WO2025201236A1 - Mct4 inhibitors and pharmaceutical composition and use thereof - Google Patents

Abstract

The present disclosure provides compounds of formula (III) or pharmaceutically acceptable salts, hydrates, solvates, stereoisomers (such as cis-trans isomers), tautomers, isotope-labelled substances (such as deuterated substances) or prodrugs thereof, or a pharmaceutical composition comprising same, which can inhibit MCT4 protein, so as to treat cancers or tumors, asthma, chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF).

Description

MCT4 inhibitors and pharmaceutical compositions and uses thereof Technical Field

The present disclosure belongs to the field of medicinal chemistry, and specifically relates to a compound of formula (I) (e.g., a compound of formula (III)), a pharmaceutical composition comprising the same, and its use as an MCT4 inhibitor and a therapeutic drug for diseases or conditions associated with abnormally elevated MCT4 levels.

Background Art

As a result of metabolic reprogramming, cancer cells exhibit a high rate of glycolysis, leading to excessive lactate production and increased extracellular acidity. Proton-linked monocarboxylate transporters (MCTs) are crucial in maintaining this metabolic phenotype and contribute to cancer cell pH regulation by mediating proton-coupled membrane lactate flux. Among the proteins encoded by the SLC16 gene family, MCT1 and MCT4 isoforms have been most studied in cancer and are overexpressed in many cancer types, ranging from solid tumors to hematological malignancies. Similar to what occurs in specific physiological contexts, MCT1 and MCT4 mediate lactate shuttling between cancer cells and between cancer cells and stromal cells in the tumor microenvironment. This form of metabolic cooperation is responsible for key cancer aggressiveness traits, such as cell proliferation, survival, angiogenesis, migration, invasion, metastasis, immune tolerance, and therapeutic resistance. The increasing understanding of MCT function and regulation provides new avenues for the design of novel inhibitors with foreseeable clinical application.

MCT4 is primarily expressed in glycolytic tissues, including white skeletal muscle, astrocytes, and leukocytes. MCT4 is overexpressed in a variety of cancer types and is closely associated with elevated levels of tumor glycolysis (Warburg effect), phenotypes associated with cell migration and invasion. For example, elevated MCT4 levels have been reported in renal cell carcinoma (RCC), gastric cancer, cervical cancer, non-small cell lung cancer, breast cancer, head and neck cancer, bladder cancer, non-Hodgkin lymphoma, and human glioblastoma. MCT4 is also overexpressed in glioblastoma (GBM) cells expressing the stemness marker CD133 and is significantly correlated with poor clinical and prognostic outcomes in GBM patients.

Asthma and chronic obstructive pulmonary disease are chronic airway inflammatory diseases involving multiple cells and cellular components. Stimuli such as aeroallergens trigger the release of inflammatory factors from airway epithelial cells, leading to recurrent acute exacerbations. Clinically, these symptoms include recurrent wheezing and shortness of breath, with or without chest tightness or cough.

Idiopathic pulmonary fibrosis (IPF) is a progressive and ultimately fatal chronic interstitial lung disease in which repetitive alveolar epithelial injury triggers the early development of fibrosis. These injuries, coupled with dysregulated wound repair and fibroblast dysfunction, lead to the persistent tissue remodeling and fibrosis seen in end-stage pulmonary fibrosis, which is associated with high mortality and limited therapeutic options. Studies have suggested that metabolic reprogramming may be a therapeutic strategy in IPF. Recent studies have demonstrated the effects of a novel metabolic strategy, namely, lactate transport inhibition, on myofibroblast differentiation and experimental pulmonary fibrosis. Sustained glycolysis in myofibroblasts is dependent on lactate secretion, which is carried out by the monocarboxylate transporter family 4 (MCT4).

Currently, no MCT4 inhibitor has entered clinical research, and the development of MCT4 inhibitors has very important clinical value and significance.

Summary of the Invention

The present disclosure provides a compound having a structure represented by formula (I) (e.g., a compound of formula (III)), which has excellent pharmacodynamic properties, good metabolic stability, and exhibits strong inhibitory activity against MCT4. Furthermore, relative to MCT1, the compound exhibits high selectivity for MCT4 and good solubility.

In one aspect, the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer (e.g., cis-trans isomer), tautomer, isotopically labeled substance (e.g., deuterated substance), or prodrug thereof,

wherein R ₁ and R ₂ are each independently hydrogen, deuterium or C _1-6 alkyl;

R ₃ is hydrogen, deuterium, C _1-6 alkyl or C _3-6 cycloalkyl;

X is CH ₂ , O, S or NH;

Y is a bond or (CH ₂ ) _n ;

n is an integer selected from 1 to 5;

Ring A and Ring B are each independently selected from a C _6-10 aryl group, or a 5-10 membered heteroaryl group containing 1-3 heteroatoms, wherein Ring A and Ring B are each independently optionally substituted by one or more selected from halogen, amino, C _1-6 alkyl, and C _1-6 alkoxy, and the heteroatom may be one or more of O, N, or S;

Ring C is selected from C _4-10 cycloalkyl, 4-10 membered heterocycloalkyl containing 1-3 heteroatoms, C _6-10 aryl, 5-10 membered heteroaryl containing 1-3 heteroatoms, wherein ring C is optionally substituted by one or more selected from halogen, amino, cyano, hydroxy, C _1-6 alkylhydroxy, amide, C _1-6 alkyl, C _1-6 alkoxy, C _1-6 haloalkoxy, and the heteroatom may be one or more of O, N or S.

In some embodiments, R ₁ and R ₂ are each independently hydrogen, deuterium, or C _1-3 alkyl. In some preferred embodiments, R ₁ and R ₂ are each independently hydrogen, deuterium, methyl, or ethyl; preferably, R ₁ and R ₂ are each independently hydrogen, deuterium, or methyl.

In some embodiments, R ₃ is hydrogen, deuterium or C _1-3 alkyl. In some preferred embodiments, R ₃ is hydrogen, deuterium or methyl; preferably, R ₃ is methyl.

In some embodiments, X is O or S. In some preferred embodiments, X is O.

In some embodiments, Y is a bond or (CH ₂ ) _n , where n is an integer selected from 1 to 3. For example, Y is a bond, CH ₂ , CH ₂ CH ₂ , or CH ₂ CH ₂ CH ₂ . In some preferred embodiments, Y is a bond, CH ₂ or CH ₂ CH ₂ .

In some embodiments, ring A and ring B are each independently selected from a C _6-10 aryl group, or a 5-8 membered heteroaryl group containing 1-3 heteroatoms, and ring A and ring B are each independently optionally substituted by one or more selected from halogen, amino, C _1-3 alkyl, and C _1-3 alkoxy groups, and the heteroatom may be one or more of O, N, or S.

In some embodiments, ring A and ring B are each independently selected from a C _6-10 aryl group, or a 5-8 membered heteroaryl group containing 1-3 heteroatoms, and ring A and ring B are each independently optionally substituted by a C _1-3 alkyl group, and the heteroatom may be one or more of O or N.

In some embodiments, ring A is selected from a C _6-10 aryl group, or a 5-8 membered heteroaryl group containing 1-3 heteroatoms, and the ring A is optionally substituted with one or more selected from halogen, amino, C _1-3 alkyl, and C _1-3 alkoxy groups, and the heteroatoms may be one or more of O, N, and S. In some preferred embodiments, ring A is selected from a phenyl group, or a 5-6 membered heteroaryl group containing 1-2 heteroatoms, and the ring A is optionally substituted with a C _1-3 alkyl group, and the heteroatoms may be one or more of O, N, and S. In some preferred embodiments, ring A is selected from a phenyl group, or a 5-6 membered heteroaryl group containing 1-2 heteroatoms, and the heteroatoms may be one or more of O or N.

In some embodiments, ring B is selected from C _6-10 aryl, or a 5-8 membered heteroaryl containing 1-3 heteroatoms, and the ring B is optionally substituted with one or more selected from halogen, amino, C _1-3 alkyl, and C _1-3 alkoxy, and the heteroatom may be one or more of O, N, or S. In some preferred embodiments, ring B is selected from phenyl, or a 5-8 membered heteroaryl containing 1-2 heteroatoms, and the ring B is optionally substituted with C _1-3 alkyl, and the heteroatom may be one or more of O, N, or S. In some preferred embodiments, ring B is selected from phenyl, or a 5-6 membered heteroaryl containing 1-2 heteroatoms, and the heteroatom may be one or more of O or N.

In some embodiments, Ring A and Ring B are each independently selected from phenyl, or a 5-6 membered heteroaryl group containing 1-2 heteroatoms. In some embodiments, Ring A and Ring B are each independently selected from phenyl, or a 6 membered heteroaryl group containing 1-2 heteroatoms.

In some embodiments, Ring A and Ring B are each independently selected from phenyl, pyridinyl, pyrazinyl, pyrimidinyl, or pyridazinyl. In some embodiments, Ring A is selected from phenyl. In some embodiments, Ring B is selected from pyridinyl, pyrazinyl, pyrimidinyl, or pyridazinyl.

In some embodiments, ring C is selected from C _4-8 cycloalkyl, 4-8 membered heterocycloalkyl containing 1-3 heteroatoms, phenyl, 5-8 membered heteroaryl containing 1-3 heteroatoms, wherein ring C is optionally substituted by one or more selected from halogen, amino, cyano, hydroxy, C _1-3 alkylhydroxy, amide, C _1-3 alkyl, C _1-3 alkoxy, C _1-3 haloalkoxy, and the heteroatom may be one or more of O, N or S.

In some embodiments, ring C is selected from C _4-8 cycloalkyl, 4-8 membered heterocycloalkyl containing 1-3 heteroatoms, wherein ring C is optionally substituted with one or more selected from halogen, amino, C _1-3 alkyl, C _1-3 alkoxy, and the heteroatom may be one or more of O, N, or S. In some embodiments, ring C is selected from phenyl or 5-6 membered heteroaryl containing 1-2 heteroatoms, and the heteroatom may be one or more of O, N, or S. In some embodiments, ring C is selected from C _4-8 cycloalkyl, 4-6 membered heterocycloalkyl containing 1-2 heteroatoms, phenyl, or 5-6 membered heteroaryl containing 1-2 heteroatoms, wherein ring C is optionally substituted with one or more selected from halogen, amino, C _1-3 alkyl, C _1-3 alkoxy, and the heteroatom may be one or more of O, N, or S.

In some embodiments, ring C is selected from _C5-7 cycloalkyl, 5-7 membered heterocycloalkyl containing 1-2 heteroatoms, wherein the heteroatoms may be one or more of O or N; preferably, ring C is optionally substituted with _C1-6 alkyl, _C1-5 alkyl, _C1-4 alkyl, _C1-3 alkyl, _C1 alkyl, _C2 alkyl, or _C3 alkyl. In some embodiments, ring C is a 5-6 membered heterocycloalkyl containing 1-2 heteroatoms (e.g., 1 heteroatom), wherein ring C is optionally substituted with _C1-3 alkyl, wherein the heteroatoms may be one or more of O or N.

In some embodiments, ring C can be selected from cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, oxetanyl, azetidinyl, tetrahydrofuranyl, pyrrolidinyl, tetrahydropyranyl, piperidinyl, morpholinyl, piperazinyl, dioxane, phenyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, oxazolyl, pyrrolyl, furanyl or imidazolyl. In some embodiments, ring C can be selected from cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, oxetanyl, tetrahydrofuranyl, pyrrolidinyl, tetrahydropyranyl, piperidinyl, morpholinyl, phenyl, pyridinyl or oxazolyl.

In some embodiments, the present disclosure provides a compound of formula (II) or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer (e.g., cis-trans isomer), tautomer, isotope-labeled substance (e.g., deuterated substance), or prodrug thereof,

wherein Y is a bond or (CH ₂ ) _n ;

n is an integer selected from 1 to 5;

Ring A and Ring B are each independently selected from a C _6-10 aryl group, or a 5-10 membered heteroaryl group containing 1-3 heteroatoms, wherein Ring A and Ring B are each independently optionally substituted by one or more selected from halogen, amino, C _1-6 alkyl, and C _1-6 alkoxy, and the heteroatom may be one or more of O, N, or S;

Ring C is selected from C _4-10 cycloalkyl, 4-10 membered heterocycloalkyl containing 1-3 heteroatoms, C _6-10 aryl, 5-10 membered heteroaryl containing 1-3 heteroatoms, wherein ring C is optionally substituted by one or more selected from halogen, amino, cyano, hydroxy, C _1-6 alkylhydroxy, amide, C _1-6 alkyl, C _1-6 alkoxy, C _1-6 haloalkoxy, and the heteroatom may be one or more of O, N or S.

In some embodiments, in the compound of formula (II), Y, n, Ring A, Ring B, and Ring C are further defined as described above.

In some embodiments, the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer (e.g., cis-trans isomer), tautomer, isotope-labeled substance (e.g., deuterated substance), or prodrug thereof, wherein:

R ₁ and R ₂ are each independently hydrogen, deuterium or C _1-3 alkyl;

R ₃ is hydrogen, deuterium or C _1-3 alkyl;

X is O or S;

Y is a bond or (CH ₂ ) _n , where n is an integer selected from 1 to 3;

Ring A and Ring B are each independently selected from a C _6-10 aryl group, or a 5-8 membered heteroaryl group containing 1-3 heteroatoms, and Ring A and Ring B are each independently optionally substituted by one or more selected from halogen, amino, C _1-3 alkyl, and C _1-3 alkoxy groups, and the heteroatoms may be one or more of O, N, and S. Preferably, Ring A and Ring B are each independently selected from a C _6-10 aryl group, or a 5-8 membered heteroaryl group containing 1-3 heteroatoms, and Ring A and Ring B are each independently optionally substituted by a C _1-3 alkyl group, and the heteroatoms may be one or more of O or N.

