CN112225734B

CN112225734B - KRAS G12C inhibitors and uses thereof

Info

Publication number: CN112225734B
Application number: CN202011153732.6A
Authority: CN
Inventors: 张军波; 朱曙灏; 齐晓昕
Original assignee: Nanjing Ruijie Pharma Co ltd
Current assignee: Nanjing Ruijie Pharma Co ltd
Priority date: 2019-10-25
Filing date: 2020-10-23
Publication date: 2021-12-07
Anticipated expiration: 2040-10-23
Also published as: CN112225734A

Abstract

The invention relates to a KRAS G12C inhibitor and application thereof, and particularly provides a compound shown as a formula (I), or a stereoisomer thereof, or an atropisomer thereof, or a pharmaceutically acceptable salt thereof, or a tautomer thereof, or a prodrug thereof.

Description

KRAS G12C inhibitors and uses thereof

Technical Field

The invention relates to the field of medicines, in particular to a KRAS G12C inhibitor.

Background

RAS represents a group of closely related 189 amino acid (molecular weight 21kDa) monomeric globular proteins that are associated with the plasma membrane and bind GDP or GTP. RAS acts as a molecular switch. When the RAS contains a bound GDP, it is in a quiescent or off state and is in an "inactive state". In response to exposure of cells to certain growth-promoting stimuli, RAS is induced to convert its bound GDP to GTP. Upon binding to GTP, RAS is "turned on" and is able to interact with and activate other proteins (their "downstream targets"). The RAS protein itself has a very low intrinsic capacity to hydrolyze GTP back to GDP, leaving itself in an off state. Shutting down RAS requires an extrinsic protein called GTPase Activating Proteins (GAPs) that interact with RAS and greatly accelerate conversion of GTP to GDP. Any mutation in RAS that affects its ability to interact with GAPs or convert GTP back to GDP will result in an extended activation time of the protein, resulting in an extended cell signaling, allowing it to continue to grow and divide. Because these signals lead to cell growth and division, hyperactive RAS signals may ultimately lead to cancer.

Structurally, the RAS protein contains a G domain responsible for the enzymatic activity of RAS-guanine nucleotide binding and hydrolysis (GTPase reaction). It also contains a C-terminal extension called CAAX box, can be post-translationally modified, and is responsible for targeting proteins to the membrane. The G domain is about 21-25kDa in size and comprises a phosphate binding ring (P-ring). The P-loop is the pocket for nucleotide binding in proteins, a rigid part of the domain with conserved amino acid residues ((glycine 12, threonine 26 and lysine 16)), which is essential for nucleotide binding and hydrolysis. The G domain also contains the so-called Switch I (residues 30-40) and Switch II (residues 60-76) regions, both of which are dynamic parts of the protein, which are commonly referred to as "spring-loaded" mechanisms because they are capable of switching between resting and loaded states. The key interaction is the hydrogen bond formed by threonine 35 and glycine 60, with the gamma-phosphate of GTP, which maintains the Switch 1 and Switch 2 regions in their active conformations, respectively. After hydrolysis of GTP and release of phosphate, the two relax into the inactive GDP conformation.

The most well-known members of the RAS subfamily are HRAS, KRAS and NRAS, primarily because of their association with various types of cancer. Mutations in any of the three major isoforms of RAS (HRAS, NRAS or KRAS) genes are most common in human tumorigenesis. About 30% of human tumors were found to carry RAS gene mutations. Notably, KRAS mutations were detected in 25-30% of tumors. In contrast, the rate of oncogenic mutations that occur in NRAS and HRAS family members is much lower (8% and 3%, respectively). The most common KRAS mutations were found at residues G12 and G13 and at residue Q61 of the P loop. G12C is a frequent mutation of the KRAS gene (glycine 12 to cysteine). This mutation has been found in about 13% of carcinogenesis, about 43% of lung carcinogenesis and about 100% of MYH-related polyposis (familial colon cancer syndrome).

As a leading-edge target, KRAS G12C mutant protein received extensive attention. Araxes (a subsidiary of Wellspring) and Janssen Biotech have signed a unique agreement to develop a leading antitumor drug, one of the companies that entered into the field of KRAS inhibitors at the earliest. It developed ARS-853 and ARS-1620 compounds in 2013 and 2016, respectively. In recent years, it has also been filed for KRAS G12C inhibitors, for example WO2016164675 and WO 2016168540. The ARS-853 compounds show good cell viability, but their pharmacokinetic properties are poor, which is not suitable for evaluating the pharmacodynamics of animal models in vivo. Ars-1620 has high efficiency and selectivity for KRAS G12C, and can achieve rapid and sustained target effect in vivo, thereby inducing tumor regression. The in vivo evidence provided by this study suggests that ARS-1620 represents a new generation of KRAS G12C-specific inhibitors with great therapeutic potential. 5.2019, Wellspring announced FDA approval for IND use by ARS-3248. Janssen full authority is responsible for the clinical development of this compound. Day 26, 7 months, JNJ-74699157(ARS-3248) was subjected to phase I clinical trial enrollment (NCT04006301) in clinical trials and KRAS G12C positive patients with advanced solid tumors were selected for study for 4 years.

Medshine Discovery Int also issued a KRAS G12C inhibitor at 25.7.2019. The earliest priority date was month 1 of 2018. Other candidate KRAS G12C inhibitors include MRTX-849 for Mirati therapy and BI-2852 for Boehringer Ingelheim, among others. The invention further provides a series of compounds as KRAS G12C inhibitors.

The multidrug resistance of the tumor can obviously reduce the sensitivity of the tumor to the antitumor drugs, thereby reducing the killing effect of the antitumor drugs to the tumor, and the existing clinical drugs lack drugs which can effectively inhibit or reverse the multidrug resistance of the tumor to the antitumor drugs, so that the development of drugs which can inhibit or reverse the multidrug resistance of the tumor to the antitumor drugs becomes a hotspot of the current research.

Therefore, there is a need in the art to develop a drug that can effectively inhibit or reverse the multidrug resistance of tumors to anti-tumor drugs, thereby improving the therapeutic effect of anti-tumor drugs on tumors.

Disclosure of Invention

The present invention provides a compound that modulates G12C mutant KRAS, HRAS and/or NRAS proteins, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, or a tautomer thereof, or a prodrug thereof. In certain embodiments, the compounds of the invention function as electrophiles capable of forming a covalent bond with the cysteine residue at position 12 of the KRAS, HRAS or NRAS G12C mutant proteins. The invention also provides methods of treating various diseases or disorders, such as cancer, preferably tumors that develop multidrug resistance to anti-tumor drugs, with the compounds of the invention.

In one aspect of the present invention, there is provided a compound represented by formula (I), or a stereoisomer thereof, or an atropisomer thereof, or a pharmaceutically acceptable salt thereof, or a tautomer thereof, or a prodrug thereof:

in another aspect of the present invention, there is provided a pharmaceutical composition comprising one or more compounds of formula (I) according to the present invention and a pharmaceutically acceptable carrier.

In another aspect of the invention, there is provided a method of inhibiting KRAS G12C in a cell, said method comprising the steps of: contacting a cell with a compound of formula (I).

In another aspect of the present invention, there is provided a method of treating cancer in a subject, said method comprising the steps of: administering to the subject a therapeutically effective amount of a compound of formula (I).

In another aspect of the invention, there is provided a compound of formula (I) according to the invention for use in the treatment of cancer in a subject.

Specifically, the method comprises the following steps:

the invention provides a compound shown as a formula (I), or a stereoisomer thereof, or an atropisomer thereof, or a pharmaceutically acceptable salt thereof, or a tautomer thereof, or a prodrug thereof:

wherein:

R¹is H, CN, F, Cl, C_1-6An alkyl group;

R⁶and R⁷Each independently H, F or Cl;

R⁸and R⁹Each independently of the others being H, F, Cl, OH, CN or NH₂；

Ring A is a substituted or unsubstituted 4-7 membered ring or a bicyclic, bridged, fused or spiro 6-11 membered ring having one or two heteroatoms in the ring, each of said substituted substituents being independently selected from alkyl, or cycloalkyl;

y is (CH)₂) p, p is any integer between 0 and 6;

z is a substituted or unsubstituted 4-7 membered ring having one or two heteroatoms in the ring, each of the substituted substituents being independently selected from alkyl or cycloalkyl.

Preferably, ring a is a substituted or unsubstituted 5-7 membered heterocycloalkyl ring, the 6 membered heterocycloalkyl ring having 2N heteroatoms, and the substitution means that 1 or 2 hydrogen atoms of the 5-7 membered heterocycloalkyl ring are substituted with a substituent selected from the group consisting of: c_1-4Alkyl, or C_3-6A cycloalkyl group.

Preferably, ring a is a substituted or unsubstituted piperazine ring, said 6-membered heterocycloalkyl ring having 2N heteroatoms, said substitution being such that 1 or 2 hydrogen atoms of the piperazine ring are substituted by a substituent selected from the group consisting of: c_1-4Alkyl, or C_3-6A cycloalkyl group.

Preferably, ring a is a substituted or unsubstituted piperazine ring.

Preferably, ring A is selected from mono-or di-substituted

Preferably, R¹Is H, CN, F, Cl, C_1-6An alkyl group.

Preferably，R⁶And R⁷Each independently H, F or Cl.

Preferably, R⁸And R⁹Each independently of the others being H, F, Cl, OH, CN or NH₂。

Preferably, p is 0, 1 or 2.

Preferably, Z is a substituted or unsubstituted 4-7 membered heterocyclic ring.

Preferably, Z is a substituted or unsubstituted pyrrole ring or pyridine ring.

Preferably, R¹H, CN or F.

Preferably, R⁶H, F or Cl.

Preferably, R⁷Is H.

Preferably, R⁸Is H, OH or NH₂。

Preferably, R⁹Is F.

Preferably, Z is a substituted or unsubstituted 5-7 membered heteroaryl, said substitution meaning that 1, 2 or 3 hydrogen atoms of the 5-7 membered heteroaryl are substituted by a substituent selected from the group consisting of: C1-C4 alkyl, or C3-C6 cycloalkyl.

Preferably, Z is a substituted or unsubstituted pyridyl group, said substitution means that 1, 2 or 3 hydrogen atoms of the pyridyl group are substituted by a substituent selected from the group consisting of: c_1-4Alkyl, or C_3-6A cycloalkyl group. Preferably, said substitution means that one or more (preferably 1, 2,3, or 4) hydrogen atoms on the group are substituted by a substituent selected from the group consisting of: c_1-4Alkyl, or C_3-6A cycloalkyl group.

