WO2021148010A1 - Pyrazolo heteroaryl ring compound and application thereof - Google Patents

Abstract

Disclosed in the present invention are a type of pyrazolo heteroaryl ring compounds and application thereof. Specifically, disclosed is a compound represented by formula (III) or a pharmaceutically acceptable salt thereof.

Description

Pyrazolo heteroaromatic ring compound and its application

This application claims the following priority

CN202010074092.3, application date: 2020.01.22;

CN202010915330.9, application date: 2020.09.03;

CN202011497060.0, application date: 2020.12.17.

Technical field

The invention relates to a class of pyrazolo heteroaromatic ring compounds and their applications. Specifically, it relates to a compound represented by formula (III) or a pharmaceutically acceptable salt thereof.

Background technique

The phosphorylation of tyrosine kinase and the dephosphorylation of tyrosine phosphatase are sufficient signal transduction mechanisms that exist in organisms, and they jointly regulate the level of tyrosine phosphorylation of intracellular proteins. Shp2 (SH2 domain-containing protein-tyrosine phosphatase-2) is a non-transmembrane protein tyrosine phosphatase that plays a role of dephosphorylation, and is one of the important members of the protein tyrosine phosphatase (PTP) family. Its molecule is encoded by the PTPN11 gene, which can either positively regulate downstream signal transduction pathways through the catalytic activity of phosphatase, or act as a phosphatase-independent adaptor protein to play a positive regulatory role. It can also play a role under specific conditions Negative regulation, which is widely involved in the regulation of cell differentiation, migration and other biological functions and related signal transduction processes. PTPN11 mutation is considered to be a high-risk factor for juvenile myelomonocytic leukemia (JMML). At the same time, it is considered to be the proto-oncogene of leukemia due to the abnormal activation and mutation of Shp2 in different types of leukemia; In cancer, pancreatic cancer, gastric cancer and glioma, Shp2 has also been reported to be over-activated; in lung cancer, Shp2, as an oncogene, promotes the occurrence and development of tumors by regulating various mechanisms. But in the process of liver cancer, Shp2 plays a role of tumor suppressor gene under the influence of specific environment. In short, as an important node molecule, Shp2 plays an important regulatory role in the process of tumorigenesis and development, and is a potential therapeutic target.

Summary of the invention

The present invention provides a compound of formula (III) or a pharmaceutically acceptable salt thereof,

in,

为

Structural units

for

为

Structural units

for

T ₃ is CR ₁ or N;

T ₄ is CR ₄ or N;

T ₅ is N or NH;

R ₁ is H, F, Cl, Br, I, OH, NH ₂ or C _1-3 alkyl;

R ₂ is H, F, Cl, Br, I, OH, NH ₂ or C _1-3 alkyl;

R ₃ is F, Cl, Br, I or C _1-3 alkyl, said C _1-3 alkyl optionally substituted with 1, 2 or 3 R _a;

R ₄ is H, F, Cl, Br, I, OH, NH ₂ or C _1-3 alkyl;

R _{5 is} each independently H, F, Cl, Br, I, OH, NH ₂ or C _1-3 alkyl, and the C _1-3 alkyl is optionally substituted with 1, 2 or 3 R _b ;

R ₆ is H, F, Cl, Br, I, or C _1-3 alkyl, and the C _1-3 alkyl is optionally substituted with 1, 2 or 3 R _c ;

R _T1 is OH, C _1-3 alkyl or C _1-3 alkoxy;

R _T2 is H or C _1-3 alkyl substituted by OH;

R _a , R _b and R _c are each independently F, Cl, Br, I, OH or NH ₂ ;

m is 1, 2 or 3;

n is 1, 2 or 3.

The present invention also provides a compound of formula (II) or a pharmaceutically acceptable salt thereof,

in,

为

Structural units

for

T ₃ is CR ₁ or N;

T ₄ is CR ₄ or N;

T ₅ is N or NH;

R ₁ is H, F, Cl, Br, I, OH, NH ₂ or C _1-3 alkyl;

R ₂ is H, F, Cl, Br, I, OH, NH ₂ or C _1-3 alkyl;

R ₃ is F, Cl, Br, I or C _1-3 alkyl, said C _1-3 alkyl optionally substituted with 1, 2 or 3 R _a;

R ₄ is H, F, Cl, Br, I, OH, NH ₂ or C _1-3 alkyl;

R _{5 is} each independently H, F, Cl, Br, I, OH, NH ₂ or C _1-3 alkyl, and the C _1-3 alkyl is optionally substituted with 1, 2 or 3 R _b ;

R _T1 is OH, C _1-3 alkyl or C _1-3 alkoxy;

R _T2 is H or OH substituted C _1-3 alkyl; R _a and R _{b are} each independently F, Cl, Br, I, OH or NH _2;

n is 1, 2 or 3.

The present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof,

in,

为

Structural units

for

R ₁ is H, F, Cl, Br, I, OH, NH ₂ or C _1-3 alkyl;

R ₂ is H, F, Cl, Br, I, OH, NH ₂ or C _1-3 alkyl;

R ₃ is F, Cl, Br, I or C _1-3 alkyl, said C _1-3 alkyl optionally substituted with 1, 2 or 3 R _a;

R _T1 is OH, C _1-3 alkyl or C _1-3 alkoxy;

R _{T2 is a C 1-3} alkyl substituted by H or OH;

R _{a is} each independently F, Cl, Br, I, OH, or NH ₂ .

In some aspects of the present invention, the above-mentioned R ₁ is H, F, Cl, Br, I, OH or NH ₂ , and other variables are as defined in the present invention.

In some aspects of the present invention, the above-mentioned R ₂ is NH ₂ , and other variables are as defined in the present invention.

Some aspects of the present invention, the above R ₃ is F, Cl, Br, I or CH _3, said alkyl optionally substituted with CH _3, two or three R _a, the other variables are as defined in the present invention.