Ring C is selected from C _4-8 cycloalkyl, 4-8 membered heterocycloalkyl containing 1-3 heteroatoms, phenyl, 5-8 membered heteroaryl containing 1-3 heteroatoms, wherein ring C is optionally substituted by one or more selected from halogen, amino, cyano, hydroxy, C _1-3 alkylhydroxy, amide, C _1-3 alkyl, C _1-3 alkoxy, C _1-3 haloalkoxy, and the heteroatom may be one or more of O, N or S; preferably, ring C is selected from C _4-8 cycloalkyl, 4-8 membered heterocycloalkyl containing 1-3 heteroatoms, wherein ring C is optionally substituted by one or more selected from halogen, amino, C _1-3 alkyl, C _1-3 alkoxy, and the heteroatom may be one or more of O, N or S.

In some embodiments, the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer (e.g., cis-trans isomer), tautomer, isotope-labeled substance (e.g., deuterated substance), or prodrug thereof, wherein:

R ₁ and R ₂ are each independently hydrogen, deuterium, methyl or ethyl; preferably, R ₁ and R ₂ are each independently hydrogen, deuterium or methyl;

R ₃ is hydrogen, deuterium or methyl; preferably, R ₃ is methyl;

X is O;

Y is a bond, CH ₂ or CH ₂ CH ₂ ;

Ring A is selected from phenyl, or a 5-6 membered heteroaryl group containing 1-2 heteroatoms, wherein the heteroatoms may be one or more of O or N; preferably, ring A is selected from phenyl;

Ring B is selected from phenyl, or a 5-6 membered heteroaryl group containing 1-2 heteroatoms, wherein the heteroatoms may be one or more of O or N; preferably, ring B is selected from pyridyl, pyrazinyl, pyrimidinyl or pyridazinyl;

Ring C is selected from _C4-8 cycloalkyl, 4-6 membered heterocycloalkyl containing 1-2 heteroatoms, phenyl or 5-6 membered heteroaryl containing 1-2 heteroatoms, and ring C is optionally substituted by _C1-6 alkyl, _C1-5 alkyl, _C1-4 alkyl, _C1-3 alkyl, _C1 alkyl, _C2 alkyl or _C3 alkyl (preferably, the 4-6 membered heterocycloalkyl containing 1-2 heteroatoms is optionally substituted by _C1-6 alkyl, _C1-5 alkyl, _C1-4 alkyl, _C1-3 alkyl, _C1 alkyl, _C2 alkyl or _C3 alkyl).

In some embodiments, the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer (e.g., cis-trans isomer), tautomer, isotope-labeled substance (e.g., deuterated substance), or prodrug thereof, wherein:

R ₁ and R ₂ are each independently hydrogen, deuterium, methyl or ethyl; preferably, R ₁ and R ₂ are each independently hydrogen, deuterium or methyl;

R ₃ is hydrogen, deuterium or methyl; preferably, R ₃ is methyl;

X is O;

Y is a bond or CH ₂ ;

Ring A is selected from phenyl, or a 5-6 membered heteroaryl group containing 1-2 heteroatoms, wherein the heteroatoms may be one or more of O or N; preferably, ring A is selected from phenyl;

Ring B is selected from phenyl, or a 5-6 membered heteroaryl group containing 1-2 heteroatoms, wherein the heteroatoms may be one or more of O or N; preferably, ring B is selected from pyridyl, pyrazinyl, pyrimidinyl or pyridazinyl;

Ring C is selected from _C5-7 cycloalkyl, 5-7 membered heterocycloalkyl containing 1-2 heteroatoms, the heteroatoms may be one or more of O or N, and ring C is optionally substituted with _C1-6 alkyl, _C1-5 alkyl, _C1-4 alkyl, _C1-3 alkyl, _C1 alkyl, _C2 alkyl or _C3 alkyl; preferably, ring C is a 5-6 membered heterocycloalkyl containing 1-2 heteroatoms (e.g., 1 heteroatom), wherein ring C is optionally substituted with _C1-3 alkyl, and the heteroatoms may be one or more of O or N.

In some embodiments, the present disclosure relates to a compound of formula (III) or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, isotopically labeled (e.g., deuterated) or prodrug thereof,

in,

Y is a bond or (CH ₂ ) _n , where n is an integer selected from 1 to 3;

Ring C is selected from C _4-8 cycloalkyl, 4-8 membered heterocycloalkyl containing 1-3 heteroatoms, phenyl, 5-8 membered heteroaryl containing 1-3 heteroatoms, wherein ring C is optionally substituted by one or more selected from halogen, amino, cyano, hydroxy, C _1-3 alkylhydroxy, amide, C _1-3 alkyl, C _1-3 alkoxy, C _1-3 haloalkoxy, and the heteroatom may be one or more of O, N or S.

In some embodiments, in the compound of formula (III),

Y is a bond or (CH ₂ ) _n , n is an integer selected from 1 to 3 (preferably, Y is a bond or CH ₂ );

Ring C is selected from C _4-6 cycloalkyl, 4-6 membered heterocycloalkyl containing 1-2 heteroatoms (e.g., 4-5 membered heterocycloalkyl containing 1 oxygen atom), phenyl, 5-6 membered heteroaryl containing 1-2 heteroatoms, wherein the heteroatom may be one or more of O, S or N, and the ring C is optionally substituted by C _1-3 alkyl.

In some embodiments, in the compound of formula (III),

Y is a bond or (CH ₂ ) _n , n is an integer selected from 1 to 3 (preferably, Y is a bond or CH ₂ );

Ring C is selected from cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, oxetanyl, azetidinyl, tetrahydrofuranyl, tetrahydrothiophenyl, tetrahydropyrrolyl, tetrahydropyranyl, piperidinyl, morpholinyl, piperazinyl, dioxanyl, phenyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, oxazolyl, pyrrolyl, furanyl or imidazolyl; preferably, ring C is selected from cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, oxetanyl, tetrahydrofuranyl, pyrrolidinyl, tetrahydropyranyl, piperidinyl, morpholinyl, phenyl, pyridinyl or oxazolyl; wherein, the ring C is optionally substituted with methyl, ethyl, n-propyl or isopropyl.

In some embodiments, in the compound of formula (III),

Y is a bond or CH ₂ ;

Ring C is selected from a 5-7 membered heterocycloalkyl group containing 1-2 heteroatoms, wherein the heteroatoms are one or more of O, S or N, and ring C is optionally substituted by a methyl group, an ethyl group, an n-propyl group or an isopropyl group.

In some embodiments, in the compound of formula (III),

Y is a bond or CH ₂ (preferably, Y is a bond);

Ring C is selected from 5-6 membered heterocycloalkyl containing 1-2 heteroatoms, wherein the heteroatoms are O, S or N (preferably, ring C is selected from 5-6 membered heterocycloalkyl containing 1 oxygen atom), and the ring C is optionally substituted by methyl or ethyl.

In some embodiments, Ring C is selected from tetrahydrofuranyl, tetrahydrothiophenyl, or tetrahydropyrrolyl (pyrrolidinyl).

In some embodiments, the present disclosure relates to a compound of Formula (IV) or Formula (V) or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, isotopically labeled (e.g., deuterated) or prodrug thereof,

wherein Y and ring C are as defined above.

In some embodiments, in the compound of formula (IV) or formula (V),

Y is a bond or (CH ₂ ) _n , n is an integer selected from 1 to 3 (preferably, Y is a bond or CH ₂ );

Ring C is selected from C _4-6 cycloalkyl, 4-6 membered heterocycloalkyl containing 1-2 heteroatoms (e.g., 4-5 membered heterocycloalkyl containing 1 oxygen atom), phenyl, 5-6 membered heteroaryl containing 1-2 heteroatoms, wherein the heteroatom may be one or more of O, S or N, and the ring C is optionally substituted by C _1-3 alkyl.

In some embodiments, in the compound of formula (IV) or formula (V),

Y is a bond or (CH ₂ ) _n , n is an integer selected from 1 to 3 (preferably, Y is a bond or CH ₂ );

Ring C is selected from cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, oxetanyl, azetidinyl, tetrahydrofuranyl, tetrahydrothiophenyl, pyrrolidinyl, tetrahydropyranyl, piperidinyl, morpholinyl, piperazinyl, dioxane, phenyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, oxazolyl, pyrrolyl, furanyl or imidazolyl; preferably, ring C is selected from cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, oxetanyl, tetrahydrofuranyl, tetrahydrothiophenyl, pyrrolidinyl, tetrahydropyranyl, piperidinyl, morpholinyl, phenyl, pyridinyl or oxazolyl; wherein, the ring C is optionally substituted with methyl, ethyl, n-propyl or isopropyl.

In some embodiments, ring C is selected from cyclobutyl, cyclopentyl, cyclohexyl, oxetanyl, tetrahydrofuranyl, tetrahydrothiophenyl, tetrahydropyrrolyl, tetrahydropyranyl, phenyl, pyridinyl or oxazolyl, and the ring C is optionally substituted with methyl, ethyl, n-propyl or isopropyl.

In some embodiments, in the compound of formula (IV) or formula (V),

Y is a bond or CH ₂ (preferably, Y is a bond);

Ring C is selected from 5-6 membered heterocycloalkyl containing 1-2 heteroatoms, wherein the heteroatoms are O, S or N (preferably, ring C is selected from 5-6 membered heterocycloalkyl containing 1 oxygen atom), and the ring C is optionally substituted by methyl or ethyl.

In some embodiments, in the compound of formula (IV) or formula (V),

Y is a bond or CH ₂ (preferably, Y is a bond);

Ring C is selected from oxetanyl, tetrahydrofuranyl, tetrahydrothienyl, tetrahydropyrrolyl or tetrahydropyranyl, and said ring C is optionally substituted with methyl or ethyl.

In some embodiments, in the compound of formula (IV) or formula (V),

Y is a bond or CH ₂ (preferably, Y is a bond);

Ring C is selected from tetrahydrofuranyl, tetrahydrothienyl or tetrahydropyrrolyl, said tetrahydropyrrolyl being optionally substituted with methyl or ethyl.

In some embodiments, Ring C is selected from

In some embodiments, the present disclosure relates to the following compounds or pharmaceutically acceptable salts, hydrates, solvates, stereoisomers (e.g., cis-trans isomers), tautomers, isotopically labeled substances (e.g., deuterated substances), or prodrugs thereof:

This application also covers solutions obtained by any combination, deletion or replacement of the above-mentioned embodiments and preferred solutions.

In one aspect, the present application also relates to a pharmaceutical composition comprising (e.g., a therapeutically effective amount of) a compound of the above-mentioned formula (I), formula (II), formula (III), formula (IV) or formula (V) or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer (e.g., cis-trans isomer), tautomer, isotope label (e.g., deuterated substance) or prodrug thereof, and optionally a pharmaceutically acceptable excipient.

In one aspect, the present application also relates to the use of the above-mentioned formula (I), formula (II), formula (III), formula (IV) or formula (V) compounds or their pharmaceutically acceptable salts, hydrates, solvates, stereoisomers (e.g., cis-trans isomers), tautomers, isotope labels (e.g., deuterated substances) or prodrugs, or pharmaceutical compositions comprising the same in the preparation of MCT4 inhibitors. Alternatively, the present application also relates to the above-mentioned formula (I), formula (II), formula (III), formula (IV) or formula (V) compounds or their pharmaceutically acceptable salts, hydrates, solvates, stereoisomers (e.g., cis-trans isomers), tautomers, isotope labels (e.g., deuterated substances) or prodrugs, or pharmaceutical compositions comprising the same for inhibiting MCT4. Alternatively, the present application also relates to a method of inhibiting MCT4 activity in a subject in need thereof, comprising administering to the subject a compound of the above-mentioned Formula (I), Formula (II), Formula (III), Formula (IV) or Formula (V) or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer (e.g., cis-trans isomer), tautomer, isotope label (e.g., deuterated substance) or prodrug thereof, or a pharmaceutical composition comprising the same.

In one aspect, the present application also relates to the use of the above-mentioned formula (I), formula (II), formula (III), formula (IV) or formula (V) compounds or their pharmaceutically acceptable salts, hydrates, solvates, stereoisomers (e.g., cis-trans isomers), tautomers, isotopic labels (e.g., deuterated substances) or prodrugs, or pharmaceutical compositions comprising the same, in the preparation of a medicament for treating a disease or condition associated with abnormally elevated MCT4 levels. Alternatively, the present application also relates to the above-mentioned formula (I), formula (II), formula (III), formula (IV) or formula (V) compounds or their pharmaceutically acceptable salts, hydrates, solvates, stereoisomers (e.g., cis-trans isomers), tautomers, isotopic labels (e.g., deuterated substances) or prodrugs, or pharmaceutical compositions comprising the same, for treating a disease or condition associated with abnormally elevated MCT4 levels. Alternatively, the present application also relates to a method for treating a disease or condition associated with abnormally elevated MCT4 levels, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of the above-mentioned formula (I), formula (II), formula (III), formula (IV) or formula (V) or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer (e.g., cis-trans isomer), tautomer, isotope label (e.g., deuterated substance) or prodrug, or a pharmaceutical composition comprising the same.

In some embodiments, the abnormally elevated MCT4 level refers to a level in which the MCT4 level is significantly elevated compared to the level in healthy subjects to a degree that may lead to the occurrence of a disease or the risk of the occurrence of a disease.

