CN112839946B - Bicyclopyrazoles as TGF-βR1 Kinase Inhibitors - Google Patents

Abstract

Disclosed are a class of bicyclopyrazole compounds as TGF-βR1 inhibitors, and their application in the preparation of TGF-βR1 inhibitor drugs. Specifically disclosed is the compound represented by formula (I), its pharmaceutically acceptable salt or its isomer.

Description

Bicyclopyrazoles as TGF-βR1 Kinase Inhibitors

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the following priority: CN201811288439.3, filed on October 31, 2018; CN201910288923.4, filed on April 11, 2019.

technical field

The present invention relates to a class of bicyclopyrazole compounds as TGF-βR1 inhibitors, and their application in the preparation of TGF-βR1 inhibitor drugs. Specifically, it relates to the compound represented by formula (I), its pharmaceutically acceptable salt or its isomer.

Background technique

Transforming growth factor-β (TGF-β) is a multifunctional growth factor superfamily with a wide range of biological activities involved in early embryonic development, cartilage and bone formation, synthesis of extracellular matrix, inflammation, Interstitial fibrosis, regulation of immune and endocrine functions, tumor formation and progression.

The TGF-β superfamily consists of a class of structurally and functionally related polypeptide growth factors, and TGF-β is one of the important members of this family. In mammals, TGF-β mainly exists in three forms: TGF-β1, TGF-β2 and TGF-β3, which are located on different chromosomes, of which TGF-β1 accounts for the highest proportion (>90%) in somatic cells, It is the most active, the most functional, and the most widely distributed.

TGF-β signaling molecules carry out signal transduction through transmembrane receptor complexes. TGF-β receptors are transmembrane proteins present on the cell surface, which are divided into type I receptors (TGF-βRI), type II receptors (TGF-βR II), and type III receptors (TGF-βRIII). -βR I is also known as activin receptor-like kinase 5 (ALK5). TGF-βRIII lacks intrinsic activity and is mainly related to the storage of TGF-β. TGF-βR I and TGF-βR II belong to the serine/threonine kinase family. Type II receptors can bind to TGF-β ligands with high affinity and form heterologous receptor complexes with type I receptors. Phosphorylation of a region rich in glycine and serine residues (GS domain) near the membrane of type I receptors initiates an intracellular signaling cascade.

Smads are important TGF-β signal transduction and regulation molecules in cells, which can directly transduce TGF-β signals from the cell membrane, such as in the nucleus. The TGF-β/Smads signaling pathway plays an important role in the occurrence and development of tumors. . In TGF-β/Smads signal transduction, activated TGF-β first binds to TGF-βR II on the cell membrane surface to form a heterodimeric complex, and TGF-βR I recognizes and binds to the binary complex.

TGF-βR II phosphorylates serine/threonine in the GS domain of the cytoplasmic domain of TGF-βR I, thereby activating TGF-βRI; the activated TGF-βR I further phosphorylates R-Smads (Smad2/Smad3) protein, and then It then combines with Co-Smad (Smad4) to form a heterotrimeric complex, which enters the nucleus and cooperates with other co-activators and co-inhibitors to regulate the target. transcription of genes. Changes in any link in the TGF-β/Smads signaling pathway will lead to abnormalities in the signal transduction pathway.

Current studies have shown that in tumor cells, TGF-β can directly affect tumor growth (extrinsic effects of TGF-β signaling), or by inducing epithelial-mesenchymal transition, blocking anti-tumor immune responses, increasing tumor-associated Fibrosis and enhanced angiogenesis indirectly affect tumor growth (intrinsic effects of TGF-beta). Meanwhile, TGF-β has a strong fibrosis-inducing effect, and it is an activator of tumor-associated fibroblasts. These fibroblasts are a major source of collagen type I and other fibrotic factors. Induced products of fibroblasts and other fibrotic factors may go on to foster a microenvironment that reduces immune response, increases drug resistance, and enhances tumor angiogenesis. In addition, TGF-β affects both ontogeny and tumor growth. Angiogenesis. For example, TGF-βR type I-deficient mouse embryos display severe defects in vascular development, demonstrating that the TGF-β signaling pathway is a key regulator in vascular endothelial and smooth muscle cell development.

Recent research reports also pointed out that TGF-β is significantly related to immune escape and has a greater impact on CD8+ T cell-mediated anti-tumor immune responses. In clinical trials for metastatic urothelial carcinoma, patients with high expression of TGF-β gene had a low response to PD-L1 monoclonal antibody and a low simulated survival rate. The basic research of TGF-β monoclonal antibody also proves that when it is used synergistically with PD-L1 monoclonal antibody, more CD8+ T cells infiltrate and play a role, revealing the activation effect of blocking TGF-β on immunity and its mechanism . Due to the immunomodulatory effect of TGF-β, small molecule TGF-βR I inhibitors alone or in combination with PD-(L)1 monoclonal antibody have great application prospects in the treatment of various solid tumors.

Eli Lilly's patent application WO2002094833A1 reported that compound A (ie LY2157299 or Galunisertib) has TGF-beta inhibitory activity. The compound can inhibit tumor cell invasion and metastasis, and simultaneously inhibit tumor cell infiltration into blood vessels. Several clinical trials are currently underway. Another patent application by Eli Lilly, WO2016057278A1, reported compound B (ie, LY3200882), which is a small molecule inhibitor of TGF-β newly developed by the company. Phase I clinical trials of the compound in combination with PD-L1 in solid tumors are ongoing.

SUMMARY OF THE INVENTION

On the one hand, the present invention provides the compound represented by formula (I), its pharmaceutically acceptable salt or its isomer,

Wherein, ring A is 5-6 membered heteroaryl, phenyl, C _5-6 cycloalkyl or 5-6 membered heterocycloalkyl;

R ₁ , R ₂ and R ₃ are each independently H, F, Cl, Br, I, -CN, -OH, C _1-6 alkoxy, C _1-6 alkyl, C _2-4 alkenyl, C _2-4 alkynyl or C _3-6 cycloalkyl, wherein said C _1-6 alkoxy, C _1-6 alkyl, C _2-4 alkenyl, C _2-4 alkynyl and C _{3- 6} Cycloalkyl is optionally substituted with 1, 2 or 3 substituents independently selected from F, Cl, Br, CN, -OH, -CH ₃ , -OCH ₃ and -NH ₂ ;

m is 1 or 2;

R ₄ is a 5-6 membered heteroaryl or phenyl, wherein the 5-6 membered heteroaryl and phenyl are optionally substituted by 1, 2 or 3 R _a ;

T ₁ is -O- or -CH ₂ -;

T ₂ is N or C(R ₅ );

_{R 5} is H, F, Cl, Br , -CN, -OH, -NH ₂ , -OCH ₃ or -CH ₃ ;

Each R _a is independently -CN, -C(=O)NR _b R _c , C _1-6 alkoxy, or optionally 1, 2 or 3 independently selected from F, Cl, Br, I, -OH , -OCH ₃ , -CN or C _1-6 alkyl substituted by a substituent with NH ₂ ;

一起形成式(B)所示结构：or the pyridyl to which R ₄ and R ₅ are attached and

Together to form the structure shown in formula (B):

L is a single bond,

R ₆ is independently H, -CN, -C(=O)NR _b R _c , C _1-4 alkyl, C _3-6 cycloalkyl or 5-6 membered heteroaryl, wherein the C _{1- 4} alkyl, C _3-6 cycloalkyl and 5-6 membered heteroaryl are optionally substituted with 1, 2 or 3 R _d ;

each _Rd is independently F, _Cl , Br, I, _-OH , -CN, _-NH2 , _-OCH3 , _-OCH2CH3 , _-CH3 , _-CF3 or -CH2CH3 _;

_Rb and _Rc are each independently H, _-CH3 , _-CH2CH3 , _-CH2CH2CH3 , _{-CH2 ( CH3 ) 2} or cyclopropyl _;

Said 5-6 membered heterocycloalkyl and 5-6 membered heteroaryl respectively contain 1, 2, 3 or 4 heteroatoms or heteroatomic groups independently selected from N, -O-, -S- and -NH- .

In some schemes of the present invention, the above-mentioned compound, its pharmaceutically acceptable salt or its isomer, has the structure represented by formula (I-A):

wherein ring A, R ₁ , R ₂ , R ₃ , R ₄ , T ₁ and m are as defined in the present invention.

In some schemes of the present invention, the above-mentioned compound, its pharmaceutically acceptable salt or its isomer have the structure shown in formula (I-B):

wherein ring A, R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , T ₁ and m are as defined in the present invention.

In some aspects of the present invention, the above R ₄ is a 5-6 membered heteroaryl or phenyl, wherein the 5-6 membered heteroaryl and phenyl are optionally substituted by 1, 2 or 3 R _a ; R5 is H, F, _Cl , Br, -CN, -OH, _-OCH3 , _-NH2 or _-CH3 , other variables are as defined in the present invention.

In some schemes of the present invention, the above-mentioned compound, its pharmaceutically acceptable salt or its isomer, has the structure represented by formula (I-C):

wherein ring A, R ₁ , R ₂ , R ₃ , T ₁ , m, R ₆ and L are as defined in the present invention.

其他变量如本发明所定义。In some aspects of the present invention, each of the above R _a is independently CN, -OCH ₃ ,

Other variables are as defined in the present invention.

In some aspects of the present invention, the above R ₄ is pyrrolyl, pyrazolyl, pyridyl, pyrimidinyl or phenyl, and said pyrrolyl, pyrazolyl, pyridyl, pyrimidinyl and phenyl are optionally substituted by 1, 2 or 3 R _a is substituted, and other variables are as defined in the present invention.

R_a及其他变量如本发明所定义。In some aspects of the present invention, the above R ₄ is

_Ra and other variables are as defined in the present invention.

其他变量如本发明所定义。In some aspects of the present invention, the above R ₄ is

Other variables are as defined in the present invention.

其他变量如本发明所定义。在本发明的一些方案中，上述化合物、其药学上可接受的盐或其异构体，其具有式(I-A1)～(I-A4)所示结构：In some aspects of the present invention, the above R ₄ is

Other variables are as defined in the present invention. In some embodiments of the present invention, the above-mentioned compounds, their pharmaceutically acceptable salts or their isomers have structures represented by formulae (I-A1) to (I-A4):

wherein Rings A, R ₁ , R ₂ , R ₃ , T ₁ , _Ra and m are as defined in the present invention.

In some embodiments of the present invention, the above-mentioned compounds, their pharmaceutically acceptable salts or their isomers have structures represented by formulas (I-A5) to (I-A12):

wherein Ring A, R ₁ , R ₂ , R ₃ and _Ra are as defined in the present invention.

In some embodiments of the present invention, the above-mentioned compounds, their pharmaceutically acceptable salts or their isomers have structures represented by formulae (I-B1) to (I-B4):

wherein Ring A, R ₁ , R ₂ , R ₃ , R ₅ , T ₁ , m and _Ra are as defined in the present invention.

In some embodiments of the present invention, the above-mentioned compounds, pharmaceutically acceptable salts or isomers thereof have structures represented by formulae (I-B5) to (I-B12):

wherein Ring A, R ₁ , R ₂ , R ₃ , R ₅ and _Ra are as defined in the present invention.

In some schemes of the present invention, the above-mentioned compound, its pharmaceutically acceptable salt or its isomer have the structure represented by formula (I-C1) or (I-C2):

wherein Rings A, R ₁ , R ₂ , R ₃ , R ₆ and L are as defined in the present invention.

In some aspects of the present invention, the above R ₁ , R ₂ and R ₃ are each independently H, F, Cl, Br, I, -CN, -OH, -OCH ₃ , -OCH ₂ CH ₃ , -CH ₃ , -CH ₂ CH ₃ , vinyl, ethynyl or cyclopropyl, wherein -OCH ₃ , -OCH ₂ CH ₃ , -CH ₃ , -CH ₂ CH ₃ , vinyl, ethynyl and cyclopropyl are any Select is substituted with 1, 2 or 3 substituents independently selected from F, Cl, Br, CN, -OH, _-CH3 , _-OCH3 and _-NH2 , other variables are as defined herein.

In some aspects of the invention, each of the above R ₁ , R ₂ and R ₃ is independently H, F, Cl, Br, -CN, -OH, -OCH ₃ , -CH ₃ , -CH ₂ CH ₃ or - _CF3 , other variables are as defined in the present invention.

In some embodiments of the present invention, the above-mentioned Ring A is thienyl, pyrrolyl, pyridyl, pyrimidinyl, phenyl or tetrahydro-2H-pyranyl, and other variables are as defined herein.

为

R₁、R₂和R₃及其他变量如本发明所定义。In some aspects of the invention, the above

for

R ₁ , R ₂ and R ₃ and other variables are as defined herein.

为

R₁及其他变量如本发明所定义。In some aspects of the invention, the above

for

R ₁ and other variables are as defined in the present invention.

为

其他变量如本发明所定义。In some aspects of the invention, the above

for

Other variables are as defined in the present invention.

为

其他变量如本发明所定义。In some aspects of the invention, the above

for

Other variables are as defined in the present invention.

In some embodiments of the present invention, the above-mentioned compounds, pharmaceutically acceptable salts or isomers thereof have the structures represented by formulae (I-A13) to (I-A16):

wherein, R ₁ and _Ra are as defined in the present invention.

In some embodiments of the present invention, the above-mentioned compounds, pharmaceutically acceptable salts or isomers thereof have the structures represented by formulae (I-B13) to (I-B36):

Wherein, R ₁ and R _a are defined in the present invention.

In some embodiments of the present invention, the above-mentioned compounds, their pharmaceutically acceptable salts or their isomers have structures represented by formulae (I-C3) to (I-C5):

Wherein, R ₁ , L and R ₆ are as defined in the present invention.

In some embodiments of the present invention, the above-mentioned compounds, pharmaceutically acceptable salts or isomers thereof have the structures represented by formulae (I-C6) to (I-C9):

Wherein, R ₁ and R ₆ are as defined in the present invention.

In some embodiments of the present invention, the above R ₆ is H, -C(=O)NH ₂ , isopropyl, cyclopropyl, cyclohexyl, pyrazolyl or pyridyl, wherein the isopropyl, cyclopropyl The radical, cyclohexyl, pyrazolyl and pyridyl are optionally substituted with 1, 2 or 3 _Rd , _Rd and other variables as defined herein.

In some aspects of the invention, the above R ₆ is independently H, -CN, -C(=O)NH ₂ , isopropyl, cyclopropyl, cyclohexyl, pyrazolyl or pyridyl, wherein the isopropyl Propyl, cyclopropyl, cyclohexyl, pyrazolyl and pyridyl are optionally substituted with 1, 2 or 3 _Rd , _Rd and other variables as defined herein.

R_d及其他变量如本发明所定义。In some aspects of the present invention, the above R ₆ is H, -C(=O)NH ₂ ,

_Rd and other variables are as defined herein.

R_d及其他变量如本发明所定义。In some aspects of the present invention, the above R ₆ is independently H, -CN, -C(=O)NH ₂ ,

_Rd and other variables are as defined herein.

其他变量如本发明所定义。In some aspects of the present invention, the above R ₆ is H, -C(=O)NH ₂ ,

Other variables are as defined in the present invention.

其他变量如本发明所定义。In some aspects of the present invention, the above R ₆ is independently H, -CN, -C(=O)NH ₂ ,

Other variables are as defined in the present invention.

There are also some solutions of the present invention that are formed by any combination of the above variables.

In some aspects of the invention, the above-mentioned compound is selected from a compound of the formula, a pharmaceutically acceptable salt thereof, or an isomer thereof,

On the other hand, the present invention also provides the use of the above compounds, pharmaceutically acceptable salts or isomers thereof in the preparation of TGF-βR1 inhibitor drugs.

The present invention also provides the use of the above compounds, pharmaceutically acceptable salts or isomers thereof in the preparation of medicaments for treating solid cancer.

technical effect

The compounds of the present invention have high selectivity for TGF-βR1 and have significant kinase inhibitory activity. Demonstrated significant inhibition of TGF-β downstream signaling in cells, while also possessing excellent pharmacokinetic, pharmacodynamic properties and in vivo efficacy.

Definition and Explanation

Unless otherwise specified, the following terms and phrases used herein are intended to have the following meanings. A particular term or phrase should not be considered indeterminate or unclear without specific definitions, but should be understood in its ordinary meaning. When a trade name appears herein, it is intended to refer to its corresponding commercial product or its active ingredient.