In some embodiments, the compound has the following structure (II),

wherein:

R²、R³、R⁴and R⁵Each independently is H, C_1-6Alkyl radical, C_1-6Alkoxy radical, C_3-6Cycloalkyl radical, C_2-6Alkenyl or C_2-6An alkynyl group;

in some embodiments, the compound has a structure represented by formula (IIa), formula (IIb), or formula (IIc):

wherein R is^10a、R^10b、R^11a、R^11b、R^12a、R^12b、R^13aAnd R^13bEach being H, halogen or C_1-3An alkyl group;

R¹、R⁶、R⁷、R⁸and R⁹As defined above.

Preferably, R²And R⁵Each independently is C_1-3Alkyl, hydrogen.

Preferably, R³And R⁴Is H.

Preferably, R²And R⁵Each independently is H, isopropyl or Me.

Preferably, R³And R⁴Is H.

Preferably, R^10a、R^10b、R^11a、R^11bEach independently is H or CH³，R^12a，R^12b，R^13aAnd R^13bIs H.

Preferably, R¹Is H, CN or F, R²And R⁵Each independently H, i-Pr or Me, R³And R⁴Is H, R⁶Is H, F or Cl, R⁷Is H, R⁸Is H, OH or NH₂，R⁹Is F.

Preferably, the alkyl group is methyl, ethyl, propyl or butyl.

In certain embodiments, the compounds have the following structure (III),

R¹⁴is H, F, Cl, CN or alkyl.

Preferably, wherein ring A is a substituted or unsubstituted piperazine ring, R¹Is H, CN or F, R⁶Is H, F or Cl, R⁷Is H, R⁸Is H, OH or NH₂，R⁹Is F, R¹⁴Is H.

In certain embodiments, wherein the compound has the structure shown in formula (IV):

R¹⁵is alkyl or cycloalkyl;

R¹⁶is H, F, Cl, CN, alkyl or cycloalkyl.

Preferably, ring a is a substituted or unsubstituted piperazine ring, R1 is H, CN or F, R6 is H, F or Cl, R7 is H, R8 is H, OH or NH2, R9 is F, R15 is Me, Et or cyclopropane, R16 is H.

Preferably, the compound of formula (I) is a compound selected from one of the following structures:

the compounds of the present invention may be in the form of pharmaceutically acceptable salts. The compounds of the present invention may be prepared as compositions (e.g., pharmaceutical compositions) comprising a compound of the present invention and a pharmaceutically acceptable carrier.

The present invention also provides a method of inhibiting KRAS G12C in a cell in vitro, said method comprising the steps of: contacting a cell with a compound or composition described herein.

The present invention also provides a method of treating cancer in a subject, said method comprising the steps of: administering to the subject a therapeutically effective amount of a compound or composition described herein.

The invention also provides an application of the compound shown in the formula (I), or a stereoisomer thereof, or an atropisomer thereof, or a pharmaceutically acceptable salt thereof, or a tautomer thereof, or a prodrug thereof, in preparing a pharmaceutical composition, wherein the pharmaceutical composition is used for: (i) preventing and/or treating tumors; (ii) inhibiting or reversing multidrug resistance in tumors to antineoplastic drugs; (iii) inhibition of P-glycoprotein; (iv) enhancing the anti-tumor activity of the anti-tumor drug; and/or (v) inhibiting KRAS G12C.

In some embodiments, the tumor is selected from the group consisting of: lung cancer, pancreatic cancer, colorectal cancer, hematologic malignancies, oral cancer, or a combination thereof.

In some embodiments, the oral cancer comprises an oral epidermoid cancer, preferably a human oral epidermoid cancer.

In some embodiments, the tumor comprises a tumor that is multidrug resistant to an anti-neoplastic drug.

In some embodiments, the anti-neoplastic agent comprises vincristine.

In some embodiments, the tumor is a P-glycoprotein high expression tumor.

In some embodiments, the multidrug resistance is multidrug resistance caused by P-glycoprotein.

In some embodiments, the multidrug resistance is multidrug resistance caused by high expression of P-glycoprotein.

In some embodiments, said inhibiting P-glycoprotein comprises inhibiting the expression of P-glycoprotein.

In some embodiments, the P-glycoprotein is tumor P-glycoprotein.

In some embodiments, the composition further comprises an additional pharmaceutically active compound.

In some embodiments, the other pharmaceutically active compound is carfilzomib or cytarabine.

In some embodiments, the pharmaceutical composition comprises vincristine.

The invention also provides a pharmaceutical composition, which comprises vincristine and the compound shown in the formula (I) of the invention, or a stereoisomer thereof, or an atropisomer thereof, or a pharmaceutically acceptable salt thereof, or a tautomer thereof, or a prodrug thereof.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments.

Detailed Description

The invention develops a compound shown in a formula (I), wherein the compound shown in the formula (I) can inhibit KRAS G12C, and has an excellent treatment effect on multidrug-resistant tumors.

Term(s) for

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

As used herein, the terms "comprising," "including," and "containing" are used interchangeably and include not only open-ended definitions, but also semi-closed and closed-ended definitions. In other words, the term includes "consisting of … …", "consisting essentially of … …".

Abbreviations: the letter abbreviations herein and their meanings are set forth in Table A below:

TABLE A

Definition of

The following terms and phrases used herein are intended to have the following meanings unless otherwise indicated. Absent a specific definition, a particular term or phrase is not to be considered as indefinite or unclear, but rather construed according to a common meaning. When a trade name appears in the present invention, it refers to the corresponding commercial product or active ingredient thereof. As used herein, the term "pharmaceutically acceptable" refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment and are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, and each separate value is incorporated into the specification as if it were individually recited herein. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

The term "alkyl" as used herein refers to straight and branched Cl-6 hydrocarbon groups including, but not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, n-pentyl, 2-methylbutyl, 3-methylbutyl, 2-dimethylpropyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2-dimethylbutyl, 2, 3-dimethylbutyl, 3-dimethylbutyl, and 2-ethylbutyl. The term "C_m-n"means that the alkyl group has" m "to" n "carbon atoms. The term "alkylene" refers to an alkyl group having a substituent. Alkyl (e.g. methyl) or alkylene (e.g. -CH2-) groupsMay be substituted by one or more, typically 1 to 3, independently selected groups, such as halogen, trifluoromethyl, trifluoromethoxy, hydroxy, alkoxy, nitro, cyano, alkylamino, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -NC, amino, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, NC, -CO2H, CO2C1-6 alkyl, -OCOC1-6 alkyl, C3-8 cycloalkyl, C3-8 heterocycloalkyl, C5-8 aryl and C5-8 heteroaryl.

As used herein, the terms "alkenyl" and "alkynyl" refer to alkyl groups that include double or triple bonds, respectively.

As used herein, the term "halogen" refers to fluorine, chlorine, bromine and iodine.

The term "alkoxy" as used herein is defined as-OR, wherein R is alkyl.

Unless otherwise specified, each substituted substituent is independently selected from F, Cl, CN, C1-3 alkyl, C1-3 alkyl, C3-5 cycloalkyl, C2-4 alkenyl, or C2-4 alkynyl. The term "heterocycloalkyl ring" refers to a fully saturated or partially unsaturated ring (including, but not limited to, e.g., a 5-7 membered monocyclic ring) in which at least one heteroatom (e.g., N, O, S) is present in the ring having at least one carbon atom. When a heterocycle is preceded by a defined number of members, this refers to the number of ring atoms of the heterocycle, for example a 5-7 membered heterocycle refers to a heterocycle having 5-7 ring atoms. Each heteroatom-containing heterocycle may carry one or more (e.g. 1, 2,3 or 4) heteroatoms each independently selected from nitrogen, oxygen or sulfur atoms. Typical monocyclic heterocycloalkyl rings include, but are not limited to, a piperidine ring, a piperazine ring, or the like.

The term "heteroaryl" refers to an aromatic heterocyclic group having one to more (preferably 1, 2,3 or 4) heteroatoms, which may be monocyclic (monocyclic) or polycyclic (bicyclic, tricyclic or polycyclic) fused together or covalently linked, and each heteroatom-containing heterocycle may carry one more (e.g., 1, 2,3, 4) heteroatoms each independently selected from the group consisting of: oxygen, sulfur and nitrogen. When a heteroaryl group is preceded by a number of members, this refers to the number of ring atoms of the heteroaryl group, for example, 5-7 membered heteroaryl refers to heteroaryl groups having 5-7 ring atoms, representative examples include, but are not limited to: pyrrolyl, pyrazolyl, or the like.

Compound (I)

As used herein, "a compound of the present invention", "a compound of formula (I)", which are used interchangeably, refer to a compound having formula (I), or a stereoisomer thereof, or an atropisomer thereof, or a pharmaceutically acceptable salt thereof, or a tautomer thereof, or a prodrug thereof:

specifically, the compound shown in the formula (I) is as described above.

The compounds of the invention are preferably specific compounds as in the examples of the invention.

Typically, the compounds of the invention are selected from the group consisting of:

provided herein are KRAS inhibitors having one of the structures of formulae (Ia) - (Ic).

The compounds disclosed herein include all pharmaceutically acceptable isotopically-labeled compounds in which one or more atoms of the compound disclosed herein are replaced by an atom having the same atomic number, but an atomic mass or atomic number different from the atomic mass or atomic number usually found in nature. Examples of isotopes that can be incorporated into the compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine and iodine, e.g.) "²H、³H、¹¹C、¹⁴C、¹³N、¹⁵N、¹⁵O、¹⁷O、¹⁸O、³¹P、³²P、³⁵S、¹⁸F and³⁶and (4) Cl. These radiolabeled compounds can be used to help determine or measure the effectiveness of a compound by characterizing, for example, the site or mode of action or binding affinity to a pharmacologically important site of action. Certain isotopically-labeled compounds of the present disclosure, for example, those incorporating a radioisotope, are useful in drug and/or substrate tissue distribution studies. The radioactive isotopes tritium, i.e. "3H and carbon-14", are particularly useful for this purpose, considering their easy binding and easy means of detection.

With heavier isotopes of, e.g. deuterium, i.e.) "²H ", may have certain therapeutic advantages due to higher metabolic stability (e.g., increased in vivo half-life or reduced dosage requirements) and is therefore preferred in some circumstances.

Using positron-emitting isotopes (e.g. of the type¹¹C、¹⁸F、¹⁵O and¹³n) substitution can be used in Positron Emission Tomography (PET) studies to examine the occupancy of substrate receptors. Isotopically-labeled compounds of structure (I) can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the preparations and examples below, using an appropriate isotopically-labeled reagent in place of the non-labeled reagent previously used.

The compounds disclosed herein may exist as stereoisomers (i.e., isomers that differ only in the spatial arrangement of the atoms), including optical isomers and conformational isomers (or conformers). The compounds disclosed herein include all stereoisomers, both pure individual stereoisomer preparations and individual enriched preparations, as well as racemic mixtures of such stereoisomers as well as individual diastereomers and enantiomers which can be separated according to methods known to those skilled in the art. In addition, the compounds disclosed herein include all tautomeric forms of the compounds.

