CN114073704B - Use of fluorine-containing heterocyclic derivative with macrocyclic structure - Google Patents

Use of fluorine-containing heterocyclic derivative with macrocyclic structure Download PDF

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CN114073704B
CN114073704B CN202110928936.0A CN202110928936A CN114073704B CN 114073704 B CN114073704 B CN 114073704B CN 202110928936 A CN202110928936 A CN 202110928936A CN 114073704 B CN114073704 B CN 114073704B
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ret
gene
cancer
fusion gene
compound
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CN114073704A (en
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李志勇
张晓东
张德伟
阳安乐
黄浩喜
何权鸿
陈垌珲
唐军
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Scinnohub Pharmaceutical Co Ltd
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems

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Abstract

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

Description

Use of fluorine-containing heterocyclic derivative with macrocyclic structure
The present application is based on and claims priority from CN application number 202010820363.5, filing date 2020, 8/14, the disclosure of which is incorporated herein by reference in its entirety.
Technical Field
The application relates to application of fluorine-containing heterocyclic derivatives with a macrocyclic structure in treating diseases, in particular to application of a compound shown in a formula (I), stereoisomer or pharmaceutically acceptable salt thereof in preparing medicines for treating RET related diseases and application in treating RET related diseases.
Background
Proto-oncogene RET (Rearranged during Transfection) is responsible for encoding 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. Physiological ligands for RET belong to the family of glial cell-derived neurotrophic factors (GDNFs). RET activation is accomplished by interactions between the four receptors for the growth factor receptor alpha 1/2/3/4 (GFRalpha 1/2/3/4) and the four ligands for GDNFs, GFRalpha specifically binds GDNFs family members, promotes phosphorylation of RET protein receptors and brings RET into an activated state, thereby activating downstream signaling pathways involved in Cell proliferation, migration and differentiation, mainly including the Ras/Raf/MEK/ERK-MAPK pathway and the PI3K/Akt/mTOR pathway as well as other pathways (PLC-. Gamma.pathway, JAK-STAT pathway), etc. (Cell S.Airaksinen.mol Neurosci.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, driving the occurrence of tumors and maintaining the proliferation and survival of tumors.
RET gene variation is observed in a variety of tumors, and abnormal activation of RET is a key driver of tumor growth and proliferation in a large number of solid tumors. About 1% -2% of non-small cell lung cancer patients, 65% of medullary thyroid cancer, 10% -20% of papillary thyroid cancer patients develop RET mutations or fusions, and in other cancers, the overall average incidence of RET mutations is less than 1%. The oncogenic RET positive variation accounts for about 71.6% of the total RET variation. 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%) (thumb Kato. Clin Cancer Res.2018.23 (8)). In preclinical and clinical studies, RET inhibitors have been shown to significantly inhibit RET gene fusion and mutated tumor cell proliferation.
RET inhibitors are mainly divided into two types at present, one type is targeted RET small molecule inhibitors, including LOXO-292, BLU-667 and the like, and the targeted RET small molecule inhibitors are the focus of research on the RET inhibitors at present; the other class is multi-kinase inhibitors, which have RET inhibiting activity. LOXO-292 (Selpercatinib) was approved by the FDA in month 5 of 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, the first marketed RET inhibitor. Another RET inhibitor BLU-667 has been currently NDA-applied to the FDA for the treatment of patients with non-small cell lung cancer positive for locally advanced or metastatic RET fusion genes. In addition, there are other small molecule inhibitors of RET that have been developed preclinically.
As protooncogenes, RET positive mutations or fusion play a key role in the proliferation and growth of tumors such as non-small cell lung cancer, medullary thyroid cancer, and the like. More and more researches show that RET inhibitors can effectively inhibit proliferation of RET fusion or mutation malignant tumors in vivo, have higher disease remission rate clinically, and are effective treatment targets without differentiating cancer species.
Disclosure of Invention
The application provides an application of a compound shown in a formula (I), a stereoisomer or a pharmaceutically acceptable salt thereof in preparing a medicament for treating RET related diseases,
the application also provides a compound shown in the formula (I), a stereoisomer or a pharmaceutically acceptable salt thereof, which is used for treating RET related diseases.
The present application also provides a method of treating a RET related disease comprising administering to a subject in need thereof an effective amount of a compound of formula (I), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.
The application also provides a composition for treating RET related diseases, which comprises a compound shown in a formula (I), a stereoisomer or a pharmaceutically acceptable salt thereof, and optionally 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 an amount effective to treat a RET related disease.
The application also provides a medicament for treating RET related diseases, which comprises a compound shown in a formula (I), a stereoisomer or a pharmaceutically acceptable salt thereof, and optionally 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 an amount effective to treat a RET related 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 related disease is a disease associated with abnormal expression, activity, or level of a RET gene or a RET kinase protein.
In certain embodiments, the RET related disease is a disease associated with mutations 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 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 (PTC 1), RET-KIF5B (Kex 15Rex 14), RET-PRKAR1A (PTC 2), RET-BCR, RET-NCOA4 (PTC 3), 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 (PTC 3).
In certain embodiments, the RET fusion gene is RET-CCDC6 (PTC 1).
In certain embodiments, the RET fusion gene is RET-KIF5B (Kex 15Rex 14).
In certain embodiments, the RET fusion gene is RET-PRKAR1A (PTC 2).
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 (a 883F), RET (S904F), 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 related disease is a cancer associated with abnormal expression, activity, or level regulation of the RET gene or RET kinase protein.
In certain embodiments, the RET related disease is a cancer associated with mutation of 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 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, 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 ganglioma, and cervical cancer.
In certain embodiments, the RET related disease is a RET gene mutation-related lung cancer.
In certain embodiments, the RET related disease is thyroid medullary carcinoma associated with mutations in the RET gene.
In certain embodiments, the RET related disease is colon cancer associated with mutations in the RET gene.
In certain embodiments, the RET related disease is one or more selected from the group consisting of RET gene mutation-related small cell lung cancer, RET gene mutation-related non-small cell lung cancer, RET gene mutation-related bronchiolar lung cell cancer, and RET gene mutation-related lung adenocarcinoma.
In certain embodiments, the RET related disease is small cell lung cancer associated with mutations in the RET gene.
In certain embodiments, the RET related disease is non-small cell lung cancer associated with mutations in the RET gene.
In certain embodiments, the RET related disease is a RET gene mutation-related bronchioloalvee cancer.
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-associated 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, 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 ganglioma, and cervical cancer.
In certain embodiments, the RET related disease is RET fusion gene related lung cancer.
In certain embodiments, the RET related disease is a RET fusion gene-related medullary thyroid carcinoma.
In certain embodiments, the RET related disease is a RET fusion gene-related colon cancer.
In certain embodiments, the RET-related disorder is RET-CCDC 6-related colon cancer.
In certain embodiments, the RET related disease is one or more selected from the group consisting of RET fusion gene-related small cell lung cancer, RET fusion gene-related non-small cell lung cancer, RET fusion gene-related bronchiolar lung cell cancer, and RET fusion gene-related lung adenocarcinoma.
In certain embodiments, the RET related disease is small cell lung cancer associated with a RET fusion gene.
In certain embodiments, the RET related disease is non-small cell lung cancer associated with a RET fusion gene.
In certain embodiments, the RET related disease is a RET fusion gene-related bronchiolar lung cell carcinoma.
In certain embodiments, the RET related disease is a RET fusion gene-related lung adenocarcinoma.
In certain embodiments, the RET related disorder is a RET related disorder in a human, and the compound of formula (I), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof is administered at a dose of from 0.5 to 4mg/kg/day.
Definition:
in the present application, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. For a better understanding of the present application, definitions and explanations of the relevant terms are provided below.
The term "stereoisomer" as used herein refers to an isomer produced by the spatial arrangement of atoms in a molecule, and can be classified into cis-trans isomers, enantiomers, and enantiomers. The isomers which are caused by the same connecting sequence of atoms or groups of atoms in the molecule but different spatial arrangements are called stereoisomers, and two types of stereoisomers exist. Stereoisomers due to bond length, bond angle, double bonds within the molecule, rings, etc. are known as configurational isomers (configuration stereo-isomers). In general, configurational isomers cannot or are difficult to interconvert. Stereoisomers that are caused by rotation of a single bond alone are referred to as conformational isomers (conformational stereo-isomers), sometimes also referred to as rotamers. Configurational isomers fall into two categories. Wherein isomers due to the inability of single bonds of double bonds or ring-forming carbon atoms to rotate freely are called geometric isomers (cis-trans isomers), stereoisomers with different optical properties due to the lack of anti-axisymmetry in the molecule are called optical isomers (optical isomers).