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

The term "pharmaceutically acceptable salts" refers to salts of the compounds of the present invention, prepared from compounds with specific substituents discovered by the present invention and relatively non-toxic acids or bases. When compounds of the present invention contain relatively acidic functional groups, base addition salts can be obtained by contacting 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 compounds of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting such compounds with a sufficient amount of acid in neat solution or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts including, for example, hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, Hydrogen sulfate, hydroiodic acid, phosphorous acid, etc.; and organic acid salts including, for example, acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, Similar acids such as fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-toluenesulfonic, citric, tartaric, and methanesulfonic acids; also include salts of amino acids such as arginine, etc. , and salts of organic acids such as glucuronic acid. Certain specific compounds of the present invention contain both basic and acidic functional groups and thus can be converted into either base or acid addition salts.

The pharmaceutically acceptable salts of the present invention can be synthesized from the acid or base containing parent compound by conventional chemical methods. Generally, such salts are prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of the appropriate 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. The present invention contemplates all such compounds, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers isomers, (D)-isomers, (L)-isomers, and racemic mixtures thereof and other mixtures, such as enantiomerically or diastereomerically enriched mixtures, all of which belong to this within the scope of the invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl. All such isomers, as well as mixtures thereof, are included within the scope of the present invention.

Unless otherwise indicated, the terms "enantiomers" or "optical isomers" refer to stereoisomers that are mirror images of each other.

Unless otherwise specified, the terms "cis-trans isomer" or "geometric isomer" result from the inability to rotate freely due to double bonds or single bonds to ring carbon atoms.

Unless otherwise indicated, the term "diastereomer" refers to a stereoisomer in which the molecule has two or more chiral centers and the molecules are in a non-mirror-image relationship.

Unless otherwise specified, "(+)" means dextrorotatory, "(-)" means levorotatory, and "(±)" means racemic.

和楔形虚线键

表示一个立体中心的绝对构型，用波浪线

表示楔形实线键

或楔形虚线键

Use solid wedge keys unless otherwise specified

and wedge-dotted keys

Indicate the absolute configuration of a solid center, with a wavy line

Represents a solid wedge key

or wedge-dotted key

连接，则表示该化合物的(Z)型异构体、(E)型异构体或两种异构体的混合物。例如下式(A)表示该化合物以式(A-1)或式(A-2)的单一异构体形式存在或以式(A-1)和式(A-2)两种异构体的混合物形式存在；下式(B)表示该化合物以式(B-1)或式(B-2)的单一异构体形式存在或以式(B-1)和式(B-2)两种异构体的混合物形式存在。下式(C)表示该化合物以式(C-1)或式(C-2)的单一异构体形式存在或以式(C-1)和式(C-2)两种异构体的混合物形式存在。Unless otherwise specified, when there is a double bond structure in the compound, such as carbon-carbon double bond, carbon-nitrogen double bond and nitrogen-nitrogen double bond, and each atom on the double bond has two different substituents attached (including nitrogen atom) In the double bond, a lone pair of electrons on the nitrogen atom is regarded as a substituent for its connection), if a wavy line is used between the atom on the double bond and its substituent in the compound

If connected, it means the (Z) isomer, (E) isomer or a mixture of both isomers of the compound. For example, the following formula (A) indicates that the compound exists as a single isomer of formula (A-1) or formula (A-2) or as two isomers of formula (A-1) and formula (A-2) The following formula (B) indicates that the compound exists in the form of a single isomer of formula (B-1) or formula (B-2) or exists in two forms of formula (B-1) and formula (B-2) exists as a mixture of isomers. The following formula (C) represents that the compound exists in the form of a single isomer of formula (C-1) or formula (C-2) or in the form of two isomers of formula (C-1) and formula (C-2) exists in the form of a mixture.

The compounds of the present invention may exist in particular. Unless otherwise specified, the term "tautomer" or "tautomeric form" refers to isomers of different functional groups that are in dynamic equilibrium and are rapidly interconverted at room temperature. A chemical equilibrium of tautomers can be achieved if tautomers are possible (eg, in solution). For example, proton tautomers (also called prototropic tautomers) include interconversions by migration of protons, such as keto-enol isomerization and imine-ene Amine isomerization. Valence isomers (valencetautomer) include some interconversions by the recombination of bonding electrons. A specific example of keto-enol tautomerization is the interconversion between two tautomers, pentane-2,4-dione and 4-hydroxypent-3-en-2-one.

Unless otherwise indicated, the terms "enriched in one isomer", "enriched in isomers", "enriched in one enantiomer" or "enriched in one enantiomer" refer to one of the isomers or pairs The enantiomer content is less than 100%, and the isomer or enantiomer content is greater than or equal to 60%, or greater than or equal to 70%, or greater than or equal to 80%, or greater than or equal to 90%, or greater than or equal to 95%, or Greater than or equal to 96%, or greater than or equal to 97%, or greater than or equal to 98%, or greater than or equal to 99%, or greater than or equal to 99.5%, or greater than or equal to 99.6%, or greater than or equal to 99.7%, or greater than or equal to 99.8%, or greater than or equal to 99.9%.

Unless otherwise indicated, the terms "isomeric excess" or "enantiomeric excess" refer to the difference between two isomers or relative percentages of two enantiomers. For example, if the content of one isomer or enantiomer is 90% and the content of the other isomer or enantiomer is 10%, the isomer or enantiomeric excess (ee value) is 80% .

Optically active (R)- and (S)-isomers, as well as D and L isomers, can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one enantiomer of a compound of the present invention is desired, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, wherein the resulting mixture of diastereomers is separated and the auxiliary group is cleaved to provide pure desired enantiomer. Alternatively, when the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), a diastereomeric salt is formed with an appropriate optically active acid or base, followed by conventional methods known in the art The diastereoisomers were resolved and the pure enantiomers recovered. In addition, separation of enantiomers and diastereomers is usually accomplished by the use of chromatography employing a chiral stationary phase, optionally in combination with chemical derivatization (eg, from amines to amino groups) formate).

Compounds of the invention may contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute the compound. For example, compounds can be labeled with radioisotopes, such as tritium ( ³ H), iodine-125 ( ¹²⁵ I) or C-14 ( ¹⁴ C). For another example, deuterated drugs can be formed by replacing hydrogen with deuterium, and the bonds formed by deuterium and carbon are stronger than those formed by ordinary hydrogen and carbon. Compared with non-deuterated drugs, deuterated drugs can reduce toxic side effects and increase drug stability. , enhance the efficacy, prolong the biological half-life of drugs and other advantages. All transformations of the isotopic composition 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 subsequently described event or circumstance may, but need not, occur, and that the description includes instances where said event or circumstance occurs and instances in which it does not.

The term "substituted" means that any one or more hydrogen atoms on a specified atom are replaced by a substituent, which may include deuterium and hydrogen variants, as long as the valence of the specified atom is normal and the substituted compound is stable of. When the substituent is oxygen (ie =O), it means that two hydrogen atoms are substituted. Oxygen substitution does not occur on aromatic groups. The term "optionally substituted" means that it may or may not be substituted, and unless otherwise specified, the type and number of substituents may be arbitrary on a chemically achievable basis.

When any variable (eg, R) occurs more than once in the composition or structure of a compound, its definition in each case is independent. Thus, for example, if a group is substituted with 0-2 Rs, the group may optionally be substituted with up to two Rs, with independent options for R in each case. Furthermore, combinations of substituents and/or variants thereof are permissible only if such combinations result in stable compounds.

When the number of a linking group is 0, such as -(CRR) ₀ -, it means that the linking group is a single bond.

When one of the variables is selected from a single bond, it means that the two groups connected to it are directly connected, for example, when L in A-L-Z represents a single bond, it means that the structure is actually A-Z.

When a substituent is vacant, it means that the substituent does not exist. For example, when X in A-X is vacant, it means that the structure is actually A. When the listed substituents do not indicate through which atom it is attached to the substituted group, such substituents may be bonded through any of its atoms, for example, pyridyl as a substituent may be through any one of the pyridine ring The carbon atom is attached to the substituted group.

中连接基团L为-M-W-，此时-M-W-既可以按与从左往右的读取顺序相同的方向连接环A和环B构成

也可以按照与从左往右的读取顺序相反的方向连接环A和环B构成

所述连接基团、取代基和/或其变体的组合只有在这样的组合会产生稳定的化合物的情况下才是被允许的。When the listed linking group does not indicate its direction of attachment, the direction of attachment is arbitrary, for example,

The linking group L in the middle is -MW-, at this time -MW- can connect ring A and ring B in the same direction as the reading order from left to right.

It is also possible to connect ring A and ring B in the opposite direction to the reading order from left to right.

Combinations of the linking groups, substituents and/or variants thereof are permissible only if such combinations result in stable compounds.

直形虚线键

或波浪线

表示。例如-OCH₃中的直形实线键表示通过该基团中的氧原子与其他基团相连；

中的直形虚线键表示通过该基团中的氮原子的两端与其他基团相连；

中的波浪线表示通过该苯基基团中的1位和2位碳原子与其他基团相连。Unless otherwise specified, when a group has one or more attachable sites, any one or more sites in the group can be linked to other groups by chemical bonds. The chemical bond connecting the site to other groups can be represented by straight solid line bonds

straight dotted key

or wavy lines

express. For example, a straight solid bond in -OCH ₃ indicates that it is connected to other groups through the oxygen atom in this group;

The straight dashed bond in the group indicates that it is connected to other groups through the two ends of the nitrogen atom in the group;

The wavy lines in the phenyl group indicate connections to other groups through the 1 and 2 carbon atoms in the phenyl group.

Unless otherwise specified, the number of atoms in a ring is generally defined as the number of ring members, eg, "5-7 membered ring" refers to a "ring" of 5-7 atoms arranged around it.

Unless otherwise specified, the term "5-6 membered ring" refers to cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, cycloalkynyl, heterocycloalkynyl, Aryl or Heteroaryl. Said ring includes a single ring, and also includes a bicyclic ring system such as a spiro ring, a paracyclic ring and a bridged ring. Unless otherwise specified, the ring optionally contains 1, 2 or 3 heteroatoms independently selected from O, S and N. The 5-6 membered ring includes 5-membered, 6-membered ring and the like. "5-6 membered ring" includes, for example, phenyl, pyridyl, piperidinyl, and the like; on the other hand, the term "5-6 membered heterocycloalkyl" includes piperidinyl and the like, but does not include phenyl. The term "ring" also includes ring systems containing at least one ring, wherein each "ring" independently meets the above definition.

Unless otherwise specified, the term "C _1-6 alkyl" is used to denote a straight or branched chain saturated hydrocarbon group consisting of 1 to 6 carbon atoms. The C _1-6 alkyl 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 and the like; it can be Is monovalent (eg methyl), divalent (eg methylene) or polyvalent (eg methine). Examples of C _1-6 alkyl include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), butyl (including n-butyl, isobutyl , s-butyl and t-butyl), pentyl (including n-pentyl, isopentyl and neopentyl), hexyl, etc.

Unless otherwise specified, the term "C _1-4 alkyl" is used to denote a straight or branched chain saturated hydrocarbon group consisting of 1 to 4 carbon atoms. The C _1-4 alkyl includes C _1-2 , C _1-3 and C _2-3 alkyl, etc.; it can be monovalent (such as methyl), divalent (such as methylene) or polyvalent ( such as methine). Examples of C _1-4 alkyl include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), butyl (including n-butyl, isobutyl , s-butyl and t-butyl) and so on.

Unless otherwise specified, the term "C _2-4 alkenyl" is used to denote a straight or branched chain hydrocarbon group consisting of 2 to 4 carbon atoms containing at least one carbon-carbon double bond, a carbon-carbon double bond. The bond can be located anywhere in the group. The C _2-4 alkenyl group includes C _2-3 , C ₄ , C ₃ and C ₂ alkenyl groups, etc.; the C _2-4 alkenyl group may be monovalent, divalent or multivalent. Examples of C _2-4 alkenyl groups include, but are not limited to, vinyl, propenyl, butenyl, butadienyl, and the like.

Unless otherwise specified, the term "C _2-4 alkynyl" is used to denote a straight or branched chain hydrocarbon group consisting of 2 to 4 carbon atoms containing at least one carbon-carbon triple bond, carbon-carbon three The bond can be located anywhere in the group. The C _2-4 alkynyl groups include C _2-3 , C ₄ , C ₃ and C ₂ alkynyl groups and the like. It can be monovalent, bivalent or multivalent. Examples of _C2-4alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, and the like.

Unless otherwise specified, the term "C1-6alkoxy" refers to those _alkyl groups containing 1 to 6 carbon atoms attached to the remainder of the molecule through an oxygen atom. The C _1-6 alkoxy groups include 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, etc. . 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) oxy, s-butoxy and t-butoxy), pentyloxy (including n-pentyloxy, isopentyloxy and neopentyloxy), hexyloxy and the like.

Unless otherwise specified, the term " _C1-4alkoxy " refers to those alkyl groups containing 1 to 4 carbon atoms attached to the remainder of the molecule through an oxygen atom. The C _1-4 alkoxy group includes C _1-3 , C _1-2 , C _2-4 , C ₄ and C ₃ alkoxy and the like. 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) oxy, s-butoxy and t-butoxy), pentyloxy (including n-pentyloxy, isopentyloxy and neopentyloxy), hexyloxy and the like.

Unless otherwise specified, the term "halogen" or "halogen" by itself or as part of another substituent means a fluorine, chlorine, bromine or iodine atom.

Unless otherwise specified, the term "C _3-6 cycloalkyl" denotes a saturated cyclic hydrocarbon group consisting of 3 to 6 carbon atoms, which are monocyclic and bicyclic ring systems, the C _3-6 cycloalkyl group Including _C3-5 , _C4-5 and _C5-6 cycloalkyl and the like; it may be monovalent, divalent or polyvalent. Examples of _C3-6 cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like.

Unless otherwise specified, the term "C _5-6 cycloalkyl" refers to a saturated cyclic hydrocarbon group consisting of 5 to 6 carbon atoms, which are monocyclic and bicyclic ring systems, the C _5-6 cycloalkyl group Include _C5 and _C6 cycloalkyl and the like; it may be monovalent, divalent or polyvalent. Examples of _C5-6 cycloalkyl groups include, but are not limited to, cyclopentyl, cyclohexyl, and the like.

Unless otherwise specified, the term "5-6 membered heterocycloalkyl" by itself or in combination with other terms denotes a saturated cyclic group consisting of 5 to 6 ring atoms, respectively, of which 1, 2, 3 or 4 ring atoms are heteroatoms independently selected from O, S, and N, and the remainder are carbon atoms, where the nitrogen atom is optionally quaternized, and the nitrogen and sulfur heteroatoms are optionally oxidized (ie, NO and S(O) _p , p is 1 or 2). It includes monocyclic and bicyclic ring systems, wherein bicyclic ring systems include spiro, paracyclic and bridged rings. Furthermore, with respect to the "5-6 membered heterocycloalkyl", a heteroatom may occupy the position of attachment of the heterocycloalkyl to the remainder of the molecule. The 5-6 membered heterocycloalkyl includes 5- and 6-membered heterocycloalkyl. Examples of 5-6 membered heterocycloalkyl include, but are not limited to, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, tetrahydrothienyl (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, isothiazole Alkyl, 1,2-oxazinyl, 1,2-thiazinyl, hexahydropyridazinyl, homopiperazinyl or homopiperidinyl and the like.

Unless otherwise specified, the terms "C _6-12 aryl ring" and "C _6-12 aryl group" can be used interchangeably, and the term "C _6-12 aryl ring" or "C _6-12 aryl group" means a range from 6 to A cyclic hydrocarbon group consisting of 12 carbon atoms with a conjugated _π -electron system, which can be a monocyclic, fused bicyclic or fused tricyclic system, wherein each ring is aromatic. It may be monovalent, divalent or polyvalent, and C _6-12 aryl groups include C _6-10 , C _6-9 , C _6-8 , C ₁₂ , C ₁₀ , and C ₆ aryl groups, and the like. Examples of _C6-12 aryl groups include, but are not limited to, phenyl, naphthyl (including 1-naphthyl and 2-naphthyl, and the like).