In some aspects of the present invention, the above-mentioned R ₃ is -CH ₃ , and other variables are as defined in the present invention.

In some aspects of the present invention, the above-mentioned R ₄ is H, F, Cl, Br, I, OH or NH ₂ , and other variables are as defined in the present invention.

In some aspects of the present invention, the above-mentioned R _{5 is} independently H, F, Cl, Br, I or CH ₃ , and the CH ₃ alkyl group is optionally substituted with 1, 2 or 3 R _b , and other variables are as in the present invention. Defined by the invention.

In some aspects of the present invention, the above-mentioned R _{5 is} independently H, F, Cl, Br, I, CH ₃ or CF ₃ , and other variables are as defined in the present invention.

In some aspects of the present invention, the above-mentioned R _{6 is} independently H, F, Cl, Br or I, and other variables are as defined in the present invention.

In some aspects of the present invention, the above-mentioned R _T1 is OH or -OCH ₃ , and other variables are as defined in the present invention.

In some aspects of the present invention, the above-mentioned R _T2 is H or -CH ₂ -OH, 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 present invention, the above-mentioned structural unit

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 present invention, the above-mentioned structural unit

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 present invention, the above-mentioned structural unit

for

Other variables are as defined in the present invention.

There are also some schemes of the present invention that come from any combination of the above-mentioned variables.

In some aspects of the present invention, the above-mentioned compound or a pharmaceutically acceptable salt thereof has the structure (II-1)

in,

如本发明所定义。 n, T ₃ , T ₄ , R ₅ and structural units

As defined in the present invention.

In some aspects of the present invention, the above-mentioned compound or a pharmaceutically acceptable salt thereof has the structure (Ⅰ-1)

in,

如本发明所定义。 R ₁ and structural unit

As defined in the present invention.

The present invention also provides the following compounds or their pharmaceutically acceptable salts,

In some aspects of the present invention, the above-mentioned compound or a pharmaceutically acceptable salt thereof,

The present invention also provides the use of the above-mentioned compound, its isomers or pharmaceutically acceptable salts thereof in the preparation of medicines for treating diseases related to SHP2 inhibitors.

Technical effect

The compound of the present invention exhibits good inhibitory activity on protein tyrosine phosphatase SHP2, and will have an excellent therapeutic effect in patients with SHP2 abnormal tumors.

Definition and description

Unless otherwise stated, the following terms and phrases used herein are intended to have the following meanings. A specific term or phrase should not be considered uncertain or unclear without a special definition, but should be understood in its ordinary meaning. When a trade name appears in this article, it is meant to refer to its corresponding commodity or its active ingredient.

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

The term "pharmaceutically acceptable salt" refers to a salt of the compound of the present invention, which is prepared from a compound with specific substituents discovered in the present invention and a relatively non-toxic acid or base. When the compound of the present invention contains a relatively acidic functional group, a base addition salt can be obtained by contacting the compound with a sufficient amount of base in a pure solution or a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine or magnesium salt or similar salts. When the compound of the present invention contains a relatively basic functional group, the acid addition salt can be obtained by contacting the compound with a sufficient amount of acid in a pure solution or 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, hydrogen carbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, Hydrogen sulfate, hydroiodic acid, phosphorous acid, etc.; and organic acid salts, the organic acid includes, for example, acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, Similar acids such as fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid and methanesulfonic acid; 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 basic and acidic functional groups, which can be converted into any base or acid addition salt.

The pharmaceutically acceptable salt of the present invention can be synthesized from the parent compound containing acid or base by conventional chemical methods. In general, such salts are prepared by reacting these compounds in free acid or base form with a stoichiometric amount of appropriate base or acid in water or organic solvent or a mixture of both.

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 their racemic mixtures and other mixtures, such as enantiomers or diastereomer-enriched mixtures, all of these mixtures belong to this Within the scope of the invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All these isomers and their mixtures are included in the scope of the present invention.

Unless otherwise specified, the term "enantiomer" or "optical isomer" refers to stereoisomers that are mirror images of each other.

Unless otherwise specified, the term "cis-trans isomer" or "geometric isomer" is caused by the inability to rotate freely because of double bonds or single bonds of ring-forming carbon atoms.

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

Unless otherwise specified, "(+)" means right-handed, "(-)" means left-handed, and "(±)" means racemic.

和楔形虚线键

表示一个立体中心的绝对构型，用直形实线键

和直形虚线键

表示立体中心的相对构型，用波浪线

表示楔形实线键

或楔形虚线键

或用波浪线

表示直形实线键

和直形虚线键

Unless otherwise specified, use wedge-shaped solid line keys

And wedge-shaped dashed key

Represents the absolute configuration of a three-dimensional center, with a straight solid line key

And straight dashed key

Indicates the relative configuration of the three-dimensional center, using wavy lines

Represents a wedge-shaped solid line key

Or wedge-shaped dashed key

Or use wavy lines

Represents a straight solid line key

And straight dashed key

还有两个互变异构体

Unless otherwise specified, the term "tautomer" or "tautomeric form" means that at room temperature, the isomers of different functional groups are in dynamic equilibrium and can be transformed into each other quickly. If tautomers are possible (such as in solution), the chemical equilibrium of tautomers can be reached. For example, proton tautomer (proton tautomer) (also called proton transfer tautomer) includes interconversion through proton migration, such as keto-enol isomerization and imine-ene Amine isomerization. Valence isomers (valence tautomer) include some recombination of bonding electrons to carry out mutual conversion. A specific example of keto-enol tautomerization is the tautomerization between two tautomers of pentane-2,4-dione and 4-hydroxypent-3-en-2-one; for example structure

There are also two tautomers

Unless otherwise specified, the term "enriched in one isomer", "enriched in isomers", "enriched in one enantiomer" or "enriched in enantiomers" refers to one of the isomers or pairs of The content of the enantiomer is less than 100%, and the content of the isomer or enantiomer 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 96% or greater, or 97% or greater, or 98% or greater, or 99% or greater, or 99.5% or greater, or 99.6% or greater, or 99.7% or greater, or 99.8% or greater, or greater than or equal 99.9%.

