter and inoculated subcutaneously in the right anterior scapula of BALB/c nude mice. The average tumor volume reaches 151mm³The grouped administration is started. The test was divided into 4 groups, group 1 was vehicle control group, group 2 was test drug low dose group (test drug was compound Ib, single dose was 10mg/kg, QD 14 days, then BID 21 days), group 3 was test drug high dose group (test drug was compound Ib, single dose was 20mg/kg, QD 14 days, then BID 21 days), group 4 was positive control group (positive control drug was BLU-667, single dose was 10mg/kg, BID 34 days), 5 of each group were orally administered by gavage, and the test was completed on day 35 after administration.

3. Tumor measurement and Experimental indices

The experimental index is to investigate whether the tumor growth is inhibited, delayed or cured. Tumor diameters were measured twice weekly using a vernier caliper. The formula for tumor volume is: v is 0.5a × b²And a and b represent the major and minor diameters of the tumor, respectively.

The tumor suppressor therapeutic effect of the compound was evaluated as TGI (%) or relative tumor proliferation rate T/C (%). TGI (%), reflecting the rate of tumor growth inhibition. Calculation of TGI (%): TGI (%) × 100% (1- (average tumor volume at the end of administration of a certain treatment group-average tumor volume at the start of administration of the treatment group)/(average tumor volume at the end of treatment of the solvent control group-average tumor volume at the start of treatment of the solvent control group)).

Relative tumor proliferation rate T/C (%): the calculation formula is as follows: T/C (%) ═ T_RTV/C_RTV×100％(T_RTV: treatment group RTV; c_RTV: vehicle control group RTV). Calculating Relative Tumor Volume (RTV) according to the tumor measurement result, wherein the calculation formula is that RTV is V_n/V₀In which V is₀The resulting tumor volume, V, was measured at the time of group administration (i.e., d0)_nTumor volume at a certain measurement, T_RTVAnd C_RTVThe same day data was taken.

The tumor weight will be detected after the experiment is over,and calculating T/C_weightPercent, T_weightAnd C_weightTumor weights of the administered group and the vehicle control group are shown, respectively.

4. Statistical analysis

The analysis results included mean and Standard Error (SEM) of tumor volume for each time point for each group. Treatment groups showed significant tumor suppression at day 35 post-dose at the end of the trial, and therefore statistical analysis was performed based on this data to assess differences between groups. The homogeneity assumption of variance between all groups was first verified using the Bartlett test. When the Bartlett test has a p value of not less than 0.05, one-way anova will be used to test whether all group means are equal. If the p-value for one-way anova was less than 0.05, we will perform pairwise comparisons between all groups using the Tukey HSD test, or between each treatment group and the control group using the Dunnett's t test. When the Bartlett test has a p-value less than 0.05, the Kruskal Wallis test will be used to test whether the median is equal for all groups. 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 treated group and the control group and make the corresponding p-value corrections based on the number of groups in the multiplex test. All statistical analysis and graphical rendering was 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

Results and discussion of anti-tumor effects in the Colon cancer CR2518 model

5.1.1 growth curves and tumor volume analysis tables for mean tumor volume in each group of mice are shown in FIGS. 1 and 2.

TABLE 2 in

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

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

5.2 test drugs Compounds Ib and BLU-667

Results and discussion of safety studies in the colon cancer CR2518 model

5.2.1 mean body weight change curves and body weight change analysis tables for the groups of mice are shown in FIGS. 2 and 3.

TABLE 3 in

Weight change of each group in colon cancer CR2518 model

Note: 1. data are expressed as "mean ± standard error"; g3-00442# mice found death on day 30 after the cohort; g3-00443# mice were found dead on day 33 post-group.