Certain compounds disclosed herein may exist as atropisomers, which are conformational stereoisomers that occur when rotation about a single bond in a molecule is prevented or greatly slowed due to steric interaction with other parts of the molecule. The compounds disclosed herein include all atropisomers, either as pure individual atropisomer preparations, as individual enriched preparations, or as a non-specific mixture of each. If the rotational barrier around a single bond is high enough and the interconversion between conformations is slow enough, separation of isomers may be allowed.

Process for the preparation of compounds

The compounds disclosed herein can be synthesized by a number of specific methods. Examples of specific synthetic routes and the following general schemes are intended to provide guidance to the ordinarily skilled synthetic chemist, and those skilled in the art will readily appreciate that solvents, concentrations, reagents, protecting groups, sequence of synthetic steps, times, temperatures, and the like may be modified as necessary within the skill and judgment of the ordinarily skilled artisan.

Compounds of formula (IIa) as disclosed herein can be synthesized as described in method 1:

compounds of formula (IIb) as disclosed herein can be synthesized as described in method 2:

compounds of formula (IIc) as disclosed herein can be synthesized as described in method 3:

use of

The present invention also provides the use of a compound of the invention for the preparation of a pharmaceutical composition for: (i) preventing and/or treating tumors; (ii) inhibiting or reversing multidrug resistance in tumors to antineoplastic drugs; (iii) inhibition of P-glycoprotein; (iv) preparing a medicament for enhancing the anti-tumor activity of the anti-tumor medicament; and/or (v) inhibiting KRAS G12C preferably, the tumor is selected from the group consisting of: lung cancer, pancreatic cancer, colorectal cancer, hematologic malignancies, oral cancer, or a combination thereof.

Typically, the oral cancer comprises oral epidermoid cancer, preferably human oral epidermoid cancer.

Preferably, the tumor comprises a tumor that has developed multidrug resistance to an anti-neoplastic drug.

Preferably, the antineoplastic drug comprises vincristine.

Preferably, the tumor is a P-glycoprotein high expression tumor.

Preferably, the multidrug resistance is multidrug resistance caused by P-glycoprotein.

Preferably, the multidrug resistance is multidrug resistance caused by high expression of P-glycoprotein.

Preferably, said inhibiting P-glycoprotein comprises inhibiting the expression of P-glycoprotein.

Preferably, the P-glycoprotein is tumor P-glycoprotein.

Preferably, the composition further comprises other pharmaceutically active compounds.

Preferably, the other pharmaceutically active compound is carfilzomib or cytarabine.

Pharmaceutical compositions, dosages and routes of administration

Also provided herein are pharmaceutical compositions comprising a compound disclosed herein and a pharmaceutically acceptable carrier, such as a diluent or carrier. The compounds of the invention and the compounds of the pharmaceutical compositions are administered in an amount effective to achieve their intended purpose. Administration of the compounds is described in more detail below.

Suitable pharmaceutical formulations can be determined by those skilled in the art depending on the route of administration and the desired dosage. See, for example, Reming-ton's Pharmaceutical Sciences, 1435-. The formulation may affect the physical state, stability, rate of in vivo release and rate of in vivo clearance of the administered agent. The appropriate dosage can be calculated according to the route of administration, body weight, body surface area or organ size. The therapeutic dosage can be routinely determined by one of ordinary skill in the art without undue experimentation, particularly in light of the dosage information and assays disclosed herein and the pharmacokinetic data that can be obtained with an animal or animal.

The term "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or human. As used herein, "pharmaceutically acceptable" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. Such excipients for pharmaceutically active substances are well known in the art. Except insofar as any conventional media or agent is incompatible with the therapeutic composition, it is contemplated that it will be used in the therapeutic compositions. Other active ingredients may also be incorporated into the composition. In exemplary embodiments, the formulation may comprise corn syrup solids, high oleic safflower oil, coconut oil, soybean oil, L-leucine, tribasic calcium phosphate, L-tyrosine, L-proline, L-lysine acetate, DATEM (emulsifier), L-glutamine, L-valine, dipotassium hydrogen phosphate, L-isoleucine, L-arginine, L-alanine, glycine, L-asparagine monohydrate, L-serine, potassium citrate, L-threonine, sodium citrate, magnesium chloride, L-histidine, L-methionine, ascorbic acid, calcium carbonate, L-glutamic acid, L-cystine dihydrochloride, L-tryptophan, L-aspartic acid, choline chloride, taurine, inositol, ferrous sulfate, calcium citrate, sodium chloride, calcium chloride, magnesium chloride, ferrous sulfate, calcium chloride, magnesium sulfate, magnesium chloride, Ascorbyl palmitate, zinc sulfate, alpha-tocopheryl acetate, sodium chloride, niacinamide, tocopherol, calcium pantothenate, copper sulfate, thiamine hydrochloride, vitamin A palmitate, manganese sulfate, riboflavin, folic acid, beta-carotene, potassium iodide, phylloquinone, biotin, sodium selenate, chronium chloride, sodium molybdate, vitamin D3, and cyanocobalamin.

The compounds of the present invention may be present in the pharmaceutical compositions in the form of pharmaceutically acceptable salts. As used herein, "pharmaceutically acceptable salts" include, for example, base addition salts and acid addition salts.

Pharmaceutically acceptable base addition salts may be formed with metals or amines, for example alkali and alkaline earth metals or organic amines. Pharmaceutically acceptable salts of the compounds may also be prepared with pharmaceutically acceptable cations. Suitable pharmaceutically acceptable cations are well known to those skilled in the art and include alkali, alkaline earth, ammonium and quaternary ammonium cations. Carbonates or bicarbonates are also possible. Examples of metals used as cations are sodium, potassium, magnesium, ammonium, calcium or ferric iron and the like. Examples of suitable amines include isopropylamine, trimethylamine, histidine, N' -dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine.

Pharmaceutically acceptable acid addition salts include inorganic or organic acid salts. Examples of suitable acid salts include hydrochloride, formate, acetate, citrate, salicylate, nitrate, phosphate. Other suitable pharmaceutically acceptable salts are well known to those skilled in the art and include, for example, formic, acetic, citric, oxalic, tartaric or mandelic acid or hydrochloric, hydrobromic, sulfuric or phosphoric acid; with organic carboxylic, sulfonic or phosphoric acids or N-substituted sulfamic acids, for example acetic acid, trifluoroacetic acid (TFA), propionic acid, glycolic acid, succinic acid, maleic acid, hydroxymaleic acid, methylmaleic acid, fumaric acid, malic acid, tartaric acid, lactic acid, oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, salicylic acid, 4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid, nicotinic acid or isonicotinic acid; and amino acids, such as the 20 alpha amino acids involved in protein synthesis in nature, e.g., glutamic acid or aspartic acid, as well as phenylacetic acid, methanesulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-methylbenzenesulfonic acid, naphthalene 2-sulfonic acid, naphthalene 1, 5-disulfonic acid, 2-or 3-phosphoglycerate, glucose-6-phosphate, N-cyclohexylsulfamic acid (cyclic amides), or in combination with other acidic organic compounds, such as ascorbic acid.

Pharmaceutical compositions containing a compound disclosed herein can be manufactured in a conventional manner, e.g., by conventional mixing, dissolving, granulating, sugarcoating, suspension-emulsifying, encapsulating, entrapping or lyophilizing processes. Suitable formulations depend on the chosen route of administration.

For oral administration, suitable compositions can be readily prepared by: the compounds disclosed herein are combined with pharmaceutically acceptable excipients such as carriers well known in the art. Such excipients and carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral administration to a patient to be treated. Pharmaceutical preparations for oral use can be obtained by the following method: the compounds disclosed herein are added together with solid excipients, optionally grinding the resulting mixture, and working up the mixture of granules, if desired with the addition of suitable auxiliaries, to obtain tablets or dragee cores. Suitable excipients include, for example, fillers and cellulose preparations. If desired, a disintegrant may be added. Pharmaceutically acceptable ingredients are well known for various types of formulations and may be, for example, binders (e.g., natural or synthetic polymers), lubricants, surfactants, sweetening and flavoring agents, coatings, preservatives, dyes, thickeners, adjuvants, antimicrobial agents, antioxidants and carriers of various formulation types.

When a therapeutically effective amount of a compound disclosed herein is administered orally, the composition is typically in the form of a solid (e.g., a tablet, capsule, pill, powder, or lozenge) or a liquid formulation (e.g., an aqueous formulation). .

When administered in tablet form, the compositions may additionally comprise functional solids and/or solid carriers, such as gelatin or adjuvants. Tablets, capsules and powders may contain from about 1 to about 95% of the compound, preferably from about 15 to about 90% by weight.

When administered as a liquid or suspension, a functional liquid and/or liquid carrier, such as water, petroleum or an oil of animal or vegetable origin, may be added. The liquid form of the composition may further comprise a physiological saline solution, a sugar alcohol solution, glucose or other sugar solution, or ethylene glycol. When administered as a liquid or suspension, the composition may comprise from about 0.5% to about 90% by weight of a compound disclosed herein, preferably from about 1 to about 50% by weight of the compound. In one embodiment, the liquid carrier is non-aqueous or substantially non-aqueous. For administration in liquid form, the composition may be provided as a rapidly dissolving solid formulation to be dissolved or suspended immediately prior to administration.

When a therapeutically effective amount of a compound disclosed herein is administered by intravenous, cutaneous or subcutaneous injection, the composition is in the form of a pyrogen-free parenterally acceptable aqueous solution. It is within the skill of the art to prepare such parenterally acceptable solutions with due consideration of pH, isotonicity, stability, and the like. Preferred compositions for intravenous, cutaneous or subcutaneous injection typically comprise an isotonic excipient in addition to the compounds disclosed herein. Such compositions may be prepared for administration as a solution of the free base or pharmacologically acceptable salt in water, mixed with a suitable surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof and oils. Under conventional conditions of storage and use, these formulations may optionally contain a preservative to prevent the growth of microorganisms.

