TW202535381A - A pyridothiophene-2-carboxylic acid derivative and its preparation method and use - Google Patents

Abstract

The present disclosure relates to a pyridinothiophene-2-carboxylic acid derivative and its preparation method and use, specifically relates to a compound as shown in formula (I) or its pharmaceutically acceptable salt, which has good inhibitory/degradation effect on BCKDK. The substituents in the formula are as defined in the specification.

Description

A pyridothiophene-2-carboxylic acid derivative, its preparation method and uses

This disclosure pertains to the field of pharmaceutical chemistry, specifically relating to a pyridothiophene-2-carboxylic acid derivative, its preparation method, and its uses.

Branched-chain ketoacid dehydrogenase kinase (BCKDK) is associated with a variety of human diseases. In addition to regulating the catabolism of branched-chain amino acids (BCAAs), BCKDK can enhance MEK/ERK signaling, which is associated with malignant proliferation in cancer.

BCAAs (leucine, isoleucine, and vortexin) are essential amino acids for the human body, accounting for approximately 40% of the essential amino acids required by healthy subjects. They must be obtained through a balanced diet and play a crucial role in nutrient sensing and cellular signaling.

While branched-chain amino acids (BCKAs) are toxic in excess, they are essential for protein synthesis and cellular signaling. BCKAs are converted to α-keto acid forms by branched-chain aminotransferases (BCAT): α-ketoisohexanoate (KIC/ketoleucine), 2-keto-3-methylvaleric acid (KMV/ketoisoleucine), and α-ketoisovaleric acid (KIV/ketovaleric acid). The BCKAs are then oxidatively decarboxylated by a branched-chain keto acid dehydrogenase (BCKDH) complex, which consists of multiple copies of the BCKDH E1a/p tetramer, BCKDH E2, and BCKDH E3 subunits. This complex is regulated by inhibitory phosphorylation, mediated by BCKDH kinase (BCKDK), which is dephosphorylated at the same phosphorylation site by the phosphatase PPM1K. Inhibition of complex phosphorylation promotes BCKDH activity, thereby promoting irreversible catabolism of BCKA (Lynch CJ, Adams SH: Branched-chain amino acids in metabolic signalling and insulin resistance. Nat Rev Endocrinol 2014, 10: 723-36.). The absence of BCKDK in mice confirms this regulation, as mice lacking BCKDK exhibit enhanced BCKDH activity in multiple tissues (Joshi MA, Jeoung NH, Obayashi M, Hattab EM, Brocken EG, Liechty EA, Kubek MJ, Vatem KM, Wek RC, Harris RA: Impaired growth and neurological abnormalities in branched-chain alpha-keto acid dehydrogenase kinase-deficient mice. Biochem J 2006, 400: 153-62.).

Inadequate metabolism of branched-chain amino acids (BCAAs) is associated with insulin resistance, diabetes, congenital heart disease, and heart failure. Circulating branched-chain amino acids and their breakdown products, branched-chain α-keto acids (BCKAs), are also indicators of diabetes development. The α-keto acid dehydrogenase complex (BCKDH) is the rate-limiting step in the breakdown and clearance of branched-chain amino acids; BCKDK phosphorylates the E1α subunit of BCKDH, thereby inhibiting its function.

In recent years, although some early studies related to BCKDK have been conducted, such as WO2020056155A, WO2020261144A, WO2020261205A, WO2022175959A, and WO2023100061A, there is still a need to develop novel and effective BCKDK inhibitors for the treatment of diseases or conditions associated with elevated BCAA levels.

This disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof,

^R1 , ^R2 , and ^R3 are each independently selected from hydrogen, fluorine, chlorine, or methyl;

The ^R4 is selected from fluorine or chlorine;

R ⁵ is selected from _C1-4 alkyl, halogenated _C1-4 alkyl, 3 to 4-membered cycloalkyl, -L1 ^-R9 _, where _L1 is selected from sulfur or oxygen, and R ⁹ is selected from _C1-4 alkyl, halogenated _C1-4 alkyl, deuterated _C1-4 alkyl, 3 to 4-membered cycloalkyl;

_X1 is selected from nitrogen or ^CR6 , and ^R6 is selected from hydrogen, fluorine, or chlorine;

^R7 and ^R8 are each independently selected from hydrogen, fluorine, and chlorine.

In some embodiments, _X1 is nitrogen in the compound of formula (I) or its pharmaceutically acceptable salt provided in this disclosure.

In some embodiments, the compound of formula (I) or its pharmaceutically acceptable salt provided in this disclosure is the compound of formula (II) or its pharmaceutically acceptable salt.

^R1 , ^R2 , ^R3 , ^R4 , ^R5 , ^R6 , ^R7 and ^R8 are defined as in equation (I).

In some embodiments, in the compounds of formulas (I) and (II) provided in this disclosure or their pharmaceutically acceptable salts, ^R5 is selected from _C1-4 alkyl or halogenated _C1-4 alkyl.

In alternative embodiments, in the compounds of formulas (I) and (II) provided in this disclosure or their pharmaceutically acceptable salts, ^R5 is selected from methyl, ethyl, or isopropyl.

In some embodiments, in the compounds of formulas (I) and (II) provided in this disclosure, or their pharmaceutically acceptable salts, ^R5 is selected from methyl or ethyl.

In some embodiments, in the compounds of formulas (I) and (II) provided in this disclosure, or their pharmaceutically acceptable salts, R ⁵ is selected from 3 to 4 cycloalkyl groups.

In an alternative embodiment, in the compounds of formulas (I) and (II) provided in this disclosure, or their pharmaceutically acceptable salts, R ⁵ is cyclopropyl.

In some embodiments, the compounds of formulas (I) and (II) provided in this disclosure, or their pharmaceutically acceptable salts, are the compounds of formula (II-1) or their pharmaceutically acceptable salts.

Among them, ^R1 , ^R2 , ^R3 , ^R4 , ^R6 , ^R7 , ^R8 , and ^R9 are defined as in equation (I).

In some embodiments, in the compounds of formulas (I), (II), (II-1) or their pharmaceutically acceptable salts provided in this disclosure, ^R9 is selected from _C1-4 alkyl, halogenated _C1-4 alkyl or deuterated _C1-4 alkyl.

In alternative embodiments, in the compounds of formulas (I), (II), and (II-1) provided in this disclosure, or their pharmaceutically acceptable salts, ^R9 is selected from methyl, ethyl, difluoromethyl, trifluoromethyl, or deuterated methyl.

In alternative embodiments, in the compounds of formulas (I), (II), and (II-1) provided in this disclosure, or their pharmaceutically acceptable salts, ^R9 is selected from methyl, difluoromethyl, trifluoromethyl, or deuterated methyl.

In some embodiments, the compounds represented by formulas (I) and (II) provided in this disclosure, or their pharmaceutically acceptable salts, are the compounds represented by formula (II-2) or their pharmaceutically acceptable salts.

Among them, ^R1 , ^R2 , ^R3 , ^R4 , ^R6 , ^R7 , ^R8 , and ^R9 are defined as in equation (I).

In some embodiments, in the compound of formula (II-2) provided in this disclosure or its pharmaceutically acceptable salt, ^R9 is selected from _C1-4 alkyl, halogenated _C1-4 alkyl or deuterated _C1-4 alkyl.

In an alternative embodiment, in the compound of formula (II-2) provided in this disclosure or its pharmaceutically acceptable salt, ^R9 is selected from methyl, ethyl, difluoromethyl, trifluoromethyl or deuterated methyl.

In an alternative embodiment, in the compound of formula (II-2) provided in this disclosure or its pharmaceutically acceptable salt, R ⁹ is methyl.

In some embodiments, in the compounds or pharmaceutically acceptable salts of formulas (I), (II), (II-1), and (II-2) provided in this disclosure, at least one of ^R1 , ^R2 , and ^R3 is selected from fluorine or chlorine.

In some embodiments, in the compounds or pharmaceutically acceptable salts of formulas (I), (II), (II-1), (II-2) provided in this disclosure, ^R1 and ^R3 are each independently selected from hydrogen, fluorine, or chlorine.

In alternative embodiments, in the compounds or pharmaceutically acceptable salts of formulas (I), (II), (II-1), and (II-2) provided in this disclosure, one of ^R1 or ^R3 is hydrogen, and the other is selected from hydrogen, fluorine, or chlorine.

In alternative embodiments, in the compounds or pharmaceutically acceptable salts of formulas (I), (II), (II-1), and (II-2) provided in this disclosure, ^R1 or ^R3 is each independently hydrogen.

In some embodiments, in the compounds or pharmaceutically acceptable salts of formulas (I), (II), (II-1), and (II-2) provided in this disclosure, ^R2 is fluorine or chlorine.

In some embodiments, in the compounds or pharmaceutically acceptable salts of formulas (I), (II), (II-1), and (II-2) provided in this disclosure, ^R2 is fluorine.

In some embodiments, in the compounds or pharmaceutically acceptable salts of formulas (I), (II), (II-1), and (II-2) provided in this disclosure, ^R2 is chlorine.

In some embodiments, in the compounds or pharmaceutically acceptable salts of formulas (I), (II), (II-1), (II-2) provided in this disclosure, ^R1 or ^R3 is each independently hydrogen; and ^R2 is fluorine or chlorine.

In some embodiments, in the compounds or pharmaceutically acceptable salts of formulas (I), (II), (II-1), (II-2) provided in this disclosure, ^R4 is selected from fluorine or chlorine.

In some embodiments, in the compounds or pharmaceutically acceptable salts of formulas (I), (II), (II-1), and (II-2) provided in this disclosure, ^R4 is fluorine.

In some embodiments, in the compounds or pharmaceutically acceptable salts of formulas (I), (II), (II-1), (II-2) provided in this disclosure, ^R1 or ^R3 is each independently hydrogen, ^R2 is fluorine or chlorine, and ^R4 is fluorine.

In some embodiments, in the compounds of formulas (I), (II), (II-1), (II-2) provided in this disclosure, or their pharmaceutically acceptable salts, at least one of ^R6 , ^R7 and ^R8 is selected from fluorine or chlorine.

In alternative embodiments, of the compounds represented by formulas (I), (II), (II-1), and (II-2) provided in this disclosure, or their pharmaceutically acceptable salts, at least two of ^R6 , ^R7, and ^R8 are selected from fluorine or chlorine.

In some embodiments, in the compounds of formulas (I), (II), (II-1), (II-2) provided in this disclosure, or their pharmaceutically acceptable salts, R ⁶ is selected from fluorine or chlorine.

In some embodiments, in the compounds or pharmaceutically acceptable salts of formulas (I), (II), (II-1), and (II-2) provided in this disclosure, at least one of ^R6 , ^R7 , and ^R8 is chlorine.

In some embodiments, in the compounds or pharmaceutically acceptable salts of formulas (I), (II), (II-1), and (II-2) provided in this disclosure, ^R7 is selected from fluorine or chlorine, and ^R8 is hydrogen.

In alternative embodiments, in the compounds or pharmaceutically acceptable salts of formulas (I), (II), (II-1), and (II-2) provided in this disclosure, R ⁷ is fluorine and R ⁸ is hydrogen.

In some embodiments, in the compounds or pharmaceutically acceptable salts of formulas (I), (II), (II-1), and (II-2) provided in this disclosure, ^R8 is selected from fluorine or chlorine, and ^R7 is hydrogen.

In some embodiments, in the compounds or pharmaceutically acceptable salts of formulas (I), (II), (II-1), and (II-2) provided in this disclosure, ^R1 or ^R3 is each independently hydrogen; ^R2 is fluorine or chlorine; ^R4 is fluorine; ^R6 is selected from fluorine or chlorine; ^R7 is selected from fluorine or chlorine and ^R8 is hydrogen, or ^R8 is selected from fluorine or chlorine and ^R7 is hydrogen; and at least one of ^R6 , ^R7 , and ^R8 is chlorine.

In some embodiments, the compounds or pharmaceutically acceptable salts of formulas (I), (II), (II-1), and (II-2) provided in this disclosure are, in fact, the compounds or pharmaceutically acceptable salts of formula (II-A).

^R2 , ^R5 , ^R6 , and ^R7 are defined as in equations (I), (II), (II-1), and (II-2), respectively.

In an alternative embodiment, in the compound of formula (II-A) provided in this disclosure or its pharmaceutically acceptable salt, ^R2 is chlorine.

In an alternative embodiment, in the compound of formula (II-A) or its pharmaceutically acceptable salt provided in this disclosure, ^R2 is fluorine.

In some embodiments, the compounds or pharmaceutically acceptable salts of formulas (I), (II), (II-1), and (II-2) provided in this disclosure are, in fact, the compounds or pharmaceutically acceptable salts of formula (II-B).

^R2 , ^R5 , ^R6 , and ^R7 are defined as in equations (I), (II), (II-1), and (II-2), respectively.

In an alternative embodiment, in the compound of formula (II-B) provided in this disclosure or its pharmaceutically acceptable salt, ^R2 is chlorine.

In an alternative embodiment, in the compound of formula (II-B) or its pharmaceutically acceptable salt provided in this disclosure, ^R2 is fluorine.

In some embodiments, in the compounds of formulas (II-A) and (II-B) provided in this disclosure, or their pharmaceutically acceptable salts, ^R5 is selected from methyl or ethyl.

In some embodiments, in the compounds of formulas (II-A) and (II-B) provided in this disclosure, or their pharmaceutically acceptable salts, ^R5 is selected from _-L1 - ^R9 , where _L1 is oxygen and ^R9 is selected from methyl, ethyl, difluoromethyl, trifluoromethyl, or deuterated methyl.

In some embodiments, in the compounds of formulas (II-A) and (II-B) provided in this disclosure, or their pharmaceutically acceptable salts, ^R5 is selected from _-L1 - ^R9 , where _L1 is oxygen and ^R9 is selected from methyl or deuterated methyl.