After 35 days of administration, the positive control group (BLU-667, 10mg/kg) had significant tumor suppression effect compared to the vehicle control group (T/C0.0%, TGI 100%, p)<0.001); the T/C values of the groups 2 (Compound Ib, 10mg/kg) and 3 (Compound Ib, 20mg/kg) were 29.3% and 9.5%, respectively, the TGI values were 70.7% and 90.5%, respectively, and the p values were 70.7% and 90.5%, respectively<0.05 and<0.001, compared with a vehicle control group, has obvious tumor inhibition effect. The test drug compound Ib had no animal death at the 10mg/kg dose used, no significant drug toxicity and was well tolerated during the treatment period. When compound Ib is administrated once a day at the application dose of 20mg/kg for 14 days continuously, the weight of the mice is not reduced, and no obvious drug toxicity is shown. The subsequent dose of compound Ib is 20mg/kgThe lower adjustment is that the administration is carried out twice a day, the administration is carried out for 21 days continuously, the weight of the mice is reduced, and some animals die, and certain drug toxicity is shown. Test drug compound Ib at the applied dose

Colon cancer CR2518 showed significant tumor growth inhibition. Tumor-bearing mice tolerated low doses of compound Ib well. The high-dose compound Ib has certain drug toxicity to tumor-bearing mice.

The dose for human administration can be estimated from the dose for mouse administration described above, and is shown in the following table.

6. Conclusion

Test drug Compound Ib and Positive control drug BLU-667 at the doses used

The colon cancer CR2518 has the function of remarkably inhibiting the growth of tumors. Tumor-bearing mice tolerated compound Ib and BLU-667 well at the doses tested and at the low doses.

Test example 3: kinase inhibitory Activity

This test example was completed by songdia pharmaceutical technology (shanghai) llc.

1. Purpose of experiment

The compounds of the present application were tested for their inhibitory activity against three kinases, TRKa, TRKA (G595R) and TRKC (G623R).

2. Experimental Material

2.2.1 reagents and consumables

Name of reagent	Suppliers of goods	Goods 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 orifice plate (white)	PerkinElmer	6007290	810712

2.2.2 instruments

Centrifuge (manufacturer: Eppendorf, model 5430); enzyme-labeling instrument (manufacturer: Perkin Elmer, model: Caliper EZ ReaderII); echo550 (manufacturer: Labcyte, model: Echo550)

3. Experimental methods

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

3.2 kinase reaction Processes

3.2.1 preparation of 1 Xkinase buffer.

3.2.2 preparation of Compound concentration gradient: test compounds were tested at an initial concentration of 1000 nM, diluted in 384 plates to 100-fold final concentration of 100% DMSO, and then diluted 3-fold to give 10 concentrations of compound in DMSO. Using a dispenser Echo550 to the reaction plate OptiPlate-384F transfer 250 nL 100 times the final concentration of compounds.

3.2.3A 2.5 fold final concentration of kinase solution was prepared using 1 Xkinase buffer.

3.2.4 Add 10. mu.L of 2.5 fold final concentration kinase solution to the compound wells and positive control wells, respectively; add 10. mu.L of 1 Xkinase buffer to the negative control wells.

3.2.5 the reaction plate was shaken well and incubated at room temperature for 10 minutes.

3.2.6 preparation with 1 Xkinase buffer

A mixed solution of ATP and kinase substrate 22 at double final concentration.

3.2.7 Add 15. mu.L of

Mixed solution of ATP and substrate at double final concentration.

3.2.8 the 384 well plates were centrifuged at 1000rpm for 30 seconds and shaken to mix and incubated at room temperature for the appropriate time.

3.2.9 the kinase reaction is stopped.

3.2.10 conversion was read using a microplate Reader Caliper EZ Reader.

4. Data analysis

Formula for calculation

Inhibition% (Inhibition) — (conversion% max-conversion% sample)/(conversion% max-conversion% min) × 100

Wherein: percent conversion samples are conversion readings of the sample; conversion% minimum: negative control well mean, representing conversion readings without enzyme live wells; maximum% conversion: positive control wells are averaged for conversion readings in wells without compound inhibition.

Fitting a dose-response curve: the log value of the concentration is taken as an X axis, the percent inhibition rate is taken as a Y axis, and a dose-effect curve (four-parameter model fitting) is fitted by adopting the log (inhibitor) vs. response-Variable slope (Variable slope) of the analytic software GraphPadprism 5, so that the IC (integrated circuit) of each compound on the enzyme activity is obtained₅₀The value is obtained.