Injectable compositions may include sterile aqueous solutions, suspensions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions, suspensions or dispersions. In all embodiments, the form must be sterile and must be fluid to allow easy injection. It must be stable under the conditions of manufacture and storage and must be resistant to contamination, exerting an antimicrobial (e.g. bacterial and fungal) action by optionally including a preservative. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures, and vegetable oils. In one embodiment, the carrier is non-aqueous or substantially non-aqueous. Proper fluidity can be maintained, for example, by the use of a coating, by the maintenance of the required particle size of the compound in the case of dispersion embodiments, and by the use of a surfactant such as lecithin. The action of microorganisms can be prevented by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many embodiments, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the embodiment of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Sustained release or sustained release formulations may also be prepared to achieve controlled release of the active compound in contact with body fluids in the gastrointestinal tract and to provide a substantially constant and effective level of the active compound in the plasma. For example, release may be controlled by one or more of dissolution, diffusion and ion exchange. In addition, sustained release methods may enhance absorption through saturation or restrictive pathways in the gastrointestinal tract. For example, for this purpose, the compound may be embedded in a polymer matrix of a biodegradable polymer, a water-soluble polymer or a mixture of both and optionally a suitable surfactant. In this case, embedding may mean incorporating the microparticles into a polymer matrix. Controlled release formulations can also be obtained by encapsulation of dispersed microparticles or emulsified microdroplets by known dispersion or emulsion coating techniques.

For administration by inhalation, the compounds of the invention are conveniently delivered in aerosol form from pressurized packs or a nebulizer, with the use of a suitable propellant. In embodiments of pressurized aerosols, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, for example, gelatin, containing a powder mix of the compound and a suitable powder base such as lactose or starch may be formulated for use in an inhaler or insufflator.

The compounds disclosed herein may be formulated for parenteral administration by injection (e.g., by bolus injection or continuous infusion). Formulations for injection may be presented in unit dosage form (e.g., in ampoules or in multi-dose containers) and with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical formulations for parenteral administration include aqueous solutions of the compounds in water-soluble form. Alternatively, suspensions of the compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils or synthetic fatty acid esters. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compound and allow for the preparation of highly concentrated solutions. Alternatively, the present compositions may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use.

The compounds disclosed herein may also be formulated in rectal compositions such as suppositories or retention enemas (e.g., containing conventional suppository bases). In addition to the foregoing formulations, these compounds may also be formulated as depot preparations. Such long acting formulations may be administered by implantation (e.g. subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated as suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, e.g., as a sparingly soluble salt.

In particular, the compounds disclosed herein may be administered orally, buccally or sublingually in the form of tablets containing excipients such as starch or lactose, or in the form of capsules or ovules, alone or in admixture with excipients, or in the form of suspensions containing flavouring or colouring agents. Such liquid formulations may be prepared with pharmaceutically acceptable additives. The compounds may also be injected parenterally, for example intravenously, intramuscularly, subcutaneously or intracoronary. For parenteral administration, the compounds are preferably used in the form of a sterile aqueous solution which may contain other substances, for example salts or sugar alcohols (e.g. mannitol) or glucose, to render the solution isotonic with blood.

For veterinary use, the compounds disclosed herein are administered in a suitably acceptable formulation according to normal veterinary practice. Veterinarians can readily determine the dosage regimen and route of administration that is most appropriate for a particular animal.

In some embodiments, all necessary components for treating KRAS-related disorders using the compounds disclosed herein, alone or in combination with another agent or intervention conventionally used to treat such diseases, may be packaged in a kit.

In particular, the invention provides a kit for therapeutic intervention in a disease, comprising a packaged drug comprising a compound disclosed herein and buffers and other components for preparing a deliverable form of the drug, and/or a device for delivery. These drugs and/or any drug used in combination therapy with the compounds disclosed herein, and/or instructions for treatment of a disease packaged with the drug. The product description may be fixed in any tangible medium, such as printed paper or a computer readable magnetic or optical medium, or may refer to a remote computer data source, such as instructions from a world wide web page accessible via the internet.

"therapeutically effective amount" refers to an amount effective to treat or prevent the development of a disease or alleviate a symptom thereof in a subject being treated. Determination of an effective amount is well within the ability of those skilled in the art, especially in light of the detailed disclosure provided herein.

Generally, a "therapeutically effective dose" refers to the amount of a compound that results in the desired effect. For example, in a preferred embodiment, a therapeutically effective amount of a compound disclosed herein reduces KRAS activity by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% as compared to a control.

The amount of the compound to be administered depends on the subject to be treated, the age, health, sex and weight of the subject and the kind of concurrent treatment (if any), severity, the desired effect, the mode and frequency of treatment and the judgment of the prescribing physician. The frequency of administration may also depend on the pharmacodynamic effect on arterial blood oxygen pressure. However, as understood and determined by those skilled in the art, the most appropriate dosage may be appropriate for an individual subject without undue experimentation. This typically involves adjusting the standard dose (e.g., reducing the dose if the patient is low in weight).

Despite individual variations in demand, it is within the skill of the art to determine the optimal range of effective amounts of a compound. For administration to humans for the treatment or prevention of the conditions and diseases described herein, for example, a typical dose of a compound of the invention may be from about 0.05 mg/kg/day to about 50 mg/kg/day, for example at least 0.05mg/kg, at least 0.08mg/kg, at least 0.1mg/kg, at least 0.2mg/kg, at least 0.3mg/kg, at least 0.4mg/kg or at least 0.5mg/kg, and preferably 50mg/kg or less, 40mg/kg or less, 30mg/kg or less, 20mg/kg or less, or 10mg/kg or less, and may be from about 2.5mg/day (0.5mg/kgx5kg) to about 5000mg/day (e.g., 50mg/kg x 100 kg). For example, the dosage of the compound may be from about 0.1 mg/kg/day to about 50 mg/kg/day, from about 0.05 mg/kg/day to about 10 mg/kg/day, from about 0.05 mg/kg/day to about 5. mg/kg/day, from about 0.05 mg/kg/day to about 3 mg/kg/day, from about 0.07 mg/kg/day to about 3 mg/kg/day, from about 0.09 mg/kg/day to about 3 mg/kg/day, from about 0.05 mg/kg/day to about 0.1 mg/kg/day, from about 0.1 mg/kg/day to about 1 mg/day, from about 1 mg/kg/day to about 10 mg/day, from about 1 mg/kg/day to about 5 mg/kg/day, from about 1 mg/kg/day to about 3 mg/kg/day, from about 3 mg/day to about 500 mg/day, from 5mg/day to about 250 mg/day, from about 10 mg/day to about 100 mg/day, from about 3 mg/day to about 10 mg/day, or from about 100 mg/day to about 250 mg/day. Such doses may be administered in a single dose or may be divided into multiple doses.

Biometric data

For the compounds of the invention, the following assay conditions were employed:

KRAS G12C-coupled nucleotide exchange method

Purified GDP-coupled KRAS protein (aa 1-169) containing G12C and C118A amino acid substituents and an N-terminal His-tag was dose-reaction titrated with compound in assay buffer (50mM HEPES PH 7.4, 0.01% BSA)) for 30 min preincubation.

After compound preincubation, purified SOS protein (aa 564-1049) and GTP were added to the assay wells and incubated for 2 hours. To determine the extent of inhibition of SOS-mediated nucleotide exchange, cRAF RBD, glutathione donor and nickel chelate acceptor microbeads were added to assay wells and incubated for 2 hours. The AlphaLisa signal on Envision was then read and the data was analyzed using the model described in the examples to calculate IC50 values.

WB (Western blot) analysis in pERK cells

NCI-H358(ATCC) cells were cultured in RPMI1640 medium (Gibco) containing 10% FBS and 1% P/S2 days before compound treatment. Cells were seeded into 384-well plates and incubated at 37 ℃ with 5% CO2 overnight before compound treatment. Compound dose-responsive titrates were diluted in growth medium, added to appropriate wells of cell culture plates, and then incubated at 37 ℃, 5% CO2 for 3 hours. After compound treatment, cells were fixed at room temperature, washed with PBS, permeabilized, and incubated in blocking buffer at room temperature. After removal of blocking buffer and addition of primary, rabbit anti-pERK, mouse anti-GAPDH, cells were washed with PBST. Then, after adding a secondary antibody, the cells were incubated at room temperature in the dark. After washing with PBST, the cell plates were centrifuged upside down and scanned with Odyssey CLx. The data were analyzed using the model in the examples to calculate IC50 values.

Biochemical and cellular activities of the compounds of table 1

Compound numbering	Coupling exchange IC50(μ M)	NCI-H 358 IC50(nM)
			I2	1852	986
I3	446.1	178
			I4	601.8	213

The main excellent technical effects of the invention

The invention unexpectedly develops a compound shown as a formula (I), which can inhibit KRAS G12C and has an excellent treatment effect on multidrug-resistant tumors.

Examples

The invention will be better understood from the following examples. However, it will be readily understood by those skilled in the art that the contents described in the embodiments are only intended to illustrate the present invention, and do not limit the present invention described in detail in the claims.

In this example, compound AMG510 was prepared according to the method of WO2018217651a 1:

EXAMPLE 1 preparation of a Compound of formula I1

Compounds of formula I1

4- (4-acryloyl-2-methylpiperazin-1-yl) -6-fluoro-7- (2-fluoro-6-hydroxyphenyl) -1- (2-isopropyl-4-methylpyridin-3-yl) -1, 8-naphthyridin-2 (1H) -one (I1 compound)

Step 1: preparation of Compound I-03

To compound I-01(10.0g, 52.36mmol) in 1, 4-dioxane/H under nitrogen₂To a solution in O (100/10mL) was added Compound I-02(13.3g, 78.53mmol), Na₂CO₃(16.6g, 157.08mmol) and Pd (PPh3)4(6.1g, 5.24mmol), and the reaction mixture was heated to 110 ℃ and stirred overnight.

The reaction was completed by LC-MS detection. The mixture was cooled to room temperature and quenched with water (50mL), and the solution was extracted with EA (50 mL. times.3). The combined organic layers were washed with brine (50mL) and Na₂SO₄Dried, filtered and concentrated to give the crude product. The crude product was purified by silica gel chromatography (PE: EA ═ 10: 1) to give compound I-03(3.6g, 24.5% yield) as a white solid.

¹H NMR(400MHz,DMSO-d₆)δ:8.77(d,J＝8.4Hz,1H),7.63-7.57(m,1H),7.08(d,J＝8.4Hz,1H),7.01(t,J＝8.8Hz,1H),3.80(s,3H).

Step 2: preparation of Compound I-05

To a solution of Compound I-03(3.6g, 12.86mmol) in toluene (50mL) under nitrogen was added Compound I-04(2.3g, 15.43mmol), CS₂CO₃(12.6g, 38.58mmol), xanthphos (1.5g, 2.58mmol) and Pd2(dba)3(1.2g, 1.29mmol), the reaction mixture was heated to 110 ℃ and stirred overnight.

The reaction was completed by LC-MS detection. The mixture was cooled to room temperature and quenched with water (100mL), and the solution was extracted with EA (50 mL. times.3). Combined organic layerWashed with brine (50mL) over Na₂SO₄Dried, filtered and concentrated to give the crude product. The crude product was purified by silica gel chromatography (PE: EA ═ 2: 1) to give compound I-05(1.5g, 30.0% yield) as a yellow solid.

