HK40067104A - Triazole three fused ring derivative, preparation method therefor and application - Google Patents

Description

Triazole tricyclic derivatives and their preparation methods and applications

Technical Field

The present invention relates to triazole tricyclic derivatives, preparation methods, and applications thereof. The present invention also relates to drugs containing triazole tricyclic derivatives and salts thereof as active ingredients, which can be used to diagnose, prevent, and/or treat diseases associated with vasopressin receptors.

Background Art

Hormones play a crucial role in regulating human internal homeostasis. Arginine vasopressin (AVP) is closely linked to the regulation of water and sodium metabolism. Disturbances in AVP metabolism can cause a variety of conditions, including hyponatremia, syndrome of inappropriate antidiuretic hormone secretion, congestive heart failure, cirrhosis, kidney disease, hypertension, and edema. Arginine vasopressin (AVP) receptor antagonists inhibit the binding of AVP to the receptor, thereby providing therapeutic benefits for these conditions. Arginine vasopressin V2 receptor antagonists, such as tolvaptan, can increase free water excretion without affecting electrolyte metabolism, making them ideal agents for treating these conditions. However, marketed AVP V2 receptor antagonists, such as tolvaptan, are metabolized by hepatic enzymes, producing a large number of metabolites in the body and leading to severe drug-induced hepatotoxicity. The FDA has issued a black box warning on the drug's label, limiting its application. Therefore, the development of novel V2 receptor antagonists with high efficacy and minimal side effects is crucial.

Summary of the Invention

One object of the present invention is to provide a novel benzazepine spirocyclic compound or a salt thereof having vasopressin V2 receptor antagonism, favorable metabolic stability and/or metabolites with reduced drug-induced hepatotoxicity, and the medical use of the compound.

The present invention provides a compound represented by formula (I), its optical isomers and pharmaceutically acceptable salts thereof.

wherein Ring B is selected from phenyl and 5-10 membered heteroaryl;

Ring A is selected from phenyl and 5-6 membered heteroaryl;

R ₁ is selected from H, OH, NH ₂ , C _1-6 alkyl and C _1-6 heteroalkyl, wherein the C _1-6 alkyl or C _1-6 heteroalkyl is optionally substituted with 1, 2 or 3 R;

R ₂ is selected from F, Cl, Br, I, CN and C _1-6 alkyl, wherein the C _1-6 alkyl is optionally substituted with 1, 2 or 3 R;

R ₃ is selected from H, F, Cl, Br, I, C _1-6 alkyl, C _1-6 alkoxy, C _3-6 cycloalkyl and 3-6 membered heterocycloalkyl, wherein the C _1-6 alkyl, C _1-6 alkoxy, C _3-6 cycloalkyl or 3-6 membered heterocycloalkyl is optionally substituted with 1, 2 or 3 R;

R ₄ is selected from H, F, Cl, Br, I, C _1-6 alkyl, phenyl, naphthyl and 5-10 membered heteroaryl, wherein the C _1-6 alkyl, phenyl, naphthyl or 5-10 membered heteroaryl is optionally substituted with 1, 2 or 3 R;

T ₁ and T ₂ are independently selected from N and CH;

R is independently selected from H, F, Cl, Br, I and C _1-6 alkyl;

m1, m2, m3, and m4 are each independently selected from 0, 1, 2, 3, or 4;

n is selected from 1 or 2;

The C _1-6 heteroalkyl, 5-10 membered heteroaryl and 3-6 membered heterocycloalkyl contain 1, 2 or 3 heteroatoms or heteroatom groups independently selected from O, NH, S, C(═O), C(═O)O, S(═O), S(═O) ₂ and N.

The present invention also provides a compound represented by formula (II), its optical isomers and pharmaceutically acceptable salts thereof.

wherein Ring B is selected from phenyl and 5-10 membered heteroaryl;

R ₁ is selected from H, OH, NH ₂ , C _1-6 alkyl and C _1-6 heteroalkyl, wherein the C _1-6 alkyl or C _1-6 heteroalkyl is optionally substituted with 1, 2 or 3 R;

R ₂ is selected from F, Cl, Br, I, CN and C _1-6 alkyl, wherein the C _1-6 alkyl is optionally substituted with 1, 2 or 3 R;

R ₃ is selected from H, F, Cl, Br, I, C _1-6 alkyl, C _1-6 alkoxy, C _3-6 cycloalkyl and 3-6 membered heterocycloalkyl, wherein the C _1-6 alkyl, C _1-6 alkoxy, C _3-6 cycloalkyl and 3-6 membered heterocycloalkyl are optionally substituted with 1, 2 or 3 R;

R ₄ is selected from H, F, Cl, Br, I, C _1-6 alkyl, phenyl, naphthyl and 5-10 membered heteroaryl, wherein the C _1-6 alkyl, phenyl, naphthyl or 5-10 membered heteroaryl is optionally substituted with 1, 2 or 3 R;

T ₁ and T ₂ are independently selected from N and CH;

R is independently selected from H, F, Cl, Br, I and C _1-6 alkyl;

X is selected from CH and N;

m2, m4 are selected from 0, 1, 2, 3 or 4;

The C _1-6 heteroalkyl, 5-10 membered heteroaryl and 3-6 membered heterocycloalkyl contain 1, 2 or 3 heteroatoms or heteroatom groups independently selected from O, NH, S, C(═O), C(═O)O, S(═O), S(═O) ₂ and N.

In some embodiments of the present invention, the ring A is selected from phenyl and pyridyl, and other variables are as defined in the present invention.

In some embodiments of the present invention, _R3 is selected from H, F, Cl, Br, I, _C1-3 alkyl, _C1-3 alkoxy, _C3-6 cycloalkyl, and 3-6 membered heterocycloalkyl, wherein the _C1-3 alkyl, _C1-3 alkoxy, _C3-6 cycloalkyl, or 3-6 membered heterocycloalkyl is optionally substituted with 1, 2, or 3 R, and the other variables are as defined herein. In some embodiments of the present invention, _R3 is selected from H, F, Cl, Br, I, methyl, ethyl, _CHF2 , and the other variables are as defined herein.

In some embodiments of the present invention, the above structural units are selected from other variables as defined in the present invention.

In some embodiments of the present invention, the ring B is selected from phenyl and 5-6 membered heteroaryl, and other variables are as defined in the present invention.

In some embodiments of the present invention, the ring B is selected from phenyl, pyridyl, thienyl and furyl, and other variables are as defined in the present invention.

In some embodiments of the present invention, the above-mentioned _R4 is selected from H, F, Cl, Br, I, _C1-3 alkyl, phenyl, pyridyl and thiazolyl, and the _C1-3 alkyl, phenyl, pyridyl and thiazolyl are optionally substituted by 1, 2 or 3 R, and other variables are as defined in the present invention.

In some embodiments of the present invention, R ₄ is selected from methyl, ethyl, F, Cl, Br, I, CF ₃ , CHF ₂ , and other variables are as defined in the present invention.

In some embodiments of the present invention, the above structural units are selected from other variables as defined in the present invention.

The present invention also provides the following compounds, their optical isomers and pharmaceutically acceptable salts thereof, which are selected from

In another aspect of the present invention, the present invention also provides the use of the aforementioned compound, its optical isomers and pharmaceutically acceptable salts thereof in the preparation of a medicament for preventing or treating diseases related to the arginine vasopressin V1a receptor, arginine vasopressin V1b receptor, arginine vasopressin V2 receptor, sympathetic nervous system or renin-angiotensin-aldosterone system.

In some embodiments of the present invention, the diseases associated with arginine vasopressin V1a receptor, arginine vasopressin V1b receptor, arginine vasopressin V2 receptor, sympathetic nervous system or renin-angiotensin-aldosterone system include: hypertension, Reye's syndrome, dysmenorrhea, premature birth, corticotropin-releasing hormone secretion disorder, adrenal hyperplasia, depression, chronic congestive heart failure, cirrhosis, syndrome of inappropriate antidiuretic hormone secretion, hyponatremia caused by chronic heart failure/cirrhosis/inappropriate antidiuretic hormone secretion, or polycystic kidney disease.

Definition and Description

Unless otherwise indicated, the following terms and phrases used herein are intended to have the following meanings. A particular term or phrase should not be construed as indefinite or unclear unless specifically defined, but rather should be understood in accordance with its ordinary meaning. When a trade name appears in this document, it is intended to refer to the corresponding commercial product or its active ingredient.

As used herein, the phrase "at least one" when referring to a list of one or more elements should be understood to mean at least one element selected from any one or more elements in the list of elements, but does not necessarily include at least one of each element specifically listed in the list of elements, and does not exclude any combination of elements in the list of elements. This definition also allows that elements other than the elements specifically identified in the list of elements to which the phrase "at least one" refers may optionally be present, whether related or unrelated to those specifically identified elements.

The term "pharmaceutically acceptable" as used herein refers to those compounds, materials, compositions and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response or other problems or complications, commensurate with a reasonable benefit/risk ratio.

The term "pharmaceutically acceptable salt" refers to salts of the compounds of the present invention, prepared by reacting the compounds of the present invention with relatively nontoxic acids or bases. When the compounds of the present invention contain relatively acidic functional groups, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of base in neat solution or in a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine, or magnesium salts, or similar salts. When the compounds of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of acid in solution or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, bisulfate, hydroiodic acid, phosphorous acid, and the like; and organic acid salts such as acetic acid, propionic acid, isobutyric acid, trifluoroacetic acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, and methanesulfonic acid; and salts of amino acids (such as arginine) and organic acids such as glucuronic acid. Certain specific compounds of the present invention contain both basic and acidic functional groups and can be converted into either base or acid addition salts.

The pharmaceutically acceptable salts of the present invention can be synthesized by conventional chemical methods from parent compounds containing acid radicals or bases. Generally, such salts are prepared by reacting these compounds in free acid or base form with a stoichiometric amount of a suitable base or acid in water or an organic solvent or a mixture of the two.

The compounds of the present invention may exist in specific geometric or stereoisomeric forms. All such compounds, including cis- and trans-isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, and racemic and other mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures, are contemplated by the present invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers and mixtures thereof are encompassed within the scope of the present invention.

The compounds of the present invention may exist in specific forms. Unless otherwise indicated, the term "tautomer" or "tautomeric form" refers to isomers with different functional groups that are in dynamic equilibrium at room temperature and can quickly convert into each other. If tautomerism is possible (such as in solution), a chemical equilibrium of tautomers can be achieved. For example, proton tautomers (also known as prototropic tautomers) include interconversions that occur through proton migration, such as keto-enol isomerization and imine-enamine isomerization. Valence isomers (valencetautomers) include interconversions that occur through the reorganization of some bonding electrons. A specific example of keto-enol tautomerization is the interconversion between two tautomers of pentane-2,4-dione and 4-hydroxypent-3-ene-2-one.

The compounds of the present invention may contain unnatural proportions of atomic isotopes on one or more atoms constituting the compound. For example, the compound may be labeled with a radioactive isotope, such as tritium ( ³ H), iodine-125 ( ¹²⁵ I), or C-14 ( ¹⁴ C). For another example, deuterated drugs may be formed by replacing hydrogen with heavy hydrogen. The bond formed by deuterium and carbon is stronger than the bond formed by ordinary hydrogen and carbon. Compared with non-deuterated drugs, deuterated drugs have advantages such as reduced toxic side effects, increased drug stability, enhanced therapeutic efficacy, and prolonged drug biological half-life. All isotopic composition changes of the compounds of the present invention, whether radioactive or not, are included within the scope of the present invention. "Optional" or "optionally" means that the event or situation described subsequently may but is not required to occur, and the description includes cases where the event or situation occurs as well as cases where the event or situation does not occur.

When a group valence is marked with a dashed line, such as in, the dashed line represents the point of attachment of the group to the rest of the molecule. When a single bond is marked with a dashed line, such as in, the dashed line represents the single bond or its absence, and also means a single bond or a double bond.

The term "substituted" or "substituted with" means that any one or more hydrogen atoms on a particular atom are replaced by a substituent, including deuterium and hydrogen variants, as long as the valence state of the particular atom is normal and the substituted compound is stable. The term "optionally substituted" or "optionally substituted with" means that the atom may or may not be substituted. Unless otherwise specified, the type and number of substituents may be any based on chemical practicability.

When any variable (e.g., R) occurs more than once in a compound's composition or structure, its definition at each occurrence is independent. Thus, for example, if a group is substituted with 1, 2, or 3 R's, the group may optionally be substituted with 1, 2, or 3 R's, with each occurrence of R' being an independent choice. Furthermore, combinations of substituents and/or their variants are permissible only if such combinations result in stable compounds.

When one of the variables is selected from a single bond, it means that the two groups it connects are directly connected. For example, when _L1 represents a single bond, it means that the structure is actually

When the substituents listed do not specify through which atom they are connected to the substituted group, such substituents can be bonded through any atom thereof. For example, a pyridyl substituent can be connected to the substituted group through any carbon atom on the pyridine ring.

When the listed linking groups do not specify their connection direction, their connection direction is arbitrary. For example, when the linking group L is _-CH2O- , _-CH2O- can be connected to form a phenyl group and a cyclopentyl group in the same direction as the reading order from left to right, or it can be connected to form a phenyl group and a cyclopentyl group in the opposite direction as the reading order from left to right. Combinations of the linking groups, substituents and/or their variations are permitted only if such combinations result in stable compounds.

Unless otherwise specified, the number of atoms in a ring is generally defined as the ring member number, for example, a "3-6 membered ring" refers to a "ring" having 3-6 atoms arranged around it.

Unless otherwise specified, the term "C _1-6 alkyl" is used to refer to a straight or branched saturated hydrocarbon group consisting of 1 to 6 carbon atoms. The C _1-6 alkyl group includes C _1-5 , C _1-4 , C _1-3 , C _1-2 , C 2-6, C _2-4 , C ₆ , and C ₅ alkyl groups, etc.; and can be monovalent (such as CH ₃ ), divalent (-CH ₂ -), or polyvalent (such as 2-). Examples of _{C 1-6} alkyl groups include, but are not limited to, CH ₃ , etc.

Unless otherwise specified, the term "C _1-4 alkyl" is used to refer to a straight or branched saturated hydrocarbon group consisting of 1 to 4 carbon atoms. The C _1-4 alkyl group includes C _1-2 , C _1-3 , C _3-4 , and C _2-3 alkyl groups, etc.; and can be monovalent (such as CH ₃ ), divalent (-CH ₂ -), or polyvalent (such as 2-). Examples of C _1-4 alkyl groups include, but are not limited to, CH ₃ , etc.

Unless otherwise specified, " _C2-3 alkenyl" refers to a linear or branched hydrocarbon group consisting of 2 to 3 carbon atoms containing at least one carbon-carbon double bond, which may be located at any position within the group. The _C2-3 alkenyl group includes _C3 and _C2 alkenyl groups; the _C2-3 alkenyl group may be monovalent, divalent, or polyvalent. Examples of _C2-3 alkenyl groups include, but are not limited to, the following.

Unless otherwise specified, " _C2-3 alkynyl" refers to a linear or branched hydrocarbon group consisting of 2 to 3 carbon atoms containing at least one carbon-carbon triple bond, which may be located at any position within the group. It may be monovalent, divalent, or polyvalent. The _C2-3 alkynyl group includes _C3 and _C2 alkynyl groups. Examples of _C2-3 alkynyl groups include, but are not limited to, the following.

Unless otherwise specified, the term "heteroalkyl" by itself or in combination with another term means a stable straight or branched chain alkyl radical or combination thereof consisting of a certain number of carbon atoms and at least one heteroatom or heteroatom group. In some embodiments, the heteroatom is selected from B, O, N, and S, wherein the nitrogen and sulfur atoms are optionally oxidized and the nitrogen heteroatom is optionally quaternized. In other embodiments, the heteroatom group is selected from -C(=O)O-, -C(=O)-, -C(=S)-, -S(=O), -S(=O) ₂ -, -C(=O)N(H)-, -N(H)-, -C(=NH)-, -S(=O) ₂ N(H)-, and -S(=O)N(H)-. In some embodiments, the heteroalkyl group is C _1-6 heteroalkyl; in other embodiments, the heteroalkyl group is C _1-3 heteroalkyl. The heteroatom or heteroatom group may be placed at any interior position of the heteroalkyl group, including the position at which the alkyl group is attached to the remainder of the molecule, but the term "alkoxy" is used conventionally to refer to those alkyl groups attached to the remainder of the molecule through an oxygen atom. Examples of heteroalkyl groups include, but are not limited to, -OCH ₃ , -OCH ₂ CH ₃ , -OCH ₂ CH ₂ CH ₃ , -OCH ₂ (CH ₃ ) ₂ , -CH ₂ -CH ₂ -O-CH ₃ , -NHCH ₃ , -N(CH ₃ ) ₂ , -NHCH ₂ CH ₃ , -N(CH ₃ )(CH ₂ CH ₃ ), -CH ₂ -CH ₂ -NH-CH ₃ , -CH ₂ -CH ₂ -N(CH ₃ )-CH ₃ , -SCH ₃ , -SCH ₂ CH ₃ , -SCH ₂ CH ₂ CH ₃ , -SCH ₂ (CH ₃ ) ₂ , -CH ₂ -S-CH ₂ -CH ₃ , -CH ₂ -CH ₂ , -S(＝O)-CH ₃ , -CH ₂ -CH ₂ -S(=O) ₂ -CH ₃ , and up to two heteroatoms may be consecutive, for example -CH ₂ -NH-OCH ₃ .

Unless otherwise specified, the term "C _1-6 alkoxy" refers to an alkyl group containing 1 to 6 carbon atoms that is attached to the rest of the molecule via an oxygen atom. The C _1-6 alkoxy group includes C _1-4 , C _1-3 , C _1-2 , C _2-6 , C _2-4 , C ₆ , C ₅ , C ₄ , and C ₃ alkoxy groups. Examples of C _1-6 alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy (including n-propoxy and isopropoxy), butoxy (including n-butoxy, isobutoxy, s-butoxy, and t-butoxy), pentoxy (including n-pentoxy, isopentoxy, and neopentoxy), hexyloxy, and the like.