The results are shown in table 4 below:

TABLE 4 IC of the Compounds of the present application for three kinase inhibitory Activity₅₀Value of

The results show that the compound Ib has high inhibitory activity on three kinases of TRKa, TRKA (G595R) and TRKC (G623R). Compared with compounds 4-6, the compound Ib has significant difference in activity, which shows that fluorine substitution at different positions has larger influence difference on the inhibitory activity of the three kinases.

Test example 4: in vitro cell Activity

The test cases were created by the Coprinus biomedical science and technology company, Inc., in Hefei, in which NTRK mutant cells were used.

1. Purpose of experiment

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/F3EVT6-NTRK3-G623R, Ba/F3 LMNA-NTRK1-G595R)

2. Reagent and consumable

Cell line:

reagent:

fetal bovine serum FBS (GBICO, Cat # 10099-141);

luminescent Cell visual Assay (CTG, Promega, Cat # G7573); 96-hole transparent flat-bottom black wall plate (

Cat#165305)；RPMI-1640(Hyclone，Cat#SH30809.01)

3. Procedure of experiment

3.1 cell culture and inoculation:

collecting cells in logarithmic growth phase, counting cells with platelet counter, and adjusting cell concentration to 3-6 × 10⁴cell/mL, 90. mu.L of cell suspension was added to a 96-well plate, and the cells in the 96-well plate were placed at 37 ℃ in 5% CO₂And cultured overnight under 95% humidity conditions.

3.2 drug dilution and dosing:

the test compound was prepared into 10-fold drug solution with a maximum concentration of 10 μ M using a culture medium containing 1% DMSO, and sequentially diluted 3-fold to obtain 9-fold drug solutions. Adding 10 mu L of the prepared drug solution into each hole of a 96-hole plate inoculated with cells, adding 90 mu L of culture medium containing 1% DMSO to obtain the drug solution with the highest concentration of 1 mu M, sequentially diluting by 3 times, and setting three multiple holes for each concentration of drug, wherein the final concentration of DMSO in each hole is 0.1%. The cells in the dosed 96-well plate were placed at 37 ℃ in 5% CO₂And further cultured under 95% humidity conditions for 72 hours, after which CTG analysis was performed.

3.3 end reading plate

Equal volume of CTG solution was added to each well, the 96-well plate was left at room temperature for 20 minutes to stabilize the luminescence signal, and the luminescence values were read.

4. Data processing

Data were analyzed using GraphPad Prism 7.0 software, fitted to the data using non-linear sigmoidal regression to derive a dose-effect curve, and IC was calculated therefrom₅₀The value is obtained.

The cell survival rate (%). is (cold light value of the drug well to be tested-cold light value of the culture solution control well)/(cold light value of the cell control well-cold light value of the culture solution control well) × 100%.

The results of the experiment are shown in table 5 below:

TABLE 5 IC of inhibitory Activity of Compounds of the present application on three lines of cells₅₀Value of

The results show that compound Ib has strong growth inhibition effect on three NTRK mutant cells (Ba/F3 LMNA-NTRK1-G667C, Ba/F3EVT6-NTRK3-G623R, Ba/F3 LMNA-NTRK 1-G595R). The inhibitory activity of the compounds 1 and 2 on the three NTRK mutant cells is not obviously different from that of the corresponding positive compound, but the inhibitory activity of the compound Ib on the three NTRK mutant cells is obviously higher than that of the corresponding positive control 2.

Test example 5: liver microsome stability test

1. Purpose of experiment

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

2. Test materials and instruments

Reagents and consumables:

name of reagent	Suppliers of goods	Goods number	Batch number
				Human liver microsomes	BiolVT	X008070	SDL
Rat liver microsome	BiolVT	M00001	TIQ
				Mouse liver microsome	Biopredic	MIC255037	BQM

3. Experimental procedure

3.1 buffer and liver microsomes were prepared as follows:

reagent	Concentration of	Volume of
			Phosphate buffer	100mM	216.25μL
Liver microsome	20mg/mL	6.25μL

3.2 the following two experiments were performed: a) incubation system with addition of coenzyme factor NADPH: adding 25 μ L ADPH (10mM) to hatching fluid (mainly containing liver microsomes, phosphate buffer) so that the final concentrations of liver microsomes and NADPH are 0.5mg/mL and 1mM, respectively; b) incubation system without addition of coenzyme factor NADPH: to the hatching fluid, 25. mu.L of phosphate buffer (100mM) was added so that the final concentration of liver microsomes was 0.5 mg/mL. The above incubation systems were preheated at 37 ℃ for 10 minutes, respectively.