In some embodiments, the disease or condition associated with abnormally elevated MCT4 levels is a cancer or tumor. In some embodiments, the cancer or tumor is selected from renal cell carcinoma (RCC), kidney cancer, gastric cancer, cervical cancer, non-small cell lung cancer, breast cancer, head and neck cancer, bladder cancer, non-Hodgkin's lymphoma, and glioblastoma.

In some embodiments, the disease or condition associated with abnormally elevated MCT4 levels is asthma, chronic obstructive pulmonary disease (COPD), and idiopathic pulmonary fibrosis.

In some embodiments, the therapeutically effective amount refers to an amount sufficient to improve, inhibit, postpone, or slow the progression of a disease or condition associated with abnormally elevated MCT4 levels, which can be determined by a clinician based on the severity of the disease, the patient's physical condition, age, sex, weight, etc., and can be administered by conventional routes of administration in the art. The compound described herein, or its pharmaceutically acceptable salt, hydrate, solvate, stereoisomer (e.g., cis-trans isomer), tautomer, isotope label (e.g., deuterated substance), or prodrug, or a pharmaceutical composition comprising the same, can be prepared into any conventional dosage form known in the art and can be administered by parenteral, oral, intravenous, intraperitoneal, or other routes.

The disclosed compounds exhibit good MCT4 inhibitory activity, good metabolic stability, and high selectivity for MCT4 relative to MCT1. They also exhibit good solubility and good pharmacokinetic properties, and can exhibit good anti-tumor, asthma-improving, chronic obstructive pulmonary disease-improving, and pulmonary fibrosis-improving effects.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Penh test results of the model group and the drug-treated group 14 days after drug administration in the asthma model of Example 8.

Figure 2: HE staining results of the model group and the drug-treated group 17 days after drug administration in the asthma model of Example 8.

Figure 3: Airway wall thickness test results of the model group and the drug-treated group 17 days after drug administration in the asthma model of Example 8.

Figure 4: HE staining score results of the model group and the drug-treated group after drug administration in the IPF model of Example 9.

Figure 5: Masson's staining scoring results of the model group and the drug-treated group after drug administration in the IPF model of Example 9.

Figure 6: α-SMA score results of the model group and the drug-treated group after drug administration in the IPF model of Example 9.

FIG7 : Penh test results of the model group and the drug-treated group after drug administration in the COPD model of Example 10.

Figure 8: HE staining lung injury scores of the model group and the drug-treated group after drug administration in the COPD model of Example 10.

DETAILED DESCRIPTION

Unless otherwise specified, the terms used in this application have the following meanings: A particular term should not be considered as ambiguous or unclear if it is not specifically defined, but should be understood according to its ordinary meaning in the art.

The term "pharmaceutically acceptable salt" refers to a salt of a compound having the structure of Formula I or Formula II that is substantially non-toxic to organisms. Pharmaceutically acceptable salts generally include, but are not limited to, salts formed by reacting the compounds of the present disclosure with pharmaceutically acceptable inorganic or organic acids, such salts being also known as acid addition salts. Common inorganic acids include, but are not limited to, hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, sulfuric acid (which can form sulfates or acid sulfates), phosphoric acid (which can form phosphates or acid phosphates), etc. Common organic acids include, but are not limited to, trifluoroacetic acid, citric acid (which can form citric acid monosalt, disalt or trisalt), maleic acid (which can form maleic acid monosalt or disalt), fumaric acid (which can form fumaric acid monosalt or disalt), succinic acid (which can form succinic acid monosalt or disalt), tartaric acid (which can form tartaric acid monosalt or disalt), oxalic acid (which can form oxalic acid monosalt or disalt), malonic acid (which can form malonic acid monosalt or disalt), malic acid (which can form malic acid monosalt or disalt), oxalic acid (which can form oxalic acid monosalt or disalt), lactic acid, pyruvic acid, salicylic acid, formic acid, acetic acid, propionic acid, benzoic acid, glycolic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, etc.

The term "hydrate" refers to a substance formed by the compound of the present disclosure or a pharmaceutically acceptable salt thereof and water through non-covalent intermolecular forces. Common hydrates include but are not limited to hemihydrates, monohydrates, dihydrates, trihydrates, etc.

The term "solvate" refers to a substance formed by the compound of the present disclosure or a pharmaceutically acceptable salt thereof and at least one solvent molecule bound by non-covalent intermolecular forces. The term "solvate" includes "hydrates." Common solvates include, but are not limited to, hydrates, ethanolates, acetonates, and the like. It should be understood that the present disclosure encompasses all solvate forms that possess MCT4 inhibitory activity.

The term "isomers" refers to compounds that have the same number and types of atoms, and therefore the same molecular weight, but differ in the arrangement or configuration of the atoms in space.

The term "stereoisomer" (or "optical isomer") refers to a stable isomer that has a perpendicular asymmetric plane due to at least one chiral factor (including a chiral center, chiral axis, chiral plane, etc.), thereby being able to rotate plane-polarized light. Since there are asymmetric centers and other chemical structures in the compounds of the present disclosure that may lead to stereoisomerism, the present disclosure also includes these stereoisomers and mixtures thereof. Since the compounds of the present disclosure and their salts include asymmetric carbon atoms, they can exist in the form of single stereoisomers, racemates, enantiomers and mixtures of diastereomers. Generally, these compounds can be prepared in the form of racemic mixtures. However, if desired, such compounds can be prepared or separated to obtain pure stereoisomers, i.e., single enantiomers or diastereomers, or mixtures enriched in a single stereoisomer (purity ≥98%, ≥95%, ≥93%, ≥90%, ≥88%, ≥85% or ≥80%). As described below, a single stereoisomer of a compound is synthesized from an optically active starting material containing the desired chiral center, or by preparing a mixture of enantiomeric products followed by separation or resolution, for example, by conversion to a mixture of diastereoisomers followed by separation or recrystallization, chromatography, use of a chiral resolving agent, or direct separation of the enantiomers on a chiral chromatographic column. Starting compounds with a specific stereochemistry are either commercially available or prepared as described below and resolved by methods well known in the art. The term "enantiomer" refers to a pair of stereoisomers that are nonsuperimposable mirror images of each other. The term "diastereomer" or "diastereomers" refers to optical isomers that are not mirror images of each other. The term "racemic mixture" or "racemate" refers to a mixture containing equal parts of a single enantiomer (i.e., an equimolar mixture of two R and S enantiomers). The term "non-racemic mixture" refers to a mixture containing unequal parts of a single enantiomer. Unless otherwise stated, all stereoisomeric forms of the compounds of the present disclosure are within the scope of the present disclosure.

The term "tautomer" (or "tautomeric form") refers to structural isomers with different energies that can be interconverted through a low energy barrier. If tautomerism is possible (such as in solution), a chemical equilibrium of the tautomers can be achieved. For example, proton tautomers (or prototropic tautomers) include, but are not limited to, interconversions via proton migration, such as keto-enol isomerization, imine-enamine isomerization, amide-iminoalcohol isomerization, and the like. Unless otherwise indicated, all tautomeric forms of the compounds of the present disclosure are within the scope of the present disclosure.

The term "cis-trans isomers" refers to stereoisomers formed by the different positions of the atoms (or groups) on either side of a double bond or ring system relative to a reference plane; in cis isomers, the atoms (or groups) are on the same side of the double bond or ring system, while in trans isomers, the atoms (or groups) are on opposite sides of the double bond or ring system. Unless otherwise indicated, all cis- and trans-isomeric forms of the compounds of the present disclosure are within the scope of the present disclosure.

The term "prodrug" refers to a derivative compound that is capable of providing a compound of the present disclosure directly or indirectly upon administration to a patient. Particularly preferred derivative compounds or prodrugs are compounds that can increase the bioavailability of a compound of the present disclosure when administered to a patient (e.g., more readily absorbed into the bloodstream), or compounds that promote delivery of the parent compound to the site of action (e.g., the lymphatic system). Unless otherwise indicated, all prodrug forms of the compounds of the present disclosure are within the scope of the present disclosure, and various prodrug forms are well known in the art.

The term "alkyl" refers to a hydrocarbon group of the general formula _{CnH2n +1} , which may be straight-chain or branched. For example, the term " _C1-6alkyl " refers to an alkyl group containing 1 to 6 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, neopentyl, hexyl, 2-methylpentyl, and the like. The term "alkoxy" refers to an -O-alkyl group.

The term "cycloalkyl" refers to a fully saturated carbocyclic ring that can exist as a monocyclic, bridged, or spirocyclic ring. The term "heterocycloalkyl" refers to a fully saturated cyclic group that can exist as a monocyclic, bridged, or spirocyclic ring. Unless otherwise indicated, the heterocycle typically contains 1 to 3 heteroatoms independently selected from sulfur, oxygen, and/or nitrogen. Non-limiting examples of heterocycloalkyl may include, but are not limited to, tetrahydrofuranyl, tetrahydropyrrolyl, tetrahydrothienyl, etc.

The term "aryl" refers to an all-carbon monocyclic or fused polycyclic aromatic ring group having a conjugated π electron system. Non-limiting examples of aryl groups include, but are not limited to, phenyl, naphthyl, and the like. The term "heteroaryl" refers to a monocyclic or fused polycyclic aromatic system containing at least one ring atom selected from N, O, and S. Non-limiting examples of heteroaryl groups include, but are not limited to, pyrrolyl, furanyl, thienyl, imidazolyl, oxazolyl, pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, quinolyl, isoquinolyl, tetrazolyl, triazolyl, triazinyl, benzofuranyl, benzothienyl, indolyl, and isoindolyl.

The term "halogen" herein encompasses F, Cl, Br or I.

Unless otherwise indicated, when referring to an "isotopically labeled" compound of formula (I) of the present application, it refers to a compound in which an atom at a certain group in the compound is substituted by its corresponding isotope. Unless otherwise indicated, the compounds of the present disclosure include various isotopes of H, C, N, O, F, P, S, and Cl, such as ² H (D), ³ H (T), ¹³ C, ¹⁴ C, ¹⁵ N, ¹⁷ O, ¹⁸ O, ¹⁸ F, ³¹ P, ³² P, ³⁵ S, ³⁶ S, and ³⁷ Cl. For example, a deuterated compound can be defined as a compound in which a hydrogen atom in the compound is substituted with deuterium (i.e., D), and the amount of deuterium at that position far exceeds (e.g., at least 1000 times) the abundance of naturally occurring deuterium.

Unless otherwise indicated, "bond" in the definition of a substituent (eg, Y) herein refers to a single bond.

The term "independently" means that when one or more substituents are selected from a number of possible groups, the corresponding groups of these substituents may be the same or different.

The term "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and the description includes both occurrence and non-occurrence of the event or circumstance. For example, when a group is referred to as "optionally substituted", this means that the group is substituted or unsubstituted; when a composition "optionally" includes a component, the composition may or may not include the component.

The term "subject" encompasses mammals, such as humans, non-human primates (eg, rhesus monkeys), pigs, cows, horses, sheep, dogs, rabbits, mice, and the like.

As used herein, unless otherwise indicated, the term " _Cmn " means that the moiety modified by the term has mn carbon atoms (n is greater than m, and both are integers). For example, _C1 - _C6 means that the moiety modified by the term has 1-6 carbon atoms, such as 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, or 6 carbon atoms.

As used herein, unless otherwise specified, “m-n membered heterocycloalkyl” and “m-n membered heteroaryl” mean that the total number of ring atoms, including heteroatoms, in the heterocycloalkyl or heteroaryl group is m-n. For example, “4-10 membered heterocycloalkyl” encompasses 4-membered heterocycloalkyl, 5-membered heterocycloalkyl, 6-membered heterocycloalkyl, 7-membered heterocycloalkyl, 8-membered heterocycloalkyl, 9-membered heterocycloalkyl, and 10-membered heterocycloalkyl; “5-10 membered heteroaryl” encompasses 5-membered heteroaryl, 6-membered heteroaryl, 7-membered heteroaryl, 8-membered heteroaryl, 9-membered heteroaryl, and 10-membered heteroaryl.

In this application, the terms "include," "comprising," and "containing" and their equivalents should be understood as having an open, non-exclusive meaning, i.e., "including but not limited to," meaning that in addition to the listed elements, components, and steps, other unspecified elements, components, and steps may also be included. In this document, unless the context clearly indicates otherwise, singular terms encompass plural referents, and vice versa. Similarly, unless the context clearly indicates otherwise, the word "or" is intended to include "and."

Unless otherwise indicated, herein, the parameter values representing the amount of ingredients or physicochemical properties or reaction conditions, etc. should be understood as being modified by the term "about" in all cases. When the term "about" is used to describe the present application, the term "about" indicates that there is an error value, for example, it indicates a change within the range of ±5%, such as ±1% or ±0.1% of a particular value.

Example

Next, the present disclosure will be further described in detail through examples, but the protection scope of the present disclosure is not limited to these examples.

Example 1: Preparation of Compound 1

(1) Synthesis of Compound 1-1

Synthesis of Compound 1-1a: 2,4-dibromoacetophenone (10.00 g, 35.98 mmol, 1 eq), acetylacetone (3.96 g, 39.55 mmol, 1.1 eq), anhydrous potassium carbonate (14.90 g, 107.81 mmol, 3 eq), and ethanol (100 mL) were added sequentially to a reaction flask. The mixture was allowed to react overnight at room temperature. Water (200 mL) was added to the reaction system, followed by extraction three times with ethyl acetate (300 mL). The combined ethyl acetate phases were dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography (eluent: PE:EA = 9:1) to obtain Compound 1-1a (4.5 g, yield: 48.91%). MS: m/z 254.8 [M+1] ⁺ .