Unless otherwise specified, the terms "C _6-10 aryl ring" and "C _6-10 aryl group" are used interchangeably, and the term "C _6-10 aryl ring" or "C _6-10 aryl group" means a range from 6 to A cyclic hydrocarbon group composed of 10 carbon atoms with a conjugated π-electron system, which can be a monocyclic, fused bicyclic or fused tricyclic system, wherein each ring is aromatic. It may be monovalent, divalent or polyvalent, and _C6-10 aryl groups include _C6-9 , _C9 , _C10 and _C6 aryl groups and the like. Examples of _C6-10 aryl groups include, but are not limited to, phenyl, naphthyl (including 1-naphthyl and 2-naphthyl, and the like).

Unless otherwise specified, the terms "5-6 membered heteroaryl" and "5-6 membered heteroaryl" are used interchangeably, and the term "5-6 membered heteroaryl" refers to a ring consisting of 5 to 6 ring atoms. A monocyclic group 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 rest are carbon atoms. Where the nitrogen atom is optionally quaternized, the nitrogen and sulfur heteroatoms may be optionally oxidized (ie, NO and S(O) _p , p is 1 or 2). A 5-6 membered heteroaryl group can be attached to the remainder of the molecule through a heteroatom or a carbon atom. The 5-6 membered heteroaryl groups include 5- and 6-membered heteroaryl groups. Examples of the 5-6 membered heteroaryl include, but are not limited to, pyrrolyl (including N-pyrrolyl, 2-pyrrolyl and 3-pyrrolyl, etc.), pyrazolyl (including 2-pyrazolyl and 3-pyrrolyl, etc.) azolyl, etc.), imidazolyl (including N-imidazolyl, 2-imidazolyl, 4-imidazolyl and 5-imidazolyl, etc.), oxazolyl (including 2-oxazolyl, 4-oxazolyl and 5- oxazolyl, etc.), 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 particular instance of n to n+ _m carbons, eg _C1-12 includes _C1 , _C2 , C3, C ₄ , C ₅ , C ₆ , C ₇ , C ₈ , C ₉ , C ₁₀ , C ₁₁ , and C ₁₂ , also including any one range from n to n+m, eg C _1-12 includes C _1-3 , C _1-6 , C _1-9 , C _3-6 , C _3-9 , C _3-12 , C _6-9 , C _6-12 , and C _9-12 , etc.; in the same way, n yuan to n +m-membered means that the number of atoms in the ring is from n to n+m, for example, 3-12-membered ring includes 3-membered ring, 4-membered ring, 5-membered ring, 6-membered ring, 7-membered ring, 8-membered ring, 9-membered ring , 10-membered ring, 11-membered ring, and 12-membered ring, also including any one range from n to n+m, for example, 3-12-membered ring includes 3-6 membered ring, 3-9 membered ring, 5-6 membered ring ring, 5-7 membered ring, 6-7 membered ring, 6-8 membered ring, and 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 (eg, affinity substitution reaction). For example, representative leaving groups include triflate; chlorine, bromine, iodine; sulfonate groups such as mesylate, tosylate, p-bromobenzenesulfonate, p-toluenesulfonic acid Esters, etc.; acyloxy, such as acetoxy, trifluoroacetoxy, and the like.

The term "protecting group" includes, but is not limited to, "amino protecting group", "hydroxy protecting group" or "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 groups, such as alkanoyl groups (eg, acetyl, trichloroacetyl, or trifluoroacetyl); alkoxycarbonyl groups, such as tert-butoxycarbonyl (Boc) ; Arylmethoxycarbonyl, such as benzyloxycarbonyl (Cbz) and 9-fluorenylmethoxycarbonyl (Fmoc); Arylmethyl, such as benzyl (Bn), trityl (Tr), 1,1-di -(4'-Methoxyphenyl)methyl; silyl groups such as trimethylsilyl (TMS) and tert-butyldimethylsilyl (TBS) and the like. The term "hydroxy protecting group" refers to a protecting group suitable for preventing hydroxyl side reactions. Representative hydroxy protecting groups include, but are not limited to: alkyl groups such as methyl, ethyl and tert-butyl; acyl groups such as alkanoyl (eg acetyl); arylmethyl groups such as benzyl (Bn), p-methyl Oxybenzyl (PMB), 9-fluorenylmethyl (Fm) and diphenylmethyl (diphenylmethyl, DPM); silyl groups such as trimethylsilyl (TMS) and tert-butyl Dimethylsilyl (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 enumerated below, embodiments formed in combination with other chemical synthesis methods, and those well known to those skilled in the art Equivalent to alternatives, preferred embodiments include, but are not limited to, the embodiments of the present invention.

The solvent used in the present invention is commercially available.

The following abbreviations are used in the present invention: _CDCl3 stands for deuterated chloroform; DMSO stands for dimethyl sulfoxide; Boc stands for tert-butoxycarbonyl; DPBS stands for Dulbecco's Phosphate Buffered Saline.

软件命名，市售化合物采用供应商目录名称。Compounds of the present invention are either used according to conventional nomenclature in the art

Software naming, commercially available compounds use supplier catalog names.

Detailed ways

The present invention will be described in detail by the following examples, but it does not mean any unfavorable limitation of the present invention. The present invention has been described in detail herein, and specific embodiments thereof have also been disclosed. For those skilled in the art, various changes and modifications can be made to the specific embodiments of the present invention without departing from the spirit and scope of the invention. will be obvious.

Example 1: Compound 1

Step A: Compound 1-1 (200 g, 1.02 mol, 1.0 equiv) was dissolved in 1 liter of dichloromethane, pyridine (84.64 g, 1.07 mol, 1.05 equiv) was added, and the reaction temperature was controlled at 30-40 degrees Celsius Next, 4-chlorobutyryl chloride (150.88 g, 1.07 mol, 1.05 equiv) was added dropwise. After the dropwise addition, the mixture was stirred at 25°C for 12 hours. 1 liter of water was added to the reaction solution at 25 degrees Celsius, and the organic phase was separated after stirring for 30 minutes. The organic phase was dried over anhydrous sodium sulfate and concentrated to obtain compound 1-2.

Step B: Compound 1-2 (319.60 g, 1.06 mol, 1.0 equiv) was dissolved in 1.8 L of tetrahydrofuran and potassium tert-butoxide (125.19 g, 1.12 mol, 1.05 equiv) was added portionwise at 25 degrees Celsius. Stir at 25°C for 8 hours. 1.8 liters of water was added to the reaction solution at 25 degrees Celsius, the organic phase was separated after stirring for 30 minutes, the aqueous phase was extracted with ethyl acetate (1.2 liters), the organic phases were combined, washed with saturated brine (1.2 liters), dried over anhydrous sodium sulfate, Concentrate to obtain compound 1-3.

Step C: Compound 1-3 (267.08 g, 1.01 mol, 1.0 equiv) was added to 1.5 L of toluene at 25 degrees Celsius, stirred, and water (18.20 g, 1.01 mol, 1.0 equiv) was added. P-toluenesulfonic acid monohydrate (176.83 g, 929.60 mmol, 0.92 equiv) was added in portions and the addition temperature was controlled between 40-45 degrees Celsius. Stir at 40-45 degrees Celsius for 3 hours after the addition is complete. It was then cooled to 15°C and stirred for 1 hour. The reaction solution was filtered, and the filter cake was rinsed with toluene (600 mL) and dried to obtain compound 1-4.

Step D: Ethyl acetate (349.71 g, 3.97 mol, 3.0 equiv) was added to 1 L of toluene and sodium ethoxide (180.07 g, 2.65 mol, 2.0 equiv) was added portionwise at 20-30 degrees Celsius. Stir at 20-30 degrees Celsius for 1 hour after the addition is complete. Compound 1-5 (200 g, 1.32 mol, 1.0 equiv) was added in three batches and stirred at 100 degrees Celsius for 12 hours after addition. The reaction solution was cooled to 25 degrees Celsius, the pH value was adjusted to 6 with glacial acetic acid, 1 liter of water was added, and the organic phase was separated after stirring for 30 minutes. The aqueous phase was extracted with toluene (600 mL), and the organic phases were combined and concentrated to give compound 1-6.

Step E: Compound 1-6 (137 g, 661.12 mol, 1.0 equiv) was added to 350 mL of pyridine followed by compound 1-4 (216.04 g, 793.34 mmol, 1.2 equiv). Stir for 12 hours at 40-45 degrees Celsius. The reaction solution was cooled to 25 degrees Celsius, 700 mL of water was added, and extracted with toluene (560 mL×2). The organic phases were combined, washed with saturated brine (560 mL), dried over anhydrous sodium sulfate, and concentrated to obtain compound 1-7.

Step F: Compound 1-7 (148.50 g, 513.26 mmol, 1.0 equiv) was dissolved in 900 mL toluene and sodium ethoxide (69.85 g, 1.03 mol, 2.0 equiv) was added portionwise. Stir at 100-110 degrees Celsius for 12 hours after the addition is complete. The reaction solution was cooled to 25 degrees Celsius, 600 ml of water was added, and the mixture was stirred for 15 minutes. The aqueous phase was separated, washed with ethyl acetate (300 mL×3), and then the pH value of the aqueous phase was adjusted to 6 with concentrated hydrochloric acid, and filtered. The filter cake was slurried with 150 mL of isopropanol, filtered and dried to obtain compound 1-8. MS (ESI) m/z: 244.0 [M+H ⁺ ].

Step G: Compound 1-8 (77.22 g, 317.44 mmol, 1.0 equiv) was dissolved in 800 mL N,N-dimethylformamide, and N-bromosuccinimide (59.32 g) was added in portions. , 333.31 mmol, 1.05 equiv), stirred at 25 degrees Celsius for 8 hours. Further N-bromosuccinimide (59.32 g, 333.31 mmol, 1.05 equiv) was added and stirred at 50°C for 12 hours. Cool the reaction solution to 0-10 degrees Celsius, add 2 liters of water, stir at 0-10 degrees Celsius for 30 minutes, and filter. The filter cake was slurried with water (500 mL), filtered and dried to give compound 1-9. MS (ESI) m/z: 277.8, 279.8 [M+H ⁺ ].

Step H: Compound 1-9 (20 g, 71.90 mmol, 1.0 equiv), triisopropyl borate (35.84 g, 190.55 mmol, 2.65 equiv) were added to 200 mL of anhydrous tetrahydrofuran and cooled to -70 to -60 degrees Celsius. n-Butyllithium (2.5 moles per liter of n-heptane solution, 68.74 mL, 2.39 equiv) was added dropwise at -70 to -60 degrees Celsius. After the addition, the mixture was stirred at -70 to -60 degrees Celsius for 2 hours. The reaction solution was poured into 500 mL of saturated ammonium chloride solution at 0-5 degrees Celsius, filtered, and the filter cake was washed with water (100 mL×2), and dried to obtain compound 1-10. MS (ESI) m/z: 244.2 [M+H ⁺ ].

Step I: Compound 1-10 (26.62 g, 109.52 mmol, 1.0 equiv) and hydrogen peroxide (37.25 g, 328.55 mmol, 30% concentration, 3.0 equiv) were added to 250 mL of tetrahydrofuran, stirred, and cooled to 0-5 degrees Celsius . Aqueous 2 molar sodium hydroxide solution (219.03 mL, 4.0 equiv) was added dropwise at 0-5 degrees Celsius. After the addition was complete, the mixture was stirred at 25°C for 12 hours. 250 mL of water was added to the reaction solution, the pH value was adjusted to 7 with concentrated hydrochloric acid, extracted with ethyl acetate (200 mL×3), washed with saturated sodium sulfite solution (100 mL×2), dried over anhydrous sodium sulfate, concentrated, and the column layer was Analytical purification gave compound 1-11. MS (ESI) m/z: 215.9 [M+H ⁺ ].

Step J: To a flask containing 100 mL of anhydrous dichloromethane, compound 1-12 (5 g, 25.38 mmol, 1.0 equiv), di-tert-butyl dicarbonate (6.65 g, 30.45 g) were added successively with constant stirring mmol, 1.2 equiv), 4-dimethylaminopyridine (310.03 mg, 2.54 mmol, 0.1 equiv) and triethylamine (7.70 g, 76.13 mmol, 3.0 equiv). The reaction solution was stirred at 25°C for 3 hours. 150 mL of water was added to the reaction solution, extracted with dichloromethane (100 mL×2), the organic phase was washed with 100 mL of saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain compound 1-13.

Step K: Compound 1-13 (2 g, 6.73 mmol, 1 equiv) was dissolved in 20 mL of 1,4-dioxane, and bispinacol boronate (2.05 g, 8.08 mmol, 1.2 equiv), [1,1'-bis(diphenylphosphino)]palladium dichloride (492.49 mg, 673.07 micromoles, 0.1 equiv) and potassium acetate (1.32 g, 13.46 mmol, 2 equiv). The reaction system was replaced with nitrogen three times, and then heated to 100 degrees Celsius under nitrogen protection and stirred for 8 hours. The reaction solution was cooled to 25 degrees Celsius, 50 mL of water was added, and ethyl acetate was extracted (40 mL×3). The organic phases were combined, washed with 50 mL of saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound 1-14. MS (ESI) m/z: 345.1 [M+H ⁺ ].

Step L: Compound 1-14 (3.26 g, 9.47 mmol, 1 equiv) was dissolved in a mixed solvent of 20 mL of acetone and 10 mL of water, and sodium periodate (6.28 g, 29.36 mmol) was added successively under constant stirring. , 1.63 mL, 3.1 equiv) and ammonium acetate (2.19 g, 28.41 mmol, 3 equiv). Stir at 25°C for 12 hours. 30 mL of water was added to the reaction solution, followed by extraction with ethyl acetate (30 mL×3). The organic phases were combined, washed with saturated brine (30 mL), and concentrated. The crude product was dissolved in 30 mL of 1 M NaOH solution and washed with ethyl acetate (30 mL x 3). The pH of the aqueous phase was adjusted to 6 with 2 moles per liter of hydrochloric acid. After filtration, the filter cake was washed with water (5 mL×2) and dried to obtain compound 1-15. MS (ESI) m/z: 263.2 [M+H ⁺ ].

分子筛(500毫克)，体系用氧气置换三次。于25摄氏度下氧气氛围中反应12小时。向反应液中加入20毫升水，二氯甲烷萃取(30毫升×3)。合并有机相，饱和食盐水洗涤(30毫升)，无水硫酸钠干燥，过滤，浓缩，柱层析纯化得到化合物1-16。Step M: Compounds 1-15 (200 mg, 763.16 μmol, 1 equiv) and 1-11 (246.41 mg, 1.14 mmol, 1.5 equiv) were dissolved in 5 mL dichloromethane and copper acetate (207.92 mg, 1.5 equiv) was added. 1.14 mmol, 1.5 equiv), triethylamine (231.67 mg, 2.29 mmol, 318.67 μl, 3 equiv) and

Molecular sieves (500 mg), and the system was replaced with oxygen three times. The reaction was carried out in an oxygen atmosphere at 25°C for 12 hours. 20 mL of water was added to the reaction solution, followed by extraction with dichloromethane (30 mL×3). The organic phases were combined, washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain compound 1-16.

Step N: Compound 1-16 (200 mg, 463.51 μmol, 1 equiv) was added to 4 mol/L hydrochloric acid in methanol (5 mL, 43.15 equiv) and stirred at 25°C for 8 hours. The reaction solution was poured into 20 ml of water, and the pH was adjusted to 8 to 9 with 4 mol/liter aqueous sodium hydroxide solution. Methanol was distilled off under reduced pressure and extracted with ethyl acetate (30 mL×3). The organic phases were combined, washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered and concentrated, and compound 1 was obtained by preparative high performance liquid phase separation. MS (ESI) m/z: 332.1 [M+H ⁺ ]; ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 11.59 (s, 1H), 7.96 (d, J=5.6 Hz, 1H), 7.58 -7.51(m, 2H), 7.32(t, J=2.8Hz, 1H), 6.97(d, J=7.2Hz, 1H), 6.41-6.39(m, 2H), 4.20(t, J=7.4Hz, 2H), 2.82 (t, J=7.2Hz, 2H), 2.62-2.55 (m, 2H), 2.05 (s, 3H).

Example 2: Compound 2

Step A: Compound 2-1 (10 g, 46.29 mmol, 1 equiv) was dissolved in tetrahydrofuran (100 mL) and methylmagnesium bromide (3 mol per liter in tetrahydrofuran) was added dropwise to the solution under nitrogen protection. solution, 33.95 mL, 2.2 equiv), maintaining the temperature between 0 and 5 degrees Celsius. After the dropwise addition, the temperature was raised to 25 degrees Celsius, and the mixture was stirred for 1 hour. The pH of the reaction solution was adjusted to 7 with 1 mole per liter of hydrochloric acid. Add water (200 mL) to dilute, extract with ethyl acetate (200 mL×3), combine the organic phases, wash with water (200 mL), dry over anhydrous sodium sulfate, filter, and concentrate to obtain compound 2-2.