3.3 to each incubation system described in "step 3.2" above, the reaction was started by adding 2.5 μ L of a positive control compound, verapamil (purchased from Sigma) or a test compound solution of the present application (100 μ M), respectively, such that the final concentration of the test compound or positive control compound of the present application was 1 μ M. The incubation solution after addition of the compound was incubated in batches in water at 37 ℃.

3.4 aliquots of 30. mu.L were removed from the reaction solution at 0.5, 5, 15, 30 and 45 minutes, respectively, and the reaction was stopped by adding 5 volumes of cold acetonitrile containing 200nM caffeine and 100nM tolbutamide. Aliquots were centrifuged at 3220g acceleration of gravity for 40 minutes and 100. mu.L of the supernatant was mixed with 100. mu.L of ultrapure water and then used for LC-MS/MS analysis.

3.5 data analysis

The peak area was determined from the extracted ion chromatogram. The slope value k is determined by linear regression of the remaining percentage of parent drug versus the natural logarithm of the incubation time curve.

Respectively calculating and determining in vitro half-life (t) according to the slope values_1/2) Conversion to intrinsic clearance in vitro (CLint, expressed in μ L/min/mg protein) is by mean half-life in vitro.

The results of the experiment 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 compound Ib of the present application has good stability in human, rat and mouse liver microsomes. The stability of the compounds 1 and 2 in human, rat and mouse liver microsomes is not obviously different from that of the corresponding positive compounds, or the stability in most species of liver microsomes is poor, but the stability of the compound Ib in the invention in human, rat and mouse liver microsomes is obviously 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. Test animal

The species are as follows: SD rats, SPF grade. The source is as follows: animals were transferred from laboratory animal stores (999M-017), Shanghai Sphall-BikeKa laboratory animals Co., Ltd. Quantity: 3 of each dosage form.

2. Preparation of test article

2.1 accurately weighing a proper amount of a sample, adding a final volume of 5% DMSO, 10% polyethylene glycol-15 hydroxystearate and 85% normal saline, and sufficiently mixing by vortex or ultrasound to obtain a 0.2mg/mL administration solution for intravenous injection administration.

2.2 accurately weighing a proper amount of the test sample, adding 5% DMSO, 10% polyethylene glycol-15 hydroxystearate and 85% normal saline in the final volume, and sufficiently and uniformly mixing by vortex or ultrasound to obtain 0.5mg/mL administration solution for oral intragastric administration.

3. Design of experiments

4. Mode of administration

The weight was weighed before administration, and the amount administered was calculated from the body weight. Administered orally by intravenous or intragastric administration.

5. Time point of blood sampling

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

6. Sample collection and disposal

Blood is collected via jugular vein or other suitable method, each sample is collected about 0.20mL, heparin sodium is anticoagulated, the blood sample is placed on ice after collection, and plasma is centrifugally separated within 2 hours (centrifugal force: 6800g, 6 minutes, 2-8 ℃). The collected plasma samples are stored in a refrigerator at minus 80 ℃ before analysis, and the residual plasma samples after analysis are continuously stored in the refrigerator at minus 80 ℃ for temporary storage.

7. Biological analysis and data processing

And (4) detecting the blood concentration of the test substance, and marking the BLQ as 0 when a blood plasma drug concentration-time curve is drawn. When calculating the drug-induced parameters, the concentration before administration is calculated according to 0; c_maxThe previous BLQ (including "No peak") is calculated as 0; BLQ (including "No peak") appearing after Cmax does not participate in the calculation uniformly. Calculation of pharmacokinetic parameters, such as AUC (0-T), T, using WinNonlin from plasma concentration data at various time points_1/2Cmax, etc. The results are shown in Table 7.

