Pyridothiophene-2-carboxylic acid derivative, method of preparation thereof and its use

Abstract

The present invention relates to a pyridothiophene-2-carboxylic acid derivative, a method for its preparation and use, and in particular to a compound as shown in formula (I) or a pharmaceutical salt thereof, which exhibits a good inhibitory / degrading effect on BCKDK. The substituents in the formula are defined as indicated in the description.

Description

A pyridothiophene-2-carboxylic acid derivative and its preparation method and use Technical Field

[0001] The present disclosure relates to the field of medicinal chemistry, and in particular to a pyridothiophene-2-carboxylic acid derivative, a preparation method, and uses thereof. Background Art

[0002] 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 also enhance MEK / ERK signaling, which is associated with cancer malignant proliferation.

[0003] BCAAs (leucine, isoleucine, and valine) are essential amino acids for the human body, accounting for approximately 40% of the essential amino acids in healthy subjects. They must be obtained through a balanced diet and play an important role in nutrient sensing and cell signaling.

[0004] Excessive amounts of branched-chain amino acids (BCAAs) are toxic but essential for protein synthesis and cell signaling. BCAAs are converted to α-ketoacid forms: α-ketoisocaproate (KIC / ketoleucine), 2-keto-3-methylvaleric acid (KMV / ketoisoleucine), and α-ketoisovaleric acid (KIV / ketovaline) by branched-chain aminotransferases (BCATs). BCKAs are then oxidatively decarboxylated by the branched-chain ketoacid dehydrogenase (BCKDH) complex, which consists of the BCKDH Ela / p tetramer and multiple copies of the BCKDH E2 and BCKDH E3 subunits. This complex is regulated by inhibitory phosphorylation mediated by BCKDH kinase (BCKDK), with the same phosphorylation site being dephosphorylated by the phosphatase PPM1K. Phosphorylation of the inhibitory complex promotes BCKDH activity, thereby promoting the irreversible catabolism of BCKA (Lynch CJ, Adams SH: Branched-chain amino acids in metabolic signaling and insulin resistance. Nat Rev Endocrinol 2014, 10: 723-36.). This regulation is confirmed by the absence of BCKDK in mice, as mice lacking BCKDK exhibit enhanced BCKDH activity in various tissues (Joshi MA, Jeoung NH, Obayashi M, Hattab EM, Brocken EG, Liechty EA, Kubek MJ, Vattem 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.).

[0005] Inadequate catabolism of branched-chain amino acids (BCAAs) is associated with insulin resistance, diabetes, congenital heart disease, and heart failure. Circulating BCAAs and their breakdown products, branched-chain α-keto acids (BCKAs), are also indicators of diabetes pathogenesis. The α-ketoacid dehydrogenase complex (BCKDH) is the rate-limiting step in the breakdown and clearance of BCAAs; BCKDK phosphorylates the E1α subunit of BCKDH, thereby inhibiting its function.

[0006] In recent years, although there have been some early studies related to BCKDK, such as WO2020056155A, WO2020261144A, WO2020261205A, WO2022175959A, WO2023100061A, etc., there is still a need to develop novel and effective BCKDK inhibitors for the treatment of diseases or conditions associated with elevated BCAA concentrations. Summary of the Invention

[0007] The present disclosure provides a compound represented by formula (I) or a pharmaceutically acceptable salt thereof,

[0008] Among them, R 1 、R 2 、R 3 are each independently selected from hydrogen, fluorine, chlorine or methyl;

[0009] The R 4 selected from fluorine or chlorine;

[0010] The R 5 Selected from C 1-4 Alkyl, halogenated C 1-4 Alkyl, 3 to 4-membered cycloalkyl, -L1-R 9 , said L1 is selected from sulfur or oxygen, said R 9 Selected from C 1-4 Alkyl, halogenated C 1-4 Alkyl, deuterated C 1-4 Alkyl, 3- to 4-membered cycloalkyl;

[0011] X1 is selected from nitrogen or CR 6 , the R 6 is selected from hydrogen, fluorine or chlorine;

[0012] The R 7 and R 8 Each is independently selected from hydrogen, fluorine and chlorine.

[0013] In some embodiments, in the compound of formula (I) or a pharmaceutically acceptable salt thereof provided by the present disclosure, X1 is nitrogen.

[0014] In some embodiments, the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, which is a compound of formula (II) or a pharmaceutically acceptable salt thereof,

[0015] Among them, the R 1 、R 2 、R 3 、R 4 、R 5 、R 6 、R 7 and R8 are respectively as defined in formula (I).

[0016] In some embodiments, in the compounds of formula (I) and (II) or pharmaceutically acceptable salts thereof provided by the present disclosure, the R 5 Selected from C 1-4 Alkyl or halogenated C 1-4 alkyl.

[0017] In an optional embodiment, in the compounds of formula (I) and (II) or pharmaceutically acceptable salts thereof provided by the present disclosure, the R 5 Selected from methyl, ethyl, isopropyl.

[0018] In some embodiments, in the compounds of formula (I) and (II) or pharmaceutically acceptable salts thereof provided by the present disclosure, the R 5 Selected from methyl or ethyl.

[0019] In some embodiments, in the compounds of formula (I) and (II) or pharmaceutically acceptable salts thereof provided by the present disclosure, the R 5 is selected from 3- to 4-membered cycloalkyl groups.

[0020] In an optional embodiment, in the compounds of formula (I) and (II) or pharmaceutically acceptable salts thereof provided by the present disclosure, the R 5 It is cyclopropyl.

[0021] In some embodiments, the compounds of formula (I) and (II) or pharmaceutically acceptable salts thereof provided by the present disclosure are compounds of formula (II-1) or pharmaceutically acceptable salts thereof,

[0022] Among them, the R 1 、R 2 、R 3 、R 4 、R 6 、R 7 、R 8 、R 9 are respectively as defined in formula (I).

[0023] In some embodiments, in the compounds of formula (I), (II), (II-1) or pharmaceutically acceptable salts thereof provided herein, the R 9 Selected from C 1-4 Alkyl, halogenated C 1-4 Alkyl or deuterated C 1-4 alkyl.

[0024] In an optional embodiment, in the compounds of formula (I), (II), (II-1) or pharmaceutically acceptable salts thereof provided by the present disclosure, the R 9 is selected from methyl, ethyl, difluoromethyl, trifluoromethyl or deuterated methyl.

[0025] In an optional embodiment, in the compounds of formula (I), (II), (II-1) or pharmaceutically acceptable salts thereof provided by the present disclosure, the R 9 is selected from methyl, difluoromethyl, trifluoromethyl or deuterated methyl.

[0026] In some embodiments, the compounds of formula (I) and (II) or pharmaceutically acceptable salts thereof provided by the present disclosure are compounds of formula (II-2) or pharmaceutically acceptable salts thereof,

[0027] Among them, the R 1 、R 2 、R 3 、R 4 、R 6 、R 7 、R 8 、R 9 are respectively as defined in formula (I).

[0028] In some embodiments, in the compound of formula (II-2) or a pharmaceutically acceptable salt thereof provided by the present disclosure, the R 9 Selected from C 1-4 Alkyl, halogenated C 1-4 Alkyl or deuterated C 1-4 alkyl.

[0029] In an optional embodiment, in the compound of formula (II-2) or a pharmaceutically acceptable salt thereof provided by the present disclosure, the R 9 is selected from methyl, ethyl, difluoromethyl, trifluoromethyl or deuterated methyl.

[0030] In an optional embodiment, in the compound of formula (II-2) or a pharmaceutically acceptable salt thereof provided by the present disclosure, the R 9 It is a methyl group.

[0031] In some embodiments, in the compounds of formula (I), (II), (II-1), (II-2) or pharmaceutically acceptable salts thereof provided herein, the R 1 、R 2 、R 3 At least one of them is selected from fluorine or chlorine.

[0032] In some embodiments, in the compounds of formula (I), (II), (II-1), (II-2) or pharmaceutically acceptable salts thereof provided herein, the R 1 and R 3 are each independently selected from hydrogen, fluorine or chlorine.

[0033] In an optional embodiment, in the compounds of formula (I), (II), (II-1), (II-2) or pharmaceutically acceptable salts thereof provided by the present disclosure, the R 1 or R 3 One of them is hydrogen and the other is selected from hydrogen, fluorine or chlorine.

[0034] In an optional embodiment, in the compounds of formula (I), (II), (II-1), (II-2) or pharmaceutically acceptable salts thereof provided by the present disclosure, the R 1 or R 3 are each independently hydrogen.

[0035] In some embodiments, in the compounds of formula (I), (II), (II-1), (II-2) or pharmaceutically acceptable salts thereof provided herein, the R 2 is fluorine or chlorine.

[0036] In some embodiments, in the compounds of formula (I), (II), (II-1), (II-2) or pharmaceutically acceptable salts thereof provided herein, the R 2 For fluorine.

[0037] In some embodiments, in the compounds of formula (I), (II), (II-1), (II-2) or pharmaceutically acceptable salts thereof provided herein, the R 2 For chlorine.

[0038] In some embodiments, in the compounds of formula (I), (II), (II-1), (II-2) or pharmaceutically acceptable salts thereof provided herein, the R 1 or R 3 are each independently hydrogen; the R 2 is fluorine or chlorine.

[0039] In some embodiments, in the compounds of formula (I), (II), (II-1), (II-2) or pharmaceutically acceptable salts thereof provided herein, the R 4 Selected from fluorine or chlorine.

[0040] In some embodiments, in the compounds of formula (I), (II), (II-1), (II-2) or pharmaceutically acceptable salts thereof provided herein, the R 4 For fluorine.

[0041] In some embodiments, in the compounds of formula (I), (II), (II-1), (II-2) or pharmaceutically acceptable salts thereof provided herein, the R 1 or R 3 are each independently hydrogen, the R 2 is fluorine or chlorine, said R 4 For fluorine.

[0042] In some embodiments, in the compounds of formula (I), (II), (II-1), (II-2) or pharmaceutically acceptable salts thereof provided herein, the R 6 、R 7 and R 8 At least one of them is selected from fluorine or chlorine.

[0043] In an optional embodiment, in the compounds of formula (I), (II), (II-1), (II-2) or pharmaceutically acceptable salts thereof provided by the present disclosure, the R 6 、R 7 and R 8 At least two of them are selected from fluorine or chlorine.

[0044] In some embodiments, in the compounds of formula (I), (II), (II-1), (II-2) or pharmaceutically acceptable salts thereof provided herein, the R 6 Selected from fluorine or chlorine.

[0045] In some embodiments, in the compounds of formula (I), (II), (II-1), (II-2) or pharmaceutically acceptable salts thereof provided herein, the R 6 、R 7 and R 8 At least one is chlorine.

[0046] In some embodiments, in the compounds of formula (I), (II), (II-1), (II-2) or pharmaceutically acceptable salts thereof provided herein, the R 7 is selected from fluorine or chlorine, said R 8 For hydrogen.

[0047] In an optional embodiment, in the compounds of formula (I), (II), (II-1), (II-2) or pharmaceutically acceptable salts thereof provided by the present disclosure, the R 7 is fluorine, the R 8 For hydrogen.

[0048] In some embodiments, in the compounds of formula (I), (II), (II-1), (II-2) or pharmaceutically acceptable salts thereof provided herein, the R 8 is selected from fluorine or chlorine, said R 7 For hydrogen.

[0049] In some embodiments, in the compounds of formula (I), (II), (II-1), (II-2) or pharmaceutically acceptable salts thereof provided herein, the R 1 or R 3 are each independently hydrogen; the R 2 is fluorine or chlorine; said R 4is fluorine; the R 6 is selected from fluorine or chlorine; said R 7 is selected from fluorine or chlorine, said R 8 is hydrogen, or the R 8 is selected from fluorine or chlorine, said R 7 is hydrogen; and said R 6 、R 7 and R 8 At least one is chlorine.

[0050] In some embodiments, the compounds of formula (I), (II), (II-1), (II-2) or pharmaceutically acceptable salts thereof provided herein are compounds of formula (II-A) or pharmaceutically acceptable salts thereof,

[0051] Among them, the R 2 、R 5 、R 6 、R 7 As defined in formula (I), (II), (II-1), and (II-2), respectively.

[0052] In an optional embodiment, in the compound of formula (II-A) or a pharmaceutically acceptable salt thereof provided by the present disclosure, the R 2 For chlorine.

[0053] In an optional embodiment, in the compound of formula (II-A) or a pharmaceutically acceptable salt thereof provided by the present disclosure, the R 2 For fluorine.