The term "RET (Rearranged during Transfection) gene" as used in the present application is a proto-oncogene, full name: RET pro-ontogene: located on the long arm of autosomal chromosome 10 (10q11.2), is 53kb in length, contains 20 exons, and encodes a 1114 amino acid tyrosine kinase receptor.
The term "RET kinase" as used in the present application is a receptor tyrosine kinase encoded by RET gene and involved in signal transduction in the processes of cell proliferation, migration, differentiation and survival of neural crest cells, formation of kidney organs, spermatogenesis, etc.
The term "RET fusion gene" as used in the present application means that RET gene is rearranged with other gene sequences, resulting in fusion after protein expression, which results in abnormal expression or activity of RET gene level or protein level. In the present application, "RET fusion gene" is denoted by "A-B" or "B-A", wherein A, B represents RET gene and other genes fused to RET gene, respectively. "A-B" or "B-A" has the same meaning and means ase:Sub>A fusion gene of the A gene and the B gene. For example, RET-CCDC6 and CCDC6-RET are the same meaning, and both are fusion genes of RET and CCDC 6.
As used herein, the term "RET gene mutation" 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 the protein.
The term "pharmaceutically acceptable salts" as used herein includes conventional salts with pharmaceutically acceptable inorganic or organic acids, or inorganic or organic bases. Methods for preparing pharmaceutically acceptable salts of the compounds of the present application are known to those skilled in the art.
The application relates to a compound, which comprises a compound shown in a formula (I), a stereoisomer 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 that results, directly or indirectly, from the combination of compounds of the present application. The pharmaceutical composition may be administered by a route such as oral or parenteral. The pharmaceutical compositions of the present application may be formulated into various dosage forms, including but not limited to tablets, capsules, solutions, suspensions, granules or injections, etc., by conventional methods in the art, and administered orally or parenterally.
The term "effective amount" as used herein refers to an amount sufficient to achieve the desired therapeutic effect, e.g., to achieve a reduction in symptoms associated with the disease to be treated.
The term "treatment" as used herein is intended to reduce or eliminate the disease state or condition for which it is intended. A subject is successfully "treated" if the subject has received a therapeutically effective amount of a compound, stereoisomer thereof, pharmaceutically acceptable salt thereof, or pharmaceutical composition thereof, in accordance with the methods of the application, and the subject exhibits an observable and/or detectable decrease or improvement in one or more of the indications and symptoms. It is also to be understood that the 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 use of the compounds of the present application will depend upon a number of factors including the age, weight, sex, natural health, nutritional status of the patient, the strength of 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 compounds described in the present application are based on the structural formula of the compound if the name is not identical to the structural formula of the same compound.
Drawings
FIG. 1 showsGrowth curves of average tumor volumes of mice in each group in a colon cancer CR2518 model;
FIG. 2 showsBody weight profile of each group of mice in colon cancer CR2518 model over treatment time.
In fig. 1 and 2: 1. data are expressed as "mean ± standard error"; g3-00442# mice found death on day 30 post-grouping; g3-00443# mice found death on day 33 post-grouping; bid stands for twice a day dosing; QD stands for once a day administration.
Detailed Description
The following examples further illustrate the application in connection with its embodiments, and it should be understood that the following examples are illustrative of the application and are not intended to limit the scope of the application. The specific conditions are not noted in the examples below, and are carried out according to conventional conditions or manufacturer's recommendations. The raw materials used are conventional products which are commercially available without identifying the manufacturer.
Although many materials and methods of operation are known in the art for use in the following examples, the application is nevertheless described in as much detail as possible. It will be clear to those skilled in the art that the materials and methods of operation used in the examples which follow are well known in the art, if not specifically described.
The compounds useful as RET inhibitors provided herein and methods for 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-oxybenzotriazol) -N, N' -tetramethyluronium hexafluorophosphate;
Cs 2 CO 3 represents cesium carbonate;
THF represents tetrahydrofuran;
POCl 3 represents phosphorus oxychloride;
DMF means N, N-dimethylformamide;
NaOH represents sodium hydroxide;
DIPEA or DIEA represents N, N-diisopropylethylamine;
H 2 o represents water;
HCl/Dioxane represents a hydrogen chloride Dioxane solution;
rt represents the reaction temperature is room temperature;
n, N-Diethyllaniline represents N, N-Diethylaniline;
CH 3 CN or ACN represents acetonitrile;
zn represents zinc powder;
NH 4 cl represents ammonium chloride;
CH 3 MgBr represents methyl magnesium bromide;
K 2 CO 3 represents potassium carbonate;
EtOH represents ethanol;
OTs represents p-toluenesulfonyloxy,
ee represents enantiomeric excess.
RET (S891A) means that serine at 891 of RET gene is mutated to alanine;
RET (L790F) means that leucine of RET gene at 790 is mutated into phenylalanine;
RET (V804M) represents the mutation of valine at position 804 of the RET gene to 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 is mutated to alanine;
RET (R912P) represents a mutation of arginine to proline at position 912 of the RET gene;
RET (Y791F) indicates that the RET gene is mutated from tyrosine at position 791 to phenylalanine;
RET (V778I) means that valine at position 778 of RET gene is mutated to isoleucine;
RET (G691S) means that glycine at position 691 of RET gene is mutated to serine;
RET (V804L) indicates that valine at position 804 of the RET gene is mutated to leucine;
RET (R813Q) indicates that the arginine at position 813 of the RET gene is mutated to glutamine;
RET (E762Q) represents a mutation of glutamic acid at position 762 of the RET gene to glutamine;
RET (V804E) indicates that valine at position 804 of the RET gene is mutated to glutamate;
RET (A883F) indicates that the alanine at position 883 of the RET gene is mutated to phenylalanine;
RET (S904F) indicates that serine at position 904 of the RET gene is mutated to phenylalanine;
RET (Y806H) indicates that the tyrosine at position 806 of RET gene is mutated to histidine;
RET (M918T) means that the methionine at position 918 of the RET gene is mutated to threonine.