TABLE 7 in vivo pharmacokinetic study data for intravenous and oral administration of test compounds in SD rats

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

Test example 7: two-way penetration study of test Compounds on MDCK-MDR1 cell line

1.1 materials

MDCK-MDR1 cells were purchased from the Netherlands cancer institute using between 10 and 20 passages.

1.2 design of the experiment

1.2.1 cell culture and plating

1) Prior to cell inoculation, 50. mu.L of cell culture medium was added to each well of the upper Transwell chamber and 25mL of cell culture medium was added to the lower plate. The plates were incubated at 37 ℃ in 5% CO₂After incubation in the incubator for 1 hour, the cells can be used for inoculation.

2) The MDCK-MDR1 cells were resuspended using medium to a final concentration of 1.56X 10⁶cells/mL. The cell suspension was added to the upper chamber of a 96-well Transwell plate at 50. mu.L per well. The incubator is set at 37 ℃ and 5% CO₂And ensuring that the culture is carried out for 4 to 8 days at the relative humidity of 95 percent. And (4) starting to change the culture medium 48 hours after inoculation, culturing for 4-8 days, and changing the culture medium every other day.

1.2.2 evaluation of cell monolayer integrity

1) The original medium in the lower plate was removed and fresh pre-warmed medium was added to the upper chamber.

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

3) After the measurement was completed, the Transwell plate was returned to the incubator.

4) Calculation of resistance value: measurement of resistance value (ohms). times.Membrane area (cm)²) TEER value (ohm cm)²) If the TEER value<42ohms·cm²Then the hole cannot be used for penetration testing.

1.2.3 solution preparation

1) Test compounds were formulated in 10mM stock using DMSO,

2) positive control compounds were prepared in DMSO as a stock solution at a concentration of 10 mM.

1.2.4 drug penetration test

1) MDCK-MDR1 Transwell plates were removed from the incubator. The cell monolayer was rinsed twice with pre-warmed HBSS (25mM HEPES, pH7.4) buffer and incubated at 37 ℃ for 30 minutes.

2) Stock solutions of control and test compounds were diluted in DMSO to give 200. mu.M solutions, which were then diluted with HBSS (10mM HEPES, pH7.4) to give 1. mu.M working solutions. The final concentration of DMSO in the incubation system was 0.5%.

3) The transport rate of the compound from the apical to the basal end was determined. To the upper chamber (top end), 125. mu.L of the working solution was added, and then from the lower chamber (bottom end), 50. mu.L of the sample solution was immediately transferred to a 96-well plate containing 200. mu.L of acetonitrile containing an internal standard as a 0-minute administration sample (A-B) for detection, and 235. mu.L of HBSS (25mM HEPES, pH7.4) buffer was added to the lower chamber. Internal standards were included (100nM alprazolam, 200nM caffeine and 100nM tosylbutamide). The above transferred 50. mu.L of sample solution was vortexed at 1000rpm for 10 min.

4) The transport rate of the compound from the basal end to the apical end was determined. 285. mu.L of the working solution was added to the lower chamber (basal end), and then 50. mu.L of the sample solution was immediately transferred to the upper chamber (top end) and added to a 96-well plate containing 200. mu.L of acetonitrile containing an internal standard as a 0-minute administration sample (B-A) for detection, and 75. mu.L of HBSS (25mM HEPES, pH7.4) buffer was added to the upper chamber. Internal standards were included (100nM alprazolam, 200nM caffeine and 100nM tosylbutamide) and 50. mu.L of the above transferred sample solution was vortexed at 1000rpm for 10 min. Experiments in the tip-to-base direction and base-to-tip direction should be performed simultaneously.

5) After adding the buffer to the lower and upper chambers, respectively, MDCK-MDR1 Transwell cultures were incubated at 37 ℃ for 2 hours.

6) After the incubation was completed, the drug was removed 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) 50 μ L of the sample solution was taken in a new 96-well plate, and 4-fold volume of ethanol containing an internal standard substance containing (100nM alprazolam, 200nM caffeine and 100nM tosylbutamide) was added to the plate, vortexed for 10 minutes, and then centrifuged at 3220g for 40 minutes. Draw 100. mu.L of supernatant and mix with equal volume of ultrapure water for LC-MS/MS analysis.