Unless otherwise specified, the term "isomer excess" or "enantiomeric excess" refers to the difference between the relative percentages of two isomers or 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 enantiomer excess (ee value) is 80% .

The optically active (R)- and (S)-isomers and D and L isomers can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If you want to obtain an enantiomer of a compound of the present invention, it can be prepared by asymmetric synthesis or derivatization with chiral auxiliary agents, in which the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide pure The 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), it forms a diastereomeric salt with an appropriate optically active acid or base, and then passes through a conventional method known in the art The diastereoisomers are resolved, and then the pure enantiomers are recovered. In addition, the separation of enantiomers and diastereomers is usually accomplished through the use of chromatography, which uses a chiral stationary phase and is optionally combined with chemical derivatization (for example, the formation of amino groups from amines). Formate).

The compound of the present invention may contain unnatural proportions of atomic isotopes on one or more of the atoms constituting 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, deuterium can be substituted for hydrogen to form deuterated drugs. The bond formed by deuterium and carbon is stronger than the bond formed by ordinary hydrogen and carbon. Compared with undeuterated drugs, deuterated drugs can reduce toxic side effects and increase drug stability. , Enhance the efficacy, extend the biological half-life of drugs and other advantages. All changes in the isotopic composition of the compounds of the present invention, whether radioactive or not, are included in the scope of the present invention. "Optional" or "optionally" means that the event or condition described later may but not necessarily occur, and the description includes a situation in which the event or condition occurs and a situation in which the event or condition does not occur.

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

When any variable (such as 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 can optionally be substituted with up to two Rs, and R has independent options in each case. In addition, combinations of substituents and/or variants thereof are only permitted 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 connected groups 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 is absent. For example, when X in A-X is vacant, it means that the structure is actually A. When the listed substituents do not indicate which atom is connected to the substituted group, such substituents can be bonded via any atom. For example, a pyridyl group can pass through any one of the pyridine ring as a substituent. 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 linking direction, its linking direction is arbitrary, for example,

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

It can also be formed by connecting 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 only permitted if such combinations result in stable compounds.

直形虚线键

或波浪线

表示。例如-OCH ₃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和2位碳原子与其他基团相连。 Unless otherwise specified, when a group has one or more connectable sites, any one or more sites of the group can be connected to other groups through chemical bonds. The chemical bond between the site and other groups can be a straight solid bond

Straight dashed key

Or wavy line

Express. For example _{, the straight solid bond in -OCH 3} means that it is connected to other groups through the oxygen atom in the group;

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

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

Unless otherwise specified, the term "C _1-3 alkyl" is used to indicate a linear or branched saturated hydrocarbon group composed of 1 to 3 carbon atoms. The C _1-3 alkyl group includes C _1-2 and C _2-3 alkyl groups, etc.; it can be monovalent (such as methyl), divalent (such as methylene) or multivalent (such as methine) . Examples of C _1-3 alkyl include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), and the like.

Unless otherwise specified, C _n-n+m or C _n -C _n+m includes any specific case of n to n+m carbons, for example, C _1-12 includes C ₁ , C ₂ , C ₃ , C ₄ , C ₅ , C ₆ , C ₇ , C ₈ , C ₉ , C ₁₀ , C ₁₁ , and C ₁₂ , including any range from n to n+m, for example, 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.; similarly, from n to n +m member 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, including any 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, 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 (for example, a nucleophilic 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 groups, 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, such as alkanoyl (such as acetyl, trichloroacetyl or trifluoroacetyl); alkoxycarbonyl, such as tert-butoxycarbonyl (Boc) ; Arylmethyloxycarbonyl, such as benzyloxycarbonyl (Cbz) and 9-fluorenylmethyloxycarbonyl (Fmoc); arylmethyl, such as benzyl (Bn), trityl (Tr), 1,1-di -(4'-Methoxyphenyl) methyl; silyl, such as trimethylsilyl (TMS), 2-(trimethylsilyl) ethoxymethyl (SEM) and tert-butyl two Methylsilyl (TBS) and so on. The term "hydroxy protecting group" refers to a protecting group suitable for preventing side reactions of the hydroxyl group. Representative hydroxy protecting groups include but are not limited to: alkyl groups, such as methyl, ethyl, and tert-butyl; acyl groups, such as alkanoyl groups (such as 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 so on.

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

扫描，收集相关数据后，进一步采用直接法(Shelxs97)解析晶体结构，便可以确证绝对构型。 The structure of the compound of the present invention can be confirmed by conventional methods well known to those skilled in the art. If the present invention relates to the absolute configuration of the compound, the absolute configuration can be confirmed by conventional technical means in the field. For example, single crystal X-ray diffraction (SXRD), the cultured single crystal is collected with the Bruker D8 venture diffractometer to collect the diffraction intensity data, the light source is CuKα radiation, and the scanning method:

After scanning and collecting relevant data, the direct method (Shelxs97) is further used to analyze the crystal structure to confirm the absolute configuration.

The solvent used in the present invention is commercially available. The present invention uses the following abbreviations: aq stands for water; DMF stands for N,N-dimethylformamide; EtOAc stands for ethyl acetate; EtOH stands for ethanol; MeOH stands for methanol; BOC stands for tert-butoxycarbonyl is an amine protection Group; HOAc stands for acetic acid; rt stands for room temperature; THF stands for tetrahydrofuran; Boc ₂ O stands for di-tert-butyl dicarbonate; TFA stands for trifluoroacetic acid; DIPEA stands for diisopropylethylamine; SOCl ₂ stands for sodium chloride Sulfone; CS ₂ stands for carbon disulfide; TsOH stands for p-toluenesulfonic acid; mp stands for melting point; LDA stands for lithium diisopropylamine; EDTA stands for ethylenediaminetetraacetic acid; SEM stands for 2-trimethylsilylethoxymethyl .