RET-CCDC6 (PTC 1) is a fusion gene of RET and CCDC6 (alias PTC1, coded-coil domain containing, coiled-coil domain protein 6, gene ID (NCBI): 8030);
RET-KIF5B (Kex 15Rex 14) is a fusion gene of RET and KIF5B (the kinesin family B gene, kinesin family 5B, gene ID (NCBI): 3799), where the fusion position begins at the 15 exon of the KIF5B gene (amino acids 1-575) and ends at the 14-21 exon of RET (amino acids 713);
RET-PRKAR1A (PTC 2) 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 B gene, kinesin family 5B, gene ID (NCBI): 3799);
RET-BCR: fusion genes of RET and BCR (alias BCR1; 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 B gene, kinesin family 5B, gene ID (NCBI): 3799);
RET-NCOA4 (PTC 3) 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, CAP-Gly domain-containing linker protein 1, gene ID (NCBI): 6249);
CCDC6-RET is a fusion gene of RET and CCDC6 (coiled-coil domain protein 6, gene ID (NCBI): 8030);
TRIM33-RET is a fusion gene of RET and TRIM33 (tripartite motif containing, tri-motif protein 33, gene ID (NCBI): 51592);
ERC1-RET is a fusion gene of RET and ERC1 (ELKS/RAB 6-interacting/CAST family member 1, ELKS/Rab6 interaction/CAST protein family member 1, gene ID (NCBI): 23085);
FGFR1OP-RET is a fusion Gene of RET and FGFR1OP (FGFR 1 oncogene partner, gene ID (NCBI): 11116);
RET-MBD1 is a fusion Gene of RET and MBD1 (methyl-CpG binding domain protein 1, cytidylylguanosine monophosphate binding protein, gene ID (NCBI): 4152);
RET-RAB6IP2 is a fusion gene of RET and RAB6IP2 (Rab 6 Interacting Protein A, alias ERC-1, gene ID (NCBI): 23085);
RET-TRIM24 is a fusion gene of RET and TRIM24 (tripartite motif containing, three 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, hooked microtubule tethered protein 3, gene ID (NCBI): 84376);
KTN1-RET is a fusion gene of RET and KTN1 (kinetin 1, kinesin binding protein 1, gene ID (NCBI): 3895);
TRIM27-RET is a fusion gene of RET and TRIM27 (tripartite motif containing, motif protein 27, gene ID (NCBI): 5987);
AKAP13-RET is a fusion gene of RET and AKAP13 (A-kinase anchoring protein 13, kinase-anchored protein 13, gene ID (NCBI): 11214);
FKBP15-RET is a fusion gene of RET and FKBP15 (FKBP prolyl isomerase family member, proline 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 frizzled domain 1like kd glycoprotein, gene ID (NCBI): 2384);
TBL1XR1-RET is a fusion gene of RET and TBL1XR1 (TBL 1X receptor 1, gene ID (NCBI): 79718);
CEP55-RET is a fusion gene of RET and CEP55 (centrosomal protein, centrosome protein 55, gene ID (NCBI): 55165);
CUX1-RET is a fusion gene of RET and CUX1 (cut like homeobox 1, homologous frame cleavage protein 1, gene ID (NCBI): 1523);
KIAA1468-RET is a fusion Gene of RET and KIAA1468 (alias RAB11 binding and LisH domain, coded-coil and HEAT repeat containing, gene ID (NCBI): 57614)
RFG8-RET is a 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, a fusion gene of RET extracellular domain exon 9 with exon 1 of CCDC6 (coiled-coil domain 6, gene ID (NCBI): 8030);
MYH13-RET is a fusion gene of RET and MYH13 (myosin heavy chain, myosin heavy chain 13, gene ID (NCBI): 8735);
PIBF1-RET is a fusion gene of RET and PIBF1 (progesterone immunomodulatory binding factor, progesterone immunoregulatory 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, myosin phosphatase-Rho acting protein, gene ID (NCBI): 23164);
HRH4-RET is a fusion Gene of RET and HRH4 (histamine receptor H, 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, regulatory subunit RIA of protein kinase A);
RET-PTC4 is a fusion Gene of RET and PTC4 (type 2C protein phosphatase,2C type protein phosphatase, gene ID (NCBI): 5108);
FRMD4A-RET is a fusion gene of RET and FRMD4A (FERM domain containing A, FERM domain protein 4A, gene ID (NCBI): 55691);
SQSTM1-RET is a fusion gene of RET and SQSTM1 (sequencer 1, autophagy linker protein 1, gene ID (NCBI): 8878);
AFAP1L2-RET is a fusion gene of RET and AFAP1L2 (actin filament associated protein like 2, actin-wire related protein 1-like protein 2, gene ID (NCBI): 84632);
PPFIBP2-RET is a fusion gene of RET and PPFIBP2 (PPFIA binding protein, 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-3 family cell polarity modulator, gene ID (NCBI): 56288);
MYH10-RET is a fusion gene of RET and MYH10 (myosin heavy chain, myosin heavy chain 10, gene ID (NCBI): 4628);
HTIF1-RET is a fusion gene of RET and HTIF1 (alias tripartite motif containing, three motif protein 24, gene ID (NCBI): 8805);
AFAP1-RET is a fusion gene of RET and AFAP1 (actin filament associated protein 1, actin-wire related protein 1, gene ID (NCBI): 60312);
RASGEF1A-RET is a fusion gene of RET and RASGEF1A (RasGEF domain family member A, rasGEF domain family member 1A, gene ID (NCBI): 221002);
TEL-RET is a 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, run and FYVE domains comprise 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, MAGUK scaffold protein 5, gene ID (NCBI): 9231);
FOXP4-RET is a fusion Gene of RET and FOXP4 (forkhead box P4, fork box P4, gene ID (NCBI): 116113);
OLFM4-RET is a fusion gene of RET and OLFM4 (Olfactor vidin-4, olfactory protein 4, gene ID (NCBI): 418826);
RRBP1-RET is a fusion Gene of RET and RRBP1 (ribosome binding protein, ribosome binding protein 1Gene ID (NCBI): 6238).
Preparation example 1: preparation of 5-chloro-6-fluoropyrazolo [1,5-a ] pyrimidine-3-carboxylic acid ethyl ester
Step 1: preparation of 2-fluoromalonic acid
Diethyl 2-fluoromalonate (5.0 g) and sodium hydroxide (17.3 g) were weighed into a mixed solution of ethanol/water (100/100 mL) at room temperature, reacted overnight, LCMS showed completion of the reaction. The reaction solution was concentrated to remove ethanol, water (50 mL) was added thereto, 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 (2.0 g) and 5-amino-1H-pyrazole-4-carboxylic acid ethyl ester (1.7 g) were weighed into phosphorus oxychloride (20 mL) at room temperature, N-dimethylformamide (2 mL) and N, N-diethylaniline (4.9 g) were added, and the temperature was raised to 110℃for 3 hours, and LCMS showed complete reaction. The reaction solution was concentrated to remove phosphorus oxychloride, then poured into a saturated sodium bicarbonate solution (100 mL), the solution was kept alkaline, extracted three times with ethyl acetate, dried over anhydrous sodium sulfate, filtered, concentrated, the obtained crude product was purified by column chromatography, and the obtained solid was washed with petroleum ether and dried to obtain the title compound 1.7g.
MS(ESI)m/z(M+H) + ＝278.0.
1 H NMR(400MHz,DMSO-d 6 )δ8.80(s,1H),4.33(q,J＝7.2Hz,2H),1.32(t,J＝7.0Hz,3H).
Step 3: preparation of 5-chloro-6-fluoropyrazolo [1,5-a ] pyrimidine-3-carboxylic acid ethyl ester
To a mixed solution of ethyl 5, 7-dichloro-6-fluoropyrazolo [1,5-a ] pyrimidine-3-carboxylate (1.14 g) and ammonium chloride (800 mg) in ethanol/tetrahydrofuran/water (30/10/20 mL) was weighed, zinc powder (1.3 g) was added during stirring, and after 5 minutes of reaction, the zinc powder was filtered, the filter cake was washed with ethyl acetate, the filtrate was collected, dried over anhydrous sodium sulfate, filtered, and concentrated, and the crude product obtained was purified by column chromatography to give 800mg of the title compound.
MS(ESI)m/z(M+H) + ＝244.0.
1 H NMR(400MHz,DMSO-d 6 )δ9.93(d,J＝4.4Hz,1H),8.68(s,1H),4.31(q,J＝7.2Hz,2H),1.31(t,J＝7.2Hz,3H).
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-methylpropan-2-sulfinamide
(R) -2-methylpropane-2-sulfinamide (12.9 g) was dissolved in tetrahydrofuran (100 mL), and 5-fluoro-2-methoxynicotinaldehyde (15.0 g) and cesium carbonate (40.9 g) were added sequentially. The resulting mixture was reacted at room temperature for 2 hours, and TLC showed complete consumption of starting material. Suction filtration, washing the filter cake with tetrahydrofuran three times, backwashing the obtained filtrate with saturated sodium chloride solution once, drying with anhydrous sodium sulfate, filtering and concentrating. The crude product was purified by column chromatography to give the title compound 23.0g.
MS(ESI)m/z(M+H) + ＝259.1.
1 H NMR(400MHz,DMSO-d 6 )δ8.67(d,J＝2.4Hz,1H),8.42(d,J＝3.2Hz,1H),8.14(dd,J＝8.4,3.2Hz,1H),3.98(s,3H),1.18(s,9H).
Step 2: preparation of (R) -N- ((R) -1- (5-fluoro-2-methoxypyridin-3-yl) ethyl) -2-methylpropan-2-sulfinamide
(R) -N- (((5-fluoro-2-methoxypyridin-3-yl) methylene) -2-methylpropane-2-sulfinamide (5.0 g) was weighed and dissolved in tetrahydrofuran (40 mL), after the resulting mixture was cooled to-78 ℃, methylmagnesium bromide (7.8 mL, 3M) was slowly added dropwise, the temperature was maintained below-65 ℃ C., after dropping, the temperature was naturally returned to room temperature, and the reaction was continued for 1 hour, TLC showed that the reaction was complete.