Step B: Compound 2-2 (10.33 g, 31.99 mmol, 1 equiv) was dissolved in aqueous ammonia (76.53 g, 611.36 mmol, 84.10 mL, 28%, 19.11 equiv), and copper powder (1.22 g, 19.19 mmol) was added. , 0.6 equiv). Stir for 1 hour at 25°C in air and 12 hours at 100°C under nitrogen protection. The reaction solution was filtered, the filtrate was lyophilized, and purified by column chromatography to obtain compound 2-3. MS (ESI) m/z: 153.3 [M+H ⁺ ].

分子筛(5克)。35摄氏度下氧气氛围中搅拌12小时。将体系温度降到20摄氏度到25摄氏度之间，缓慢加入水(300毫升)稀释，乙酸乙酯(200毫升×3)萃取，合并有机相，饱和食盐水(200毫升)洗涤，无水硫酸钠干燥，浓缩，柱层析纯化得到化合物2-5。MS(ESI)m/z：327.1[M+H⁺]。Step C: Compound 1-11 (5.3 g, 21.80 mmol, 1 equiv) and compound 2-4 (6.86 g, 43.60 mmol, 2 equiv) were dissolved in dichloromethane (250 mL), followed by stirring Add anhydrous copper acetate (5.94 g, 32.70 mmol, 1.5 equiv), triethylamine (6.62 g, 65.40 mmol, 9.10 mL, 3 equiv),

Molecular sieves (5 g). Stir in an oxygen atmosphere at 35°C for 12 hours. Lower the temperature of the system to between 20 and 25 degrees Celsius, slowly add water (300 ml) to dilute, extract with ethyl acetate (200 ml × 3), combine the organic phases, wash with saturated brine (200 ml), and wash with anhydrous sodium sulfate. Drying, concentration, and purification by column chromatography gave compound 2-5. MS (ESI) m/z: 327.1 [M+H ⁺ ].

Step D: Compound 2-5 (1.5 g, 4.59 mmol, 1 equiv) was dissolved in 1,4-dioxane (30 mL), followed by compound 2-3 (1.05 g, 6.89 mmol, 1.5 equiv), bis(dibenzylideneacetone)palladium (527.89 mg, 918.05 μmol, 0.2 equiv), 4,5-bis(diphenylphosphine)-9,9-dimethylxanthene (531.20 mg , 918.05 μmol, 0.2 equiv), sodium phenate (2.13 g, 18.36 mmol, 4 equiv). After replacing nitrogen three times, the mixture was heated to 100 degrees Celsius and stirred for 12 hours. The reaction solution was filtered, water (50 mL) was added to the filtrate, extracted with ethyl acetate (50 mL×3), the organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, concentrated, and HPLC Chromatographic purification affords compound 2. MS(ESI) m/z: 443.2 [M+H+];

¹ H NMR (400 MHz, DMSO-d ₆ ) δ=9.35 (s, 1H), 8.17 (d, J=5.6 Hz, 1H), 8.10 (d, J=5.6 Hz, 1H), 7.68 (s, 1H) , 7.65-7.61 (m, 2H), 7.56 (d, J=8.0Hz, 1H), 7.04 (d, J=7.2Hz, 1H), 6.59 (dd, J=2.0, 5.6Hz, 1H), 6.39 ( d, J=2.0Hz, 1H), 5.08 (s, 1H), 4.20 (t, J=7.2Hz, 2H), 2.86 (t, J=7.6Hz, 2H), 2.63-2.56 (m, 2H), 2.22 (s, 3H), 1.39 (s, 6H).

Example 3: Compound 3

Step A: in a solution of 3-1 (5 g, 32.34 mmol, 4.17 mL, 1 equiv) in diethyl formate (39.00 g, 330.14 mmol, 40 mL, 10.21 equiv) at 5 degrees C in portions Sodium hydrogen (2.68 g, 66.95 mmol, purity: 60%, 2.07 equiv) was added, the temperature was slowly raised to 85 degrees Celsius and reacted at this temperature for 2 hours, 100 mL of water was added, and ethyl acetate (100 mL×2) was added for extraction. The organic phases were combined, washed with saturated brine (100 mL×2), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain compound 3-2. MS (ESI) m/z: 227.1 [M+H ⁺ ].

Step B: To a solution of compound 3-2 (5.3 g, 23.38 mmol, 1 equiv) in pyridine (15 mL) at 25 degrees C was added portionwise 1-4 (7.75 g, 28.06 mmol, 1.2 equiv, p. Tosylate), react at 40 degrees Celsius for 16 hours, add 50 ml of water, ethyl acetate (100 ml × 3). The organic phases were combined, washed with saturated brine (100 mL×2), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain compound 3-3. MS (ESI) m/z: 309.1 [M+H ⁺ ].

Step C: To a solution of compound 3-3 (4.3 g, 13.93 mmol, 1 equiv) in toluene (25 mL) was added sodium ethoxide (1.90 g, 27.85 mmol, 2 equiv) in portions at 100-110 degrees Celsius After 16 hours of reaction, 150 ml of ethyl acetate and 100 ml of water were added, and the aqueous phase was washed twice with 25 ml of ethyl acetate, and then the pH of the aqueous phase was adjusted to 4 with 36% hydrochloric acid, and ethyl acetate (50 ml × 2) extraction. The organic phases were combined, washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound 3-4.

MS (ESI) m/z: 263.1 [M+H ⁺ ].

Step D: To a solution of compound 3-4 (1.27 g, 4.83 mmol, 1 equiv) in N,N-dimethylformamide (50 mL) was added N-bromosuccinimide (860.48 mg in portions) , 4.83 mmol, 1 equiv), reacted at 40 degrees Celsius for 12 hours, added 100 mL of water, and extracted with ethyl acetate (100 mL×2). The organic phases were combined, washed with saturated brine (50 mL×2), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain compound 3-5. MS (ESI) m/z: 298.9 [M+H ⁺ ].

Step E: A solution of 3-5 (763 mg, 2.56 mmol, 1 equiv) and boron isopropoxypinacol ester (1.26 g, 6.79 mmol, 1.39 mL, 2.65 equiv) in tetrahydrofuran ( 20 mL) was cooled to -70 degrees Celsius, then n-butyllithium (2.5 moles per liter, 2.45 mL, 2.39 equiv) was added dropwise, and the temperature was slowly raised to 20 degrees Celsius. After 12 hours of reaction under nitrogen protection and 20 degrees Celsius, 50 The reaction was quenched with mL of saturated ammonium chloride solution, diluted with 50 mL of ethyl acetate, and extracted with ethyl acetate (100 mL×2). The organic phases were combined, washed with saturated brine (100 mL×2), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound 3-6.

MS (ESI) m/z: 345.1 [M+H ⁺ ].

Step F: To a solution of compound 3-6 (1.65 g, 4.79 mmol, 1 equiv) in tetrahydrofuran (30 mL) was added dropwise hydrogen peroxide (2.17 g, 19.15 mmol, 1.84 mL) at 0 degrees Celsius, purity: 30%, 4 equiv), then 5 mL aqueous solution of sodium hydroxide (383.01 mg, 9.58 mmol, 2 equiv) was added, and after 12 hours of reaction at 25 degrees Celsius, the pH was adjusted to 6 with 1 mol/L hydrochloric acid solution , 20 mL of ethyl acetate was added, and extracted with ethyl acetate (50 mL×2). The organic phases were combined, washed with saturated brine (50 mL×2), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound 3-7. MS (ESI) m/z: 235.1 [M+H ⁺ ].

分子筛(5克)，25摄氏度下反应12小时后，加入50毫升水和50毫升二氯甲烷，用二氯甲烷(50毫升×2)萃取。合并有机相，饱和食盐水(50毫升×2)洗，无水硫酸钠干燥，过滤，浓缩，柱层析纯化得到化合物3-8。MS(ESI)m/z：346.0[M+H⁺]。Step G: To a solution of compounds 3-7 (1.09 g, 4.64 mmol, 1 equiv) and 2-4 (1.46 g, 9.29 mmol, 2 equiv) in dichloromethane (70 mL) was added copper acetate (1.27 g) , 6.97 mmol, 1.5 equiv), triethylamine (1.41 g, 13.93 mmol, 1.94 mL, 3 equiv) and

Molecular sieves (5 g), reacted at 25 degrees Celsius for 12 hours, added 50 mL of water and 50 mL of dichloromethane, and extracted with dichloromethane (50 mL×2). The organic phases were combined, washed with saturated brine (50 mL×2), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain compound 3-8. MS (ESI) m/z: 346.0 [M+H ⁺ ].

Step H: To a solution of compounds 3-8 (200 mg, 577.68 μmol, 1 equiv) and 2-3 (131.88 mg, 866.53 μmol, 1.5 equiv) in 1,4-dioxane (10 mL) was added 4,5-Bis(diphenylphosphine)-9,9-dimethylxanthene (66.85 mg, 115.54 μmol, 0.2 equiv), bis(dibenzylideneacetone)palladium (66.43 mg, 115.54 μmol , 0.2 equiv) and cesium carbonate (752.88 mg, 2.31 mmol, 4 equiv), react under nitrogen protection and 100 degrees Celsius for 12 hours, add 10 mL of water and 30 mL of ethyl acetate, ethyl acetate (25 mL × 2 )extraction. The organic phases were combined, washed with saturated brine (25 mL×2), dried over anhydrous sodium sulfate, filtered, concentrated, purified by preparative thin layer chromatography and then purified by preparative high performance liquid chromatography (formic acid system) to obtain compound 3. MS (ESI) m/z: 462.3 [M+H ⁺ ]; ¹ H NMR (400 MHz, CDCl ₃ ): δ 9.42 (br s, 1H), 8.52 (br s, 1H), 8.18 (br d, J =5.50Hz, 2H), 7.85-7.56(m, 4H), 7.32-7.17(m, 3H), 6.75-6.63(m, 2H), 4.22(brt, J=6.66Hz, 2H), 2.84(br d , J=6.85Hz, 2H), 2.65 (brd, J=6.72Hz, 2H), 1.53 (br s, 6H).

Example 4: Formate salt of compound 4

Step A: Compound 4-1 (5 g, 26.58 mmol, 3.97 mL, 1 equiv) was added to diethyl carbonate (34.13 g, 288.87 mmol, 35 mL, 10.87 equiv), cooled to 0°C, and then Sodium hydrogen (2.13 g, 53.16 mmol, 60%, 2 equiv) was added portionwise at 0 degrees Celsius. The mixture was heated to 85°C and stirred for 2 hours. The reaction solution was quenched with hydrochloric acid (2 mol per liter), and the pH was adjusted to 2 with hydrochloric acid, then water (50 mL) was added, extracted with ethyl acetate (30 mL×3), and the combined organic phases were washed with saturated brine (30 mL). mL), washed with anhydrous sodium sulfate, filtered and concentrated to give compound 4-2. MS (ESI) m/z: 261.0 [M+H ⁺ ].

Step B: To a solution of compound 4-2 (7.81 g, 30.01 mmol, 1 equiv) in pyridine (15 mL) was added 1-4 (9.81 g, 36.02 mmol, 1.2 equiv, p-toluenesulfonate), The mixture was heated to 40 degrees Celsius and stirred for 12 hours. Water (50 mL) was added to the reaction solution, extracted with ethyl acetate (50 mL×4), the combined organic phases were washed with saturated brine (50 mL×2), dried over anhydrous sodium sulfate, filtered and concentrated to obtain Compound 4-3. MS (ESI) m/z: 343.1 [M+H ⁺ ].

Step C: To a solution of compound 4-3 (1 g, 2.92 mmol, 1 equiv) in toluene (15 mL) was added sodium ethoxide (596.39 mg, 8.76 mmol, 3 equiv) and the mixture was heated to 100 to 110 reaction for 12 hours between degrees Celsius. The reaction solution was poured into water (60 mL) and the pH was adjusted to between 6 and 7 with hydrochloric acid (2 mol per liter). The aqueous phase was extracted with ethyl acetate (60 mL×2), the combined organic phases were washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered and concentrated to obtain compound 4-4.

MS (ESI) m/z: 297.0 [M+H ⁺ ].

Step D: To a solution of compound 4-4 (0.78 g, 2.63 mmol, 1 equiv) in N,N-dimethylformamide (12 mL) was added N-bromosuccinimide (445.19 mg, 2.50 mmol, 0.95 equiv), the mixture was heated to 50 degrees Celsius and stirred for 4 hours. The reaction solution was poured into water (60 mL), the aqueous phase was extracted with ethyl acetate (60 mL×2), the combined organic phases were washed with saturated brine (60 mL×2), dried over anhydrous sodium sulfate, filtered, Concentrated and purified by column chromatography to obtain compound 4-5.

Step E: To compound 4-5 (360 mg, 1.09 mmol, 1 equiv) and isopropoxy pinacol boronate (405.60 mg, 2.18 mmol, 444.74 μl, 2 equiv) at -78 degrees Celsius To the tetrahydrofuran solution (10 mL) was added n-butyllithium (2.5 mol/L, 872.00 μL, 2 equiv.), the reaction solution was naturally heated to 25°C and stirred for 12 hours. Saturated aqueous ammonium chloride solution (60 mL) was poured into the reaction solution, the aqueous phase was extracted with ethyl acetate (60 mL×2), the combined organic phases were washed with saturated brine (60 mL), and dried over anhydrous sodium sulfate , filtered and concentrated to give compound 4-6. MS (ESI) m/z: 297.1 [M+H ⁺ ].

Step F: To a solution of compound 4-6 (450 mg, 1.52 mmol, 1 equiv) in tetrahydrofuran (10 mL) was added hydrogen peroxide (689.37 mg, 6.08 mmol, 584.21 μL, 30%, 4 equiv.) After cooling to between 0 and 5 degrees Celsius, additional sodium hydroxide (4 moles per liter, 1.52 mL, 4 equiv.) was added and the mixture was stirred at 25 degrees Celsius for 12 hours. The reaction solution was poured into water (60 mL), the aqueous phase was extracted with ethyl acetate (80 mL), the combined organic phases were washed with saturated brine (80 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and thin-layered Chromatographic purification affords compound 4-7. MS (ESI) m/z: 269.0 [M+H ⁺ ].

分子筛(1克)。该体系用氧气置换若干次，然后该混合物在25摄氏度反应12小时。将反应液过滤，滤液浓缩，薄层色谱纯化得到化合物4-8。Step G: To a solution of compound 4-7 (240 mg, 894.74 μmol, 1 equiv) and compound 2-4 (211.20 mg, 1.34 mmol, 1.5 equiv) in dichloromethane (10 mL) was added copper acetate (243.77 g mg, 1.34 mmol, 1.5 equiv), triethylamine (271.62 mg, 2.68 mmol, 373.61 μl, 3 equiv) and

Molecular sieves (1 g). The system was replaced with oxygen several times, and then the mixture was reacted at 25 degrees Celsius for 12 hours. The reaction solution was filtered, the filtrate was concentrated, and purified by thin layer chromatography to obtain compound 4-8.

MS (ESI) m/z: 380.0, 382.0 [M+H ⁺ ].

Step H: To a solution of compound 4-8 (130 mg, 342.32 μmol, 1 equiv) and compound 2-3 (104.20 mg, 684.64 μmol, 2 equiv) in 1,4-dioxane (6 mL) Add bis(dibenzylideneacetone)palladium (19.68 mg, 34.23 μmol, 0.1 equiv), 4,5-bis(diphenylphosphine)-9,9-dimethylxanthene (19.81 mg, 34.23 μmol) moles, 0.1 equiv) and cesium carbonate (223.07 mg, 684.64 micromoles, 2 equiv). The system was replaced with nitrogen and then heated to 100 degrees Celsius for 12 hours. The reaction solution was concentrated, purified by column chromatography, and then purified by preparative high performance liquid chromatography (formic acid system) to obtain the formate salt of compound 4. MS (ESI) m/z: 496.3 [M+H ⁺ ]; ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 9.48 (s, 1H), 8.24-8.13 (m, 3H), 8.02-7.95 ( m, 2H), 7.72-7.59 (m, 4H), 6.68 (dd, J=5.6Hz, 2.4Hz, 1H), 6.48 (d, J=2.4Hz, 1H), 4.23 (t, J=7.2Hz, 2H), 2.84 (t, J=7.2Hz, 2H), 2.64-2.57 (m, 2H), 1.40 (s, 6H).