Synthesis of Compounds 1-1b and 1-1c: Compound 1-1a (8.3 g, 32.66 mmol, 1 eq) was added to a reaction flask and dissolved in acetic acid (80 mL). Hydrazine hydrate (2.04 g, 80%, 32.60 mmol, 1 eq) was added at 5°C and allowed to react for 1 h to produce Compound 1-1b. DDQ (11.12 g, 48.99 mmol, 1.5 eq) was then slowly added and allowed to react for 2 h. The reaction system was concentrated in vacuo, water (500 mL) was added, and the pH was adjusted to a weak base with sodium bicarbonate. The mixture was extracted three times with ethyl acetate (500 mL). The ethyl acetate phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography (eluent: PE:EA = 3:2) to afford Compound 1-1c (3.00 g, yield: 36.72%). MS: m/z 248.8 [M+1] ⁺ .

Synthesis of compound 1-1: To a reaction flask were added trichloroisocyanuric acid (2.10 g, 9.04 mmol, 1 eq), compound 1-1c (3.00 g, 12.10 mmol, 1.3 eq), and dichloromethane (35 mL) in sequence. The mixture was stirred at room temperature for 0.5 h to produce compound 1-1 (1.5 g, yield: 43.99%), MS: m/z 283.0 [M+1] ⁺ .

(2) Synthesis of Compound 1-2

Synthesis of compound 1-2a: Benzoic acid (100.0 g, 819.7 mmol, 1 eq), potassium hydroxide (50.5 g, 901 mmol, 1.1 eq), and N,N-dimethylformamide (1 L) were added sequentially to a reaction flask at room temperature. The reaction mixture was stirred at 50°C for 1 h, followed by the addition of 3-chloro-2,4-dipentanone (110 g, 819 mmol, 1 eq). The mixture was stirred at 50°C overnight. The reaction system was cooled to room temperature and extracted three times with ethyl acetate (1 L). The organic phase was then washed sequentially with water (1 L x 2), saturated ammonium chloride (500 mL), and saturated brine (500 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. Purification by silica gel column chromatography (PE:EA=8:1) afforded compound 1-2a (131.2 g, yield: 72.8%) as a yellow oil; MS: m/z 221.2 [M+1] ⁺ .

Synthesis of Compound 1-2b: Compound 1-2a (58.00 g, 263.64 mmol, 1 eq), 3-amino-5-methyl-4H-1,2,4-triazole (27.10 g, 276.31 mmol, 1.05 eq), and acetic acid (300 mL) were added sequentially to a reaction flask at room temperature and stirred at 90°C for 10 h. After cooling to room temperature, the reaction system was concentrated and the pH was adjusted to between 7 and 8 with aqueous sodium bicarbonate. The mixture was extracted three times with ethyl acetate (500 mL). The combined organic phases were washed with saturated brine, filtered, and concentrated to afford Compound 1-2b (53.2 g, yield: 71.6%), which was retained for later use; MS: m/z 283.2 [M+1] ⁺ .

Synthesis of Compound 1-2c: 1M NaOH (177 mL, 177.11 mmol, 1 eq), Compound 1-2b (50 g, 177 mmol, 1 eq), and ethanol (500 mL) were added sequentially to a reaction flask at room temperature. The mixture was stirred at room temperature for 3 hours, then concentrated in vacuo to remove the ethanol and slowly acidified with 6M HCl until a precipitate formed (approximately pH = 7). The precipitate was isolated by filtration, washed with water, and the filter cake dried to afford Compound 1-2c (24.00 g, 76% yield); MS: m/z 179.2 [M+1] ⁺ .

Synthesis of Compound 1-2d: Compound 1-2c (5.00 g, 28.09 mmol, 1 eq), (S)-tert-butyl 3-hydroxypyrrolidine-1-carboxylate (5.80 g, 31.02 mmol, 1.1 eq), triphenylphosphine (8.83 g, 33.67 mmol, 1.2 eq), and THF (100 mL) were placed in a reaction flask. Diisopropyl azodicarboxylate (6.82 g, 33.73 mmol, 1.2 eq) was slowly added dropwise. The mixture was allowed to react at room temperature for 2 h, then concentrated in vacuo. Purification by silica gel column chromatography (PE:EA = 1:2) afforded Compound 1-2d (8.00 g, yield: 82.05%), MS: m/z 348.4 [M+1] ⁺ .

Synthesis of compound 1-2: To a reaction flask were added compound 1-2d (8.00 g, 23.06 mmol, 1 eq) and methanol (32 mL), followed by 4 M dioxane hydrochloride (48 ml). The mixture was stirred at room temperature for 2 h. The reaction system was concentrated in vacuo to afford compound 1-2 (4.5 g, yield: 78.95%); MS: m/z 248.2 [M+1] ⁺ .

(3) Preparation of Compound 1

Synthesis of Compound 1a: Compound 1-1 (190.00 mg, 0.67 mmol, 1.0 eq), tetrahydrofurfuryl alcohol (137.50 mg, 1.35 mmol, 2.0 eq), triethylamine (204.56 mg, 2.01 mmol, 3 eq), and dichloromethane (10 mL) were added sequentially to a reaction flask at room temperature. The reaction mixture was reacted at 40°C for 24 h. The system was then concentrated in vacuo and purified by silica gel column chromatography (petroleum ether:ethyl acetate = 2:1) to afford Compound 1a (150.22 mg, 59.5% yield); MS: m/z 376.2 [M+1] ⁺ .

Synthesis of Compound 1: Compound 1a (200.00 mg, 0.53 mmol, 1.0 eq), compound 1-2 (263.47 mg, 1.07 mmol, 2.0 eq), cesium carbonate (1.74 g, 5.31 mmol, 10 eq), RuPhos Pd G3 (61.98 mg, 0.075 mmol, 0.14 eq), RuPhos (34.62 mg, 0.075 mmol, 0.14 eq), methyltetrahydrofuran (10 ml) and water (5 ml) were added to a reaction flask at room temperature. The system was fully purged with _N2 , and the reaction mixture was reacted at 100°C for 20 h. The reaction system was poured into water (10 ml), extracted three times with ethyl acetate (10 ml), washed with saturated brine (30 mL), and the ethyl acetate phase was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. Purification by silica gel column chromatography (dichloromethane:methanol=10:1) gave Compound 1 (24.91 mg, yield: 8.62%); MS: m/z 516.4 [M+1] ⁺ . ¹ H NMR (400MHz, DMSO-d ₆ )δ8.12(d,J＝8.9Hz,1H),8.05(d,J＝8.5Hz,2H),7.65(d,J＝8.9Hz,1H),6. 74(d,J＝8.5Hz,2H),4.97(s,1H),4.78(s,2H),4.06–3.96(m,1H),3.80–3 .41(m,8H),2.58(s,3H),2.52(s,3H),2.47(s,3H),2.44–2.27(m,2H),1. 92(m,J＝19.5,9.8,6.3Hz,1H),1.80(m,J＝7.0Hz,2H),1.63–1.52(m,1H). ¹³ C NMR (101MHz, DMSO-d ₆ )δ167.08,160.10,157.77,157.48,151.99,148.50,141.47,138.91,127.78,125.96,122.96,122.71 ,111.88,82.78,77.18,72.94,71.61,67.34,51.97,45.65,31.24,27.66,25.17,20.66,14.73,11.99.

Example 2: Preparation of Compound 2

The synthesis method was the same as Example 1 except that tetrahydrofurfuryl alcohol was replaced by 3-tetrahydrofuranmethanol to obtain compound 2 (3.28 mg); MS: m/z 516.4 [M+1] ⁺ . ¹ H NMR (400 MHz, DMSO-d ₆ )δ8.16(d,J＝8.9Hz,1H),8.08–8.01(m,2H),7.69(d,J＝8.9Hz,1H),6.78–6. 72(m,2H),4.97(m,J＝3.1,2.3Hz,1H),4.76(s,2H),3.77–3.39(m,11H),2.58 (s,3H),2.52(s,3H),2.47(s,3H),2.40(d,J＝6.8Hz,1H),2.32(m,J＝8.7,4. 3Hz, 1H), 1.94 (m, J＝12.6, 8.1, 5.6Hz, 1H), 1.54 (m, J＝12.6, 7.7, 6.2Hz, 1H). ¹³ C NMR (101MHz, DMSO-d ₆ )δ164.68,160.13,157.72,157.39,151.95,148.64,140.06,138.92,127.92,126.54,123.52,122.19 ,111.92,82.77,72.31,71.30,70.01,66.84,51.97,45.67,38.54,31.23,28.55,20.66,14.72,11.98.

Example 3: Preparation of Compound 3

The synthesis method was the same as Example 1 except that tetrahydrofurfuryl alcohol was replaced by 1-methyl-3-pyrrolidinemethanol to obtain compound 3 (5.24 mg); MS: m/z 529.4 [M+1] ⁺ . ¹ H NMR (400 MHz, DMSO-d ₆ )δ8.27(m,J＝8.9,2.7Hz,1H),8.17–8.10(m,2H),7.91–7.84(m,1H),6.77(d, J＝8.9Hz,2H),4.98(d,J＝5.2Hz,1H),4.91(d,J＝2.8Hz,1H),4.84(s,1H),3.6 6–3.42(m,9H),3.28(m,J＝11.9,8.5Hz,1H),2.87–2.66(m,2H),2.58(s,3H), 2.51(s,3H), 2.52(s,3H), 2.47(s,3H), 2.38–2.20(m,2H), 2.04–1.87(m,1H). ¹³ C NMR (101MHz, DMSO-d ₆ )δ168.84, 165.09, 160.92, 160.35, 158.48, 151.98, 150.56, 139.46, 130.24, 128.18, 121.80, 118.95, 11 1.98, 85.84, 77.36, 70.15, 64.14, 61.62, 51.96, 48.18, 45.67, 38.29, 37.40, 31.33, 20.65, 14.74, 11.99.

Example 4: Preparation of Compound 4

The synthesis method was the same as Example 1, except that tetrahydrofurfuryl alcohol was replaced by tetrahydropyran-4-ol, to obtain compound 4 (2.62 mg); MS: m/z 516.4 [M+1] ⁺ . ¹ H NMR (400 MHz, DMSO-d ₆ ) δ8.10 (d, J=8.9 Hz, 1H), 8.04 (d, J=8.7 Hz, 2H), 7.67 (d, J=8.9 Hz, 1H), 6.74 (d, J=8.8 Hz, 2H), 4.97 (s, 1H), 4.80 (s, 2H), 3.83 (m, J=11.7, 4.3 Hz, 2H), 3.73–3.50 (m, 4H), 3.45 (m, J= 12.1,4.0Hz,1H),3.36(m,J＝11.7,9.8,2.6Hz,2H),2.58(s,3H),2.52(s,3H),2.46(s,3H) ,2.41(m,J＝7.7Hz,1H),2.33(m,J＝8.9,4.2Hz,1H),2.05–1.88(m,2H),1.56–1.42(m,2H). ¹³ C NMR (101MHz, DMSO-d ₆ )δ164.71,160.07,157.87,157.74,151.98,148.48,140.02,138.89,127.75,126.09,122.94, 122.71,111.86,82.77,73.55,68.18,64.78,51.97,45.65,32.19,31.23,20.66,14.73,11.98.

Example 5: Preparation of Compound 5 (2,5,7-trimethyl-6-((1-(4-(6-(((tetrahydrofuran-3-yl)oxy)methyl)pyridazin-3-yl)phenyl)pyrrolidin-3-yl)oxy)-[1,2,4]triazolo[1,5-a]pyrimidine)

Synthesis of Compound 5a: Compound 1-1 (19.00 g, 67.39 mmol, 1.0 eq), 3-hydroxytetrahydrofuran (35.62 g, 404.31 mmol, 6.0 eq), DMSO (190 mL), and sodium ethoxide (9.17 g, 134.78 mmol, 2.0 eq) were added sequentially to a reaction flask at room temperature under nitrogen for 2 h. After completion, the reaction system was poured into water (1 L) and extracted with ethyl acetate (300 mL x 3). The combined organic phases were washed with saturated brine (500 mL) and dried over anhydrous sodium sulfate. The organic phase was then spin-dried to dryness, and the crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 2:1) to obtain Compound 5a (15.00 g, yield: 66.7%); MS: m/z 335.0 [M+1] ⁺ . ¹ H NMR (400MHz, DMSO-d ₆ )δ8.22(d,J＝8.8Hz,1H),8.12(d,J＝8.6Hz,2H),7.76(dd,J＝9.8,8.6Hz,3H),4.80(d,J＝2.5Hz,2H ), 4.36 (m, J = 4.3, 1.8 Hz, 1H), 3.88–3.79 (m, 2H), 3.775–3.66 (m, 2H), 2.03 (m, J = 7.6, 4.4 Hz, 2H). ¹³ C NMR (101MHz, DMSO-d ₆ ) δ 159.48, 156.86, 135.13, 132.00, 128.80, 126.52, 124.62, 123.75, 79.44, 72.01, 69.22, 66.21, 32.06.