MS(ESI)m/z(M+H) + ＝275.2.
1 H NMR(400MHz,DMSO-d 6 )δ8.04(d,J＝2.8Hz,1H),7.74(dd,J＝9.2,3.2Hz,1H),5.80(d,J＝8.8Hz,1H),4.57-4.50(m,1H),3.88(s,3H),1.33(d,J＝6.8Hz,3H),1.11(s,9H).
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 (4.5 g) was dissolved in a hydrogen chloride-dioxane solution (30 mL) at room temperature and reacted overnight, LCMS showed complete consumption of starting material. The reaction solution was concentrated to give 3.1g of crude product, more than 95% ee, which was used directly in the next step without purification.
MS(ESI)m/z(M+H) + ＝171.2.
1 H NMR(400MHz,DMSO-d 6 )δ8.80-8.66(m,3H),8.18(d,J＝2.8Hz,1H),8.04-8.00(m,1H),7.09–6.60(m,1H),4.51–4.45(m,1H),3.90(s,3H),1.49(d,J＝6.4Hz,3H).
Example 1: (3 1 S,3 3 S,6 3 E,6 4 E,8R)-1 5 ,6 6 -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) -cyclobutaneoctan-5-one (Compound Ib)
Step 1: preparation of (R) -6-fluoro-5- ((1- (5-fluoro-2-methoxypyridin-3-yl) ethyl) amino) pyrazolo [1,5-a ] pyrimidine-3-carboxylic acid ethyl ester
(R) -1- (5-fluoro-2-methoxypyridin-3-yl) ethyl-1-amine hydrochloride (1.0 g) was dissolved in acetonitrile (20 mL), and N, N-diisopropylethylamine (1.9 g) and ethyl 5-chloro-6-fluoropyrazolo [1,5-a ] pyrimidine-3-carboxylate (1.2 g) were sequentially added, and the resulting mixture was reacted at 60℃for 3 hours, and TLC showed complete reaction. 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 crude product was purified by column chromatography to give the title compound 1.1g.
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: preparation of (R) -6-fluoro-5- (((1- (5-fluoro-2-methoxypyridin-3-yl) ethyl) amino) pyrazolo [1,5-a ] pyrimidine-3-carboxylic acid
(R) -6-fluoro-5- ((1- (5-fluoro-2-methoxypyridin-3-yl) ethyl) amino) pyrazolo [1,5-a ] pyrimidine-3-carboxylic acid ethyl ester (1.1 g) was dissolved in a mixed solution of ethanol/water (5/15 mL) at room temperature, sodium hydroxide (584 mg) was added, the resulting mixture was reacted overnight at 50℃and TLC showed that the reaction was complete. The reaction solution was concentrated to remove ethanol, the residual solution was poured into water (20 mL), the pH was adjusted to about 5 with hydrogen chloride solution (2M), dichloromethane extraction was performed, the organic phases were combined, the saturated sodium chloride solution was backwashed, dried over anhydrous sodium sulfate, filtered, and 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.
1 H NMR(400MHz,DMSO-d 6 )δ11.68(s,1H),9.13(d,J＝6.0Hz,1H),8.51(d,J＝7.6Hz,1H),8.14(s,1H),8.01(d,J＝2.8Hz,1H),7.72(dd,J＝9.0,3.0Hz,1H),5.59-5.52(m,1H),3.92(s,3H),1.50(d,J＝6.8Hz,3H).
Step 3: preparation of (1R, 3R) -3- (6-fluoro-5- (((R) -1- (5-fluoro-2-methoxypyridin-3-yl) ethyl) amino) pyrazolo [1,5-a ] pyrimidine-3-carboxamide) -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 (800 mg), 2- (7-azobenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate (1.0 g) and N, N-diisopropylethylamine (886 mg) were dissolved in dry tetrahydrofuran (10 mL), and after the resulting mixture was reacted at room temperature for 1 hour, (3-hydroxycyclobutyl) carbamic acid tert-butyl hydrochloride (953 mg) was added and the reaction was continued for 1 hour, TLC showed completion of the reaction, the reaction solution was poured into water (30 mL), extracted with ethyl acetate, the organic phases were combined, a saturated sodium chloride solution was backwashed, dried over anhydrous sodium sulfate, filtered, and concentrated.
MS(ESI)m/z(M+H) + ＝573.2.
1 H NMR(400MHz,DMSO-d 6 )δ9.21(d,J＝6.0Hz,1H),8.57(d,J＝7.6Hz,1H),8.09(s,1H),8.05(d,J＝2.8Hz,1H),7.80–7.78(m,2H),7.66(dd,J＝8.8,2.8Hz,1H),7.60(d,J＝6.8Hz,1H),7.47(d,J＝8.0Hz,2H),5.44-5.37(m,1H),4.96–4.92(m,1H),4.33-4.28(m,1H),3.80(s,3H),2.47–2.38(m,5H),2.24-2.18(m,1H),2.12–2.08(m,1H),1.52(d,J＝6.8Hz,3H).
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-carboxamide) -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-carboxamide) -4-methylbenzenesulfonic acid cyclobutyl ester (800 mg) was dissolved in dioxane (4M, 10 mL) of hydrogen chloride and the resulting mixture was reacted overnight at 55℃until TLC showed completion of the reaction. The reaction solution was directly concentrated to remove most of dioxane to obtain 600mg of crude product, which was directly used in the next step without purification.
MS(ESI)m/z(M+H) + ＝559.2.
Step 5: (3 1 S,3 3 S,6 3 E,6 4 E,8R)-1 5 ,6 6 -difluoro-8-methyl-2-oxa-4, 7-diaza-6 (3, 5) -pyrazolo [1,5-a]Preparation of pyrimidine-1 (2, 3) -pyridin 3-3 (1, 3) -cyclobutaneoctan-5-one
(1R, 3R) -3- (6-fluoro-5- (((R) -1- (5-fluoro-2-hydroxypyridin-3-yl) ethyl) amino) pyrazolo [1,5-a ] pyrimidine-3-carboxamide) -4-methylbenzenesulfonate cyclobutyl hydrochloride (250 mg) was dissolved in N, N-dimethylformamide (6 mL), potassium carbonate (232 mg) was added, and the resulting mixture was reacted at room temperature for 5 hours, and TLC showed completion of the reaction. 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 crude product obtained is purified by preparative thin layer chromatography and preparative high performance liquid chromatography to give 58.0mg of the title compound with 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 the test examples 1 to 7 according to the application include:
compound Ib, prepared according to inventive example 1;
LOXO-195, a second generation TRK inhibitor, has the chemical structure:/>
positive control 1, a compound of example 5 of patent WO2019210835, has the chemical structure:
positive control 2, a compound of example 6 of patent WO2019210835, having the chemical structure:
compound 1, structural formula:
compound 2, having the structural formula:
Compound 4, structural formula:
compound 5, structural formula:/>
compound 6, having the structural formula:
LOXO-195 and control compounds 1-2 are commercially available and can also be prepared synthetically according to conventional routes, e.g., LOXO-195 is commercially available and positive controls 1 and 2 can be synthesized as described in WO 2019210835. Compounds 1, 2 and 4-6 can all be prepared synthetically according to conventional routes, and can also be synthesized by the methods described in WO2021115401A 1.
Test example 1: inhibition of RET kinase by Compound Ib prepared in example 1 at a dose of 1. Mu.M.
2.1 preparation of test samples
2.2 Compound dose
Test sample concentration: 1 mu M
Compound incubation time: 20 minutes
ATP concentration: 10 mu M
Reaction time: for 2 hours
2.3: detection conditions and protocol
Detection conditions:
buffer solution preparation: 20mM HEPES (pH 7.5), 10mM MgCl 2 ,1mM EGTA,0.01％Brij35 0.02mg/mL
BSA,0.1mM Na 3 VO 4 ,2mM DTT,1％DMSO
The desired cofactor is added separately to the corresponding kinase reaction
The reaction process comprises the following steps:
the test subjects were subjected to single dose experiments (2 multiplex wells) at a concentration of 1. Mu.M, with initial concentration of control Staurosporine (Med Chem, MC-2104) of 20. Mu.M or 100. Mu.M, at a ratio of 1:4 to give 10 reaction concentrations. The test will determine the single concentration inhibition of the test agent IC for comparison product 50 Values.