MS Calcd.:394.4；MS Found:395.4[M+H]^+.

And step 3: preparation of Compound I-07

To a solution of compound I-05(800.0mg, 3.0mmol) in pyridine (10mL) was added compound I-06(10 mL). The reaction mixture was heated to 130 ℃ and stirred for 24 hours.

The reaction was completed by LC-MS detection. The mixture was poured into 50mL of water and the product was extracted with EA (50 mL. times.3). The organic layer was washed with water (50 mL. times.2) and 50mL brine, then Na₂SO₄And (5) drying. After filtration, the solvent was removed under reduced pressure. The residue was dried to give Compound I-07 as a brown oil (800mg, crude).

MS Calcd.:436.5；MS Found:437.4[M+H]+.

And 4, step 4: preparation of Compound I-08

To a solution of compound I-07(500.0mg, 1.15mmol) in DMF (10mL) was added t-BuOK (257.0mg, 2.29 mmol). The reaction mixture was stirred at room temperature for 2 hours.

The reaction was completed by LC-MS detection. The mixture was poured into 50mL of water and the product was extracted with EA (50 mL. times.3). The organic layer was washed with water (50 mL. times.2) and 50mL brine, then Na₂SO₄And (5) drying. After filtration, the solvent was removed under reduced pressure. The residue was purified by silica gel chromatography (PE: EA ═ 5: 1-1: 2) to give compound I-08(320.0mg, 64.0% yield) as a red solid.

MS Calcd.:436.5；MS Found:437.4[M+H]⁺.

And 5: preparation of Compound I-09

To compound I-08(2800.0mg, 0.64mmol) in DCM/H under an ice-water bath and nitrogen atmosphere₂To a solution of O (10/10mL) were added BrCCl3(381.0mg, 1.93mmol) and NaNO2(443.0mg, 6.42 mmol). The reaction mixture was stirred at 35 ℃ for 4 hours. HOAc (385.0mg, 6.42mmol) was then added to the mixture. The mixture was stirred at room temperature for 16 hours.

The reaction was completed by LC-MS detection. The reaction was diluted with water (40mL) and extracted with DCM (40 mL. times.3). The combined organic layers were washed with brine (20 mL. times.2) and Na₂SO₄Dried, filtered and concentrated. The residue was purified by silica gel chromatography (PE: EA ═ 5: 1-2: 1) to give compound I-09(130.0mg, 40.5% yield) as a yellow solid.

MS Calcd.:500.3；MS Found:502.2[M+H]⁺.

Step 6: preparation of Compound I-11

A vial was charged with Compound I-09(300.0mg, 0.60mmol), Compound I-10(360.0mg, 1.80mmol), t-Buona (173.0mg, 1.80mmol), BINAP (187.0mg, 0.30mmol), Pd (OAc)₂(135.0mg, 0.60mmol) and toluene (8 mL). The sealed vial was irradiated in a microwave at 120 ℃ for 2 hours.

The reaction was completed by LC-MS detection. The reaction was diluted with water (20mL) and extracted with EA (20 mL. times.2). The combined organic layers were washed with brine (20mL) and Na₂SO₄Dried, filtered and concentrated. The residue was purified by HPLC to give Compound I-11 as a yellow solid (40.0mg, 10.8% yield).

MS Calcd.:619.7；MS Found:620.5[M+H]⁺.

And 7: preparation of Compound I-12

To a solution of compound I-11(30.0mg, 0.06mmol) in DCM (5mL) at-78 deg.C was added BBr3(0.3mL, 1M). The reaction mixture was allowed to warm to room temperature and stirred for 16 hours.

The reaction was completed by LC-MS detection. The reaction mixture was quenched with water (20 mL). The mixture was extracted with DCM (15 mL. times.2). The combined aqueous phases were passed over Na₂CO₃The aqueous solution was basified to pH 7-8 and the aqueous layer was extracted with DCM (15mL × 2). The organic layer was washed with Na₂SO₄Drying, filtration and concentration gave compound I-12(40.0mg) as a yellow solid.

MS Calcd.:505.6；MS Found:506.5[M+H]⁺.

And 8: preparation of Compounds of formula I1

To a solution of compound I-12(40.0mg, 0.08mmol) in DCM (5mL) under nitrogen at 0 deg.C was added TEA (30.0mg, 0.30mmol) and acryloyl chloride (21.0mg, 0.24 mmol). The reaction mixture was allowed to warm to room temperature for 1 hour. The reaction was diluted with water (10mL) and extracted with DCM (10 mL. times.2). The combined organic layers were concentrated in vacuo. The residue was dissolved in THF/H2O (5/1mL), and LiOH (6.0mg, 0.24mmol) was added. The mixture was stirred at room temperature for 2 hours.

The reaction was completed by LC-MS detection. The mixture was concentrated in vacuo and purified by HPLC to give Compound I1(20.0mg, 45.5% yield) as a yellow solid

¹H NMR(400MHz,CDCl₃)δ:9.37(d,J＝6.8Hz,1H),8.65(dd,J＝1.2,4.4Hz,1H),8.00(d,J＝9.2Hz,1H),7.25-7.20(m,2H),6.71-6.66(m,3H),6.44-6.39(m,1H),6.33(d,J＝3.2Hz,1H),5.82(d,J＝10.4,1H),4.58-4.52(m,0.5H),4.38-4.24(m,0.5H),4.13-3.46(m,5H),3.12-3.06(m,1H),2.73-2.54(m,1H),2.06(s,1.5H),1.99(s,1.5H),1.26-1.18(m,6H),1.04(d,J＝6.8Hz,3H).

MS Calcd.:559.6；MS Found:559.9[M+H]⁺ _.

EXAMPLE 2 preparation of the Compound of formula I2

Compounds of formula I2

4- (4-acryloyl-2-methylpiperazin-1-yl) -1- (2, 4-dimethylpyridin-3-yl) -6-fluoro-7- (2-fluoro-6-hydroxyphenyl) -3-isocyano-1, 8-naphthyridin-2 (1H) -one (I2)

The preparation method comprises the following steps:

step 1: synthesis of I2-3

To a solution of I2-1(10.0g, 52.36mmol) in 1, 4-dioxane/H under nitrogen₂To a solution of O (100mL/10mL) was added compound I2-2(13.3g, 78.53mmol), Na₂CO₃(16.6g, 157.08mmol) and Pd (PPh)₃)₄(6.1g, 5.24 mmol). The reaction mixture was heated to 110 ℃ and stirred overnight.

The reaction mixture was detected by LC-MS. The reaction mixture was cooled to room temperature, quenched with water (50mL), and extracted with EA (50mLx 3). The combined organic layers were washed with brine (50mL) and Na₂SO₄Dried, filtered and concentrated. The residue was purified by silica gel chromatography (PE: EA ═ 10: 1) to give compound I2-3(3.6g, 24.5% yield) as a white solid.

Step 2: i2-5 Synthesis:

to a solution of compound I2-3(3.6g, 12.86mmol) in toluene (50mL) under nitrogen was added compound I2-4(2.4g, 19.29mmol), CS₂CO₃(12.6g, 38.58mmol), xanthphos (2.98g, 5.14mmol) and Pd₂(dba)₃(2.35g, 2.57 mmol). The reaction mixture was heated to 110 ℃ and stirred overnight.

The reaction mixture was detected by LC-MS. The reaction mixture was cooled to room temperature, quenched with water (100mL), and extracted with EA (50mLx 3). The combined organic layers were washed with brine (50mL) and Na₂SO₄Dried, filtered and concentrated. The residue was purified by silica gel chromatography (PE/EA ═ 4/1) to give compound I2-5(3.8g, 80.8% yield) as a yellow solid. MS calculated: 366.1; MS measurement: 367.4[ M + H]+。

¹H NMR(400MHz,DMSO-d₆)δ:8.83(s,1H),8.25(d,J＝8.8Hz,1H),8.16(d,J＝4.8Hz,1H),7.47-7.41(m,1H),7.10(d,J＝4.8Hz,1H),6.94(d,J＝8.4Hz,1H),6.87(t,J＝8.8Hz,1H),3.71(s,3H),2.32(s,3H),2.15(s,3H).

And step 3: synthesis of I2-7

To a solution of compound I2-5(1.1g, 3.0mmol) in THF/DMF (30mL/10mL) at 0 deg.C was added NaH (1.2g, 30.0 mmol). After stirring for 30 min, Compound I2-6(3.4g, 30.0mmol) was slowly added to the mixture. The reaction mixture was then heated to 80 ℃ and stirred overnight.

The reaction mixture was detected by LC-MS. The reaction mixture was quenched with water (100mL) and stirred for 30 min. The suspension was filtered. The filter cake was washed with water (30 mL. times.3) and dried to give compound I2-7(1.3g, crude) as a yellow solid.

MS calculated: 433.1; MS measurement: 434.4[ M + H ] +.

And 4, step 4: synthesis I2-8

To a solution of compound I2-7(500mg, 1.154mmol) ACN (40mL) was added CuBr (495mg, 3.461mmol) and t-BuNO under ice-water bath and nitrogen conditions₂(594mg, 5.768 mmol). The reaction mixture was stirred at room temperature for 4 hours.

The reaction mixture was detected by LC-MS. The reaction mixture was diluted with water (40mL) and extracted with EA (40 mL. times.3). The combined organic layers were washed with brine (20mLx2) and Na₂SO₄Dried, filtered and concentrated. The residue was purified by silica gel chromatography (PE/EA ═ 5/1) to give compound I2-8(300mg, 52.3% yield) as a yellow solid.

MS calculated: 496.0, respectively; MS measurement: 497.3[ M + H ] +

And 5: i2-10 Synthesis

A vial was charged with Compound I2-8(200mg, 0.402mmol), DMSO (5mL), tert-butyl 3-methylpiperazine-1-carboxylate (241mg, 1.206mmol) and DIEA (156mg, 1.206 mmol)). The sealed vial was irradiated in a microwave at 110 ℃ for 1 hour.

The reaction mixture was detected by LC-MS. The reaction was cooled to room temperature, diluted with water (20mL), and extracted with EA (20 mL. times.2). The combined organic layers were washed with brine (20mL) and Na₂SO₄Dried, filtered and concentrated. The residue was purified by silica gel chromatography (PE/EA ═ 5/1) to give compound I2-10(120mg, 48.4% yield) as a yellow solid.

MS calculated: 616.2, respectively; MS measurement: 617.7[ M + H ] +.

Step 6: i2-11 Synthesis

To a solution of compound I2-10(150mg, 0.29mmol) in DCM (5mL) at-78 deg.C was added BBr3(0.2mL) and the reaction mixture was warmed to room temperature and stirred overnight.