软件命名，市售化合物采用供应商目录名称。 Compounds are based on conventional naming principles in the field or use

The software is named, and the commercially available compounds use the supplier catalog name.

Detailed ways

The present invention will be described in detail through the following examples, but it is not meant to impose any disadvantageous restriction on the present invention. The present invention has been described in detail herein, and its specific embodiments are also disclosed. For those skilled in the art, various changes and improvements can be made to the specific embodiments of the present invention without departing from the spirit and scope of the present invention. Will be obvious.

Example 1 and Example 2:

synthetic route:

Step 1: Synthesis of compound 001-2

Compound 001-1 (4g, 26.30mmol, 1eq) was added to phosphorus oxychloride (199.66g, 1.30mol, 121.01mL, 49.52eq) at 10°C, and then N,N-dimethyl was added at 0°C Benzylaniline (5.10g, 34.19mmol, 5.47mL, 1.3eq), stirred and refluxed at 106°C for 16 hours. The reaction solution was concentrated by distillation and added to 50mL ice water, extracted with 50mL ethyl acetate 3 times, the combined organic phase was washed with 100mL saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, column chromatography (ethyl acetate /Petroleum ether = 1:1-pure ethyl acetate) to obtain compound 001-2. MS (ESI) m/z: 188.9 [M+H] ⁺ . ¹ H NMR (400MHz, DMSO-d ₆ ) δppm 8.58 (s, 1H)

Step 2: Synthesis of compound 001-3

Compound 001-2 (1.5g, 7.94mmol, 1eq) was added to tetrahydrofuran (15mL), sodium methoxide (643.13mg, 11.90mmol, 1.5eq) in methanol (2.25mL) was added, and the reaction was stirred at 25°C. 1.5 hours. The reaction solution was concentrated under reduced pressure, and the residue was subjected to column chromatography (petroleum ether/ethyl acetate=20:1-3:1) to obtain compound 001-3. MS (ESI) m/z: 184.9 [M+H] ⁺ . ¹ H NMR (400 MHz, CH ₃ OH-d ₄ ) δ ppm 4.25 (s, 3H) 8.15 (s, 1H).

Step 3: Synthesis of compound 001-4

Compound 001-3 (0.2g, 1.08mmol, 1eq) , compound 001-4A (181.93mg, 1.30mmol, 1.2eq) , copper acetate (196.80mg, 1.08mmol, 1eq), 2,2 '- bipyridine ( 338.46mg, 2.17mmol, 2eq) was added to N,N-dimethylformamide (4mL), oxygen gas (15psi) was introduced with an oxygen balloon, and the reaction was stirred at 80°C for 16 hours. The reaction solution was added with 10 mL of water to precipitate solids, filtered, and the filter cake was washed with 10 mL of water three times to obtain a crude filter cake. Purification by column chromatography (petroleum ether-petroleum ether/ethyl acetate=5:1). Compound 001-4 was obtained. MS (ESI) m/z: 278.9 [M+H] ⁺ . ¹ H NMR (400MHz, METHANOL-d ₄ ) δ ppm 4.13 (s, 3H) 7.26-7.35 (m, 2H) 7.65-7.72 (m, 2H) 8.30 (s, 1H).

Step 4: Synthesis of compound 001-5 and compound 001-6

N,N-Diisopropylethylamine (1.48g, 11.48mmol, 2mL, 41.56eq) was added to compound 001-4 (110mg, 276.31μmol, 1eq), compound 001-4A (161.00mg, 414.47μmol, 1.5 eq, TFA), the reaction was heated and stirred at 130°C for 2 hours, and then concentrated under reduced pressure to obtain the crude product 001-5 (MS(ESI)m/z:503.3[M+H] ⁺ ) and the crude product 001-6 (MS (ESI) m/z: 517.3 [M+H] ⁺ ).

Step 5: Synthesis of compound 001 and compound 002

The crude products of compound 001-5 and compound 001-6 (130.00mg, 258.65μmol, 1eq) were added to the reaction flask, hydrochloric acid/methanol (1mL, 4M) was added, and the reaction was stirred at 20°C for 1 hour. The reaction solution was decompressed Concentrate, add 5 mL of anhydrous methanol, concentrate under reduced pressure, and separate by reverse preparation column (separation column: Welch Xtimate C18 100*40mm*3μm; mobile phase: [water (0.225% formic acid)-acetonitrile]; acetonitrile%: 10%- 40%, 8 minutes). The formate salt of compound 001 and the formate salt of compound 002 were obtained.

The formate of compound 001: MS (ESI) m/z: 398.1 [M+H] ⁺ , ¹ H NMR (400MHz, CD ₃ OD) δ ppm 1.33 (d, J = 6.63 Hz, 3H) 1.35 (s, 1H ) 1.71 (br d, J = 12.76 Hz, 1H) 1.77-2.00 (m, 3H) 3.09-3.27 (m, 2H) 3.42 (br d, J = 3.88 Hz, 1H) 3.84 (d, J = 9.13 Hz, 1H)3.97(d,J＝9.13Hz,1H)4.05(br d,J＝13.88Hz,1H)4.14(br d,J＝13.76Hz,1H)4.24-4.36(m,1H)7.23(t,J = 8.69 Hz, 2H) 7.61-7.78 (m, 2H) 7.83 (s, 1H) 8.52 (br s, 1H).