1. Preparing a reaction buffer solution, and preparing a corresponding substrate by using the newly prepared reaction buffer solution;
2. transferring a desired coenzyme factor into the substrate solution;
3. adding the corresponding kinase into the substrate solution, and gently and uniformly mixing;
4. transferring the test sample dissolved in DMSO into the kinase reaction mixture using an acoustic pipette (Echo 550);
5. will be 10 mu M 33 P-ATP is transferred to the above solution, at which point the reaction begins;
6. incubating for 120 minutes at room temperature;
7. spotting the reaction solution on P81 ion exchange chromatography paper (Whatman # 3698-915);
8. washing the chromatographic paper with a large amount of 0.75% phosphoric acid solution;
9. the remaining radioactively phosphorylated substrate on the ion exchange chromatography paper is detected.
Data analysis:
kinase activity is expressed as a percentage of kinase activity after reaction of the corresponding kinase with compound Ib and after reaction with vehicle group (DMSO). 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 strong inhibition activity on various RET mutations or fusion kinases, can reach more than 85% inhibition rate at 1 mu M, and can reach more than 95% inhibition rate on certain RET mutations and fusion kinases.
Test example 2: compounds Ib inIn vivo pharmacodynamics study on colon cancer CR2518 subcutaneous xenograft model BALB/c nude mouse model
1. Experimental materials and reagents
1.1 test animals: BALB/c Nude mice, females, 6-8 weeks (week of age of the mice at tumor cell inoculation), body weights 21-25g,25, purchased from Beijing An Kaiyi Bo Biotechnology Co., ltd., animal eligibility number: 110330201100091758. feeding environment: SPF stage.
1.2 test Compounds
1.3 model information
Colon cancer allograft model CR2518 information
CR2518 is established by colonic tumors originating in a female patientXenograft model. The model is a CCDC6-RET fusion mutation, has a tumor bursting tendency, and can cause slight weight loss of animals.
Model ID Type of cancer Subtype type Race and race Sex (sex) Age of Pathological diagnosis Characteristics (1)
CR2518 Colon cancer ADC Asian-type Female 82 Has the following components RET-CCDC6 fusion
2. Test method
Animal inoculation and grouping: from the slaveTumor tissues are collected from a tumor-bearing mouse of a colon cancer xenograft model CR2518 (R14P 5), and tumor blocks with diameters of 2-3mm are cut and inoculated under the skin at the right anterior shoulder blade of a BALB/c nude mouse. The average tumor volume reaches 151mm 3 The administration of the packets was started at that time. The test was divided into 4 groups, group 1 was a vehicle control group, group 2 was a low dose group of the test drug (test drug was compound Ib, single dose was 10mg/kg, QD was 14 days, then BID was 21 days), group 3 was a high dose group of the test drug (test drug was compound Ib, single dose was 20mg/kg, QD was 14 days, then BID was 21 days), group 4 was a positive control group (positive control drug was BLU-667, single dose was 10mg/kg, BID was 34 days), 5 groups each, oral gavage administration was completed, and the experiment was ended on day 35 after administration.
3. Tumor measurement and experimental index
The experimental index is to examine whether the tumor growth is affectedInhibit, delay or cure. Tumor diameters were measured twice weekly with vernier calipers. The calculation formula of the tumor volume is: v=0.5a×b 2 A and b represent the major and minor diameters of the tumor, respectively.
The tumor-inhibiting effect of the compound was evaluated by TGI (%) or relative tumor proliferation rate T/C (%). TGI (%) reflects the tumor growth inhibition rate. Calculation of TGI (%): TGI (%) = [1- (mean tumor volume at the end of dosing of a treatment group-mean tumor volume at the beginning of dosing of a treatment group)/(mean tumor volume at the end of treatment of solvent control group-mean tumor volume at the beginning of treatment of solvent control group) ]x100%.
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 (C) RTV : vehicle control RTV). Relative tumor volume (relative tumor volume, RTV) is calculated from the result of the tumor measurement, calculated as rtv=v n /V 0 Wherein V is 0 Is the tumor volume obtained by measurement of group administration (i.e. d 0), V n For tumor volume at a certain measurement, T RTV And C RTV The same day data was taken.
Tumor weights will be measured after the end of the experiment and T/C calculated weight Percent, T weight And C weight Tumor weights are shown for the dosing group and the vehicle control group, respectively.
4. Statistical analysis
The analysis results included mean and Standard Error (SEM) of tumor volumes for each time point for each group. Treatment groups exhibited significant tumor inhibition at day 35 post-dosing at the end of the trial, and thus statistical analysis was performed based on this data to assess inter-group differences. The Bartlett test was first used to verify all inter-group variance homogeneity assumptions. When the Bartlett test p-value is not less than 0.05, a one-way anova will be used to test if all group means are equal. If the p-value of the one-way anova is less than 0.05, we will either make a pairwise comparison 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 p-value of the Bartlett test is less than 0.05, the Kruskal Wallis test will be used to test whether the median of all groups is equal. If the p-value of the Kruskal Wallis test is less than 0.05, we will use the over test to make a pairwise comparison between all groups or between each treatment group and the control group, and make a corresponding p-value correction based on the number of groups tested in multiple. All statistical analyses and graphic renderings were performed in the R language environment (version 3.3.1). All tests were double-tailed and p-values less than 0.05 were considered statistically significant.
5. Test results
5.1 test drug Compound Ib inResults and discussion of anti-tumor effects in colon cancer CR2518 model
5.1.1 the growth curve and tumor volume analysis of the average tumor volume of each group of mice are shown in fig. 1 and table 2.
TABLE 2 atTumor volume analysis table of each group in colon cancer CR2518 model
Note that: 1. data are expressed as "mean ± standard error"; T/C% = T RTV /C RTV X 100%; TGI% = (1-T/C) ×100%;3. a comparison between each treatment group and the control group was made using the over test.
5.2 test drug Compounds Ib and BLU-667 inSafety study results and discussion in colon cancer CR2518 model
5.2.1 average body weight change curves and body weight change analyses for each group of mice are shown in fig. 2 and table 3.
TABLE 3 atBody weight change conditions of all groups in colon cancer CR2518 model
Note that: 1. data are expressed as "mean ± standard error"; g3-00442# mice found death on day 30 post-grouping; the G3-00443# mice found death on day 33 post-grouping.
35 days after administration, the positive control group (BLU-667, 10 mg/kg) had a significant tumor inhibiting effect (T/C=0.0%, TGI=100%, p) compared with the vehicle control group<0.001 A) is provided; T/C values of group 2 (compound Ib,10 mg/kg) and group 3 (compound Ib,20 mg/kg) are 29.3% and 9.5%, respectively, TGI values are 70.7% and 90.5%, respectively, p values, respectively <0.05 sum<0.001, has remarkable tumor inhibiting effect compared with the vehicle control group. The test drug compound Ib has no animal death at the dosage of 10mg/kg, no obvious drug toxicity and good tolerance during treatment. Compound Ib was administered once daily at a dose of 20mg/kg for 14 days continuously without loss of body weight and without apparent drug toxicity in mice. Compound Ib was then adjusted to be administered twice daily at 20mg/kg for 21 consecutive days, and mice developed weight loss with some animal death, exhibiting some drug toxicity. Test drug Compound Ib at the dose usedColon cancer CR2518 showed a significant tumor growth inhibiting effect. Tumor-bearing mice are well tolerated by low dose compound Ib. The high-dose compound Ib has certain drug toxicity to tumor-bearing mice.
The dose of the human can be calculated from the dose of the mice, as shown in the following table.
6. Conclusion(s)
Test drug chemistryCompound Ib and the positive control agent BLU-667 are paired at the dosages usedColon cancer CR2518 all had a significant tumor growth inhibiting effect. Tumor-bearing mice are well tolerated by compounds Ib and BLU-667 at the test doses and at low doses.
Test example 3: kinase inhibitory Activity
The test case entrusts the completion of the Sandinia medical technology (Shanghai) Limited liability company.