In some embodiments, in the compounds of formulas (II-A) and (II-B) provided in this disclosure, or their pharmaceutically acceptable salts, ^R5 is selected from _-L1 - ^R9 , where _L1 is oxygen and ^R9 is selected from difluoromethyl or trifluoromethyl.

In some embodiments, in the compounds of formulas (II-A) and (II-B) provided in this disclosure, or their pharmaceutically acceptable salts, ^R5 is selected from _-L1 - ^R9 , where _L1 is sulfur and ^R9 is selected from methyl, ethyl, difluoromethyl, trifluoromethyl, or deuterated methyl.

In some embodiments, in the compounds of formula (II-A) and (II-B) provided in this disclosure or their pharmaceutically acceptable salts, ^R5 is selected from _-L1 - ^R9 , where _L1 is sulfur and ^R9 is methyl or deuterated methyl.

In some embodiments, in the compounds or pharmaceutically acceptable salts of formulas (II-A) and (II-B) provided in this disclosure, ^R6 is fluorine or chlorine, and ^R7 is hydrogen.

In alternative embodiments, in the compounds of formulas (II-A) and (II-B) provided in this disclosure, or their pharmaceutically acceptable salts, ^R6 is fluorine and ^R7 is hydrogen.

In some embodiments, the compounds or pharmaceutically acceptable salts of formulas (I), (II), (II-1), and (II-2) provided in this disclosure are, in fact, the compounds or pharmaceutically acceptable salts of formula (II-C).

^R2 , ^R5 , ^R6 , and ^R7 are defined as in equations (I), (II), (II-1), and (II-2), respectively.

In an alternative embodiment, in the compound of formula (II-C) or its pharmaceutically acceptable salt provided in this disclosure, ^R2 is chlorine.

In an alternative embodiment, in the compound of formula (II-C) provided in this disclosure or its pharmaceutically acceptable salt, ^R2 is fluorine.

In some embodiments, in the compound of formula (II-C) provided in this disclosure or its pharmaceutically acceptable salt, ^R5 is selected from methyl or ethyl.

In some embodiments, in the compound of formula (II-C) provided in this disclosure or its pharmaceutically acceptable salt, ^R5 is selected from _-L1 - ^R9 , where _L1 is oxygen and ^R9 is selected from methyl, ethyl, difluoromethyl, trifluoromethyl or deuterated methyl.

In some embodiments, in the compound of formula (II-C) provided in this disclosure or its pharmaceutically acceptable salt, ^R5 is selected from _-L1 - ^R9 , where _L1 is oxygen and ^R9 is selected from methyl or deuterated methyl.

In some embodiments, in the compound of formula (II-C) provided in this disclosure or its pharmaceutically acceptable salt, ^R5 is selected from _-L1 - ^R9 , where _L1 is oxygen and ^R9 is selected from difluoromethyl or trifluoromethyl.

In some embodiments, in the compound of formula (II-C) provided in this disclosure or its pharmaceutically acceptable salt, ^R5 is selected from _-L1 - ^R9 , where _L1 is sulfur and ^R9 is selected from methyl, ethyl, difluoromethyl, trifluoromethyl or deuterated methyl.

In some embodiments, in the compound of formula (II-C) provided in this disclosure or its pharmaceutically acceptable salt, ^R5 is selected from _-L1 - ^R9 , where _L1 is sulfur and ^R9 is methyl or deuterated methyl.

In some embodiments, in the compound of formula (II-C) provided in this disclosure or its pharmaceutically acceptable salt, at least one of ^R6 and ^R7 is selected from fluorine or chlorine.

In an alternative embodiment, in the compound of formula (II-C) provided in this disclosure or its pharmaceutically acceptable salt, ^R6 is selected from fluorine or chlorine.

In an alternative embodiment, in the compound of formula (II-C) provided in this disclosure or its pharmaceutically acceptable salt, ^R6 is selected from fluorine and ^R7 is selected from fluorine or chlorine.

In an alternative embodiment, in the compound of formula (II-C) provided in this disclosure or its pharmaceutically acceptable salt, ^R6 is selected from chlorine and ^R7 is selected from fluorine or chlorine.

This disclosure further provides for compounds selected from the following or their pharmaceutically acceptable salts:

This disclosure further provides an isotopic substitution of the aforementioned compound or its pharmaceutically acceptable salt, optionally, the isotopic substitution being a deuterated derivative.

This disclosure provides a pharmaceutical composition comprising the aforementioned compounds and one or more pharmaceutically acceptable excipients.

In some implementation schemes, the unit dose of this pharmaceutical composition ranges from 0.001 mg to 1000 mg.

In some embodiments, based on the total weight of the composition, the pharmaceutical composition contains 0.01-99.99% of the aforementioned compound or its pharmaceutically acceptable salt or its isotopic substitutions. In some embodiments, the pharmaceutical composition contains 0.1-99.9% of the aforementioned compound or its pharmaceutically acceptable salt or its isotopic substitutions. In some embodiments, the pharmaceutical composition contains 0.5%-99.5% of the aforementioned compound or its pharmaceutically acceptable salt or its isotopic substitutions. In some embodiments, the pharmaceutical composition contains 1%-99% of the aforementioned compound or its pharmaceutically acceptable salt or its isotopic substitutions. In some embodiments, the pharmaceutical composition contains 2%-98% of the aforementioned compound or its pharmaceutically acceptable salt or its isotopic substitutions.

In some embodiments, the pharmaceutical composition contains 0.01%-99.99% pharmaceutically acceptable excipients based on the total weight of the composition. In some embodiments, the pharmaceutical composition contains 0.1%-99.9% pharmaceutically acceptable excipients. In some embodiments, the pharmaceutical composition contains 0.5%-99.5% pharmaceutically acceptable excipients. In some embodiments, the pharmaceutical composition contains 1%-99% pharmaceutically acceptable excipients. In some embodiments, the pharmaceutical composition contains 2%-98% pharmaceutically acceptable excipients.

This disclosure further provides the use of the aforementioned compound or its pharmaceutically acceptable salt or its isotopic substitutes, or the aforementioned pharmaceutical composition, as a medicine.

This disclosure further provides the use of the aforementioned compound or its pharmaceutically acceptable salt or isotopic substitute, or the aforementioned pharmaceutical composition, in the preparation of a medicament for the prevention and/or treatment of diseases related to BCKDK.

This disclosure further provides the use of the aforementioned compound or its pharmaceutically acceptable salt or isotopic substitute or the aforementioned pharmaceutical composition in the preparation of a medicament for the prevention and/or treatment of diseases, including but not limited to disorders of glucose metabolism or abnormalities of blood sugar, heart disease, or kidney disease.

This disclosure further provides a method for preventing and/or treating diseases related to BCKDK, comprising administering to a patient the aforementioned compound or its pharmaceutically acceptable salt or isotopic substitute, or the aforementioned pharmaceutical composition.

This disclosure further provides a method for preventing and/or treating a disease comprising administering to a patient the aforementioned compound or its pharmaceutically acceptable salt or isotopic substitute or the aforementioned pharmaceutical composition, the disease including, but not limited to, disorders of glucose metabolism or abnormal blood sugar, heart disease or kidney disease.

In alternative embodiments, the method provided in this disclosure administers to the patient a therapeutically effective amount of the aforementioned compound or its pharmaceutically acceptable salt or its isotopic substitute, or the aforementioned pharmaceutical composition.

In some implementation protocols, BCKDK-related disorders include, but are not limited to, disorders of glucose metabolism or abnormal blood sugar levels, heart disease, or kidney disease.

In some implementation protocols, this glucose metabolism disorder or abnormal blood sugar condition is defined as diabetes.

In some implementation protocols, the heart condition is defined as heart failure.

The aforementioned compounds, or their pharmaceutically acceptable salts or isotopic substitutes, or the aforementioned pharmaceutical compositions disclosed herein exhibit good inhibitory/degradative effects on BCKDK.

This disclosure, in another aspect, provides a method for preparing a compound of formula (I) or a pharmaceutically acceptable salt thereof, comprising the step of removing the protecting group PG from the compound of formula (I-a) or a pharmaceutically acceptable salt thereof under alkaline conditions, and further comprising the step of adjusting the pH to acidic conditions.

Wherein, PG is a carboxyl protecting group; ^R1 , ^R2 , ^R3 , ^R4 , ^R5 , _X1 , ^R7 and ^R8 are defined as in formula (I).

In an alternative embodiment, in the method for preparing the compound of formula (I) or its pharmaceutically acceptable salt provided in this disclosure, the PG is a _C1-6 alkyl group.

This disclosure, in another aspect, provides a compound of formula (I-a) or a pharmaceutically acceptable salt thereof,

Wherein, PG is a carboxyl protecting group; ^R1 , ^R2 , ^R3 , ^R4 , ^R5 , _X1 , ^R7 and ^R8 are defined as in formula (I).

In an alternative embodiment, the PG in the compound of formula (Ia) or its pharmaceutically acceptable salt is a _C1-6 alkyl group.

Definition of Terms

Without specifying a particular configuration, the compounds disclosed herein may exist in specific geometric or stereoisomeric forms. This disclosure envisions all such compounds, including cis and trans isomers, (-)- and (+)- enantiomers, (R)- and (S)- enantiomers, diastereomers, (D)- isomers, (L)- isomers, rotation-blocked isomers (i.e., rotationally blocked stereoisomers), their racemic mixtures, and other mixtures, such as mixtures enriched with enantiomers or diastereomers, all of which are within the scope of this disclosure. Additional asymmetrical carbon atoms may be present in substituents such as alkyl groups. All such isomers and mixtures thereof are included within the scope of this disclosure.

Furthermore, the compounds and intermediates disclosed herein can exist in various tautomer forms, and all such forms are included within the scope of this disclosure. The terms "tautomer" or "tautomer form" refer to structural isomers of different energies that can interconvert via low-energy barriers.

The compounds disclosed herein may be asymmetric, for example, having one or more stereoisomers. Unless otherwise stated, all stereoisomers include, such as enantiomers and diastereomers. The compounds containing asymmetric carbon atoms disclosed herein can be isolated in optically active pure form or in racemic form. The optically active pure form can be resolved from racemic mixtures or synthesized using chiral starting materials or chiral reagents.

Optically active (R)- and (S)- isomers, as well as D- and L- isomers, can be prepared by chiral synthesis, chiral reagents, or other conventional techniques. To obtain an enantiomer of a compound disclosed herein, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, wherein the resulting diastereomeric mixture is separated, and auxiliary groups are cleaved to provide a pure desired enantiomer. Alternatively, when the molecule contains a basic functional group (such as an amine) or an acidic functional group (such as a carboxyl group), a salt of the diastereomeric isomer is formed with a suitable optically active acid or base, followed by diastereomeric resolution using conventional methods known in the art, and then the pure enantiomer is recovered. Furthermore, the separation of enantiomers and diastereomers is typically accomplished using chromatography with a chiral stationary phase, and, as needed, in conjunction with chemical derivatization (e.g., the formation of aminoformates from amines).

In the chemical structure of the compound disclosed herein, the bond " "Indicates that no configuration is specified, meaning that if a chiral isomer exists in the chemical structure, the key " "Can be" "or" , or containing " "and" "Two configurations. In the chemical structure of the compound disclosed herein, the bond..." "No configuration specified, i.e., key " The configuration of “” can be E type or Z type, or include both E and Z configurations.

This disclosure also includes compounds identical to those described herein, but in which one or more atoms are labeled with isotopes whose atomic weights or mass numbers differ from those normally found in nature. Examples of isotopes that can be incorporated into the compounds of this disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as ^2H , ^3H , ^11C , ^13C , ^14C , ^13N , ^15N , ^15O , ^17O , ^18O , ^31P , ^32P , ^35S , ^18F , ^123I , ^125I , and ^36Cl , respectively.

Unless otherwise specified, when a position is specifically designated as deuterium (D), that position should be understood as deuterium having an abundance at least 1000 times greater than the natural abundance of deuterium (which is 0.015%) (i.e., at least 10% deuterium doping). The natural abundance of deuterium in the example compounds can be at least 1000 times, at least 2000 times, at least 3000 times, at least 4000 times, at least 5000 times, at least 6000 times, or even higher. This disclosure also includes compounds in various deuterated forms. Each available hydrogen atom bonded to a carbon atom can be independently replaced by a deuterium atom. Those skilled in the art can synthesize the deuterated form of the compound by referring to relevant literature. Commercially available deuterated starting materials can be used in the preparation of the deuterated form of the compound, or they can be synthesized using conventional techniques with deuterating reagents, including but not limited to deuterboranes, trideuteronane tetrahydrofuran solutions, deuterated aluminum lithium hydroxide, deuterated iodoethane, and deuterated iodomethane, etc.

The term "alkyl" refers to a saturated aliphatic hydrocarbon group, preferably an alkyl group with 1 to 6 carbon atoms (e.g., 1, 2, 3, 4, 5, or 6 carbons). Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tributyl, dibutyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, and 2,3-dimethylbutyl.

The term "cycloalkyl" or "carbocyclic" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent containing 3 to 20 carbon atoms, preferably 3 to 6 carbon atoms. Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, etc.; polycyclic cycloalkyl groups include spirocyclic, fused-ring, and bridged cycloalkyl groups.

The term "alkoxy" refers to -O- (alkyl) and -O- (unsubstituted cycloalkyl), where alkyl and cycloalkyl are defined as described above. Non-limiting examples of alkoxy groups include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentoxy, and cyclohexoxy.

The term "hydroxyl" refers to -OH.

The term "halogen" refers to fluorine, chlorine, bromine, or iodine.

The term "halogenated alkyl" refers to an alkyl group substituted with a halogen, where the alkyl group is defined as above.

The term "cyano" refers to -CN.