The reaction mixture was detected by LC-MS. The reaction mixture was quenched with water (10mL) and extracted with DCM (15 mL. times.2). The combined organic phases are passed over Na₂SO₄Drying, filtration and concentration gave compound 12-11 as a yellow solid (60mg, crude).

MS calculated: 502.2; MS measurement: 503.6[ M + H ] +

And 7: i2 Synthesis

To a solution of compound I2-11(50mg, 0.10mmol) in DCM (5mL) was added TEA (30mg, 0.30mmol) and acryloyl chloride (27mg, 0.30mmol) at 0 deg.C under nitrogen. The reaction mixture was allowed to warm to room temperature for 1 hour. Reacting with H₂O (10mL) was diluted and extracted with DCM (10mLx 2). The combined organic layers were concentrated in vacuo. The residue was dissolved in THF/H₂O (5mL/1mL), then LiOH (7mg, 0.30mmol) was added. The reaction mixture was stirred at room temperature for 2 hours.

The reaction mixture was detected by LC-MS. The reaction mixture was concentrated in vacuo. The residue was purified by TLC (MeOH/DCM ═ 1/20) to give compound I2(25mg, 45.5% yield) as a yellow solid.

MS calculated: 556.2, respectively; MS measurement: 557.2[ M + H ].

¹H NMR(400MHz，CD₃OD)δ：8.33(d，J＝4.4Hz，1H)，8.25(d，J＝9.2Hz，1H)，7.30-7.21(m，2H)，6.95-6.81(m，1H)，6.67-6.57(m，2H)，6.33(d，J＝16.0Hz，1H)，5.84(dd，J＝10.4，1.6Hz，1H)，4.60-4.38(m，2H)，4.29-4.03(m，3H)，3.76-3.30(m，2H)，2.26(s，1.6H)，2.14(s，1.4H)，2.12(s，1.4H)，2.01(s，1.6H)，1.45-1.29(m，3H).

EXAMPLE 3 preparation of the Compound of formula I3

Compounds of formula I3

4- (4-acryloyl-2-methylpiperazin-1-yl) -6-fluoro-7- (2-fluoro-6-hydroxyphenyl) -3-isocyano-1- (2-isopropyl-4-methylpyridin-3-yl) -1, 8-naphthyridin-2 (1H) -one

Preparation method

Step 1: synthesis I3-3

To compound I3-1(10g, 52.36mmol) dioxane/H₂To a solution of O (100mL/10mL) was added compound I3-2(10.68g, 62.83mmol), Na₂CO₃(16.6g, 157.08mmol) and Pd (PPh)₃)₄(6.1g, 5.24 mmol). The resulting mixture was stirred at 110 ℃ overnight.

The reaction mixture was cooled to room temperature and washed with H₂O (100mL) was diluted and extracted with EA (200mLx 2). The combined organic layers were washed with brine (100mLx2) and Na₂SO₄Dried, filtered and concentrated. The residue was purified by flash chromatography on silica gel (PE/EA ═ 10/1) to give compound I3-3(4.96g, yield 34%) as a yellow solid.

MS calculated: 280 parts of; MS measurement: 281[ M + H ] +.

Step 2: synthesis I3-5:

to a solution of Compound I3-3(2g, 7.13mmol) in toluene (30mL) was added Compound I3-4(1.39g, 9.26mmol), CS₂CO₃(6.97g, 21.39mmol), xanthphos (1.65g, 2.85mmol) and Pd₂(dba)₃(1.31g, 1.43 mmol). The resulting mixture is in N₂The mixture was stirred at 110 ℃ overnight under these conditions.

The reaction mixture was cooled to room temperature and concentrated. The residue was purified by flash chromatography on silica gel (PE/EA ═ 6/1-3/1) to give compound I3-5(2.52g, 90% yield) as a yellow oil.

MS calculated: 394; MS measurement: 395[ M + H ] +.

And step 3: synthesis I3-6:

to a solution of compound I3-5(0.50g, 1.27mmol) in THF (20mL) at 0 deg.C was added NaH (508mg, 12.7 mmol). The reaction mixture was stirred at this temperature for 30 minutes and ethyl 2-cyanoacetate (1.43g, 12.7mmol) was added. The mixture was then stirred at 80 ℃ overnight.

The reaction mixture was cooled to room temperature and washed with H₂O (10mL) was diluted and extracted with EA (15mLx 2). The combined organic layers were washed with brine (20mLx2) over Na₂SO₄Dried, filtered and concentrated. The residue was purified by flash reverse phase silica gel chromatography (ACN/H)₂O ═ 5% to 95%, 254nm, 30 min) to give compound I3-6(140mg, 24% yield) as a yellow solid.

MS calculated: 461; MS measurement: 462[ M + H ] +.

And 4, step 4: synthesis I3-7:

to a solution of compound I3-6(200mg, 1.818mmol) in ACN (20mL) in an ice-water bath were added CuBr (311mg, 2.17mmol) and t-BuNO₂(224mg, 2.17 mmol). The reaction mixture was stirred at room temperature overnight.

Reacting with H₂O (15mL) was diluted and extracted with EA (20mLx 2). The combined organic layers were washed with brine (20mLx2) and Na₂SO₄Dried, filtered and concentrated. The residue was purified by flash chromatography on silica gel (PE/EA ═ 5/1) to give compound I3-7(91mg, 40% yield) as a yellow solid.

MS calculated: 524; MS measurement: 525[ M + H ] +.

And 5: synthesis I3-8:

to a solution of compound I3-7(150mg, 0.29mmol) in DMF (10mL) was added tert-butyl 3-methylpiperazine-1-carboxylate (85.8mg, 0.43mmol) and DIEA (112.2mg, 0.87 mmol). The reaction mixture was heated to 110 ℃ for 3 h.

The reaction was cooled to room temperature and quenched with H₂O (20mL) was diluted and extracted with EA (20mLx 2). The combined organic layers were washed with brine (20mL) and Na₂SO₄Dried, filtered and concentrated. The residue was purified by flash chromatography on silica gel (PE/EA ═ 5/1) to give compound I3-8(120mg, yield 65%) as a white solid.

MS calculated: 644; MS measurement: 645M + H +.

Step 6: synthesis I3-9:

to a solution of compound I3-8(50mg, 0.08mmol) in DCM (5mL) at 0 deg.C was added BBr3(0.2 mL). The reaction mixture was stirred at room temperature overnight.

With Na₂CO₃The solution adjusts the pH of the reaction mixture to>7, then extracted with DCM (15mLx 2). The combined organic phases were washed with Na₂SO₄Drying, filtration and concentration gave compound I3-9(38mg, 92% yield) as a yellow solid.

MS calculated: 530; MS measurement: 531[ M + H ] +.

And 7: synthesis I3:

to a solution of compound I3-9(20mg, 0.04mmol) in DCM (5mL) was added DIEA (10.3mg, 0.08mmol) and acryloyl chloride (3.4mg, 0.08mmol) at 0 deg.C. The reaction mixture was stirred at room temperature for 1 hour.

Reacting with H₂O (5mL) was diluted and extracted with DCM (10mLx 2). The combined organic layers were washed with brine (10mLx2) and Na₂SO₄Dried, filtered and concentrated. The residue was purified by Pre-HPLC to give compound I3(3mg, 13.6% yield) as a yellow solid.

MS calculated: 584.2, respectively; MS measurement: 585.0[ M + H ] +.

¹H NMR(400MHz，CD₃OD)δ：8.33-8.31(m，1H)，8.14(d，J＝9.2Hz，1H)，7.17-7.10(m，2H)，6.85-6.74(m，1H)，6.55-6.46(m，2H)，6.23(d，J＝16.4Hz，1H)，5.75(d，J＝10.4Hz，1H)，4.51-4.34(m，2H)，4.29-3.96(m，3H)，3.66-3.51(m，2H)，1.98(s，1.5H)，1.95-1.89(m，1H)，1.84(s，1.5H)，1.28-1.23(m，3H)，1.11-1.03(m，3H)，0.93-0.78(m，3H).

Example 4 Compounds of formula I4

Compounds of formula I4

4- ((S) -4-acryloyl-2-methylpiperazin-1-yl) -6-fluoro-7- (2-fluoro-6-methoxyphenyl) -1- (2-isopropyl-4-methylpyridin-3-yl) -2-oxo-1, 2-dihydro-1, 8-naphthyridine-3-carbonitrile

Obtained by referring to the preparation method of example 1 and changing the preparation of the corresponding reagent.

Example 5: compounds of formula I5

Compounds of formula I5

4- (4-acryloyl-2-methylpiperazin-1-yl) -3, 6-difluoro-7- (2-fluoro-6-hydroxyphenyl) -1- (2-isopropyl-4-methylpyridin-3-yl) -1, 8-naphthyridin-2 (1H) -one

Step 1: synthesis of I5-03

To a solution of compound I5-01(10g, 52.36mmol) in dioxane/H2O (100mL/10mL) was added compound I5-02(10.68g, 62.83mmol), Na₂CO₃(16.6g, 157.08mmol) and Pd (pph3)4(6.1g, 5.24 mmol). The resulting mixture was stirred at 110 ℃ overnight.

By H₂The reaction solution was diluted with O (100mL) and extracted with EA (200 mL. times.2). The combined organic layers were washed with brine (100 mL. times.2) and Na₂SO₄Dried, filtered and concentrated. The residue was purified by silica gel chromatography (PE/EA ═ 10/1) to give compound I5-03(4.96g, 34% yield) as a yellow solid.

MS Calcd.:280；MS Found:281[M+H]⁺.

Step 2: synthesis of I5-05

To a solution of compound I5-03(2g, 7.13mmol) in toluene (30mL) was added compound I5-04(1.39g, 9.26mmol), CS₂CO₃(6.97g, 21.39mmol), xanthphos (1.65g, 2.85mmol) and Pd₂(dba)₃(1.31g, 1.43 mmol). The resulting mixture is stirred under N₂And stirred at 110 ℃ overnight.

The reaction was concentrated and the residue was purified by silica gel chromatography (PE/EA ═ 6/1-3/1) to give compound I5-05 as a yellow solid (2.52g, 90% yield).

MS Calcd.:394；MS Found:395[M+H]⁺.

And step 3: synthesis of I5-07

To a solution of compound I5-05(2.6g, 6.77mmol) in pyridine (20mL) was added compound I5-06(20 mL). The reaction mixture was heated to 130 ℃ and stirred for 24 hours.

The reaction was completed by LC-MS detection. The mixture was poured into 50mL of water and the product was extracted with EA (50 mL. times.3). The organic layer was washed with water (50 mL. times.2) and 50mL brine, over Na₂SO₄And (5) drying. After filtration, the solvent was removed under reduced pressure. The residue was dried to give Compound I5-07(3.6g) as a brown oil.