The formate of compound 002: MS (ESI) m/z: 413.1 [M+H] ⁺ , ¹ H NMR (400MHz, CD ₃ OD) δ ppm 1.33 (br d, J = 6.25 Hz, 3H) 1.71 (br d ,J=12.13Hz,1H)1.85(br s,3H)3.15-3.31(m,2H)3.40(br s,1H)3.82-3.92(m,1H)3.93-4.02(m,1H)4.04(s, 3H) 4.32 (br d, J = 4.38 Hz, 1H) 4.43-4.65 (m, 2H) 7.26 (br t, J = 8.50 Hz, 2H) 7.61 (br dd, J = 8.32, 4.69 Hz, 2H) 7.95 ( s, 1H) 8.53 (br s, 1H).

Example 3:

synthetic route:

Step 1: Synthesis of compound 003-2

Compound 003-1 (10g, 88.44mmol, 1eq), compound 003-1A (24.75g, 176.87mmol, 2eq), copper acetate (16.06g, 88.44mmol, 1eq) and pyridine (34.98g, 442.18mmol, 35.69mL , 5eq) was dissolved in anhydrous dichloromethane (500mL), replaced with oxygen three times, and stirred under an oxygen balloon (15psi) for 12 hours at 25°C. The reaction solution was filtered through celite, and the celite was washed with dichloromethane (100 mL×2). The filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column (petroleum ether: ethyl acetate=20:1, 10:1, 5:1) to obtain compound 003-2. ¹ H NMR (400MHz, CD ₃ Cl) δ ppm 8.51 (s, 1H), 8.19 (s, 1H), 7.56-7.69 (m, 2H), 7.12 to 7.22 (m, 2H).

Step 2: Synthesis of compound 003-3

Compound 003-2 (10 g, 48.27 mmol, 1 eq) was dissolved in anhydrous tetrahydrofuran (100 mL) and replaced with nitrogen three times. Cool to -60°C, add dropwise lithium bis(trimethylsilyl)amide (1M, 57.93mL, 1.2eq), and stir for 0.5 hours. At -60°C, a solution of hexachloroethane (12.57g, 53.10mmol, 6.01mL, 1.1eq) in anhydrous tetrahydrofuran (50mL) was added dropwise and stirred for 1 hour. The reaction solution was poured into 200 mL saturated aqueous ammonium chloride solution, and extracted with ethyl acetate (50 mL*2). The organic phases were combined, washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column (petroleum ether: ethyl acetate=30:1, 10:1, 5:1) to obtain compound 003-3. ¹ H NMR (400MHz, CD ₃ Cl) δ ppm 8.28 (s, 1H), 7.42-7.53 (m, 2H), 7.12-7.27 (m, 2H).

Step 3: Synthesis of compound 003-4

Compound 003-3 (3.67g, 15.19mmol, 1eq), compound 003-3A (4.06g, 45.57mmol, 3eq) and sodium bicarbonate (10.21g, 121.52mmol, 4.73mL, 8eq) were dissolved in absolute ethanol ( 100mL) and water (100mL), heated to 80°C, and stirred for 16 hours. The reaction solution was concentrated under reduced pressure to remove ethanol, and the aqueous phase was extracted with dichloromethane (50 mL). The aqueous phase was adjusted to pH=4~5 with 3N hydrochloric acid aqueous solution, filtered, and the filter cake was washed with water (50 mL). The filter cake was collected and dried to obtain compound 003-4. ¹ H NMR (400MHz, (CD ₃ ) ₂ SO) δppm 13.20 (s, 1H), 8.29 (s, 1H), 7.66 (dd, J = 8.8, 4.8 Hz, 2H), 7.35 to 7.56 (m, 3H ), 3.66-3.91 (m, 1H), 1.20 (d, J=7.2 Hz, 3H).

Step 4: Synthesis of compound 003-5

The hydrogenation flask was replaced with nitrogen, and Pd/C (600mg, 5%) and compound 003-4 (2.9g, 9.86mmol, 1eq) in methanol (120mL) were sequentially added, replaced with hydrogen three times, and reacted for 60 hours at 25°C and 50psi hydrogen. . The reaction solution was filtered through Celite and washed with methanol (20 mL). The filtrate was concentrated under reduced pressure to obtain compound 003-5, which was directly used in the next reaction.

Step 5: Synthesis of compound 003-6

Compound 003-5 (2.3g, 9.34mmol, 1eq) was dissolved in anhydrous dioxane (50mL), manganese dioxide (2.44g, 28.02mmol, 3eq) was added, and stirred at 25°C for 24 hours. The reaction solution was filtered through Celite and washed with 50 mL of methanol. The filtrate was concentrated under reduced pressure to obtain compound 003-6, which was directly used in the next reaction.

Step 6: Synthesis of compound 003-7

Compound 003-6 (1 g, 4.09 mmol, 1 eq) was dissolved in phosphorus oxychloride (10 mL), replaced with nitrogen three times, heated to 80° C., and stirred for 16 hours. The reaction solution was concentrated under reduced pressure, and the residue was dissolved in ethyl acetate (15 mL), poured into a saturated aqueous sodium bicarbonate solution (30 mL), and separated. The aqueous phase was extracted with ethyl acetate (10 mL×2), the organic phases were combined, washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column (petroleum ether: ethyl acetate=30:1, 10:1, 5:1) to obtain compound 003-7. ¹ H NMR (400MHz, (CD ₃ ) ₂ SO) δ ppm 8.67 (s, 1H), 8.10-8.29 (m, 2H), 7.47 (t, J=8.8 Hz, 2H), 2.76 (s, 3H).

Step 7: Synthesis of compound 003-8

Compound 003-7 (801mg, 3.05mmol, 1eq), N-bromosuccinimide (1.09g, 6.10mmol, 2eq) and azobisisobutyronitrile (150.22mg, 914.84μmol, 0.3eq) were dissolved Replace carbon tetrachloride (10 mL) with nitrogen three times, and heat to 80° C. and stir for 6 hours. The reaction solution was concentrated under reduced pressure. The crude product was purified by silica gel column (petroleum ether: ethyl acetate=50:1, 30:1, 10:1) to obtain compound 003-8. ¹ H NMR (400MHz, CD ₃ Cl) δ ppm 8.39 (s, 1H), 8.12-8.28 (m, 2H), 7.20-7.36 (m, 2H), 4.85 (s, 2H).