Unless otherwise specified, the term "C _1-3 alkoxy" refers to an alkyl group containing 1 to 3 carbon atoms that is attached to the rest of the molecule via an oxygen atom. The C _1-3 alkoxy group includes C _1-3 , C _1-2 , C _2-3 , C ₁ , C ₂ , and C ₃ alkoxy groups. Examples of C _1-3 alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy (including n-propoxy and isopropoxy), and the like.

Unless otherwise specified, the term "C _1-6 alkylamino" refers to those alkyl groups containing 1 to 6 carbon atoms that are attached to the rest of the molecule through an amino group. The C _1-6 alkylamino group includes C _1-4 , C _1-3 , C _1-2 , C _2-6 , C _2-4 , C ₆ , C ₅ , C ₄ , C ₃ and C ₂ alkylamino groups, etc. Examples of _{C 1-6} alkylamino groups include, but are not limited to, -NHCH ₃ , -N(CH ₃ ) ₂ , -NHCH ₂ CH ₃ , -N(CH ₃ )CH ₂ CH ₃ , -N(CH ₂ CH ₃ )(CH ₂ CH ₃ ), -NHCH ₂ CH ₂ CH ₃ , -NHCH ₂ (CH ₃ ) ₂ , -NHCH ₂ CH ₂ CH ₂ CH ₃ , and the like.

Unless otherwise specified, the term "C _1-3 alkylamino" refers to an alkyl group containing 1 to 3 carbon atoms that is attached to the rest of the molecule through an amino group. The C _1-3 alkylamino group includes C _1-3 , C _1-2 , C _2-3 , C ₁ , C ₂ , and C ₃ alkylamino groups. Examples of C _1-3 alkylamino groups include, but are not limited to, -NHCH ₃ , -N(CH ₃ ) ₂ , -NHCH ₂ CH ₃ , -N(CH ₃ )CH ₂ CH ₃ , -NHCH ₂ CH ₂ CH ₃ , -NHCH ₂ (CH ₃ ) _{2 ,} and the like.

Unless otherwise specified, the term "C _1-6 alkylthio" refers to an alkyl group containing 1 to 6 carbon atoms that is attached to the rest of the molecule through a sulfur atom. The C _1-6 alkylthio group includes C _1-4 , C _1-3 , C _1-2 , C 2-6, C _2-4 , C ₆ , C ₅ , C ₄ , C ₃ and C ₂ alkylthio groups. Examples of _{C 1-6 alkylthio} groups include, but are not limited to, -SCH ₃ , -SCH ₂ CH ₃ , -SCH ₂ CH ₂ CH ₃ , -SCH ₂ (CH ₃ ) ₂ and the like.

Unless otherwise specified, the term "C _1-3 alkylthio" refers to an alkyl group containing 1 to 3 carbon atoms that is attached to the rest of the molecule through a sulfur atom. The C _1-3 alkylthio group includes C _1-3 , C _1-2 , C _2-3 , C ₁ , C ₂ , and C ₃ alkylthio groups. Examples of C _1-3 alkylthio groups include, but are not limited to, -SCH ₃ , -SCH ₂ CH ₃ , -SCH ₂ CH ₂ CH ₃ , -SCH ₂ (CH ₃ ) ₂ , and the like.

Unless otherwise specified, " _C3-6 cycloalkyl" refers to a saturated cyclic hydrocarbon group consisting of 3 to 6 carbon atoms, including monocyclic and bicyclic ring systems. Such _C3-6 cycloalkyl groups include _C3-5 , _C4-5 , and _C5-6 cycloalkyl groups, and may be monovalent, divalent, or polyvalent. Examples of _C3-6 cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

Unless otherwise specified, the term "3-6 membered heterocycloalkyl" by itself or in combination with other terms refers to a saturated cyclic group consisting of 3 to 6 ring atoms, 1, 2, 3 or 4 of which are heteroatoms independently selected from O, S and N, and the rest are carbon atoms, wherein the nitrogen atom is optionally quaternized, and the nitrogen and sulfur heteroatoms are optionally oxidized (i.e., NO and S(O) _p , p is 1 or 2). It includes monocyclic and bicyclic ring systems, wherein the bicyclic ring system includes spirocyclic, fused and bridged rings. In addition, with respect to the "3-6 membered heterocycloalkyl", heteroatoms can occupy the position at which the heterocycloalkyl is connected to the rest of the molecule. The 3-6 membered heterocycloalkyl includes 4-6 membered, 5-6 membered, 4 membered, 5 membered and 6 membered heterocycloalkyl, etc. Examples of 3-6 membered heterocycloalkyl groups include, but are not limited to, azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, tetrahydrothiophenyl (including tetrahydrothiophen-2-yl and tetrahydrothiophen-3-yl, etc.), tetrahydrofuranyl (including tetrahydrofuran-2-yl, etc.), tetrahydropyranyl, piperidinyl (including 1-piperidinyl, 2-piperidinyl and 3-piperidinyl, etc.), piperazinyl (including 1-piperazinyl and 2-piperazinyl, etc.), morpholinyl (including 3-morpholinyl and 4-morpholinyl, etc.), dioxanyl, dithianyl, isoxazolidinyl, isothiazolidinyl, 1,2-oxazinyl, 1,2-thiazinyl, hexahydropyridazinyl, homopiperazinyl or homopiperidinyl, etc.

Unless otherwise specified, the terms "5-6 membered heteroaromatic ring" and "5-6 membered heteroaryl" are used interchangeably herein, and the term "5-6 membered heteroaryl" refers to a monocyclic group consisting of 5 to 6 ring atoms with a conjugated π electron system, wherein 1, 2, 3 or 4 ring atoms are heteroatoms independently selected from O, S and N, and the remainder are carbon atoms. The nitrogen atom is optionally quaternized, and the nitrogen and sulfur heteroatoms are optionally oxidized (i.e., NO and S(O) _p , where p is 1 or 2). The 5-6 membered heteroaryl group may be attached to the rest of the molecule via a heteroatom or a carbon atom. The 5-6 membered heteroaryl group includes 5-membered and 6-membered heteroaryl groups. Examples of the 5-6 membered heteroaryl group include, but are not limited to, pyrrolyl (including N-pyrrolyl, 2-pyrrolyl and 3-pyrrolyl), pyrazolyl (including 2-pyrazolyl and 3-pyrazolyl), imidazolyl (including N-imidazolyl, 2-imidazolyl, 4-imidazolyl and 5-imidazolyl), oxazolyl (including 2-oxazolyl, 4-oxazolyl and 5-oxazolyl), triazolyl (1H-1,2,3-triazolyl, 2H-1,2,3-triazolyl, 1H-1,2,4-triazolyl), and 4H-1,2,4-triazolyl, etc.), tetrazolyl, isoxazolyl (3-isoxazolyl, 4-isoxazolyl and 5-isoxazolyl, etc.), thiazolyl (including 2-thiazolyl, 4-thiazolyl and 5-thiazolyl, etc.), furyl (including 2-furyl and 3-furyl, etc.), thienyl (including 2-thienyl and 3-thienyl, etc.), pyridyl (including 2-pyridyl, 3-pyridyl and 4-pyridyl, etc.), pyrazinyl or pyrimidinyl (including 2-pyrimidinyl and 4-pyrimidinyl, etc.).

Unless otherwise specified, Cn _-n+m or _Cn - _Cn+m includes any specific case of n to n+m carbon atoms, for example, _C1-12 includes _C1 , _C2 , C3, _C4 , _{C5 , C6} , _C7 , _C8 , _C9 , _C10 , _C11 , and _C12 , and also includes any range from n to n+m, for example, _C1-12 includes _C1-3 , _C1-6 , _C1-9 , _C3-6 , _C3-9 , _C3-12 , _C6-9 , _C6-12 , and C13. _9-12 , etc.; similarly, n-membered to n+m-membered means that the number of atoms in the ring is n to n+m, for example, a 3-12-membered ring includes a 3-membered ring, a 4-membered ring, a 5-membered ring, a 6-membered ring, a 7-membered ring, an 8-membered ring, a 9-membered ring, a 10-membered ring, an 11-membered ring, and a 12-membered ring, and also includes any range from n to n+m, for example, a 3-12-membered ring includes a 3-6-membered ring, a 3-9-membered ring, a 5-6-membered ring, a 5-7-membered ring, a 5-10-membered ring, a 6-7-membered ring, a 6-8-membered ring, a 6-9-membered ring and a 6-10-membered ring, etc.

The term "leaving group" refers to a functional group or atom that can be replaced by another functional group or atom through a substitution reaction (e.g., an affine substitution reaction). For example, representative leaving groups include trifluoromethanesulfonate; chloro, bromo, iodo; sulfonate groups such as methanesulfonate, toluenesulfonate, p-bromobenzenesulfonate, p-toluenesulfonate, etc.; acyloxy groups such as acetoxy and trifluoroacetoxy, etc.

The term "protecting group" includes, but is not limited to, an "amino protecting group," a "hydroxy protecting group," or a "thiol protecting group." The term "amino protecting group" refers to a protecting group suitable for preventing side reactions at the amino nitrogen position. Representative amino protecting groups include, but are not limited to, formyl; acyl, such as alkanoyl (e.g., acetyl, trichloroacetyl, or trifluoroacetyl); alkoxycarbonyl, such as tert-butyloxycarbonyl (Boc); arylmethoxycarbonyl, such as benzyloxycarbonyl (Cbz) and 9-fluorenylmethoxycarbonyl (Fmoc); arylmethyl, such as benzyl (Bn), trityl (Tr), 1,1-bis-(4'-methoxyphenyl)methyl; silyl, such as trimethylsilyl (TMS) and tert-butyldimethylsilyl (TBS), and the like. The term "hydroxy protecting group" refers to a protecting group suitable for preventing side reactions at the hydroxyl group. Representative hydroxy protecting groups include, but are not limited to, alkyl groups such as methyl, ethyl and tert-butyl; acyl groups such as alkanoyl (e.g., acetyl); arylmethyl groups such as benzyl (Bn), p-methoxybenzyl (PMB), 9-fluorenylmethyl (Fm) and diphenylmethyl (diphenylmethyl, DPM); silyl groups such as trimethylsilyl (TMS) and tert-butyldimethylsilyl (TBS), and the like.

The compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthesis methods, and equivalent substitutions well known to those skilled in the art. Preferred embodiments include but are not limited to the examples of the present invention.

The solvent used in the present invention is commercially available.

Compounds were named according to conventional nomenclature in the art or using software, and commercially available compounds were named according to the supplier's catalog.

DETAILED DESCRIPTION

The present application is described in detail below by way of examples, but this does not necessarily mean that there are any adverse limitations on the present application. The present application has been described in detail herein, and specific embodiments thereof have been disclosed. It will be apparent to those skilled in the art that various changes and modifications can be made to the specific embodiments of the present application without departing from the spirit and scope of the present application.

Unless otherwise specified, the experimental materials and reagents used in the following examples can be obtained from commercial channels.

Preparation of intermediates

Reference Example 1: Preparation of Intermediate I-1

Methyl 2-amino-5-chlorobenzoate (5.80 g, 31.2 mmol) was dissolved in tetrahydrofuran (80 mL) at room temperature, and pyridine (2.97 g, 37.5 mmol) and ethyl succinate chloride (7.70 g, 46.8 mmol) were added sequentially. After the addition was complete, the reaction mixture was stirred at room temperature for 2 hours. Water (100 mL) was added to the reaction mixture, and the mixture was extracted with ethyl acetate (100 mL x 3). The organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to remove the organic solvent to obtain intermediate I-1, which was used in the next reaction without further purification.

LC-MS (ESI) [M+H] ⁺ 314.0.

Reference Example 2: Preparation of Intermediate I-2

Sodium hydride (2.78 g, 60% wt, 69.5 mmol) was added to dimethyl sulfoxide (30 mL) at room temperature, and a mixture of intermediate I-1 (10.9 g, 34.7 mmol) in dimethyl sulfoxide (30 mL) and tetrahydrofuran (10 mL) was slowly added dropwise. After the addition was complete, the reaction mixture was stirred at room temperature for 1 hour. Acetic acid (30 mL) was added and stirring continued for 30 minutes. Water (200 mL) was added to the reaction mixture, causing a large amount of solid to precipitate. The solid was filtered, and the filter cake was dissolved in dichloromethane (400 mL), washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to remove the organic solvent to yield intermediate I-2. This compound, a mixture of methyl and ethyl esters, was used in the next reaction without further purification.

LC-MS (ESI) [M+H] ⁺ 282.1 (ethyl ester)/268.0 (methyl ester).

Reference Example 3: Preparation of Intermediate I-3

At room temperature, intermediate I-2 (5.03 g) was dissolved in dimethyl sulfoxide (50 mL) and water (5 mL) was added. After the addition was complete, the reaction mixture was stirred at 150°C for 5 hours. The reaction mixture was cooled to room temperature, poured into saturated brine (300 mL), and extracted with ethyl acetate (150 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to remove the organic solvent to obtain intermediate I-3.

LC-MS (ESI) [M+H] ⁺ 210.1.

Reference Example 4: Preparation of Intermediate I-4

At room temperature, intermediate I-3 (3.63 g, 17.3 mmol) was dissolved in tetrahydrofuran (50 mL), and sodium borohydride (1.97 g, 52.1 mmol) was added. After the addition was complete, the reaction mixture was stirred at room temperature for 1 hour. Water (100 mL) was added to the reaction mixture, and the mixture was extracted with ethyl acetate (100 mL x 3). The organic phases were combined, washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was separated and purified by silica gel chromatography to obtain intermediate I-4.

LC-MS (ESI) [M+H] ⁺ 212.0.

Reference Example 5: Preparation of Intermediate I-5

At room temperature, intermediate I-4 (1.66 g, 7.84 mmol) was dissolved in N,N-dimethylformamide (15 mL), and imidazole (1.07 g, 15.7 mmol) and tert-butyldimethylsilyl chloride (1.76 g, 11.7 mmol) were added sequentially. After the addition was complete, the reaction mixture was stirred at 120°C for 3 hours. The reaction mixture was cooled to room temperature, water (150 mL) was added, and extraction was performed with ethyl acetate (50 mL x 3). The organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was separated and purified by silica gel chromatography to obtain intermediate I-5.

LC-MS (ESI) [M+H] ⁺ 326.2.

Reference Example 6: Preparation of Intermediate I-6

At room temperature, intermediate I-5 (150 mg, 0.460 mmol) was dissolved in toluene (10 mL), and Lawesson's reagent (93.1 mg, 0.230 mmol) was added. The reaction solution was stirred at 120°C for 5 hours. After cooling to room temperature, the mixture was diluted with water (20 mL) and extracted with ethyl acetate (15 mL x 2). The organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was separated and purified by silica gel chromatography to obtain intermediate I-6.

LC-MS (ESI) [M+H] ⁺ 342.2.

Reference Example 7: Preparation of Intermediate I-7

At room temperature, sulfuric acid (3 mL) was added to a solution of 2-methyl-4-nitrobenzoic acid (2.0 g, 11.0 mmol) in ethanol (20 mL). The reaction mixture was stirred at 80°C for 16 hours. After cooling to room temperature, the mixture was concentrated under reduced pressure to remove most of the solvent, diluted with water (20 mL), and extracted with ethyl acetate (20 mL x 2). The organic phases were combined and washed sequentially with water (30 mL), saturated aqueous sodium bicarbonate (30 mL), and saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to remove the organic solvent to yield the ethyl ester product. The resulting product was dissolved in ethanol (30 mL), and 80% hydrazine hydrate (10 mL) was added. The reaction mixture was stirred at 80°C for 5 hours. The crude product was concentrated under reduced pressure to yield the product, which was purified by silica gel chromatography to yield intermediate I-7.

LC-MS (ESI) [M+H] ⁺ 196.2.

Reference Example 8: Preparation of Intermediate I-8

Intermediate I-6 (120 mg, 0.351 mmol) and intermediate I-7 (82.2 mg, 0.421 mmol) were dissolved in cyclohexanol (3 mL) at room temperature, and the reaction mixture was stirred at 160°C for 5 hours. After cooling to room temperature, the mixture was diluted with ethyl acetate (15 mL), washed sequentially with water (100 mL × 8) and saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was separated and purified by silica gel chromatography to obtain intermediate I-8.

LC-MS (ESI) [M+H] ⁺ 485.2.

Reference Example 9: Preparation of Intermediate I-9

Iron powder (28.8 mg, 0.516 mmol) and ammonium chloride (54.8 mg, 1.02 mmol) were added to a solution of intermediate I-8 (50 mg, 0.103 mmol) in methanol/water (5 mL/2 mL) at room temperature. Under argon, the reaction mixture was stirred at 70°C for 3 hours. The mixture was cooled to room temperature, filtered, and the filtrate was concentrated under reduced pressure to remove most of the methanol. The mixture was diluted with water (10 mL) and extracted with ethyl acetate (10 mL x 3). The organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude intermediate I-9. The crude product was used directly in the next reaction without further purification.

LC-MS (ESI) [M+H] ⁺ 455.3.

Reference Example 10: Preparation of Intermediate I-10

Thionyl chloride (9.00 g, 75.7 mmol) was added to a solution of 2-phenylbenzoic acid (3.0 g, 15.1 mmol) in dichloromethane (20 mL) at room temperature. N,N-dimethylformamide (1 drop) was then added. The reaction mixture was stirred at room temperature for 5 hours. The solvent was removed by concentration under reduced pressure to afford crude intermediate I-10, which was used directly in the next reaction.

Reference Example 11: Preparation of Intermediate I-11

Intermediate I-10 (40.0 mg) was added to a solution of Intermediate I-9 (70 mg, 0.154 mmol) and triethylamine (31.2 mg, 0.308 mmol) in dichloromethane (5 mL) at room temperature, and the reaction mixture was stirred at room temperature for 5 hours. Dichloromethane (10 mL) was added to the reaction mixture, which was then washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was separated and purified by silica gel chromatography to obtain Intermediate I-11.