1. Purpose of experiment
The inhibitory activity of the compounds of the present application against three kinases TRKa, TRKa (G595R) and TRKC (G623R) was determined.
2. Experimental materials
2.2.1 reagents and consumables
Reagent name Suppliers of goods Goods number Lot 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 pore plate (white) PerkinElmer 6007290 810712
2.2.2 instruments
Centrifuge (manufacturer: eppendorf, model: 5430); enzyme label instrument (manufacturer: perkin Elmer, model: caliper EZ ReaderII); echo 550 (manufacturer: labcyte, model: echo 550)
3. Experimental method
3.1 test compounds were precisely weighed and dissolved in 100% dmso to make up a 10 mM solution.
3.2 kinase reaction procedure
3.2.1 preparation of 1 Xkinase buffer.
3.2.2 preparation of compound concentration gradient: the test compounds were tested at an initial concentration of 1000 nM, diluted 100-fold in 384 plates to 100% DMSO at final concentration, and then 3-fold diluted to give 10 concentrations of compound in DMSO. The final concentration of compound was transferred to the reaction plate OptiPlate-384F at 250 nL 100-fold using a dispenser Echo 550.
3.2.3A 2.5-fold final concentration of kinase solution was prepared with 1 Xkinase buffer.
3.2.4 adding 10. Mu.L of 2.5-fold final concentration kinase solution to each of the compound well and the positive control well; mu.L of 1 Xkinase buffer was added to the negative control wells.
3.2.5 the plates were mixed with shaking and incubated at room temperature for 10 minutes.
3.2.6 preparation with 1 Xkinase bufferMixed solution of ATP and kinase substrate 22 at a double final concentration.
3.2.7 Add 15. Mu.LMixed solution of ATP and substrate at multiple final concentrations.
3.2.8 the 384-well plates were centrifuged at 1000 rpm for 30 seconds, and after shaking and mixing, incubated at room temperature for a corresponding period of time.
3.2.9 terminates the kinase reaction.
3.2.10 the conversion was read with a microplate reader Caliper EZ Reader.
4. Data analysis
Calculation formula
Inhibition = (% conversion max-% conversion sample)/(% conversion max-% conversion min) x 100
Wherein: the% conversion sample is a conversion reading of the sample; the percent conversion is the smallest: negative control Kong Junzhi, representing conversion reading without enzyme wells; conversion% is maximum: positive control Kong Bizhi mean represents conversion readings for wells without compound inhibition.
Fitting dose-response curve: the log of concentration is taken as the X axis, the percent inhibition is taken as the Y axis, the log (inhibitor) vs. response-Variable slope (Variable slope) fit of analytical software GraphPadprism 5 is adopted (four-parameter model fitting) to obtain the IC of each compound to the enzyme activity 50 Values.
The results are shown in table 4 below:
TABLE 4 IC of the inhibitory Activity of the compounds of the application against three kinases 50 Value of
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The results show that compound Ib has high inhibitory activity against three kinases, TRKa (G595R) and TRKC (G623R). Compared with the compounds 4-6, the activity of the compound Ib is obviously different, which shows that the fluorine substitution at different positions has larger difference on the inhibition activity of three kinases.
Test example 4: in vitro cell Activity
The test example was completed by the Co-fertilizer Koehne Biotechnology Co.Ltd, wherein the NTRK mutant cells used were constructed by the Co.
1. Purpose of experiment
Determination of the inhibitory Effect of the Compounds of the application on the growth of three NTRK mutant cells (Ba/F3 LMNA-NTRK1-G667C, ba/F3EVT6-NTRK3-G623R, ba/F3 LMNA-NTRK 1-G595R)
2. Reagent and consumable
Cell line:
reagent:
fetal bovine serum FBS (GBICO, cat#10099-141);luminescent Cell Viability Assay (CTG, promega, cat#G7573); 96-well transparent flat bottom black wall plate (+)>Cat#165305)；RPMI-1640(Hyclone，Cat#SH30809.01)
3. Experimental procedure
3.1 cell culture and seeding:
collecting cells in logarithmic growth phase, counting with platelet counter, and adjusting cell concentration to 3-6X10 4 cell/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% CO 2 Incubated overnight at 95% humidity.
3.2 drug dilution and dosing:
the test compound is prepared into a 10-time drug solution with a culture medium containing 1% of DMSO, and the highest concentration is 10 mu M, and the drug solutions with the concentration of 9 concentrations are obtained by 3 times dilution in sequence. More than 10 mu L of prepared drug solution is added into each hole of a 96-well plate inoculated with cells, 90 mu L of culture medium containing 1% of DMSO is added to obtain the drug solution with the highest concentration of 1 mu M, the drug solution is diluted 3 times in sequence, the total concentration is 9, the final concentration of DMSO in each hole is 0.1%, and three compound holes are arranged for drugs with each concentration. Cells in the dosed 96-well plates were placed at 37℃with 5% CO 2 The culture was continued for 72 hours at 95% humidity, after which CTG analysis was performed.
3.3 end point reading board
An equal volume of CTG solution was added to each well, and the 96-well plate was left at room temperature for 20 minutes to stabilize the luminescence signal, and the luminescence value was read.
4. Data processing
Analysis of data using GraphPad Prism 7.0 software, fitting data to derive dose-response curves using nonlinear S-curve regression, and calculating IC therefrom 50 Values.
Cell viability (%) = (test drug Kong Lengguang value-culture broth control Kong Lengguang value)/(cell control Kong Lengguang value-culture broth control Kong Lengguang value) ×100%.
The experimental results are shown in table 5 below:
TABLE 5 IC of the inhibitory Activity of the compounds of the application against three strains of cells 50 Value of
The results show that the compound Ib has strong growth inhibition effect on three NTRK mutant cells (Ba/F3 LMNA-NTRK1-G667C, ba/F3EVT6-NTRK3-G623R and Ba/F3 LMNA-NTRK 1-G595R). The inhibitory activity of compounds 1 and 2 against three NTRK mutant cells was not significantly different from the corresponding positive compounds, but the inhibitory activity of compound Ib against three NTRK mutant cells was significantly higher than the corresponding positive control 2.
Test example 5: liver microsome stability test
1. Purpose of experiment
Determination of the stability of the Compounds of the application in human, rat and mouse liver microsomes
2. Test material and instrument
Reagent and consumable:
reagent name Suppliers (suppliers) Goods number Lot number
Human liver microsome 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 to prepare hatching fluid:
reagent(s) Concentration of Volume of
Phosphate buffer 100mM 216.25μL
Liver microsome 20mg/mL 6.25μL
3.2 the following two experiments were performed separately: a) Hatching system with addition of coenzyme factor NADPH: 25 μLNADPH (10 mM) was added to hatching fluid (mainly containing liver microsomes, phosphate buffer) so that the final concentrations of liver microsomes and NADPH were 0.5mg/mL and 1mM, respectively; b) Incubation system without addition of cofactor NADPH: to the hatching fluid 25. Mu.L of phosphate buffer (100 mM) was added so that the final concentration of liver microsomes was 0.5mg/mL. The incubation systems were preheated at 37℃for 10 minutes, respectively.
3.3 to each hatching system described in the foregoing "step 3.2", the reaction was started by adding 2.5. Mu.L of positive control compound or test compound solution of the present application (100. Mu.M), respectively, which was verapamil (purchased from Sigma), so that the final concentration of the test compound of the present application or the positive control compound was 1. Mu.M. The hatching solution after adding the compound is hatched in batches in water at 37 ℃.
3.4 30. Mu.L aliquots were removed from the reaction solution at 0.5, 5, 15, 30 and 45 minutes, respectively, and the reaction was quenched by the addition of 5 volumes of cold acetonitrile containing 200nM caffeine and 100nM tolbutamide. Aliquots were centrifuged at 3220g gravity for 40 min, 100 μl of supernatant was mixed with 100 μl of ultrapure water, and then used for LC-MS/MS analysis.
3.5 data analysis
The peak area is determined from the extracted ion chromatogram. The slope value k is determined by linear regression of the remaining percentage of parent drug against the natural logarithm of the incubation time curve.
Determination of in vitro half-life (t) by separate calculation from slope values 1/2 ) Conversion to in vitro intrinsic clearance (CLint, expressed in μl/min/mg protein) was by in vitro half-life mean.