The term "amine" refers to _-NH2 .

"As needed" or "as required" means that the event or environment described thereafter may or may not occur, and the description includes situations in which the event or environment may or may not occur. For example, " _C1-6 alkyl groups substituted with halogen or cyano groups as required" means that halogen or cyano groups may or may not be present, and the description includes cases where the alkyl group is substituted with halogen or cyano groups and cases where the alkyl group is not substituted with halogen or cyano groups.

The term "substituted" refers to one or more hydrogen atoms in a group, preferably up to five, and more preferably one to three hydrogen atoms, independently substituted by a corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and those with ordinary knowledge in the relevant art can determine possible or impossible substitutions without much effort (through experiment or theory).

"Being replaced by one or more..." means that the object can be replaced by one or more substituents. When replaced by multiple substituents, the substituents can be a plurality of identical substituents or a combination of one or more different substituents.

In this disclosure, the terms "contains" and "including" can be replaced with "composed of".

The term "composition" refers to a mixture of a drug containing one or more of the compounds described herein, or their physiologically pharmaceutically acceptable salts or prodrugs, with other chemical components, such as physiologically pharmaceutically acceptable carriers and excipients. The purpose of a composition is to facilitate drug delivery to the body, enhance the absorption of the active ingredient, and thereby exert its biological activity.

The term "pharmaceutical-acceptable excipient" includes, but is not limited to, any adjuvant, carrier, excipient, flow aid, sweetener, thinner, preservative, dye/coloring agent, flavoring agent, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isointense agent, solvent, or emulsifier that has been approved by the U.S. Food and Drug Administration for use in humans or livestock.

Unless otherwise specified, the "compounds" disclosed herein may exist in the form of salts, mixed salts, or non-salts (e.g., free acids or free bases). When present as salts or mixed salts, they may be pharmaceutically acceptable or medicinally usable salts.

The terms "pharmaceutically acceptable salt" and "medicinal salt" are used interchangeably to refer to both pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

As used herein, the term “inhibition” may be used interchangeably with “reduction,” “silencing,” “downregulation,” “blocking,” and other similar terms, and includes any level of inhibition. Inhibition can be assessed by a reduction in one or more of these variables at an absolute or relative level compared to a control level. This control level can be any type of control level used in the art, such as a baseline level before administration or a level determined from similarly untreated or controlled (e.g., buffer-only or inert) subjects, cells, or samples.

"Effective dose," "effective dosage," "effective therapeutic dose," or "therapeuticly effective dose" refers to the amount of a drug, compound, or pharmaceutical composition necessary to achieve any one or more beneficial or desired therapeutic outcomes. For preventative use, beneficial or desired outcomes include eliminating or reducing the risk, mitigating the severity of the condition, or delaying the onset of the condition, including the condition itself, its complications, and the biochemical, histological, and/or behavioral symptoms of intermediate pathological phenotypes present during the development of the condition.

As used herein, the terms “subject,” “patient,” “subject,” or “individual” are used interchangeably and include human or non-human animals, such as mammals, such as humans or monkeys.

The present disclosure is further described below with reference to embodiments, but these embodiments do not limit the scope of the disclosure. Experimental methods in the embodiments of this disclosure that do not specify specific conditions are generally performed under conventional conditions or as recommended by the raw material or product manufacturer. Reagents whose specific source is not specified are obtained from any supplier of molecular biology reagents with the quality/purity required for molecular biology applications.

Unless otherwise stated, all reagents used in the following examples are commercially available products.

The analytical conditions used for all mass spectrometry data in the following examples are as follows: Column type: ACQUITY UPLC BEH C18 1.7 μM, 2.1 x 50 mm column; Mobile phase A: 0.05% ammonia + 0.01% formic acid aqueous solution; Mobile phase B: 0.01% formic acid acetonitrile solution; Mobile phase B gradient was 5%–95% from 0 to 1.7 minutes; the gradient of mobile phase B was maintained at 95% from 1.7 to 2.5 minutes.

The instruments used for all hydrogen spectral data in the following embodiments are: a Bruker 400MHz NMR spectrometer; the instruments used for all fluorine spectral data are: a Bruker 376MHz NMR spectrometer.

Implementation Example 1

6-Chloro-3-(2,4,5-trifluoro-3-methoxyphenyl)thieno[3,2-b]pyridine-2-carboxylic acid ( 1 )

Step 1: Synthesis of methyl 3-amino-6-chlorothiopheno[3,2-b]pyridine-2-carboxylate 1c

At 0°C, methyl 2-aminoacetate 1b (2.4 mL, 27.24 mmol) was slowly added dropwise to a solution of 5-chloro-3-nitropyridine-2-onitrile 1a (5.00 g, 27.24 mmol) in N,N-dimethylformamide (60 mL), followed by the addition of potassium hydroxide (3.06 g, 54.50 mmol) in water (10.0 mL). The reaction mixture was stirred at 0–5°C for 1 hour, then ice water (60 mL) was added, and the mixture was extracted with ethyl acetate (2 × 100 mL). The organic phases were combined, washed with saturated brine (4 × 80 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give crude product 1c (6.5 g, 98% yield).

MS(ESI)m/z=243.2[M+H] ⁺ .

Step 2: Synthesis of methyl 3-bromo-6-chlorothiophene[3,2-b]pyridine-2-carboxylic acid ester 1d

At 25°C under a nitrogen atmosphere, tributyl nitrite (3.92 g, 1.3 mL, 10.7 mmol) was added dropwise to a three-necked flask containing copper(II) bromide (2.05 g, 9.13 mmol) and acetonitrile (15.0 mL). Then, a suspension of compound 1c (2.0 g, 8.3 mmol) in acetonitrile (5.0 mL) was added at 20°C. The reaction mixture was stirred at 25°C for 2 hours, then slowly poured into hydrochloric acid solution (2N, 20mL), extracted with ethyl acetate (2×70mL), the organic phases were combined, washed with saturated brine (2×100mL), the organic phase was dried with anhydrous sodium sulfate, filtered, concentrated under reduced pressure to obtain crude product, purified by column chromatography (petroleum ether/ethyl acetate) to obtain the title compound 1d (1.0g, yield 39%).

MS(ESI)m/z=306.1[M+H] ⁺ .

Step 3: Synthesis of methyl 6-chloro-3-(2,4,5-trifluoro-3-methoxyphenyl)thiopheno[3,2-b]pyridine-2-carboxylate 1f

At room temperature, compounds 1d (75 mg, 0.25 mmol) and 1e (50 mg, 0.25 mmol) were dissolved in 1,4-dioxane (2 mL) and water (0.4 mL), and 1,1'-di-tert-butylphosphinoferrocene palladium dichloride (15.9 mg, 0.024 mmol) and potassium carbonate (101 mg, 0.73 mmol) were added. The reaction mixture was stirred at 100 °C for 1 h under a nitrogen atmosphere. After cooling to room temperature, ethyl acetate (60 mL) was added, followed by washing with saturated brine (2 × 20 mL), drying with anhydrous sodium sulfate, filtration, and concentration under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (petroleum ether/ethyl acetate) to obtain compound 1f (60 mg, yield 61%).

MS(ESI)m/z=388.2[M+H] ⁺ .

Step 4: Synthesis of 6-chloro-3-(2,4,5-trifluoro-3-methoxyphenyl)thiopheno[3,2-b]pyridine-2-carboxylic acid 1

Compound 1f (60 mg, 0.16 mmol) was dissolved in a mixed solvent of tetrahydrofuran (1 mL), methanol (0.5 mL), and water (0.5 mL) at room temperature. Lithium hydroxide (37 mg, 1.6 mmol) was added, and the reaction mixture was stirred at room temperature for 1 h. Water (30 mL) was added, and the pH was adjusted to 4-5 with hydrochloric acid (1 N). The mixture was extracted with ethyl acetate (2 × 10 mL), and the organic phases were combined. The mixture was washed with saturated brine (2 × 10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by preparative high-performance liquid chromatography (HPLC) (column: Waters Xbridge, 30 × 250 mm. Mobile phase A: 0.1% formic acid aqueous solution; mobile phase B: acetonitrile; gradient: 40%-95%). Compound 1 (15 mg, yield 25%) was obtained at a flow rate of 30 ml/min and a elution time of 11.06 min (18 min).

MS(ESI)m/z=374.2[M+H] ⁺ .

¹ H-NMR (400MHz, DMSO- d ₆ ) δ 8.87 (s, 1H), 8.78 (s, 1H), 7.44-7.37 (m, 1H), 4.02 (s, 3H).

¹⁹ F-NMR (376MHz, DMSO- d ₆ ) δ -132.28(dd,1F), -142.60(dd,1F), -151.65(dd,1F).

Implementation Example 2

6-Chloro-3-(6-chloro-2,4-difluoro-3-methoxyphenyl)thieno[3,2-b]pyridine-2-carboxylic acid ( 2 ); 6-Chloro-3-(6-chloro-2,4-difluoro-3-methoxyphenyl)thieno[3,2-b]pyridine-2-carboxylic acid ( 2-P1 ); 6-Chloro-3-(6-chloro-2,4-difluoro-3-methoxyphenyl)thieno[3,2-b]pyridine-2-carboxylic acid ( 2-P2 )

Step 1: Synthesis of methyl 2b of 6-chloro-3-(6-chloro-2,4-difluoro-3-methoxyphenyl)thienro[3,2-b]pyridine-2-carboxylate

At room temperature, compounds 1d (60 mg, 0.20 mmol) and 2a (89 mg, 0.30 mmol) were dissolved in toluene (3 mL) and water (0.3 mL), and tris(dibenzylacetone)dipalladium (18 mg, 0.02 mmol), 2-biscyclohexylphosphine-2',6'-dimethoxybiphenyl (16 mg, 0.04 mmol), and potassium phosphate (125 mg, 0.59 mmol) were added. The reaction mixture was stirred at 70 °C for 2 h under a nitrogen atmosphere. After cooling to room temperature, ethyl acetate (40 mL) was added, followed by washing with saturated brine (2 × 20 mL), drying with anhydrous sodium sulfate, filtration, and concentration under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (petroleum ether/ethyl acetate) to obtain the title compound 2b (50 mg, yield 61%).

MS(ESI)m/z=404.2[M+H] ⁺ .

Step 2: Synthesis of 6-chloro-3-(6-chloro-2,4-difluoro-3-methoxyphenyl)thiopheno[3,2-b]pyridine- 2 -carboxylic acid

Compound 2b (50 mg, 0.12 mmol) was dissolved in a mixture of tetrahydrofuran (2 ml), methanol (1 ml) and water (1 ml) at room temperature, and lithium hydroxide (30 mg, 1.2 mmol) was added. The reaction mixture was stirred at room temperature for 1 h. Add water (30 mL), adjust pH to 4-5 with hydrochloric acid (1 N), extract with ethyl acetate (2 × 10 mL), combine organic phases, wash with saturated brine (2 × 10 mL), dry with anhydrous sodium sulfate, filter, concentrate under reduced pressure to obtain crude product, and purify by preparative high performance liquid chromatography (column: Waters Xbridge, 30 × 250 mm. Mobile phase A: 0.1% formic acid aqueous solution; mobile phase B: acetonitrile; gradient: 40%-95% for 18 min, flow rate: 30 mL/min. Elution time: 11.06 min) to obtain title compound 2 (18 mg, yield 38%).

MS(ESI)m/z=390.1[M+H] ⁺ .

¹ H-NMR (400MHz, DMSO- d ₆ ) δ 8.88 (s, 1H), 8.76 (s, 1H), 7.63-7.60 (m, 1H), 3.96 (s, 3H).

¹⁹ F-NMR (376MHz, DMSO- d ₆ ) δ -124.92 (d, 1F), -126.84 (d, 1F).

Step 3: 6-Chloro-3-(6-chloro-2,4-difluoro-3-methoxyphenyl)thieno[3,2-b]pyridine-2-carboxylic acid ( 2-P1 ) and 6-chloro-3-(6-chloro-2,4-difluoro-3-methoxyphenyl)thieno[3,2-b]pyridine-2-carboxylic acid ( 2-P2 )

The purified product 2 from the second step was further separated using SFC. Separation method: Column: ChiralPak AD, 250×30mm ID, 5μm; Mobile phase: A: _CO2 ; B: Isopropanol [0.1% _NH3 (7M MeOH solution)]; Gradient: B 30%; Flow rate: 100mL/min; Back pressure: 100bar; Column temperature: 35℃; Wavelength: 214nm; Cycle time: ~2 minutes. Analytical methods: Column: ChiralPak AD, 100×4.6mm ID, 3μm; Mobile phase: A: _CO2 ; B: Isopropanol (0.1%DEA); Gradient: B 5-45%, 4 min; Flow rate: 3.0 mL/min; Back pressure: 2000 psi; Column temperature: 40℃; Wavelength: 214 nm; Cycle time: ~2 min.

2-P1 , retention time t=2.985 min, 1.7 mg;

2-P2 , retention time t=2.807 min, 2.1 mg.

Implementation Example 3

6-Chloro-3-(2,4,5-trifluoro-3-methylphenyl)thieno[3,2-b]pyridine-2-carboxylic acid ( 3 )

Step 1: Synthesis of methyl 6-chloro-3-(2,4,5-trifluoro-3-methylphenyl)thiopheno[3,2-b]pyridine-2-carboxylate 3b

At room temperature, compound 1d (100 mg, 0.33 mmol) and compound 3a (133 mg, 0.49 mmol) were dissolved in 1,4-dioxane (2 mL) and water (0.40 mL), and 1,1'-di-tertiary-butylphosphinoferrocene palladium dichloride (21.3 mg, 0.03 mmol) and potassium carbonate (135 mg, 0.09 mmol) were added. The reaction mixture was stirred at 70 °C for 1 h under nitrogen atmosphere. After cooling to room temperature, ethyl acetate (40 mL) was added, and the mixture was washed with saturated brine (2 × 20 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure to obtain crude product, and purified by column chromatography (petroleum ether/ethyl acetate) to obtain title compound 3b (80 mg, yield 65%).