[0054] In some embodiments, the compounds of formula (I), (II), (II-1), (II-2) or pharmaceutically acceptable salts thereof provided herein are compounds of formula (II-B) or pharmaceutically acceptable salts thereof,

[0055] Among them, the R 2 、R 5 、R 6 、R 7 As defined in formula (I), (II), (II-1), and (II-2), respectively.

[0056] In an optional embodiment, in the compound of formula (II-B) or a pharmaceutically acceptable salt thereof provided by the present disclosure, the R 2 For chlorine.

[0057] In an optional embodiment, in the compound of formula (II-B) or a pharmaceutically acceptable salt thereof provided by the present disclosure, the R 2 For fluorine.

[0058] In some embodiments, in the compounds of formula (II-A) and (II-B) or pharmaceutically acceptable salts thereof provided herein, the R 5 Selected from methyl or ethyl.

[0059] In some embodiments, in the compounds of formula (II-A) and (II-B) or pharmaceutically acceptable salts thereof provided herein, the R 5 Selected from -L1-R 9 , the L1 is oxygen, the R 9 is selected from methyl, ethyl, difluoromethyl, trifluoromethyl or deuterated methyl.

[0060] In some embodiments, in the compounds of formula (II-A) and (II-B) or pharmaceutically acceptable salts thereof provided herein, the R 5 Selected from -L1-R 9 , the L1 is oxygen, the R 9 is selected from methyl or deuterated methyl.

[0061] In some embodiments, in the compounds of formula (II-A) and (II-B) or pharmaceutically acceptable salts thereof provided herein, the R 5 Selected from -L1-R 9 , the L1 is oxygen, the R 9 Selected from difluoromethyl or trifluoromethyl.

[0062] In some embodiments, in the compounds of formula (II-A) and (II-B) or pharmaceutically acceptable salts thereof provided herein, the R 5 Selected from -L1-R 9 , the L1 is sulfur, the R 9 is selected from methyl, ethyl, difluoromethyl, trifluoromethyl or deuterated methyl.

[0063] In some embodiments, in the compounds of formula (II-A) and (II-B) or pharmaceutically acceptable salts thereof provided herein, the R 5 Selected from -L1-R 9 , the L1 is sulfur, the R 9 It is methyl or deuterated methyl.

[0064] In some embodiments, in the compounds of formula (II-A) and (II-B) or pharmaceutically acceptable salts thereof provided herein, the R 6 is fluorine or chlorine, said R 7 For hydrogen.

[0065] In an optional embodiment, in the compounds of formula (II-A) and (II-B) or pharmaceutically acceptable salts thereof provided by the present disclosure, the R 6 is fluorine, the R 7 For hydrogen.

[0066] In some embodiments, the compounds of formula (I), (II), (II-1), (II-2) or pharmaceutically acceptable salts thereof provided herein are compounds of formula (II-C) or pharmaceutically acceptable salts thereof,

[0067] Among them, the R 2 、R 5 、R 6 、R 7 As defined in formula (I), (II), (II-1), and (II-2), respectively.

[0068] In an optional embodiment, in the compound of formula (II-C) or a pharmaceutically acceptable salt thereof provided by the present disclosure, the R 2 For chlorine.

[0069] In an optional embodiment, in the compound of formula (II-C) or a pharmaceutically acceptable salt thereof provided by the present disclosure, the R 2 For fluorine.

[0070] In some embodiments, in the compound of formula (II-C) or a pharmaceutically acceptable salt thereof provided by the present disclosure, the R 5 Selected from methyl or ethyl.

[0071] In some embodiments, in the compound of formula (II-C) or a pharmaceutically acceptable salt thereof provided by the present disclosure, the R 5 Selected from -L1-R 9 , the L1 is oxygen, the R 9 is selected from methyl, ethyl, difluoromethyl, trifluoromethyl or deuterated methyl.

[0072] In some embodiments, in the compound of formula (II-C) or a pharmaceutically acceptable salt thereof provided by the present disclosure, the R 5 Selected from -L1-R 9 , the L1 is oxygen, the R 9 is selected from methyl or deuterated methyl.

[0073] In some embodiments, in the compound of formula (II-C) or a pharmaceutically acceptable salt thereof provided by the present disclosure, the R 5 Selected from -L1-R 9 , the L1 is oxygen, the R 9 Selected from difluoromethyl or trifluoromethyl.

[0074] In some embodiments, in the compound of formula (II-C) or a pharmaceutically acceptable salt thereof provided by the present disclosure, the R 5 Selected from -L1-R 9 , the L1 is sulfur, the R 9is selected from methyl, ethyl, difluoromethyl, trifluoromethyl or deuterated methyl.

[0075] In some embodiments, in the compound of formula (II-C) or a pharmaceutically acceptable salt thereof provided by the present disclosure, the R 5 Selected from -L1-R 9 , the L1 is sulfur, the R 9 It is methyl or deuterated methyl.

[0076] In some embodiments, in the compound of formula (II-C) or a pharmaceutically acceptable salt thereof provided by the present disclosure, the R 6 and R 7 At least one is selected from fluorine or chlorine.

[0077] In an optional embodiment, in the compound of formula (II-C) or a pharmaceutically acceptable salt thereof provided by the present disclosure, the R 6 Selected from fluorine or chlorine.

[0078] In an optional embodiment, in the compound of formula (II-C) or a pharmaceutically acceptable salt thereof provided by the present disclosure, the R 6 is selected from fluorine, said R 7 Selected from fluorine or chlorine.

[0079] In an optional embodiment, in the compound of formula (II-C) or a pharmaceutically acceptable salt thereof provided by the present disclosure, the R 6 is selected from chlorine, said R 7 Selected from fluorine or chlorine.

[0080] The present disclosure further provides a compound selected from the following or a pharmaceutically acceptable salt thereof:

[0081] The present disclosure further provides an isotope substitution of the aforementioned compound or a pharmaceutically acceptable salt thereof. Optionally, the isotope substitution is a deuterated compound.

[0082] The present disclosure provides a pharmaceutical composition comprising the above compound and one or more pharmaceutically acceptable excipients.

[0083] In some embodiments, the unit dose of the pharmaceutical composition is 0.001 mg-1000 mg.

[0084] In certain embodiments, the pharmaceutical composition contains 0.01-99.99% of the aforementioned compound or its pharmaceutically acceptable salt or its isotopic substitution, based on the total weight of the composition. In certain embodiments, the pharmaceutical composition contains 0.1-99.9% of the aforementioned compound or its pharmaceutically acceptable salt or its isotopic substitution. In certain embodiments, the pharmaceutical composition contains 0.5%-99.5% of the aforementioned compound or its pharmaceutically acceptable salt or its isotopic substitution. In certain embodiments, the pharmaceutical composition contains 1%-99% of the aforementioned compound or its pharmaceutically acceptable salt or its isotopic substitution. In certain embodiments, the pharmaceutical composition contains 2%-98% of the aforementioned compound or its pharmaceutically acceptable salt or its isotopic substitution.

[0085] In certain embodiments, the pharmaceutical composition comprises 0.01% to 99.99% of a pharmaceutically acceptable excipient, based on the total weight of the composition. In certain embodiments, the pharmaceutical composition comprises 0.1% to 99.9% of a pharmaceutically acceptable excipient. In certain embodiments, the pharmaceutical composition comprises 0.5% to 99.5% of a pharmaceutically acceptable excipient. In certain embodiments, the pharmaceutical composition comprises 1% to 99% of a pharmaceutically acceptable excipient. In certain embodiments, the pharmaceutical composition comprises 2% to 98% of a pharmaceutically acceptable excipient.

[0086] The present disclosure further provides use of the aforementioned compound, or a pharmaceutically acceptable salt thereof, or an isotope substituted product thereof, or the aforementioned pharmaceutical composition as a medicine.

[0087] The present disclosure further provides use of the aforementioned compound, or a pharmaceutically acceptable salt thereof, or an isotope substituted product thereof, or the aforementioned pharmaceutical composition in the preparation of a medicament for preventing and / or treating diseases associated with BCKDK.

[0088] The present disclosure further provides a use of the aforementioned compound or its pharmaceutically acceptable salt or isotope substitute or the aforementioned pharmaceutical composition in the preparation of a medicament for preventing and / or treating a disease, wherein the disease includes but is not limited to glucose metabolism disorder or abnormal blood sugar disease, heart disease or kidney disease.

[0089] The present disclosure further provides a method for preventing and / or treating diseases related to BCKDK, comprising administering the aforementioned compound or its pharmaceutically acceptable salt or isotope substitute or the aforementioned pharmaceutical composition to a patient.

[0090] The present disclosure further provides a method for preventing and / or treating a disease, comprising administering the aforementioned compound or its pharmaceutically acceptable salt or isotope substitute or the aforementioned pharmaceutical composition to a patient. The disease includes but is not limited to glucose metabolism disorder or abnormal blood sugar disease, heart disease or kidney disease.

[0091] In an optional embodiment, in the method provided by the present disclosure, the aforementioned compound or its pharmaceutically acceptable salt or its isotope substitute or the aforementioned pharmaceutical composition administered to the patient is a therapeutically effective amount.

[0092] In some embodiments, the BCKDK-related disease includes but is not limited to glucose metabolism disorders or abnormal blood sugar diseases, heart diseases, or kidney diseases.

[0093] In some embodiments, the carbohydrate metabolism disorder or dysglycemic disease is diabetes.

[0094] In some embodiments, the cardiac disease is heart failure.

[0095] The aforementioned compound or its pharmaceutically acceptable salt or its isotope substitute or the aforementioned pharmaceutical composition provided by the present disclosure has a good inhibitory / degradation effect on BCKDK.

[0096] Another aspect of the present disclosure provides a method for preparing a compound of formula (I) or a pharmaceutically acceptable salt thereof, comprising the step of removing the protective group PG from the compound of formula (Ia) or a pharmaceutically acceptable salt thereof under alkaline conditions, and further comprising the step of adjusting the pH to acidic.

[0097] Wherein, the PG is a carboxyl protecting group; the R 1 、R 2 、R 3 、R 4 、R 5 , X1, R 7 and R 8 are respectively as defined in formula (I).

[0098] In an optional embodiment, in the method for preparing the compound of formula (I) or a pharmaceutically acceptable salt thereof provided by the present disclosure, the PG is C 1-6 alkyl.

[0099] Another aspect of the present disclosure provides a compound represented by formula (Ia) or a pharmaceutically acceptable salt thereof,

[0100] Wherein, the PG is a carboxyl protecting group; the R 1 、R 2 、R 3 、R 4 、R 5 , X1, R 7 and R 8 are respectively as defined in formula (I).

[0101] In an optional embodiment, in the compound represented by formula (Ia) or a pharmaceutically acceptable salt thereof, the PG is C 1-6 alkyl.

[0102] Definition of terms

[0103] Where the present disclosure does not limit a particular configuration, the compounds of the present disclosure may exist in specific geometric or stereoisomeric forms. The present disclosure contemplates all such compounds, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, atropisomers (i.e., stereoisomers with hindered rotation), and racemic mixtures thereof, and other mixtures, such as enantiomerically or diastereomerically enriched mixtures, all of which are within the scope of the present disclosure. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All of these isomers and their mixtures are included within the scope of the present disclosure.

[0104] Additionally, the compounds and intermediates of the present disclosure may also exist in different tautomeric forms, and all such forms are encompassed within the scope of the present disclosure.The term "tautomer" or "tautomeric form" refers to structural isomers of different energies that are interconvertible via a low energy barrier.

[0105] The compounds of the present disclosure may be asymmetric, for example, having one or more stereoisomers. Unless otherwise indicated, all stereoisomers are included, such as enantiomers and diastereomers. The compounds of the present disclosure containing asymmetric carbon atoms can be isolated in optically pure form or racemic form. Optically pure forms 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 or chiral reagents or other conventional techniques. If one enantiomer of a compound of the present disclosure is desired, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, wherein the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide the pure desired enantiomer. Alternatively, when the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), diastereomeric salts are formed with an appropriate optically active acid or base, and then the diastereoisomers are resolved by conventional methods well known in the art, and then the pure enantiomers are recovered. In addition, the separation of enantiomers and diastereomers is usually achieved by using chromatography using a chiral stationary phase and optionally combined with a chemical derivatization method (such as the formation of carbamates from amines).

[0106] In the chemical structures of the compounds disclosed herein, the bond Indicates that the configuration is not specified, that is, if chiral isomers exist in the chemical structure, the bond Can be or or include both and Two configurations. In the chemical structure of the compound disclosed in the present invention, the bond No configuration is specified, i.e., the bond The configuration can be E-type or Z-type, or include both E and Z configurations.