LC-MS (ESI) [M+H] ⁺ 635.2.

Reference Example 12: Preparation of Intermediate I-12

2-Fluorophenylboronic acid (534 mg, 3.82 mmol) was dissolved in dioxane/water (10.0 mL/5.00 mL) at room temperature. Ethyl 2-iodobenzoate (1.00 g, 3.62 mmol), tetrakis(triphenylphosphine)palladium (441 mg, 0.382 mmol), and potassium carbonate (1.60 g, 11.5 mmol) were added sequentially. Under nitrogen, the reaction mixture was stirred at 75°C for 3 hours. The reaction system was cooled to room temperature, poured into water (50.0 mL), and extracted with ethyl acetate (50 mL x 3). The organic phases were combined, washed with saturated brine (15 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was separated and purified by silica gel chromatography to obtain intermediate I-12.

¹ H NMR (400MHz, CDCl ₃ )δ7.98(dd,J＝7.8,1.2Hz,1H),7.56(td,J＝7.5,1.4Hz,1H),7.45(td,J＝7.6,1.3Hz,1H),7.38–7.24( m,3H),7.19(td,J＝7.5,1.1Hz,1H),7.13–7.03(m,1H),4.14(q,J＝7.1Hz,2H),1.06(t,J＝7.1Hz,3H).

Reference Example 13: Preparation of Intermediate I-13

At room temperature, intermediate I-12 (500 mg, 2.05 mmol) was dissolved in methanol/water (5.00 mL/2.00 mL), and lithium hydroxide monohydrate (344 mg, 8.20 mmol) was added. The reaction mixture was stirred at room temperature for 3 hours. The system was poured into water (30 mL), and the pH was adjusted to 5 with dilute hydrochloric acid (1N). It was extracted with ethyl acetate (15 mL × 3). The organic phases were combined, washed with saturated brine (15 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to dryness to obtain intermediate I-13.

¹ H NMR (400MHz, CDCl ₃ )δ8.06(dd,J＝7.8,1.2Hz,1H),7.61(td,J＝7.6,1.4Hz,1H),7.47(td,J＝7.7,1 .2Hz,1H),7.39–7.27(m,3H),7.19(td,J＝7.5,1.1Hz,1H),7.11–7.02(m,1H).

Reference Example 14: Preparation of Intermediate I-14

At room temperature, intermediate I-13 (100 mg, 0.463 mmol) was dissolved in dichloromethane (3.00 mL), and intermediate I-9 (211 mg, 0.463 mmol), O-(7-azabenzotriazol-1-yl)-N,N,N'-tetramethyluronium hexafluorophosphate (176 mg, 0.463 mmol), and N,N-diisopropylethylamine (180 mg, 1.39 mmol) were added in sequence. The reaction mixture was stirred at room temperature for 3 hours. The reaction solution was concentrated under reduced pressure and purified by silica gel chromatography to obtain intermediate I-14.

LC-MS (ESI) [M+H] ⁺ 653.0.

Reference Example 15: Preparation of Intermediate I-15

Methyl 2-bromo-5-fluorobenzoate (500 mg, 2.15 mmol), 2-fluorophenylboronic acid (361 mg, 2.58 mmol), sodium carbonate (456 mg, 4.30 mmol), and tetrakis(triphenylphosphine)palladium (125 mg, 0.108 mmol) were mixed in dioxane (10 mL) and water (2 mL). The reaction mixture was purged with argon three times at room temperature and stirred at 90°C under argon for 3 hours. The mixture was cooled to room temperature and filtered. Water (20 mL) was added to the filtrate, and the dioxane was removed under reduced pressure. The aqueous phase was extracted with ethyl acetate (10 mL x 2). The combined organic phases were washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure. The residue was purified by silica gel chromatography to afford Intermediate I-15.

LC-MS (ESI) [M+H] ⁺ 249.1.

Reference Example 16: Preparation of Intermediate I-16

Intermediate I-15 (410 mg, 1.65 mmol) was dissolved in tetrahydrofuran (15 mL). A solution of lithium hydroxide monohydrate (692 mg, 16.5 mmol) in water (5 mL) was added dropwise with stirring at room temperature. The reaction mixture was heated under reflux for 2 hours. The mixture was cooled to room temperature and the tetrahydrofuran was removed under reduced pressure. The aqueous phase was adjusted to pH 1 with dilute hydrochloric acid (1N) and extracted with ethyl acetate (10 mL x 3). The organic phases were combined, washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain crude intermediate I-16, which was used directly in the next reaction without purification.

Reference Example 17: Preparation of Intermediate I-17

Intermediate I-9 (60.0 mg, 0.132 mmol), intermediate I-16 (40.3 mg, 0.172 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (50.6 mg, 0.264 mmol), and 4-dimethylaminopyridine (32.3 mg, 0.264 mmol) were mixed in 1,2-dichloroethane (5 mL). The reaction mixture was stirred at 60°C overnight. The reaction mixture was cooled to room temperature and the solvent was removed under reduced pressure. The residue was separated and purified by silica gel chromatography to obtain intermediate I-17.

LC-MS (ESI) [M+H] ⁺ 671.1.

Reference Example 18: Preparation of Intermediate I-18

Methyl 2-bromo-4-fluorobenzoate (500 mg, 2.14 mmol), phenylboronic acid (288 mg, 2.36 mmol), 1,1'-bis(diphenylphosphinoferrocenepalladium) dichloride (156 mg, 0.21 mmol), and potassium carbonate (886 mg, 6.42 mmol) were mixed in 1,4-dioxane/water (10 mL/1 mL) at room temperature. The reaction mixture was stirred at 100°C under nitrogen for 16 hours. The reaction system was cooled to room temperature, diluted with water (20 mL), and extracted with ethyl acetate (30 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was separated and purified by silica gel chromatography to obtain intermediate I-18.

LCMS (ESI) [M+H] ⁺ 231.2.

Reference Example 19: Preparation of Intermediate I-19

Intermediate I-18 (425 mg, 1.85 mmol) was dissolved in tetrahydrofuran/methanol/water (10 mL/10 mL/10 mL) at room temperature. Sodium hydroxide (740 mg, 18.5 mmol) was added at room temperature, and the reaction mixture was stirred at room temperature for 16 hours. The mixture was diluted with water (20 mL), and the pH was adjusted to less than 7 with hydrochloric acid. The mixture was extracted with ethyl acetate (30 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was separated and purified by silica gel chromatography to obtain intermediate I-19.

LCMS (ESI) [M+H] ⁺ 217.0.

Reference Example 20: Preparation of Intermediate I-20

Intermediate I-19 (80 mg, 0.37 mmol) was dissolved in dichloromethane (5 mL) at room temperature, and 1 drop of N,N-dimethylformamide was added. Oxalyl chloride (0.5 mL) was added to the reaction mixture at 0°C under nitrogen protection, and stirring was continued at 0°C for 1 hour. The mixture was concentrated to dryness to obtain crude intermediate I-20, which was used directly in the next reaction.

Reference Example 21: Preparation of Intermediate I-21

Compound I-9 (77 mg, 0.17 mmol) was dissolved in dichloromethane (5 mL) at room temperature, and triethylamine (103 mg, 1.02 mmol) was added. Intermediate I-20 (80 mg) was added at 0°C under nitrogen, and the reaction mixture was stirred at room temperature for 16 hours. The mixture was diluted with water (10 mL) and extracted with dichloromethane (20 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was separated and purified by silica gel chromatography to obtain intermediate I-21.

LCMS (ESI) [M+H] ⁺ 653.6.

Reference Example 22: Preparation of Intermediate I-22

At room temperature, tri-n-butyl 2-pyridyltin (1.19 g, 3.23 mmol) was added to a solution of methyl 2-bromo-5-fluorobenzoate (500 mg, 2.15 mmol) and tetrakis(triphenylphosphine)palladium (74.5 mg, 0.0645 mmol) in N,N-dimethylformamide (10 mL). Under argon, the reaction mixture was stirred at 100°C for 16 hours. After cooling to room temperature, the mixture was diluted with water (20 mL) and extracted with ethyl acetate (10 mL x 2). The organic phases were combined, washed sequentially with water (10 mL) and saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was purified by silica gel chromatography to obtain intermediate I-22.

LC-MS (ESI) [M+H] ⁺ 232.1.

Reference Example 23: Preparation of Intermediate I-23

At room temperature, sodium hydroxide (203 mg, 5.08 mmol) was added to a solution of intermediate I-22 (390 mg, 1.69 mmol) in tetrahydrofuran/water (10 mL/3 mL). The reaction mixture was refluxed for 3 hours. After cooling to room temperature, water (10 mL) was added, and the pH was adjusted to 7 with hydrochloric acid (1 mol/L). The mixture was extracted with ethyl acetate (10 mL x 8). The organic phases were combined, washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to remove the organic solvent to obtain intermediate I-23. This intermediate was used directly in the next reaction without further purification.

LC-MS (ESI) [M+H] ⁺ 218.1.

Reference Example 24: Preparation of Intermediate I-24

1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (42.2 mg, 0.220 mmol) and 4-dimethylaminopyridine (40.3 mg, 0.330 mmol) were added to a solution of Intermediate I-9 (50 mg, 0.110 mmol) and Intermediate I-23 (47.8 mg, 0.220 mmol) in 1,2-dichloroethane (5 mL) at room temperature. The reaction mixture was stirred at room temperature for 5 hours. The reaction mixture was diluted with dichloromethane (10 mL), washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was separated and purified by silica gel chromatography to obtain Intermediate I-24.

LC-MS (ESI) [M+H] ⁺ 654.0.

Reference Example 25: Preparation of Intermediate I-25

Methyl o-bromobenzoate (0.50 g, 2.33 mmol), 2-chlorophenylboronic acid (0.44 g, 2.79 mmol), 1,1'-bis(diphenylphosphinoferrocenedichloropalladium) (0.17 g, 0.23 mmol), and potassium carbonate (0.96 g, 6.98 mmol) were mixed in 1,4-dioxane/water (10 mL/3 mL) at room temperature. The reaction mixture was stirred at 80°C under nitrogen for 16 hours. The reaction system was cooled to room temperature, diluted with water (20 mL), and extracted with ethyl acetate (30 mL x 2). The organic phases were combined, washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to remove the organic solvent to obtain the crude product. The crude product was separated and purified by silica gel chromatography to obtain intermediate I-25.

LCMS (ESI) [M+H] ⁺ 247.2.

Reference Example 26: Preparation of Intermediate I-26

Intermediate I-25 (0.50 g, 2.03 mmol) was dissolved in methanol/water (10 mL/3 mL) at room temperature. Sodium hydroxide (0.24 g, 6.03 mmol) was added, and the reaction mixture was stirred at room temperature for 16 hours. The mixture was diluted with water (20 mL), adjusted to a pH of less than 7 with hydrochloric acid, and extracted with ethyl acetate (30 mL x 2). The combined organic phases were washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was separated and purified by silica gel chromatography to obtain intermediate I-26.

Reference Example 27: Preparation of Intermediate I-27

Intermediate I-26 (0.20 g, 0.86 mmol) was dissolved in dichloromethane (5 mL) at room temperature, and 1 drop of N,N-dimethylformamide was added. Oxalyl chloride (0.33 g, 2.58 mmol) was added at 0°C under nitrogen, and the reaction mixture was stirred at 0°C for 1 hour. The organic solvent was removed by concentration under reduced pressure to obtain crude intermediate I-27. This intermediate was used directly in the next reaction without further purification.

Reference Example 28: Preparation of Intermediate I-28

At room temperature, intermediate I-9 (70.00 mg, 0.15 mmol) was dissolved in dichloromethane (5 mL) and triethylamine (0.16 g, 1.59 mmol) was added. Under nitrogen protection, intermediate I-27 (0.20 g) was added at 0°C, and the reaction solution was slowly warmed to room temperature and stirred for 5 hours. Dilute with water (10 mL) and extract with dichloromethane (20 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to remove the organic solvent to obtain the crude product intermediate I-28, which was used directly in the next reaction.

LCMS (ESI) [M+H] ⁺ 669.2.

Reference Example 29: Preparation of Intermediate I-29

Methyl 2-bromo-5-fluorobenzoate (300 mg, 1.29 mmol), phenylboronic acid (157 mg, 1.29 mmol), sodium carbonate (410 mg, 3.87 mmol), and tetrakis(triphenylphosphine)palladium (149 mg, 0.129 mmol) were mixed in dioxane (10 mL) and water (2 mL). The reaction mixture was purged with argon three times at room temperature and stirred at 90°C under argon for 4 hours. The mixture was cooled to room temperature and filtered. Water (20 mL) was added to the filtrate, and the dioxane was removed under reduced pressure. The aqueous phase was extracted with ethyl acetate (10 mL x 2). The combined organic phases were washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure. The residue was purified by silica gel chromatography to yield intermediate I-29.

Reference Example 30: Preparation of Intermediate I-30

Intermediate I-29 (220 mg, 0.956 mmol) was dissolved in tetrahydrofuran (15 mL). A solution of lithium hydroxide monohydrate (401 mg, 9.56 mmol) in water (5 mL) was added dropwise with stirring at room temperature. The reaction mixture was stirred at 60°C for 2 hours. The mixture was cooled to room temperature and the tetrahydrofuran was removed under reduced pressure. The aqueous phase was adjusted to pH 1 with 1N dilute hydrochloric acid and extracted with ethyl acetate (10 mL x 3). The organic phases were combined, washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain crude intermediate I-30. The crude product was used directly in the next reaction without purification.

Reference Example 31: Preparation of Intermediate I-31

Intermediate I-9 (60.0 mg, 0.132 mmol), intermediate I-30 (100 mg, 0.463 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (50.6 mg, 0.264 mmol), and 4-dimethylaminopyridine (32.3 mg, 0.264 mmol) were mixed in 1,2-dichloroethane (5 mL). The reaction mixture was stirred at 60°C overnight. The mixture was cooled to room temperature and the solvent was removed under reduced pressure. The residue was separated and purified by silica gel chromatography to obtain intermediate I-31.

LC-MS (ESI) [M+H] ⁺ 653.3.

Reference Example 32: Preparation of Intermediate I-32

At room temperature, intermediate I-9 (40.0 mg, 0.0879 mmol) and 2-(pyridin-2-yl)benzoic acid (30.0 mg, 0.151 mmol) were added to a tetrahydrofuran (5.00 mL) solution. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (33.7 mg, 0.176 mmol) and 4-dimethylaminopyridine (21.5 mg, 0.176 mmol) were then added. The reaction mixture was stirred at room temperature for 2 hours, then water (20 mL) was added and extracted with ethyl acetate (10 mL x 3). The organic phases were combined, washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was separated and purified by silica gel chromatography to obtain intermediate I-32.

LC-MS (ESI) [M+H] ⁺ 636.3.

Reference Example 33: Preparation of Intermediate I-33

Tetrakis(triphenylphosphine)palladium (63.0 mg, 0.0545 mmol) was added to a solution of 2-ethoxycarbonylphenylboronic acid pinacol ester (300 mg, 1.09 mmol) and 2-bromothiazole (179 mg, 1.09 mmol) in ethylene glycol dimethyl ether (15 mL) at room temperature, followed by the addition of cesium carbonate aqueous solution (3 mL, 2 mol/L). Under argon, the reaction mixture was stirred at 90°C for 16 hours. After cooling to room temperature, the mixture was diluted with water (25 mL) and extracted with ethyl acetate (20 mL x 2). The organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was purified by silica gel chromatography to obtain intermediate I-33.

LC-MS (ESI) [M+H] ⁺ 234.1.

Reference Example 34: Preparation of Intermediate I-34

At room temperature, sodium hydroxide (55.8 mg, 1.395 mmol) was added to a solution of intermediate I-33 (65.0 mg, 0.279 mmol) in tetrahydrofuran/water (5 mL/1 mL). The reaction mixture was stirred at 70°C for 3 hours. The mixture was cooled to room temperature, adjusted to pH 6 with hydrochloric acid (3 mol/L), and extracted with ethyl acetate (10 mL x 5). The organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to remove the organic solvent to afford intermediate I-34, which was used directly in the next step without purification.

LC-MS (ESI) [M+H] ⁺ 205.9.

Reference Example 35: Preparation of Intermediate I-35

Thionyl chloride (69.6 mg, 0.585 mmol) and a drop of N,N-dimethylformamide were added sequentially to a solution of intermediate I-34 (40 mg, 0.195 mmol) in dichloromethane (10 mL) at room temperature. The reaction mixture was stirred at room temperature for 3 hours. The organic solvent was removed by concentration under reduced pressure to afford intermediate I-35, which was used directly in the next reaction without purification.

Reference Example 36: Preparation of Intermediate I-36

Triethylamine (36.2 mg, 0.358 mmol) was added to a solution of Intermediate I-9 (81.4 mg, 0.179 mmol) and Intermediate I-35 (40 mg) in dichloromethane (5 mL) at room temperature. The reaction mixture was stirred at room temperature for 2 hours. The organic solvent was removed by concentration under reduced pressure to obtain a crude product, which was then purified by silica gel chromatography to obtain Intermediate I-36.

LC-MS (ESI) [M+H] ⁺ 642.3.

Reference Example 37: Preparation of Intermediate I-37

At room temperature, o-chlorobenzoyl chloride (15.4 mg, 0.0880 mmol) was added to a solution of intermediate I-9 (40.0 mg, 0.0879 mmol) and triethylamine (26.7 mg, 0.264 mmol) in dichloromethane (2.00 mL). The reaction mixture was stirred at room temperature for 2 hours. Water (10 mL) was added, and the mixture was extracted with dichloromethane (5 mL x 3). The organic phases were combined, washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was separated and purified by silica gel chromatography to obtain intermediate I-37.

LC-MS (ESI) [M+H] ⁺ 593.2.