MS Calcd.:436；MS Found:437[M+H]+.

And 4, step 4: synthesis of I5-08

To a solution of compound I5-07(2.88g, 6.6mmol) in DMF (20mL) was added t-BuOK (2.26g, 19.8 mmol). The reaction mixture was stirred at room temperature for 2 hours.

The reaction was completed by LC-MS detection. The mixture was poured into 50mL of water and the product was extracted with EA (50 mL. times.3). The organic layer was washed with water (50 mL. times.2) and 50mL brine, then Na₂SO₄And (5) drying. After filtration, the solvent was removed under reduced pressure. The residue was purified by silica gel chromatography (PE/EA ═ 5/1-1/2) to give compound I5-08(1.22g, 42% yield) as a yellow solid.

MS Calcd.:436；MS Found:437[M+H]+.

And 5: synthesis of I5-09

To a solution of compound I5-08(800mg, 1.83mmol) in ACN (20mL) was added selectfluor (779.3mg, 2.2 mmol). The reaction mixture was heated to 80 ℃ overnight.

The reaction was diluted with water (20mL) and extracted with EA (20 mL. times.2). The combined organic layers were washed with brine (20mL) and Na₂SO₄Dried, filtered and concentrated. The residue was purified by reverse phase silica gel chromatography (ACN/H)₂O ═ 5% to 95%, 254nm, 30 min) to give compound I5-09(185mg, 22% yield) as a yellow solid.

MS Calcd.:454；MS Found:455[M+H]⁺.

Step 6: synthesis of I5-10

To a solution of compound I5-09(185mg, 0.41mmol) in ACN (15mL) at 0 deg.C were added CuBr (294mg, 2.05mmol) and t-BuONO (211.4mg, 2.05 mmol). The reaction mixture was stirred at room temperature overnight.

The reaction was diluted with water (15mL) and extracted with EA (20 mL. times.2). The combined organic layers were washed with brine (20 mL. times.2) and Na₂SO₄Dried, filtered and concentrated. The residue was purified by reverse phase silica gel chromatography (ACN/H2O ═ 5% to 95%, 254nm, 30 min) to give compound I5-10(27mg, 13% yield) as a yellow solid.

MS Calcd.:518；MS Found:519[M+H]⁺.

And 7: synthesis of I5-11

A vial was charged with compound I5-10(170mg, 0.33mmol), 3-methylpiperazine-1-carboxylic acid (R) -tert-butyl ester (132mg, 0.66mmol), t-BuONa (95mg, 0.99mmol), BINAP (103mg, 0.17mmol), Pd (OAc)₂(74mg, 0.33mmol) and toluene (5 mL). The sealed vial was irradiated in a microwave at 120 ℃ for 2 hours.

The reaction was diluted with water (20mL) and extracted with EA (20 mL. times.2). The combined organic layers were washed with brine (20mL) and Na₂SO₄Dried, filtered and concentrated. The residue was purified by reverse phase silica gel chromatography (ACN/H)₂O ═ 5% to 95%, 254nm, 30 min), compound I5-11 was obtained as a yellow solid (31.4mg, 15% yield).

MS Calcd.:637；MS Found:638[M+H]⁺。

And 8: synthesis of I5-12

To a solution of compound I5-11(50mg, 0.08mmol) in DCM (5mL) at-78 deg.C was added BBr3(0.3mL, 1M). The reaction mixture was warmed to room temperature and stirred for 2 hours.

The reaction mixture was quenched with water (20 mL). The mixture was extracted with DCM (15 mL. times.2). The combined aqueous phases were passed over Na₂CO₃The aqueous solution was basified to pH 7-8 and the aqueous layer was extracted with DCM (15mL × 2). The organic layer was washed with Na₂SO₄Drying, filtration and concentration gave compound I5-12(40mg) as a yellow solid.

MS Calcd.:523；MS Found:524[M+H]⁺.

And step 9: synthesis of I5

To a solution of compound I5-12(30mg, 0.06mmol) in DCM (5mL) under nitrogen and at 0 deg.C was added TEA (9.1mg, 0.09mmol) and acryloyl chloride (12mg, 0.13 mmol). The reaction mixture was allowed to warm to room temperature for 1 hour.

The reaction was diluted with water (10mL) and extracted with DCM (10 mL. times.2). The combined organic layers were concentrated in vacuo. The residue was dissolved in THF/H₂O (5/1mL), and LiOH. H₂O (8mg, 0.18 mmol). The mixture was stirred at room temperature for 2 hours.

The reaction solution was concentrated in vacuo and purified by HPLC to give compound I5 as a solid (3mg, 11% yield).

¹H NMR(400MHz,CDCl₃)δ:9.09(d,J＝5.2Hz,1H),8.67(d,J＝4.8Hz,1H),8.22(brs,0.5H),7.24-7.22(m,2H),6.71-6.62(m,2.5H),6.42-6.38(m,1H),5.82-5.79(m,1H),5.36-5.33(m,1H),3.72-3.69(m,1H),3.53-3.40(m,1H),3.28-3.19(m,1H),2.27-2.17(m,1H),2.05-1.98(m,4H),1.31-1.17(m,6H),1.04(d,J＝6.8Hz,3H),0.88(t,J＝6.8Hz,3H).

MS Calcd.:577；MS Found:578[M+H]⁺ _.

Example 6: compounds of formula I6

Compounds of formula I6

4- ((2S, 5R) -4-acryloyl-2, 5-dimethylpiperazin-1-yl) -6-fluoro-7- (2-fluoro-6-hydroxyphenyl) -1- (2-isopropyl-4-methylpyridin-3-yl) -1, 8-naphthyridin-2 (1H) -one

Example 7: compounds of formula I7

Compounds of formula I7

4- (4-acryloyl-2, 2-dimethylpiperazin-1-yl) -6-fluoro-7- (2-fluoro-6-hydroxyphenyl) -1- (2-isopropyl-4-methylpyridin-3-yl) -1, 8-naphthyridin-2 (1H) -one

Example 8: compounds of formula I8

Compounds of formula I8

4- ((S) -4-acryloyl-2-methylpiperazin-1-yl) -7- (2-amino-6-fluorophenyl) -6-fluoro-1- (2-isopropyl-4-methylpyridin-3-yl) -1, 8-naphthyridin-2 (1H) -one

Example 9: compounds of formula I9

Compounds of formula I9

4- ((S) -4-acryloyl-2-methylpiperazin-1-yl) -6-chloro-7- (2-fluoro-6-hydroxyphenyl) -1- (2-isopropyl-4-methylpyridin-3-yl) -1, 8-naphthyridin-2 (1H) -one

Example 10: compounds of formula I10

Compounds of formula I10

4- ((S) -4-acryloyl-2-methylpiperazin-1-yl) -1- (2, 4-diisopropylpyridin-3-yl) -6-fluoro-7- (2-fluoro-6-hydroxyphenyl) -1, 8-naphthyridin-2 (1H) -one

Example 11: compounds of formula I11

Compounds of formula I11

4- ((S) -4-acryloyl-2-methylpiperazin-1-yl) -7- (2-amino-6-fluorophenyl) -6-chloro-1- (2-isopropyl-4-methylpyridin-3-yl) -1, 8-naphthyridin-2 (1H) -one

Example 12: compounds of formula I12

Compounds of formula I12

4- ((2S, 5R) -4-acryloyl-2, 5-dimethylpiperazin-1-yl) -7- (2-amino-6-fluorophenyl) -6-fluoro-1- (2-isopropyl-4-methylpyridin-3-yl) -1, 8-naphthyridin-2 (1H) -one

Example 13: compounds of formula I13

Compounds of formula I13

4- ((2S, 5R) -4-acryloyl-2, 5-dimethylpiperazin-1-yl) -6-chloro-7- (2-fluoro-6-hydroxyphenyl) -1- (2-isopropyl-4-methylpyridin-3-yl) -1, 8-naphthyridin-2 (1H) -one

Example 14: compounds of formula I14

Compounds of formula I14

4- (4-acryloyl-2-oxopiperazin-1-yl) -6-fluoro-7- (2-fluoro-6-hydroxyphenyl) -1- (2-isopropyl-4-methylpyridin-3-yl) -1, 8-naphthyridin-2 (1H) -one

Example 15: compounds of formula I15

Compounds of formula I15

4- (6-acryloyl-2, 6-diazaspiro [3.3] hept-2-yl) -6-fluoro-7- (2-fluoro-6-hydroxyphenyl) -1- (2-isopropyl-4-methylpyridin-3-yl) -1, 8-naphthyridin-2 (1H) -one

Step 1: synthesis of I15-02

A vial was charged with Compound I15-01(100.0mg, 0.20mmol), Compound RJ(231.0mg，0.80mmol)，t-BuONa(96.0mg，0.80mmol)，BINAP(62.0mg，0.10mmol)，Pd(OAc)₂(45.0mg, 0.20mmol) and toluene (3 mL). The sealed vial was irradiated in a microwave at 120 ℃ for 2 hours.

The reaction was completed by LC-MS detection. The reaction was diluted with water (20mL) and extracted with EA (20 mL. times.2). The combined organic layers were washed with brine (20mL) and Na₂SO₄Dried, filtered and concentrated. The residue was purified by HPLC to give compound I15-02(100.0mg, 80.9% yield) as a yellow solid.

MS Calcd.:617.7；MS Found:618.4[M+H]⁺.

Step 2: synthesis of I15-03

To a solution of compound I15-02(10.0mg, 0.16mmol) in DCM (10mL) at-78 deg.C was added BBr3(1.6mL, 1M). The reaction mixture was allowed to warm to room temperature and stirred for 16 hours.

The reaction was completed by LC-MS detection. The reaction mixture was quenched with water (20 mL). The mixture was extracted with DCM (15 mL. times.2). The combined aqueous phases were passed over Na₂CO₃The aqueous solution was basified to pH 7-8 and the aqueous layer was extracted with DCM (15mL × 2). The organic layer was washed with Na₂SO₄Drying, filtration and concentration gave compound I15-03(80.0mg) as a yellow solid.

MS Calcd.:503.5；MS Found:504.3[M+H]⁺.

And step 3: synthesis of I15

To a solution of compound I15-03(80.0mg, 0.16mmol) in DCM (5mL) under nitrogen and at 0 deg.C were added TEA (48.0mg, 0.48mmol) and acryloyl chloride (43.0mg, 0.48 mmol). The reaction mixture was allowed to warm to room temperature for 1 hour. The reaction was diluted with water (10mL) and extracted with DCM (10 mL. times.2). The combined organic layers were concentrated in vacuoAnd (4) shrinking. The residue was dissolved in THF/H₂O (5/1mL), and LiOH (24.0mg, 0.48mmol) was added. The mixture was stirred at room temperature for 2 hours.