[0107] The present disclosure also includes isotopically labeled compounds of the present disclosure that are identical to those described herein, but where one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into the compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as 2 H. 3 H. 11 C. 13 C. 14 C. 13 N. 15 N. 15 O. 17 O. 18 O. 31 P. 32 P. 35 S. 18 F. 123 I. 125 I and 36 Cl et al.

[0108] Unless otherwise stated, when a position is specifically designated as deuterium (D), the position is understood to have deuterium (i.e., at least 10% deuterium incorporation) at least 1000 times greater than the natural abundance of deuterium (which is 0.015%). In the example, the compound has a natural abundance greater than deuterium of at least 1000 times the abundance of deuterium, at least 2000 times the abundance of deuterium, at least 3000 times the abundance of deuterium, at least 4000 times the abundance of deuterium, at least 5000 times the abundance of deuterium, at least 6000 times the abundance of deuterium, or more. The present disclosure also includes compounds in various deuterated forms. Each available hydrogen atom connected to a carbon atom can be independently replaced by a deuterium atom. Those skilled in the art can synthesize compounds in deuterated forms with reference to relevant literature. In preparing deuterated forms of the compounds, commercially available deuterated starting materials may be used, or they may be synthesized using conventional techniques with deuterated reagents including, but not limited to, deuterated borane, trideuterated borane in tetrahydrofuran, deuterated lithium aluminum hydride, deuterated iodoethane, deuterated iodomethane, and the like.

[0109] The term "alkyl" refers to a saturated aliphatic hydrocarbon group, preferably an alkyl group of 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, tert-butyl, sec-butyl, 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.

[0110] The term "cycloalkyl" or "carbocycle" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent, wherein the cycloalkyl ring contains 3 to 20 carbon atoms, preferably 3 to 6 carbon atoms. Non-limiting examples of monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, and the like; polycyclic cycloalkyls include spirocyclic, fused, and bridged cycloalkyls.

[0111] The term "alkoxy" refers to -O-(alkyl) and -O-(unsubstituted cycloalkyl), wherein alkyl and cycloalkyl are as defined above. Non-limiting examples of alkoxy include methoxy, ethoxy, propoxy, butoxy, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, and cyclohexyloxy.

[0112] The term "hydroxy" refers to -OH.

[0113] The term "halogen" refers to fluorine, chlorine, bromine or iodine.

[0114] The term "haloalkyl" refers to an alkyl group substituted with a halogen, wherein alkyl is as defined above.

[0115] The term "cyano" refers to -CN.

[0116] The term "amino" refers to -NH2.

[0117] "Optionally" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs or does not occur. For example, "C 1-6 The term "alkyl" means that halogen or cyano may but need not be present, and the description includes both the case where the alkyl is substituted by halogen or cyano and the case where the alkyl is not substituted by halogen and cyano.

[0118] The term "substituted" means that one or more hydrogen atoms, preferably up to 5, more preferably 1 to 3 hydrogen atoms in a group are replaced independently of one another by a corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions and a person skilled in the art can determine (by experiment or theory) which substitutions are possible or impossible without undue effort.

[0119] "Substituted by one or more..." means that the compound may be substituted by a single or multiple substituents. When substituted by multiple substituents, the substituents may be multiple identical substituents or a combination of one or more different substituents.

[0120] In the present disclosure, the terms "comprising" and "including" can be replaced with "consisting of".

[0121] The term "composition" refers to a mixture of one or more compounds described herein, or their physiologically acceptable salts or precursors, with other chemical components, as well as other components such as physiologically acceptable carriers and excipients. The purpose of a composition is to facilitate administration to an organism, facilitating absorption of the active ingredient and thereby exerting its biological activity.

[0122] The term "pharmaceutically acceptable excipient" or "pharmaceutically acceptable excipient" includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye / colorant, flavoring agent, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent or emulsifier that has been approved by the U.S. Food and Drug Administration as acceptable for use by humans or domestic animals.

[0123] Unless otherwise specified, the compounds of the present invention may exist in the form of salts, mixed salts or non-salts (e.g., free acids or free bases). When present in the form of salts or mixed salts, they may be pharmaceutically acceptable salts or pharmaceutically usable salts.

[0124] The terms "pharmaceutically acceptable salts" and "pharmaceutically usable salts" are used interchangeably and are meant to include pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

[0125] As used herein, the term "inhibit" can be used interchangeably with "reduce," "silence," "downregulate," "suppress," and other similar terms, and includes any level of inhibition. Inhibition can be assessed by a reduction in the absolute or relative level of one or more of these variables compared to a control level. The control level can be any type of control level used in the art, such as a pre-dose baseline level or a level determined from a similar untreated or controlled (e.g., buffer-only or inert agent-only) treated subject, cell, or sample.

[0126] "Effective amount," "effective dose," "therapeutically effective amount," or "therapeutically effective amount" refers to the amount of a drug, compound, or pharmaceutical composition necessary to achieve any one or more beneficial or desired therapeutic results. For prophylactic uses, beneficial or desired results include eliminating or reducing the risk, lessening the severity, or delaying the onset of a condition, including biochemical, histological, and / or behavioral symptoms of the condition, its complications, and intermediate pathological phenotypes present during the course of the condition.

[0127] As used herein, "subject," "patient," "subject," or "individual" are used interchangeably and include humans or non-human animals, such as mammals, eg, humans or monkeys. DETAILED DESCRIPTION

[0128] The present disclosure is further described below with reference to the following examples, but these examples are not intended to limit the scope of this disclosure. Experimental methods in the examples disclosed herein, where specific conditions are not specified, were generally performed under conventional conditions or as recommended by the raw material or commercial manufacturer. Reagents whose sources are not specified can be obtained from any molecular biology reagent supplier of a quality / purity suitable for molecular biology applications.

[0129] Unless otherwise specified, the reagents used in the following examples are commercially available products.

[0130] The analytical conditions used for all mass spectrometric data in the following examples are as follows: Column model: ACQUITY UPLC BEH C18 1.7 μM, 2.1 x 50 mm Column; Mobile phase A: 0.05% ammonia + 0.01% formic acid in water; Mobile phase B: 0.01% formic acid in acetonitrile; Mobile phase B gradient: 5% to 95% from 0 to 1.7 minutes; Mobile phase B gradient: 95% from 1.7 to 2.5 minutes.

[0131] The instrument used for all hydrogen spectrum data in the following examples is a Bruker 400 MHz nuclear magnetic resonance instrument; the instrument used for all fluorine spectrum data is a Bruker 376 MHz nuclear magnetic resonance instrument.

[0132] Example 1

[0133] 6-Chloro-3-(2,4,5-trifluoro-3-methoxyphenyl)thieno[3,2-b]pyridine-2-carboxylic acid (1)

[0134] Step 1: Synthesis of methyl 3-amino-6-chlorothieno[3,2-b]pyridine-2-carboxylate 1c

[0135] To a solution of 5-chloro-3-nitropyridine-2-carbonitrile 1a (5.00 g, 27.24 mmol) in N,N-dimethylformamide (60 mL) at 0°C was slowly added dropwise methyl 2-mercaptoacetate 1b (2.4 mL, 27.24 mmol), followed by a solution 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 extracted with ethyl acetate (2 x 100 mL). The organic phases were combined, washed with saturated brine (4 x 80 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to afford crude product 1c (6.5 g, 98% yield).

[0136] MS (ESI) m / z = 243.2 [M+H] + .

[0137] Step 2: Synthesis of methyl 3-bromo-6-chlorothieno[3,2-b]pyridine-2-carboxylate 1d

[0138] To a three-necked flask containing copper(II) bromide (2.05 g, 9.13 mmol) and acetonitrile (15.0 mL) was added dropwise tert-butyl nitrite (3.92 g, 1.3 mL, 10.7 mmol) at 25°C under a nitrogen atmosphere. A suspension of compound 1c (2.0 g, 8.3 mmol) in acetonitrile (5.0 mL) was then added at 20°C. The reaction mixture was stirred at 25°C for 2 hours and then slowly poured into a 2N hydrochloric acid solution (20 mL). The mixture was extracted with ethyl acetate (2 × 70 mL). The organic phases were combined, washed with saturated brine (2 × 100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product, which was purified by column chromatography (petroleum ether / ethyl acetate) to afford the title compound 1d (1.0 g, 39% yield).

[0139] MS (ESI) m / z = 306.1 [M+H] + .

[0140] Step 3: Synthesis of methyl 6-chloro-3-(2,4,5-trifluoro-3-methoxyphenyl)thieno[3,2-b]pyridine-2-carboxylate 1f

[0141] Compound 1d (75 mg, 0.25 mmol) and compound 1e (50 mg, 0.25 mmol) were dissolved in 1,4-dioxane (2 mL) and water (0.4 mL) at room temperature. 1,1'-di-tert-butylphosphinoferrocenepalladium 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 under a nitrogen atmosphere for 1 h. After cooling to room temperature, ethyl acetate (60 mL) was added, and the mixture was washed with saturated brine (2 x 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 afford the title compound 1f (60 mg, 61% yield).

[0142] MS (ESI) m / z = 388.2 [M+H] + .

[0143] Step 4: Synthesis of 6-chloro-3-(2,4,5-trifluoro-3-methoxyphenyl)thieno[3,2-b]pyridine-2-carboxylic acid 1

[0144] 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 (1N). The mixture was extracted with ethyl acetate (2 × 10 mL). The combined organic phases were washed with saturated brine (2 × 10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product, which was separated and purified by preparative HPLC (column: Waters Xbridge, 30 × 250 mm. Mobile phase A: 0.1% formic acid in water; mobile phase B: acetonitrile; gradient: 40%-95% over 18 min, flow rate: 30 ml / min. Peak elution time: 11.06 min) to obtain the title compound 1 (15 mg, yield 25%).

[0145] MS (ESI) m / z = 374.2 [M+H] + .

[0146] 1 H-NMR (400MHz, DMSO-d6) δ8.87(s,1H),8.78(s,1H),7.44–7.37(m,1H),4.02(s,3H).

[0147] 19 F-NMR (376MHz, DMSO-d6) δ-132.28(dd,1F),-142.60(dd,1F),-151.65(dd,1F).

[0148] Example 2

[0149] 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)

[0150] Step 1: Synthesis of methyl 6-chloro-3-(6-chloro-2,4-difluoro-3-methoxyphenyl)thieno[3,2-b]pyridine-2-carboxylate 2b

[0151] Compound 1d (60 mg, 0.20 mmol) and compound 2a (89 mg, 0.30 mmol) were dissolved in toluene (3 mL) and water (0.3 mL) at room temperature. Tris(dibenzylideneacetone)dipalladium (18 mg, 0.02 mmol), 2-dicyclohexylphosphino-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 under a nitrogen atmosphere for 2 h. After cooling to room temperature, ethyl acetate (40 mL) was added, and the mixture was washed with saturated brine (2 x 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 afford the title compound 2b (50 mg, 61% yield).

[0152] MS (ESI) m / z = 404.2 [M+H] + .

[0153] Step 2: Synthesis of 6-chloro-3-(6-chloro-2,4-difluoro-3-methoxyphenyl)thieno[3,2-b]pyridine-2-carboxylic acid 2

[0154] 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. Lithium hydroxide (30 mg, 1.2 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). The combined organic phases were washed with saturated brine (2 × 10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product, which was separated and purified by preparative HPLC (column: Waters Xbridge, 30 × 250 mm; mobile phase A: 0.1% formic acid in water; mobile phase B: acetonitrile; gradient: 40%-95% over 18 minutes, flow rate: 30 ml / min, peak elution time: 11.06 min) to obtain the title compound 2 (18 mg, 38% yield).

[0155] MS (ESI) m / z = 390.1 [M+H] + .

[0156] 1 H-NMR (400MHz, DMSO-d6) δ8.88(s,1H),8.76(s,1H),7.63–7.60(m,1H),3.96(s,3H).

[0157] 19 F-NMR(376MHz,DMSO-d6)δ-124.92(d,1F),-126.84(d,1F).

[0158] 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)

[0159] The purified product 2 from the second step was further resolved by SFC using the following separation method: Column: ChiralPak AD, 250×30 mm ID, 5 μm; Mobile phase: A: CO2; B: Isopropanol [0.1% NH3 (7 M in MeOH)]; Gradient: B 30%; Flow rate: 100 mL / min; Back pressure: 100 bar; Column temperature: 35°C; Wavelength: 214 nm; Cycle time: ~2 minutes. Analytical method: Column: ChiralPak AD, 100×4.6 mm ID, 3 μm; Mobile phase: A: CO2; B: Isopropanol (0.1% DEA); Gradient: B 5-45% over 4 minutes; Flow rate: 3.0 mL / min; Back pressure: 2000 psi; Column temperature: 40°C; Wavelength: 214 nm; Cycle time: ~2 minutes.