MS(ESI)m/z=372.2[M+H] ⁺ .

Step 2: Synthesis of 6-chloro-3-(2,4,5-trifluoro-3-methylphenyl)thiopheno[3,2-b]pyridine-2-carboxylic acid 3

Compound 3b (80 mg, 0.22 mmol) was dissolved in a mixture of tetrahydrofuran (2 ml), methanol (1 ml) and water (1 ml) at room temperature, and lithium hydroxide (52 mg, 2.2 mmol) was added. The reaction mixture was stirred at room temperature for 1 h. Add water (30 mL), adjust pH to 4-5 with hydrochloric acid (1 N), extract with ethyl acetate (2 × 10 mL), combine organic phases, wash with saturated brine (2 × 20 mL), dry with anhydrous sodium sulfate, filter, concentrate under reduced pressure to obtain crude product, and purify by preparative high performance liquid chromatography (column: Waters Xbridge, 30 × 250 mm. Mobile phase A: 0.1% formic acid aqueous solution; mobile phase B: acetonitrile; gradient: 35%-85% for 18 min, flow rate: 30 mL/min. Elution time: 13.50 min), to obtain title compound 3 (18 mg, yield 22%).

MS(ESI)m/z=358.2[M+H] ⁺ .

¹ H-NMR (400MHz, DMSO- d ₆ ) δ 8.86 (s, 1H), 8.77 (s, 1H), 7.54-7.48 (m, 1H), 2.26 (s, 3H).

¹⁹ F-NMR (376MHz, DMSO- d ₆ ) δ -118.78(dd,1F), -137.93(dd,1F), -144.56(dd,1F).

Implementation Example 4

6-Fluoro-3-[2,4,5-trifluoro-3-(methylthio)phenyl]thieno[3,2-b]pyridine-2-carboxylic acid ( 4 )

Step 1: Synthesis of methyl 4b of 3-amino-6-fluorothiopheno[3,2-b]pyridine-2-carboxylate

At 0°C, methyl methacrylate (1.1 mL, 12.57 mmol) was added dropwise to a solution of compound 4a (2 g, 11.97 mmol) in N,N-dimethylformamide (20 mL), followed by the slow addition of an aqueous solution of potassium hydroxide (5 M, 4.8 mL, 23.94 mmol), and the reaction was maintained at 0°C for 30 minutes. The reaction solution was poured into ice water (60 mL), extracted with ethyl acetate (3 × 50 mL), the organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the crude product was purified by column chromatography (petroleum ether/ethyl acetate) to give compound 4b (2 g, 73% yield).

MS(ESI)m/z=227.2[M+H] ⁺ .

Step 2: Synthesis of methyl 6-fluoro-3-iodothiopheno[3,2-b]pyridine-2-carboxylate 4c

At room temperature, compound 4b (1 g, 4.42 mmol) and diiodomethane (0.54 mL, 6.63 mmol) were dissolved in acetonitrile (20 mL), and the mixture was heated to 70 °C. Isoamyl nitrite (0.89 mL, 6.63 mmol) was then slowly added dropwise to the system, maintaining the temperature at 70-80 °C during the addition. After the addition was completed, the reaction was continued at 70 °C for 2 hours. The reaction solution was directly concentrated to dryness under reduced pressure, and the residue was purified by column chromatography (petroleum ether/ethyl acetate) to give crude product 1c (350 mg, yield 23%).

MS(ESI)m/z=338.2[M+H] ⁺ .

Step 3: Synthesis of methyl 6-fluoro-3-(2,4,5-trifluoro-3-hydroxyphenyl)thiopheno[3,2-b]pyridine-2-carboxylate 4e

At room temperature, compounds 4c (300 mg, 0.89 mmol) and 4d (366 mg, 1.34 mmol) were dissolved in 1,4-dioxane (2 mL) and water (0.40 mL), and 1,1'-di-tert-butylphosphinoferrocene palladium dichloride (58 mg, 0.09 mmol) and potassium carbonate (369 mg, 2.67 mmol) were added. The reaction mixture was stirred at 100 °C for 1 h under nitrogen atmosphere. After cooling to room temperature, ethyl acetate (20 mL) was added, followed by washing with saturated brine (2 × 10 mL), drying with anhydrous sodium sulfate, filtration, and concentration under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (petroleum ether/ethyl acetate) to obtain compound 4e (100 mg, yield 31%).

MS(ESI)m/z=358.2[M+H] ⁺ .

Step 4: Synthesis of 2,5,6-trifluoro-3-[6-fluoro-2-(methoxycarbonyl)thiopheno[3,2-b]pyridin-3-yl]phenyl ester 4f

At room temperature, triethylamine (85 mg, 0.84 mmol) and trifluoromethanesulfonic anhydride (118 mg, 0.42 mmol) were added to a dichloromethane (4 mL) solution of compound 4e (100 mg, 0.28 mmol), and the reaction was carried out at room temperature for 1 h. Dichloromethane was removed by depressurization concentration, and the residue was added to ethyl acetate (20 mL), washed with saturated brine (2 × 10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under depressurization to obtain the crude product. The crude product was purified by column chromatography (petroleum ether/ethyl acetate) to obtain the title compound 4f (98 mg, yield 71%).

MS(ESI)m/z=490.2[M+H] ⁺ .

Step 5: Synthesis of 4g of methyl 6-fluoro-3-[2,4,5-trifluoro-3-(methylthio)phenyl]thiopheno[3,2-b]pyridine-2-carboxylate

Compound 4f (80 mg, 0.16 mmol), sodium methanethiol (57 mg, 0.82 mmol), palladium acetate (7.3 mg, 0.033 mmol), 1,1-binaphthyl-2,2-bis(diphenylphosphine) (20.4 mg, 0.033 mmol), and toluene (2 mL) were added to a round-bottom flask at room temperature. The mixture was heated to 120 °C and reacted overnight under a nitrogen atmosphere. After cooling the reaction mixture to room temperature, ethyl acetate (20 mL) was added, and the mixture was washed with saturated brine (2 × 10 mL). The solution was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (petroleum ether/ethyl acetate) to obtain 4 g (50 mg, yield 79%) of the title compound.

MS(ESI)m/z=388.2[M+H] ⁺ .

Step 6: Synthesis of 6-fluoro-3-[2,4,5-trifluoro-3-(methylthio)phenyl]thiopheno[3,2-b]pyridine- 2 -carboxylic acid

At room temperature, 4 g (50 mg, 0.13 mmol) of the compound was dissolved in a mixture of tetrahydrofuran (2 ml), methanol (1 ml) and water (1 ml), and lithium hydroxide (31 mg, 1.3 mmol) was added. The reaction mixture was stirred at room temperature for 1 h. Add water (30 mL), adjust pH to 4-5 with hydrochloric acid (1 N, 4 mL), extract with ethyl acetate (2 × 20 mL), combine organic phases, wash with saturated brine (2 × 20 mL), dry with anhydrous sodium sulfate, filter, concentrate under reduced pressure to obtain crude product, and purify by preparative high performance liquid chromatography (column: Waters Xbridge, 30 × 250 mm. Mobile phase A: 0.1% formic acid aqueous solution; mobile phase B: acetonitrile; gradient: 40%-95% for 18 min, flow rate: 30 mL/min. Elution time: 10.99 min) to obtain title compound 4 (18 mg, yield 37%).

MS(ESI)m/z=374.2[M+H] ⁺ .

¹ H-NMR (400MHz, DMSO- d ₆ ) δ 8.80 (s, 1H), 8.65 (d, 1H), 2.52 (s, 3H).

¹⁹ F-NMR (376MHz, DMSO- d ₆ ) δ -110.02(d,1F), -127.38(s,1F), -129.29(d,1F), -142.40(dd,1F).

Implementation Example 5

6-Fluoro-3-(2,4,5-trifluoro-3-methoxyphenyl)thieno[3,2-b]pyridine-2-carboxylic acid ( 5 )

Using the synthetic method in Example 1, 6-fluoro-3-(2,4,5-trifluoro-3-methoxyphenyl)thiopheno[3,2-b]pyridine-2-carboxylic acid ( 5 ) was prepared by replacing compound 1d with compound 4b .

Preparation of Compound 5 : High-performance liquid chromatography (HPLC) separation and purification conditions: Column: Waters Xbridge, 30×250mm. Mobile phase A: 0.1% formic acid aqueous solution; Mobile phase B: acetonitrile; Gradient: 40%-90% for 18 min, flow rate: 30 ml/min. Elution time: 10.5 min.

MS(ESI)m/z=358.30[M+H] ⁺ .

¹ H-NMR (400MHz, DMSO- d ₆ ) δ 8.80 (d, 1H), 8.64 (dd, 1H), 7.44-7.38 (m, 1H), 4.01 (s, 3H).

¹⁹ F-NMR (376MHz, DMSO- d ₆ ) δ -127.42(s,1F), -132.32(dd,1F), -142.65(dd,1F), -151.72(dd,1F).

Implementation Example 6

3-(3-ethyl-2,4,5-trifluorophenyl)-6-fluorothieno[3,2-b]pyridine-2-carboxylic acid ( 6 )

Step 1: Synthesis of 6 g of methyl 6-fluoro-3-(2,4,5-trifluoro-3-vinylphenyl)thiopheno[3,2-b]pyridine-2-carboxylate

Compound 4f (160 mg, 0.32 mmol), potassium trifluoroborate (127 mg, 0.95 mmol), tris(dibenzylacetone)dipalladium (28.9 mg, 0.032 mmol), 2-biscyclohexylphosphine-2',6'-dimethoxybiphenyl (13 mg, 0.032 mmol), and potassium phosphate (168 mg, 0.79 mmol) were dissolved in toluene (5 mL) at room temperature and reacted at 100 °C for 1 hour under nitrogen atmosphere. The reaction solution was poured into water (50 mL), extracted with ethyl acetate (3 × 30 mL), the organic phases were combined, washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (petroleum ether/ethyl acetate) to give 6 g (112 mg, yield 95%).

MS(ESI)m/z=368.2[M+H] ⁺ .

Step 2: Synthesis of methyl 3-(3-ethyl-2,4,5-trifluorophenyl)-6-fluorothiopheno[3,2-b]pyridine-2-carboxylate 6h

At room temperature, 6 g (100 mg, 0.26 mmol) of the compound and 10% palladium carbon (29 mg, 0.27 mmol) were dissolved in methanol (15 mL) and stirred for 2 hours under a hydrogen atmosphere. The reaction solution was directly filtered, and the filtrate was concentrated under reduced pressure to give crude product 6 h (95 mg, 98%).

MS(ESI)m/z=370.2[M+H] ⁺ .

Step 3: Synthesis of 3-(3-ethyl-2,4,5-trifluorophenyl)-6-fluorothiopheno[3,2-b]pyridine-2-carboxylic acid 6

At room temperature, compound 6h (100 mg, 0.27 mmol) was dissolved in a mixture of tetrahydrofuran (6 ml), methanol (3 ml) and water (3 ml), and lithium hydroxide (114 mg, 2.71 mmol) was added. The reaction mixture was stirred at room temperature for 1 h. Add water (30 mL), adjust pH to 4-5 with hydrochloric acid (1 N, 4 mL), extract with ethyl acetate (3 × 30 mL), combine organic phases, wash with saturated brine (2 × 20 mL), dry with anhydrous sodium sulfate, filter, concentrate under reduced pressure to obtain crude product, and purify by preparative high performance liquid chromatography (column: Waters Xbridge, 30 × 250 mm. Mobile phase A: 0.1% formic acid aqueous solution; mobile phase B: acetonitrile; gradient: 40%-95% for 18 min, flow rate: 30 mL/min. Elution time: 11.52 min) to obtain title compound 6 (26.8 mg, yield 27%).

MS(ESI)m/z=356.2[M+H] ⁺ .

¹ H-NMR (400MHz, DMSO- d ₆ ) δ 8.79 (s, 1H), 8.63 (d, 1H), 7.52 (dd, 1H), 2.73 (q, 2H), 1.19 (q, 3H).

¹⁹ F-NMR (376MHz, DMSO- d ₆ ) δ -120.81 (dd, 1F), -127.60 (s, 1F), -140.39 (dd, 1F) - 144.30 (dd, 1F).

Implementation Example 7

6-Chloro-3-(3-ethyl-2,4,5-trifluorophenyl)thieno[3,2-b]pyridine-2-carboxylic acid ( 7 )

Step 1: Synthesis of methyl 6-chloro-3-(2,4,5-trifluoro-3-hydroxyphenyl)thiopheno[3,2-b]pyridine-2-carboxylate 7e

At room temperature, compounds 1d (500 mg, 1.64 mmol) and 4d (676 mg, 2.47 mmol) were dissolved in 1,4-dioxane (5 mL) and water (0.80 mL), and 1,1'-di-tert-butylphosphinoferrocene palladium dichloride (103 mg, 0.16 mmol) and potassium carbonate (680 mg, 4.92 mmol) were added. The reaction mixture was stirred at 100 °C for 1 h under nitrogen atmosphere. After cooling to room temperature, ethyl acetate (40 mL) was added, followed by washing with saturated brine (2 × 15 mL), drying with anhydrous sodium sulfate, filtration, and concentration under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (petroleum ether/ethyl acetate) to obtain compound 7e (171 mg, yield 28%).

MS(ESI)m/z=374.1[M+H] ⁺ .