The reaction was completed by LC-MS detection. The mixture was concentrated in vacuo and purified by HPLC to give compound I15 as a white solid (5.8mg, 6.6% yield).

¹H NMR(400MHz,CDCl₃)δ:9.32(brs,1H),8.62(d,J＝4.2Hz,1H),7.89(d,J＝10.0Hz,1H),7.25-7.19(m,2H),6.67(t,J＝8.8Hz,2H),6.39(dd,J＝1.6,16.8Hz,1H),6.24-6.17(m,1H),5.75(dd,J＝1.6,10.0Hz,1H),5.68(s,1H),4.55-4.37(m,8H),2.68-2.61(m,1H),2.01(s,3H),1.20(d,J＝6.8Hz,3H),1.02(d,J＝6.8Hz,3H).

MS Calcd.:557.6；MS Found:558.2[M+H]⁺。

Example 16: compounds of formula I16

Compounds of formula I16

4- ((3R) -1-acryloyl-3-methylpiperidin-4-yl) -6-fluoro-7- (2-fluoro-6-hydroxyphenyl) -1- (2-isopropyl-4-methylpyridin-3-yl) pyrido [2,3-d ] pyrimidin-2 (1H) -one

Example 17 Compounds of formula I17

Compounds of formula I17

4- ((S) -4-acryloyl-2-methylpiperazin-1-yl) -6-fluoro-7- (2-fluoro-6-hydroxyphenyl) -1- (2- (pyrrolidin-1-yl) ethyl) -1, 8-naphthyridin-2 (1H) -one

Example 18 Compounds of formula I18

Compounds of formula I18

4- ((S) -4-acryloyl-2-methylpiperazin-1-yl) -1- ((((R) -1-cyclopropylpyrrolidin-2-yl) methyl) -6) -fluoro-7- (2-fluoro-6-hydroxyphenyl) -1, 8-naphthyridin-2 (1H) -one

Example 19KRAS G12C coupled nucleotide exchange assay

1. Purpose of the experiment: compounds were evaluated for their inhibitory effect on SOS 1-mediated nucleotide exchange.

2. Reagents, consumables and apparatus

3. Compound information and processing

Table 2 compound information and treatment

4. Experimental procedures

a) At 1mM Mg₂+ pre-incubate KRAS G12C and GDP in buffer for 30 min, then stop the assay using 10mM Mg2+ buffer.

b) KRAS G12C-GDP was desalted by chromatography column and then tested for KRAS G12C-GDP concentration using the Brandford method.

c) Compounds were diluted 3-fold in DMSO (11 spots). Then 2. mu.L of the compound was transferred to 38. mu.L of assay buffer to give a compound solution.

d) 4 μ L of KRASG12C-GDP at the indicated concentration was added to the 384 assay plates.

e) mu.L of compound solution (prepared in step c) was transferred to the assay plate and pre-incubated with enzyme for 15 minutes at 25 ℃.

f) Add 4 μ L of SOS1 and GTP mixture to assay wells and incubate for 2 hours.

g) Add 10. mu.L of cRAF RBD, glutathione donor and nickel chelate acceptor mixture to the assay well.

h) Centrifuge at 1000RPM for 1 minute and incubate at 25 ℃ for 2 h.

i) The AlphaLisa signal is read on Envision.

j) The raw data was analyzed using the formula (see "5. data analysis" below).

5. Data analysis

a) For each screening plate, mean data and Standard Deviation (SD) of DMSO were calculated as high and low controls.

b) Percent inhibition (% inh) of compound wells was 100 × (average high control-cpd wells)/(average high control-average low control).

c) Assay robustness analysis with low control and high control data:

S/B ═ average high control/average low control.

CV% (low control) 100 × (SD low control/average low control).

CV% (high control) ═ 100 × (SD high control/average high control).

Z ═ 1-3 ═ (SD low control + SD high control)/(AVE high control-AVE low control).

d) Fitting cpd IC according to non-linear regression equation₅₀。

Y ═ bottom + (top-bottom)/(1 +10^ ((LogIC)₅₀-X)*HillSlope))。

X: log of compound concentration.

Y: percent inhibition (% inh).

Top and bottom: same as the Y unit.

logIC₅₀: log unit same as X.

Hill slope is the coefficient of slope or slope.

TABLE 3 summary of the analyses

Example 20: pERK intracellular WB assay

1. Purpose of the experiment: evaluation of the proliferation inhibitory Effect of Compounds on KRAS Gl2C mutant NCI-H358 human non-Small cell Lung cancer cells

2. Reagents, consumables and apparatus

3. Compound information and processing

4. Experimental procedures

4.1 cell culture

a) On day 1, seed cells were placed in T75 flasks as shown in the following table:

cell lines	Basal growth medium	Cell # T75	Growth cycle
				NCI-H358	RPMI1640+10％FBS+1％P/S	3.5-4x10⁶	2 days

b) On day 3, the medium was removed and washed once with DPBS,

c) at Room Temperature (RT) or 37 ℃ with 2mL TrypLE^TMExpress enzyme trypsinizes cells until cells detach.

d) 5mL of fresh medium was added, the cells were suspended, and then centrifuged at 1000rpm for 5 minutes at room temperature.

e) The supernatant was discarded and the cells were resuspended in 5mL fresh medium and counted by Cell counting I.

f) Cells were seeded back into T75 flasks for further culture or placed into assay plates for intracellular Western analysis.

4.2 Western analysis in pERK cells

a) Cells were seeded in 384-well plates and 5% CO at 37 ℃₂And incubated overnight.

b) 200nL serial dilutions of compound (final 0.5% DMSO) were added via Echo 550 at 37 deg.C, 5% CO₂And cells were incubated for 3 hours.

c) Cells were fixed at room temperature.

d) The cells were washed once with 40. mu.LPBS and then permeabilized.

e) Wash once with 40. mu.L/well PBS.

f) Blocking buffer was added and incubated at room temperature.

g) Blocking buffer was removed and primary antibody, rabbit anti-pERK, mouse anti-GAPDH was added.

h) Wash with 40 μ L/well PBST 3 times.

i) Secondary antibody was added and incubated at room temperature in the dark.

j) Wash with 40 μ L/well PBST 3 times.

k) Centrifuge at 1000rpm for 1 minute, then scan well plate with Odyssey CLx.

5. Data analysis

a) Assay stability check was performed using DMSO control data:

relative signal is signal value (total channel 800)/signal value (total channel 700).

H ═ Ave (dmso), L ═ Ave (compound).

Sd (h) ═ STDEV (dmso), sd (l) ═ STDEV (compound).

CV% (DMSO) ═ 100 (SD _ H/Ave _ H), CV% (compound) ═ 100 SD _ L/Ave _ L.

Z'＝1-3*(SD_H+SD_L)/(Ave_H-Ave_L)。

Relative pERK ═ Sample-Ave _ L)/(Ave _ H-Ave _ L).

b) Fitting cpdIC according to non-Linear regression equation₅₀：

Y ═ bottom + (top-bottom)/(1 +10^ ((Logic50-X) } HillSlope))

X: log cpd concentration.

Y: ave (relative pERK).

Top and bottom: same units as Y.

logIC 50: log unit same as X:

hill slope coefficient or slope

6. Results

The antiproliferative activity of the compounds of the invention against NCI-H358 cells, IC50, is shown in Table 4.

TABLE 4

Example 21 examination of the reversing Effect of the Compounds of the present invention on the multidrug resistance in tumors

Test compounds: compounds of the formulae AMG510 and I1-I18 of the examples of the invention.

Parental cell KB cell is a human oral epidermoid cancer cell;

p-glycoprotein (P-gp) high expression cell (KB)_V200) KB cells are used as parents, stimulated by mutagen ethyl methanesulfonate, and then added with increasing concentrations of growthThe vincristine is obtained by induction and has drug resistance to vincristine.

Respectively inoculating parent cell KB cells and P-gp high-expression cells KBV200 cells into a 96-well plate (5000 cells/well), after the cells are completely attached to the wall, adding vincristine into corresponding wells in a concentration gradient manner, filling the difference of the drug volume with Phosphate Buffer Solution (PBS), adding 10 mu l of 0.5mg/ml thiazole blue (MTT) into each well after 72 hours, incubating for 4 hours at 37 ℃, sucking out culture solution, adding 50 mu l of dimethyl sulfoxide (DMSO) into each well after air drying to dissolve crystals, and measuring the OD value at 570nm of an enzyme reader; combining different compounds with vincristine with gradient concentration, repeating the above process, setting 3 multiple wells for each concentration, repeating for 3 times, calculating Mean + -SD, and calculating each group of IC with Graphpad Prism software₅₀(half inhibitory concentration, i.e., the concentration of the inhibitor at which 50% inhibition was achieved), the results are shown in Table 5.

TABLE 5 inhibition of KB cells and KBV200 cells with the use of different compounds in combination with vincristine

As can be seen from Table 5, KB of the present compounds capable of reversing the high expression of P-gp_V200The drug resistance to vincristine, in particular to the compound shown as the formula I1, can obviously inhibit KB_V200Multidrug resistance of cells to vincristine.

The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A compound of formula (I), or a stereoisomer thereof, or an atropisomer thereof, or a pharmaceutically acceptable salt thereof, or a tautomer thereof:

the compound of formula (I) is a compound selected from one of the following structures:

2. a pharmaceutical composition comprising (1) a compound of claim 1; and (2) a pharmaceutically acceptable carrier.

3. Use of a compound according to claim 1 for the preparation of a pharmaceutical composition for: (ii) inhibiting or reversing multidrug resistance in tumors to antineoplastic drugs; (iii) inhibition of P-glycoprotein; and/or (iv) enhance the anti-tumor activity of the anti-tumor drug.

4. The use of claim 3, wherein the neoplasm comprises a neoplasm that is multidrug resistant to an antineoplastic drug.

5. The use of claim 3, wherein the tumor is selected from the group consisting of: lung cancer, pancreatic cancer, colorectal cancer, hematologic malignancies, oral cancer, or a combination thereof.

6. The use of claim 3, wherein said antineoplastic drug comprises vincristine.

7. The use according to claim 3, wherein the tumor is a P-glycoprotein-highly expressed tumor.

8. The use of claim 3, wherein said multidrug resistance is multidrug resistance caused by high expression of P-glycoprotein.

9. The use of claim 5, wherein the oral cancer comprises oral epidermoid cancer.

10. The use of claim 3, wherein the pharmaceutical composition is for: (ii) inhibiting or reversing multidrug resistance in tumors to antineoplastic drugs;

the tumor is a P-glycoprotein high expression tumor;

the tumor is oral epidermoid carcinoma;

the anti-tumor drug is vincristine.