[0160] 2-P1, retention time t = 2.985 min, 1.7 mg;

[0161] 2-P2, retention time t=2.807 minutes, 2.1 mg.

[0162] Example 3

[0163] 6-Chloro-3-(2,4,5-trifluoro-3-methylphenyl)thieno[3,2-b]pyridine-2-carboxylic acid (3)

[0164] Step 1: Synthesis of methyl 6-chloro-3-(2,4,5-trifluoro-3-methylphenyl)thieno[3,2-b]pyridine-2-carboxylate 3b

[0165] 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) at room temperature. 1,1'-di-tert-butylphosphinoferrocenepalladium 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 under a nitrogen atmosphere for 1 h. After cooling to room temperature, ethyl acetate (40 mL) was added, and the mixture was washed with saturated brine (2 x 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 afford the title compound 3b (80 mg, 65% yield).

[0166] MS (ESI) m / z = 372.2 [M+H] + .

[0167] Step 2: Synthesis of 6-chloro-3-(2,4,5-trifluoro-3-methylphenyl)thieno[3,2-b]pyridine-2-carboxylic acid 3

[0168] 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. Lithium hydroxide (52 mg, 2.2 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). The combined organic phases were washed with saturated brine (2 × 20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product, which was separated and purified by preparative HPLC (column: Waters Xbridge, 30 × 250 mm; mobile phase A: 0.1% formic acid in water; mobile phase B: acetonitrile; gradient: 35%-85% over 18 minutes; flow rate: 30 ml / min; peak elution time: 13.50 min) to obtain the title compound 3 (18 mg, yield 22%).

[0169] MS (ESI) m / z = 358.2 [M+H] + .

[0170] 1 H-NMR (400MHz, DMSO-d6) δ8.86(s,1H),8.77(s,1H),7.54–7.48(m,1H),2.26(s,3H).

[0171] 19 F-NMR (376MHz, DMSO-d6) δ-118.78(dd,1F),-137.93(dd,1F),-144.56(dd,1F).

[0172] Example 4

[0173] 6-Fluoro-3-[2,4,5-trifluoro-3-(methylthio)phenyl]thieno[3,2-b]pyridine-2-carboxylic acid (4)

[0174] Step 1: Synthesis of methyl 3-amino-6-fluorothieno[3,2-b]pyridine-2-carboxylate 4b

[0175] To a solution of compound 4a (2 g, 11.97 mmol) in N,N-dimethylformamide (20 mL) at 0°C was added methyl thioglycolate (1.1 mL, 12.57 mmol) dropwise, followed by the slow addition of an aqueous solution of potassium hydroxide (5 M, 4.8 mL, 23.94 mmol). The reaction was maintained at 0°C for 30 minutes. The reaction solution was poured into ice water (60 mL) and extracted with ethyl acetate (3 × 50 mL). The organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography (petroleum ether / ethyl acetate) to afford compound 4b (2 g, 73% yield).

[0176] MS (ESI) m / z = 227.2 [M+H] + .

[0177] Step 2: Synthesis of methyl 6-fluoro-3-iodothieno[3,2-b]pyridine-2-carboxylate 4c

[0178] Compound 4b (1 g, 4.42 mmol) and diiodomethane (0.54 mL, 6.63 mmol) were dissolved in acetonitrile (20 mL) at room temperature. The temperature was raised to 70°C, and isoamyl nitrite (0.89 mL, 6.63 mmol) was slowly added dropwise. The temperature was maintained at 70-80°C during the addition. The reaction was continued at 70°C for 2 hours. The reaction solution was concentrated to dryness under reduced pressure, and the residue was purified by column chromatography (petroleum ether / ethyl acetate) to obtain crude product 1c (350 mg, 23% yield).

[0179] MS (ESI) m / z = 338.2 [M+H] + .

[0180] Step 3: Synthesis of methyl 6-fluoro-3-(2,4,5-trifluoro-3-hydroxyphenyl)thieno[3,2-b]pyridine-2-carboxylate 4e

[0181] Compound 4c (300 mg, 0.89 mmol) and compound 4d (366 mg, 1.34 mmol) were dissolved in 1,4-dioxane (2 mL) and water (0.40 mL) at room temperature. 1,1'-di-tert-butylphosphinoferrocenepalladium dichloride (58 mg, 0.09 mmol) and potassium carbonate (369 mg, 2.67 mmol) were added. The reaction mixture was stirred at 100°C under a nitrogen atmosphere for 1 h. After cooling to room temperature, ethyl acetate (20 mL) was added, and the mixture was washed with saturated brine (2 x 10 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 afford the title compound 4e (100 mg, 31% yield).

[0182] MS (ESI) m / z = 358.2 [M+H] + .

[0183] Step 4: Synthesis of 2,5,6-trifluoro-3-[6-fluoro-2-(methoxycarbonyl)thieno[3,2-b]pyridin-3-yl]phenyl trifluoromethanesulfonate 4f

[0184] To a solution of compound 4e (100 mg, 0.28 mmol) in dichloromethane (4 mL) were added triethylamine (85 mg, 0.84 mmol) and trifluoromethanesulfonic anhydride (118 mg, 0.42 mmol) at room temperature. The mixture was allowed to react for 1 h. The dichloromethane was removed by concentration under reduced pressure, and the residue was added with ethyl acetate (20 mL). The residue 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 product was purified by column chromatography (petroleum ether / ethyl acetate) to afford the title compound 4f (98 mg, 71% yield).

[0185] MS (ESI) m / z = 490.2 [M+H] + .

[0186] Step 5: Synthesis of 4 g of methyl 6-fluoro-3-[2,4,5-trifluoro-3-(methylthio)phenyl]thieno[3,2-b]pyridine-2-carboxylate

[0187] To a round-bottom flask at room temperature, compound 4f (80 mg, 0.16 mmol), sodium thiomethoxide (57 mg, 0.82 mmol), palladium acetate (7.3 mg, 0.033 mmol), 1,1-binaphthyl-2,2-bisdiphenylphosphine (20.4 mg, 0.033 mmol), and toluene (2 mL) were added. The mixture was heated to 120°C under a nitrogen atmosphere and allowed to react overnight. The reaction mixture was cooled to room temperature, added with ethyl acetate (20 mL), washed with saturated brine (2 × 10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product, which was purified by column chromatography (petroleum ether / ethyl acetate) to afford 4 g of the title compound (50 mg, 79% yield).

[0188] MS (ESI) m / z = 388.2 [M+H] + .

[0189] Step 6: Synthesis of 6-fluoro-3-[2,4,5-trifluoro-3-(methylthio)phenyl]thieno[3,2-b]pyridine-2-carboxylic acid 4

[0190] Compound 4g (50 mg, 0.13 mmol) was dissolved in a mixture of tetrahydrofuran (2 ml), methanol (1 ml), and water (1 ml) at room temperature. Lithium hydroxide (31 mg, 1.3 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 (1N, 4 mL). The mixture was extracted with ethyl acetate (2 x 20 mL). The combined organic phases were washed with saturated brine (2 x 20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product, which was separated and purified by preparative HPLC (column: Waters Xbridge, 30 x 250 mm; mobile phase A: 0.1% formic acid in water; mobile phase B: acetonitrile; gradient: 40%-95% over 18 minutes; flow rate: 30 ml / min; peak elution time: 10.99 minutes) to obtain the title compound 4 (18 mg, 37% yield).

[0191] MS (ESI) m / z = 374.2 [M+H] + .

[0192] 1 H-NMR (400MHz, DMSO-d6) δ8.80(s,1H),8.65(d,1H),2.52(s,3H).

[0193] 19 F-NMR(376MHz, DMSO-d6)δ-110.02(d,1F),-127.38(s,1F),-129.29(d,1F),-142.40(dd,1F).

[0194] Example 5

[0195] 6-Fluoro-3-(2,4,5-trifluoro-3-methoxyphenyl)thieno[3,2-b]pyridine-2-carboxylic acid (5)

[0196] The synthetic method in Example 1 was used to replace compound 1d with compound 4b to prepare 6-fluoro-3-(2,4,5-trifluoro-3-methoxyphenyl)thieno[3,2-b]pyridine-2-carboxylic acid (5).

[0197] Preparative HPLC separation and purification of compound 5: Column: Waters Xbridge, 30 × 250 mm. Mobile phase A: 0.1% formic acid in water; Mobile phase B: acetonitrile. Gradient: 40% to 90% over 18 minutes. Flow rate: 30 ml / min. Peak elution time: 10.5 minutes.

[0198] MS (ESI) m / z = 358.30 [M+H] + .

[0199] 1 H-NMR (400MHz, DMSO-d6) δ8.80(d,1H),8.64(dd,1H),7.44-7.38(m,1H),4.01(s,3H).

[0200] 19 F-NMR (376MHz, DMSO-d6) δ-127.42(s,1F),-132.32(dd,1F),-142.65(dd,1F),-151.72(dd,1F).

[0201] Example 6

[0202] 3-(3-Ethyl-2,4,5-trifluorophenyl)-6-fluorothieno[3,2-b]pyridine-2-carboxylic acid (6)

[0203] Step 1: Synthesis of 6g of methyl 6-fluoro-3-(2,4,5-trifluoro-3-vinylphenyl)thieno[3,2-b]pyridine-2-carboxylate

[0204] Compound 4f (160 mg, 0.32 mmol), potassium ethylene trifluoroborate (127 mg, 0.95 mmol), tris(dibenzylideneacetone)dipalladium (28.9 mg, 0.032 mmol), 2-dicyclohexylphosphino-2',6'-dimethoxybiphenyl (13 mg, 0.032 mmol), and potassium phosphate (168 mg, 0.79 mmol) were dissolved in toluene (5 mL) and reacted at 100°C under a nitrogen atmosphere for 1 hour at room temperature. The reaction solution was poured into water (50 mL) and extracted with ethyl acetate (3 x 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 yield 6 g (112 mg, 95% yield).

[0205] MS (ESI) m / z = 368.2 [M+H] + .

[0206] Step 2: Synthesis of methyl 3-(3-ethyl-2,4,5-trifluorophenyl)-6-fluorothieno[3,2-b]pyridine-2-carboxylate 6h

[0207] Compound 6g (100 mg, 0.26 mmol) and 10% palladium on carbon (29 mg, 0.27 mmol) were dissolved in methanol (15 mL) at room temperature and stirred under a hydrogen atmosphere for 2 hours. The reaction solution was directly filtered and the filtrate was concentrated under reduced pressure to obtain crude product 6h (95 mg, 98%).

[0208] MS (ESI) m / z = 370.2 [M+H] + .

[0209] Step 3: Synthesis of 3-(3-ethyl-2,4,5-trifluorophenyl)-6-fluorothieno[3,2-b]pyridine-2-carboxylic acid 6

[0210] Compound 6h (100 mg, 0.27 mmol) was dissolved in a mixture of tetrahydrofuran (6 ml), methanol (3 ml), and water (3 ml) at room temperature. Lithium hydroxide (114 mg, 2.71 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 (1N, 4 mL). The mixture was extracted with ethyl acetate (3 × 30 mL). The combined organic phases were washed with saturated brine (2 × 20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product, which was separated and purified by preparative HPLC (column: Waters Xbridge, 30 × 250 mm; mobile phase A: 0.1% formic acid in water; mobile phase B: acetonitrile; gradient: 40%-95% over 18 minutes, flow rate: 30 ml / min, peak elution time: 11.52 minutes) to obtain the title compound 6 (26.8 mg, 27% yield).

[0211] MS (ESI) m / z = 356.2 [M+H] + .

[0212] 1 H-NMR (400MHz, DMSO-d6) δ8.79(s,1H),8.63(d,1H),7.52(dd,1H),2.73(q,2H),1.19(q,3H).

[0213] 19 F-NMR(376MHz, DMSO-d6)δ-120.81(dd,1F),-127.60(s,1F),-140.39(dd,1F)-144.30(dd,1F).