7) The integrity of the cell monolayer membranes after 2 hours incubation was evaluated by leakage of fluorescein. The fluorescein stock was diluted to a final concentration of 100. mu.M with HBSS (10mM HEPES, pH7.4), and 100. mu.L of fluorescein solution was added to each well of the upper chamber (top end) and 300. mu.L of HBSS (25mM HEPES, pH7.4) to each well of the substrate of the lower chamber (bottom end). After incubation at 37 ℃ for 30 min, 80. mu.L of the solution was aspirated from each well, both above and below, into a new 96-well plate. Fluorescence measurement was carried out using a microplate reader under excitation wavelength of 485nm and emission wavelength of 530 nm.

1.2.5 analytical conditions

LC system:Shimadzu

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

1) LC parameter

Column temperature 40 deg.C

Chromatographic column Waters XSelect HSS T3C 18, 2.5. mu.M, 2.1X50mm

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

Sample introduction volume of 5 muL

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 gas 35L/min

Collision gas 9L/min

Carrier gas 50L/min

Auxiliary gas 50L/min

The temperature is 500 DEG C

Voltage of ion spray +5500V (positive)

1.3 data analysis

The peak area was calculated from the ion chromatography results. Apparent permeability coefficient of the compound (Papp, unit: cm/s.times.10)^-6) Calculated using the following formula:

in the formula: v_AThe volume of the receiving end solution (Ap → Bl is 0.3mL, Bl → Ap is 0.1mL) and Area is the Area of the Transwell-96 well plate membrane (0.143 cm)²) (ii) a time is incubation time (unit: s); [ drug]Is the drug concentration.

The results of the experiment are shown in table 8 below:

TABLE 8 Bi-Permeability data of test compounds on the same lot of MDCK-MDR1 cell line

Ratio of outer row ═ P_app(B-A)/P_app(A-B)

The results show the permeability (P) of Compound Ib_app(A-B)) The compound Ib is superior to a positive control 2, shows that the compound Ib is easier to absorb and enter cells, and simultaneously has a lower excretion ratio, shows that the compound Ib is not easy to be excreted, so that higher drug concentration can be maintained in the cells, and better drug effect is generated. Therefore, combining with the in vivo pharmacokinetic study of SD rat, it is more fully demonstrated that the compound Ib of the present application has a significant improvement in the in vivo pharmacokinetic parameters (AUC/CL/F%) compared with the positive control 2.

Test example 8: in vitro cell Activity assessment

The test cases were completed by the Coprinus biomedical science and technology Co.Ltd, in which the cell lines used were constructed.

1. Purpose of experiment

The compounds of the present application were tested for their antiproliferative effect on the 6 strain BaF3 cell line in vitro.

2. Reagent and consumable

Cell line:

cell lines	Cell type	Cell number/well	Culture medium
				Ba/F3-LMNA-NTRK1	Suspended in water	2000	RPMI 1640+10％FBS+1％PS
Ba/F3-LMNA-NTRK1-V573I	Suspended in water	2000	RPMI 1640+10％FBS+1％PS
				Ba/F3-LMNA-NTRK1-F589L	Suspended in water	2000	RPMI 1640+10％FBS+1％PS
Ba/F3-LMNA-NTRK1-G667S	Suspended in water	2000	RPMI 1640+10％FBS+1％PS
				Ba/F3-TEL-NTRK2	Suspended in water	2000	RPMI 1640+10％FBS+1％PS
Ba/F3-TEL-NTRK3	Suspended in water	2000	RPMI 1640+10％FBS+1％PS

Materials:

3. procedure of experiment

Cell treatment and administration

Cell culture conditions

The 6 Ba/F3 cell lines were cultured using RPMI 1640(Biological Industries, Israel) + 10% fetal bovine serum (Biological Industries, Israel) + 1% double antibody (penillilin Streptomycin solution, Coring, USA), and two generations were cultured after cell recovery and tested.