Step 8: Synthesis of compound 003-9

Compound 003-8 (150 mg, 439.15 μmol, 1 eq) was dissolved in DMSO (2 mL), replaced with nitrogen three times, heated to 140° C., and stirred for 4 hours. The reaction solution was poured into 10 mL ice water, the pH was adjusted to 8-9 with saturated sodium bicarbonate aqueous solution, and extracted with ethyl acetate (2 mL×3). The organic phases were combined, washed with saturated brine (2 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain compound 003-9. Used directly in the next reaction. ¹ H NMR (400MHz, (CD ₃ ) ₂ SO) δ ppm 10.29 (s, 1H), 8.92 (s, 1H), 8.18-8.28 (m, 3H), 7.53 (t, J=8.8 Hz, 3H).

Step 9: Synthesis of compound 003-10

Compound 003-9 (230 mg, 831.37 μmol, 1 eq) and potassium persulfate complex salt (766.65 mg, 1.25 mmol, 1.5 eq) were dissolved in DMF (3 mL) and stirred at 20° C. for 3 hours. The reaction solution was poured into water (10 mL), extracted with ethyl acetate (2 mL×3), and the organic phases were combined, washed with saturated brine (2 mL), dried with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain compound 003-10. Used directly in the next reaction. ¹ H NMR (400MHz, (CD ₃ ) ₂ SO) δ ppm 8.84 (s, 1H), 8.05-8.21 (m, 2H), 7.45-7.55 (m, 2H).

Step 10: Synthesis of compound 003-11

Compound 003-10 (210mg, 717.57μmol, 1eq), compound 001-4A (295.38mg, 1.08mmol, 1.5eq), potassium carbonate (297.52mg, 2.15mmol, 3eq) and cesium fluoride (109.00mg, 717.57μmol) , 26.46μL, 1eq) was dissolved in dimethylsulfoxide (5mL), replaced with nitrogen three times, heated to 110°C, and stirred for 16 hours. The reaction solution was poured into a saturated aqueous ammonium chloride solution (5 mL), and extracted with ethyl acetate (1 mL*2). The organic phases were combined, washed with saturated brine (1 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (dichloromethane: methanol = 50:1, 30:1, 10:1) to obtain compound 003-11.

Step 11: Synthesis of compound 003-12

Compound 003-11 (209mg, 393.88μmol, 1eq), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU) ( 179.72mg, 472.66μmol, 1.2eq) and triethylamine (59.79mg, 590.83μmol, 82.24μL, 1.5eq) were dissolved in N,N-dimethylformamide (2mL) and stirred at 20°C for 10 minutes. Anhydrous methanol (63.10mg, 1.97mmol, 79.70μL, 5eq) was added dropwise at 20°C and stirred for 12 hours. The reaction solution was poured into a saturated aqueous ammonium chloride solution (10 mL), and extracted with ethyl acetate (2 mL*3). The organic phases were combined, washed with saturated brine (2 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column (dichloromethane: methanol=50:1, 30:1, 10:1) to obtain compound 003-12. ¹ H NMR (400MHz, CD ₃ Cl) δppm 7.99-8.19 (m, 3H), 7.09-7.18 (m, 2H), 4.11-4.22 (m, 1H), 3.98 (s, 3H), 3.78 (d, J =8.8Hz,1H),3.54-3.71(m,3H),3.41-3.51(m,1H), 3.26(d,J=10.0Hz,1H),2.96-3.16(m,2H),1.92-2.11( m, 2H), 1.52-1.72 (m, 2H), 1.13-1.23 (m, 12H).

Step 12: Synthesis of compound 003-13

At 20°C, compound 003-12 (50 mg, 91.80 μmol, 1 eq) was dissolved in anhydrous tetrahydrofuran (2 mL), replaced with nitrogen three times, lithium borohydride (6.00 mg, 275.41 μmol, 3 eq) was added at 20°C, and stirred for 3.5 hours. The three batches of reaction solution were poured into 15 mL of saturated aqueous ammonium chloride solution, and extracted with ethyl acetate (4 mL*3). The organic phases were combined, washed with saturated brine (4 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was separated and purified by a preparation plate (dichloromethane: methanol = 10:1) to obtain compound 003-1.

Step 13: Synthesis of compound 003

At 20°C, compound 003-13 (57 mg, 110.33 μmol, 1 eq) was dissolved in 1N hydrochloric acid methanol solution (1 mL), and stirred for 1 hour. The reaction solution was cooled to 0°C, adjusted to pH=8-9 with ammonia water, and concentrated under reduced pressure. The crude product was separated by a reverse preparation column (separation column: Waters Xbridge BEH C18 100*30mm*10μm; mobile phase: [water (10mM bicarbonate)-acetonitrile]; acetonitrile%: 17%-53%, 8 minutes) to obtain Compound 003. ¹ H NMR (400MHz, CD ₃ OD) δppm 8.27-8.36 (m, 2H), 8.22 (s, 1H), 7.27 (t, J = 8.6 Hz, 2H), 4.88 (s, 2H), 4.18-4.31 ( m, 1H), 3.69-3.90 (m, 2H), 3.35-3.48 (m, 2H), 2.95-3.18 (m, 3H), 1.89-2.07 (m, 2H), 1.71-1.85 (m, 2H), 1.23 (d, J=6.4 Hz, 3H).