[0214] Example 7

[0215] 6-Chloro-3-(3-ethyl-2,4,5-trifluorophenyl)thieno[3,2-b]pyridine-2-carboxylic acid (7)

[0216] Step 1: Synthesis of methyl 6-chloro-3-(2,4,5-trifluoro-3-hydroxyphenyl)thieno[3,2-b]pyridine-2-carboxylate 7e

[0217] At room temperature, compound 1d (500 mg, 1.64 mmol) and compound 4d (676 mg, 2.47 mmol) were dissolved in 1,4-dioxane (5 mL) and water (0.80 mL). 1,1'-di-tert-butylphosphinoferrocenepalladium dichloride (103 mg, 0.16 mmol) and potassium carbonate (680 mg, 4.92 mmol) were added. The reaction mixture was stirred at 100°C under a nitrogen atmosphere for 1 h. After cooling to room temperature, ethyl acetate (40 mL) was added, and the mixture was washed with saturated brine (2 × 15 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 afford the title compound 7e (171 mg, 28% yield).

[0218] MS (ESI) m / z = 374.1 [M+H] + .

[0219] Step 2: Synthesis of 2,5,6-trifluoro-3-[6-chloro-2-(methoxycarbonyl)thieno[3,2-b]pyridin-3-yl]phenyl trifluoromethanesulfonate 7f

[0220] To a solution of compound 7e (171 mg, 0.46 mmol) in dichloromethane (6 mL) were added triethylamine (255 mg, 1.38 mmol) and trifluoromethanesulfonic anhydride (177 mg, 0.69 mmol) at room temperature. The mixture was allowed to react for 1 h. The dichloromethane was removed by concentration under reduced pressure, and the residue was added with ethyl acetate (40.0 mL). The residue was 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 afford the title compound 7f (200 mg, 86% yield).

[0221] MS (ESI) m / z = 506.1 [M+H] + .

[0222] Using the synthesis method in Example 6, 6-chloro-3-(3-ethyl-2,4,5-trifluorophenyl)thieno[3,2-b]pyridine-2-carboxylic acid (7) was synthesized from compound 7f in three steps.

[0223] Preparative HPLC separation and purification of compound 7: Column: Waters Xbridge, 30 × 250 mm. Mobile phase A: 0.1% formic acid in water; Mobile phase B: acetonitrile. Gradient: 45% to 95% over 18 minutes. Flow rate: 30 ml / min. Peak elution time: 12.37 minutes.

[0224] MS (ESI) m / z = 372.1 [M+H] + .

[0225] 1 H-NMR (400MHz, DMSO-d6) δ8.86(s,1H),8.78(s,1H),7.52(q,1H),2.73(q,2H),1.19(t,3H).

[0226] 19 F-NMR(376MHz, DMSO-d6)δ-120.80(dd,1F),-140.28(dd,1F),-144.24(dd,1F).

[0227] Example 8

[0228] 3-{6-chloro-3-[(difluoromethyl)oxy]-2,4-difluorophenyl}-6-fluorothieno[3,2-b]pyridine-2-carboxylic acid (8)

[0229] Step 1: Synthesis of tert-butyl (4-chloro-2,6-difluorophenoxy)dimethylsilyl ester 8b

[0230] To a solution of compound 8a (10 g, 60.8 mmol) in dichloromethane (200 mL) was added triethylamine (9.2 g, 91.2 mmol) under ice-cooling. After stirring for 10 minutes, tert-butyldimethylsilyl trifluoromethanesulfonate (24.1 g, 91.2 mmol) was added dropwise. Stirring was continued at room temperature for 1 hour. The reaction mixture 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 afford 8b as an oil (15.1 g, 89% yield).

[0231] Step 2: Synthesis of (3-((tert-butyldimethylsilyl)oxy)-6-chloro-2,4-difluorophenyl)boronic acid 8c

[0232] To a solution of compound 8b (15 g, 53.8 mmol) in anhydrous tetrahydrofuran (200 mL) at -78°C under a nitrogen atmosphere was slowly added n-butyllithium (2.5 M, 23.6 mL, 59.1 mmol) dropwise. The mixture was stirred at -78°C for 1 hour. A solution of trimethyl borate (7.3 g, 70 mmol) in anhydrous tetrahydrofuran (20 mL) was then slowly added dropwise. The mixture was stirred at -78°C for an additional hour. The reaction mixture was slowly added to a saturated ammonium chloride solution (200 mL) stirred under an ice bath. After 5 minutes, ethyl acetate (100 mL) was added. After mixing thoroughly, the organic phase was separated, 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 afford 8c (11.2 g, 64% yield) as an oil.

[0233] 1 H-NMR (400MHz, DMSO-d6) δ8.71(s,2H),7.28(d,1H),0.98(s,9H),0.18(s,6H).

[0234] Step 3: Synthesis of methyl 3-(3-((tert-butyldimethylsilyl)oxy)-6-chloro-2,4-difluorophenyl)-6-fluorothieno[3,2-b]pyridine-2-carboxylate 8d

[0235] Compound 8c (334 mg, 1.03 mmol) and compound 5d (300 mg, 1.03 mmol) were dissolved in toluene (5 mL) and water (1 mL) at room temperature. Tris(dibenzylideneacetone)dipalladium (94.6 mg, 0.10 mmol), 2-dicyclohexylphosphino-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 under a nitrogen atmosphere for 1 h. After cooling to room temperature, ethyl acetate (40 mL) was added, and the mixture was washed with saturated brine (2 x 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 afford the title compound 8d (197 mg, 39% yield).

[0236] MS (ESI) m / z = 488.4 [M+H] + .

[0237] Step 4: Synthesis of methyl 3-(6-chloro-2,4-difluoro-3-hydroxyphenyl)-6-fluorothieno[3,2-b]pyridine-2-carboxylate 8e

[0238] 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. The reaction mixture was poured into water (30 mL) and extracted with ethyl acetate (3 × 20 mL). The combined organic phases were washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to afford crude product 8e (201 mg, 145% crude yield).

[0239] MS (ESI) m / z = 374.2 [M+H] + .

[0240] Step 5: Synthesis of methyl 3-{6-chloro-3-[(difluoromethyl)oxy]-2,4-difluorophenyl}-6-fluorothieno[3,2-b]pyridine-2-carboxylate 8f

[0241] 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 under a nitrogen atmosphere for 2 hours. The reaction solution was poured into water (30 mL) and 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 obtain the crude product, which was then purified by column chromatography (petroleum ether / ethyl acetate) to obtain compound 8f (100 mg, 49%).

[0242] MS (ESI) m / z = 424.2 [M+H] + .

[0243] Step 6: Synthesis of 3-{6-chloro-3-[(difluoromethyl)oxy]-2,4-difluorophenyl}-6-fluorothieno[3,2-b]pyridine-2-carboxylic acid 8

[0244] 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. 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 × 30 mL). The combined organic phases were washed with saturated brine (2 × 20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product, which was separated and purified by preparative HPLC (column: Waters Xbridge, 30 × 250 mm; mobile phase A: 0.1% formic acid in water; mobile phase B: acetonitrile; gradient: 35%-90% over 18 minutes, flow rate: 30 ml / min, peak elution time: 11.37 min) to obtain the title compound 8 (27.8 mg, 28% yield).

[0245] MS (ESI) m / z = 410.1 [M+H] + .

[0246] 1 H-NMR (400MHz, DMSO-d6) δ8.78(s,1H),8.67(d,1H),7.84(d,1H),7.34(t,1H).

[0247] 19 F-NMR(376MHz, DMSO-d6)δ-82.76(t,2F),-121.98(dd,1F),-124.08(s,1F),-126.95(s,1F).

[0248] Example 9

[0249] 3-{3-[(difluoromethyl)oxy]-2,4,5-trifluorophenyl}-6-fluorothieno[3,2-b]pyridine-2-carboxylic acid (9)

[0250] The synthetic method of Example 8 was used to prepare 3-{3-[(difluoromethyl)oxy]-2,4,5-trifluorophenyl}-6-fluorothieno[3,2-b]pyridine-2-carboxylic acid (9) by substituting compound 4e for compound 8e.

[0251] Preparative HPLC separation and purification of compound 9: Column: Waters Xbridge, 30 × 250 mm. Mobile phase A: 0.1% formic acid in water; Mobile phase B: acetonitrile. Gradient: 25% to 70% over 18 minutes. Flow rate: 30 ml / min. Peak elution time: 11.72 minutes.

[0252] MS (ESI) m / z = 392.1 [MH] - .

[0253] 1 H-NMR (400MHz, DMSO-d6) δ8.81(s,1H),8.65(d,1H),7.76(dd,1H),7.36(t,1H).

[0254] 19 F-NMR(376MHz, DMSO-d6)δ-82.48(s,2F),-127.30(3,1F),-129.43~-129.50(m,1F),-141.61(dd,1F),-147.72(d,1F).

[0255] Example 10

[0256] 6-Chloro-3-{3-[(difluoromethyl)oxy]-2,4,5-trifluorophenyl}thieno[3,2-b]pyridine-2-carboxylic acid (10)

[0257] The synthetic method of Example 8 was used to prepare 6-chloro-3-{3-[(difluoromethyl)oxy]-2,4,5-trifluorophenyl}thieno[3,2-b]pyridine-2-carboxylic acid (10) by substituting compound 7e for compound 8e.

[0258] Preparative HPLC separation and purification of compound 10: Column: Waters Xbridge, 30 × 250 mm. Mobile phase A: 0.1% formic acid in water; Mobile phase B: acetonitrile. Gradient: 45% to 90% over 18 minutes. Flow rate: 30 ml / min. Peak elution time: 11.28 minutes.

[0259] MS (ESI) m / z = 392.1 [MH] - .

[0260] 1 H-NMR (400MHz, DMSO-d6) δ8.81(s,1H),8.65(d,1H),7.76(dd,1H),7.36(t,1H).

[0261] 19 F-NMR (376MHz, DMSO-d6) δ-82.48(s,2F),-127.30(3,1F),-129.43~-129.50(m,1F),-141.61(dd,1F),-147.72(d,1F).

[0262] Example 11

[0263] 6-Chloro-3-{6-chloro-3-[(difluoromethyl)oxy]-2,4-difluorophenyl}thieno[3,2-b]pyridine-2-carboxylic acid (11)

[0264] The synthetic method of Example 8 was used to prepare 6-chloro-3-{6-chloro-3-[(difluoromethyl)oxy]-2,4-difluorophenyl}thieno[3,2-b]pyridine-2-carboxylic acid (11) by replacing compound 5d with compound 1d.

[0265] Preparative HPLC separation and purification of compound 11: Column: Waters Xbridge, 30 × 250 mm. Mobile phase A: 0.1% formic acid in water; Mobile phase B: acetonitrile. Gradient: 50% to 95% over 18 minutes. Flow rate: 30 ml / min. Peak elution time: 10.18 minutes.

[0266] MS (ESI) m / z = 426.1 [M+H] + .

[0267] 1 H-NMR (400MHz, DMSO-d6) δ8.89(s,1H),8.77(s,1H),7.83(d,1H),7.33(t,1H).

[0268] 19F-NMR(376MHz, DMSO-d6)δ-82.75(t,2F),-121.93(d,1F),-124.02(s,1F).

[0269] Example 12

[0270] 6-Chloro-3-(4-chloro-2-fluoro-3-methoxyphenyl)thieno[3,2-b]pyridine-2-carboxylic acid (12)

[0271] The synthetic method of Example 8 was used to prepare 6-chloro-3-(4-chloro-2-fluoro-3-methoxyphenyl)thieno[3,2-b]pyridine-2-carboxylic acid (12) by replacing compound 5d with compound 1d and compound 8c with compound 12a.

[0272] Preparative HPLC separation and purification of compound 12: Column: Waters Xbridge, 30 × 250 mm. Mobile phase A: 0.1% formic acid in water; Mobile phase B: acetonitrile. Gradient: 50% to 90% over 18 minutes. Flow rate: 30 ml / min. Peak elution time: 10.65 minutes.

[0273] MS (ESI) m / z = 372.2 [M+H] + .

[0274] 1 H-NMR (400MHz, DMSO-d6) δ8.86(s,1H),8.76(s,1H),7.43(d,1H),7.24(t,1H),3.90(s,3H).

[0275] 19 F-NMR(376MHz,DMSO-d6)δ-127.61(s,1F).

[0276] Example 13

[0277] 6-Chloro-3-(4-chloro-2-fluoro-3-methylphenyl)thieno[3,2-b]pyridine-2-carboxylic acid (13)

[0278] The synthesis method in Example 8 was used to synthesize 6-chloro-3-(4-chloro-2-fluoro-3-methylphenyl)thieno[3,2-b]pyridine-2-carboxylic acid (13).

[0279] Preparative HPLC separation and purification of compound 13: Column: Waters Xbridge, 30 × 250 mm. Mobile phase A: 0.1% formic acid in water; Mobile phase B: acetonitrile. Gradient: 50% to 95% over 18 minutes. Flow rate: 30 ml / min. Peak elution time: 11.22 minutes.