Reference Example 38: Preparation of Intermediate I-38

Methyl 4-nitro-2-chlorobenzoate (1.00 g, 4.65 mmol) was dissolved in ethanol (2 mL) at room temperature, and 80% hydrazine hydrate (10 mL) was added. The reaction mixture was stirred at 80°C for 16 hours. The reaction mixture was concentrated to dryness and purified by silica gel chromatography to afford Intermediate I-38. LCMS (ESI) [M+H] ⁺ 185.9.

Reference Example 39: Preparation of Intermediate I-39

At room temperature, intermediate I-38 (271 mg, 0.73 mmol) and intermediate I-6 (250 mg, 0.73 mmol) were dissolved in n-butanol (10 mL), and the reaction mixture was stirred at 160°C for 7 hours. The reaction system was cooled to room temperature, diluted with water (20 mL), and extracted with dichloromethane (20 mL × 3). The organic phases were combined, washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was separated and purified by preparative HPLC (formic acid system) to obtain intermediate I-39.

LCMS (ESI) [M+H] ⁺ 475.2.

Reference Example 40: Preparation of Intermediate I-40

At room temperature, intermediate I-39 (146 mg, 0.31 mmol) was dissolved in dichloromethane (10 mL). o-Chlorobenzoyl chloride (162 mg, 0.92 mmol) and triethylamine (93.3 mg, 0.92 mmol) were added sequentially. The reaction mixture was stirred at room temperature for 4 hours. Diluted with water (10 mL), extracted with dichloromethane (20 mL x 3), and the combined organic phases were washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was separated and purified by silica gel chromatography to obtain intermediate I-40.

LCMS (ESI) [M+H] ⁺ 615.1 (isotope peak).

Reference Example 41: Preparation of Intermediate I-41

Methyl 4-nitro-2-fluorobenzoate (1.00 g, 5.02 mmol) was dissolved in ethanol (2 mL) at room temperature, and 80% hydrazine hydrate (10 mL) was added. The reaction mixture was stirred at 80°C for 16 hours. The reaction mixture was concentrated to dryness and purified by silica gel chromatography to obtain intermediate I-41. LCMS (ESI) [M+H] ⁺ 170.0.

Reference Example 42: Preparation of Intermediate I-42

At room temperature, intermediate I-41 (248 mg, 1.46 mmol) and intermediate I-6 (250 mg, 0.73 mmol) were dissolved in n-butanol (10 mL), and the reaction mixture was stirred at 160°C for 7 hours. The reaction system was cooled to room temperature, diluted with water (20 mL), and extracted with dichloromethane (20 mL × 3). The organic phases were combined, washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was separated and purified by preparative HPLC (formic acid) to obtain intermediate I-42.

LCMS (ESI) [M+H] ⁺ 459.2.

Reference Example 43: Preparation of Intermediate I-43

At room temperature, intermediate I-42 (150 mg, 0.33 mmol) was dissolved in dichloromethane (10 mL). o-Chlorobenzoyl chloride (171.8 mg, 0.98 mmol) and triethylamine (99.1 mg, 0.98 mmol) were added sequentially. The reaction mixture was stirred at room temperature for 5 hours. Diluted with water (10 mL), extracted with dichloromethane (20 mL x 3), and the combined organic phases were washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was separated and purified by silica gel chromatography to obtain intermediate I-43.

LCMS (ESI) [M+H] ⁺ 597.2.

Reference Example 44: Preparation of Intermediate I-44

At room temperature, methyl 4-nitro-2-bromobenzoate (1.00 g, 3.86 mmol), isopropenyl borate (1.90 g, 11.58 mmol), 1,1'-bis(diphenylphosphinoferrocenepalladium) dichloride (282 mg, 0.39 mmol), and potassium carbonate (1.60 g, 11.58 mmol) were mixed in 1,4-dioxane/water (30 mL/1.2 mL). The reaction mixture was stirred at 100°C under nitrogen for 16 hours. The reaction system was cooled to room temperature, diluted with water (30 mL), and extracted with ethyl acetate (50 mL x 3). The organic phases were combined, washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to remove the organic solvent to obtain the crude product. The crude product was purified by silica gel chromatography to obtain intermediate I-44.

LCMS (ESI) [M+H] ⁺ 222.0.

Reference Example 45: Preparation of Intermediate I-45

At room temperature, intermediate I-44 (700 mg, 3.17 mmol) was dissolved in ethanol (2 mL), and 80% hydrazine hydrate (10 mL) was added. The reaction solution was stirred at 80°C for 16 hours. The reaction solution was concentrated to dryness and purified by silica gel chromatography to obtain intermediate I-45.

LCMS (ESI) [M+H] ⁺ 192.2.

Reference Example 46: Preparation of Intermediate I-46

Intermediate I-45 (290 mg, 1.52 mmol) was dissolved in methanol (5 mL) at room temperature, and platinum dioxide (29 mg) was added. The reaction mixture was stirred under a hydrogen atmosphere (hydrogen balloon) at room temperature for 1 hour. The mixture was filtered, and the filtrate was concentrated under reduced pressure to remove the organic solvent to obtain crude intermediate I-46, which was directly used in the next step without purification.

LCMS (ESI) [M+H] ⁺ 194.0.

Reference Example 47: Preparation of Intermediate I-47

Intermediate I-46 (135 mg, 0.70 mmol) and intermediate I-6 (120 mg, 0.35 mmol) were dissolved in n-butanol (5 mL) at room temperature, and the reaction mixture was stirred at 130°C for 16 hours. The reaction system was cooled to room temperature and concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was separated and purified by preparative HPLC (formic acid system) to obtain intermediate I-47.

LCMS (ESI) [M+H] ⁺ 483.2.

Reference Example 48: Preparation of Intermediate I-48

Intermediate I-47 (79 mg, 0.16 mmol) was dissolved in dichloromethane (5 mL) at room temperature. o-Chlorobenzoyl chloride (85.8 mg, 0.49 mmol) and triethylamine (49.5 mg, 0.49 mmol) were added sequentially. The reaction mixture was stirred at room temperature for 2 hours. The mixture was diluted with water (10 mL) and extracted with dichloromethane (20 mL x 3). The organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was separated and purified by silica gel chromatography to obtain intermediate I-48.

LCMS (ESI) [M+H] ⁺ 621.2.

Reference Example 49: Preparation of Intermediate I-49

At room temperature, o-chlorobenzoyl chloride (579 mg, 3.31 mmol) was added to a solution of methyl 6-amino-4-methylnicotinate (500 mg, 3.01 mmol) in pyridine (10.0 mL). The reaction mixture was stirred at room temperature for 1 hour, poured into water (50 mL), filtered, and the filter cake was washed with water (20 mL) and dried to obtain Intermediate I-49.

LC-MS (ESI) [M+H] ⁺ 305.0.

Reference Example 50: Preparation of Intermediate I-50

80% hydrazine hydrate (410 mg) was added to a solution of intermediate I-49 (250 mg, 0.820 mmol) in methanol/water (5 mL/0.5 mL) at room temperature, and the reaction mixture was stirred at 75°C for 6 hours. The reaction solution was concentrated under reduced pressure to remove the organic solvent, and the residue was purified by silica gel chromatography to obtain intermediate I-50.

LC-MS (ESI) [M+H] ⁺ 304.9.

Reference Example 51: Preparation of Intermediate I-51

At room temperature, intermediate I-50 (196 mg, 0.643 mmol) and intermediate I-6 (242 mg, 0.707 mmol) were added to cyclohexanol (5.00 mL) and heated to 160°C with stirring for 8 hours. The reaction system was cooled to room temperature and diluted with ethyl acetate (20 mL). The mixture was washed with water (50 mL × 5) and saturated brine (50 mL × 2), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was separated and purified by silica gel chromatography to obtain intermediate I-51.

LC-MS (ESI) [M+H] ⁺ 593.9.

Reference Example 52: Preparation of Intermediate I-52

Methyl 4-nitro-2-bromobenzoate (200 mg, 0.77 mmol), potassium cyclopropyltrifluoroborate (227 mg, 1.54 mmol), 1,1'-bis(diphenylphosphinoferrocenepalladium) dichloride (56.2 mg, 0.077 mmol), and potassium carbonate (318.7 mg, 2.31 mmol) were mixed in 1,4-dioxane/water (10 mL/1 mL) at room temperature. The reaction mixture was stirred at 100°C under nitrogen for 16 hours. The reaction system was cooled to room temperature, diluted with water (20 mL), and extracted with ethyl acetate (30 mL x 3). The organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to remove the organic solvent to obtain the crude product. The crude product was purified by silica gel chromatography to obtain intermediate I-52.

LCMS (ESI) [M+H] ⁺ 222.0.

Reference Example 53: Preparation of Intermediate I-53

At room temperature, intermediate I-52 (120 mg, 0.54 mmol) was dissolved in ethanol (2 mL), and 80% hydrazine hydrate (10 mL) was added. The reaction solution was stirred at 80°C for 16 hours. The reaction solution was concentrated to dryness and purified by silica gel chromatography to obtain intermediate I-53.

LCMS (ESI) [M+H] ⁺ 192.0.

Reference Example 54: Preparation of Intermediate I-54

Intermediate I-53 (78 mg, 0.42 mmol) and intermediate I-6 (120 mg, 0.35 mmol) were dissolved in n-butanol (5 mL) at room temperature. The reaction mixture was stirred at 130°C for 16 hours, then heated to 160°C and stirred for 4 hours. The reaction system was cooled to room temperature and concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was separated and purified by preparative HPLC (formic acid system) to obtain intermediate I-54.

LCMS (ESI) [M+H] ⁺ 481.2.

Reference Example 55: Preparation of Intermediate I-55

At room temperature, intermediate I-54 (90 mg, 0.19 mmol) was dissolved in dichloromethane (10 mL). o-Chlorobenzoyl chloride (98 mg, 0.56 mmol) and triethylamine (56.6 mg, 0.56 mmol) were added sequentially. The reaction mixture was stirred at room temperature for 2 hours. Diluted with water (10 mL), extracted with dichloromethane (20 mL x 3), and the organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was separated and purified by silica gel chromatography to obtain intermediate I-55.

LCMS (ESI) [M+H] ⁺ 619.2.

Reference Example 56: Preparation of Intermediate I-56

Methyl 4-nitro-2-bromobenzoate (2.00 g, 7.69 mmol), potassium vinyl trifluoroborate (3.00 g), 1,1'-bis(diphenylphosphinoferrocenepalladium) dichloride (564 mg, 0.77 mmol), and potassium carbonate (3.20 g, 23.16 mmol) were mixed in 1,4-dioxane/water (50 mL/2 mL) at room temperature. The reaction mixture was stirred at 100°C under nitrogen for 16 hours. The reaction system was cooled to room temperature, diluted with water (20 mL), and extracted with ethyl acetate (100 mL x 3). The organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to remove the organic solvent to obtain the crude product. The crude product was separated and purified by silica gel chromatography to obtain intermediate I-56.

LCMS (ESI) [M+H] ⁺ 208.0.

Reference Example 57: Preparation of Intermediate I-57

At room temperature, intermediate I-56 (400 mg, 1.93 mmol) was dissolved in ethanol (2 mL), and 80% hydrazine hydrate (10 mL) was added. The reaction solution was stirred at 80°C for 16 hours. The reaction solution was concentrated to dryness and purified by silica gel chromatography to obtain intermediate I-57.

LCMS (ESI) [M+H] ⁺ 180.0.

Reference Example 58: Preparation of Intermediate I-58

At room temperature, intermediate I-57 (62 mg, 0.35 mmol) and intermediate I-6 (120 mg, 0.35 mmol) were dissolved in n-butanol (5 mL). The reaction mixture was stirred at 130°C for 16 hours, then heated to 160°C and stirred for 5 hours. The reaction system was cooled to room temperature and concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was separated and purified by preparative HPLC (formic acid system) to obtain intermediate I-58.

LCMS (ESI) [M+H] ⁺ 469.2.

Reference Example 59: Preparation of Intermediate I-59

At room temperature, intermediate I-58 (65 mg, 0.14 mmol) was dissolved in dichloromethane (5 mL). o-Chlorobenzoyl chloride (72.7 mg, 0.42 mmol) and triethylamine (41.9 mg, 0.42 mmol) were added sequentially. The reaction mixture was stirred at room temperature for 2 hours. Diluted with water (10 mL), extracted with dichloromethane (20 mL x 3), and the combined organic phases were washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was separated and purified by silica gel chromatography to obtain intermediate I-59.

LCMS (ESI) [M+H] ⁺ 607.2.

Reference Example 60: Preparation of Intermediate I-60

2-Hydroxy-4-nitrobenzoic acid (200 mg, 1.09 mmol) and dimethyl sulfate (343 mg, 2.72 mmol) were mixed in N,N-dimethylformamide (10 mL) at room temperature and stirred for 1 hour. The reaction system was cooled to room temperature, diluted with water (20 mL), and extracted with ethyl acetate (20 mL x 2). The organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was separated and purified by silica gel chromatography to obtain intermediate I-60.

LCMS (ESI) [M+H] ⁺ 212.2.

Reference Example 61: Preparation of Intermediate I-61

At room temperature, intermediate I-60 (178 mg, 0.84 mmol) was dissolved in ethanol (2 mL), and 80% hydrazine hydrate (10 mL) was added. The reaction solution was stirred at 80°C for 2 hours. The reaction solution was concentrated to dryness and purified by silica gel chromatography to obtain intermediate I-61.

LCMS (ESI) [M+H] ⁺ 182.2.

Reference Example 62: Preparation of Intermediate I-62

Intermediate I-61 (95 mg, 0.53 mmol) and intermediate I-6 (120 mg, 0.35 mmol) were dissolved in n-butanol (10 mL) at room temperature. The reaction mixture was stirred at 130°C for 16 hours, then heated to 160°C and stirred for 4 hours. The reaction system was cooled to room temperature and concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was separated and purified by preparative HPLC (formic acid system) to obtain intermediate I-62.

LCMS (ESI) [M+H] ⁺ 471.4.

Reference Example 63: Preparation of Intermediate I-63

Intermediate I-62 (140 mg, 0.297 mmol) was dissolved in dichloromethane (10 mL) at room temperature. o-Chlorobenzoyl chloride (156 mg, 0.891 mmol) and triethylamine (90 mg, 0.89 mmol) were added sequentially. The reaction mixture was stirred at room temperature for 2 hours. Diluted with water (10 mL), extracted with dichloromethane (20 mL x 2), and the combined organic phases were washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was separated and purified by silica gel chromatography to obtain Intermediate I-63.

LCMS (ESI) [M+H] ⁺ 609.2.

Reference Example 64: Preparation of Intermediate I-64

o-Toluoyl chloride (27.2 mg, 0.176 mmol) was added to a solution of intermediate I-9 (40.0 mg, 0.0879 mmol) and triethylamine (44.5 mg, 0.440 mmol) in dichloromethane (3 mL) at room temperature. The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was washed with saturated brine (3 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to remove the organic solvent to obtain the crude product, which was used directly in the next reaction without purification.

LC-MS (ESI) [M+H] ⁺ 573.2.

Reference Example 65: Preparation of Intermediate I-65

At room temperature, intermediate I-56 (500 mg, 2.41 mmol) and potassium osmate dihydrate (44.4 mg, 0.12 mmol) were mixed in tetrahydrofuran/water (20 mL/10 mL). Sodium periodate (2.05 g, 9.66 mmol) was added, and the reaction mixture was stirred at room temperature for 0.5 hours. The mixture was diluted with water (10 mL) and extracted with ethyl acetate (50 mL x 3). The organic phases were combined, washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to remove the organic solvent to obtain the crude product. The crude product was separated and purified by silica gel chromatography to obtain intermediate I-65.

LCMS (ESI) [M+H] ⁺ 210.0.

Reference Example 66: Preparation of Intermediate I-66

Intermediate I-65 (230 mg, 1.10 mmol) was dissolved in dichloromethane (10 mL). Diethylaminosulfur trifluoride (888.5 mg, 5.50 mmol) was added at -78°C. The reaction solution was slowly warmed from -78°C to room temperature and stirred for 2 hours. Diluted with water (10 mL), extracted with ethyl acetate (20 mL x 3), and the organic phases were combined, washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was separated and purified by silica gel chromatography to obtain intermediate I-66.

LCMS (ESI) [M+H] ⁺ 232.0.

Reference Example 67: Preparation of Intermediate I-67

At room temperature, intermediate I-66 (150 mg, 0.65 mmol) was dissolved in ethanol (2 mL), and 80% hydrazine hydrate (10 mL) was added. The reaction solution was stirred at 80°C for 6 hours. The mixture was concentrated to dryness under reduced pressure and purified by silica gel chromatography to obtain intermediate I-67.

LCMS (ESI) [M+H] ⁺ 202.0.

Reference Example 68: Preparation of Intermediate I-68

Intermediate I-67 (61 mg, 0.29 mmol) and intermediate I-6 (100 mg, 0.29 mmol) were dissolved in n-butanol (7 mL) at room temperature. The reaction mixture was stirred at 160°C for 5 hours and then at 130°C for 12 hours. The reaction system was cooled to room temperature and concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was separated and purified by preparative HPLC (formic acid system) to obtain intermediate I-68.

LCMS (ESI) [M+H] ⁺ 491.2.

Reference Example 69: Preparation of Intermediate I-69

At room temperature, intermediate I-68 (62 mg, 0.13 mmol) was dissolved in dichloromethane (5 mL). o-Chlorobenzoyl chloride (66 mg, 0.38 mmol) and triethylamine (38 mg, 0.38 mmol) were added sequentially. The reaction mixture was stirred at room temperature for 2 hours. Diluted with water (10 mL), extracted with dichloromethane (20 mL x 3), and the combined organic phases were washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was separated and purified by silica gel chromatography to obtain intermediate I-69.

LCMS (ESI) [M+H] ⁺ 629.2.