The experimental results are shown in table 6 below:
TABLE 6 stability data of the compounds of the application in human, rat and mouse liver microsomes
The results show that the compound Ib 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 significantly different from the corresponding positive compounds, or the stability in liver microsomes of most species is poor, but the stability of the compound Ib of the application in human, rat and mouse liver microsomes is significantly better than that of the positive control 2.
Test example 6: in vivo pharmacokinetic study of SD rats intravenously and orally administered test compounds
1. Test animals
Species: SD rats, SPF grade. The source is as follows: animals were transferred from a laboratory animal store (999M-017), shanghai Sipuler-BiKai laboratory animal Co. Quantity: 3 each dosage form.
2. Test sample preparation
2.1 accurately weighing a proper amount of test sample, adding 5% DMSO, 10% polyethylene glycol-15 hydroxystearate and 85% physiological saline into the test sample, and mixing the test sample with vortex or ultrasonic wave to obtain 0.2mg/mL of administration solution for intravenous injection.
2.2 accurately weighing a proper amount of test sample, adding 5% DMSO, 10% polyethylene glycol-15 hydroxystearate and 85% physiological saline in the final volume, and mixing thoroughly by vortex or ultrasonic to obtain 0.5mg/mL administration solution for oral administration by gastric lavage.
3. Design of experiment
4. Administration mode
The weight of the patient was weighed before administration, and the amount of the drug administered was calculated from the weight of the patient. Oral administration is by intravenous or intragastric administration.
5. Blood collection time point
Pre-and post-dosing 0.083h,0.25h,0.5h,1h,2h,4h,6h,8h,24h.
6. Sample collection and handling
Blood was collected via jugular vein or other suitable means, about 0.20mL of each sample was collected, heparin sodium was anticoagulated, the blood samples were placed on ice after collection, and plasma was centrifuged over 2 hours (centrifugation conditions: centrifugal force 6800g,6 minutes, 2-8deg.C). The collected plasma sample is stored in a refrigerator at the temperature of minus 80 ℃ before analysis, and the residual plasma sample after analysis is stored in the refrigerator at the temperature of minus 80 ℃ for temporary storage.
7. Biological analysis and data processing
When the blood concentration of the test object is detected and the plasma drug concentration-time curve is drawn, BLQ is marked as 0. When the drug generation parameter calculation is carried out, the concentration before drug administration is calculated according to 0; c (C) max Previous BLQ (including "No peak") was calculated as 0; BLQ (including "No peak") occurring after Cmax does not participate in the calculation. Calculation of pharmacokinetic parameters, such as AUC (0-T), T using WinNonlin, by blood concentration data at different time points 1/2 Cmax, etc. The results are shown in Table 7.
TABLE 7 in vivo pharmacokinetic study data for SD rats intravenously and orally administered test compounds
The results show that compound 2 had poorer in vivo pharmacokinetic parameters (AUC/CL/F%) in SD rats compared to the positive compound and compound Ib had significantly improved in vivo pharmacokinetic parameters (AUC/CL/F%) in SD rats compared to positive control 2.
Test example 7: bi-directional permeability studies of test compounds on MDCK-MDR1 cell lines
1.1 materials
MDCK-MDR1 cells were purchased from the Netherlands cancer institute and used between 10 and 20 passages of cells.
1.2 design of experiments
1.2.1 cell culture and seed plates
1) Before cell seeding, 50. Mu.L of cell culture medium was added to each well of the Transwell upper chamber, and 25mL of cell culture medium was added to the lower plate. The plates were placed at 37℃with 5% CO 2 After incubation in the incubator for 1 hour, it can be used to inoculate cells.
2) MDCK-MDR1 cells were resuspended using medium to a final concentration of 1.56X10 6 cells/mL. The cell suspension was added to the 96-well Transwell plate upper chamber at 50 μl per well. Cultivation methodThe incubator is set at 37 ℃ and 5% CO 2 Ensuring the relative humidity of 95 percent for 4 to 8 days. The medium was changed 48 hours after inoculation, and the culture was started for 4-8 days with medium change every other day.
1.2.2 evaluation of cell monolayer membrane integrity
1) The original medium in the lower plate was removed and fresh pre-warmed medium was added to the upper chamber.
2) The single layer film resistance was measured with a resistance meter (Millipore, USA) and the resistance per well was recorded.
3) After the measurement, the Transwell plates were returned to the incubator.
4) Calculating the resistance value: measurement of resistance value (ohms). Times.membrane area (cm) 2 ) TEER value (ohm cm) 2 ) If TEER value<42ohms·cm 2 The well cannot be used for penetration testing.
1.2.3 preparation of solutions
1) Test compounds were formulated as a 10mM stock solution using DMSO,
2) Positive control compounds were formulated with DMSO as a stock solution at a concentration of 10 mM.
1.2.4 drug penetration test
1) The MDCK-MDR1 Transwell plates were removed from the incubator. The cell monolayer membranes were rinsed twice with pre-warmed HBSS (25mM HEPES,pH 7.4) buffer and incubated for 30 min at 37 ℃.
2) Stock solutions of control compound and test compound were diluted in DMSO to give 200. Mu.M solution, which was then diluted with HBSS (10 mM HEPES, pH 7.4) to give 1. Mu.M working solution. The final concentration of DMSO in the incubation system was 0.5%.
3) The transport rate of the compound from the apical to basal end was determined. 125. Mu.L of working solution was added to the upper chamber (top end), then 50. Mu.L of sample solution was immediately transferred from the lower chamber (base end), and the mixture was added 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,pH 7.4) buffer was added to the lower chamber. The internal standard contained (100 nM alprazolam, 200nM caffeine and 100nM toluene-sulbutamide). The transferred 50. Mu.L sample solution was vortexed at 1000rpm for 10min.
4) The rate of transport of the compound from the substrate end to the top end was determined. 285 μl of working solution was added to the lower chamber (base end), then 50 μl of the upper chamber (top end) sample solution was immediately transferred to ase:Sub>A 96-well plate containing 200 μl of acetonitrile containing internal standard as ase:Sub>A 0 minute dosing sample (B-ase:Sub>A) 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, 200nM caffeine and 100nM toluene-sulfobutyramide) and 50. Mu.L of the above-transferred sample solution was vortexed at 1000rpm for 10min. The tip-to-base end direction and base end-to-tip experiments should be performed simultaneously.
5) After addition of buffer to the lower and upper chambers, respectively, MDCK-MDR1 Transwell cultures were incubated at 37℃for 2 hours.
6) After the incubation, the drug 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 μl of sample solution was taken into a new 96-well plate, 4 volumes of ethanol containing internal standard substance including (100 nM alprazolam, 200nM caffeine and 100nM tosylamide) were added to the plate, vortexed for 10 minutes, and centrifuged at 3220g for 40 minutes. 100. Mu.L of the supernatant was aspirated and mixed with an equal volume of ultrapure water for LC-MS/MS analysis.
7) The integrity of the cell monolayer after incubation for 2 hours was assessed by leakage of fluorescein. The yellow stock solution was diluted with HBSS (10mM HEPES,pH 7.4) to a final concentration of 100. Mu.M, 100. Mu.L of yellow solution was added to each well of the upper cell (top end), and 300. Mu.L of HBSS (25mM HEPES,pH 7.4) was added to each well of the lower cell (base end) substrate. After incubation at 37 ℃ for 30 minutes, 80 μl of solution was aspirated from each well upper and lower layer into a new 96-well plate. Fluorescence measurement was performed using an enzyme-labeled instrument at excitation wavelength 485nm and emission wavelength 530 nm.