[0280] MS (ESI) m / z = 356.1 [M+H] + .

[0281] 1 H-NMR (400MHz, DMSO-d6) δ8.85(s,1H),8.74(s,1H),7.40(d,1H),7.33(t,1H),2.31(s,3H).

[0282] 19 F-NMR (376MHz, DMSO-d6) δ-112.58 (s, 1F).

[0283] Example 14

[0284] 6-Chloro-3-{6-chloro-3-[(difluoromethyl)oxy]-2,4-difluorophenyl}thieno[3,2-b]pyridine-2-carboxylic acid (11-P1);

[0285] 6-Chloro-3-{6-chloro-3-[(difluoromethyl)oxy]-2,4-difluorophenyl}thieno[3,2-b]pyridine-2-carboxylic acid (11-P2)

[0286] Compound 11 (60 mg) was separated by SFC using the following separation method: column: ChiralPak IC, 250×30 mm ID, 5 μm; mobile phase: A: CO 2 ; B: isopropanol [0.1% NH 3 (7 M in MeOH)]; gradient: B 20%; flow rate: 100 mL / min; back pressure: 100 bar; column temperature: 35° C.; wavelength: 214 nm; cycle time: ~2.5 minutes.

[0287] 11-P1, retention time t = 2.862 minutes, 24 mg;

[0288] 11-P2, retention time t=2.263 minutes, 18 mg.

[0289] Example 15

[0290] 6-Chloro-3-(4-chloro-2,5-difluoro-3-methylphenyl)thieno[3,2-b]pyridine-2-carboxylic acid (15)

[0291] Step 1: Synthesis of 2,5-difluoro-3-methylaniline 15b

[0292] 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 under a nitrogen atmosphere. The reaction mixture 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 obtain compound 15b (700 mg, 84% yield).

[0293] MS (ESI) m / z = 144.1 [M+H] + .

[0294] Step 2: Synthesis of 4-chloro-2,5-difluoro-3-methylaniline 15c

[0295] Compound 15b (650 mg, 4.54 mmol) and N-chlorosuccinimide (606 mg, 4.54 mmol) were dissolved in dichloromethane (10 mL) at room temperature and stirred at 50°C overnight under a nitrogen atmosphere. The reaction solution was concentrated under reduced pressure, and the residue was purified by column chromatography (petroleum ether / ethyl acetate) to obtain compound 15c (300 mg, 37% yield).

[0296] Step 3: Synthesis of 1-bromo-4-chloro-2,5-difluoro-3-methylbenzene 15d

[0297] At room temperature, tert-butyl 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 portionwise to the mixture and stirred at room temperature for 1 hour. The reaction mixture 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 afford compound 15d (300 mg, 88% yield).

[0298] Step 4: Synthesis of methyl 6-chloro-3-(4-chloro-2,5-difluoro-3-methylphenyl)thieno[3,2-b]pyridine-2-carboxylate 15e

[0299] Compound 15d (200 mg, 0.83 mmol), bis(pinacol boronate) (210 mg, 0.83 mmol), 1,1'-bis(diphenylphosphino)ferrocenepalladium dichloride (61.44 mg, 0.08 mmol), and potassium acetate (203 mg, 2.07 mmol) were dissolved in 1,4-dioxane (5 mL) at room temperature and stirred at 80°C overnight under a nitrogen atmosphere. Compound 1d (100 mg, 0.33 mmol), potassium carbonate (239 mg, 1.73 mmol), 1,1'-di-tert-butylphosphinoferrocenepalladium dichloride (45 mg, 0.07 mmol), and water (1 mL) were then added to the mixture and stirred at 80°C under a nitrogen atmosphere for 1 hour. The reaction mixture was poured into water (30 mL), extracted with ethyl acetate (3 x 30 mL), washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate concentrated under reduced pressure. The residue was purified by column chromatography (petroleum ether / ethyl acetate) to give compound 15e (120 mg, yield 44%).

[0300] Step 5: Synthesis of 6-chloro-3-(4-chloro-2,5-difluoro-3-methylphenyl)thieno[3,2-b]pyridine-2-carboxylic acid 15

[0301] 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. Water (30 mL) was added, and the pH was adjusted to 4-5 with hydrochloric acid (1N, 4 mL). The mixture was extracted with ethyl acetate (2 × 20 mL). The combined organic phases were washed with saturated brine (2 × 20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product, which was separated and purified by preparative HPLC (Waters Xbridge column, 30 × 250 mm; mobile phase A: 0.1% formic acid in water; mobile phase B: acetonitrile; gradient: 45%-95% over 15 minutes; flow rate: 30 mL / min; peak elution time: 9.85 minutes) to obtain the title compound 15 (54 mg, 46% yield).

[0302] MS (ESI) m / z = 374.1 [M+H] + .

[0303] 1 H-NMR (400MHz, DMSO-d6) δ8.86(s,1H),8.76(s,1H),7.50(t,1H),2.35(s,3H).

[0304] 19F-NMR(376MHz,DMSO-d6)δ-117.41(d,1F),-120.46(d,1F).

[0305] Example 16

[0306] 6-Chloro-3-(5-chloro-2,4-difluoro-3-methoxyphenyl)thieno[3,2-b]pyridine-2-carboxylic acid (16)

[0307] The synthetic method in Example 1 was used to replace compound 1e with compound 16a to prepare 6-chloro-3-(5-chloro-2,4-difluoro-3-methoxyphenyl)thieno[3,2-b]pyridine-2-carboxylic acid (16).

[0308] Preparative HPLC separation and purification of compound 16: Column: Waters Xbridge, 30 x 250 mm. Mobile phase A: 0.1% formic acid in water, mobile phase B: acetonitrile; gradient: 40% to 95% over 18 minutes, flow rate: 30 ml / min. Peak elution time: 12.26 minutes.

[0309] MS (ESI) m / z = 390.1 [M+H] + .

[0310] 1 H-NMR (400MHz, DMSO-d6) δ8.87(s,1H),8.79(s,1H),7.50(t,1H),3.99(s,3H).

[0311] 19 F-NMR(376MHz,DMSO-d6)δ-127.96(s,1F),-128.99(s,1F).

[0312] Example 17

[0313] 6-Chloro-3-(5-chloro-2,4-difluoro-3-methylphenyl)thieno[3,2-b]pyridine-2-carboxylic acid (17)

[0314] Step 1: Synthesis of 6-chloro-2,4-difluoro-3-methylaniline 17b

[0315] Compound 17a (2 g, 13.97 mmol) and N-chlorosuccinimide (1.96 mg, 14.67 mmol) were dissolved in dichloromethane (20 mL) at room temperature and stirred at 50°C overnight. The solvent was removed by concentration, and the residue was added with ethyl acetate (50 mL), 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 purified by column chromatography (petroleum ether / ethyl acetate) to afford 17b (700 mg, 28% yield).

[0316] Step 2: Synthesis of 2-bromo-1-chloro-3,5-difluoro-4-methylbenzene 17c

[0317] To a three-necked flask at room temperature, copper bromide (973 mg, 4.34 mmol) and acetonitrile (15 mL) were added dropwise. Tert-butyl 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. Stirring was continued at room temperature for 2 hours. Dilute hydrochloric acid (1N, 10 mL) was added, and the mixture was extracted with ethyl acetate (2 × 20 mL). The mixture was washed sequentially with water (1 × 10 mL) and saturated brine (2 × 10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product, which was then purified by column chromatography (petroleum ether / ethyl acetate) to afford compound 17c (450 mg, 47% yield).

[0318] Step 3: Synthesis of 2-(6-chloro-2,4-difluoro-3-methylphenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane 17d

[0319] Under a nitrogen atmosphere, a solution of compound 17c (450 mg, 1.86 mmol) in tetrahydrofuran (5 mL) was slowly added dropwise with isopropylmagnesium chloride in tetrahydrofuran (2M, 2.8 mL) at dry ice-acetonitrile temperature. After stirring for 3 hours, 4,4,5,5-tetramethyl-2-(propan-2-yloxy)-1,3,2-dioxaborolane (0.76 mL, 3.73 mmol) was slowly added dropwise to the reaction mixture. The temperature was slowly raised to 20°C and stirring continued for 2 hours. Dilute hydrochloric acid (1N, 10 mL) was added to the reaction mixture, and the mixture was extracted with ethyl acetate (2 × 20 mL). The mixture was washed sequentially with water (1 × 10 mL) and saturated brine (2 × 10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product, which was then purified by column chromatography (petroleum ether / ethyl acetate) to obtain compound 17d (140 mg, 26% yield).

[0320] Step 4: Synthesis of methyl 6-chloro-3-(6-chloro-2,4-difluoro-3-methylphenyl)thieno[3,2-b]pyridine-2-carboxylate 17e

[0321] Compound 17d (100 mg, 0.32 mmol) and compound 1d (140 mg, 0.48 mmol) were dissolved in toluene / water (2 ml / 0.5 ml) at room temperature. Tris(dibenzylideneacetone)dipalladium (30 mg, 0.032 mmol), 2-dicyclohexylphosphino-2',6'-dimethoxybiphenyl (29 mg, 0.064 mmol), and potassium phosphate (216 mg, 0.96 mmol) were then added. The mixture was stirred at 100°C under a nitrogen atmosphere for 4 hours. Ethyl acetate (60 ml) was added to the reaction system, and the mixture was washed sequentially with water (1 x 10 ml) and saturated brine (2 x 10 ml), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product, which was then purified by column chromatography (petroleum ether / ethyl acetate) to afford compound 17e (90 mg, 72% yield).

[0322] MS (ESI) m / z = 388.2 [M+H] + .

[0323] Step 5: Synthesis of 6-chloro-3-(5-chloro-2,4-difluoro-3-methylphenyl)thieno[3,2-b]pyridine-2-carboxylic acid 17

[0324] Compound 17e (90 mg, 0.23 mmol) was dissolved in a mixture of tetrahydrofuran (2 ml), methanol (1 ml), and water (1 ml) at room temperature. 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). Ethyl acetate (40 ml) was added, and the mixture was washed sequentially with water (1 x 10 ml) and saturated brine (2 x 10 ml), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to afford crude product 17. This was then separated and purified by preparative HPLC (column: Waters Xbridge, 30 x 250 mm; mobile phase A: 0.1% formic acid in water; mobile phase B: acetonitrile; flow rate: 30 ml / min, 18 min; gradient: 40%-95%; retention time: 11.99 min) to afford the title compound 17 (37 mg, 43% yield).

[0325] MS (ESI) m / z = 374.1 [M+H] + .

[0326] 1 H-NMR (400MHz, DMSO-d6) δ8.82(s,1H),8.74(s,1H),7.48(s,1H),2.19(s,3H).

[0327] 19F-NMR (376MHz, DMSO-d6) δ-111.12 (s, 1F), δ = -112.74 (s, 1F).

[0328] Example 18

[0329] 6-Chloro-3-{6-chloro-2,4-difluoro-3-[(trideuteriomethyl)oxy]phenyl}thieno[3,2-b]pyridine-2-carboxylic acid (18);

[0330] 6-Chloro-3-{6-chloro-2,4-difluoro-3-[(trideuteriomethyl)oxy]phenyl}thieno[3,2-b]pyridine-2-carboxylic acid (18-P1); 6-Chloro-3-{6-chloro-2,4-difluoro-3-[(trideuteriomethyl)oxy]phenyl}thieno[3,2-b]pyridine-2-carboxylic acid (18-P2)

[0331] Step 1: Synthesis of methyl 6-chloro-3-(6-chloro-2,4-difluoro-3-hydroxyphenyl)thieno[3,2-b]pyridine-2-carboxylate 18

[0332] Compound 2b (5 g, 12.4 mmol) was dissolved in dichloromethane (10 mL) under ice-cooling. A dichloromethane solution of boron tribromide (1 M, 50 mL) was slowly added dropwise. The mixture was stirred at room temperature for 2 hours. Methanol was slowly added under ice-cooling, and the mixture was stirred for 10 minutes. The mixture was then concentrated under reduced pressure to afford crude product 18a (4 g, 82% yield), which was used directly in the next step.

[0333] MS (ESI) m / z = 390.3 [M+H] + .

[0334] Step 2: Synthesis of methyl 6-chloro-3-(6-chloro-2,4-difluoro-3-(methoxy-d3)phenyl)thieno[3,2-b]pyridine-2-carboxylate 18b

[0335] 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) at room temperature and stirred at 50°C overnight. Water (30 mL) was added, and the mixture was extracted with ethyl acetate (3 × 10 mL). The combined organic phases were washed with saturated brine (2 × 10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to afford crude product 18b (3.5 g, 83% yield), which was used directly in the next step.