Synthesis of compound 5: Compound 5a (20.00 g, 59.87 mmol, 1.0 eq), compound 1-2 (33.90 g, 119.75 mmol, 2.0 eq), cesium carbonate (195.07 g, 598.70 mmol, 10.0 eq), RuPhos Pd G3 (7.02 g, 8.38 mmol, 0.14 eq), RuPhos (3.91 g, 8.38 mmol, 0.14 eq), methyltetrahydrofuran (200 mL) and water (100 mL) were added sequentially to a reaction flask at room temperature. The system was fully replaced with _N2 , and the reaction mixture was reacted at 100°C for 8 h. The reaction system was poured into water (400 mL) and extracted three times with ethyl acetate (200 mL). The organic phases were combined and washed with saturated brine (300 mL). The ethyl acetate phase was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The crude product was purified by silica gel column chromatography (dichloromethane:methanol=40:1) to give compound 5 (11.00 g, yield: 36.7%); MS: m/z 502.4 [M+1] ⁺ ; ¹ H NMR (400 MHz, DMSO-d ₆ )δ8.10(d,J＝8.9Hz,1H),8.04(d,J＝8.8Hz,2H),7.65(d,J＝8.9Hz,1H),6.74(d, J＝7.12Hz,2H),4.97(m,J＝4.8,2.7Hz,1H),4.75(d,J＝2.4Hz,2H),4.31(m,J＝4. 3,1.9Hz,1H),3.83–3.75(m,2H),3.74–3.49(m,5H),3.45(m,J＝12.0,4.1Hz,1H ),2.58(s,3H),2.52(s,3H),2.47(s,3H),2.45–2.25(m,2H),2.04–1.95(m,2H); ¹³ C NMR (101MHz, DMSO-d ₆ )δ164.71,160.07,157.79,157.41,151.98,148.49,140.02,138.89,127.77,126.19,122.93,122 .69,111.86,82.77,79.29,72.03,69.51,66.20,51.97,45.65,32.09,31.23,20.66,14.73,11.98.

Example 6: Preparation of Compound 6 (2,5,7-trimethyl-6-(((R)-1-(4-(6-((((S)-tetrahydrofuran-3-yl)oxy)methyl)pyridazin-3-yl)phenyl)pyrrolidin-3-yl)oxy)-[1,2,4]triazolo[1,5-a]pyrimidine)

Synthesis of Compound 6a: Compound 1-1 (20.00 g, 70.93 mmol, 1.0 eq), (S)-(+)-3-hydroxytetrahydrofuran (37.47 g, 425.59 mmol, 6.0 eq), DMSO (200 mL), and sodium ethoxide (9.66 g, 141.86 mmol, 2.0 eq) were added sequentially to a reaction flask at room temperature under nitrogen for 2 h. After completion, the reaction system was poured into water (1 L) and extracted with ethyl acetate (300 mL x 3). The organic phases were combined, washed with saturated brine (500 mL), and dried over anhydrous sodium sulfate. The organic phase was then spin-dried to dryness, and the crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 2:1) to obtain Compound 6a (17 g, yield: 72%); MS: m/z 335.0 [M+1] ⁺ . ¹ H NMR (400MHz, DMSO-d ₆ )δ8.27(d,J＝8.8Hz,1H),8.12(d,J＝8.6Hz,2H),7.78(m,J＝9.8,8.6Hz,3H),4.82(d,J＝2.5Hz,2H ), 4.34 (m, J = 4.3, 1.8 Hz, 1H), 3.84–3.77 (m, 2H), 3.77–3.68 (m, 2H), 2.01 (m, J = 7.6, 4.4 Hz, 2H). ¹³ CNMR (101MHz, DMSO-d ₆ ) δ159.40,156.82,135.12,132.00,128.82,126.50,124.67,123.79,79.47,72.03,69.28,66.20,32.08.

Synthesis of compound 6: Compound 6a (10.00 g, 29.93 mmol, 1.0 eq), compound 1-2 (16.95 g, 59.87 mmol, 2.0 eq), cesium carbonate (97.52 g, 299.3 mmol, 10.0 eq), RuPhos Pd G3 (3.51 g, 4.19 mmol, 0.14 eq), RuPhos (1.96 g, 4.19 mmol, 0.14 eq), methyltetrahydrofuran (100 mL) and water (50 mL) were added sequentially to a reaction flask at room temperature. The system was fully replaced with _N2 , and the reaction mixture was reacted at 100°C for 8 h. The reaction system was poured into water (200 mL) and extracted three times with ethyl acetate (100 mL). The organic phases were combined and washed with saturated brine (150 mL). The ethyl acetate phase was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The crude product was purified by silica gel column chromatography (dichloromethane:methanol=40:1) to give compound 6 (6.00 g, yield: 40%); MS: m/z 502.2 [M+1] ⁺ ; ¹ H NMR (400 MHz, DMSO-d ₆ )δ8.10(d,J＝8.9Hz,1H),8.07–8.01(m,2H),7.64(d,J＝8.9Hz,1H),6.77 –6.70(m,2H),4.96(m,J＝4.6,2.5Hz,1H),4.75(d,J＝2.4Hz,2H),4.31(m, J＝4.4,2.0Hz,1H),3.83–3.50(m,7H),3.45(dd,J＝12.0,4.1Hz,1H),2.58 (s,3H),2.52(s,3H),2.46(s,3H),2.44–2.26(m,2H),2.05–1.95(m,2H). ¹³ C NMR (101MHz, DMSO-d ₆ )δ164.69,160.02,157.76,157.37,151.95,148.47,139.96,138.88,127.74,126.14,122.89,122 .68,111.84,82.75,79.28,72.01,69.50,66.18,51.97,45.62,32.08,31.21,20.63,14.70,11.94.

Example 7: Preparation of Compound 7 (2,5,7-trimethyl-6-(((R)-1-(4-(6-((((R)-tetrahydrofuran-3-yl)oxy)methyl)pyridazin-3-yl)phenyl)pyrrolidin-3-yl)oxy)-[1,2,4]triazolo[1,5-a]pyrimidine)

Synthesis of Compound 7a: Compound 1-1 (2.00 g, 7.09 mmol, 1.0 eq), (R)-(-)-3-hydroxytetrahydrofuran (3.75 g, 42.56 mmol, 6.0 eq), DMSO (20 mL), and sodium ethoxide (0.96 g, 14.18 mmol, 2.0 eq) were added sequentially to a reaction flask at room temperature under nitrogen for 2 h. After completion, the reaction system was poured into water (100 mL) and extracted with ethyl acetate (30 mL x 3). The combined organic phases were washed with saturated brine (100 mL) and dried over anhydrous sodium sulfate. The organic phase was then spin-dried to dryness, and the crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 2:1) to obtain Compound 7a (1.20 g, yield: 51%); MS: m/z 335.1 [M+1] ⁺ . ¹ H NMR (400MHz, DMSO-d ₆ )δ8.26(d,J＝8.8Hz,1H),8.10(d,J＝8.6Hz,2H),7.79(m,J＝9.8,8.6Hz,3H),4.85(d,J＝2.5Hz,2H ), 4.39 (m, J = 4.3, 1.8 Hz, 1H), 3.80–3.74 (m, 2H), 3.75–3.70 (m, 2H), 2.03 (m, J = 7.6, 4.4 Hz, 2H). ¹³ CNMR (101MHz, DMSO-d ₆ ) δ 159.46, 156.85, 135.11, 132.03, 128.81, 126.55, 124.64, 123.81, 79.50, 72.09, 69.30, 66.26, 32.06.

Synthesis of compound 7: Compound 7a (1.2 g, 3.59 mmol, 1.0 eq), compound 1-2 (2.03 g, 7.18 mmol, 2.0 eq), cesium carbonate (11.71 g, 35.92 mmol, 10.0 eq), RuPhos Pd G3 (0.42 g, 0.50 mmol, 0.14 eq), RuPhos (0.23 g, 0.50 mmol, 0.14 eq), methyltetrahydrofuran (12 mL) and water (6 mL) were added sequentially to a reaction flask at room temperature. The system was fully replaced with _N2 , and the reaction mixture was reacted at 100°C for 24 h. The reaction system was poured into water (50 mL) and extracted three times with ethyl acetate (20 mL). The organic phases were combined and washed with saturated brine (60 mL). The ethyl acetate phase was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The crude product was purified by silica gel column chromatography (dichloromethane:methanol=40:1) to give compound 7 (110 mg, yield: 6%); MS: m/z 502.2 [M+1] ⁺ ; ¹ H NMR (400 MHz, DMSO-d ₆ )δ8.10(d,J＝8.9Hz,1H),8.04(d,J＝8.6Hz,2H),7.64(d,J＝8.9Hz,1H),6.74(d,J ＝8.6Hz,2H),5.00–4.93(m,1H),4.75(d,J＝2.5Hz,2H),4.31(m,J＝4.4,1.9Hz,1H ),3.83–3.50(m,7H),3.45(m,J＝12.0,4.0Hz,1H),2.58(s,3H),2.52(s,3H),2.4 6(s,3H),2.44–2.37(m,1H),2.32(m,J=9.0,8.6,4.1Hz,1H),2.05–1.95(m,2H). ¹³ C NMR (101MHz, DMSO-d ₆ )δ164.69,160.03,157.76,157.38,151.95,148.47,139.96,138.88,127.74,126.14,122.89,122 .69,111.84,82.76,79.27,72.01,69.50,66.18,51.97,45.63,32.08,31.21,20.63,14.69,11.95.

Example 8: Preparation of Compound 8 ((R)-6-((1-(4-(6-((phenyl)methyl)pyridazin-3-yl)phenyl)pyrrolidin-3-yl)oxy)-2,5,7-trimethyl-[1,2,4]triazolo[1,5-a]pyrimidine)

The synthesis method was the same as Example 5, except that 3-hydroxytetrahydrofuran was replaced by benzyl alcohol, to give Compound 8 (195 mg); MS: m/z 521.8 [M+1] ⁺ . ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.19 (d, J = 8.9 Hz, 1H), 8.05 (d, J = 8.5 Hz, 2H), 7.76 (d, J = 9.0 Hz, 1H), 7.43–7.36 (m, 4H), 7.33 (m, J = 5.9, 2.7 Hz, 1H), 6.75 (d, J = 8.7 Hz, 2H), 4.97 (q, J = 3.2, 2.3 Hz, 1H), 4.82(s,2H),4.64(s,2H),3.71–3.50(m,3H),3.45(m,J＝12.1,4.0Hz,1H),2.58(s,3 H), 2.52 (s, 3H), 2.46 (s, 3H), 2.40 (d, J = 6.8Hz, 1H), 2.32m, J = 13.6, 8.8, 4.1Hz, 1H). ¹³ C NMR (101MHz, DMSO-d ₆ )δ164.64,160.15,157.67,157.34,151.19,148.73,140.07,138.93,137.94,128.32,128.01,127.70,1 27.63,126.90,123.80,121.92,111.95,82.76,71.94,70.60,51.98,45.67,31.22,20.66,14.70,11.98.

Example 9: Preparation of Compound 9 ((R)-2,5,7-trimethyl-6-((1-(4-(6-((pyridin-2-ylmethoxy)methyl)pyridazin-3-yl)phenyl)pyrrolidin-3-yl)oxy)-[1,2,4]triazolo[1,5-a]pyrimidine)

The synthesis method was the same as Example 5, except that 3-hydroxytetrahydrofuran was replaced by 2-pyridinemethanol, to obtain compound 9 (21 mg); MS: m/z 522.8 [M+1] ⁺ . ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.55 (d, J = 4.6 Hz, 1H), 8.12 (d, J = 8.9 Hz, 1H), 8.05 (d, J = 8.8 Hz, 2H), 7.83 (td, J = 7.7, 1.8 Hz, 1H), 7.74 (d, J = 8.9 Hz, 1H), 7.53 (d, J = 7.8 Hz, 1H), 7.36–7.28 (m, 1H), 6.74 (d, J = 8.8 Hz, 2H ),4.96(m,J＝4.8,2.8Hz,1H),4.90(s,2H),4.73(s,2H),3.71–3.49(m,3H),3.45(dd,J＝12.0 ,4.1Hz,1H),2.58(s,3H),2.52(s,3H),2.47(s,3H),2.40(s,1H),2.33(m,J=8.9,4.2Hz,1H). ¹³ C NMR (101MHz, DMSO-d ₆ )δ164.70,160.05,157.85,157.73,157.13,151.96,148.93,148.50,139.99,138.89,136.79,127.79,126.1 8,122.95,122.68,122.65,121.42,111.86,82.77,73.02,71.31,51.97,45.64,31.23,20.66,14.73,11.97.

Example 10: Preparation of Compound 10 ((R)-2,5,7-trimethyl-6-((1-(4-(6-((pyridin-3-ylmethoxy)methyl)pyridazin-3-yl)phenyl)pyrrolidin-3-yl)oxy)-[1,2,4]triazolo[1,5-a]pyrimidine)

The synthesis method was the same as Example 5, except that 3-hydroxytetrahydrofuran was replaced with 3-pyridinemethanol, to obtain compound 10 (120 mg); MS: m/z 522.8 [M+1] ⁺ . ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.61 (d, J = 2.1 Hz, 1H), 8.53 (dd, J = 4.8, 1.7 Hz, 1H), 8.12 (d, J = 8.9 Hz, 1H), 8.05 (d, J = 8.8 Hz, 2H), 7.82 (m, J = 7.9, 2.0 Hz, 1H), 7.71 (d, J = 8.9 Hz, 1H), 7.41 (m, J = 7.8, 4.8 Hz, 1H), 6.74 (d, J = 8. 8Hz,2H),4.97(m,J＝3.2,2.4Hz,1H),4.84(s,2H),4.68(s,2H),3.71–3.49(m,3H),3.45(m,J＝12 .0,4.1Hz,1H),2.58(s,3H),2.52(s,3H),2.47(s,3H),2.40(s,1H),2.33(m,J＝8.9,4.2Hz,1H). ¹³ C NMR (101MHz, DMSO-d ₆ )δ164.73, 160.09, 157.88, 157.11, 151.99, 149.00, 148.92, 148.53, 140.04, 138.91, 135.58, 133.47, 127.8 1,126.26,123.52,122.99,122.65,111.89,82.78,71.09,69.50,51.97,45.65,31.24,20.66,14.73,11.98.