Step 2: Synthesis of 2,5,6-trifluoro-3-[6-chloro-2-(methoxycarbonyl)thiopheno[3,2-b]pyridin-3-yl]phenyl ester 7f

At room temperature, triethylamine (255 mg, 1.38 mmol) and trifluoromethanesulfonic anhydride (177 mg, 0.69 mmol) were added to a dichloromethane (6 mL) solution of compound 7e (171 mg, 0.46 mmol), and the reaction was carried out at room temperature for 1 h. Dichloromethane was removed by reduced pressure concentration, and the residue was added to ethyl acetate (40.0 mL), washed with saturated brine (2 × 20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (petroleum ether/ethyl acetate) to obtain compound 7f (200 mg, yield 86%).

MS(ESI)m/z=506.1[M+H] ⁺ .

Using the synthetic method in Example 6, 6-chloro-3-(3-ethyl-2,4,5-trifluorophenyl)thiopheno[3,2-b]pyridine-2-carboxylic acid ( 7 ) was synthesized from compound 7f in three steps.

Preparation of Compound 7 : High-performance liquid chromatography (HPLC) separation and purification conditions: Column: Waters Xbridge, 30×250mm. Mobile phase A: 0.1% formic acid aqueous solution; Mobile phase B: acetonitrile; Gradient: 45%-95% for 18 min, flow rate: 30 ml/min. Elution time: 12.37 min.

MS(ESI)m/z=372.1[M+H] ⁺ .

¹ H-NMR (400MHz, DMSO- d ₆ ) δ 8.86 (s, 1H), 8.78 (s, 1H), 7.52 (q, 1H), 2.73 (q, 2H), 1.19 (t, 3H).

¹⁹ F-NMR (376MHz, DMSO- d ₆ ) δ -120.80(dd,1F), -140.28(dd,1F), -144.24(dd,1F).

Implementation Example 8

3-{6-chloro-3-[(difluoromethyl)oxy]-2,4-difluorophenyl}-6-fluorothieno[3,2- b ]pyridine-2-carboxylic acid ( 8 )

Step 1: Synthesis of (4-chloro-2,6-difluorophenoxy)dimethylsilane tributyl ester 8b

Under ice bath conditions, triethylamine (9.2 g, 91.2 mmol) was added to a dichloromethane (200 mL) solution of compound 8a (10 g, 60.8 mmol). After stirring for 10 minutes, tributyldimethylsilyltrifluoromethanesulfonate (24.1 g, 91.2 mmol) was added dropwise, and the mixture was stirred at room temperature for 1 hour after the addition was complete. The reaction solution was washed with saturated brine (3 × 80 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (petroleum ether/ethyl acetate) to give an oily compound 8b (15.1 g, 89% yield).

Step 2: Synthesis of (3-((tributyldimethylsilyl)oxy)-6-chloro-2,4-difluorophenyl)boronic acid 8c

At -78°C under a nitrogen atmosphere, n-butyllithium (2.5M, 23.6mL, 59.1mmol) was slowly added dropwise to an anhydrous tetrahydrofuran (200mL) solution of compound 8b (15g, 53.8mmol). After the addition was completed, the mixture was stirred at -78°C for 1 hour. Then, an anhydrous tetrahydrofuran (20mL) solution of trimethyl borate (7.3g, 70mmol) was slowly added dropwise. After the addition was completed, the mixture was stirred at -78°C for another 1 hour. The reaction solution was slowly added to a saturated ammonium chloride solution (200 mL) stirred in an ice bath. After 5 minutes, ethyl acetate (100 mL) was added, and the mixture was thoroughly mixed. The organic phase was then separated and washed with saturated brine (3 × 80 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (petroleum ether/ethyl acetate) to give an oily substance 8c (11.2 g, yield 64%).

¹ H-NMR (400MHz, DMSO- d ₆ ) δ 8.71 (s, 2H), 7.28 (d, 1H), 0.98 (s, 9H), 0.18 (s, 6H).

Step 3: Synthesis of methyl 3-(3-((tributyldimethylsilyl)oxy)-6-chloro-2,4-difluorophenyl)-6-fluorothieno[3,2-b]pyridine-2-carboxylate 8d

At room temperature, compounds 8c (334 mg, 1.03 mmol) and 5d (300 mg, 1.03 mmol) were dissolved in toluene (5 mL) and water (1 mL), and tris(dibenzylacetone)dipalladium (94.6 mg, 0.10 mmol), 2-biscyclohexylphosphine-2',6'-dimethoxybiphenyl (84.9 mg, 0.20 mmol), and potassium phosphate (548 mg, 2.6 mmol) were added. The reaction mixture was stirred at 100 °C for 1 h under a nitrogen atmosphere. After cooling to room temperature, ethyl acetate (40 mL) was added, followed by washing with saturated brine (2 × 20 mL), drying with anhydrous sodium sulfate, filtration, and concentration under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (petroleum ether/ethyl acetate) to obtain the title compound 8d (197 mg, yield 39%).

MS(ESI)m/z=488.4[M+H] ⁺ .

Step 4: Synthesis of methyl 3-(6-chloro-2,4-difluoro-3-hydroxyphenyl)-6-fluorothiopheno[3,2-b]pyridine-2-carboxylate 8e

Compound 8d (180 mg, 0.37 mmol) and tetrabutylammonium fluoride (1 M, 0.6 mL, 0.6 mmol) were dissolved in tetrahydrofuran (10 mL) at room temperature and stirred for 2 hours at room temperature. The reaction solution was poured into water (30 mL), extracted with ethyl acetate (3 × 20 mL), the organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give crude product 8e (201 mg, crude product yield 145%).

MS(ESI)m/z=374.2[M+H] ⁺ .

Step 5: Synthesis of methyl 3-{6-chloro-3-[(difluoromethyl)oxy]-2,4-difluorophenyl}-6-fluorothieno[3,2-b]pyridine-2-carboxylate 8f

Compound 8e (180 mg, 0.48 mmol), sodium difluorochloroacetate (147 mg, 0.96 mmol), and potassium carbonate (80 mg, 0.58 mmol) were dissolved in N,N-dimethylformamide (5 mL) at room temperature and reacted at 100 °C for 2 hours under a nitrogen atmosphere. The reaction solution was poured into water (30 mL), extracted with ethyl acetate (3 × 20 mL), and the organic phases were combined. The mixture was washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure to obtain a crude product, and then purified by column chromatography (petroleum ether/ethyl acetate) to obtain compound 8f (100 mg, 49%).

MS(ESI)m/z=424.2[M+H] ⁺ .

Step 6: Synthesis of 3-{6-chloro-3-[(difluoromethyl)oxy]-2,4-difluorophenyl}-6-fluorothieno[3,2-b]pyridine-2-carboxylic acid 8

Compound 8f (100 mg, 0.236 mmol) was dissolved in a mixture of tetrahydrofuran (4 ml), methanol (2 ml) and water (2 ml) at room temperature. Lithium hydroxide (100 mg, 2.4 mmol) was added, and the reaction mixture was stirred at room temperature for 2 h. Add water (30 mL), adjust pH to 4-5 with hydrochloric acid (1 N), extract with ethyl acetate (2 × 30 mL), combine organic phases, wash with saturated brine (2 × 20 mL), dry with anhydrous sodium sulfate, filter, concentrate under reduced pressure to obtain crude product, and purify by preparative high performance liquid chromatography (column: Waters Xbridge, 30 × 250 mm. Mobile phase A: 0.1% formic acid aqueous solution; mobile phase B: acetonitrile; gradient: 35%-90% for 18 min, flow rate: 30 mL/min. Elution time: 11.37 min) to obtain title compound 8 (27.8 mg, yield 28%).

MS(ESI)m/z=410.1[M+H] ⁺ .

¹ H-NMR (400MHz, DMSO- d ₆ ) δ 8.78 (s, 1H), 8.67 (d, 1H), 7.84 (d, 1H), 7.34 (t, 1H).

¹⁹ F-NMR (376MHz, DMSO- d ₆ ) δ -82.76 (t, 2F), -121.98 (dd, 1F), -124.08 (s, 1F), -126.95 (s, 1F).

Implementation Example 9

3-{3-[(difluoromethyl)oxy]-2,4,5-trifluorophenyl}-6-fluorothieno[3,2-b]pyridine-2-carboxylic acid

Using the synthetic method in Example 8, compound 4e was used instead of compound 8e to prepare 3-{3-[(difluoromethyl)oxy]-2,4,5-trifluorophenyl}-6-fluorothieno[3,2-b]pyridine-2-carboxylic acid ( 9 ).

Preparation of Compound 9 : High-performance liquid chromatography (HPLC) separation and purification conditions: Column: Waters Xbridge, 30 × 250 mm. Mobile phase A: 0.1% formic acid aqueous solution; Mobile phase B: acetonitrile; Gradient: 25%-70% for 18 min, flow rate: 30 ml/min. Elution time: 11.72 min.

MS(ESI)m/z=392.1[MH] ^- .

¹ H-NMR (400MHz, DMSO- d ₆ ) δ 8.81 (s, 1H), 8.65 (d, 1H), 7.76 (dd, 1H), 7.36 (t, 1H).

¹⁹ F-NMR (376MHz, DMSO- d ₆ ) δ -82.48(s,2F),-127.30(3,1F),-129.43~-129.50(m,1F),-141.61(dd,1F),-147.72(d,1F).

Implementation Example 10

6-Chloro-3-{3-[(difluoromethyl)oxy]-2,4,5-trifluorophenyl}thieno[3,2-b]pyridine-2-carboxylic acid ( 10 )

Using the synthetic method in Example 8, compound 7e was used instead of compound 8e to prepare 6-chloro-3-{3-[(difluoromethyl)oxy]-2,4,5-trifluorophenyl}thiopheno[3,2-b]pyridine-2-carboxylic acid ( 10 ).

Preparation of Compound 10 : High-performance liquid chromatography (HPLC) separation and purification conditions: Column: Waters Xbridge, 30×250mm. Mobile phase A: 0.1% formic acid aqueous solution; Mobile phase B: acetonitrile; Gradient: 45%-90% for 18 min, flow rate: 30 ml/min. Elution time: 11.28 min.

MS(ESI)m/z=392.1[MH] ^- .

¹ H-NMR (400MHz, DMSO- d ₆ ) δ 8.81 (s, 1H), 8.65 (d, 1H), 7.76 (dd, 1H), 7.36 (t, 1H).

¹⁹ F-NMR (376MHz, DMSO- d ₆ ) δ -82.48(s,2F),-127.30(3,1F),-129.43~-129.50(m,1F),-141.61(dd,1F),-147.72(d,1F).

Implementation Example 11

6-Chloro-3-{6-chloro-3-[(difluoromethyl)oxy]-2,4-difluorophenyl}thieno[3,2-b]pyridine-2-carboxylic acid ( 11 )

Using the synthesis method in Example 8, compound 1d was used instead of compound 5d to prepare 6-chloro-3-{6-chloro-3-[(difluoromethyl)oxy]-2,4-difluorophenyl}thieno[3,2-b]pyridine-2-carboxylic acid ( 11 ).

Preparation of Compound 11 : High-performance liquid chromatography (HPLC) separation and purification conditions: Column: Waters Xbridge, 30×250mm. Mobile phase A: 0.1% formic acid aqueous solution; Mobile phase B: acetonitrile; Gradient: 50%-95% for 18 min, flow rate: 30 ml/min. Elution time: 10.18 min.

MS(ESI)m/z=426.1[M+H] ⁺ .

¹ H-NMR (400MHz, DMSO- d ₆ ) δ 8.89 (s, 1H), 8.77 (s, 1H), 7.83 (d, 1H), 7.33 (t, 1H).

¹⁹ F-NMR (376MHz, DMSO- d ₆ ) δ -82.75 (t, 2F), -121.93 (d, 1F), -124.02 (s, 1F).

Implementation Example 12

6-Chloro-3-(4-chloro-2-fluoro-3-methoxyphenyl)thieno[3,2-b]pyridine-2-carboxylic acid ( 12 )

Using the synthetic method in Example 8 , 6-chloro-3-(4-chloro-2-fluoro-3-methoxyphenyl)thieno[3,2-b]pyridine-2-carboxylic acid ( 12 ) was prepared by replacing compound 5d with compound 1d and compound 8c with compound 12a .

Preparation of Compound 12 : High-performance liquid chromatography (HPLC) separation and purification conditions: Column: Waters Xbridge, 30×250mm. Mobile phase A: 0.1% formic acid aqueous solution; Mobile phase B: acetonitrile; Gradient: 50%-90% for 18 min, flow rate: 30 ml/min. Elution time: 10.65 min.

MS(ESI)m/z=372.2[M+H] ⁺ .

¹ H-NMR (400MHz, DMSO- d ₆ ) δ 8.86 (s, 1H), 8.76 (s, 1H), 7.43 (d, 1H), 7.24 (t, 1H), 3.90 (s, 3H).

¹⁹ F-NMR (376MHz, DMSO- d ₆ ) δ -127.61 (s, 1F).

Implementation Example 13

6-Chloro-3-(4-chloro-2-fluoro-3-methylphenyl)thieno[3,2-b]pyridine-2-carboxylic acid ( 13 )

6-chloro-3-(4-chloro-2-fluoro-3-methylphenyl)thieno[3,2-b]pyridine-2-carboxylic acid ( 13 ) was synthesized using the synthetic method described in Example 8.

Preparation of Compound 13 : High-performance liquid chromatography (HPLC) separation and purification conditions: Column: Waters Xbridge, 30×250mm. Mobile phase A: 0.1% formic acid aqueous solution; Mobile phase B: acetonitrile; Gradient: 50%-95% for 18 min, flow rate: 30 ml/min. Elution time: 11.22 min.

MS(ESI)m/z=356.1[M+H] ⁺ .

¹ H-NMR (400MHz, DMSO- d ₆ ) δ 8.85 (s, 1H), 8.74 (s, 1H), 7.40 (d, 1H), 7.33 (t, 1H), 2.31 (s, 3H).