[0336] MS (ESI) m / z = 407.3 [M+H] + .

[0337] Step 3: Synthesis of 6-chloro-3-(6-chloro-2,4-difluoro-3-(methoxy-d3)phenyl)thieno[3,2-b]pyridine-2-carboxylic acid 18

[0338] 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. Lithium hydroxide (2.1 g, 87 mmol) was added, and the reaction mixture was stirred at room temperature for 2 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 × 30 mL). The combined organic phases were washed with saturated brine (2 × 10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product, which was separated and purified by preparative HPLC (column: Waters Xbridge, 30 × 250 mm; mobile phase A: 0.1% formic acid in water; mobile phase B: acetonitrile; gradient: 35%-85% over 15 min; flow rate: 30 mL / min; peak elution time: 9.82 min) to obtain the title compound 18 (1.8 g, 52% yield).

[0339] MS (ESI) m / z = 393.3 [M+H] +

[0340] 1 H-NMR (400MHz, DMSO-d6) δ8.87(d,1H),8.76(d,1H),7.67(d,1H).

[0341] 19 F-NMR(376MHz,DMSO-d6)δ-124.93(s,1F),-126.95(s,1F).

[0342] Step 4: 6-chloro-3-{6-chloro-2,4-difluoro-3-[(trideuteriomethyl)oxy]phenyl}thieno[3,2-b]pyridine-2-carboxylic acid (18-P1); 6-chloro-3-{6-chloro-2,4-difluoro-3-[(trideuteriomethyl)oxy]phenyl}thieno[3,2-b]pyridine-2-carboxylic acid (18-P2)

[0343] Compound 18 (92 mg) was separated by SFC using the following separation method: column: ChiralPak AD, 250×30 mm ID, 5 μm; mobile phase: A: CO 2 ; B: isopropanol [0.1% NH 3 (7 M in MeOH)]; gradient: B 20%; flow rate: 100 mL / min; back pressure: 100 bar; column temperature: 35° C.; wavelength: 214 nm; cycle time: ~5 minutes.

[0344] 18-P1, retention time t = 2.819 min, 32 mg;

[0345] 18-P2, retention time t=2.626 minutes, 34 mg.

[0346] Example 19

[0347] 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)

[0348] Step 1: Synthesis of 3-(6-chloro-2,4-difluoro-3-methoxyphenyl)-6-fluorothieno[3,2-b]pyridine-2-carboxylic acid (19)

[0349] The synthetic method of Example 2 was used to prepare 3-(6-chloro-2,4-difluoro-3-methoxyphenyl)-6-fluorothieno[3,2-b]pyridine-2-carboxylic acid (19) by substituting compound 5d for compound 1d.

[0350] Preparative HPLC separation and purification of compound 19: Column: Waters Xbridge, 30 × 250 mm. Mobile phase A: 0.1% formic acid in water; Mobile phase B: acetonitrile. Gradient: 35% to 95% over 18 minutes. Flow rate: 30 mL / min. Peak elution time: 10.89 minutes.

[0351] MS (ESI) m / z = 374.2 [M+H] + .

[0352] 1 H-NMR (400MHz, DMSO-d6) δ8.77(d,1H),8.65(dd,1H),7.61(dd,1H),3.95(s,3H).

[0353] 19 F-NMR(376MHz, DMSO-d6)δ-124.96(d,1F),-126.96(d,1F),-127.19(s,1F).

[0354] 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)

[0355] The product 19 (520 mg) was separated by SFC using the following 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; cycle time: ~2 minutes.

[0356] 19-P1, retention time t = 2.409 minutes, 240 mg;

[0357] 19-P2, retention time t=1.993 minutes, 234 mg.

[0358] Biological evaluation

[0359] The following test examples further illustrate and explain the present invention, but these examples are not intended to limit the scope of the present invention.

[0360] Test Example 1: Detection of the inhibitory effect of compounds on BCKDK protein in HEK293 cells

[0361] 1. Experimental Materials

[0362] 2. Test methods

[0363] HEK293 cells were seeded at 10,000 cells / well in 384-well black, clear-bottom plates overnight. The cells were treated with various compound concentrations (100 μM maximum dose, 3-fold dilutions, 10 concentrations) for 4 hours at 37°C under 5% CO2. 50 μL of pre-chilled 8% paraformaldehyde was added to the plate and fixed for 1 hour in the dark. The fixative was aspirated and discarded. 50 μL of permeabilization solution was added per well and allowed to stand for 5 minutes each time, repeated six times. 50 μL of blocking buffer was then added per well and incubated on a shaker at room temperature for 1 hour. The primary antibody was incubated with Phospho-BCKDH-E1α (Ser293) (1000X) at 4°C for 24 hours. After repeating the permeabilization step again, the secondary antibodies Cell Tag520 (500X) and IRDye 800CW (1000X) were incubated for 1 hour in the dark at room temperature. The permeabilization step was repeated once more, and the permeabilization solution was aspirated a final time. Signals were detected using the Odyssey Imager M imaging system, and data were processed and IC calculated using XL fit. 50 .

[0364] 3. Experimental results

[0365] The inhibition of the disclosed compounds on the BCKDK protein of HEK293 cells can be determined by the above test, and the measured IC 50See Table 1.

[0366] Table 1. IC values ​​of the disclosed compounds against HEK293A 50 (μM)

[0367] PF-07328948 is the compound of Example 9 in patent WO2023100061A1, and its structure is

[0368] Test Example 2: Inhibitory activity of compounds against human CYP450 (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A)

[0369] 1. Equipment, Materials, and Reagents

[0370] 1) Human liver microsomes (HLM)

[0371] Human liver microsomes (source: BioIVT) were stored at -80°C. Before use, human liver microsomes were taken out of the refrigerator and thawed in a 37°C water bath, then placed on ice.

[0372] 2) Substrate

[0373] The preparation details of the substrate mixed solution are as follows: the substrate mixed solution consists of 50% pure water and 50% organic solvent. The prepared substrate mixed solution is stored in a -20°C refrigerator. Before use, it is taken out of the refrigerator and warmed to room temperature, and vortexed for 30 seconds before use.

[0374] CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A substrate mixture

[0375] 3) Phosphate buffer (100 mmol / L, pH 7.4)

[0376] Disodium hydrogen phosphate (analytical grade) and potassium dihydrogen phosphate (analytical grade) were purchased from a local supplier. Solution A was prepared by accurately weighing 7.098 g of disodium hydrogen phosphate, adding it to 500 ml of pure water, and sonicating. Solution B was prepared by accurately weighing 3.400 g of potassium dihydrogen phosphate, adding it to 250 ml of pure water, and sonicating. Solution B was added to Solution A to a final pH of 7.4.

[0377] 4) 10 mmol / L NADPH solution

[0378] NADPH (MW: 833.4 g / mol) was purchased from MCE and dissolved in phosphate buffer at 8.334 mg / mL.

[0379] 2. Experimental process

[0380] 1) Preparation of incubation solution

[0381] The incubation system for the substrate and human liver microsome mixture is as follows:

[0382] 2) Compound dilution

[0383] Add 15 μL of a 10 mmol / L DMSO solution of the test substance to a sequentially labeled compound deep-well plate. The dilution steps are as follows:

[0384] 3) Incubation

[0385] Each well of a 96-well deep-well plate contained 179 μL of a mixture of substrate and human liver microsomes in phosphate-buffered saline, and 1 μL of the test compound or blank solution. The plate was preincubated in a 37°C water bath for 5 minutes, followed by the addition of 20 μL of 10 mM NADPH to initiate the reaction. Following the addition of NADPH, the plate was incubated at 37°C for an additional 5 minutes.

[0386] 4) Reaction quenching

[0387] 300 μL of an acetonitrile solution containing 3% formic acid, 200 nM tolbutamide, 200 nM alprazolam, and 200 nM labetalol was added for quenching, followed by centrifugation at 3,220 g for 50 minutes, and 200 μL of the supernatant was collected for LC / MS / MS analysis.

[0388] 3. Data processing

[0389] The peak areas of all samples and internal standard peak areas were automatically calculated and imported into Excel software.

[0390] The percentage of remaining activity was calculated according to the following formula:

[0391] Area ratio = peak area 受试物 / peak area 内标

[0392] Remaining activity percentage (%) = area ratio 受试物 / Area ratio 空白 ×100

[0393] IC calculation using Excel XLfit 5.3.1.3 50 value (the concentration of the test substance that produces 50% inhibition).

[0394] Table 2. IC values ​​of the compounds disclosed herein for CYP450 inhibition 50 (μM)

[0395] Reference 1 is the compound of Example 16 in patent WO2023100061A1.

[0396] From the results, compound 19-P1 had no inhibitory effect on CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A (IC 50 >30μM), PF-07328948 has a strong inhibitory effect on CYP2C9 ((IC 50 =1.09μM), and the reference substance 1 had an effect on CYP1A2 (IC 50 =5.0 μM), CYP2C9 (IC 50 >2.32μM)、CYP2C19(IC 50 =15.76 μM) showed strong inhibition.

[0397] Test Example 3: Stability test of compounds in hepatocytes

[0398] 1. Experimental steps

[0399] 1) Prepare a high concentration (10 mM) stock solution of the test substance with DMSO and dilute it to a 100 μM working solution with DMSO before use. The final concentration of the test substance is 1 μM.

[0400] 2) Take a tube of cryopreserved hepatocytes (rat, canine, and human hepatocytes from BioIVT, monkey hepatocytes from RILD) and ensure they remain frozen before thawing. Quickly place the hepatocytes 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.

[0401] 3) Pour the contents of the hepatocyte tubules from different species into a centrifuge tube containing 50 mL of recovery medium and centrifuge at 100 g for 10 minutes. After centrifugation, aspirate the recovery medium and add enough incubation medium to obtain a cell density of approximately 1.0 × 10 6 cells / mL of cell suspension.

[0402] 4) Count the hepatocytes and determine the viable cell density using a 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 × 10 6 cells / mL.

[0403] 5) Transfer 198 μL of the 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 the experiment in duplicate.

[0404] 6) Add 2 μL of 100 μM test substance to each well to initiate the reaction, and place the deep-well plate back on the incubator vortexer.

[0405] 7) Incubate the sample. At 0, 15, 30, 60, 90, and 120 minutes, aspirate 25 μL of the suspension and terminate the reaction by adding 150 μL of acetonitrile containing the internal standard. Vortex for 10 minutes and centrifuge at 3220 g at 4°C for 45 minutes. Transfer 100 μL of the supernatant to the injection plate and mix thoroughly with 100 μL of purified water for UPLC-MS / MS analysis.

[0406] 2. Data Analysis

[0407] All data calculations were performed using Microsoft Excel. Peak areas were determined by extracted ion chromatograms. In vitro clearance of the parent drug was determined by linear fitting the natural logarithm of the parent drug elimination percentage versus time.

[0408] In vitro clearance (μL / min / 10 6 cells) were calculated using the following formula:

[0409] In vitro CL int =kV / N

[0410] V = incubation volume per well (0.2 mL);

[0411] N = number of cells per well (0.1 × 10 6 cells)

[0412] Table 3. Metabolic clearance CL of compounds in hepatocytes int (μL / min / 10 6 cells)

[0413] Judging from the results, compound 19-P1 has significant advantages in metabolic stability in dog, monkey and human liver cells compared with PF-07328948 and reference 1.

[0414] Test Example 4: Pharmacokinetics in rats and dogs

[0415] 1. Pharmacokinetics experiment in rats

[0416] SD rats (Source: Zhejiang Weitong Lihua Laboratory Animal Technology Co., Ltd.) were used as test animals. LC / MS / MS was used to determine the plasma concentration of the test substance at different time points after oral administration. The pharmacokinetic behavior of the test substance in rats was studied and its pharmacokinetic characteristics were evaluated.

[0417] Experimental animals: Two healthy 6- to 8-week-old male rats (200-300 g) were used in each group.

[0418] Drug preparation: Weigh a certain amount of drug and prepare a 2 mg / mL colorless clear solution (solvent: 100% normal saline and 1 mol / L hydrochloric acid and sodium hydroxide to adjust the final solution to pH 7).

[0419] Administration: Rats were fasted overnight and then gavage was performed to administer the test substance at a dose of 20 mg / kg.

[0420] Procedure: Rats were orally administered with the test substance. Approximately 0.2 mL of blood was collected from the jugular vein at 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours after administration. The blood was placed in a tube containing EDTA-K2 and centrifuged at 4000 g per minute for 5 minutes at about 4°C to separate the plasma, which was then stored at -75±15°C.