Reference Example 70: Preparation of Intermediate I-70

At room temperature, o-fluorobenzoyl chloride (13.9 mg, 0.0877 mmol) was added to a solution of intermediate I-9 (40.0 mg, 0.0879 mmol) and triethylamine (26.7 mg, 0.264 mmol) in dichloromethane (2.00 mL). The reaction mixture was stirred at room temperature for 2 hours. Water (10 mL) was added, and the mixture was extracted with dichloromethane (5 mL x 3). The organic phases were combined, washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was separated and purified by silica gel chromatography to obtain intermediate I-70.

LC-MS (ESI) [M+H] ⁺ 577.3.

Reference Example 71: Preparation of Intermediate I-71

At room temperature, o-trifluoromethylbenzoyl chloride (36.7 mg, 0.176 mmol) was added to a solution of intermediate I-9 (80.0 mg, 0.176 mmol) and triethylamine (35.6 mg, 0.352 mmol) in 1,2-dichloroethane (3.00 mL). The reaction mixture was stirred at 70°C for 16 hours. Water (10 mL) was added, and the mixture was extracted with dichloromethane (5 mL x 3). The organic phases were combined, washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was separated and purified by silica gel chromatography to obtain intermediate I-71.

LC-MS (ESI) [M+H] ⁺ 627.2.

Reference Example 72: Preparation of Intermediate I-72

Methyl 2-formylbenzoate (2.50 g, 15.2 mmol) was dissolved in dichloromethane (30.0 mL) at room temperature, and diethylaminosulfur trifluoride (2.93 g, 18.2 mmol) and methanol (0.10 mL) were added sequentially. The reaction mixture was stirred at room temperature for 10 hours, then poured into a saturated aqueous sodium bicarbonate solution (100 mL) and extracted with dichloromethane (50 mL x 3). The organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was separated and purified by silica gel chromatography to obtain intermediate I-72.

¹ H NMR (400MHz, DMSO-d ₆ ) δ8.01–7.96(m,1H),7.79(dd,J＝6.7,1.1Hz,2H),7.72–7.67(m,1H),7.53(t,J＝55.2Hz,1H),3.88(s,3H).

Reference Example 73: Preparation of Intermediate I-73

At room temperature, intermediate I-72 (600 mg, 3.22 mmol) was dissolved in tetrahydrofuran/water (5.00 mL/1.00 mL). Sodium hydroxide (516 mg, 12.9 mmol) was added to the reaction system, and the reaction mixture was stirred at room temperature for 2 hours. The pH of the reaction solution was adjusted to 6-7 with hydrochloric acid solution (0.5 mol/L), the system was poured into water (30 mL), and extracted with ethyl acetate (10 mL x 3). The organic phases were combined, washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain intermediate I-73.

¹ H NMR (400MHz, DMSO-d ₆ ) δ13.55 (s, 1H), 7.99 (d, J = 7.8Hz, 1H), 7.82–7.74 (m, 2H), 7.73–7.45 (m, 2H).

Reference Example 74: Preparation of Intermediate I-74

Intermediate I-73 (500 mg, 2.90 mmol) was dissolved in thionyl chloride (5.00 mL) at room temperature, and the reaction mixture was stirred at room temperature for 3 hours. The reaction system was concentrated under reduced pressure to obtain the crude intermediate I-74, which was used directly in the next reaction.

Reference Example 75: Preparation of Intermediate I-75

At room temperature, intermediate I-74 (80.0 mg, 0.420 mmol) was dissolved in dichloromethane (5.00 mL), and intermediate I-9 (191 mg, 0.420 mmol) and triethylamine (127 mg, 1.26 mmol) were added sequentially. The reaction mixture was stirred at room temperature for 3 hours. The reaction solution was concentrated under reduced pressure, and the residue was separated and purified by silica gel chromatography to obtain intermediate I-75.

LC-MS (ESI) [M+H] ⁺ 609.1.

Reference Example 76: Preparation of Intermediate I-76

2-Ethylbenzoic acid (500 mg, 3.33 mmol) was dissolved in thionyl chloride (10.0 mL) at room temperature, and the reaction mixture was stirred at room temperature for 3 hours. The reaction system was concentrated under reduced pressure to dryness to obtain the crude intermediate I-76, which was used directly in the next reaction.

Reference Example 77: Preparation of Intermediate I-77

At room temperature, intermediate I-9 (270 mg, 0.593 mmol) was dissolved in dichloromethane (2.00 mL), and intermediate I-76 (100 mg) and triethylamine (180 mg, 1.78 mmol) were added sequentially. The reaction mixture was stirred at room temperature for 4 hours. The reaction solution was concentrated under reduced pressure, and the residue was separated and purified by silica gel chromatography to obtain intermediate I-77.

LC-MS (ESI) [M+H] ⁺ 587.1.

Reference Example 78: Preparation of Intermediate I-78

At room temperature, intermediate I-9 (50 mg, 0.11 mmol) was dissolved in 1,2-dichloroethane (10 mL). 2-(Trifluoromethyl)nicotinic acid (63 mg, 0.33 mmol), 4-dimethylaminopyridine (67 mg, 0.55 mmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (106 mg, 0.55 mmol) were added. The reaction mixture was stirred at 70°C for 4 hours. The reaction mixture was cooled to room temperature, diluted with water (10 mL), and allowed to stand for separation. The aqueous phase was extracted with dichloromethane (10 mL x 3). The organic phases were combined, washed with water (10 mL x 3), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel chromatography to obtain intermediate I-78.

LC-MS (ESI) [M+H] ⁺ 628.3.

Reference Example 79: Preparation of Intermediate I-79

At room temperature, intermediate I-9 (50.0 mg, 0.110 mmol) and 3-chloro-2-methylbenzoic acid (37.5 mg, 0.220 mmol) were added to tetrahydrofuran (3.00 mL). 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (42.2 mg, 0.220 mmol) and 4-dimethylaminopyridine (26.9 mg, 0.220 mmol) were also added. The reaction mixture was stirred at 60°C for 8 hours, then water (10 mL) was added and the mixture was extracted with ethyl acetate (5 mL x 3). The organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel chromatography to obtain intermediate I-79.

LC-MS (ESI) [M+H] ⁺ 607.0.

Reference Example 80: Preparation of Intermediate I-80

At room temperature, m-methylbenzoyl chloride (13.6 mg, 0.0879 mmol) was added to a solution of Intermediate I-9 (40.0 mg, 0.0879 mmol) and triethylamine (26.7 mg, 0.264 mmol) in dichloromethane (2.00 mL). The reaction mixture was stirred at room temperature for 2 hours, then water (5 mL) was added and extracted with dichloromethane (5 mL x 3). The combined organic phases were washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel chromatography to yield Intermediate I-80.

LC-MS (ESI) [M+H] ⁺ 573.3.

Reference Example 81: Preparation of Intermediate I-81

m-Fluorobenzoyl chloride (20.9 mg, 0.132 mmol) was added to a solution of Intermediate I-9 (60.0 mg, 0.132 mmol) and triethylamine (40.1 mg, 0.396 mmol) in 1,2-dichloroethane (3.00 mL) at room temperature. The reaction mixture was stirred at 70°C for 16 hours. Water (10 mL) was added, and the mixture was extracted with dichloromethane (10 mL x 2). The combined organic phases were washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel chromatography to afford Intermediate I-81.

LC-MS (ESI) [M+H] ⁺ 577.0.

Reference Example 82: Preparation of Intermediate I-82

5-Fluoro-2-methylbenzoyl chloride (22.8 mg, 0.132 mmol) was added to a solution of Intermediate I-9 (60.0 mg, 0.132 mmol) and triethylamine (40.1 mg, 0.396 mmol) in dichloromethane (3.00 mL) at room temperature. The reaction mixture was stirred at room temperature for 16 hours. Water (10 mL) was added, and the mixture was extracted with dichloromethane (10 mL x 2). The combined organic phases were washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel chromatography to afford Intermediate I-82.

LC-MS (ESI) [M+H] ⁺ 591.0.

Reference Example 83: Preparation of Intermediate I-83

2-Chloro-3-fluorobenzoic acid (200 mg, 1.15 mmol) was dissolved in thionyl chloride (3.50 mL) at room temperature, and the reaction mixture was stirred at room temperature for 3 hours. The reaction solution was concentrated under reduced pressure to dryness to obtain the crude intermediate I-83, which was used directly in the next reaction.

Reference Example 84: Preparation of Intermediate I-84

At room temperature, intermediate I-83 (100 mg) was dissolved in dichloromethane (5.00 mL), and intermediate I-9 (236 mg, 0.518 mmol) and triethylamine (157 mg, 1.55 mmol) were added sequentially. The reaction mixture was stirred at room temperature for 3 hours. The reaction solution was concentrated under reduced pressure, and the residue was separated and purified by silica gel chromatography to obtain intermediate I-84.

LC-MS (ESI) [M+H] ⁺ 611.0.

Reference Example 85: Preparation of Intermediate I-85

1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (42.2 mg, 0.220 mmol) and 4-dimethylaminopyridine (40.3 mg, 0.330 mmol) were added to a solution of Intermediate I-9 (50 mg, 0.110 mmol) and 3-fluoro-2-methylbenzoic acid (33.9 mg, 0.220 mmol) in dichloroethane (5 mL) at room temperature. The reaction mixture was stirred at 60°C for 16 hours. The reaction mixture was cooled to room temperature, diluted with dichloromethane (5 mL), washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel chromatography to obtain Intermediate I-85.

LC-MS (ESI) [M+H] ⁺ 591.1.

Reference Example 86: Preparation of Intermediate I-86

1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (42.2 mg, 0.220 mmol) and 4-dimethylaminopyridine (40.3 mg, 0.330 mmol) were added to a solution of Intermediate I-9 (50 mg, 0.110 mmol) and 3-chloro-2-fluorobenzoic acid (38.4 mg, 0.220 mmol) in 1,2-dichloroethane (5 mL) at room temperature. The reaction mixture was stirred at 50°C for 16 hours. The reaction mixture was cooled to room temperature, diluted with dichloromethane (10 mL), washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel chromatography to obtain Intermediate I-86.

LC-MS (ESI) [M+H] ⁺ 611.3.

Reference Example 87: Preparation of Intermediate I-87

At room temperature, intermediate I-9 (50 mg, 0.11 mmol) was dissolved in 1,2-dichloroethane (10 mL). 4,5-difluoro-2-methylbenzoic acid (57 mg, 0.33 mmol), 4-dimethylaminopyridine (67 mg, 0.55 mmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (106 mg, 0.55 mmol) were added. The reaction mixture was stirred at 70°C for 2 hours. The reaction mixture was cooled to room temperature, diluted with water (10 mL), and allowed to stand for separation. The aqueous phase was extracted with dichloromethane (10 mL × 3). The combined organic phases were washed with water (10 mL × 3), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel chromatography to obtain intermediate I-87.

LC-MS (ESI) [M+H] ⁺ 609.1.

Reference Example 88: Preparation of Intermediate I-88

Intermediate I-9 (60.00 mg, 0.13 mmol) was dissolved in N,N-dimethylformamide (5 mL) at room temperature. 3-Methyl-2-thiophenecarboxylic acid (28.12 mg, 0.20 mmol), N,N-diisopropylethylamine (51.12 mg, 0.40 mmol), and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (75.20 mg, 0.20 mmol) were added sequentially. The reaction mixture was stirred at 90°C for 16 hours. The reaction mixture was cooled to room temperature, water (10 mL) was added, and the mixture was extracted with ethyl acetate (10 mL x 3). The organic phases were combined, washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain crude intermediate I-88, which was used directly in the next reaction.

LC-MS (ESI) [M+H] ⁺ 579.4.

Reference Example 89: Preparation of Intermediate I-89

At room temperature, intermediate I-9 (80.00 mg, 0.18 mmol) was dissolved in N,N-dimethylformamide (10 mL). 2-Methyl-3-furoic acid (26.60 mg, 0.21 mmol), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (100.27 mg, 0.26 mmol), and N,N-diisopropylethylamine (68.16 mg, 0.53 mmol) were added sequentially. The reaction mixture was stirred at 90°C for 12 hours. The reaction mixture was cooled to room temperature, diluted with ethyl acetate (20 mL), washed sequentially with water (20 mL) and saturated brine (10 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to remove the organic solvent to obtain the crude product. The crude product was separated and purified by silica gel chromatography to obtain intermediate I-89.

LC-MS (ESI) [M+H] ⁺ 563.2.

Reference Example 90: Preparation of Intermediate I-90

At room temperature, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (42.2 mg, 0.220 mmol) and 4-dimethylaminopyridine (40.3 mg, 0.330 mmol) were added to a solution of intermediate I-9 (50 mg, 0.110 mmol) and 4-fluoro-2-chlorobenzoic acid (38.4 mg, 0.220 mmol) in dichloroethane (5 mL). The reaction mixture was stirred at 50°C for 5 hours. The reaction mixture was cooled to room temperature, diluted with dichloromethane (10 mL), washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was separated and purified by silica gel chromatography to obtain intermediate I-90.

LC-MS (ESI) [M+H] ⁺ 611.3.

Reference Example 91: Preparation of Intermediate I-91

At room temperature, intermediate I-9 (80.0 mg, 0.176 mmol) was dissolved in anhydrous 1,2-dichloroethane (50.0 mL). 2-Chloro-4,5-difluorobenzoic acid (67.8 mg, 0.352 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (101 mg, 0.528 mmol), and 4-dimethylaminopyridine (64.5 mg, 0.528 mmol) were added sequentially. The reaction mixture was stirred at 60°C under argon for 24 hours. The reaction mixture was cooled to room temperature and concentrated under reduced pressure. The residue was purified by silica gel chromatography to obtain intermediate I-91.

LC-MS (ESI) [M+H] ⁺ 628.9.

Reference Example 92: Preparation of Intermediate I-92

Methyl 5-bromoaminobenzoate (15.00 g, 65.20 mmol) was dissolved in tetrahydrofuran (300 mL) at room temperature. Pyridine (10.31 g, 130.40 mmol) and ethyl succinate chloride (12.88 g, 78.24 mmol) were added sequentially and stirred at room temperature for 1 hour. Water (300 mL) was added and the mixture was extracted with ethyl acetate (300 mL x 3). The organic phases were combined, washed with saturated brine (300 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was separated and purified by silica gel chromatography to obtain Intermediate I-92.

LC-MS (ESI) [M+H] ⁺ 359.8 (isotope peak).

Reference Example 93: Preparation of Intermediate I-93

Intermediate I-92 (23.00 g, 64.21 mmol) was dissolved in tetrahydrofuran (300 mL) at room temperature, and potassium tert-butoxide (15.85 g, 141.27 mmol) was added. The mixture was stirred at room temperature for 2 hours. Water (300 mL) was added, and the pH was adjusted to 5-6 with 2N hydrochloric acid. A large amount of solid precipitated, which was filtered and the filter cake was washed with water (100 mL). The filter cake was collected and dried to obtain Intermediate I-93.

LC-MS (ESI) [M+H] ⁺ 325.80.

Reference Example 94: Preparation of Intermediate I-94

At room temperature, intermediate I-93 (16.00 g, 49.06 mmol) was dissolved in dimethyl sulfoxide (160 mL). Water (16 mL) was added, and the mixture was heated to 150°C and stirred for 5 hours. The reaction solution was cooled to room temperature, water (200 mL) was added, and extracted with ethyl acetate (200 mL x 3). The organic phases were combined, washed with saturated brine (200 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to dryness to obtain intermediate I-94.

LC-MS (ESI) [M+H] ⁺ 253.8.

Reference Example 95: Preparation of Intermediate I-95

At room temperature, intermediate I-94 (11.00 g, 43.29 mmol) was dissolved in tetrahydrofuran (100 mL), sodium borohydride (1.64 g, 43.29 mmol) was added, and the mixture was stirred at room temperature for 2 hours. Water (100 mL) was added, and the mixture was extracted with ethyl acetate (100 mL x 3). The organic phases were combined, washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to dryness to obtain intermediate I-95.

LC-MS (ESI) [M+H] ⁺ 257.8 (isotope peak).

Reference Example 96: Preparation of Intermediate I-96

At room temperature, intermediate I-95 (10.00 g, 39.05 mmol) was dissolved in N,N-dimethylformamide (100 mL). Imidazole (5.32 g, 78.09 mmol) and tert-butyldimethylsilyl chloride (11.77 g, 78.09 mmol) were added sequentially, and the mixture was stirred at 80°C for 3 hours. The reaction mixture was cooled to room temperature, water (100 mL) was added, and the mixture was extracted with ethyl acetate (100 mL x 3). The organic phases were combined, washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to dryness to obtain the crude product, which was then purified by slurrying with petroleum ether (100 mL) to obtain intermediate I-96.

LC-MS (ESI) [M+H] ⁺ 371.8 (isotope peak).

Reference Example 97: Preparation of Intermediate I-97

At room temperature, intermediate I-96 (2.00 g, 5.40 mmol) was dissolved in toluene (30 mL), and Lawesson's reagent (2.62 g, 6.48 mmol) was added. The reaction mixture was stirred at 90°C for 2 hours. The reaction mixture was cooled to room temperature, water (50 mL) was added, and the mixture was extracted with ethyl acetate (50 mL x 3). The organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was separated and purified by silica gel chromatography to obtain intermediate I-97.

LC-MS (ESI) [M+H] ⁺ 385.8.

Reference Example 98: Preparation of Intermediate I-98

At room temperature, intermediate I-97 (1.10 g, 2.85 mmol) was dissolved in n-butanol (10 mL), and intermediate I-7 (666.72 mg, 3.42 mmol) was added. The reaction mixture was stirred at 130°C for 16 hours. The reaction mixture was concentrated to dryness under reduced pressure, and the residue was purified by silica gel chromatography to obtain intermediate I-98.

LC-MS (ESI) [M+H] ⁺ 531.0 (isotope peak).