Example 4:

synthetic route:

Step 1: Synthesis of compound 004-2

Compound 004-1A (4.3g, 26.05mmol, 1eq) and compound 004-1 (3.24g, 28.65mmol, 1.1eq), potassium carbonate (4.32g, 31.26mmol, 1.2eq) were added to dimethyl sulfoxide (50mL ), then the temperature is raised to 90°C for 16 hours. The reaction solution was added with 50 mL of saturated brine and the aqueous phase was extracted 3 times with 50 mL of ethyl acetate, and most of the organic solvents were removed by spinning off. The organic phase was washed twice with 50 mL of saturated brine, and the organic phase was concentrated under reduced pressure to obtain a crude product. Purified by chromatography column (petroleum ether/ethyl acetate=20:1-4:1). Compound 004-2 was obtained. ¹ H NMR (400MHz, CDCl ₃ ) δppm 7.77 (dd, J = 8.16, 4.64 Hz, 1H) 7.99-8.04 (m, 1H) 8.34 (s, 1H) 8.47 (s, 1H) 8.87-8.96 (m, 1H) ).

Step 2: Synthesis of compound 004-3

Compound 004-2 (5.6 g, 21.69 mmol, 1 eq) was dissolved in tetrahydrofuran (50 mL) and replaced with nitrogen three times. Cool to -60°C, add dropwise lithium bis(trimethylsilyl)amide (1M, 32.54mL, 1.5eq), gradually increase to -30°C, and stir for 1 hour. At -60°C, a solution of hexachloroethane (5.65g, 23.86mmol, 2.70mL, 1.1eq) in tetrahydrofuran (50mL) was added dropwise and stirred for 1 hour. The reaction solution was added with 50 mL of saturated brine, and then the aqueous phase was extracted 3 times with 50 mL of ethyl acetate, and the organic phase was collected and concentrated under reduced pressure to obtain a crude product. The crude product was purified by a chromatography column (petroleum ether/ethyl acetate=10:1). Compound 004-3 was obtained. ¹ H NMR (400 MHz, CDCl ₃ ) δ ppm 7.75-7.84 (m, 1H) 7.85-7.92 (m, 1H) 8.41 (s, 1H) 8.99 (d, J = 3.76 Hz, 1H).

Step 3: Synthesis of compound 004-4

Compound 004-3 (0.9g, 3.08mmol, 1eq), compound 004-3A (1.57g, 9.23mmol, 3eq) and sodium bicarbonate (2.07g, 24.61mmol, 957.05μL, 8eq) were dissolved in ethanol (10mL) And water (10 mL) was heated to 80°C and stirred for 16 hours. The reaction solution was concentrated under reduced pressure to remove ethanol, and the aqueous phase was extracted with dichloromethane (10 mL). The aqueous phase was adjusted to pH=2-3 with concentrated hydrochloric acid, and extracted with ethyl acetate (10 mL×3). The organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Compound 004-4 was obtained. ¹ H NMR(400MHz,CD ₃ OD)δppm 3.40-3.94(m,3H)7.82-7.92(m,1H)7.98-8.13(m,1H)8.20(s,1H)8.93(d,J=4.52Hz, 1H).

Step 4: Synthesis of compound 004-5

Under the protection of argon, add palladium/carbon (0.2g, 2.49mmol, 10% purity, 1eq) and compound 004-4 (0.9g, 2.49mmol, 1eq) in methanol (20mL) solution to the hydrogenation flask, and replace with hydrogen. Three times, 30°C, stirring under hydrogen (50 psi) for 40 hours. It was filtered through Celite, and the filtrate was concentrated under reduced pressure. A mixture of compounds 004-5A and 004-5B is obtained. ¹ H NMR (400MHz, CD ₃ OD) δppm 3.64-3.72 (m, 1H) 3.83 (dd, J = 11.04, 5.77 Hz, 1H) 4.00 (t, J = 4.64 Hz, 1H) 4.72 (s, 2H) 7.24 (s, 1H) 7.81-7.93 (m, 3H) 8.09 (br d, J = 7.78 Hz, 1H) 8.19 (br d, J = 8.03 Hz, 1H) 8.83 (d, J = 4.27 Hz, 1H) 8.88 (br d,J=4.52Hz,1H)

Step 5: Synthesis of compound 004-6

Compound 004-5A&004-5B (100mg, 319.25μmol, 1eq) was added to dichloromethane (5mL), and then 2,6-lutidine (171.04mg, 1.60mmol, 185.91μL, 5eq) was added at 20°C , Tert-butyldimethylsilyl trifluoromethanesulfonate ((253.17mg, 957.75μmol, 220.15μL, 3eq), react for 2 hours. The mixture is directly added to dichloromethane (20mL), and then manganese dioxide (233.87mg, 2.69mmol, 10eq), react at 20°C for 1 hour. Filter the reaction solution, add 50mL ethyl acetate to the filtrate, wash with saturated brine (15mL x 2), dry with anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure The crude product compound 004-6 was obtained, and the crude product was directly subjected to the next reaction.

Step 6: Synthesis of compound 004-7

Compound 004-6 (0.12g, 282.03μmol, 1eq) was added to N,N-dimethylformamide (6mL), then triethylamine (114.16mg, 1.13mmol, 157.02μL, 4eq), N-benzene Bis(trifluoromethanesulfonyl)imide (201.51mg, 564.07μmol, 2eq) was reacted at 20°C for 1.5 hours. The reaction solution was diluted with ethyl acetate (60 mL), washed with saturated brine (15 mL x 3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (ethyl acetate in petroleum ether 0-5%) to obtain compound 004-7. MS(ESI)m/z:558.2[M+H] ⁺

Step 7: Synthesis of compound 004-8

Compound 001-4A (175.57mg, 451.98μmol, 1.4eq, trifluoroacetate) was added to acetonitrile (4mL), and then N,N-diisopropylethylamine (125.18mg, 968.54μmol, 168.70μL) , 3eq) to make the solution alkaline, add 004-7 (0.18g, 322.85μmol, 1eq), stir and react at 65°C for 18 hours. The reaction solution was directly concentrated under reduced pressure, and then 5 mL of saturated brine was added, the aqueous phase was extracted 3 times with 5 mL of ethyl acetate, and the organic phase was collected and concentrated under reduced pressure to obtain the product. No further purification. Compound 004-8 was obtained. MS(ESI)m/z:682.2[M+H] ⁺