Example 11: Preparation of Compound 11 ((R)-2-(((6-(4-(3-((2,5,7-trimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)oxy)pyrrolidin-1-yl)phenyl)pyridazin-3-yl)methoxy)methyl)oxazole)

The synthesis method was the same as Example 5 except that 3-hydroxytetrahydrofuran was replaced by 2-hydroxymethyloxazole to obtain compound 11 (50 mg); MS: m/z 513.1 [M+1] ⁺ . ¹ H NMR (400 MHz, DMSO-d ₆ )δ8.18–8.09(m,2H),8.05(d,J＝8.6Hz,2H),7.66(d,J＝8.9Hz,1H),7.25(d,J＝ 0.8Hz,1H),6.74(d,J＝8.6Hz,2H),4.97(s,1H),4.85(s,2H),4.74(s,2H),3.66 (m,J＝8.8,8.3Hz,1H),3.61–3.50(m,2H),3.45(m,J＝12.1,4.0Hz,1H),2.58(s ,3H),2.52(s,3H),2.47(s,3H),2.44–2.38(m,1H),2.32(m,J＝8.8,4.4Hz,1H). ¹³ C NMR (101MHz, DMSO-d ₆ )δ164.73,160.28,160.10,157.91,156.67,151.98,148.55,140.55,140.04,138.91,127.82,127.2 1,126.24,122.95,122.60,111.89,82.78,71.29,63.89,51.97,45.65,31.23,20.66,14.73,11.99.

Example 12: Preparation of Compound 12 ((R)-2,5,7-trimethyl-6-((1-(4-(6-((oxetane-3-oxy)methyl)pyridazin-3-yl)phenyl)pyrrolidin-3-yl)oxy)-[1,2,4]triazolo[1,5-a]pyrimidine)

The synthesis method was the same as Example 5, except that 3-hydroxytetrahydrofuran was replaced by 3-hydroxyoxetane, to give Compound 12 (31 mg); MS: m/z 488.1 [M+1] ⁺ . ¹ H NMR (400MHz, DMSO-d ₆ )δ8.17–8.08(m,1H),8.05(d,J＝8.5Hz,2H),7.70(d,J＝8.9Hz,1H),6.75(d,J＝8.6Hz,2H),4.71(m,J＝5.9Hz,4H),4.50– 4.41(m,2H),3.75–3.53(m,5H),3.45(m,J＝12.0,4.1Hz,1H),2.58(s,3H),2.52(s,3H),2.47(s,3H),2.42–2.27(m,2H). ¹³ C NMR (101MHz, DMSO-d ₆ )δ164.70,160.15,157.77,157.34,151.99,148.65,140.05,138.96,127.83,126.68,123.36, 122.37,111.88,82.71,77.48,72.07,69.47,52.91,45.66,31.25,21.83,20.66,14.74,11.99.

Example 13: Preparation of Compound 13 ((R)-6-((1-(4-(6-(cyclobutyloxymethyl)pyridazin-3-yl)phenyl)pyrrolidin-3-yl)oxy)-2,5,7-trimethyl-[1,2,4]triazolo[1,5-a]pyrimidine)

The synthesis method was the same as Example 5, except that 3-hydroxytetrahydrofuran was replaced by hydroxycyclobutane, to give compound 13 (200 mg); MS: m/z 485.8 [M+1] ⁺ . ¹ H NMR (400 MHz, DMSO-d ₆ ) δ8.14 (d, J=8.9 Hz, 1H), 8.05 (d, J=8.6 Hz, 2H), 7.68 (d, J=8.9 Hz, 1H), 6.75 (d, J=8.7 Hz, 2H), 4.97 (s, 1H), 4.64 (s, 2H), 4.07 (p, J=7.3 Hz, 1H), 3.66 (m, J=9.4, 6.9 Hz, 1H), 3.62–3.52 (m, 2H), 3.45 (m, J=12 .1,4.0Hz,1H),2.58(s,3H),2.52(s,3H),2.47(s,3H),2.42-2.38(m,1H),2.32(m,J＝8.9,4.5Hz,1H), 2.23–2.11(m,2H),1.90(m,J＝12.4,10.1,8.1Hz,2H),1.64(m,J＝11.4,8.6Hz,1H),1.50–1.42(m,1H). ¹³ C NMR (101MHz, DMSO-d ₆ )δ164.69,160.12,157.67,157.44,151.96,148.62,140.06,138.92,127.89,126.65,123.34, 122.27,111.91,82.77,72.64,68.30,51.97,45.66,31.23,29.98,20.66,14.72,12.08,11.98.

Example 14: Preparation of Compound 14 (2,5,7-trimethyl-6-(((3R)-1-(4-(6-((2-(tetrahydrofuran-3-yl)ethoxy)methyl)pyridazin-3-yl)phenyl)pyrrolidin-3-yl)oxy)-[1,2,4]triazolo[1,5-a]pyrimidine)

The synthesis method was the same as Example 5, except that 3-hydroxytetrahydrofuran was replaced by 2-(tetrahydrofuran-3-yl)ethanol, to give compound 14 (27 mg); MS: m/z 530.1 [M+1] ⁺ . ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.28 (d, J = 9.2 Hz, 1H), 8.06 (d, J = 8.9 Hz, 2H), 7.79 (d, J = 8.9 Hz, 1H), 6.77 (d, J = 9.0 Hz, 2H), 4.98 (q, J = 3.3, 2.2 Hz, 1H), 4.75 (s, 2H), 3.79 (m, J = 8.2, 7.2 Hz, 1H), 3.73–3.54 (m, 7H), 3.46 (m, J = 12 .2,4.0Hz,1H),3.24(m,J＝7.8Hz,1H),2.58(s,3H),2.52(s,3H),2.47(s,3H),2.44–2.29(m,2H), 2.23(p,J＝7.4Hz,1H), 1.98(m,J＝12.3,7.5,4.8Hz,1H), 1.66(m,J＝6.7Hz,2H), 1.54–1.41(m,1H). ¹³ C NMR (101MHz, DMSO-d ₆ )δ164.53,160.24,157.57,157.43,151.81,148.99,140.14,138.98,128.26,124.87,120.96,119.32,11 2.02,82.75,72.42,70.80,69.48,66.83,51.99,45.71,36.02,32.59,31.89,31.22,20.67,14.66,11.98.

Example 15: Preparation of Compound 15 (2,5,7-trimethyl-6-(((3R)-1-(4-(6-(((1-methylpyrrolidin-3-yl)oxy)methyl)pyridazin-3-yl)phenyl)pyrrolidin-3-yl)oxy)-[1,2,4]triazolo[1,5-a]pyrimidine)

The synthesis method was the same as Example 5 except that 3-hydroxytetrahydrofuran was replaced by 3-hydroxy-1-methyltetrahydropyrrole to obtain compound 15 (78 mg); MS: m/z 515.1 [M+1] ⁺ . ¹ H NMR (400 MHz, DMSO-d ₆ )δ8.16(d,J＝8.9Hz,1H),8.06(d,J＝8.9Hz,2H),7.74(d,J＝8.9Hz,1H),6.75(d,J ＝9.0Hz,2H),4.98(s,1H),4.80(s,2H),4.48-4.44(m,1H),3.79(d,J＝11.6Hz,1H ),3.67–3.55(m,3H),3.45(m,J＝12.1,4.0Hz,1H),3.33-3.00(m,3H),2.87(s,3H ),2.58(s,3H),2.52(s,3H),2.47(s,3H),2.42–2.24(m,3H),2.12-2.08(m,1H). ¹³ C NMR (101MHz, DMSO-d ₆ )δ164.69, 160.15, 158.41, 157.87, 151.95, 148.64, 140.08, 138.92, 132.04, 127.93, 126.68, 122.33 , 111.91, 82.78, 77.26, 69.38, 59.65, 53.99, 51.97, 45.68, 40.57, 31.24, 30.08, 20.66, 14.72, 11.99.

Example 16: Preparation of Compound 16 ((R)-6-((1-(4-(6-((cyclohexyloxy)methyl)pyridazin-3-yl)phenyl)pyrrolidin-3-yl)oxy)-2,5,7-trimethyl-[1,2,4]triazolo[1,5-a]pyrimidine)

The synthesis method was the same as Example 5, except that 3-hydroxytetrahydrofuran was replaced by hydroxycyclohexane, to give Compound 16 (58 mg); MS: m/z 514.1 [M+1] ⁺ . ¹ H NMR (400MHz, DMSO-d ₆ )δ8.14(d,J＝8.9Hz,1H),8.04(d,J＝8.4Hz,2H),7.68(d,J＝9.1Hz,1H),6.75(d,J＝8.4Hz,2H),4.97(s,1H),4.77(s,2H),3.72–3. 50(m,3H),3.50–3.39(m,2H),2.58(s,3H),2.53,(s,3H),2.47(s,3H),2.42–2.28(m,2H),1.96–1.85(m,2H),1.41–1.20(m,8H). ¹³ C NMR (101MHz, DMSO-d ₆ )δ164.68,160.12,157.99,156.15,151.76,148.65,140.38,138.62,127.87,126.44,123.43,122 .27,111.90,82.78,79.66,70.38,51.96,45.67,31.68,23.37,22.17,21.71,20.66,14.72,11.98.

Example 17: Preparation of Compound 17 ((R)-6-((1-(4-(6-((cyclooctyloxy)methyl)pyridazin-3-yl)phenyl)pyrrolidin-3-yl)oxy)-2,5,7-trimethyl-[1,2,4]triazolo[1,5-a]pyrimidine)

The synthesis method was the same as Example 5, except that 3-hydroxytetrahydrofuran was replaced by hydroxycyclooctane, to give Compound 17 (13 mg); MS: m/z 542.1 [M+1] ⁺ . ¹ H NMR (400MHz, DMSO-d ₆ )δ8.19(d,J＝8.9Hz,1H),8.04(d,J＝8.6Hz,2H),7.72(d,J＝9.0Hz,1H),6.75(d,J＝8.7Hz,2H),4.97(s,1H),4.74(s,2H),3.72–3.50(m,4H),3 .45(dd,J＝12.1,4.0Hz,1H),2.58(s,3H),2.52(s,3H),2.47(s,3H),2.41-2.31(m,2H), 1.69(m,J＝14.1,8.4,6.0Hz,5H),1.57–1.38(m,9H). ¹³ C NMR (101MHz, DMSO-d ₆ )δ164.64,160.14,157.99,157.34,151.76,148.75,140.08,138.92,128.03,126.65,123.34,122.27 ,111.95, 82.75, 79.01, 68.42, 51.98, 45.66, 31.22, 30.74, 26.89, 24.79, 22.33, 20.66, 14.70, 11.98.

Activity test

Experimental Example 1: MCT4 lactate efflux assay

I. Experimental Methods

1. SKBr3 cells (ATCC, Cat#HTB-30) were digested and harvested, resuspended in McCoy's 5A medium (Gibco) supplemented with 10% FBS (AusGeneX, Cat#FBS500-S), and counted. Cells were then plated at 4000 cells/well in a 384-well cell culture plate (Greiner). 5 μL of the test compound diluted in Opti-MEM (Invitrogen, Cat#31985070) was then added to each well. The final concentrations of the test compound were 500 nM, 100 nM, 20 nM, 4 nM, 0.8 nM, 0.16 nM, 0.032 nM, 0.0064 nM, and 0.00128 nM. A control group (5 μL of Opti-MEM) and a positive drug group (AZD0095 at the same concentration series as the test compound) were set up. The cell culture plate was incubated at 37°C, 5% CO2 for ₂ h.

2. The cell culture plate was removed, and the supernatant was diluted with D-PBS (Solarbio). Then, 5 μL of the diluted supernatant was added to a new 384-well cell culture plate (Greiner), and the prepared lactate detection reagent (Lactate-Glo ^™ Assay, Promega) was added thereto.

3. Shake the plate on a plate shaker for 30-60 seconds to mix. Then, incubate the plate in the dark at room temperature for 60 minutes.

4. After standing, read the luminescence value using a microplate reader.

II. The inhibition rate is calculated as follows:

Inhibition rate (%) = (fluorescence value of the control group - fluorescence value of the test compound group) / (fluorescence value of the control group) × 100%

III. Calculation of compound _IC50 using GraphPad nonlinear fitting formula

The log value of compound concentration was used as the X-axis and Activity% as the Y-axis. The log(agonist) vs. response—Variable slop analysis software GraphPad was used to fit the dose-effect relationship, thereby obtaining the inhibitory activity of each compound on MCT4.

The fitting formula is: Y = Bottom + (Top-Bottom) / (1 + 10^((LogIC50-X)*HillSlope))

Among them: Top represents the top platform, and the Top standard of the curve is generally between 80% and 120%; Bottom represents the bottom platform, and the Bpttom of the curve is generally between -20% and 20%.

The results of the in vitro activity test are shown in Table 1 below:

Table 1

The above experimental results show that the compounds of the embodiments of the present disclosure have strong inhibitory activity on MCT4 lactate efflux.

(AZD0095 comes from the literature Journal of Medicinal Chemistry, 2023, 66, 1, 384-397).