Reference Example 99: Preparation of Intermediate I-99

At room temperature, intermediate I-98 (200.00 mg, 0.38 mmol) was dissolved in N,N-dimethylformamide (100 mL). Zinc cyanide (133.86 mg, 1.14 mmol) and tetrakis(triphenylphosphine)palladium (87.82 mg, 0.076 mmol) were added sequentially. The nitrogen atmosphere was replaced three times, and the reaction solution was stirred at 150°C for 3 hours. The reaction solution was cooled to room temperature, water (20 mL) was added, and the mixture was extracted with ethyl acetate (20 mL x 3). The organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to remove the organic solvent to obtain the crude product. The crude product was separated and purified by silica gel chromatography to obtain intermediate I-99.

LC-MS (ESI) [M+H] ⁺ 476.0.

Reference Example 100: Preparation of Intermediate I-100

At room temperature, intermediate I-99 (150.00 mg, 0.32 mmol) was dissolved in tetrahydrofuran (5 mL). Glacial acetic acid (5 mL) and reduced iron powder (89.36 mg, 1.60 mmol) were added. The reaction mixture was stirred at 50°C for 3 hours. Filtered, the filter cake was washed with ethyl acetate (20 mL), and the filtrate was collected and washed sequentially with water (10 mL) and saturated brine (10 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to dryness to obtain intermediate I-100.

LC-MS (ESI) [M+H] ⁺ 446.0.

Reference Example 101: Preparation of Intermediate I-101

At room temperature, intermediate I-100 (120.00 mg, 0.27 mmol) was dissolved in dichloromethane (5 mL). Triethylamine (54.64 mg, 0.54 mmol) and o-chlorobenzoyl chloride (94.25 mg, 0.54 mmol) were added sequentially. The reaction mixture was stirred at room temperature for 1 hour. Water (10 mL) was added, and the mixture was extracted with dichloromethane (10 mL x 3). The organic phases were combined, washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to remove the organic solvent to obtain the crude product. The crude product was separated and purified by silica gel chromatography to obtain intermediate I-101.

LC-MS (ESI) [M+H] ⁺ 584.0.

Reference Example 102: Preparation of Intermediate I-102

Intermediate I-96 (0.94 g, 2.54 mmol) was dissolved in a mixture of dioxane and water (4:1, 14 mL) at room temperature. Potassium methyltrifluoroborate (1.76 g, 14.7 mmol) and potassium carbonate (1.05 g, 7.62 mmol) were added sequentially. The atmosphere was replaced with nitrogen three times and stirred at 85°C under a nitrogen balloon for 16 hours. The reaction mixture was cooled to room temperature and concentrated under reduced pressure. Ethyl acetate (100 mL) was added, the mixture was washed with water (30 mL x 3), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to remove the organic solvent to obtain the crude product. The crude product was purified by silica gel chromatography to obtain Intermediate I-102.

LC-MS (ESI) [2M+H] ⁺ 611.4.

Reference Example 103: Preparation of Intermediate I-103

At room temperature, intermediate I-102 (0.57 g, 1.87 mmol) was dissolved in toluene (10 mL). Lawesson's reagent (1.51 g, 3.74 mmol) was added. The atmosphere was replaced with argon three times, and the mixture was stirred at 90°C under an argon atmosphere (balloon) for 2 hours. The reaction solution was cooled to room temperature and concentrated under reduced pressure. The residue was purified by silica gel chromatography to obtain intermediate I-103.

LC-MS (ESI) [M+H] ⁺ 322.2.

Reference Example 104: Preparation of Intermediate I-104

At room temperature, intermediate I-103 (0.35 g, 1.10 mmol) was dissolved in n-butanol (5 mL), and intermediate I-7 (0.33 g, 1.65 mmol) was added. The mixture was stirred overnight at 130°C in a microwave reaction tube. The reaction solution was cooled to room temperature and concentrated under reduced pressure. The residue was separated and purified by silica gel chromatography to obtain intermediate I-104.

LC-MS (ESI) [M+H] ⁺ 465.2.

Reference Example 105: Preparation of Intermediate I-105

At room temperature, intermediate I-104 (240.00 mg, 0.52 mmol) was dissolved in methanol (3 mL). Palladium on carbon (10% wt, 24.00 mg) was added. The atmosphere was replaced with hydrogen three times, and the mixture was stirred under a hydrogen atmosphere (balloon) at room temperature for 16 hours. The reaction mixture was filtered through a pad of Celite, and the filtrate was concentrated under reduced pressure to obtain intermediate I-105.

LC-MS (ESI) [M+H] ⁺ 435.3.

Reference Example 106: Preparation of Intermediate I-106

At room temperature, intermediate I-105 (100.00 mg, 0.23 mmol) was dissolved in dichloromethane (2 mL). Triethylamine (70.00 mg, 0.69 mmol) and o-chlorobenzoyl chloride (80.00 mg, 0.46 mmol) were added and stirred at room temperature for 2 hours. The mixture was diluted with dichloromethane (20 mL), washed with water (10 mL x 2), dried over sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain crude intermediate I-106. The crude product was used directly in the next reaction without purification.

LC-MS (ESI) [M+H] ⁺ 573.2.

Preparation of Example:

Example 1: Preparation of Compound 1

A solution of tetrabutylammonium fluoride in tetrahydrofuran (0.323 mL, 1 M) was added to a solution of intermediate I-11 (41.0 mg, 0.0645 mmol) in tetrahydrofuran (5 mL) at room temperature. The reaction mixture was stirred at room temperature for 2 hours. The organic solvent was removed by concentration under reduced pressure, and the product was separated and purified by silica gel chromatography and then by preparative HPLC to obtain compound 1.

LC-MS (ESI) [M+H] ⁺ 521.2.

¹ H NMR (400MHz, CD ₃ OD)δ7.74(s,1H),7.65–7.54(m,2H),7.53–7.43(m,4H),7.41–7.27(m,5H),7.26–7.16(m,2H),6.69(d, J＝8.5Hz,1H),4.78–4.65(m,1H),3.17–2.97(m,1H),2.74–2.54(m,2H),2.22–2.07(m,1H),2.01(s,3H).

Example 2: Preparation of Compound 2

Intermediate I-14 (60.0 mg, 0.0918 mmol) was dissolved in tetrahydrofuran (5.00 mL) at room temperature, and a solution of tetrabutylammonium fluoride in tetrahydrofuran (1.38 mL, 0.1 M) was added dropwise. The reaction mixture was stirred at room temperature for 2 hours. Methanol (0.1 mL) and water (30 mL) were added to the reaction system, and the mixture was extracted with ethyl acetate (15 mL x 3). The organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was separated and purified by preparative HPLC to obtain compound 2.

LC-MS (ESI) [M+H] + 539.0.

¹ H NMR (400MHz, CD ₃ OD)δ7.86–7.64(m,2H),7.60(td,J＝7.6,1.4Hz,1H),7.53(td,J＝7.5,1.3Hz,1H),7.49–7.29(m,5H),7.22(ddd,J＝11.8,7.3,5.0H z,3H),7.09(dd,J＝14.0,4.6Hz,1H),6.70(d,J＝8.5Hz,1H),4.73(s,1H),3.24–2.93(m,1H),2.86–2.46(m,2H),2.28–1.81(m,4H).

Example 3: Preparation of Compound 3

Intermediate I-17 (40.0 mg, 0.0596 mmol) was dissolved in tetrahydrofuran (0.5 mL) at room temperature. A solution of tetrabutylammonium fluoride in tetrahydrofuran (0.1 mL, 1 M) was added, and the reaction mixture was stirred at room temperature for 1 hour. The solvent was removed by concentration under reduced pressure, and the residue was separated and purified by silica gel chromatography and preparative HPLC (ammonia system) to obtain compound 3.

LC-MS (ESI) [M+H] ⁺ 557.0.

¹ H NMR (400MHz, CD ₃ OD)δ7.74(s,1H),7.52–7.31(m,7H),7.27–7.17(m,3H),7.13–7.06(m,1H),6.71(d,J＝8.5Hz, 1H),4.79–4.68(m,1H),3.16–3.01(m,1H),2.73–2.56(m,2H),2.22–2.09(m,1H),1.99(s,3H).

Example 4: Preparation of Compound 4

At room temperature, intermediate I-21 (62 mg, 0.094 mmol) was dissolved in tetrahydrofuran (5 mL). A solution of tetrabutylammonium fluoride in tetrahydrofuran (0.28 mL, 1 M, 0.28 mmol) was added, and the reaction mixture was stirred at room temperature for 0.5 hours. The mixture was diluted with water (10 mL) and extracted with ethyl acetate (30 mL x 2). The combined organic phases were washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was separated and purified by preparative HPLC (formic acid system) to obtain compound 4.

LCMS (ESI) [M+H] ⁺ 539.2.

¹ H NMR (400MHz, DMSO-d ₆ )δ10.40(s,1H),7.69-7.63(m,2H),7.51-7.29(m,10H),7.16-7.15(d,J＝6.4Hz,1H),6.67-6.65(d,J＝ 8.8Hz,1H),5.86-5.84(m,1H),4.59-4.59(m,1H),3.03-2.98(m,1H),2.62-2.52(m,2H),1.91(m,4H).

Example 5: Preparation of Compound 5

A solution of tetrabutylammonium fluoride in tetrahydrofuran (0.0917 mL, 1 M) was added to a solution of intermediate I-24 (60 mg, 0.0917 mmol) in tetrahydrofuran (3 mL) at room temperature. The reaction mixture was stirred at room temperature for 1 hour. The organic solvent was removed by concentration under reduced pressure, and the residue was purified by silica gel chromatography and then by preparative HPLC to obtain compound 5.

LC-MS (ESI) [M+H] ⁺ 540.2.

¹ H NMR (400MHz, CD ₃ OD)δ8.54–8.47(m,1H),7.89–7.81(m,1H),7.80–7.68(m,2H),7.65(d,J＝7.9Hz,1H),7.50–7.41(m,3H),7.40–7.35(m,1H),7.34–7 .29(m,1H),7.28–7.19(m,2H),6.72(d,J=8.5Hz,1H),4.79–4.64(m,1H),3.18–2.92(m,1H),2.78–2.41(m,2H),2.32–1.82(m,4H).

Example 6: Preparation of Compound 6

At room temperature, intermediate I-28 (0.10 g) was dissolved in tetrahydrofuran (5 mL), and a solution of tetrabutylammonium fluoride in tetrahydrofuran (0.45 mL, 1 M, 0.45 mmol) was added. The reaction mixture was stirred at room temperature for 0.5 hours. The mixture was diluted with water (10 mL) and extracted with ethyl acetate (30 mL x 2). The organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was separated and purified by preparative HPLC (formic acid system) to obtain compound 6.

LCMS (ESI) [M+H] ⁺ 555.2.

¹ H NMR (400MHz, DMSO-d ₆ )δ10.43(s,1H),7.73–7.52(m,5H),7.49–7.43(m,1H),7.39(s,1H),7.37–7.28(m,5H),7.16(s,1 H),6.68(d,J＝8.5Hz,1H),5.86(s,1H),4.60(s,1H),3.03(s,1H),2.50(m,5H),2.12–1.93(m,1H).

Example 7: Preparation of Compound 7

Intermediate I-31 (41.0 mg, 0.063 mmol) was dissolved in tetrahydrofuran (0.5 mL). A solution of tetrabutylammonium fluoride in tetrahydrofuran (0.1 mL, 1 M) was added at room temperature. The reaction mixture was stirred at room temperature for 0.5 hours. The organic solvent was removed by concentration under reduced pressure. The residue was purified by silica gel chromatography and then by preparative HPLC (formic acid system) to obtain compound 7.

LC-MS (ESI) [M+H] ⁺ 539.1.

¹ H NMR (400MHz, DMSO-d ₆ )δ10.46(s,1H),7.67(s,1H),7.54–7.35(m,9H),7.34–7.28(m,2H),7.16(d,J＝7.3Hz,1H),6.67(d,J＝8 .5Hz,1H),5.85(s,1H),4.60(s,1H),3.18(m,1H),3.02(s,1H),2.51(m,1H),2.00(m,1H),1.93(s,3H).

Example 8: Preparation of Compound 8

A solution of tetrabutylammonium fluoride in tetrahydrofuran (0.338 mL, 1 M, 0.338 mmol) was added to a solution of intermediate I-32 (43.0 mg, 0.0676 mmol) in tetrahydrofuran (3.00 mL) at room temperature. The reaction mixture was stirred at room temperature for 1 hour. The organic solvent was removed by concentration under reduced pressure, and the residue was purified by silica gel chromatography and then by preparative HPLC (ammonia system) to obtain compound 8.

LC-MS (ESI) [M+H] ⁺ 522.1.

¹ H NMR (400MHz, CD ₃ OD)δ8.55–8.49(m,1H),7.85(td,J＝7.8,1.7Hz,1H),7.78–7.54(m,6H),7.47(d,J＝9.4Hz,2H),7.35–7.30(m,1H),7.24(dd,J ＝8.5,2.4Hz,2H),6.72(d,J＝8.5Hz,1H),4.74(s,1H),3.18–3.03(m,1H),2.75–2.55(m,2H),2.21–2.09(m,1H),2.01(s,3H).

Example 9: Preparation of Compound 9

A solution of tetrabutylammonium fluoride in tetrahydrofuran (0.195 mL, 1 M) was added to a solution of intermediate I-36 (25.0 mg, 0.0389 mmol) in tetrahydrofuran (2 mL) at room temperature. The reaction mixture was stirred at room temperature for 1 hour. The organic solvent was removed by concentration under reduced pressure, and the residue was purified by silica gel chromatography and then by preparative HPLC to afford compound 9.

LC-MS (ESI) [M+H] ⁺ 528.1.

¹ H NMR (400MHz, CD ₃ OD)δ7.88–7.80(m,2H),7.76(s,1H),7.66–7.51(m,6H),7.33–7.22(m,2H),6 .75–6.73(m,1H),4.76(m,1H),3.13(m,1H),2.66(m,2H),2.24–1.91(m,4H).

Example 10: Preparation of Compound 10

A solution of tetrabutylammonium fluoride in tetrahydrofuran (0.245 mL, 1 M) was added to a solution of intermediate I-37 (29.0 mg, 0.0489 mmol) in tetrahydrofuran (2.00 mL) at room temperature. The reaction mixture was stirred at room temperature for 1 hour. The organic solvent was removed by concentration under reduced pressure, and the residue was purified by silica gel chromatography and then by preparative HPLC (formic acid system) to obtain compound 10.

LC-MS (ESI) [M+H] ⁺ 479.1.

¹ H NMR (400MHz, CD ₃ OD)δ7.72(s,1H),7.68–7.60(m,2H),7.59–7.41(m,4H),7.33(d,J＝8.0Hz,1H),7.27–7.24 (m,1H),6.75(d,J＝8.5Hz,1H),4.73(m,1H),3.13(s,1H),2.66(m,2H),2.28–1.87(m,4H).

Example 11: Preparation of Compound 11

Intermediate I-40 (120 mg, 0.19 mmol) was dissolved in tetrahydrofuran (5 mL) at room temperature, and a solution of tetrabutylammonium fluoride in tetrahydrofuran (0.98 mL, 1 M) was added. The reaction mixture was stirred at room temperature for 1 hour. The mixture was diluted with water (10 mL) and extracted with ethyl acetate (20 mL x 3). The combined organic phases were washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to remove the organic solvent, and the residue was purified by preparative HPLC (formic acid system) to obtain compound 11.

LCMS (ESI) [M+H] ⁺ 499.0.

¹ H NMR (400MHz, DMSO-d ₆ )δ10.92(s,1H),7.94(s,1H),7.77-7.46(m,7H),7.36-7.33(dd,J＝10.8,2.4Hz,1H),6.76-6.74 (d,J＝8.4Hz,1H),5.84(s,1H),4.57(m,1H),2.67(m,1H),2.56-2.52(m,2H),1.98-1.94(m,1H).

Example 12: Preparation of Compound 12

Intermediate I-43 (120 mg, 0.20 mmol) was dissolved in tetrahydrofuran (5 mL) at room temperature. A solution of tetrabutylammonium fluoride in tetrahydrofuran (1.00 mL, 1 M) was added, and the reaction mixture was stirred at room temperature for 1 hour. The mixture was diluted with water (10 mL) and extracted with ethyl acetate (20 mL x 3). The combined organic phases were washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was isolated and purified by preparative HPLC (formic acid system) to obtain compound 12.

LCMS (ESI) [M+H] ⁺ 483.0.

¹ H NMR (400MHz, DMSO-d ₆ )δ10.95(s,1H),7.70-7.46(m,8H),7.38-7.35(dd,J＝10.8,2.4Hz,1H),6.87-6.85(d,J＝ 8.0Hz,1H),5.90(s,1H),4.56(m,1H),3.05(m,1H),2.50-2.50(m,2H),1.97-1.95(m,1H).

Example 13: Preparation of Compound 13

Intermediate I-48 (83 mg, 0.13 mmol) was dissolved in tetrahydrofuran (5 mL) at room temperature. A solution of tetrabutylammonium fluoride in tetrahydrofuran (0.27 mL, 1 M) was added, and the reaction mixture was stirred at room temperature for 1 hour. The mixture was diluted with water (10 mL) and extracted with ethyl acetate (20 mL x 3). The combined organic phases were washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was isolated and purified by preparative HPLC (formic acid system) to obtain compound 13.

LCMS (ESI) [M+H] ⁺ 507.4.

¹ H NMR (400MHz, DMSO-d ₆ )δ10.65(s,1H),7.77(s,1H),7.67-7.50(m,4H),7.48-7.44(m,2H),7.36-7.33(dd,J＝10.8,2.4Hz,1H),7.23(s,1H),6.68-6.66(d,J＝ 8.4Hz,1H),5.74(s,1H),4.57(m,1H),3.02(m,1H),2.56-2.51(m,2H),2.49-2.49(m,1H),1.97(m,1H),1.02-1.01(m,3H),0.77(m,3H).

Example 14: Preparation of Compound 14

A solution of tetrabutylammonium fluoride in tetrahydrofuran (2.10 mL, 1 M, 2.10 mmol) was added to a solution of intermediate I-51 (250 mg, 0.420 mmol) in tetrahydrofuran (5.00 mL) at room temperature. The reaction mixture was stirred at room temperature for 1 hour. The organic solvent was removed by concentration under reduced pressure, and the residue was purified by silica gel chromatography and then by preparative HPLC (formic acid system) to obtain compound 14.