1000 preparation of the Compounds

Test compounds were dissolved in 10mM stock solution prepared in DMSO and diluted to 1mM in DMSO. Prepared as 3-fold dilutions to 1.0000mM, 0.3333mM, 0.1111mM, 0.03704mM, 0.01235mM, 0.00412mM, 0.00137mM, 0.00046mM, 0.00015mM, 0.00005mM were stored in 96-well plates (Beaver, Suzhou) for a total of 10 concentration gradients with equal volumes of DMSO solvent as negative control.

20X preparation of the Compound

The prepared 10 test compounds with 1000 × concentration gradient were diluted 50-fold to 20 × compounds with complete medium, respectively, and stored in 96-well plates (Beaver, Suzhou) for 10 concentration gradients, while the same volume of DMSO solvent was used as negative control.

Floor board

The logarithmic phase cell suspension was seeded in 96-well white cell culture plates (Corning 3917, NY, USA) in a volume of 95. mu.l per well (about 2000 cells/well).

Mu.l of 20 Xtest compound was added to the above plate containing 95. mu.l of cell suspension, and mixed well, 2 wells per concentration gradient. The final detection concentrations of the test compounds were 1.0000. mu.M, 0.3333. mu.M, 0.1111. mu.M, 0.03704. mu.M, 0.01235. mu.M, 0.00412. mu.M, 0.00137. mu.M, 0.00046. mu.M, 0.00015. mu.M, and 0.00005. mu.M, respectively.

The culture plate was incubated at 37 ℃ with 5% CO₂Incubate in incubator for 72 hours.

Reading board

The following procedure was performed according to the instructions of Promega CellTiter-Glo luminescence cell activity assay kit (Promega-G7573).

(1) The CellTiter-Glo buffer was thawed and left to stand at room temperature.

(2) The CellTiter-Glo substrate was allowed to stand to room temperature.

(3) The CellTiter-Glo working solution was prepared by adding CellTiter-Glo buffer to a flask of CellTiter-Glo substrate to dissolve the substrate.

(4) Slow vortex to dissolve well.

(5) The cell culture plate was removed and allowed to equilibrate to room temperature for 10 minutes.

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

(7) Plates were shaken on an orbital shaker for 2 minutes to induce cell lysis.

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

(9) Detecting the luminescence signal on an MD spectra max Paradigm plate reader.

4. Data analysis

Spectra max Paradigm readings give the corresponding per well fluorescence value RLU. Cell viability data (Cell viability) was processed using the following formula:

Cell viability(％)＝(RLU_Drug-RLU_Min)/(RLU_Max-RLU_Min) 100 percent. Cell viability was calculated in EXCEL for different concentrations of compound, and GraphPad Prism software was used to plot cell viability and calculate relevant parameters including cell maximal and minimal viability, IC₅₀The value is obtained.

The results of the experiment are shown in table 9 below:

TABLE 9 IC of inhibitory Activity of the Compounds of the present application on the same batch of 6 BaF3 cell line₅₀Value of

From the bioactivity data of the compounds of the specific examples, the compound Ib has strong growth inhibition effect on 6 BaF3 cell lines. The inhibitory activity of compound 2 on 6 strains of BaF3 cell lines is not obviously different from that of the corresponding positive compound, but the inhibitory activity of compound Ib on 6 strains of BaF3 cell lines is obviously higher than that of the positive control 2.

Claims

1. The use of a compound of formula (I), a stereoisomer thereof or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of RET related diseases,

2. the use according to claim 1, wherein the compound of formula (I) is a compound of formula (Ia),

3. the use according to claim 1, wherein the compound of formula (I) is a compound of formula (Ib),

4. the method of claim 1, wherein the RET-associated disease is a disease associated with dysregulation of the expression, activity or level of RET gene or RET kinase protein.

5. The use according to claim 1, wherein the RET-associated disease is a disease associated with mutation of RET gene or RET kinase protein or a disease associated with RET fusion gene,

preferably, the RET gene or RET kinase protein mutation comprises a mutation at one or more sites.