Experimental Example 2: MCT1 lactate influx assay

I. Experimental Procedure

1. K562 cells (ATCC, CCL-243) were digested and harvested, resuspended in IMDM medium (Gibco), and counted. The cells were then plated at 50,000 cells/well in a 384-well cell culture plate (Greiner). 5 μL of test compound diluted in Opti-MEM (Invitrogen, Cat# 31985070) was then added to each well to final concentrations of 10 μM, 2.5 μM, 0.625 μM, 0.156 μM, 0.039 μM, 9.8 nM, 2.4 nM, 0.60 nM, 0.15 nM, and 0.038 nM. A blank control was set up in wells containing only cells. The cell culture plates were incubated at 37°C, 5% _CO₂ for 1 h.

2. Remove the cell culture plate, remove the supernatant and wash with D-PBS (Solarbio). After washing, add 20 μL of cell lysis buffer (Sigma-Aldrich, C2360) to a 96-well cell culture plate (PerkinElmer), centrifuge, and place at 4°C for 30 minutes for lysis.

3. Remove the cell culture plate, add 5 μL of supernatant diluted with D-PBS (Solarbio) into a new 384-well cell plate, and add the prepared lactate detection reagent (Lactate-Glo™ Assay, Promega).

4. Incubate the tube in the dark at room temperature for 60 minutes. After the incubation period, read the luminescence value using a microplate reader.

II. The inhibition rate is calculated as follows:
Inhibition rate (%) = (fluorescence value of the test compound group - fluorescence value of the blank control group) / (fluorescence value of the test compound group) × 100%

III. Calculation of compound IC50 using GraphPad nonlinear fitting formula

The log value of compound concentration was used as the X-axis and Activity% as the Y-axis. The log(agonist) vs. response—Variable slop analysis software GraphPad was used to fit the dose-effect relationship, thereby obtaining the inhibitory activity of each compound on MT1.

The fitting formula is: Y = Bottom + (Top-Bottom) / (1 + 10^((LogIC50-X)*HillSlope))

Among them: Top represents the top platform, and the Top standard of the curve is generally between 80% and 120%; Bottom represents the bottom platform, and the Bpttom of the curve is generally between -20% and 20%.

The results of the in vitro activity test are shown in Table 2 below:

Table 2

The experimental results in Table 2 above show that the compounds of the present invention have weak inhibitory activity on the lactate influx of MCT1, indicating that the compounds of the present invention have weak selectivity for MCT1. Compared with MCT1, the compounds of the present invention have high selectivity for MCT4 and can specifically target the MCT4 target, so they are safe.

Experimental Example 3: Study on the metabolic stability of compounds in human liver microsomes

1. Experimental Materials

Human liver microsomes, catalog number 452117, were purchased from Corning;

NADPH, product number BD11658, was purchased from Bidler.

2. Experimental Methods

Microsomes (20 mg/mL) were stored in a -80°C freezer, thawed in a 37°C water bath before use, and then placed on ice.

To a 96-well plate, 10 μL of microsomes (20 mg/mL, final concentration 0.5 mg/mL), 200 μL of phosphate buffer (200 mM, final concentration 100 mM), 40 μL of magnesium chloride (50 mM, final concentration 5 mM), and 106 μL of purified water were added. The system was preincubated in a 37°C water bath for 10 minutes. 40 μL of NADPH solution (10 mM, final concentration 1 mM) was added to the reaction system; 40 μL of ultrapure water was used in place of the NADPH solution as a negative control.

The reaction was initiated by adding 4 μL of 100 mM test compound and control drug (Verapamil) to the reaction, and the final concentration of the drug was 1 μM.

At 0, 5, 15, 30, and 60 min, 50 μL of the reaction sample was removed and quenched with four volumes of cold acetonitrile containing internal standards (3% formic acid, 100 nM alprazolam, 200 nM labetalol, 2 μM ketoprofen, and 200 nM caffeine). The sample was centrifuged at 3220 g for 45 min. After centrifugation, 100 μL of the supernatant was mixed with 100 μL of ultrapure water for LC-MS/MS analysis.

3. Data Analysis

The peak area was determined by extracting ion chromatograms, and the in vitro half-life (t _1/2 ) of the parent drug was determined by linear fitting the disappearance percentage of the parent drug with time.

The in vitro half-life (t _1/2 ) was calculated from the slope:
In vitro t _1/2 = 0.693/k (k = - slope value)

The in vitro t _1/2 was converted to in vitro clearance (CL _int ) (μL/min/mg protein) using the following formula:

The incubation volume was 400 μL and the protein amount was 0.2 mg.

4. Conclusion

The results in Table 3 show that the compounds disclosed herein have a low clearance rate in human liver microsomes, indicating that the compounds have good stability.

Table 3

Experimental Example 4: Plasma kinetic study of the compound in CD-1 mice

1. Experimental Animals

CD-1 mice, SPF grade, male, were purchased from Sibeifu (Beijing) Biotechnology Co., Ltd.

2. Experimental Methods

The pharmacokinetic characteristics of the compounds in rodents were tested following oral and intravenous administration using standard protocols.

In the experiments, the candidate compounds were formulated into clear solutions and administered orally and intravenously to mice in a vehicle of 5% DMSO, 45% PEG400, and 50% acetate buffer, pH 4.5. Six mice were administered each compound orally and intravenously, with an oral dose of 10 mg/kg (1 mg/mL concentration in a 10 μL/g volume) and an intravenous dose of 3 mg/kg (0.3 mg/mL concentration in a 10 μL/g volume).

Sampling was performed at 0.0833 h, 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, and 24 h after administration. Whole blood samples were immediately placed on ice after collection and centrifuged at 4 °C and 2000 g for 5 min within 0.5 h after collection to separate the plasma. The upper layer of the sample was collected into a sample tube, frozen in a -10 to -30 °C refrigerator within 0.5 h, and transferred to a -60 to -90 °C refrigerator within 24 h.

3. Data Analysis

The concentration of each compound in plasma samples at each time point was determined by LC-MS/MS. PK parameters including plasma clearance (CL), time to peak clearance (T _max ), half-life (t _1/2 ), peak concentration (C _max ), area under the concentration-time curve (AUC _0-t ), tissue distribution volume (Vd), mean residence time (MRT _0-t ) and bioavailability (F) were calculated using WinNonlin software.

4. Experimental Results

The pharmacokinetic test results of the compounds disclosed herein are shown in the following table.

Table 4. Pharmacokinetic experimental results of the compounds disclosed herein

As shown in Table 4, the compounds of the present disclosure have the characteristics of low plasma clearance, long drug half-life, good exposure and high bioavailability, and have good pharmacokinetic properties.

Experimental Example 5: Plasma kinetic study of the compound in SD rats

1. Experimental Animals

SD rats, SPF grade, male, were purchased from Sibeifu (Beijing) Biotechnology Co., Ltd.

2. Experimental Methods

The pharmacokinetic characteristics of the compounds in rodents were tested following oral and intravenous administration using standard protocols.

In the experiment, the candidate compounds were formulated into clear solutions and administered orally and intravenously to rats in a single dose in a pH 4.5 acetate buffer solution containing 5% DMSO, 45% PEG400, and 50% water. Six rats were administered each compound orally and intravenously at doses of 10 mg/kg (1 mg/mL, 10 μL/g) and 50 mg/kg (5 mg/mL, 10 μL/g) for oral administration; and 3 mg/kg (0.3 mg/mL, 10 μL/g) for intravenous administration for intravenous administration.

Blood samples were collected at 0.0833 h, 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, and 24 h after administration. Whole blood samples were immediately placed on ice after collection and centrifuged at 4 ° C and 2000 g for 5 min within 0.5 h after collection to separate the plasma. The upper layer sample was collected into a sample tube, frozen in a -10 to -30 ° C refrigerator within 0.5 h, and transferred to a -60 to -90 ° C refrigerator within 24 h.

3. Data Analysis

The concentration of each compound in plasma samples at each time point was determined by LC-MS/MS. PK parameters including plasma clearance (CL), time to peak clearance (T _max ), half-life (t _1/2 ), peak concentration (C _max ), area under the concentration-time curve (AUC _0-t ), tissue distribution volume (Vd), mean residence time (MRT _0-t ) and bioavailability (F) were calculated using WinNonlin software.

4. Experimental Results

The pharmacokinetic test results of the compounds disclosed herein are shown in Table 5 below.

Table 5. Pharmacokinetic experimental results of the compounds disclosed herein

As shown in Table 5, compound 6 of the present disclosure has the characteristics of low plasma clearance rate, long drug half-life, good exposure and high bioavailability in rats, and has good pharmacokinetic properties.

Experimental Example 6: Plasma kinetic study of the compound in cynomolgus monkeys

1. Experimental Animals

Cynomolgus macaque, SPF grade, male.

2. Experimental Methods

The compounds were tested for pharmacokinetic properties in non-rodent animals following oral and intravenous administration using standard protocols.

In the experiment, the candidate compounds were formulated into clear solutions and administered orally and intravenously to cynomolgus monkeys in a single dose in 5% DMSO, 45% PEG400, and 50% water in a pH 4.5 acetate buffer. Three cynomolgus monkeys received each compound orally and intravenously at doses of 10 mg/kg (1 mg/mL, 10 μL/g) and 50 mg/kg (5 mg/mL, 10 μL/g) for oral administration; and 3 mg/kg (0.3 mg/mL, 10 μL/g) for intravenous administration for intravenous administration.

Blood samples were collected at 0.0833 h, 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, and 24 h after administration. Whole blood samples were immediately placed on ice after collection and centrifuged at 4 ° C and 2000 g for 5 min within 0.5 h after collection to separate the plasma. The upper layer sample was collected into a sample tube, frozen in a -10 to -30 ° C refrigerator within 0.5 h, and transferred to a -60 to -90 ° C refrigerator within 24 h.

3. Data Analysis

The concentration of each compound in plasma samples at each time point was determined by LC-MS/MS. PK parameters including plasma clearance (CL), time to peak clearance (T _max ), half-life (t _1/2 ), peak concentration (C _max ), area under the concentration-time curve (AUC _0-t ), tissue distribution volume (Vd), mean residence time (MRT _0-t ) and bioavailability (F) were calculated using WinNonlin software.

4. Experimental Results

The pharmacokinetic test results of the compounds disclosed herein are shown in Table 6 below.

Table 6. Pharmacokinetic experimental results of the compounds disclosed herein

As shown in Table 6, compound 6 of the present disclosure has the characteristics of low plasma clearance, long drug half-life, good exposure and high bioavailability in cynomolgus monkeys, and has good pharmacokinetic properties.

Experimental Example 7: In vivo pharmacodynamic study of the disclosed compounds in a human non-small cell lung cancer NCI-H358 subcutaneous xenograft tumor model

1. Experimental Animals

NCG mice, SPF grade, female, were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.

2. Experimental Methods

NCI-H358 tumor cells (5×10 ⁷ /mL, 100 μL/mouse) in the logarithmic growth phase were inoculated subcutaneously into 6-8 week-old female NCG nude mice (weighing approximately 20 g). Mice were maintained in an SPF-grade laboratory environment and had free access to a commercially certified standard diet.

When the average tumor volume of mice grew to approximately 130 mm ³ , they were divided into groups of 6 and orally administered daily with the test compound (the drug solvent was 5% DMSO + 5% Tween 80 + 40% PEG400 + 50% water) (see Table 7 below). AZD0095 was used as the positive drug. Simultaneously, a blank group was established, in which mice were administered only the same volume of the vehicle.

Tumor volume was measured every two days using a two-dimensional caliper and animals were weighed every day. After 21 consecutive days of administration, the inhibition rate (TGI, %) was calculated based on the final tumor volume.

The tumor volume was calculated as follows:
V = 1/2 × a × b ² ;

Where: a represents the long diameter of the tumor, and b represents the short diameter of the tumor.

Table 7. In vivo pharmacodynamics test results of the compounds disclosed herein

As shown in Table 7, compounds 5 and 6 of the present disclosure have good in vivo efficacy.

Experimental Example 8: Pharmacodynamic Study of the Disclosed Compounds in Mouse Asthma Model

1. Purchase and breeding of animals

C57BL/6j mice (male, 9–10 weeks old) were purchased from a certified animal supplier and acclimated for 1 week prior to the experiment. Throughout the experimental period, animals were housed in reinforced and ventilated cages. Animal cages were changed at least weekly. Animals were housed in groups of five under a normal 12-h light cycle (lights off at 8:00 p.m.), at 22 ± 2°C, and at a relative humidity of 50 ± 10%, with food and water provided. All in vivo experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the Institute of Laboratory Animals, Chinese Academy of Medical Sciences.

2 Construction of animal model

Mice were weighed and divided into groups of 5 per group. Mice were sensitized (25 μg house dust mite (HDM) protein) on days 0 and 7 and challenged (25 μg HDM protein) on day 14. All HDM administration was intratracheal (i.t.).