LC-MS (ESI) [M+H] ⁺ 480.0.

¹ H NMR (400MHz, CD ₃ OD)δ8.22(s,2H),7.78(s,1H),7.59(dd,J＝7.4,1.4Hz,1H),7.54–7.47(m,2H),7.43(td,J＝7.3,1.7Hz,1H),7.30(dd,J＝8 .5,2.4Hz,1H),6.82(d,J＝8.5Hz,1H),4.79(s,1H),3.23–3.02(m,1H),2.78–2.54(m,2H),2.38–2.19(m,1H),2.14(s,3H).

Example 15: Preparation of Compound 15

Intermediate I-55 (56 mg, 0.090 mmol) was dissolved in tetrahydrofuran (5 mL) at room temperature. A solution of tetrabutylammonium fluoride in tetrahydrofuran (0.27 mL, 1 M, 0.27 mmol) was added, and the reaction mixture was stirred at room temperature for 1 hour. The mixture was diluted with water (10 mL) and extracted with ethyl acetate (20 mL x 3). The organic phases were combined, washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was separated and purified by preparative HPLC (formic acid system) to obtain compound 15.

LCMS (ESI) [M+H] ⁺ 505.4.

¹ H NMR (400MHz, DMSO-d ₆ )δ10.61(s,1H),7.68-7.65(m,2H),7.60-7.56(m,2H),7.54-7.44(m,2H),7.35-7.33(m,2H),7.26-7.26(m,1H),6.71-6.69(d,J＝8.4Hz,1 H),4.71-4.41(m,2H),3.09-3.05(m,1H),2.56-2.56(m,2H),2.49-2.45(m,1H),1.98-1.97(m,1H),1.39-1.39(m,1H),0.70-0.53(m,3H).

Example 16: Preparation of Compound 16

Intermediate I-59 (45 mg, 0.074 mmol) was dissolved in tetrahydrofuran (5 mL) at room temperature. A solution of tetrabutylammonium fluoride in tetrahydrofuran (0.22 mL, 1 M, 0.22 mmol) was added, and the reaction was stirred at room temperature for 1 hour. The mixture was diluted with water (10 mL) and extracted with ethyl acetate (20 mL x 3). The combined organic phases were washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was separated and purified by preparative HPLC (formic acid system) to obtain compound 16.

LCMS (ESI) [M+H] ⁺ 493.2.

¹ H NMR (400MHz, DMSO-d ₆ )δ10.66(s,1H),7.74(s,1H),7.68(s,1H),7.60-7.46(m,5H),7.34-7.32(dd,J＝10.4,2.0Hz,1H),7.32(s,1H),6.69-6.67(d,J＝8.4 Hz,1H),5.87(s,1H),4.59(m,1H),3.05(m,1H),2.50-2.50(m,1H),2.49-2.41(m,2H),2.24(m,1H),1.97(m,1H),0.95-0.92(m,3H).

Example 17: Preparation of Compound 17

Intermediate I-63 (94 mg, 0.15 mmol) was dissolved in tetrahydrofuran (10 mL) at room temperature. A solution of tetrabutylammonium fluoride in tetrahydrofuran (0.46 mL, 1 M, 0.46 mmol) was added, and the reaction mixture was stirred at room temperature for 1 hour. The mixture was diluted with water (10 mL) and extracted with ethyl acetate (20 mL x 2). The organic phases were combined, washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was separated and purified by preparative HPLC (formic acid system) to obtain compound 17.

LCMS (ESI) [M+H] ⁺ 495.4.

¹ H NMR (400MHz, DMSO-d ₆ )δ10.72(s,1H),7.71-7.70(d,J＝2.0Hz,1H),7.61-7.30(m,7H),7.30-7.30(d,J＝2.4Hz,1H),6.75-6.73(d,J =8.4Hz,1H),4.54(m,1H),3.19(s,3H),3.00(m,1H),2.54-2.51(m,2H),2.49-2.32(m,1H),1.96-1.94(m,1H).

Example 18: Preparation of Compound 18

A solution of tetrabutylammonium fluoride in tetrahydrofuran (0.44 mL, 1 M) was added to a solution of intermediate I-64 (50 mg) in tetrahydrofuran (10 mL) at room temperature. The reaction mixture was stirred at room temperature for 2 hours. The organic solvent was removed by concentration under reduced pressure, and the residue was purified by silica gel chromatography and then by preparative HPLC to obtain compound 18.

LC-MS (ESI) [M+H] ⁺ 459.1.

¹ H NMR (400MHz, CD ₃ OD)δ8.78–8.34(m,1H),7.76(s,1H),7.70–7.61(m,2H),7.47(d,J＝7.0Hz,1H),7.41–7.36(m,1H),7.28(dd,J＝19.0,8 .8Hz,4H),6.75(d,J＝8.5Hz,1H),4.79–4.69(m,1H),3.08(s,1H),2.67(s,2H),2.45(s,3H),2.15(s,1H),2.06(s,3H).

Example 19: Preparation of Compound 19

At room temperature, intermediate I-69 (61 mg, 0.096 mmol) was dissolved in tetrahydrofuran (5 mL). A solution of tetrabutylammonium fluoride in tetrahydrofuran (0.29 mL, 1 M, 0.29 mmol) was added, and the reaction mixture was stirred at room temperature for 10 minutes. The mixture was diluted with water (10 mL) and extracted with ethyl acetate (20 mL x 3). The organic phases were combined, washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was separated and purified by preparative HPLC (formic acid system) to obtain compound 19.

LCMS (ESI) [M+H] ⁺ 515.4.

¹ H NMR (400 MHz, DMSO-d ₆ )δ10.93(s,1H),8.29-8.29(d,J＝1.2Hz,1H),8.27(s,1H),7.77-7.75(d,J＝8. 4Hz,1H),7.69-7.58(m,2H),7.54-7.46(m,2H),7.37-7.35(dd,J＝10.8,2.4Hz ,1H),7.28-7.02(m,2H),6.75-6.73(d,J＝8.4Hz,1H),5.84(s,1H),4.73(m,1H ),3.07-3.01(m,1H),2.56-2.52(m,1H),2.49-2.44(m,1H),1.99-1.96(m,1H).

Example 20: Preparation of Compound 20

A solution of tetrabutylammonium fluoride in tetrahydrofuran (0.303 mL, 1 M, 0.303 mmol) was added to a solution of intermediate I-70 (35.0 mg, 0.0606 mmol) in tetrahydrofuran (2.00 mL) at room temperature. The reaction mixture was stirred at room temperature for 1 hour. The organic solvent was removed by concentration under reduced pressure, and the residue was purified by silica gel chromatography and then by preparative HPLC (formic acid system) to obtain compound 20.

LC-MS (ESI) [M+H] ⁺ 463.1.

¹ H NMR (400MHz, CD ₃ OD)δ7.88–7.61(m,4H),7.61–7.53(m,1H),7.38–7.20(m,4H),6.75(d,J =8.5Hz,1H),4.76(s,1H),3.13(m,1H),2.66(m,2H),2.30–1.85(m,4H).

Example 21: Preparation of Compound 21

A solution of tetrabutylammonium fluoride in tetrahydrofuran (0.560 mL, 1 M, 0.560 mmol) was added to a solution of intermediate I-71 (70.0 mg, 0.112 mmol) in tetrahydrofuran (2.00 mL) at room temperature. The reaction mixture was stirred at room temperature for 1 hour. The organic solvent was removed by concentration under reduced pressure, and the residue was purified by silica gel chromatography and then by preparative HPLC (formic acid system) to obtain compound 21.

LC-MS (ESI) [M+H] ⁺ 513.1.

¹ H NMR (400MHz, CD ₃ OD)δ7.85–7.72(m,3H),7.72–7.58(m,4H),7.33(d,J＝8.0Hz,1H),7.26(dd,J＝8.5,2.4Hz,1H),6.76( d,J＝8.5Hz,1H),4.77(s,1H),3.18–3.03(m,1H),2.75–2.55(m,2H),2.26–2.13(m,1H),2.06(s,3H).

Example 22: Preparation of Compound 22

Intermediate I-75 (50.0 mg, 0.0821 mmol) was dissolved in tetrahydrofuran (2.00 mL) at room temperature. A solution of tetrabutylammonium fluoride in tetrahydrofuran (0.246 mL, 1.0 M) was added, and the reaction system was stirred at room temperature for 3 hours. Methanol (0.2 mL) was added to the reaction system, which was then poured into water (15 mL) and extracted with ethyl acetate (10 mL x 3). The organic phases were combined, washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was separated and purified by preparative HPLC to obtain compound 22.

LC-MS (ESI) [M+H] ⁺ 495.0.

¹ H NMR (400MHz, CD ₃ OD)δ7.80–7.57(m,7H),7.37–7.07(m,3H),6.75(d,J＝8.5Hz,1H),4.76(m,1H),3.21–2.97(m,1H),2.81–2.45(m,2H),2.27–1.86(m,4H).

Example 23: Preparation of Compound 23

Intermediate I-77 (80.0 mg, 0.136 mmol) was dissolved in tetrahydrofuran (2.00 mL) at room temperature. A solution of tetrabutylammonium fluoride in tetrahydrofuran (0.408 mL, 1.0 M) was added to the reaction system, and the reaction mixture was stirred at room temperature for 3 hours. The reaction system was poured into water (20 mL) and extracted with ethyl acetate (10 mL x 4). The organic phases were combined, washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was separated and purified by preparative HPLC to obtain compound 23.

LC-MS (ESI) [M+H] ⁺ 473.0.

¹ H NMR (400MHz, CD ₃ OD)δ7.86–7.68(m,1H),7.68–7.56(m,2H),7.45–7.37(m,2H),7.35–7.22(m,4H),6.75(d,J＝8.5Hz,1H),4.80–4. 69(m,1H),3.19–2.93(m,1H),2.81(q,J＝7.6Hz,2H),2.75–2.46(m,2H),2.27–1.93(m,4H),1.24(t,J＝7.6Hz,3H).

Example 24: Preparation of Compound 24

Intermediate I-78 (50 mg, 0.080 mmol) was dissolved in tetrahydrofuran (2 mL) at room temperature, and a solution of tetrabutylammonium fluoride in tetrahydrofuran (0.2 mL, 1 M) was added. The reaction mixture was stirred at room temperature for 30 minutes. The organic solvent was removed by concentration under reduced pressure, and the residue was purified by silica gel chromatography and then by preparative HPLC to obtain compound 24.

LC-MS (ESI) [M+H] ⁺ 514.0.

¹ H NMR (400MHz, CD ₃ OD)δ8.81(d,J＝4.0Hz,1H),8.12(d,J＝7.0Hz,1H),7.77(dd,J＝7.8,4.8Hz,2H),7.61(d,J＝9.1Hz,2H),7.34(d,J＝7 .9Hz,1H),7.25(dd,J＝8.5,2.4Hz,1H),6.75(d,J＝8.5Hz,1H),4.76(s,1H),3.13(m,1H),2.66(m,2H),2.06(m,4H).

Example 25: Preparation of Compound 25

A solution of tetrabutylammonium fluoride in tetrahydrofuran (0.494 mL, 1 M, 0.494 mmol) was added to a solution of intermediate I-79 (60.0 mg, 0.0987 mmol) in tetrahydrofuran (5.00 mL) at room temperature. The reaction mixture was stirred at room temperature for 1 hour. The organic solvent was removed by concentration under reduced pressure, and the residue was isolated and purified by preparative HPLC (formic acid system) to obtain compound 25.

LC-MS (ESI) [M+H] ⁺ 492.9.

¹ H NMR (400MHz, CD ₃ OD)δ7.72(s,1H),7.69–7.60(m,2H),7.51(d,J＝8.0Hz,1H),7.40(d,J＝6.8Hz,1H),7.36–7.22(m,3H),6.75( d,J＝8.5Hz,1H),4.76(s,1H),3.13(m,1H),2.83–2.51(m,2H),2.46(s,3H),2.23–2.11(m,1H),2.06(s,3H).

Example 26: Preparation of Compound 26

A solution of tetrabutylammonium fluoride in tetrahydrofuran (0.262 mL, 1 M, 0.262 mmol) was added to a solution of intermediate I-80 (30.0 mg, 0.0523 mmol) in tetrahydrofuran (2.00 mL) at room temperature. The reaction mixture was stirred at room temperature for 1 hour. The organic solvent was removed by concentration under reduced pressure, and the residue was purified by silica gel chromatography and then by preparative HPLC (formic acid system) to obtain compound 26.

LC-MS (ESI) [M+H] ⁺ 459.1.

¹ H NMR (400MHz, CD ₃ OD)δ7.84–7.62(m,5H),7.46–7.37(m,2H),7.33(d,J＝8.1Hz,1H),7.24(dd,J＝8.5,2.4Hz,1H),6.75(d,J＝8 .5Hz,1H),4.77(s,1H),3.22–2.95(m,1H),2.86–2.52(m,2H),2.44(s,3H),2.24–2.12(m,1H),2.07(s,3H).

Example 27: Preparation of Compound 27

A solution of tetrabutylammonium fluoride in tetrahydrofuran (0.520 mL, 1 M, 0.520 mmol) was added to a solution of intermediate I-81 (60.0 mg, 0.104 mmol) in tetrahydrofuran (2.00 mL) at room temperature. The reaction mixture was stirred at room temperature for 1 hour. The organic solvent was removed by concentration under reduced pressure, and the residue was purified by silica gel chromatography and then by preparative HPLC (formic acid system) to obtain compound 27.

LC-MS (ESI) [M+H] ⁺ 463.1.

¹ H NMR (400MHz, CD ₃ OD)δ7.89–7.62(m,5H),7.58–7.51(m,1H),7.40–7.29(m,2H),7.23(dd,J＝8.5,2.4Hz,1H),6.75(d ,J＝8.5Hz,1H),4.77(s,1H),3.20–2.99(m,1H),2.76–2.51(m,2H),2.27–2.13(m,1H),2.07(s,3H).

Example 28: Preparation of Compound 28

A solution of tetrabutylammonium fluoride in tetrahydrofuran (0.590 mL, 1 M, 0.590 mmol) was added to a solution of intermediate I-82 (70.0 mg, 0.118 mmol) in tetrahydrofuran (5.00 mL) at room temperature. The reaction mixture was stirred at room temperature for 1 hour. The organic solvent was removed by concentration under reduced pressure, and the residue was purified by silica gel chromatography and then by preparative HPLC (formic acid system) to obtain compound 28.

LC-MS (ESI) [M+H] ⁺ 477.1.

¹ H NMR (400MHz, CD ₃ OD)δ7.76(s,1H),7.70–7.59(m,2H),7.38–7.28(m,2H),7.28–7.19(m,2H),7.13(td,J＝8.5,2.7Hz,1H),6.75(d ,J＝8.5Hz,1H),4.76(s,1H),3.19–3.00(m,1H),2.78–2.54(m,2H),2.41(s,3H),2.26–2.12(m,1H),2.06(s,3H).

Example 29: Preparation of Compound 29

Intermediate I-84 (60.0 mg, 0.0981 mmol) was dissolved in tetrahydrofuran (3.00 mL) at room temperature. A solution of tetrabutylammonium fluoride in tetrahydrofuran (0.294 mL, 1.0 M) was added to the reaction system, and the reaction mixture was stirred at room temperature for 2 hours. The reaction system was poured into water (20 mL) and extracted with ethyl acetate (10 mL x 4). The organic phases were combined, washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain the crude product, which was then separated and purified by preparative HPLC to obtain compound 29.

LC-MS (ESI) [M+H] ⁺ 496.9.

¹ H NMR (400MHz, CD ₃ OD)δ7.86–7.57(m,3H),7.51–7.31(m,4H),7.25(dd,J＝8.4,2.4Hz,1H),6.75(d,J＝8. 5Hz,1H),4.77–4.67(m,1H),3.19–3.01(m,1H),2.81–2.53(m,2H),2.33–1.96(m,4H).

Example 30: Preparation of Compound 30

A solution of tetrabutylammonium fluoride in tetrahydrofuran (0.127 mL, 1 M) was added to a solution of intermediate I-85 (25 mg, 0.0423 mmol) in tetrahydrofuran (5 mL) at room temperature. The reaction mixture was stirred at room temperature for 1 hour. The organic solvent was removed by concentration under reduced pressure, and the residue was purified by silica gel chromatography and then by preparative HPLC to obtain compound 30.

LC-MS (ESI) [M+H] ⁺ 477.0.

¹ H NMR (400MHz, CD ₃ OD)δ7.76(s,1H),7.69–7.61(m,2H),7.36–7.28(m,3H),7.25(dd,J=8.5,2.4Hz,1H),7.22–7.17(m, 1H),6.75(d,J＝8.5Hz,1H),4.77(s,1H),3.13(m,1H),2.67(m,2H),2.35(s,3H),2.25–1.94(m,4H).

Example 31: Preparation of Compound 31

A solution of tetrabutylammonium fluoride in tetrahydrofuran (0.088 mL, 1 M) was added to a solution of intermediate I-86 (18 mg, 0.0294 mmol) in tetrahydrofuran (2 mL) at room temperature. The reaction mixture was stirred at room temperature for 1 hour. The organic solvent was removed by concentration under reduced pressure, and the residue was purified by silica gel chromatography and then by preparative HPLC to obtain compound 31.

LC-MS (ESI) [M+H] ⁺ 497.0.

¹ H NMR (400MHz, CD ₃ OD)δ7.76(s,1H),7.67–7.60(m,2H),7.54(dd,J＝8.9,4.7Hz,1H),7.40–7.31(m,2H),7.30–7.22(m,2H ),6.75(d,J=8.5Hz,1H),4.80–4.68(m,1H),3.18–3.00(m,1H),2.75–2.49(m,2H),2.26–1.96(m,4H).