¹⁹ F-NMR (376MHz, DMSO- d ₆ ) δ -112.58 (s, 1F).

Implementation Example 14

6-Chloro-3-{6-chloro-3-[(difluoromethyl)oxy]-2,4-difluorophenyl}thieno[3,2-b]pyridine-2-carboxylic acid ( 11-P1 ); 6-Chloro-3-{6-chloro-3-[(difluoromethyl)oxy]-2,4-difluorophenyl}thieno[3,2-b]pyridine-2-carboxylic acid ( 11-P2 )

Compound 11 (60 mg) was separated using SFC. Separation method: column: ChiralPak IC, 250 × 30 mm ID, 5 μm; mobile phase: A: _CO2 ; B: isopropanol [0.1% _NH3 (7 M MeOH solution)]; gradient: B 20%; flow rate: 100 mL/min; back pressure: 100 bar; column temperature: 35 °C; wavelength: 214 nm; circulation time: ~2.5 min.

11-P1 , retention time t=2.862 min, 24 mg;

11-P2 , retention time t=2.263 min, 18 mg.

Implementation Example 15

6-Chloro-3-(4-chloro-2,5-difluoro-3-methylphenyl)thieno[3,2-b]pyridine-2-carboxylic acid ( 15 )

Step 1: Synthesis of 2,5-difluoro-3-methylaniline 15b

Compound 15a (1.00 g, 5.78 mmol), iron powder (0.97 g, 17.37 mmol), and ammonium chloride (1.54 g, 28.88 mmol) were added to ethanol (10 mL) and water (3 mL) at room temperature and stirred overnight at room temperature under a nitrogen atmosphere. The reaction solution was filtered, and the residue was washed with ethyl acetate (3 × 30 mL). The filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (petroleum ether/ethyl acetate) to give compound 15b (700 mg, yield 84%).

MS(ESI)m/z=144.1[M+H] ⁺ .

Step 2: Synthesis of 4-chloro-2,5-difluoro-3-methylaniline 15c

Compound 15b (650 mg, 4.54 mmol) and N-chlorobutylideneimide (606 mg, 4.54 mmol) were dissolved in dichloromethane (10 mL) at room temperature and stirred overnight at 50 °C under nitrogen atmosphere. The reaction solution was concentrated under reduced pressure, and the residue was purified by column chromatography (petroleum ether/ethyl acetate) to give compound 15c (300 mg, yield 37%).

Step 3: Synthesis of 1-bromo-4-chloro-2,5-difluoro-3-methylbenzene 15d

At room temperature, tributyl nitrite (0.25 mL, 2.11 mmol) and copper bromide (408 mg, 1.83 mmol) were dissolved in acetonitrile (10 mL). Compound 15c (250 mg, 1.41 mmol) was then added in portions to the system, and the mixture was stirred at room temperature for 1 hour. The reaction solution was poured into water (30 mL), extracted with ethyl acetate (3 × 30 mL), washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (petroleum ether/ethyl acetate) to give compound 15d (300 mg, 88% yield).

Step 4: Synthesis of methyl 6-chloro-3-(4-chloro-2,5-difluoro-3-methylphenyl)thieno[3,2-b]pyridine-2-carboxylate 15e

At room temperature, compound 15d (200 mg, 0.83 mmol), bis(diphenylphosphino)ferrocene palladium dichloride (210 mg, 0.83 mmol), and potassium acetate (203 mg, 2.07 mmol) were dissolved in 1,4-dioxane (5 mL) and stirred overnight at 80 °C under nitrogen atmosphere. Then, compound 1d (100 mg, 0.33 mmol), potassium carbonate (239 mg, 1.73 mmol), 1,1'-di-tert-butylphosphinoferrocene palladium dichloride (45 mg, 0.07 mmol), and water (1 mL) were added to the above system and stirred for 1 hour at 80 °C under nitrogen atmosphere. The reaction solution was poured into water (30 mL), extracted with ethyl acetate (3 × 30 mL), washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (petroleum ether/ethyl acetate) to give compound 15e (120 mg, yield 44%).

Step 5: Synthesis of 6-chloro-3-(4-chloro-2,5-difluoro-3-methylphenyl)thiopheno[3,2-b]pyridine-2-carboxylic acid 15

Compound 15e (120 mg, 0.31 mmol) and lithium hydroxide (130 mg, 3.09 mmol) were dissolved in a mixture of tetrahydrofuran (5 mL), methanol (2 mL) and water (2 mL) at room temperature and stirred for 1 hour at room temperature. Add water (30 mL), adjust pH to 4-5 with hydrochloric acid (1N, 4 mL), extract with ethyl acetate (2 × 20 mL), combine organic phases, wash with saturated brine (2 × 20 mL), dry with anhydrous sodium sulfate, filter, concentrate the filtrate under reduced pressure to obtain crude product, and purify by preparative high performance liquid chromatography (column: Waters Xbridge, 30 x 250 mm. Mobile phase A: 0.1% formic acid aqueous solution, mobile phase B: acetonitrile; gradient: 45%-95% for 15 min, flow rate: 30 mL/min. Elution time: 9.85 min) to obtain title compound 15 (54 mg, yield 46%).

MS(ESI)m/z=374.1[M+H] ⁺ .

¹ H-NMR (400MHz, DMSO- d ₆ ) δ 8.86 (s, 1H), 8.76 (s, 1H), 7.50 (t, 1H), 2.35 (s, 3H).

¹⁹ F-NMR (376MHz, DMSO- d ₆ ) δ -117.41 (d, 1F), -120.46 (d, 1F).

Implementation Example 16

6-Chloro-3-(5-chloro-2,4-difluoro-3-methoxyphenyl)thieno[3,2-b]pyridine-2-carboxylic acid ( 16 )

Using the synthetic method in Example 1, 6-chloro-3-(5-chloro-2,4-difluoro-3-methoxyphenyl)thieno[3,2-b]pyridine-2-carboxylic acid ( 16 ) was prepared by replacing compound 1e with compound 16a .

Preparation of Compound 16 : High-performance liquid chromatography (HPLC) separation and purification conditions: Column: Waters Xbridge, 30x250mm. Mobile phase A: 0.1% formic acid aqueous solution; Mobile phase B: acetonitrile; Gradient: 40%-95% for 18 min; Flow rate: 30 ml/min. Elution time: 12.26 min.

MS(ESI)m/z=390.1[M+H] ⁺ .

¹ H-NMR (400MHz, DMSO- d ₆ ) δ 8.87 (s, 1H), 8.79 (s, 1H), 7.50 (t, 1H), 3.99 (s, 3H).

¹⁹ F-NMR (376MHz, DMSO- d ₆ ) δ -127.96 (s, 1F), -128.99 (s, 1F).

Implementation Example 17

6-Chloro-3-(5-chloro-2,4-difluoro-3-methylphenyl)thieno[3,2-b]pyridine-2-carboxylic acid ( 17 )

Step 1: Synthesis of 6-chloro-2,4-difluoro-3-methylaniline 17b

Compound 17a (2 g, 13.97 mmol) and N-chlorobutylideneimide (1.96 mg, 14.67 mmol) were dissolved in dichloromethane (20 mL) at room temperature and stirred overnight at 50 °C. The solvent was removed by concentration, and the residue was added to ethyl acetate (50 mL), then washed with water (1 × 10 mL) and saturated brine (2 × 10 mL), and dried over anhydrous sodium sulfate. The crude product was concentrated under reduced pressure and then purified by column chromatography (petroleum ether/ethyl acetate) to give 17b (700 mg, yield 28%).

Step 2: Synthesis of 2-bromo-1-chloro-3,5-difluoro-4-methylbenzene 17c

At room temperature, copper bromide (973 mg, 4.34 mmol) and acetonitrile (15 mL) were added to a three-necked flask, and tributyl nitrite (528 mg, 5.12 mmol) was added dropwise under a nitrogen atmosphere. A suspension of compound 17b (700 mg, 3.94 mmol) in acetonitrile (4 mL) was added dropwise at 20 °C. After addition, the mixture was stirred at room temperature for 2 hours. Extraction was performed with dilute hydrochloric acid (1 N, 10 mL) and ethyl acetate (2 × 20 mL). The mixture was washed successively with water (1 × 10 mL) and saturated brine (2 × 10 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure to obtain a crude product, and then purified by column chromatography (petroleum ether/ethyl acetate) to give compound 17c (450 mg, yield 47%).

Step 3: Synthesis of 2-(6-chloro-2,4-difluoro-3-methylphenyl)-4,4,5,5-tetramethyl-1,3,2-dioxoborane-cyclopentane 17d

Under a nitrogen atmosphere and at the dry ice-acetonitrile system temperature, isopropyl magnesium chloride tetrahydrofuran solution (2M, 2.8mL) was slowly added dropwise to a tetrahydrofuran solution of compound 17c (450mg, 1.86mmol) (5mL). After stirring for 3 hours, 4,4,5,5-tetramethyl-2-(prop-2-yloxy)-1,3,2-dioxoborane (0.76mL, 3.73mmol) was slowly added dropwise to the reaction solution. After the addition was completed, the temperature was slowly raised to 20°C, and stirring was continued for 2 hours. Dilute hydrochloric acid (1N, 10mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (2 × 20mL). The sample was washed successively with water (1×10 ml) and saturated brine (2×10 ml), dried with anhydrous sodium sulfate, filtered, concentrated under reduced pressure to obtain a crude product, and then purified by column chromatography (petroleum ether/ethyl acetate) to obtain compound 17d (140 mg, yield 26%).

Step 4: Synthesis of methyl 6-chloro-3-(6-chloro-2,4-difluoro-3-methylphenyl)thienro[3,2-b]pyridine-2-carboxylate 17e

At room temperature, compounds 17d (100 mg, 0.32 mmol) and 1d (140 mg, 0.48 mmol) were dissolved in toluene/water (2 ml/0.5 ml), followed by the addition of tris(dibenzylacetone)dipalladium (30 mg, 0.032 mmol), 2-biscyclohexylphosphine-2',6'-dimethoxybiphenyl (29 mg, 0.064 mmol), and potassium phosphate (216 mg, 0.96 mmol). The mixture was stirred at 100 °C under nitrogen atmosphere for 4 hours. Ethyl acetate (60 ml) was added to the reaction system, and the mixture was washed successively with water (1 × 10 ml) and saturated brine (2 × 10 ml), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure to obtain a crude product, and then purified by column chromatography (petroleum ether/ethyl acetate) to obtain compound 17e (90 mg, yield 72%).

MS(ESI)m/z=388.2[M+H] ⁺ .

Step 5: Synthesis of 6-chloro-3-(5-chloro-2,4-difluoro-3-methylphenyl)thieno[3,2-b]pyridine-2-carboxylic acid 17

At room temperature, compound 17e (90 mg, 0.23 mmol) was dissolved in a mixture of tetrahydrofuran (2 ml), methanol (1 ml), and water (1 ml), and lithium hydroxide (56 mg, 2.3 mmol) was added and stirred for 1 hour. The pH was adjusted to 4-5 with dilute hydrochloric acid (1 N). Add ethyl acetate (40 ml), wash successively with water (1 × 10 ml) and saturated brine (2 × 10 ml), dry with anhydrous sodium sulfate, filter, concentrate under reduced pressure to obtain crude product 17 , and then purify by preparative high performance liquid chromatography (column: Waters Xbridge, 30 x 250 mm. Mobile phase A: 0.1% formic acid aqueous solution, mobile phase B: acetonitrile; flow rate: 30 ml/min, 18 min. Gradient: 40%-95%, retention time: 11.99 min) to obtain title compound 17 (37 mg, yield 43%).

MS(ESI)m/z=374.1[M+H] ⁺ .

¹ H-NMR (400MHz, DMSO- d ₆ ) δ 8.82 (s, 1H), 8.74 (s, 1H), 7.48 (s, 1H), 2.19 (s, 3H).

¹⁹ F-NMR (376MHz, DMSO- d ₆ ) δ -111.12 (s, 1F), δ = -112.74 (s, 1F).

Implementation Example 18

6-Chloro-3-{6-chloro-2,4-difluoro-3-[(trideuterylmethyl)oxy]phenyl}thieno[3,2-b]pyridine-2-carboxylic acid ( 18 ); 6-chloro-3-{6-chloro-2,4-difluoro-3-[(trideuterylmethyl)oxy]phenyl}thieno[3,2-b]pyridine-2-carboxylic acid ( 18-P1 ); 6-chloro-3-{6-chloro-2,4-difluoro-3-[(trideuterylmethyl)oxy]phenyl}thieno[3,2-b]pyridine-2-carboxylic acid ( 18-P2 )

Step 1: Synthesis of methyl 6-chloro-3-(6-chloro-2,4-difluoro-3-hydroxyphenyl)thiopheno[3,2-b]pyridine-2-carboxylate 18

Under ice bath conditions, compound 2b (5 g, 12.4 mmol) was dissolved in dichloromethane (10 mL), and a dichloromethane solution of boron tribromide (1 M, 50 mL) was slowly added dropwise. After addition, the mixture was stirred at room temperature for 2 hours. Methanol was then slowly added to the system under ice bath conditions, and the mixture was stirred for 10 minutes. The mixture was then concentrated under reduced pressure to obtain crude product 18a (4 g, 82% yield), which was used directly in the next step.

MS(ESI)m/z=390.3[M+H] ⁺ .

Step 2: Synthesis of methyl 18b of 6-chloro-3-(6-chloro-2,4-difluoro-3-(methoxy-d3)phenyl)thiopheno[3,2-b]pyridine-2-carboxylate

At room temperature, crude product 18a (4 g, 10.3 mmol), deuterated iodomethane (0.71 mL, 11.3 mmol), and potassium carbonate (2.1 g, 15.4 mmol) were dissolved in N,N-dimethylformamide (5 mL) and stirred overnight at 50 °C. Water (30 mL) was added, and the mixture was extracted with ethyl acetate (3 × 10 mL). The organic phases were combined, washed with saturated brine (2 × 10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain crude product 18b (3.5 g, yield 83%), which was used directly in the next step.