3. Group setting and drug administration in animal models

The animal dosing groups are shown in Table 8:

Table 8

4Test results

(1) Penh test: On the 14th day, Penh index was tested 2 hours after stimulation with 25 μg of house dust mite protein HDM. Compared with the model group (G1 group), the compound 6 (10 mpk, BID) and dexamethasone (2.5 mpk, QD) administration groups (respectively referred to as G2 group and G3 group) significantly reduced Penh values (as shown in Figure 1), indicating that compound 6 can significantly improve airway hyperresponsiveness. The scoring results are shown in Table 9 below:

Table 9

(2) HE staining: On the 17th day, the left lung tissue of the mice was collected and fixed for staining. Compared with the model group, the compound 6 (10 mpk, BID) and dexamethasone (2.5 mpk, QD) administration groups significantly improved the HE staining results (as shown in Figure 2), indicating that compound 6 can significantly improve inflammation. The scoring results are shown in Table 10 below:

Table 10

(3) Airway wall thickness test: On day 17, the left lung tissue of the mice was collected, fixed and stained. Compared with the model group, the compound 6 (10 mpk, BID) and dexamethasone (2.5 mpk, QD) administration groups both significantly improved airway wall thickness (as shown in Figure 3), indicating that compound 6 can significantly improve asthma symptoms. The results of the airway wall thickness test are shown in Table 11 below:

Table 11

The results of the asthma model experiment showed that the disclosed compound 6 can effectively improve the asthma symptoms of animals.

Experimental Example 9: Pharmacodynamic Study of the Disclosed Compounds in a Mouse Idiopathic Pulmonary Fibrosis Model

1. Purchase and breeding of animals

C57BL/6j mice (male, 9–10 weeks old) were purchased from a certified animal supplier and acclimated for 1 week prior to the experiment. Throughout the experimental period, animals were housed in reinforced and ventilated cages. Animal cages were changed at least weekly. Animals were housed in groups of five under a normal 12-h light cycle (lights off at 8:00 p.m.), at 22 ± 2°C, and at a relative humidity of 50 ± 10%, with food and water provided. All in vivo experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the Institute of Laboratory Animals, Chinese Academy of Medical Sciences.

2 Construction of animal model

The mice were divided into groups according to body weight, with 5 mice in each group. The mice were given bleomycin (0.66 mg/kg) from day 1 to day 7 to establish the model, and the above administration method was intratracheal (i.t.).

3. Group setting and drug administration in animal models

The animal dosing groups are shown in Table 12:

Table 12

4Test results

(1) HE staining score: On day 21, the left lung tissue of the mice was collected, fixed and stained. Compared with the model group (G1 group), compound 6 (10 mpk, BID) (G2 group) significantly improved the HE staining results (as shown in Figure 4), indicating that compound 6 can significantly improve inflammation. The scoring results are shown in Table 13 below:

Table 13

(2) Masson staining score: On day 21, the left lung tissue of the mice was collected and fixed for staining. Compared with the model group, the compound 6 (10 mpk, BID) administration group significantly improved the Masson staining score of the lung tissue (as shown in Figure 5), indicating that compound 6 can significantly improve lung tissue fibrosis. The scoring results are shown in Table 14 below:

Table 14

(3) α-SMA score: On day 21, the left lung tissue of the mice was collected, fixed and stained. Compared with the model group, the compound 6 (10 mpk, BID) administration group significantly improved the α-SMA score (as shown in Figure 6), indicating that compound 6 can significantly improve lung tissue fibrosis. The scoring results are shown in Table 15 below:

Table 15

The results of the idiopathic pulmonary fibrosis animal model experiment showed that the disclosed compound 6 can effectively improve idiopathic pulmonary fibrosis in animals.

Experimental Example 10: Pharmacodynamic Study of the Disclosed Compounds in a Mouse Chronic Obstructive Pulmonary Disease Model

This study used a mouse chronic obstructive pulmonary disease (COPD) model induced by short-term smoke exposure combined with lipopolysaccharide (LPS) to simulate the inflammatory changes characteristic of acute exacerbation of COPD.

1 Animal acclimation and grouping

After the mice were adapted to feeding, they were randomly divided into 5 groups according to their body weight on D0 (day 0), with 8 mice in each group.

2 Modeling plan

Mice in group G1 served as healthy controls and were exposed to room air from D1 to D35.

Mice in the G2-G5 groups were exposed to smoke twice daily (>3 hours apart) for 40 minutes each time on Days 1-D14, 16-D28, and 30-D35. On Days 15 and 29, 50 μL of 0.45 mg/mL LPS was injected into the airways. The experiment was terminated on Day 36.

3. Group setting and drug administration in animal models

The animal dosing groups are shown in Table 16:

Table 16

Solvent: 5% DMSO+45% PEG400+50% saline.

4Test results

(1) Penh test: On day 35, mice were placed in the WBP plethysmography chamber for Penh testing. Compared with the model group (G2 group), compound 6 and roflumilast-treated groups (G3 and G4 groups) significantly reduced Penh values (Figure 7), indicating that compound 6 can significantly improve airway hyperresponsiveness. The scoring results are shown in Table 17 below:

Table 17

(2) HE staining lung injury score: On day 36, the left lung tissue of the mice was collected and fixed for staining. Compared with the roflumilast-treated group (Group G4) and the ensifentrine-treated group (Group G5), Compound 6 (Group G3) had a better improvement on inflammation (Figure 8), indicating that Compound 6 is superior in improving lung injury. The scoring results are shown in Table 18 below:

Table 18

Experimental Example 11: Toxicity Dose Exploration Test

Twenty-one SD rats were randomly divided into three dose groups according to body weight, namely, vehicle control group, low-dose compound 6 group, and high-dose compound 6 group (dosage was 0 (i.e., only an equal volume of vehicle was administered), 200 mg/kg/day, and 300 mg/kg/day, twice a day), and divided into toxicity test group and toxicity satellite group. There were 2 rats/group/sex in the toxicity test group; there were 3 males/group in the toxicity satellite group. Both the low-dose compound 6 and high-dose compound 6 groups were orally gavaged with the corresponding concentration of compound 6 preparation (prepared with the following solvent: 5% DMSO + 5% Tween 80 + 40% PEG400 + 50% water, pH 4.5 acetate buffer) at a dosing volume of 10 mL/kg, and the vehicle control group was given the vehicle (the acetate buffer) at the same dosing volume. The drugs were administered twice a day for 14 consecutive days (28 times in total), and the day of the first administration was defined as the first day of the experiment (D1).

During the experiment, the general condition of the animals in each group was observed every day, and their body weight was measured twice a week. The food intake of the animals in the toxicity test group was measured once a week. At the end of administration (D15), the hematology, coagulation, and blood biochemistry of the animals in the toxicity test group were tested, and the animals in each administration group were dissected and euthanized for gross anatomical observation, organ weighing, and histopathological examination.

Blood was collected from the animals in the low-dose and high-dose compound 6 groups before the first and last doses, and 0.5 h, 1 h, 2 h, 4 h, 4.5 h, 5 h, 8 h, and 24 h after administration. Blood was collected from the animals in the vehicle control group before the first and last doses, and 1 h after administration. The plasma concentration of compound 6 was measured, and toxicokinetic parameters such as C _max , T _max , and AUC _last were calculated using Phoenix WinNonlin.

Under the experimental conditions, SD rats were orally gavaged twice daily for 2 consecutive weeks (28 doses total) at doses of 200 mg/kg/day and 300 mg/kg/day of Compound 6. No animals in the groups experienced moribundity, death, or other severe toxic reactions. No significant abnormal changes in the animals' general condition, body weight, food intake, hematology, coagulation, blood biochemistry, organ weights, or coefficients at any test point during the experiment were observed, indicating toxicological significance related to Compound 6.

The toxicokinetic results showed that after the first and last administration, the AUC _last and C _max of compound 6 in SD rats increased with the increase of the administration dose.

The tissue distribution results showed that at the end of the dosing period, the concentrations of compound 6 in the liver, kidney, colon, pancreas, lung, heart, spleen and stomach of SD rats increased with increasing dose, and the distribution order was: liver > colon > pancreas > stomach > kidney > lung > heart > spleen.

Gross autopsy showed that there were no obvious test substance-related histopathological changes in the heart, liver, spleen, lung, kidney, stomach and pancreas of the animals in the high-dose group of compound 6 in the toxicity test group of this experiment.

In summary, after SD rats were orally gavaged with compound 6 at doses of 200 mg/kg/day and 300 mg/kg/day twice a day for 2 consecutive weeks (28 times in total), their maximum tolerated dose (MTD) was > 600 mg/kg/day.

The above embodiments of the present disclosure are merely examples for the purpose of clearly illustrating the present disclosure, and are not intended to limit the embodiments of the present disclosure. For those skilled in the art, other variations or modifications in different forms can be made based on the above description. It is not necessary and impossible to list all the embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present disclosure shall be included within the scope of protection of the claims of the present disclosure.

Claims (15)

A compound of formula (III) or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, isotope-labeled substance or prodrug thereof,
in, Y is a bond or (CH ₂ ) _n , where n is an integer selected from 1 to 3; Ring C is selected from C _4-8 cycloalkyl, 4-8 membered heterocycloalkyl containing 1-3 heteroatoms, phenyl, 5-8 membered heteroaryl containing 1-3 heteroatoms, wherein ring C is optionally substituted by one or more selected from halogen, amino, cyano, hydroxy, C _1-3 alkylhydroxy, amide, C _1-3 alkyl, C _1-3 alkoxy, C _1-3 haloalkoxy, and the heteroatom may be one or more of O, N or S. The compound according to claim 1 or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, isotope-labeled substance or prodrug thereof, wherein: Y is a bond or CH ₂ ; Ring C is selected from a 5-7 membered heterocycloalkyl group containing 1-2 heteroatoms, wherein the heteroatoms are one or more of O, N or S, and is optionally substituted by methyl, ethyl, n-propyl or isopropyl; or, ring C is a C _4-6 cycloalkyl group; or, ring C is selected from a phenyl group or a 5-6 membered heteroaryl group containing 1-2 heteroatoms, wherein the heteroatoms are one or more of O, N or S. The compound according to claim 1 or 2, or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, isotope-labeled substance, or prodrug thereof, wherein the compound is a compound of formula (IV) or formula (V):
The compound according to claim 1 or 3, or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, isotope-labeled substance or prodrug thereof, wherein: Y is a bond or (CH ₂ ) _n , where n is an integer selected from 1 to 3; Ring C is selected from C _4-6 cycloalkyl, 4-6 membered heterocycloalkyl containing 1-2 heteroatoms, phenyl, 5-6 membered heteroaryl containing 1-2 heteroatoms, wherein the heteroatoms are one or more of O, S or N, and the ring C is optionally substituted by C _1-3 alkyl. The compound according to any one of claims 1 and 3-4, or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, isotopically labeled substance, or prodrug thereof, wherein Y is a bond or (CH ₂ ) _n , and n is an integer selected from 1-3; Ring C is selected from cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, oxetanyl, azetidinyl, tetrahydrofuranyl, tetrahydrothiophenyl, tetrahydropyrrolyl, tetrahydropyranyl, piperidinyl, morpholinyl, piperazinyl, dioxanyl, phenyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, oxazolyl, pyrrolyl, furanyl or imidazolyl, and the ring C is optionally substituted with methyl, ethyl, n-propyl or isopropyl. The compound according to claim 5, or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, isotope-labeled substance, or prodrug thereof, wherein ring C is selected from cyclobutyl, cyclopentyl, cyclohexyl, oxetanyl, tetrahydrofuranyl, tetrahydrothiophenyl, tetrahydropyrrolyl, tetrahydropyranyl, phenyl, pyridyl, or oxazolyl, and the ring C is optionally substituted with methyl, ethyl, n-propyl, or isopropyl. The compound according to any one of claims 4 to 6, or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, isotope-labeled substance or prodrug thereof, wherein Y is a bond or CH ₂ . The compound according to any one of claims 1 to 4, or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, isotopically labeled substance, or prodrug thereof, wherein Y is a bond or CH ₂ ; Ring C is selected from 5-6 membered heterocycloalkyl containing 1-2 heteroatoms, wherein the heteroatoms are O, S or N, and the ring C is optionally substituted by methyl or ethyl. The compound according to any one of claims 1 to 8, or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, isotope-labeled substance, or prodrug thereof, wherein ring C is selected from oxetanyl, tetrahydrofuranyl, tetrahydrothienyl, tetrahydropyrrolyl, or tetrahydropyranyl, and the ring C is optionally substituted with a methyl group or an ethyl group. The compound according to any one of claims 1 to 9, or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, isotope-labeled substance or prodrug thereof, wherein: Y is a bond or CH ₂ ; Ring C is selected from tetrahydrofuranyl, tetrahydrothienyl or tetrahydropyrrolyl, said tetrahydropyrrolyl being optionally substituted with methyl or ethyl. The compound according to any one of claims 1 to 10, or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, isotope-labeled substance, or prodrug thereof, wherein the compound is:

A pharmaceutical composition comprising a compound of formula (III) according to any one of claims 1 to 11, or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, isotope-labeled substance or prodrug thereof, and optionally a pharmaceutically acceptable excipient. A compound of formula (III) according to any one of claims 1 to 11, or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, isotope-labeled substance or prodrug thereof, or a pharmaceutical composition comprising the same, for inhibiting MCT4 or for treating a disease or condition associated with abnormally elevated MCT4 levels. The compound of formula (III) for use according to claim 13, or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, isotope-labeled substance or prodrug thereof, or a pharmaceutical composition comprising the same, wherein the disease or condition associated with abnormally elevated MCT4 levels is cancer or tumor; or The diseases or conditions associated with abnormally elevated MCT4 levels are asthma, chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF). The compound of formula (III) for use according to claim 14, or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, isotope label or prodrug thereof, or a pharmaceutical composition comprising the same, wherein the cancer or tumor is selected from renal cell carcinoma (RCC), kidney cancer, gastric cancer, cervical cancer, non-small cell lung cancer, breast cancer, head and neck cancer, bladder cancer, non-Hodgkin's lymphoma and glioblastoma.