Example 5: Compound 5

Step A: Compound 5-1 (3.13 g, 20.71 mmol, 1 equiv) and ethyl acetate (5.47 g, 62.14 mmol, 6.08 mL, 3 equiv) were dissolved in tetrahydrofuran (20 mL) and cooled to 0°C , and sodium hydrogen (1.66 g, 41.43 mmol, 60%, 2 equiv) was added portionwise at 0 degrees Celsius. The mixture was heated to 70 degrees Celsius and stirred for 4 hours. The reaction solution was quenched with a mixed solution of acetic acid and water (acetic acid: water = 1: 1), and the pH was adjusted to 7, the organic phase was separated, the aqueous phase was extracted with ethyl acetate (30 mL × 3), and the combined organic phases were Washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered and concentrated to obtain compound 5-2.

MS (ESI) m/z: 208.1 [M+H ⁺ ].

Step B: To a solution of compound 5-2 (5.52 g, 26.64 mmol, 1 equiv) in pyridine (15 mL) was added 1-4 (8.70 g, 31.97 mmol, 1.2 equiv, p-toluenesulfonate), The mixture was heated to 40 degrees Celsius and stirred for 12 hours. Water (50 mL) was added to the reaction solution, extracted with ethyl acetate (50 mL×4), the combined organic phases were washed with saturated brine (50 mL×2), dried over anhydrous sodium sulfate, filtered and concentrated to obtain Compound 5-3. MS (ESI) m/z: 290.1 [M+H ⁺ ].

Step C: To a solution of compound 5-3 (5.6 g, 19.36 mmol, 1 equiv) in toluene (50 mL) was added sodium ethoxide (3.95 g, 58.07 mmol, 3 equiv), and the mixture was heated to 110 degrees Celsius to react 12 hours. Then water (3.00 g, 166.53 mmol, 3 mL, 8.60 equiv) was added and the reaction was continued at 100 degrees Celsius for 12 hours. The reaction solution was poured into water (50 mL), and its pH was adjusted to 4 with hydrochloric acid (2 mol per liter). The organic phase was separated, the aqueous phase was extracted with isopropanol/dichloromethane (1/10, 50 mL×4), the combined organic phases were washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered and concentrated , to obtain compound 5-4. MS (ESI) m/z: 244.0 [M+H ⁺ ].

Step D: To a solution of compound 5-4 (3.97 g, 16.32 mmol, 1 equiv) in N,N-dimethylformamide (50 mL) was added N-bromosuccinimide (3.49 g, 19.58 g mmol, 1.2 equiv), the mixture was heated to 50 degrees Celsius and stirred for 12 hours. The reaction solution was poured into water (200 mL), the aqueous phase was extracted with ethyl acetate (100 mL×3), the combined organic phases were washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. Column chromatography gave compound 5-5. MS (ESI) m/z: 277.9, 279.9 [M+H ⁺ ].

Step E: To compound 5-5 (2.82 g, 10.14 mmol, 1 equiv) and isopropoxy pinacol boronate (3.77 g, 20.28 mmol, 4.14 mL, 2 equiv) at -78 to -65 degrees Celsius ) in tetrahydrofuran (30 mL) was added n-butyllithium (2.5 mol/L, 8.11 mL, 2 equiv.), the reaction solution was naturally heated to 25°C and stirred for 12 hours. Saturated aqueous ammonium chloride solution (50 mL) was poured into the reaction solution, the aqueous phase was extracted with ethyl acetate (50 mL×3), the combined organic phases were washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, Filtration and concentration gave compound 5-6. MS (ESI) m/z: 325.8 [M+H ⁺ ].

Step F: To a solution of compound 5-6 (1 g, 3.07 mmol, 1 equiv) in tetrahydrofuran (15 mL) was added hydrogen peroxide (1.39 g, 12.30 mmol, 1.18 mL, 30%, 4 equiv), the system was cooled To between 0 and 5 degrees Celsius, additional sodium hydroxide (4 moles per liter, 3.07 mL, 4 equiv) was added and the mixture was stirred at 25 degrees Celsius for 12 hours. Saturated sodium bisulfite (5 mL) was added to the reaction solution, the mixture was poured into water (60 mL), the aqueous phase was extracted with ethyl acetate (60 mL×2), the organic phases were combined and dried over anhydrous sodium sulfate , filtered, concentrated, and purified by column chromatography to obtain compound 5-7. MS (ESI) m/z: 216.1 [M+H ⁺ ].

分子筛(1克)。该体系用氧气置换若干次，然后该混合物在25摄氏度反应12小时。将反应液倒入水(60毫升)中，水相用乙酸乙酯(60毫升×2)萃取，合并的有机相用食盐水(60毫升)洗涤，无水硫酸钠干燥，过滤，浓缩，薄层色谱纯化得到化合物5-8。Step G: To a solution of compound 5-7 (300 mg, 1.39 mmol, 1 equiv) and compound 2-4 (328.98 mg, 2.09 mmol, 1.5 equiv) in dichloromethane (15 mL) was added copper acetate (379.72 mg, 2.09 mmol, 1.5 equiv), triethylamine (423.09 mg, 4.18 mmol, 581.97 μl, 3 equiv) and

Molecular sieves (1 g). The system was replaced with oxygen several times, and then the mixture was reacted at 25 degrees Celsius for 12 hours. The reaction solution was poured into water (60 mL), the aqueous phase was extracted with ethyl acetate (60 mL×2), the combined organic phases were washed with brine (60 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and thinned. Purification by layer chromatography gave compound 5-8.

MS (ESI) m/z: 326.9, 328.9 [M+H ⁺ ].

Step H: To a solution of compound 5-8 (200 mg, 612.03 μmol, 1 equiv) and compound 2-3 (93.15 mg, 612.03 μmol, 1 equiv) in 1,4-dioxane (8 mL) Add bis(dibenzylideneacetone)palladium (35.19 mg, 61.20 μmol, 0.1 equiv), 4,5-bis(diphenylphosphine)-9,9-dimethylxanthene (35.41 mg, 61.20 μg) moles, 0.1 equiv) and cesium carbonate (398.82 mg, 1.22 mmol, 2 equiv). The system was replaced with nitrogen and then heated to 90 degrees Celsius for 3 hours. The reaction solution was concentrated and purified by preparative high performance liquid chromatography (formic acid system first, then basic system) to obtain compound 5. MS (ESI) m/z: 443.4 [M+H ⁺ ];

¹ H NMR (400 MHz, DMSO-d ₆ ) δ=9.43 (s, 1H), 8.64 (d, J=2.0 Hz, 1H), 8.30 (d, J=1.6 Hz, 1H), 8.19 (d, J= 5.6Hz, 1H), 8.16 (d, J=6.0Hz, 1H), 7.86 (s, 1H), 7.71-7.61 (m, 2H), 6.67 (dd, J=2.0, 6.0Hz, 1H), 6.45 ( d, J=2.0Hz, 1H), 5.09(br s, 1H), 4.21(t, J=7.2Hz, 2H), 2.88-2.77(m, 2H), 2.60(m, 2H), 2.28(s, 3H), 1.39 (s, 6H).

Example 6: Compound 6

Step A: Ethyl acetate (17.6 g, 199.77 mmol, 3.0 equiv) was added to toluene (50 mL) and sodium ethoxide (9.06 g, 133.18 mmol, 2.0 equiv) was added at 20 degrees Celsius. After the addition was complete, the mixture was stirred at 20°C for 1 hour. Compound 6-1 (10 g, 66.59 mmol, 1.0 equiv) was added in three batches and stirred at 100 degrees Celsius for 12 hours after the addition was complete. The reaction solution was cooled to 25°C, adjusted to pH 6 with glacial acetic acid, water (50 mL) was added, extracted with toluene (30 mL×2), the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated to obtain compound 6-2.

Step B: Compound 6-2 (10.05 g, 48.73 mmol, 1.0 equiv) was added to pyridine (25 mL) followed by compound 1-4 (16.10 g, 58.48 mmol, 1.2 equiv). Stir at 40 degrees Celsius for 12 hours. The reaction solution was cooled to 20 degrees Celsius, water (50 mL) was added, and the mixture was extracted with toluene (40 mL×2). The organic phases were combined, washed with water (50 mL), dried over anhydrous sodium sulfate, and concentrated to obtain compound 6-3.

Step C: Compound 6-3 (12.54 g, 43.49 mmol, 1.0 equiv) was dissolved in toluene (80 mL) and sodium ethoxide (8.88 g, 130.47 mmol, 3.0 equiv) was added portionwise with constant stirring. Stir at 100-110 degrees Celsius for 12 hours after the addition is complete. The reaction solution was cooled to 20 degrees Celsius, and the pH was adjusted to 6 with 1 M hydrochloric acid per liter. 50 mL of water was added, extracted with ethyl acetate (50 mL×3), the organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, and concentrated to obtain compound 6-4. MS (ESI) m/z: 271.2 [M+H ⁺ ].

Step D: Sodium hydroxide (16.69 g, 417.27 mmol, 10 equiv) was dissolved in water (25 mL) and the resulting solution was added to compound 6-4 (11.28 g, 41.73 mmol, 1.0 equiv) in methanol (50 ml) solution. Stir at 40°C for 12 hours. Methanol was removed, water (25 mL) was added and washed with ethyl acetate (25 mL x 3). The pH of the aqueous phase was adjusted to 4 with concentrated hydrochloric acid and filtered. The filter cake was washed with water (25 mL×3) and dried to obtain compound 6-5. MS (ESI) m/z: 243.1 [M+H ⁺ ].

Step E: Compound 6-5 (10.01 g, 41.32 mmol, 1.0 equiv) was dissolved in N,N-dimethylformamide (120 mL) and bromosuccinimide (7.35 g, 41.32 mmol, 1.0 equiv) and sodium hydroxide (1.98 g, 49.58 mmol, 1.2 equiv). Stir at 40°C for 12 hours. The reaction solution was cooled to 20-25 degrees Celsius, water (300 mL) was added, stirred for 10 minutes, filtered, and the filter cake was washed with water (20 mL×2) and dried to obtain compound 6-6. MS (ESI) m/z: 277.2 [M+H ⁺ ].

Step F: Compound 6-6 (1.4 g, 5.05 mmol, 1.0 equiv), boron isopropoxy pinacol ester (2.49 g, 13.39 mmol, 2.65 equiv) was added to dry tetrahydrofuran (30 mL), Replace nitrogen three times, stir, and cool to -70 to -60 degrees Celsius. n-Butyllithium (2.5 moles per liter of n-heptane solution, 4.83 mL, 2.39 equiv) was added dropwise at -70 to -60 degrees Celsius. After the addition, the mixture was stirred at -70 to -60 degrees Celsius for 1 hour. It was then stirred at 25 degrees Celsius for 12 hours. The reaction solution was added dropwise to 60 mL of saturated ammonium chloride solution, extracted with ethyl acetate (40 mL×3), the organic phases were combined, washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, and concentrated to obtain compound 6- 7.

Step G: Compound 6-7 (2.08 g, 6.42 mmol, 1.0 equiv) and hydrogen peroxide (2.91 g, 25.66 mmol, 30% concentration, 4 equiv) were added to tetrahydrofuran (20 mL), stirred, and cooled to 0~ 5 degrees Celsius. 4 mol/L aqueous sodium hydroxide solution (6.42 mL, 4.0 equiv) was added dropwise at 0-5 degrees Celsius. After the addition was complete, the mixture was stirred at 25°C for 12 hours. Saturated aqueous sodium bisulfite solution (20 mL) was added to the reaction solution, the pH was adjusted to 7 with 2 mol/L hydrochloric acid, extracted with ethyl acetate (30 mL×2), washed with saturated brine (30 mL), and anhydrous Dry over sodium sulfate, filter, concentrate, and purify by column chromatography to obtain compound 6-8. MS (ESI) m/z: 215.1 [M+H ⁺ ].

分子筛(3.5克)。在25摄氏度下氧气氛围搅拌12小时。过滤，滤液加水(30毫升)稀释，乙酸乙酯萃取(30毫升×3)，合并有机相，饱和食盐水洗(40毫升)，无水硫酸钠干燥，浓缩，柱层析纯化得到化合物6-9。MS(ESI)m/z：326.0[M+H⁺]。Step H: Compound 6-8 (336 mg, 1.57 mmol, 1 equiv), compound 2-4 (494.12 mg, 3.14 mmol, 2 equiv) were dissolved in dichloromethane (20 mL), followed by copper acetate (427.75 mg, 2.36 mmol, 1.5 equiv), triethylamine (476.60 mg, 4.71 mmol, 655.58 μl, 3 equiv) and

Molecular sieves (3.5 g). It was stirred at 25°C for 12 hours under an oxygen atmosphere. The filtrate was filtered, diluted with water (30 mL), extracted with ethyl acetate (30 mL×3), the organic phases were combined, washed with saturated brine (40 mL), dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to obtain compound 6-9 . MS (ESI) m/z: 326.0 [M+H ⁺ ].

Step I: Compound 6-9 (200 mg, 613.89 μmol, 1 equiv), compound 2-3 (186.86 mg, 1.23 mmol, 2 equiv) were dissolved in 1,4-dioxane (20 mL) , followed by adding bis(dibenzylideneacetone)palladium (70.60 mg, 112.78 μmol, 0.2 equiv), 4,5-bis(diphenylphosphine)-9,9-dimethylxanthene (71.04 mg, 122.78 μmol, 0.2 equiv), cesium carbonate (800.07 mg, 2.46 mmol, 4 equiv). The nitrogen was replaced three times, and the temperature was raised to 100 degrees Celsius under nitrogen protection and stirred for 12 hours. Cool to 25 degrees Celsius, add water (50 mL) to quench, extract with ethyl acetate (50 mL×2), combine the organic phases, wash with saturated brine (40 mL), dry over anhydrous sodium sulfate, and concentrate to obtain crude product, crude product After separation by preparative high performance liquid chromatography and lyophilization, compound 6 was obtained.

MS (ESI) m/z: 442.2 [M+H ⁺ ].

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 9.50 (s, 1H), 8.21-8.15 (m, 3H), 7.71-7.67 (m, 2H), 7.55 (s, 1H), 7.47 (d, J =8.0Hz, 1H), 7.21(t, J=7.8Hz, 1H), 7.06(d, J=7.6Hz, 1H), 6.67(dd, J=2.4, 5.6Hz, 1H), 6.46(d, J =2.0Hz, 1H), 4.17(t, J=7.0Hz, 2H), 2.80(t, J=7.2Hz, 2H), 2.61-2.54(m, 2H), 2.26(s, 3H), 1.40(s , 6H).

Example 7: Compound 7

Step A: Lithium hexamethyldisilazide (1 mole per liter, 728.32 ml, 3 equiv.) was added to a three-necked flask, the reaction solution was cooled to -78 degrees Celsius, and then ethyl acetate (64.17 g, 728.32 g) was added dropwise. mmol, 71.30 mL, 3 equiv). The reaction solution was stirred at -78°C for half an hour. 7-1 (35 g, 242.77 mmol, 32.41 mL, 1 equiv) was dissolved in 400 mL tetrahydrofuran at -78 degrees Celsius and added dropwise to the reaction. The reaction was stirred at -78°C for 1 hour. The reaction was quenched with 150 mL of saturated ammonium chloride solution at -70 degrees Celsius, diluted with 400 mL of water and extracted with ethyl acetate (500 mL x 2). The organic phases were combined, washed with saturated brine (400 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound 7-2.

Step B: 7-2 (14.5 g, 72.42 mmol, 1 equiv) was dissolved in pyridine (140 mL) and 1-4 (31.55 g, 115.87 mmol, 1.6 equiv, p-toluenesulfonate) was added. The mixture was replaced with nitrogen three times and stirred at 40°C for 12 hours. Add 300 mL of water to dilute, and extract with ethyl acetate (300 mL×2). The organic phases were combined, washed with saturated brine (100 mL×2), dried over anhydrous sodium sulfate, filtered and concentrated in vacuo to obtain compound 7-3. MS (ESI) m/z: 283.1 [M+H ⁺ ].