[0421] Determination of the test compound content in rat plasma after oral administration of the test substance: 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 internal standard dexamethasone, vortex mix for 30 seconds, centrifuge for 15 minutes (3900 rpm), take 100 μL of the supernatant of the plasma sample and add 200 μL of ultrapure water to mix evenly, then take 5 μL of the dilution with a syringe for LC / MS / MS analysis.

[0422] Table 4. Pharmacokinetic parameters of the compound after oral administration to rats (20 mpk)

[0423] Note: The values ​​listed are the average of two animals.

[0424] Judging from the results, the oral exposure of compound 19-P1 in rats has a significant advantage over PF-07328948.

[0425] 2. Pharmacokinetics experiments in dogs

[0426] Beagle dogs (Source: Beijing Masi Biotechnology Co., Ltd.) were used as test animals. LC / MS / MS was used to determine plasma drug concentrations at different times after oral administration of the test substance. The pharmacokinetic behavior of the test substance in beagle dogs was studied and its pharmacokinetic characteristics were evaluated.

[0427] Experimental animals: Two healthy male beagle dogs aged 8 months to 3 years (6.0-13.0 kg) were used in each group.

[0428] Drug preparation: Weigh a certain amount of drug and prepare a 0.6 mg / mL colorless clear solution (solvent: 100% normal saline and 1 mol / L hydrochloric acid and sodium hydroxide to adjust the final solution to pH 7).

[0429] Administration: Beagle dogs were fasted overnight and then gavage was performed to administer the test substance at a dose of 3 mg / kg.

[0430] Procedure: Beagle dogs were orally administered with the test substance. Approximately 0.6 mL of blood was collected by peripheral venipuncture before administration and 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours after administration. The blood was placed in a tube containing EDTA-K2 and centrifuged at 2000 g per minute for 10 minutes at about 4°C to separate the plasma, which was then stored at -75±15°C.

[0431] Determination of the test compound content in beagle dog plasma after oral administration of the test substance: 50 μL of beagle dog plasma at each time point after administration was taken, 5 μL of blank solution and 200 μL of acetonitrile solution containing internal standard dexamethasone were added, and the mixture was vortexed for 30 seconds and centrifuged for 15 minutes (3900 rpm). 100 μL of the supernatant of the plasma sample was added to 200 μL of ultrapure water and mixed evenly. Then, 5 μL of the dilution was aspirated with an injection syringe for LC / MS / MS analysis.

[0432] Table 5. Pharmacokinetic parameters of the compound after oral administration to dogs (3 mpk)

[0433] Note: The values ​​listed are the average of two animals.

[0434] Judging from the results, the oral exposure of compound 19-P1 in dogs has a significant advantage over PF-07328948.

Claims

1. A compound of formula (I) or a pharmaceutically acceptable salt thereof, Among them, R 1 、R 2 、R 3 each independently selected from hydrogen, fluorine, chlorine or methyl; R 4 selected from fluorine or chlorine; R 5 selected from C 1-4 alkyl, halo C 1-4 alkyl, 3- to 4-membered cycloalkyl, -L1-R 9 , where L1 is selected from sulfur or oxygen, and R 9 is selected from C 1-4 alkyl, halo C 1-4 alkyl, deuterated C 1-4 alkyl, 3- to 4-membered cycloalkyl; X1 is selected from nitrogen or CR 6 , R 6 is selected from hydrogen, fluorine or chlorine; R 7 and R 8 are each independently selected from hydrogen, fluorine, and chlorine.

2. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, wherein X1 is nitrogen.

3. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, which is a compound of formula (II) or a pharmaceutically acceptable salt thereof, Among them, R 1 、R 2 、R 3 、R 4 、R 5 、R 6 、R 7 and R 8 are respectively defined as in claim 1.

4. A compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof, wherein R 5 is selected from C 1-4 alkyl or halo-C 1-4 alkyl; preferably, R 5 is selected from methyl, ethyl, isopropyl; most preferably, R 5 is selected from methyl or ethyl.

5. The compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof, wherein R 5 is selected from C3-C4 cycloalkyl, and preferably, R 5 is cyclopropyl.

6. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, which is the compound represented by formula (II-1) or a pharmaceutically acceptable salt thereof, Among them, R 1 、R 2 、R 3 、R 4 、R 6 、R 7 、R 8 、R 9 are respectively defined as in claim 1.

7. The compound or a pharmaceutically acceptable salt thereof according to claim 6, wherein R 9 is selected from C 1-4 alkyl, halo C 1-4 alkyl or deuterated C 1-4 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.

8. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, which is a compound of formula (II-2) or a pharmaceutically acceptable salt thereof, Among them, R 1 、R 2 、R 3 、R 4 、R 6 、R 7 、R 8 、R 9 are respectively defined as in claim 1.

9. The compound or its pharmaceutically acceptable salt according to claim 8, wherein R 9 is selected from C 1-4 alkyl, halo-C 1-4 alkyl or deuterated C 1-4 alkyl; preferably, R 9 is selected from methyl, ethyl, difluoromethyl, trifluoromethyl or deuterated methyl; most preferably, R 9 is methyl.

10. The compound according to any one of claims 1 to 9 or a pharmaceutically acceptable salt thereof, wherein R 1 , R 2 , R 3 is at least one selected from fluorine or chlorine.

11. The compound according to any one of claims 1 to 10 or a pharmaceutically acceptable salt thereof, wherein R 1 and R 3 are each independently selected from hydrogen, fluorine or chlorine; preferably, one of R 1 or R 3 is hydrogen and the other is selected from hydrogen, fluorine or chlorine; most preferably, each of R 1 or R 3 is independently hydrogen.

12. The compound according to any one of claims 1 to 11 or a pharmaceutically acceptable salt thereof, wherein R 2 is fluorine or chlorine; preferably, R 2 is fluorine; preferably, R 2 is chlorine.

13. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 12, wherein R 1 or R 3 is each independently hydrogen; R 2 is fluorine or chlorine.

14. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 13, wherein R 4 is selected from fluorine or chlorine; preferably, R 4 is fluorine.

15. The compound according to any one of claims 1 to 14 or a pharmaceutically acceptable salt thereof, wherein R 1 or R 3 is independently hydrogen, R 2 is fluorine or chlorine, and R 4 is fluorine.

16. The compound according to any one of claims 1 to 15 or a pharmaceutically acceptable salt thereof, wherein R 6 , R 7 and R 8 are at least one selected from fluorine or chlorine; preferably, at least two of R 6 , R 7 and R 8 are selected from fluorine or chlorine.

17. A compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 16, wherein R 6 is selected from fluorine or chlorine.

18. The compound according to any one of claims 1 to 17 or a pharmaceutically acceptable salt thereof, wherein R 6 , R 7 and R 8 at least one is chlorine.

19. The compound according to any one of claims 1 to 18 or a pharmaceutically acceptable salt thereof, wherein R 7 is selected from fluorine or chlorine, and R 8 is hydrogen; preferably, R 7 is fluorine and R 8 is hydrogen.

20. The compound according to any one of claims 1 to 19 or a pharmaceutically acceptable salt thereof, wherein R 8 is selected from fluorine or chlorine, and R 7 is hydrogen.

21. The compound according to any one of claims 1 to 20 or a pharmaceutically acceptable salt thereof, wherein R 1 or R 3 is each independently hydrogen; R 2 is fluorine or chlorine; R 4 is fluorine; R 6 is selected from fluorine or chlorine; R 7 is selected from fluorine or chlorine, R 8 is hydrogen, or R 8 is selected from fluorine or chlorine, R 7 is hydrogen; and at least one of R 6 , R 7 and R 8 is chlorine.

22. The compound or its pharmaceutically acceptable salt according to any one of claims 1 to 21, which is a compound represented by formula (II-A) or formula (II-B) or its pharmaceutically acceptable salt, preferably a compound represented by formula (II-B) or its pharmaceutically acceptable salt, Among them, R 2 、R 5 、R 6 、R 7 are each defined as in claims 1 to 21. Preferably, R 2 is chlorine; or preferably, R 2 is fluorine.

23. The compound or a pharmaceutically acceptable salt thereof according to claim 22, wherein R 5 is selected from methyl or ethyl.

24. The compound or a pharmaceutically acceptable salt thereof according to claim 22, wherein R 5 is selected from -L1-R 9 , L1 is oxygen, and R 9 is selected from methyl, ethyl, difluoromethyl, trifluoromethyl or deuteromethyl; preferably, R 9 is selected from methyl or deuteromethyl; or preferably, R 9 is selected from difluoromethyl or trifluoromethyl.

25. The compound or a pharmaceutically acceptable salt thereof according to claim 22, wherein R 5 is selected from -L1-R 9 , L1 is sulfur, and R 9 is selected from methyl, ethyl, difluoromethyl, trifluoromethyl or deuteromethyl; preferably, R 5 is selected from -L1-R 9 , L1 is sulfur, and R 9 is methyl or deuteromethyl.

26. The compound according to claim 22 or a pharmaceutically acceptable salt thereof, R 6 is fluorine or chlorine, and R 7 is hydrogen; preferably, R 6 is fluorine and R 7 is hydrogen.

27. A compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 16, 18, 19, 21, which is a compound of formula (II-C) or a pharmaceutically acceptable salt thereof, Among them, R 2 、R 5 、R 6 、R 7 are respectively defined in claims 1 to 16, 18, 19, 21. Preferably, R 2 is chlorine; or preferably, R 2 is fluorine.

28. The compound or its pharmaceutically acceptable salt according to claim 27, wherein R 5 is selected from methyl or ethyl.

29. The compound according to claim 27 or a pharmaceutically acceptable salt thereof, wherein R 5 is selected from -L1-R 9 , L1 is oxygen; R 9 is selected from methyl, ethyl, difluoromethyl, trifluoromethyl or deuteromethyl, preferably, R 9 is selected from methyl or deuteromethyl, or preferably, R 9 is selected from difluoromethyl or trifluoromethyl.

30. The compound or a pharmaceutically acceptable salt thereof according to claim 27, wherein R 5 is selected from -L1-R 9 ; L1 is sulfur; R 9 is selected from methyl, ethyl, difluoromethyl, trifluoromethyl or deuteromethyl, preferably, R 9 is methyl or deuteromethyl.

31. The compound or a pharmaceutically acceptable salt thereof according to claim 27, wherein R 6 and R 7 are each independently selected from fluorine or chlorine; preferably, R 6 is selected from fluorine or chlorine; most preferably, R 6 is selected from fluorine, R 7 is selected from fluorine or chlorine; or, R 6 is selected from chlorine, R 7 is selected from fluorine or chlorine.

32. The compound or its pharmaceutically acceptable salt according to any one of claims 1 to 31, which is selected from the following compounds or their pharmaceutically acceptable salts:

33. An isotopically substituted compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 32, preferably, the isotopically substituted compound is a deuterated compound.

34. A pharmaceutical composition comprising the compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 32, or the isotopically substituted compound according to claim 33, and one or more pharmaceutically acceptable excipients.

35. Use of the compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 32, or the isotopically substituted compound according to claim 33, or the pharmaceutical composition according to claim 34 in the preparation of a medicament for preventing and / or treating diseases related to BCKDK; preferably, the diseases related to BCKDK are selected from disorders of glucose metabolism or blood glucose abnormalities, heart diseases or kidney diseases; more preferably, the disorder of glucose metabolism or blood glucose abnormality is diabetes, and the heart disease is heart failure.

36. Use of the compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 32, or the isotopically substituted compound according to claim 33, or the pharmaceutical composition according to claim 34 in the preparation of a medicament for preventing and / or treating diseases selected from disorders of glucose metabolism or blood glucose abnormalities, heart diseases or kidney diseases; preferably, the disorder of glucose metabolism or blood glucose abnormality is diabetes, and the heart disease is heart failure.

37. A method for preparing a compound according to any one of claims 1 to 32 or a pharmaceutically acceptable salt thereof, comprising the step of removing a protecting group PG from the compound of formula (I-a) or a pharmaceutically acceptable salt thereof under basic conditions, and further comprising the step of adjusting the pH to acidic, Among them, PG is a carboxyl protecting group; R 1 , R 2 , R 3 , R 4 , R 5 , X1, R 7 and R 8 are respectively defined in claims 1 to 32; preferably, PG is C 1-6 alkyl.

38. A compound of formula (I-a) or a pharmaceutically acceptable salt thereof, Among them, PG is a carboxyl protecting group; R 1 、R 2 、R 3 、R 4 、R 5 、X1, R 7 and R 8 are respectively defined in claims 1 to 32; preferably, PG is C 1-6 alkyl.