MS(ESI)m/z=407.3[M+H] ⁺ .

Step 3: Synthesis of 6-chloro-3-(6-chloro-2,4-difluoro-3-(methoxy-d3)phenyl)thiopheno[3,2-b]pyridine-2-carboxylic acid 18

Compound 18b (3.5 g, 8.6 mmol) was dissolved in a mixture of tetrahydrofuran (6 mL), methanol (3 mL) and water (3 mL) at room temperature, and lithium hydroxide (2.1 g, 87 mmol) was added. The reaction mixture was stirred at room temperature for 2 h. Add water (30 mL), adjust pH to 4-5 with hydrochloric acid (1 N), extract with ethyl acetate (2 × 30 mL), combine organic phases, wash with saturated brine (2 × 10 mL), dry with anhydrous sodium sulfate, filter, concentrate under reduced pressure to obtain crude product, and purify by preparative high performance liquid chromatography (column: Waters Xbridge, 30 × 250 mm. Mobile phase A: 0.1% formic acid aqueous solution; mobile phase B: acetonitrile; gradient: 35%-85% for 15 min, flow rate: 30 mL/min. Elution time: 9.82 min) to obtain title compound 18 (1.8 g, yield 52%).

MS(ESI)m/z=393.3[M+H] ⁺

¹ H-NMR (400MHz, DMSO- d ₆ ) δ 8.87 (d, 1H), 8.76 (d, 1H), 7.67 (d, 1H).

¹⁹ F-NMR (376MHz, DMSO- d ₆ ) δ -124.93 (s, 1F), -126.95 (s, 1F).

Step 4: 6-Chloro-3-{6-chloro-2,4-difluoro-3-[(trideuterylmethyl)oxy]phenyl}thieno[3,2-b]pyridine-2-carboxylic acid ( 18-P1 ); 6-Chloro-3-{6-chloro-2,4-difluoro-3-[(trideuterylmethyl)oxy]phenyl}thieno[3,2-b]pyridine-2-carboxylic acid ( 18-P2 )

Compound 18 (92 mg) was resolved by SFC. Resolution method: column: ChiralPak AD, 250×30 mm ID, 5 μm; mobile phase: A: _CO2 ; B: isopropanol [0.1% _NH3 (7 M MeOH solution)]; gradient: B 20%; flow rate: 100 mL/min; back pressure: 100 bar; column temperature: 35 °C; wavelength: 214 nm; circulation time: ~5 min.

18-P1 , retention time t=2.819 min, 32 mg;

18-P2 , retention time t=2.626 min, 34 mg.

Implementation Example 19

3-(6-chloro-2,4-difluoro-3-methoxyphenyl)-6-fluorothieno[3,2-b]pyridine-2-carboxylic acid ( 19 ); 3-(6-chloro-2,4-difluoro-3-methoxyphenyl)-6-fluorothieno[3,2-b]pyridine-2-carboxylic acid ( 19-P1 ); 3-(6-chloro-2,4-difluoro-3-methoxyphenyl)-6-fluorothieno[3,2-b]pyridine-2-carboxylic acid ( 19-P2 )

Step 1: Synthesis of 3-(6-chloro-2,4-difluoro-3-methoxyphenyl)-6-fluorothieno[3,2-b]pyridine-2-carboxylic acid ( 19 )

Using the synthetic method in Example 2 , 3-(6-chloro-2,4-difluoro-3-methoxyphenyl)-6-fluorothieno[3,2-b]pyridine-2-carboxylic acid ( 19 ) was prepared by replacing compound 1d with compound 5d .

Preparation of Compound 19 : High-performance liquid chromatography (HPLC) separation and purification conditions: Column: Waters Xbridge, 30 × 250 mm. Mobile phase A: 0.1% formic acid aqueous solution; Mobile phase B: acetonitrile; Gradient: 35%-95% for 18 min, flow rate: 30 mL/min. Elution time: 10.89 min.

MS(ESI)m/z=374.2[M+H] ⁺ .

¹ H-NMR (400MHz, DMSO- d ₆ ) δ 8.77 (d, 1H), 8.65 (dd, 1H), 7.61 (dd, 1H), 3.95 (s, 3H).

¹⁹ F-NMR (376MHz, DMSO- d ₆ ) δ -124.96(d,1F), -126.96(d,1F), -127.19(s,1F).

Step 2: 3-(6-chloro-2,4-difluoro-3-methoxyphenyl)-6-fluorothieno[3,2-b]pyridine-2-carboxylic acid ( 19-P1 ); 3-(6-chloro-2,4-difluoro-3-methoxyphenyl)-6-fluorothieno[3,2-b]pyridine-2-carboxylic acid ( 19-P2 )

Product 19 (520 mg) was separated using SFC. Separation method: column: ChiralPak IC, 250×30 mm ID, 5 μm; mobile phase: A: _CO2 ; B: methanol [0.2% TFA]; gradient: B 20%; flow rate: 100 mL/min; back pressure: 100 bar; column temperature: 35 °C; wavelength: 210 nm; circulation time: ~2 min.

19-P1 , retention time t=2.409 min, 240 mg;

19-P2 , retention time t=1.993 min, 234 mg.

Biological evaluation

The following test examples further illustrate the invention, but these examples are not intended to limit the scope of the invention.

Test Example 1: Detection of the inhibitory effect of the compound on BCKDK protein in HEK293 cells

1. Experimental Materials

2. Testing Methods

HEK293 cells were seeded at 10,000 cells/well in 384-well black permeable plates overnight. Cells were treated with different concentrations of the compound (100 μM maximum dose, 3-fold dilution, 10 concentrations) at 37°C and 5% CO2 for 4 hours. Pre-chilled 8% paraformaldehyde (50 μL/well) was added to each well, and the plates were fixed in the dark for 1 hour. The fixative was then removed and discarded. Permeabilization solution (50 μL/well) was added, and the plates were incubated for 5 minutes each time, repeated 6 times. Then, 50 μL/well blocking buffer was added, and the plates were incubated on a shaker at room temperature for 1 hour. Primary antibody was incubated with Phospho-BCKDH-E1α (Ser293) (1000X) at 4°C for 24 hours. After repeating the permeabilization procedure again, the secondary antibody Cell Tag520 (500X) & IRDye 800CW (1000X) were incubated at room temperature in the dark for 1 hour. The permeabilization procedure was repeated again, and the permeabilization solution was aspirated for the last time. The signal was detected using an Odyssey imager M imaging system, and the data were processed and _IC50 was calculated using XL fit.

3. Experimental Results

The inhibitory effect of the disclosed compound on the BCKDK protein in HEK293 cells can be determined by the above experiments, and the measured _IC50 is shown in Table 1.

PF-07328948 is compound of embodiment 9 in patent WO2023100061A1, with the following structure: .

Test Example 2: Inhibitory activity of compounds against human CYP450 (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A)

1. Equipment, materials, and reagents

1) Human liver microsomes (HLM)

Human liver microsomes (source: BioIVT) are stored at -80°C. Before use, remove the human liver microsomes from the freezer and preheat them in a 37°C water bath until thawed, then place them on ice until ready to use.

2) Acceptance

The preparation details of the acceptor-subject mixture are as follows: the acceptor-subject mixture consists of 50% pure water and 50% organic solvent. The prepared acceptor-subject mixture is stored in a -20°C freezer. Before use, it is removed from the freezer and warmed to room temperature, then vortexed for 30 seconds before use.

Mixed solutions of CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A substrates

3) Phosphate buffer (100 mmol/L, pH 7.4)

Sodium bihydrogen phosphate (analytical grade) and potassium dihydrogen phosphate (analytical grade) were purchased from a local supplier. Solution A preparation: Accurately weigh 7.098 g of sodium bihydrogen phosphate and add it to 500 ml of purified water for ultrasonic treatment. Solution B preparation: Accurately weigh 3.400 g of potassium dihydrogen phosphate and add it to 250 ml of purified water for ultrasonic treatment. Add solution B to solution A to achieve a final pH of 7.4.

4) 10 mmol/L NADPH solution

NADPH (MW: 833.4 g/mol) was purchased from MCE and dissolved in phosphate buffer at a concentration of 8.334 mg/mL.

2. Experimental Procedure

1) Preparation of incubation solution

The incubation system for the mixture of the receptor and human liver microsomes is as follows:

2) Compound dilution

15 μL of a 10 mmol/L DMSO solution of the test compound was added to a sequentially labeled deep-well plate of the compound. The dilution procedure is as follows:

3) Incubation

Each well of the 96-well deep-well plate contains 179 μL of a mixture of the test sample and human liver microsomal phosphate buffer, and 1 μL of either the test sample or a blank solution. The plate is pre-incubated in a 37°C water bath for 5 minutes, then 20 μL of 10 mM NADPH is added to initiate the reaction. After adding the NADPH solution, the plate is incubated at 37°C for another 5 minutes.

4) Reaction quenching

Quenching was performed by adding 300 μL of acetonitrile solution containing 3% formic acid and 200 nM tolbutamide, 200 nM alprazolam, and 200 nM labetalol. The mixture was then centrifuged at 3,220 g for 50 min, and 200 μL of the supernatant was analyzed by LC/MS/MS.

3. Data Processing

The system automatically calculates the peak area and internal standard peak area of all samples and imports them into Excel.

Calculate the percentage of remaining activity using the following formula:

Area ratio = Peak area of _{test sample} / Peak area of _{internal standard}

Residual activity percentage (%) = Area ratio of _{test substance} / Area ratio of _blank × 100

Use Excel XLfit 5.3.1.3 to calculate the _IC50 value (the concentration of the test substance at which 50% inhibition is achieved).

Reference 1 is compound 16 of embodiment 1 in patent WO2023100061A1.

The results showed that compound 19-P1 did not inhibit CYP1A2, CYP2C9, CYP2C19, CYP2D6, or CYP3A (IC ₅₀ > 30 μM), while PF-07328948 strongly inhibited CYP2C9 (IC ₅₀ = 1.09 μM). Reference compound 1 showed strong inhibition against CYP1A2 (IC ₅₀ = 5.0 μM), CYP2C9 (IC ₅₀ > 2.32 μM), and CYP2C19 (IC ₅₀ = 15.76 μM), respectively.

Test Example 3: Stability test of the compound in hepatocytes

1. Experimental Procedure

1) Prepare a high-concentration (10 mM) stock solution of the test substance using DMSO. Dilute to a working solution of 100 μM with DMSO before use. The final concentration of the test substance is 1 μM.

2) Take a tube of cryopreserved liver cells (rat, dog, and human liver cells derived from BioIVT; monkey liver cells derived from RILD), ensuring the liver cells remain cryopreserved before resuscitation. Quickly place the liver cells in a 37°C water bath and gently shake until all ice crystals are dispersed. Spray with 70% ethanol and transfer to a biosafety cabinet.

3) Pour the contents of hepatocyte tubules from different species into a centrifuge tube containing 50 mL of resuscitation medium and centrifuge at 100 g for 10 minutes. After centrifugation, aspirate the resuscitation medium and add sufficient incubation medium to obtain a cell suspension with a cell density of approximately 1.0 × ^10⁶ cells/mL.

4) Count the hepatocytes and determine the viable cell density using Cellometer Vision. The viability of the hepatocytes must be greater than 75%. Dilute the hepatocyte suspension with incubation medium to a viable cell density of 0.5 × ^10⁶ cells/mL.

5) Transfer 198 μL of live cell suspension to a 96-well deep-well plate, place the plate on a vortex, and preheat in an incubator for 10 minutes. Perform double-parallel incubation.

6) Add 2 μL of 100 μM test substance to each well to initiate the reaction, then place the deep-well plate back onto the incubator vortex generator.

7) Incubate the sample at 0, 15, 30, 60, 90, and 120 minutes. Take 25 μL of the suspension and add 150 μL of acetonitrile containing the internal standard to terminate the reaction. Vortex for 10 minutes, then centrifuge at 3220 g and 4°C for 45 minutes. Transfer 100 μL of the supernatant to the sample plate, add 100 μL of pure water, mix well, and use for UPLC-MS/MS analysis.

2. Data Analysis

All data calculations were performed using Microsoft Excel. Peak areas were detected by extracting ion spectra. The in vitro clearance rate of the parent drug was determined by linearly fitting the natural logarithm of the percentage of elimination of the parent drug to time.

In vitro clearance (μL/min/ ^10⁶ cells) was calculated using the following formula:

In vitro CL _int = kV/N

V = Incubation volume per well (0.2 mL);

N = Number of cells per pore (0.1 × ^10⁶ cells)

The results showed that compound 19-P1 exhibited significantly better metabolic stability in canine, monkey, and human liver cells compared to PF-07328948 and reference compound 1 .

Test Example 4: Drug Metabolic Kinetics Experiments in Rats and Dogs

1. Drug metabolism kinetics experiment in rats

SD rats (source: Zhejiang Vital River Laboratory Animal Technology Co., Ltd.) were used as test animals. The plasma concentrations of the test drug at different time points after gavage administration were determined using LC/MS/MS. The pharmacokinetic behavior of the test drug in rats was investigated to evaluate its pharmacokinetic characteristics.

Experimental animals: Two healthy male rats (200-300g) aged 6-8 weeks in each group.

Drug preparation: Weigh a certain amount of the drug and prepare a colorless, clear solution at a concentration of 2 mg/mL (solvent: 100% physiological saline and 1 mol/L hydrochloric acid and sodium hydroxide; adjust the final solution to ~pH 7).

Administration: After fasting overnight, rats were administered the drug via gavage at a dose of 20 mg/kg.