6. The use of claim 5, wherein the RET fusion gene is selected from the group consisting of: BCR-RET, CLIP-RET, KIF 5-RET, CCDC-RET, NCOA-RET, TRIM-RET, ERC-RET, FGFR 1-RET, RET-MBD, RET-RAB6IP, RET-PRKAR1, RET-TRIM, RET-GOLGA, HOOK-RET, KTN-RET, TRIM-RET, AKAP-RET, FKBP-RET, SPECC 1-RET, TBL1 XR-RET, CEP-RET, CUX-RET, KIAA1468-RET, RFG-RET, ACBD-RET, PTC1 ex-RET, MYH-RET, PIBF-RET, AFKIAA 1217-RET, MPRIP-RET, Ria-RET, RET-RET, FRAP 4-RET, SQMD-RET, SQIF 1-RET, SGEF-RET, SAF-RET, MAG-RET, MYAA 1217-RET, MPIP-RET, HRH-RET, RIA-RET, RET-RET, CRIF-RET, SMIF-RET, SMAP 1-RET, SMIF-RET, SMIF-RET, SMIF-RET, SMIF-RET, TEL-RET, RUFY1-RET, UEVLD-RET, DLG5-RET, FOXP4-RET, OFLM4-RET, RRBP1-RET, and any combination thereof.

7. The use of claim 5, wherein 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.

8. The use of claim 5, wherein the RET fusion gene is selected from the group consisting of: RET (V804L) -KIF5B, RET (V804M) -KIF5B, and any combination thereof.

9. The use of claim 5, wherein 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.

10. The use of claim 1, wherein the RET-associated disease is a cancer associated with dysregulation of expression, activity or level of RET genes or RET kinase proteins.

11. The use of claim 10, wherein the RET-associated disease is a cancer associated with mutation of RET gene or RET kinase protein, or a cancer associated with RET fusion gene,

12. The use of claim 11, wherein:

the RET gene mutation-associated cancer is selected from: RET gene mutation-associated one or more of lung cancer, papillary thyroid carcinoma, medullary thyroid carcinoma, differentiated thyroid carcinoma, recurrent thyroid carcinoma, refractory differentiated thyroid carcinoma, multiple endocrine neoplasia type 2A or 2B (MEN 2A or MEN2B, respectively), pheochromocytoma, parathyroid hyperplasia, breast cancer, colon cancer, colorectal cancer, papillary renal cell carcinoma, gastrointestinal mucosal ganglion tumor, and cervical cancer,

the RET fusion gene-associated cancer is selected from: RET fusion gene associated lung cancer, papillary thyroid carcinoma, medullary thyroid carcinoma, differentiated thyroid carcinoma, recurrent thyroid carcinoma, refractory differentiated thyroid carcinoma, multiple endocrine neoplasia type 2A or 2B (MEN 2A or MEN2B, respectively), pheochromocytoma, parathyroid hyperplasia, breast cancer, colon cancer, colorectal cancer, papillary renal cell carcinoma, gastrointestinal mucosal ganglion tumor, and cervical cancer.

13. The use of claim 12, wherein: the RET-associated disease is RET gene mutation-associated lung cancer, RET gene mutation-associated medullary thyroid cancer, or RET gene mutation-associated colon cancer, or the RET-associated disease is RET fusion gene-associated lung cancer, RET fusion gene-associated medullary thyroid cancer, or RET fusion gene-associated colon cancer,

preferably, the RET-associated disease is RET-CCDC 6-associated colon cancer,

preferably, the RET-associated disease is one or more selected from the group consisting of small cell lung cancer associated with RET gene mutation, non-small cell lung cancer associated with RET gene mutation, bronchiolar lung cell cancer associated with RET gene mutation, and lung adenocarcinoma associated with RET gene mutation,

preferably, the RET-associated disease is one or more selected from the group consisting of RET fusion gene-associated small cell lung cancer, RET fusion gene-associated non-small cell lung cancer, RET fusion gene-associated bronchiolar lung cell carcinoma, or RET fusion gene-associated lung adenocarcinoma.

14. The use according to any one of claims 1 to 13, wherein the RET related disease is a human RET related disease and the compound of formula (I), a stereoisomer thereof or a pharmaceutically acceptable salt thereof is administered at a dose of 0.5 to 4 mg/kg/day.