Example 32: Preparation of Compound 32

Intermediate I-87 (52 mg, 0.085 mmol) was dissolved in tetrahydrofuran (2 mL) at room temperature, and a solution of tetrabutylammonium fluoride in tetrahydrofuran (0.2 mL, 1 M) was added. The reaction mixture was stirred at room temperature for 30 minutes, and the organic solvent was removed under reduced pressure. The residue was purified by silica gel chromatography and then by preparative HPLC to obtain compound 32.

LC-MS (ESI) [M+H] ⁺ 495.0.

¹ H NMR (400MHz, CD ₃ OD)δ7.75(s,1H),7.64(d,J＝13.6Hz,2H),7.43(dd,J＝10.5,8.1Hz,1H),7.32(d,J＝8.0Hz,1H),7.23(dt,J＝7. 3,4.1Hz,2H),6.74(d,J＝8.5Hz,1H),4.75(s,1H),3.13(m,1H),2.62(m,2H),2.42(s,3H),2.23–1.95(m,4H).

Example 33: Preparation of Compound 33

Intermediate I-88 (60.00 mg) was dissolved in tetrahydrofuran (5 mL) at room temperature. A solution of tetrabutylammonium fluoride in tetrahydrofuran (0.16 mL, 1 M, 0.16 mmol) was added, and the reaction mixture was stirred at room temperature for 1 hour. Water (10 mL) was added, and the mixture was extracted with ethyl acetate (10 mL x 3). The organic phases were combined, washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, and filtered. The organic solvent was removed from the filtrate under reduced pressure, and the residue was separated and purified by preparative HPLC (ammonium bicarbonate system) to obtain compound 33.

LC-MS (ESI) [M+H] ⁺ 465.0.

¹ H NMR (400MHz, CD ₃ OD)δ7.66(s,1H),7.57–7.47(m,2H),7.43(d,J＝5.0Hz,1H),7.22(d,J＝8.3Hz,1H),7.14(dd,J＝8.5,2.4Hz,1H),6.90(d,J＝5 .0Hz,1H),6.65(d,J=8.5Hz,1H),4.67–4.60(m,1H),3.12–2.92(m,1H),2.72–2.47(m,2H),2.41(s,3H),2.02–1.86(m,4H).

Example 34: Preparation of Compound 34

A solution of tetrabutylammonium fluoride in tetrahydrofuran (0.22 mL, 1 M) was added to a solution of intermediate I-89 (50 mg, 0.09 mmol) in tetrahydrofuran (5 mL) at room temperature. The reaction mixture was stirred at room temperature for 2 hours. The organic solvent was removed by concentration under reduced pressure, and the residue was purified by preparative HPLC (formic acid system) to obtain compound 34.

LC-MS (ESI) [M+H] ⁺ 449.2.

¹ H NMR (400MHz, DMSO-d ₆ )δ9.79(s,1H),7.65-7.70(m,2H),7.57-7.64(m,2H),7.30(dd,J＝2.50,8.50Hz,1H),7.16-7.25(m,1H),7.06(d,J＝2.00Hz,1H),6. 67(d,J＝8.50Hz,1H),5.73-5.95(m,1H),4.53-4.72(m,1H),2.94-3.14(m,1H),2.55(s,3H),2.52-2.52(m,1H),1.90-2.05(m,5H).

Example 35: Preparation of Compound 35

A solution of tetrabutylammonium fluoride in tetrahydrofuran (0.327 mL, 1 M) was added to a solution of intermediate I-90 (40 mg, 0.0654 mmol) in tetrahydrofuran (5 mL) at room temperature. The reaction mixture was stirred at room temperature for 1 hour. The organic solvent was removed by concentration under reduced pressure, and the residue was purified by preparative HPLC to afford compound 35.

LC-MS (ESI) [M+H] ⁺ 497.1.

¹ H NMR (400MHz, CD ₃ OD)δ7.89–7.67(m,3H),7.63(dd,J＝8.6,5.9Hz,1H),7.43–7.29(m,3H),7.26–7.19(m,1 H),6.86(m,1H),4.89(s,1H),3.23–3.00(m,2H),2.77–2.65(m,1H),2.45–1.91(m,4H).

Example 36: Preparation of Compound 36

A solution of tetrabutylammonium fluoride in tetrahydrofuran (0.50 mL, 1 M) was added to a solution of intermediate I-91 (45.0 mg, 0.0716 mmol) in tetrahydrofuran (2.00 mL) at room temperature. The reaction mixture was stirred at room temperature for 2 hours, and the organic solvent was removed under reduced pressure. The residue was purified by silica gel chromatography and then by preparative HPLC (formic acid system) to obtain compound 36.

LC-MS (ESI) [M+H] ⁺ 515.1.

¹ H NMR (400MHz, CD ₃ OD)δ7.76(s,1H),7.68–7.52(m,4H),7.34(d,J＝8.0Hz,1H),7.24(dd,J＝8.5,2.4Hz,1 H),6.75(d,J＝8.5Hz,1H),4.76(s,1H),3.13(s,1H),2.66(m,2H),2.28–1.90(m,4H).

Example 37: Preparation of Compound 37

At room temperature, intermediate I-101 (80.00 mg, 0.14 mmol) was dissolved in tetrahydrofuran (5 mL). A solution of tetrabutylammonium fluoride in tetrahydrofuran (0.21 mL, 1 M, 0.21 mmol) was added, and the reaction mixture was stirred at room temperature for 1 hour. Water (10 mL) was added, and the mixture was extracted with ethyl acetate (10 mL x 3). The organic phases were combined, washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, and filtered. The organic solvent was removed by concentration under reduced pressure, and the residue was isolated and purified by preparative HPLC (ammonium bicarbonate system) to obtain compound 37.

LC-MS (ESI) [M+H] ⁺ 470.2.

¹ H NMR (400MHz, DMSO-d ₆ )δ10.66(s,1H),8.06(d,J＝1.9Hz,1H),7.76(dd,J＝8.2,2.0Hz,1H),7.70( d,J＝2.3Hz,1H),7.61–7.55(m,3H),7.51(dd,J＝7.9,1.9Hz,1H),7.47(td, J＝7.3,1.5Hz,1H),7.24(d,J＝8.3Hz,1H),6.86(d,J＝8.2Hz,1H),5.92(s,1 H),4.72(s,1H),3.23–2.89(m,1H),2.62–2.51(m,2H),2.22–1.85(m,4H).

Example 38: Preparation of Compound 38

Intermediate I-106 (161.00 mg) was dissolved in tetrahydrofuran (1 mL) at room temperature, and a solution of tetrabutylammonium fluoride in tetrahydrofuran (0.46 mL, 1 M, 0.46 mmol) was added. The mixture was stirred at room temperature for 2 hours. Ethyl acetate (15 mL) was added, and the mixture was washed with water (10 mL x 3). The organic solvent was removed by concentration under reduced pressure, and the residue was separated and purified by preparative HPLC (formic acid system) to obtain compound 38.

LC-MS (ESI) [M+H] ⁺ 459.2.

¹ H NMR (400MHz, DMSO-d ₆ )δ10.63(s,1H),7.66(d,J＝2.2Hz,1H),7.64–7.41(m,6H),7.24(d,J＝8.3Hz,1H),7.02(dd,J＝8.0,2.0Hz,1H) ,6.56(d,J＝8.0Hz,1H),5.68(s,1H),4.60(s,1H),3.00(m,1H),2.51–2.50(m,2H),2.33(s,3H),1.96(m,4H).

Experimental Example 1: IC ₅₀ test of the inhibition of compounds on vasopressin-induced vasopressin receptor V2R activation

(1) Cells

HeLa cell line stably expressing human vasopressin receptor V2R (HeLa-V2R): constructed by Shanghai GeneChem Technology Co., Ltd. using lentiviral infection method, and verified by qPCR to stably express human V2R.

(2) Reagents

DMEM cell culture medium: Brand: Gibco, Catalog Number: 11995065; Fetal bovine serum: Brand: Jitai, Catalog Number: FND500; 0.25% trypsin: Brand: Gibco, Catalog Number: 25200072; Puromycin Dihydrochloride: Brand: Gibco, Catalog Number: A1113803; cAMP-GS HIRANGE KIT: Brand: Cisbio, Catalog Number: 62AM6PEC; IBMX: Brand: Sigma, Catalog Number: i5879; Vasopressin AVP: Customized by Jier Biochemical (Shanghai) Co., Ltd.

(3) Test method

HeLa-V2R cells were cultured in DMEM supplemented with 10% fetal bovine serum at 37°C and 5% CO₂ _. 2 μg/mL puromycin was added to the culture medium to continuously select for V2R expression. On the day of the experiment, cells were trypsinized, washed twice with the stimulation buffer provided in the cAMP-GS HIRANGE kit, resuspended, counted, and diluted to ^1.6 x 10⁶ cells/mL. IBMX was added to a final concentration of 0.5 mM. 5 μL of cell suspension was transferred per well of a 384-well plate. 2.5 μL of test compound (starting at 10 μM, with a 3-fold dilution series of 10) or DMSO (minimum and maximum control) were added to the corresponding wells. After incubation at room temperature for 30 minutes, 2.5 μL of vasopressin AVP solution was added to the test compound wells and the maximum wells to a final concentration of 2.25 nM. 2.5 μL of stimulation buffer was added to the minimum wells, and the plates were incubated at 25°C for 60 minutes. At the same time, prepare cAMP standard samples (starting at 5.6 uM and diluted 3-fold, with 10 concentration points). Transfer 10 uL of cAMP standard to the corresponding wells of a 384-well plate. Dilute the cAMP-d2 fluorescent and anti-cAMP antibody probes provided in the cAMP-GS HIRANGE kit 20-fold with the lysis buffer in the kit. Add 5 uL of each to each well of the 384-well plate, mix thoroughly, and briefly centrifuge. Incubate at 25°C for 2 hours before testing. Samples are assayed using the HTRF method in an Envision microplate reader, measuring fluorescence intensity at 615 nm and 665 nm. Prepare two replicate wells for each sample to be tested, and 32 replicate wells for each of the Min and Max wells.

(4) Data processing

Calculate the ratio of fluorescence intensities at 665 nm to 615 nm for each sample in each well (FI _{665/615 )} . Use the logarithm of the standard concentration as X and FI _665/615 × 1000 as Y to create a standard curve using the "log (inhibitor) vs response–variable slope (four parameters)" model in Prism 8.0 software. Calculate the cAMP concentration for each sample in Prism 8.0 software based on this standard curve, using FI _665/615 × 1000 as Y.

The formula for calculating %Inhibition is as follows:

Wherein: is the average calculated value of cAMP concentration in all maximum value wells; is the average calculated value of cAMP concentration in all minimum value wells; Ccmpd is the calculated value of cAMP concentration of the test compound.

IC50 was calculated using nonlinear regression using the "log (inhibitor) vs response – variable slope (four parameters)" model in Prism 8.0 software, with % Inhibition as Y and the logarithm of compound concentration as X, where Y = Bottom + (Top - Bottom) / (1 + 10^((LogIC50 - X) * Hill Slope)).

The experimental results are shown in Table 1:

Table 1: Evaluation of the compounds' inhibition of cAMP increase in human cervical cancer cells (Human V2R Hela-Stable cell line OE2)

Compound number Compound number 1 73 2 twenty four 3 twenty four 4 37 5 48 6 40 7 57 8 73 9 58 10 27 11 57 12 83 13 29 14 65 15 42 16 49 17 49 19 88 twenty one 32 twenty two 35 twenty four 42 25 75 26 62 27 86 29 42 30 47 31 78 32 58 33 55 34 43 35 80 37 54 38 80

Experimental Example 2: In vivo pharmacokinetic study of the compounds of the present invention

In this study, the pharmacokinetics of the drug were evaluated in mice via intravenous and oral administration.

Experimental Methods and Conditions: Male CD1 mice, 6-8 weeks old, with free access to food and water, were given a single intravenous injection of the test compound (1 mg/kg) in 5% DMSO/10% Solutol/85% Saline. Blood samples were collected retro-orbitally 5, 15, 30, 1, 2, 4, 8, or 24 hours after administration, or 10 mg/kg in 5% DMSO/10% Solutol/85% Saline (6, 8, or 24 hours after administration). Blood samples (≥50 μL each) were collected and anticoagulated with sodium heparin. Plasma was centrifuged within 1 hour for analysis. Plasma concentrations were determined by liquid chromatography-tandem mass spectrometry (LC/MS/MS), and pharmacokinetic parameters were calculated using Phoenix WinNonlin software. Tofaptane was used as the control substance 1. The experimental results are shown in Tables 2 and 3.

Table 2: Pharmacokinetics of oral administration (10 mg/kg)

Compound F(%) Compound 2 0.93 5400 12347 61 Compound 10 1.72 6030 32504 121 Reference substance 1 1.58 1307 1613 44

Table 3: Pharmacokinetics of intravenous administration (1 mg/kg)

Compound Cl (ml/min/kg) Compound 2 0.80 2022 8.24 Compound 10 2.51 2687 6.20 Reference substance 1 0.53 367 45.5

Experimental data show that the pharmacokinetic results of the compound of the present invention after intravenous and oral administration to mice show a lower in vivo metabolic clearance rate Cl and a higher in vivo exposure amount AUC _0-inf .

Claims (12)

1. The compound shown in formula (I) and its pharmacologically acceptable salt, in, Ring B is selected from phenyl and 5-10 heteroaryl groups; Ring A is selected from phenyl and 5-6-membered heteroaryl groups; _R1 is selected from H, OH, _NH2 and _C1-6 alkyl groups, wherein the _C1-6 alkyl groups are optionally substituted with 1, 2 or 3 R groups; _R2 is selected from F, Cl, Br, I, CN and _C1-6 alkyl groups, wherein the _C1-6 alkyl groups are optionally substituted with 1, 2 or 3 R groups; _R3 is selected from H, F, Cl, Br, I, _C1-6 alkyl and _C3-6 cycloalkyl, wherein the _C1-6 alkyl or _C3-6 cycloalkyl is optionally substituted with 1, 2 or 3 Rs; _R4 is selected from H, F, Cl, Br, I, _C1-6 alkyl, phenyl and 5-10 heteroaryl, wherein the _C1-6 alkyl, phenyl or 5-10 heteroaryl is optionally substituted by 1, 2 or 3 Rs; _T1 and _T2 are independently selected from N and CH, respectively; R is independently selected from H, F, Cl, Br, I and _C1-6 alkyl groups; m1, m2, m3, and m4 are each independently selected from 0, 1, 2, 3, or 4; n is selected from 1 or 2; The 5-10 membered heteroaryl group comprises 1, 2 or 3 heteroatoms or heterogroups independently selected from O, NH, S, C (=O), S (=O) and N. 2. The compound shown in formula (II) and its pharmacologically acceptable salt, in, Ring B is selected from phenyl and 5-10 heteroaryl groups; _R1 is selected from H, OH, _NH2 and _C1-6 alkyl groups, wherein the _C1-6 alkyl groups are optionally substituted with 1, 2 or 3 R groups; _R2 is selected from F, Cl, Br, I, CN and _C1-6 alkyl groups, wherein the _C1-6 alkyl groups are optionally substituted with 1, 2 or 3 R groups; _R3 is selected from H, F, Cl, Br, I, _C1-6 alkyl and _C3-6 cycloalkyl, wherein the _C1-6 alkyl or _C3-6 cycloalkyl is optionally substituted with 1, 2 or 3 Rs; _R4 is selected from H, F, Cl, Br, I, _C1-6 alkyl, phenyl and 5-10 heteroaryl, wherein the _C1-6 alkyl, phenyl or 5-10 heteroaryl is optionally substituted by 1, 2 or 3 Rs; _T1 and _T2 are independently selected from N and CH, respectively; R is independently selected from H, F, Cl, Br, I and _C1-6 alkyl groups; X is selected from CH and N; m2 and m4 are selected from 0, 1, 2, 3 or 4; The 5-10 membered heteroaryl group comprises 1, 2 or 3 heteroatoms or heterogroups independently selected from O, NH, S, C (=O), S (=O) and N. 3. The compound according to claim 1 and its pharmacologically acceptable salt, wherein ring A is selected from phenyl and pyridyl. 4. The compound according to claim 1 or 2 and its pharmacologically acceptable salt, wherein _R3 is selected from H, F, Cl, Br, I, methyl, ethyl, _CHF2 , 5. The compound according to claim 1 and its pharmacologically acceptable salt, wherein the structural unit is selected from... 6. The compound according to claim 1 or 2 and its pharmacologically acceptable salt, wherein ring B is selected from phenyl, pyridyl, thiophenyl, and furanyl. 7. The compound according to claim 1 or 2 and its pharmacologically acceptable salt, wherein _R4 is selected from H, F, Cl, Br, I, _C1-3 alkyl, phenyl, pyridyl and thiazolyl, wherein the _C1-3 alkyl, phenyl, pyridyl and thiazolyl are optionally substituted by 1, 2 or 3 Rs. 8. The compound according to claim 1 or 2 and its pharmacologically acceptable salt, wherein _R4 is selected from methyl, ethyl, F, Cl, Br, I, _CF3 , _CHF2 , 9. The compound according to claim 1 or 2 and its pharmacologically acceptable salt, wherein the structural unit is selected from... 10. Compounds of the following formula and their pharmacologically acceptable salts, selected from... 11. The use of the compound according to any one of claims 1-10 and its pharmacologically acceptable salt in the preparation of a medicament for the prevention or treatment of diseases associated with arginine vasopressin V2 receptor. 12. The application according to claim 11, wherein the diseases associated with the arginine vasopressin V2 receptor include: hypertension, Reye's syndrome, dysmenorrhea, premature birth, adrenocorticotropic hormone-releasing hormone secretion disorder, adrenal hyperplasia, depression, chronic congestive heart failure, cirrhosis, antidiuretic hormone secretion disorder syndrome, hyponatremia caused by chronic heart failure/cirrhosis/antidiuretic hormone secretion disorder, or polycystic kidney disease.