Step C: Compound 7-3 (6.3 g, 22.31 mmol, 1 equiv) was dissolved in toluene (80 mL), sodium hydride (2.68 g, 66.94 mmol, 60% pure, 3 equiv) was added, and the mixture was heated under nitrogen atmosphere. The mixture was heated to 110 degrees Celsius for 12 hours. To the mixture were added 250 mL of ethyl acetate and 250 mL of water, the organic phase was washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered and concentrated. The aqueous phase was adjusted to pH 4 with 4 mol/L hydrochloric acid, extracted with ethyl acetate (100 mL×2), the organic phases were combined, washed with saturated brine (80 mL), dried over anhydrous sodium sulfate, filtered and concentrated. Combining the two parts yields 7-4. MS (ESI) m/z: 265.1 [M+H ⁺ ].

Step D: 7-4 (7 g, 26.48 mmol, 1 equiv) was dissolved in water (20 mL) and methanol (40 mL) and sodium hydroxide (3.18 g, 79.45 mmol, 3 equiv) was added under nitrogen atmosphere ) and stirred at 60°C for 5 hours. The solution was adjusted to pH 6 with 4 mol/L hydrochloric acid, filtered, and the filter cake was dried to obtain compound 7-5. MS (ESI) m/z: 237.2 [M+H ⁺ ].

Step E: Dissolve 7-5 (5 g, 21.16 mmol, 1 equiv) in dimethylformamide (20 mL) and add bromosuccinimide (4.52 g, 25.40 mmol, 1.2 equivalents). The mixture was stirred at 25 degrees Celsius for 12 hours. Add water (100 mL) to dilute, and extract with ethyl acetate (100 mL×2). The organic phases were combined, washed with saturated brine (50 mL×2), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain compound 7-6. MS (ESI) m/z: 271, 273 [M+H ⁺ ].

Step F: Compound 7-6 (4.26 g, 15.71 mmol, 1 equiv) was dissolved in tetrahydrofuran (40 mL), and n-butyllithium (2.5 mol/L, 18.85 mL, 3 equivalents). The mixture was stirred at -70 degrees Celsius for 1 hour, isopropoxy pinacol boron ester (7.60 g, 40.85 mmol, 8.33 mL, 2.6 equiv) was added dropwise maintaining the temperature. Warm slowly to 0 degrees Celsius and stir for 1 hour. The reaction solution was poured into saturated aqueous ammonium chloride solution (100 mL) at 0°C, extracted with ethyl acetate (100 mL×2), the organic phases were combined, washed with saturated brine (50 mL×2), and dried over anhydrous sodium sulfate. , filtered and concentrated to obtain compound 7-7.

MS (ESI) m/z: 319.1 [M+H ⁺ ].

Step G: 7-7 (5.2 g, 16.34 mmol, 1 equiv) was dissolved in tetrahydrofuran (50 mL) followed by hydrogen peroxide (4.63 g, 40.85 mmol, 3.92 mL, 30% pure, 2.5 equiv) . The reaction was cooled to 0-5 degrees Celsius, and an aqueous solution of sodium hydroxide (4 moles per liter, 10.21 mL, 2.5 equiv) was added dropwise. The reaction solution was stirred at 25 degrees Celsius for 12 hours. Sodium sulfite (1 g) was added to quench, the pH was adjusted to 8 with 4 moles of hydrochloric acid per liter, and the mixture was stirred at 20 degrees Celsius for 1 hour. Add 200 mL of water to dilute, extract with ethyl acetate (300 mL×2), wash the organic phase with saturated brine (200 mL), dry over anhydrous sodium sulfate, filter and concentrate to obtain compound 7-8.

MS (ESI) m/z: 209.1 [M+H ⁺ ].

Step H: Compounds 7-8 (800 mg, 3.84 mmol, 1 equiv) and 7-9 (757.92 mg, 5.76 mmol, 1.5 equiv) were dissolved in N,N-dimethylformamide (8 mL) , potassium carbonate (1.59 g, 11.52 mmol, 3 equiv) was added under nitrogen. The mixture was reacted at 120 degrees Celsius for 12 hours. Add 60 mL of water to dilute, extract with ethyl acetate (80 mL×2), wash the organic phase with 60 mL of saturated brine, dry over anhydrous sodium sulfate, filter, concentrate, and purify by column chromatography to obtain compound 7-10. MS (ESI) m/z: 320.1 [M+H ⁺ ].

Step I: Compound 7-10 (1.05 g, 3.28 mmol, 1 equiv), 7-11 (426.68 mg, 3.61 mmol, 96.31 μl, 1.1 equiv), 4,5-bis(diphenylphosphine) -9,9-Dimethylxanthene (189.99 mg, 328.35 μmol, 0.1 equiv), cesium carbonate (3.21 g, 9.85 mmol, 3 equiv) and palladium acetate (73.72 mg, 328.35 μmol, 0.1 equiv) Dissolved in dioxane (10 mL). The reaction was carried out at 100 degrees Celsius for 3 hours under nitrogen atmosphere. Add 40 mL of water to dilute, extract with ethyl acetate (70 mL×2), wash the organic phase with 60 mL of saturated brine, dry over anhydrous sodium sulfate, filter and concentrate. Add 70 ml of ethyl acetate to dissolve and add 1 g of mercapto silica gel, and stir at 25 degrees Celsius for 1 hour. Filtration and concentration gave compound 7-12. MS (ESI) m/z: 402.1 [M+H ⁺ ].

Step J: Dissolve sodium hydroxide (179.35 mg, 4.48 mmol, 1 equiv) in water (1.8 mL), add ethanol (18 mL) at 25°C, 7-12 (1.80 g, 4.48 mmol, 1 equiv) and dimethyl sulfoxide (525.46 mg, 6.73 mmol, 525.46 μl, 1.5 equiv). Hydrogen peroxide (762.44 mg, 6.73 mmol, 646.14 μL, 30% purity, 1.5 equiv) was diluted with water (0.8 mL) and added dropwise to the reaction. Stir at 25°C for 2 hours. Add 100 mL of water to dilute, extract with ethyl acetate (150 mL×2), wash the organic phase with 100 mL of saturated brine, dry over anhydrous sodium sulfate, filter, concentrate, and purify by preparative high performance liquid chromatography (formic acid condition) to obtain compound 7. MS (ESI) m/z: 420.1 [M+H ⁺ ];

¹ H NMR (400 MHz, DMSO-d ₆ ): δ 9.11 (s, 1H), 8.08-7.98 (m, 2H), 7.91-7.77 (m, 2H), 7.39-7.21 (m, 3H), 6.47 ( dd, J=2.0, 6.0Hz, 1H), 6.29 (d, J=2.0Hz, 1H), 4.05 (br t, J=7.2Hz, 2H), 3.88-3.77 (m, 2H), 3.29 (br s , 4H), 2.78-2.64 (m, 3H), 1.73-1.59 (m, 4H).

Example 8: Compound 8

Step A: Compound 8-1 (3 g, 26.53 mmol, 1 equiv), compound 8-2 (5.74 g, 79.59 mmol, 7.07 mL, 3 equiv), cesium carbonate (17.29 g, 53.06 mmol, 2 equiv.) was dissolved in N,N-dimethylformamide (40 mL) and the resulting mixture was purged with nitrogen three times and stirred at 100 degrees Celsius for 5 hours. Water (50 mL) was added to quench the reaction and extracted with ethyl acetate (50 mL x 2). The organic phases were combined, washed with saturated sodium chloride solution (50 mL×2), dried over anhydrous sodium sulfate, filtered and concentrated to obtain compound 8-3. MS (ESI) m/z: 186.2 [M+H ⁺ ].

Step B: Compound 8-3 (3 g, 16.20 mmol, 1 equiv) was dissolved in methanol (30 mL), added wet palladium on carbon (500 mg, 10%), replaced with hydrogen, at 25°C under hydrogen atmosphere The reaction was carried out for 12 hours. Filtration and concentration gave compound 8-4.

Step C: Compound 1-11 (0.3 g, 1.39 mmol, 1 equiv) was dissolved in N,N-dimethylformamide (20 mL), to the solution was added 7-9 (219.99 mg, 1.67 mmol) , 1.2 equiv), potassium carbonate (577.86 mg, 4.18 mmol, 3 equiv), and the resulting mixture was stirred at 80 degrees Celsius for 12 hours. Water (50 mL) was added to quench the reaction. Ethyl acetate (20 mL×2) was extracted, the organic phase was washed with saturated sodium chloride solution (30 mL×2), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column separation to obtain compound 2-5.

MS (ESI) m/z: 327.1 [M+H ⁺ ].

Step D: Compound 2-5 (100 mg, 0.306 mmol, 1 equiv) and compound 8-4 (49.87 mg, 0.321 mmol, 1.05 equiv), bis(dibenzylideneacetone)palladium (17.60 mg, 30.6 micromolar, 0.1 equiv), 4,5-bis(diphenylphosphine)-9,9-dimethylxanthene (35.41 mg, 61.20 micromolar, 0.2 equiv) and cesium carbonate (199.41 mg, 612.03 micromolar) , 2 equiv) was dissolved in 1,4-dioxane (15 mL). The system was replaced with nitrogen, then heated to 100 degrees Celsius and reacted under a nitrogen atmosphere for 12 hours. Water (50 mL) was added to quench the reaction, followed by extraction with ethyl acetate (30 mL×2). The organic phase was washed with saturated sodium chloride solution (30 mL×2), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by preparative high performance liquid chromatography (hydrochloric acid condition) to obtain compound 8. MS (ESI) m/z: 446.4 [M+H ⁺ ];

¹ H NMR (400 MHz, DMSO-d ₆ ): δ 10.49 (s, 1H), 8.16-8.14 (m, 1H), 7.97-7.95 (m, 1H), 7.86 (s, 1H), 7.81 (d, J=7.6Hz, 1H), 7.53 (d, J=7.2Hz, 1H), 7.48 (s, 1H), 6.86-6.83 (m, 1H), 6.72 (s, 1H), 4.30-4.19 (m, 2H) ), 4.01 (s, 1H), 2.96-2.93 (m, 2H), 2.67-2.62 (m, 2H), 2.33 (s, 3H), 1.08 (s, 6H).

Example 9: Formate salt of compound 9

Step A: To a solution of compound 2-5 (200 mg, 612.03 μmol, 1 equiv) and compound 9-1 (120.27 mg, 795.64 μmol, 1.3 equiv) in 1,4-dioxane (8 mL) Add bis(dibenzylideneacetone)palladium (35.19 mg, 61.20 μmol, 0.1 equiv), 4,5-bis(diphenylphosphine)-9,9-dimethylxanthene (35.41 mg, 61.20 μg) moles, 0.1 equiv) and cesium carbonate (398.82 mg, 1.22 mmol, 2 equiv). The system was replaced with nitrogen and then heated to 100 degrees Celsius for 12 hours. The reaction solution was poured into water (80 mL) and stirred for 3 minutes. The aqueous phase was extracted with ethyl acetate (80 mL×2), and the organic phases were combined, washed with saturated brine (80 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound 9-2. MS (ESI) m/z: 442.3 [M+H ⁺ ].

Step B: Ammonia gas was bubbled into ethylene glycol (15 mL) at -78°C and kept ventilated for 5 minutes. Compound 9-2 (390 mg, 883.39 μmol, 1 equiv) was dissolved in the above solution. The mixture was then poured into a teflon jar, heated to 80 degrees Celsius and stirred for 12 hours. The reaction solution was poured into water (60 mL) and stirred for 3 minutes. The aqueous phase was extracted with ethyl acetate (60 mL×2), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by preparative high performance liquid chromatography (formic acid system) to obtain the formate salt of compound 9.

MS (ESI) m/z: 427.3 [M+H ⁺ ];

¹ H NMR (400 MHz, DMSO-d ₆ ): δ 9.01 (s, 1H), 8.24 (s, 1H), 8.05-7.98 (m, 2H), 7.88-7.75 (m, 2H), 7.63 (t, J=8.0Hz, 1H), 7.54 (d, J=8.0Hz, 1H), 7.34-7.21 (m, 3H), 7.05 (d, J=7.2Hz, 1H), 6.49 (dd, J=6.0Hz, 2.0Hz, 1H), 6.29(s, 1H), 4.19(t, J=7.2Hz, 2H), 2.86(t, J=7.2Hz, 2H), 2.68-2.54(m, 2H), 2.25(s, 3H).

Example 10: Compound 10

Step A: To a solution of compound 2-5 (100 mg, 306.02 μmol, 1 equiv) and compound 10-1 (60.66 mg, 367.22 μmol, 1.2 equiv) in 1,4-dioxane (3 mL) Add bis(dibenzylideneacetone)palladium (17.6 mg, 30.6 μmol, 0.1 equiv), 4,5-bis(diphenylphosphine)-9,9-dimethylxanthene (17.71 mg, 30.60 μg) moles, 0.1 equiv) and cesium carbonate (199.41 mg, 612.03 micromoles, 2 equiv). The system was replaced with nitrogen, heated to 65 degrees Celsius for 4 hours, and then heated to 100 degrees Celsius for 12 hours. Add water (20 mL) to dilute, extract with ethyl acetate (20 mL×3), combine the organic phases, extract with 2 mol/L hydrochloric acid (10 mL×4), combine the aqueous phases, use 4 mol/L sodium hydroxide The aqueous solution was adjusted to pH 7 and extracted with ethyl acetate (20 mL x 3). The organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound 10-2.

MS (ESI) m/z: 456.1 [M+H ⁺ ].

Step B: Bubble ammonia gas into 10 mL of ethylene glycol at -78 degrees Celsius for 15 minutes, then add compound 10-2 (147.54 mg, 323.91 μmol, 1.2 equiv), add this solution to a stuffy tank, and add After reacting at 100 degrees Celsius for 12 hours, add 20 ml of water and 50 ml of ethyl acetate at 25 degrees Celsius, extract twice with 50 ml of ethyl acetate, combine the organic phases, wash with saturated brine (25 ml × 2) , dried over anhydrous sodium sulfate, filtered, concentrated, and purified by preparative high performance liquid chromatography (basic) to obtain compound 10.

MS (ESI) m/z: 427.2 [M+H ⁺ ];

¹ H NMR (400 MHz, DMSO-d ₆ ) δ=9.01 (s, 1H), 8.02 (d, J=6.0 Hz, 1H), 7.73 (t, J=1.6 Hz, 1H), 7.65-7.61 (m, 1H), 7.55-7.52(m, 2H), 7.24(t, J=8.0Hz, 1H), 7.04(d, J=7.2Hz, 1H), 6.82(d, J=7.6Hz, 1H), 6.50( dd, J=2.4, 6.0Hz, 1H), 6.27 (d, J=2.0Hz, 1H), 4.19 (t, J=7.2Hz, 2H), 3.00-2.84 (m, 8H), 2.63-2.55 (m , 2H), 2.24 (s, 3H).

Example 11: Compound 11

Compound 2-5 (200 mg, 612.03 μmol, 1 equiv) was dissolved in 1,4-dioxane (5 mL), followed by compound 11-1 (150.75 mg, 918.05 μmol, 1.5 equiv), bis(dibenzylideneacetone)palladium (35.19 mg, 61.20 μmol, 0.1 equiv), 4,5-bis(diphenylphosphine)-9,9-dimethylxanthene (70.83 mg, 122.41 μmol, 0.2 equiv), cesium carbonate (598.24 mg, 1.84 mmol, 3 equiv). The nitrogen was replaced three times and then stirred at 100 degrees Celsius for 2 hours under nitrogen protection. Cool to 25 degrees Celsius, add 1 g of silica gel powder, stir for 15 minutes, and filter through a diatomaceous earth pad. The filtrate was adjusted to pH 4 with 1 M hydrochloric acid per liter, diluted with water (20 mL), and washed with ethyl acetate (20 mL×3). The aqueous phase was adjusted to pH 9 with 1 mol/liter aqueous sodium hydroxide solution, extracted with ethyl acetate (20 mL×3), the organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, concentrated, and prepared Compound 11 was purified by high performance liquid chromatography. MS (ESI) m/z: 455.3 [M+H ⁺ ];

¹ H NMR (400 MHz, DMSO-d ₆ ) δ=9.01 (s, 1H), 8.02 (d, J=6.0 Hz, 1H), 7.73 (t, J=1.6 Hz, 1H), 7.65-7.61 (m, 1H), 7.55-7.52(m, 2H), 7.24(t, J=8.0Hz, 1H), 7.04(d, J=7.2Hz, 1H), 6.82(d, J=7.6Hz, 1H), 6.50( dd, J=2.4, 6.0Hz, 1H), 6.27 (d, J=2.0Hz, 1H), 4.19 (t, J=7.2Hz, 2H), 3.00-2.84 (m, 8H), 2.63-2.55 (m , 2H), 2.24 (s, 3H).