Procedure: Rats were administered the test drug by gavage. Approximately 0.2 mL of blood was collected via jugular vein at 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours post-administration. The plasma was placed in EDTA-K2-containing test tubes and centrifuged at approximately 4°C, 4000 g/min for 5 minutes to obtain plasma, which was then stored at -75±15°C.

Determination of the analyte concentration in rat plasma after oral administration: Take 50 μL of rat plasma at each time point after administration, add 5 μL of blank solution and 200 μL of acetonitrile solution containing dexamethasone as internal standard, vortex mix for 30 seconds, centrifuge for 15 minutes (3900 rpm), and then add 100 μL of the supernatant to 200 μL of ultrapure water and mix thoroughly. Finally, inject 5 μL of the diluted solution using a syringe for LC/MS/MS analysis.

The results showed that the oral exposure of compound 19-P1 in rats was significantly lower than that of PF-07328948 .

2. Experiments on the pharmacokinetic dynamics of drugs in dogs

Beagle dogs (source: Beijing Mas Biotechnology Co., Ltd.) were used as test animals. The plasma concentrations of the test drug at different time points after gavage administration were determined using LC/MS/MS. The pharmacokinetic behavior of the test drug in beagle dogs was investigated, and its pharmacokinetic characteristics were evaluated.

Experimental animals: Two healthy male Beagles aged 8 months to 3 years (6.0-13.0 kg) per group.

Drug preparation: Weigh a certain amount of the drug and prepare a colorless, clear solution at a concentration of 0.6 mg/mL (solvent: 100% physiological saline and 1 mol/L hydrochloric acid and sodium hydroxide; adjust the final solution to ~pH 7).

Administration: After the beagle has been fasted overnight, administer the drug via gavage at a dose of 3 mg/kg.

Procedure: The test drug was administered to beagle dogs via gavage. Approximately 0.6 mL of blood was collected via peripheral vein puncture before administration and at 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours after administration. The plasma was placed in a test tube containing EDTA-K2 and centrifuged at approximately 2000 g/min for 10 minutes at around 4°C. The plasma was then stored at -75±15°C.

Determination of the analyte concentration in beagle dog plasma after oral administration: Take 50 μL of beagle dog plasma at each time point after administration, add 5 μL of blank solution and 200 μL of acetonitrile solution containing dexamethasone as internal standard, vortex mix for 30 seconds, centrifuge for 15 minutes (3900 rpm), and then add 100 μL of the supernatant to 200 μL of ultrapure water and mix thoroughly. Finally, inject 5 μL of the diluted solution using a syringe for LC/MS/MS analysis.

The results showed that compound 19-P1 had a significantly lower oral exposure in dogs than PF-07328948 .

Claims (38)

A compound of formula (I) or a pharmaceutically acceptable salt thereof, ^R1 , ^R2 , and ^R3 are each independently selected from hydrogen, fluorine, chlorine, or methyl; ^R4 is selected from fluorine or chlorine; R ⁵ is selected from _C1-4 alkyl, halogenated _C1-4 alkyl, 3 to 4-membered cycloalkyl, -L1 ^-R9 _, where _L1 is selected from sulfur or oxygen, and R ⁹ is selected from _C1-4 alkyl, halogenated _C1-4 alkyl, deuterated _C1-4 alkyl, 3 to 4-membered cycloalkyl; _X1 is selected from nitrogen or ^CR6 , and ^R6 is selected from hydrogen, fluorine or chlorine; ^R7 and ^R8 are each independently selected from hydrogen, fluorine, and chlorine. The compound of formula (I) as described in claim 1, or a pharmaceutically acceptable salt thereof, wherein _X1 is nitrogen. The compound of formula (I) as described in claim 1, or a pharmaceutically acceptable salt thereof, is the compound of formula (II) or a pharmaceutically acceptable salt thereof. ^R1 , ^R2 , ^R3 , ^R4 , ^R5 , ^R6 , ^R7 and ^R8 are defined as in Request 1. The compound or its pharmaceutically acceptable salt as described in any of claims 1 to 3, wherein R ⁵ is selected from _C1-4 alkyl or halogenated _C1-4 alkyl; preferably, R ⁵ is selected from methyl, ethyl, or isopropyl; most preferably, R ⁵ is selected from methyl or ethyl. The compound ^or its pharmaceutically acceptable salt as claimed in any one of claims 1 to 3, wherein R ⁵ is selected from a 3 to 4-membered cycloalkyl group, preferably cyclopropyl. The compound or its pharmaceutically acceptable salt described in any of claims 1 to 3 is the compound or its pharmaceutically acceptable salt represented by formula (II-1), ^R1 , ^R2 , ^R3 , ^R4 , ^R6 , ^R7 , ^R8 , and ^R9 are defined as in Request 1. The compound as claimed in claim 6 or its pharmaceutically acceptable salt, wherein R ⁹ is selected from _C1-4 alkyl, halogenated _C1-4 alkyl, or deuterated _C1-4 alkyl; preferably, R ⁹ is selected from methyl, ethyl, difluoromethyl, trifluoromethyl, or deuterated methyl; most preferably, R ⁹ is selected from methyl, difluoromethyl, trifluoromethyl, or deuterated methyl. The compound or its pharmaceutically acceptable salt described in any of claims 1 to 3 is the compound or its pharmaceutically acceptable salt represented by formula (II-2), ^R1 , ^R2 , ^R3 , ^R4 , ^R6 , ^R7 , ^R8 , and ^R9 are defined as in Request 1. The compound as claimed in claim 8 or its pharmaceutically acceptable salt, wherein R ⁹ is selected from _C1-4 alkyl, halogenated _C1-4 alkyl or deuterated _C1-4 alkyl; preferably, R ⁹ is selected from methyl, ethyl, difluoromethyl, trifluoromethyl or deuterated methyl; most preferably, R ⁹ is methyl. The compound or its pharmaceutically acceptable salt as described in any of claims 1 to 9, wherein at least one of ^R1 , ^R2 , and ^R3 is selected from fluorine or chlorine. The compound or its pharmaceutically acceptable salt as described in any of claims 1 to 10, wherein ^R1 and ^R3 are each independently selected from hydrogen, fluorine or chlorine; preferably, one of ^R1 or ^R3 is hydrogen and the other is selected from hydrogen, fluorine or chlorine; most preferably, ^R1 or ^R3 is each independently hydrogen. The compound or its pharmaceutically acceptable salt as described in any of claims 1 to 11, wherein ^R2 is fluorine or chlorine; preferably, ^R2 is fluorine; preferably, ^R2 is chlorine. The compound or its pharmaceutically acceptable salt as described in any of claims 1 to 12, wherein ^R1 or ^R3 is independently hydrogen; and ^R2 is fluorine or chlorine. The compound or its pharmaceutically acceptable salt as described in any of claims 1 to 13, wherein ^R4 is selected from fluorine or chlorine; preferably, ^R4 is fluorine. The compound or its pharmaceutically acceptable salt as described in any of claims 1 to 14, wherein ^R1 or ^R3 is hydrogen, ^R2 is fluorine or chlorine, and ^R4 is fluorine. The compound or its pharmaceutically acceptable salt as described in any of claims 1 to 15, wherein at least one of ^R6 , ^R7 and ^R8 is selected from fluorine or chlorine; preferably, at least two of ^R6 , ^R7 and ^R8 are selected from fluorine or chlorine. The compound or its pharmaceutically acceptable salt as described in any of claims 1 to 16, wherein ^R6 is selected from fluorine or chlorine. The compound or its pharmaceutically acceptable salt as described in any of claims 1 to 17, wherein at least one of ^R6 , ^R7 and ^R8 is chlorine. The compound or its pharmaceutically acceptable salt as described in any of claims 1 to 18, wherein ^R7 is selected from fluorine or chlorine, and ^R8 is hydrogen; preferably, ^R7 is fluorine and ^R8 is hydrogen. The compound or its pharmaceutically acceptable salt as described in any of claims 1 to 19, wherein ^R8 is selected from fluorine or chlorine, and ^R7 is hydrogen. The compound or its pharmaceutically acceptable salt as claimed in any of claims 1 to 20, wherein ^R1 or ^R3 is each independently hydrogen; ^R2 is fluorine or chlorine; ^R4 is fluorine; ^R6 is selected from fluorine or chlorine; ^R7 is selected from fluorine or chlorine and ^R8 is hydrogen, or ^R8 is selected from fluorine or chlorine and ^R7 is hydrogen; and at least one of ^R6 , ^R7 and ^R8 is chlorine. The compound or its pharmaceutically acceptable salt as described in any of claims 1 to 21 is a compound or its pharmaceutically acceptable salt represented by formula (II-A) or formula (II-B), preferably a compound or its pharmaceutically acceptable salt represented by formula (II-B). ^R2 , ^R5 , ^R6 , and ^R7 are defined as in claims 1 to 21, respectively. Preferably, ^R2 is chlorine; or preferably, ^R2 is fluorine. The compound or pharmaceutically acceptable salt thereof as claimed in claim 22, wherein ^R5 is selected from methyl or ethyl. The compound or pharmaceutically acceptable salt thereof as claimed in claim 22, wherein ^R5 is selected from _-L1 - ^R9 , _L1 is oxygen, and ^R9 is selected from methyl, ethyl, difluoromethyl, trifluoromethyl or deuterated methyl; preferably, ^R9 is selected from methyl or deuterated methyl; or preferably, ^R9 is selected from difluoromethyl or trifluoromethyl. The compound or pharmaceutically acceptable salt thereof as claimed in claim 22, wherein ^R5 is selected from _-L1 - ^R9 , _L1 is sulfur, and ^R9 is selected from methyl, ethyl, difluoromethyl, trifluoromethyl or deuterated methyl; preferably, ^R5 is selected from _-L1 - ^R9 , _L1 is sulfur, and ^R9 is methyl or deuterated methyl. As in claim 22, in the compound or pharmaceutically acceptable salt thereof, ^R6 is fluorine or chlorine, and ^R7 is hydrogen; preferably, ^R6 is fluorine and ^R7 is hydrogen. The compound or its pharmaceutically acceptable salt described in any of claims 1 to 16, 18, 19, and 21 is a compound or its pharmaceutically acceptable salt represented by formula (II-C). ^R2 , ^R5 , ^R6 , and ^R7 are defined as in claims 1 to 16, 18, 19, and 21, respectively. Preferably, ^R2 is chlorine; or preferably, ^R2 is fluorine. The compound or pharmaceutically acceptable salt thereof as claimed in claim 27, wherein ^R5 is selected from methyl or ethyl. The compound or pharmaceutically acceptable salt thereof as claimed in claim 27, wherein ^R5 is selected from _-L1 - ^R9 , _L1 being oxygen; ^R9 is selected from methyl, ethyl, difluoromethyl, trifluoromethyl or deuterated methyl, preferably ^R9 is selected from methyl or deuterated methyl, or more preferably ^R9 is selected from difluoromethyl or trifluoromethyl. The compound or pharmaceutically acceptable salt thereof as claimed in claim 27, wherein ^R5 is selected from _-L1 - ^R9 ; _L1 is sulfur; ^R9 is selected from methyl, ethyl, difluoromethyl, trifluoromethyl or deuterated methyl, preferably, ^R9 is methyl or deuterated methyl. The compound or pharmaceutically acceptable salt thereof as claimed in claim 27, wherein at least one of ^R6 and ^R7 is selected from fluorine or chlorine; preferably, ^R6 is selected from fluorine or chlorine; most preferably, ^R6 is selected from fluorine and ^R7 is selected from fluorine or chlorine; or, ^R6 is selected from chlorine and ^R7 is selected from fluorine or chlorine. The compound or its pharmaceutically acceptable salt as described in any of claims 1 to 31 is selected from the following compounds or their pharmaceutically acceptable salts: A compound as described in any one of claims 1 to 32, or an isotopic substitute of the pharmaceutically acceptable salt thereof, preferably a deuterated derivative. A pharmaceutical composition comprising a compound or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 32, or an isotopic substitute as described in claim 33, and one or more pharmaceutically acceptable excipients. Use of a compound as described in any one of claims 1 to 32, or a pharmaceutically acceptable salt thereof, or an isotopic substitute as described in claim 33, or a pharmaceutical composition as described in claim 34, in the preparation of a medicament for the prevention and/or treatment of BCKDK-related diseases; preferably, the BCKDK-related disease is selected from a glucose metabolism disorder or abnormal glucose level, a heart disease, or a kidney disease; more preferably, the glucose metabolism disorder or abnormal glucose level is diabetes mellitus, and the heart disease is heart failure. Use of a compound as described in any one of claims 1 to 32, or a pharmaceutically acceptable salt thereof, or an isotopic substitute as described in claim 33, or a pharmaceutical composition as described in claim 34, in the preparation of a medicament for the prevention and/or treatment of a disease selected from disorders of glucose metabolism or abnormalities of blood sugar, heart disease, or kidney disease; preferably, the disorder of glucose metabolism or abnormality of blood sugar is diabetes, and the heart disease is heart failure. A method for preparing a compound or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 32, comprising the step of removing the protecting group PG from the compound or pharmaceutically acceptable salt thereof represented by formula (I-a) under alkaline conditions, and further comprising the step of adjusting the pH to acidic conditions. Wherein, PG is a carboxyl protecting group; ^R1 , ^R2 , ^R3 , ^R4 , ^R5 , _X1 , ^R7 and ^R8 are as defined in claims 1 to 32, respectively; preferably, PG is a _C1-6 alkyl group. A compound of formula (I-a) or a pharmaceutically acceptable salt thereof, Wherein, PG is a carboxyl protecting group; ^R1 , ^R2 , ^R3 , ^R4 , ^R5 , _X1 , ^R7 and ^R8 are defined as in claims 1 to 32, respectively; preferably, PG is a _C1-6 alkyl group.