1.2.5 analysis conditions
LC system:Shimadzu
MS analysis:Triple Quad 5500instrument from AB Inc with an ESI interface
1) LC parameters
Column temperature of 40 DEG C
Waters XSelect HSS T3 column, C18, 2.5. Mu.M, 2.1X50mm
Mobile phase 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B)
Sample volume 5. Mu.L
Elution rate of 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
Scanning type multiple reaction detection (MRM)
Curtain gas of 35L/min
Collision gas 9L/min
Carrier gas 50L/min
Auxiliary gas 50L/min
The temperature is 500 DEG C
Ion spray voltage +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 (V) A To the volume of the receiving side solution (Ap. Fwdarw.Bl is 0.3mL, bl. Fwdarw.Ap is 0.1 mL), area is the Transwell-96 well plate membrane Area (0.143 cm) 2 ) The method comprises the steps of carrying out a first treatment on the surface of the time is 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 MDCK-MDR1 cell line
Ratio of outer row = P app(B-A) /P app(A-B)
The results show that the permeability of compound Ib (P app(A-B) ) Compared with positive control 2, the compound Ib is easier to absorb into cells, and meanwhile, the compound Ib has lower efflux ratio, which indicates 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. Thus, in conjunction with the SD rat in vivo pharmacokinetic study, it is more fully demonstrated that compound Ib of the application has a significant improvement in vivo pharmacokinetic parameters (AUC/CL/F%) compared to positive control 2.
Test example 8: in vitro cell Activity assessment
The test example was completed by the Co-fertilizer Utility model of the science and technology of biological medicine, inc., wherein the cell line used was constructed by the company.
1. Purpose of experiment
The in vitro antiproliferative effect of the compounds of the application on 6 BaF3 cell lines was determined.
2. Reagent and consumable
Cell line:
cell lines Cell type Cell number/well Culture medium
Ba/F3-LMNA-NTRK1 Suspending 2000 RPMI 1640+10％FBS+1％PS
Ba/F3-LMNA-NTRK1-V573I Suspending 2000 RPMI 1640+10％FBS+1％PS
Ba/F3-LMNA-NTRK1-F589L Suspending 2000 RPMI 1640+10％FBS+1％PS
Ba/F3-LMNA-NTRK1-G667S Suspending 2000 RPMI 1640+10％FBS+1％PS
Ba/F3-TEL-NTRK2 Suspending 2000 RPMI 1640+10％FBS+1％PS
Ba/F3-TEL-NTRK3 Suspending 2000 RPMI 1640+10％FBS+1％PS
Materials:
3. experimental procedure
Cell treatment and administration
Cell culture conditions
The 6 Ba/F3 cell lines were cultured with RPMI 1640 (Biological Industries, israel) +10% fetal bovine serum (Biological Industries, israel) +1% diabody (Penicillin Streptomycin solution, coring, USA), and after cell recovery, were cultured for two generations and tested.
Preparation of 1000 x Compounds
The test compound was dissolved in DMSO to prepare a 10mM stock solution, which was then diluted to 1mM with DMSO. 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) in a total of 10 concentration gradients while using the same volume of DMSO solvent as a negative control.
Preparation of 20X Compounds
The prepared 10 compounds to be tested with concentration gradient of 1000 x are diluted 50-fold to 20-fold with complete culture medium respectively, and stored in 96-well plates (Beaver, suzhou) for 10 concentration gradients, and simultaneously, DMSO solvent with the same volume is used as a negative control.
Floor board
Cell suspensions in the logarithmic growth phase were inoculated into 96-well white cell culture plates (Corning 3917, NY, USA) with a volume of 95. Mu.l per well (about 2000 cells/well).
Mu.l of 20 Xtest compound was added to the above-mentioned culture plates containing 95. Mu.l of the cell suspension, and mixed well, 2 wells per concentration gradient. The final assay 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% CO 2 Incubate in incubator for 72 hours.
Reading board
The following steps were performed according to the instructions of the Promega CellTiter-Glo luminescence cell activity assay kit (Promega-G7573).
(1) CellTiter-Glo buffer was thawed and left to stand to room temperature.
(2) Place CellTiter-Glo substrate to room temperature.
(3) CellTiter-Glo working solution was prepared by adding CellTiter-Glo buffer to a bottle of CellTiter-Glo substrate to dissolve the substrate.
(4) Slowly vortex to dissolve thoroughly.
(5) The cell culture plates were removed and allowed to stand for 10 minutes to equilibrate to room temperature.
(6) Add 50. Mu.l CellTiter-Glo working fluid to each well.
(7) The plates were shaken on an orbital shaker for 2 minutes to induce cell lysis.
(8) The plates were left at room temperature for 10 minutes to stabilize the luminescence signal.
(9) And detecting the luminous signal on a MD SpectraMax Paradigm board reader.
4. Data analysis
SpectraMax Paradigm reads gave the corresponding fluorescence per well values RLU. Cell viability (Cell viability) data were processed using the following formula:
Cell viability(％)＝(RLU Drug -RLU Min )/(RLU Max -RLU Min ) 100% of the total. Calculating cell viability corresponding to different concentrations of the compound in EXCEL, using GraphPad Prism software as cell viability graph, and calculating related parameters including maximum and minimum cell viability, IC 50 Values.
The experimental results are shown in table 9 below:
TABLE 9 IC of inhibitory Activity of the inventive compounds against the same batch of 6 BaF3 cell lines 50 Value of
From the biological activity data of the compounds in the specific examples, the compound Ib has strong growth inhibition effect on 6 BaF3 cell lines. The inhibitory activity of the compound 2 on the 6 BaF3 cell lines is not obviously different from that of the corresponding positive compound, but the inhibitory activity of the compound Ib of the application on the 6 BaF3 cell lines is obviously higher than that of the positive control 2.

Claims (15)

1. The use of a compound of formula (I) or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of RET related disorders,
wherein the RET-related disease is a cancer related to RET gene or RET kinase protein mutation, or a cancer related to RET fusion gene.
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 use of claim 1, wherein the RET gene or RET kinase protein mutation comprises a mutation at one or more sites.
5. The use of claim 1, wherein 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.
6. The use of claim 1, wherein the RET fusion gene is selected from the group consisting of: RET-CCDC6, RET-KIF5B (Kex 15Rex 14), RET-PRKAR1A, RET-BCR, RET-NCOA4, and any combination thereof.
7. The use of claim 1, wherein the RET fusion gene is selected from the group consisting of: RET (V804L) -KIF5B, RET (V804M) -KIF5B, and any combination thereof.
8. The use of claim 1, 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 (a 883F), RET (S904F), RET (V804M), RET (Y806H), RET (M918T), and any combination thereof.
9. The use of claim 1, wherein:
the RET gene mutation-related cancer is selected from the group consisting of: one or more of RET gene mutation-related lung cancer, papillary thyroid cancer, medullary thyroid cancer, differentiated thyroid cancer, recurrent thyroid cancer, multiple endocrine neoplasia type 2A or type 2B, pheochromocytoma, parathyroid hyperplasia, breast cancer, colon cancer, colorectal cancer, papillary renal cell carcinoma, gastrointestinal mucosal ganglioma and cervical cancer,
the RET fusion gene-related cancer is selected from the group consisting of: RET fusion gene related lung cancer, papillary thyroid cancer, medullary thyroid cancer, differentiated thyroid cancer, recurrent thyroid cancer, multiple endocrine neoplasia type 2A or type 2B, pheochromocytoma, parathyroid hyperplasia, breast cancer, colon cancer, colorectal cancer, papillary renal cell carcinoma, gastrointestinal mucosal ganglioma, and cervical cancer.
10. The use of claim 9, wherein the differentiated thyroid cancer is refractory differentiated thyroid cancer.
11. The use of claim 9, wherein: the RET related disease is RET gene mutation related lung cancer, RET gene mutation related medullary thyroid cancer or RET gene mutation related colon cancer, or RET fusion gene related lung cancer, RET fusion gene related medullary thyroid cancer or RET fusion gene related colon cancer.
12. The use of claim 11, wherein the RET related disease is RET-CCDC6 related colon cancer.
13. The use of claim 11, wherein the RET related disease is one or more selected from the group consisting of RET gene mutation-associated small cell lung cancer, RET gene mutation-associated non-small cell lung cancer, RET gene mutation-associated bronchioloalvee lung cancer, and RET gene mutation-associated lung adenocarcinoma.
14. The use of claim 11, wherein the RET related disease is one or more selected from the group consisting of RET fusion gene-related small cell lung cancer, RET fusion gene-related non-small cell lung cancer, RET fusion gene-related bronchiolar lung cell cancer, and RET fusion gene-related lung adenocarcinoma.
15. The use according to any one of claims 1 to 14, wherein the RET related disease is a RET related disease in humans and the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered at a dose of 0.5-4mg/kg/day.
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