Example 12: Compound 12

Compound 2-5 (200 mg, 612.03 μmmol, 1 equiv) was dissolved in 1,4-dioxane (5 mL), followed by compound 12-1 (113.06 mg, 918.05 μmol, 1.5 equiv), bis(dibenzylideneacetone)palladium (35.19 mg, 61.20 μmol, 0.1 equiv), 4,5-bis(diphenylphosphine)-9,9-dimethylxanthene (70.83 mg, 122.41 μmol, 0.2 equiv), cesium carbonate (598.24 mg, 1.84 mmol, 3 equiv). The nitrogen was replaced three times and then stirred at 100 degrees Celsius for 12 hours under nitrogen protection. Cool to 25 degrees Celsius, add 1 g of silica gel powder, stir for 15 minutes, and filter through a pad of celite. The filtrate was adjusted to pH 4 with 1 M hydrochloric acid per liter, diluted with water (20 mL), and washed with ethyl acetate (20 mL×3). The aqueous phase was adjusted to pH 9 with 1 mol/liter aqueous sodium hydroxide solution, extracted with ethyl acetate (20 mL×3), the organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, concentrated, and prepared Compound 12 was obtained after purification by high performance liquid chromatography. MS (ESI) m/z: 414.4 [M+H ⁺ ];

¹ H NMR (400 MHz, DMSO-d ₆ ) δ=8.85 (s, 1H), 8.00 (d, J=5.6 Hz, 1H), 7.63 (t, J=7.6 Hz, 1H), 7.54 (d, J= 7.6Hz, 1H), 7.33-7.32(m, 1H), 7.11-7.02(m, 3H), 6.47(dd, J=2.0, 6.0Hz, 1H), 6.44-6.39(m, 1H), 6.26(d , J=2.0Hz, 1H), 4.19 (t, J=7.2Hz, 2H), 3.68 (s, 3H), 2.85 (d, J=7.2Hz, 2H), 2.63-2.55 (m, 2H), 2.25 (s, 3H).

Example 13: Compound 13

Compound 2-5 (4.7 g, 14.38 mmol, 1 equiv), compound 7-11 (1.87 g, 15.82 mmol, 1.1 equiv), palladium acetate (322.90 mg, 1.44 mmol, 0.1 equiv), 4,5 - Bis(diphenylphosphine)-9,9-dimethylxanthene (832.21 mg, 1.44 mmol, 0.1 equiv) and cesium carbonate (14.06 g, 43.15 mmol, 3 equiv) were added to 1,4 dimethine in oxane (50 mL). Stir for 10 hours at 100°C under nitrogen protection. The temperature of the system was lowered to room temperature, 100 mL of water was added to dilute, and the mixture was extracted with ethyl acetate (100 mL×3). The organic phases were combined, washed with saturated brine (100 mL×3), dried over anhydrous sodium sulfate, concentrated, and purified by preparative high performance liquid chromatography (basic system) to obtain compound 13. MS (ESI) m/z: 409.1 [M+H ⁺ ];

¹ H NMR (400 MHz, DMSO-d ₆ ) δ=9.28 (s, 1H), 8.30 (s, 1H), 8.09 (d, J=6.0 Hz, 1H), 7.73 (dd, J=1.2, 8.4 Hz, 1H), 7.67-7.60 (m, 1H), 7.59-7.53 (m, 1H), 7.41 (s, 1H), 7.26 (d, J=7.6Hz, 1H), 7.05 (d, J=7.6Hz, 1H) ), 6.59(dd, J=2.2, 5.9Hz, 1H), 6.29(d, J=2.2Hz, 1H), 4.25-4.16(m, 2H), 2.91-2.83(m, 2H), 2.65-2.58( m, 2H), 2.23 (s, 3H).

Example 14: Compound 14

Compound 2-5 (200 mg, 612.03 μmol, 1 equiv) was dissolved in 1,4-dioxane (5 mL), followed by compound 14-1 (150.75 mg, 918.05 μmol, 1.5 equiv), bis(dibenzylideneacetone)palladium (35.19 mg, 61.20 μmol, 0.1 equiv), 4,5-bis(diphenylphosphine)-9,9-dimethylxanthene (70.83 mg, 122.41 μmol, 0.2 equiv), cesium carbonate (598.24 mg, 1.84 mmol, 3 equiv). The nitrogen was replaced three times and then stirred at 100 degrees Celsius for 12 hours under nitrogen protection. Cool to 25 degrees Celsius, add 1 g of silica gel powder, stir for 15 minutes, and filter through a diatomaceous earth pad. The filtrate was adjusted to pH 4 with 1 M hydrochloric acid per liter, diluted with water (20 mL), and washed with ethyl acetate (20 mL×3). The aqueous phase was adjusted to pH 9 with 1 mol/liter aqueous sodium hydroxide solution, extracted with ethyl acetate (20 mL×3), the organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and concentrated. Compound 14 was obtained by preparative high performance liquid chromatography. MS (ESI) m/z: 455.3 [M+H ⁺ ];

¹ H NMR (400 MHz, DMSO-d ₆ ) δ=9.10 (s, 1H), 8.03 (d, J=6.0 Hz, 1H), 7.65-7.61 (m, 3H), 7.5 (d, J=8.0 Hz, 1H), 7.28 (d, J=8.8Hz, 2H), 7.04 (d, J=7.6Hz, 1H), 6.52 (dd, J=2.4, 6.0Hz, 1H), 6.29 (d, J=2.4Hz, 1H), 4.19 (t, J=7.2Hz, 2H), 2.94 (s, 6H), 2.86 (t, J=7.2Hz, 2H), 2.63-2.56 (m, 2H), 2.23 (s, 3H).

Example 15: Compound 15

Step A: To a solution of compound 2-5 (200 mL, 612.03 μmol, 1 equiv) and compound 15-1 (121.06 mg, 795.64 μmol, 1.3 equiv) in 1,4-dioxane (8 mL) Add bis(dibenzylideneacetone)palladium (35.19 mg, 61.20 μmol, 0.1 equiv), 4,5-bis(diphenylphosphine)-9,9-dimethylxanthene (35.41 mg, 61.20 μg) moles, 0.1 equiv) and cesium carbonate (398.82 mg, 1.22 mmol, 2 equiv). The system was replaced with nitrogen and then heated to 90 degrees Celsius for 12 hours. The reaction solution was poured into water (60 mL), the aqueous phase was extracted with ethyl acetate (80 mL), and the aqueous phase was concentrated to obtain compound 15-2. MS (ESI) m/z: 429.1 [M+H+].

Step B: To a solution of compound 15-2 (200 mg, 466.81 μmol, 1 equiv) in dichloromethane (3 mL) was added oxalyl chloride (177.75 mg, 1.40 mmol, 122.59 μL, 3 equiv), reaction 1 After hours, the mixture was added to stirred aqueous ammonia (5.60 g, 47.94 mmol, 6.15 mL, 30%, 102.69 equiv) and stirred at 25 degrees Celsius for 15 minutes. The reaction solution was poured into ice water (60 mL), extracted with ethyl acetate (60 mL×2), the organic phases were combined, washed with saturated brine (60 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to prepare high efficiency. Compound 15 was isolated by liquid chromatography (neutral). MS (ESI) m/z: 428.4 [M+H ⁺ ];

¹ H NMR (400 MHz, DMSO-d ₆ ) δ=9.59 (s, 1H), 8.29 (d, J=5.6 Hz, 1H), 8.23 (s, 1H), 8.14 (d, J=5.6 Hz, 1H) , 7.98(d, J=2.8Hz, 1H), 7.81(d, J=5.6Hz, 1H), 7.65-7.60(m, 1H), 7.58-7.48(m, 2H), 7.04(d, J=7.2 Hz, 1H), 6.65(d, J=6.0Hz, 1H), 6.38(s, 1H), 4.20(t, J=7.2Hz, 2H), 2.87(t, J=7.2Hz, 2H), 2.60( t, J=7.2 Hz, 2H), 2.21 (s, 3H).

Example 16: Compound 16

Step A: To a solution of compound 1-11 (300 mg, 1.39 mmol, 1 equiv) in N,N-dimethylformamide (10 mL) was added potassium carbonate (385.24 mg, 2.79 mmol, 2 equiv) and Compound 16-1 (249.16 mg, 1.67 mmol, 1.2 equiv) was reacted at 80 degrees Celsius for 12 hours, 40 mL of water was added, filtered, and the filter cake was dried to obtain compound 16-2. MS (ESI) m/z: 328.1 [M+H ⁺ ].

Step B: To a solution of compounds 16-2 (350 mg, 1.07 mmol, 1 equiv) and 7-11 (138.76 mg, 1.17 mmol, 1.1 equiv) in 1,4-dioxane (10 mL) was added Cesium carbonate (1.04 g, 3.20 mmol, 3 equiv), 4,5-bis(diphenylphosphine)-9,9-dimethylxanthene (123.57 mg, 213.57 μmol, 0.2 equiv) and tris( Dibenzylideneacetone) dipalladium (97.78 mg, 106.78 μmol, 0.1 equiv), after reaction under nitrogen protection and 100 degrees Celsius for 12 hours, 40 ml of ethyl acetate was added, filtered, and the filtrate was washed with saturated sodium chloride solution (40 ml × 3) washing, the organic phase was added to 20 ml of 1 mol per liter hydrochloric acid, the aqueous phase obtained by separation was washed with ethyl acetate (40 ml), and then the pH of the aqueous phase was adjusted to 9 with saturated sodium carbonate solution, It was extracted once with 40 mL of ethyl acetate, the organic phase was washed with brine (40 mL×2), dried over anhydrous sodium sulfate, and concentrated to obtain compound 16-3. MS (ESI) m/z: 410.2 [M+H ⁺ ].

Step C: To a solution of compound 16-3 (350 mg, 854.82 μmol, 1 equiv) in ethanol (5 mL) and water (5 mL) was added dimethyl sulfoxide (133.58 mg, 1.71 mmol, 133.58 μl). , 2 equiv) and sodium hydroxide (68.38 mg, 1.71 mmol, 2 equiv), followed by hydrogen peroxide (166.15 mg, 1.71 mmol, 140.81 μl, 35%, 2 equiv), and reacted at 25 degrees Celsius for 12 hours , ethyl acetate (20 mL) was added, the organic phase was washed with saturated sodium chloride solution (20 mL×3), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by preparative-grade high performance liquid chromatography (formic acid system) to obtain compound 16 . MS (ESI) m/z: 428.2 [M+H ⁺ ];

¹ H NMR (400 MHz, DMSO-d ₆ ): δ 9.56 (s, 1H), 8.32 (d, J=5.6 Hz, 1H), 7.99 (s, 1H), 7.80 (s, 1H), 7.72 (d , J=8.0Hz, 1H), 7.63-7.52 (m, 2H), 7.36 (d, J=7.6Hz, 1H), 7.28 (s, 1H), 7.20 (t, J=8.0Hz, 1H),, 7.02 (d, J=7.2Hz, 1H), 6.48 (d, J=5.6Hz, 1H), 4.20 (t, J=7.2Hz, 2H), 2.87 (brt, J=7.2Hz, 2H), 2.61- 2.52 (m, 2H), 2.21 (s, 3H).

Example 17: Formate salt of compound 17

Step A: To compound 1 (3 g, 9.05 mmol, 1 equiv), triethylamine (1.10 g, 10.86 mmol, 1.51 mL, 1.2 equiv) and 4-dimethylaminopyridine (55.30 mg, 452.67 μmol, 0.05 equiv) in dichloromethane (20 mL) was added benzenesulfonyl chloride (1.92 g, 10.86 mmol, 1.39 mL, 1.2 equiv). React at 25 degrees Celsius for 12 hours. Water (100 mL) was added and extracted with dichloromethane (300 mL×3). The organic phases were combined, washed with saturated brine (300 mL×2), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column separation to obtain compound 17-1. MS (ESI) m/z: 472.2 [M+H ⁺ ].

Step B: Compound 17-1 (100 mg, 212.08 μmol, 1 equiv.) was dissolved in tetrahydrofuran (5 mL), and lithium diisopropylamide (2 mol per liter, 159.06 mol/L) was added dropwise at -78°C under nitrogen atmosphere. microliters, 318.11 micromoles, 1.5 equiv). The reaction was held at -78°C for 1 hour, at which temperature a solution of iodine (80.74 mg, 318.11 μmol, 1.5 equiv) in tetrahydrofuran (5 mL) was added and the reaction continued for 2 hours. Water (30 mL) was added and extracted with ethyl acetate (50 mL×3). The organic phases were combined, washed with saturated brine (50 mL×2), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound 17-2. MS (ESI) m/z: 598.1 [M+H ⁺ ].

Step C: Compound 17-2 (150 mg, 251.08 μmol, 1 equiv), 1-methylpyrazole-4-boronic acid (33.20 mg, 263.63 μmol, 1.05 equiv), potassium carbonate (104.10 mg, 753.23 μg) moles, 3 equiv) and [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride (18.37 mg, 25.11 μmol, 0.1 equiv) in 1,4-dioxane (5 mL) and water (1 mL) in a nitrogen atmosphere at 80°C for 12 hours. The reaction solution was filtered, the filtrate was diluted with water (30 mL), and extracted with ethyl acetate (50 mL×3). The organic phases were combined, washed with saturated brine (50 mL×2), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound 17-3. MS (ESI) m/z: 552.3 [M+H ⁺ ].

Step D: To a solution of compound 17-3 (120 mg, 217.54 μmol, 1 equiv) in methanol (5 mL) was added aqueous sodium hydroxide (2 mol per L, 543.85 μl, 1.09 mmol, 5 equiv) . 60 degrees Celsius for 4 hours. Water (50 mL) was added and extracted with ethyl acetate (50 mL×3). The organic phases were combined, washed with saturated brine (50 mL×2), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by preparative high performance liquid chromatography (formic acid condition) to obtain the formate salt of compound 17. MS (ESI) m/z: 412.2 [M+H ⁺ ];

¹ H NMR (300 MHz, DMSO-d ₆ ) δppm 2.08 (s, 3H), 2.56-2.64 (m, 2H), 2.79-2.90 (m, 2H), 3.89 (s, 3H), 4.22 (br t, J =7.04Hz, 2H), 6.36(d, J=5.49Hz, 1H), 6.62(s, 1H), 6.99(d, J=7.23Hz, 1H), 7.50-7.55(m, 1H), 7.57(br d, J=7.32Hz, 1H), 7.89 (d, J=5.49Hz, 1H), 7.97 (s, 1H), 8.16 (s, 1H), 8.25 (br s, 1H), 11.90 (br s, 1H) ).

activity test

1. In vitro enzyme inhibitory activity assay

Experimental Materials:

TGF-βR1, TGF-βR1 (T204D), LCK, p38α Kinase Enzyme System (Kinase System) were purchased from Promega.

Envision Multilabel Analyzer (PerkinElmer).

experimental method:

Dilute enzymes, substrates, ATP (adenosine triphosphates) and inhibitors using the buffer solution in the kit.

TGF-βR1:

The compound to be tested was diluted 5 times to the 8th concentration with a row gun, that is, from 50 micromoles per liter to 0.65 nanomoles per liter, and the final concentration of dimethyl sulfoxide was 5%, and a double-well experiment was set up. Add 1 µl of each concentration gradient of inhibitor, 2 µl of TGF-βR1 enzyme (30 ng), 2 µl of a mixture of substrate and ATP (50 µmol per liter ATP, 0.2 µg per µl) to the microtiter plate. TGF-βR1 peptide), at which point the final compound concentration gradient was diluted from 10 micromoles per liter to 0.13 nanomoles per liter. The reaction system was placed at 30 degrees Celsius for 120 minutes. After the reaction, 5 microliters of ADP-Glo reagent was added to each well, and the reaction was continued at 30 degrees Celsius for 40 minutes. After the reaction was completed, 10 microliters of kinase detection reagent was added to each well, and the reaction was performed at 30 degrees Celsius for 30 minutes with a PerkinElmer Envision multi-label analyzer for reading. Chemiluminescence, integration time 0.5 seconds.

TGF-βR1(T204D):

The compound to be tested was diluted 5 times to the 8th concentration with a row gun, that is, from 50 micromoles per liter to 0.65 nanomoles per liter, and the final concentration of dimethyl sulfoxide was 5%, and a double-well experiment was set up. Add 1 µl of each concentration gradient of inhibitor, 2 µl of TGF-βR1 (T204D) enzyme (15 ng), 2 µl of a mixture of substrate and ATP (50 µmol per liter ATP, 0.2 µg) to the microtiter plate. per microliter of TGF-βR1 peptide), at which time the final compound concentration gradient was 10 micromolar per liter diluted to 0.13 nanomolar per liter. The reaction system was placed at 30 degrees Celsius for 120 minutes. After the reaction, 5 microliters of ADP-Glo reagent was added to each well, and the reaction was continued at 30 degrees Celsius for 40 minutes. After the reaction was completed, 10 microliters of kinase detection reagent was added to each well, and the reaction was performed at 30 degrees Celsius for 30 minutes with a PerkinElmer Envision multi-label analyzer for reading. Chemiluminescence, integration time 0.5 seconds.

LCK:

The compound to be tested was diluted 5 times to the 8th concentration with a row gun, that is, from 500 micromoles per liter to 6.5 nanomoles per liter, and the final concentration of dimethyl sulfoxide was 5%, and a double-well experiment was set up. Add 1 µl of each concentration gradient of inhibitor, 2 µl of LCK enzyme (1.55 ng), 2 µl of a mixture of substrate and ATP (30 µmol per liter ATP, 0.4 µg per µl Poly E to the microtiter plate). ₄ Y ₁ ), at which time the final compound concentration gradient was 100 micromoles per liter and diluted to 1.3 nanomoles per liter. The reaction system was placed at 30 degrees Celsius for 60 minutes. After the reaction, 5 microliters of ADP-Glo reagent was added to each well, and the reaction was continued at 30 degrees Celsius for 40 minutes. After the reaction was completed, 10 microliters of kinase detection reagent was added to each well. After 30 minutes of reaction at 30 degrees Celsius, the PerkinElmerEnvision multi-label analyzer was used to read the chemistry. Lights up, integration time 0.5 seconds.

p38α:

The compound to be tested was diluted 5 times to the 8th concentration with a row gun, that is, from 50 micromoles per liter to 0.65 nanomoles per liter, and the final concentration of dimethyl sulfoxide was 5%, and a double-well experiment was set up. Add 1 µl of each concentration gradient of inhibitor, 2 µl of p38α enzyme (4 ng), 2 µl of a mixture of substrate and ATP (150 µmol per liter ATP, 0.2 µg per µl p38 peptide) to the microtiter plate. ), at which time the final compound concentration gradient was 10 micromoles per liter diluted to 0.13 nanomoles per liter. The reaction system was placed at 30 degrees Celsius for 60 minutes. After the reaction, 5 microliters of ADP-Glo reagent was added to each well, and the reaction was continued at 30 degrees Celsius for 40 minutes. After the reaction was completed, 10 microliters of kinase detection reagent was added to each well. After 30 minutes of reaction at 30 degrees Celsius, a PerkinElmerEnvision multi-label analyzer was used to read the chemistry. Lights up, integration time 0.5 seconds.

data analysis:

The raw data is converted into inhibition rate, and the IC ₅₀ value can be obtained by curve fitting with four parameters. Table 1 provides the enzymatic inhibitory activities of the compounds of the examples of the present invention on TGF-βR1, TGF-βR1 (T204D), LCK and p38α.

Experimental results: see Table 1:

Table 1

-- means not tested

Conclusion: The compounds of the present invention have excellent in vitro inhibitory activity against TGF-βR1 and TGF-βR1 (T204D), while weak in vitro inhibitory activity against LCK and p38α, showing excellent in vitro activity and selectivity.

2. pSMAD response unit inhibition experiment

Experimental principle:

This experiment integrated an SMAD-responsive element-controlled luciferase gene into HEK293 cells. This cell line was validated by stimulatory response to human TGFβ1 and treatment with inhibitors of the TGFβ/SMAD signaling pathway.

Experimental operation:

1. Culture HEK293 cells in growth medium. In a 96-well plate, approximately 35,000 seeded cells and 100 microliters of genomycin-free growth medium were placed in each well. and incubated at 37 degrees Celsius.

2. After 24 hours, add 90 microliters of assay medium solution with a three-fold concentration gradient of the analyte to each well. Incubate at 37°C for 4-5 hours in a CO2 incubator. For the control group, only 90 microliters of assay medium without analyte was added.

3. Each concentration point should contain three groups of experiments. a. Add 10 microliters of assay medium solution of human TGFβ1 to the medium (final TGFβ1 concentration is 20 ng/mL). b. Add 10 microliters of blank medium to the non-stimulated group. c. Add 100 microliters of blank medium to the cell-free group.

4. Incubate for 18 hours at 37 degrees Celsius in a carbon dioxide incubator.

5. Assay using the Luciferase Assay System: Add 100 μl of ONE-Step ^™ Luciferase Reagent to each well at room temperature for 15 to 30 minutes at room temperature. Fluorescence was measured using a luminometer.

6. Data Analysis: The mean background luminescence (cell-free group of wells) was subtracted from the luminescence readings for all wells.

Experimental results: see Table 2:

Table 2

Conclusion: The compounds of the present invention have excellent pSmad inhibitory activity. It is proved that the compounds of the present invention can inhibit the TGF-β/SMAD signaling pathway.

3. In vivo antitumor efficacy of mouse colon cancer CT-26 cells BALB/c mouse subcutaneous allograft model

Purpose:

The main purpose of this study is to study the antitumor efficacy of the tested compounds on the CT26 mouse allograft tumor model.

Experimental operation:

Cell culture: Mouse colon cancer CT-26 cells (cell bank of the Type Culture Collection, Chinese Academy of Sciences) were cultured in vitro in monolayer, and the culture conditions were RPMI-1640 medium (manufacturer: Gibco) with 10% fetal bovine serum, 37 Cultivated in a 5% carbon dioxide incubator. Conventional digestion treatment and passage with trypsin-EDTA (Ethylene Diamine Tetraacetic Acid, ethylene diamine tetraacetic acid) were performed twice a week. When the cell saturation is 80%-90% and the number reaches the requirement, the cells are collected, counted, and seeded.

Animals: BALB/c mice, female, 6-8 weeks old.

Tumor inoculation: 0.1 ml of DPBS cell suspension containing 3×10 ⁵ CT26 cells was subcutaneously inoculated into the right groin of each mouse, and the administration started on the day of inoculation.

Experimental index: The experimental index is to examine whether tumor growth is inhibited, delayed or cured. Tumor diameters were measured with vernier calipers twice a week. The calculation formula of tumor volume is: V=0.5L×W ² , L and W represent the long and short diameters of the tumor, respectively.

Experimental results: The tumor inhibitory effects of the compounds are shown in Table 3.

Table 3 Results of CT26 allotransplantation experiments

Experimental conclusion: The compound of the present invention has obvious antitumor efficacy in vivo in the mouse colon cancer CT-26 cell BALB/c mouse subcutaneous allograft tumor model. At the same dose (50 mg/kg, twice a day), it exhibited significantly better tumor suppressive effect than Compound A.

Claims (30)

1. A compound shown in a formula (I), pharmaceutically acceptable salt or isomer thereof,

wherein ring A is 5-6 membered heteroaryl, phenyl, C_5-6Cycloalkyl or 5-6 membered heterocycloalkyl;

R₁、R₂and R₃Each independently is H, F, Cl, Br, I, -CN、-OH、C_1-6Alkoxy radical, C_1-6Alkyl radical, C_2-4Alkenyl radical, C_2-4Alkynyl or C_3-6Cycloalkyl, wherein said C_1-6Alkoxy radical, C_1-6Alkyl radical, C_2-4Alkenyl radical, C_2-4Alkynyl and C_3-6Cycloalkyl is optionally substituted with 1, 2 or 3 substituents independently selected from F, Cl, Br, CN, -OH, -CH₃、-OCH₃and-NH₂Substituted with the substituent(s);

m is 1 or 2;

R₄is 5-6 membered heteroaryl or phenyl, wherein said 5-6 membered heteroaryl and phenyl are optionally substituted with 1, 2 or 3R_aSubstituted;

T₁is-O-or-CH₂-；

T₂Is N or C (R)₅)；

R₅Is H, F, Cl, Br, -CN, -OH, -NH₂、-OCH₃or-CH₃；

Each R_aIndependently is-CN, -C (═ O) NR_bR_c、C_1-6Alkoxy or optionally substituted by 1, 2 or 3 groups independently selected from F, Cl, Br, I, -OH, -OCH₃-CN and NH₂C substituted by a substituent of (3)_1-6An alkyl group;

or R₄And R₅Pyridyl group attached thereto and

together form a structure represented by formula (B):

l is a single bond,

R₆Independently H, -CN, -C (═ O) NR_bR_c、C_1-4Alkyl radical, C_3-6Cycloalkyl or 5-6 membered heteroaryl, wherein said C_1-4Alkyl radical、C_3-6Cycloalkyl and 5-6 membered heteroaryl optionally substituted with 1, 2 or 3R_dSubstituted;

each R_dIndependently F, Cl, Br, I, -OH, -CN, -NH₂、-OCH₃、-OCH₂CH₃、-CH₃、-CF₃or-CH₂CH₃；

R_bAnd R_cEach independently is H, -CH₃、-CH₂CH₃、-CH₂CH₂CH₃、-CH₂(CH₃)₂Or cyclopropyl;

the 5-6 membered heterocycloalkyl and 5-6 membered heteroaryl each contain 1, 2, 3 or 4 heteroatoms or groups of heteroatoms independently selected from N, -O-, -S-, and-NH-.

2. The compound of claim 1, a pharmaceutically acceptable salt thereof, or an isomer thereof, having a structure represented by formula (I-a):

wherein, ring A, R₁、R₂、R₃、R₄、T₁And m is as defined in claim 1.

3. The compound of claim 1, a pharmaceutically acceptable salt thereof, or an isomer thereof, having a structure represented by formula (I-B):

wherein, ring A, R₁、R₂、R₃、R₄、R₅、T₁And m is as defined in claim 1.

4. The compound according to claim 3, a pharmaceutically acceptable salt thereof, or an isomer thereof,wherein said R₄Is 5-6 membered heteroaryl or phenyl, wherein said 5-6 membered heteroaryl and phenyl are optionally substituted with 1, 2 or 3R_aSubstituted; r₅Is H, F, Cl, Br, -CN, -OH or-OCH₃、-NH₂or-CH₃。

5. The compound of claim 1, a pharmaceutically acceptable salt thereof, or an isomer thereof, having a structure represented by formula (I-C):

wherein, ring A, R₁、R₂、R₃、T₁、m、R₆And L is as defined in claim 1.

6. The compound of claim 1, a pharmaceutically acceptable salt thereof, or an isomer thereof, wherein each R_aIndependently is CN, -OCH₃、

7. A compound, pharmaceutically acceptable salt or isomer thereof according to any one of claims 1-4 or 6, wherein R₄Is pyrrolyl, pyrazolyl, pyridinyl, pyrimidinyl or phenyl, wherein said pyrrolyl, pyrazolyl, pyridinyl, pyrimidinyl and phenyl are optionally substituted by 1, 2 or 3R_aAnd (4) substituting.

8. The compound of claim 7, a pharmaceutically acceptable salt thereof, or an isomer thereof, wherein said R₄Is composed of

9. The compound of claim 8, a pharmaceutically acceptable salt thereof, or an isomer thereof, wherein said R₄Is composed of

10. The compound of claim 8, a pharmaceutically acceptable salt thereof, or an isomer thereof, having a structure represented by formulas (I-a1) to (I-a 4):

wherein, ring A, R₁、R₂、R₃、T₁And m is as defined in claim 1; r_aAs defined in claim 1 or 6.

11. The compound of claim 10, a pharmaceutically acceptable salt thereof, or an isomer thereof, having a structure represented by formulas (I-a5) to (I-a 12):

wherein, ring A, R₁、R₂、R₃And R_aAs defined in claim 10.

12. The compound of claim 8, a pharmaceutically acceptable salt thereof, or an isomer thereof, having a structure represented by formulas (I-B1) to (I-B4):

wherein, ring A, R₁、R₂、R₃、R₅、T₁And m is as defined in claim 1; r_aAs defined in claim 1 or 6.

13. The compound of claim 12, a pharmaceutically acceptable salt thereof, or an isomer thereof, having a structure represented by formulas (I-B5) to (I-B12):

wherein, ring A, R₁、R₂、R₃、R₅And R_aAs defined in claim 12.

14. The compound of claim 5, a pharmaceutically acceptable salt thereof, or an isomer thereof, having a structure represented by formula (I-C1) or (I-C2):

wherein, ring A, R₁、R₂、R₃、R₆And L is as defined in claim 5.

15. A compound, pharmaceutically acceptable salt thereof or isomer thereof according to any of claims 1-5 or 10-14, wherein R₁、R₂And R₃Each independently is H, F, Cl, Br, I, -CN, -OH, -OCH₃、-OCH₂CH₃、-CH₃、-CH₂CH₃Vinyl, ethynyl or cyclopropyl, whereinthe-OCH₃、-OCH₂CH₃、-CH₃、-CH₂CH₃Vinyl, ethynyl and cyclopropyl are optionally substituted by 1, 2 or 3 substituents independently selected from F, Cl, Br, CN, -OH, -CH₃、-OCH₃and-NH₂Substituted with the substituent(s).

16. The compound of claim 15, a pharmaceutically acceptable salt thereof, or an isomer thereof, wherein said R₁、R₂And R₃Each independently is H, F, Cl, Br, -CN, -OH, -OCH₃、-CH₃、-CH₂CH₃or-CF₃。

17. The compound, pharmaceutically acceptable salt thereof, or isomer thereof according to any one of claims 1-5 or 10-14, wherein the ring a is thienyl, pyrrolyl, pyridyl, pyrimidinyl, phenyl, or tetrahydro-2H-pyranyl.

18. The compound, pharmaceutically acceptable salt thereof, or isomer thereof according to any of claims 1-5, 10-14, or 16, wherein the compound

Is composed of

19. A compound according to claim 18, a pharmaceutically acceptable salt thereof, or an isomer thereof, wherein the compound

Is composed of

20. A compound according to claim 19, a pharmaceutically acceptable salt thereof, or an isomer thereof, wherein the compound

Is composed of

21. The compound of claim 19, a pharmaceutically acceptable salt thereof, or an isomer thereof, having a structure represented by formulas (I-a13) - (I-a 16):

wherein R is₁As defined in claim 1 or 16; r_aAs defined in claim 1.

22. The compound of claim 19, a pharmaceutically acceptable salt thereof, or an isomer thereof, having a structure represented by formulas (I-B13) to (I-B36):

wherein R is₁As defined in claim 1 or 16; r_aAs defined in claim 1.

23. The compound of claim 19, a pharmaceutically acceptable salt thereof, or an isomer thereof, having a structure represented by formulas (I-C3) to (I-C5):

wherein R is₁As defined in claim 1 or 16; l and R₆As defined in claim 1.

24. The compound of claim 23, a pharmaceutically acceptable salt thereof, or an isomer thereof, having a structure represented by formulas (I-C6) to (I-C9):

wherein R is₁And R₆As defined in claim 23.

25. The compound, pharmaceutically acceptable salt thereof, or isomer thereof according to any of claims 1, 5, 14, 23, or 24, wherein R₆Independently is H, -CN, -C (═ O) NH₂Isopropyl, cyclopropyl, cyclohexyl, pyrazolyl or pyridyl wherein said isopropyl, cyclopropyl, cyclohexyl, pyrazolyl and pyridyl are optionally substituted by 1, 2 or 3R_dAnd (4) substituting.

26. The compound of claim 25, a pharmaceutically acceptable salt thereof, or an isomer thereof, wherein said R₆Independently is H, -CN, -C (═ O) NH₂、

27. The compound of claim 26, a pharmaceutically acceptable salt, or an isomer thereof, wherein said R₆Independently H, -CN, -C (═ O)NH₂、

28. A compound of the formula:

29. use of a compound according to any one of claims 1 to 28, a pharmaceutically acceptable salt or isomer thereof for the manufacture of a TGF- β R1 inhibitor medicament.

30. Use of a compound according to any one of claims 1 to 28, a pharmaceutically acceptable salt thereof or an isomer thereof for the manufacture of a medicament for solid tumors.