Step 8: Synthesis of compound 004

Compound 004-8 (210 mg, 307.97 μmol, 1 eq) was added to hydrochloric acid/methanol (2 mL), and the reaction was stirred at 25° C. for 0.5 hours. The reaction solution was directly concentrated under reduced pressure to obtain a crude product. The crude product was purified by preparative HPLC (reverse column: Phenomenex Genimi NX C18150*40mm*5μm; mobile phase: [water (0.225% formic acid)-acetonitrile]; acetonitrile ratio%: 5%-35%, 10 minutes). The formate salt of compound 004 was obtained. ¹ H NMR (400MHz, CD ₃ OD) δppm 1.33 (d, J = 6.27 Hz, 3H) 1.79 (br d, J = 12.55 Hz, 1H) 1.89-1.96 (m, 1H) 1.98-2.13 (m, 2H) 2.94-3.12(m,2H)3.45(br d,J＝3.51Hz,1H)3.47-3.60(m,2H)3.86(d,J＝9.29Hz,1H)3.97(d,J＝9.03Hz,1H) 4.27-4.35(m,1H)4.81(s,2H)7.90(dd,J＝8.03,4.77Hz,1H)8.22(d,J＝7.78Hz,1H)8.33(s,1H)8.53(br s,1H ) 8.88 (d, J = 4.27 Hz, 1H). MS(ESI)m/z:464.2[M+H] ⁺

Example 5:

Step 1: Synthesis of compound 005-2:

Compound 005-1 (5g, 44.22mmol, 1eq) was dissolved in N,N-dimethylformamide (40mL), and 4-methoxybenzyl chloride (6.93g, 44.22mmol, 6.02mL, 1eq) and potassium carbonate were added (6.11g, 44.22mmol, 1eq), react at 80°C for 3 hours. The reaction solution was dropped into 200 mL of water dropwise, diluted with 300 mL of ethyl acetate, and separated, the aqueous phase was washed with ethyl acetate (200 mL), the organic phases were combined, washed with saturated sodium chloride (200 mL×4), and the organic phase was anhydrous sulfuric acid Dry with sodium, filter, and concentrate the filtrate under reduced pressure to obtain compound 005-2. ¹ H NMR(400MHz, CDCl ₃ )δppm 3.75(s,3H)5.12-5.20(m,2H)6.82-6.89(m,2H)7.16-7.19(m,2H)7.92(s,1H)7.99-8.08( m,1H).

Step 2: Synthesis of compound 005-3:

Compound 005-2 (5.2g, 22.30mmol, 1eq) was dissolved in tetrahydrofuran (150mL), and lithium bis(trimethylsilyl)amide (1M, 26.76mL, 1.2eq) was added dropwise at -60°C, and reacted for 1 hour, -60 Hexachloroethane (6.33g, 26.76mmol, 3.03mL, 1.2eq) dissolved in 30mL of tetrahydrofuran was added dropwise at ℃, and reacted at -60℃ for 1 hour. Add 100mL saturated ammonium chloride solution, gradually raise to 20°C, dilute with 300mL ethyl acetate, separate the layers, wash with ethyl acetate (200mL) in the aqueous phase, combine the organic phases, wash with saturated sodium chloride solution (100mL), anhydrous sulfuric acid Drying with sodium, filtering, and concentrating the filtrate under reduced pressure, the obtained crude product is purified by column chromatography (petroleum ether/ethyl acetate=100:1-10:1) to obtain compound 005-3. ¹ H NMR (400MHz, CDCl ₃ ) δ ppm 3.82 (s, 3H) 5.33 (s, 2H) 6.77-7.01 (m, 2H) 7.12-7.42 (m, 2H) 8.08-8.47 (m, 1H).

Step 3: Synthesis of compound 005-4:

Compound 005-3 (5g, 18.68mmol, 1eq) and compound 004-3A (9.50g, 56.04mmol, 3eq) were dissolved in ethanol (75mL) and water (75mL), and sodium bicarbonate (12.55g, 149.44mmol, 5.81 mL, 8eq), react at 90°C for 60 hours. The reaction solution was concentrated under reduced pressure to remove ethanol, the aqueous phase was washed with dichloromethane (200mL×2), concentrated hydrochloric acid was added dropwise to the aqueous phase to pH<4, diluted with 200mL of ethyl acetate, separated, the aqueous phase was washed with 200mL of ethyl acetate, and combined organic Phase, dry with anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure to obtain compound 005-4. ¹ H NMR(400MHz,DMSO-d ₆ )δppm 3.18(s,1H)3.65(br d,J＝3.26Hz,1H)3.73(s,3H)3.82(br dd,J＝11.04,3.51Hz,2H) 4.45(dt,J＝9.79,3.39Hz,1H)5.22-5.36(m,2H)6.91-6.94(m,1H)7.11(d,J＝8.53Hz,2H)7.31(d,J＝9.79Hz,1H ) 8.07-8.20 (m, 1H).

Step 4: Synthesis of compound 005-5:

Compound 005-4 (4g, 11.89mmol, 1eq) was dissolved in methanol (100mL), Pd/C was added to replace hydrogen (400mg, 11.89mmol, 10% purity, 1eq), and reacted at 50Psi at 30°C for 88 hours. Suction filtration (diatomite filtration aid), the filtrate was decompressed to remove the solvent to obtain compound 005-5, and the crude product was directly subjected to the next reaction. MS(ESI) m/z: 307.1 [M+H] ⁺ .

Step 5: Synthesis of compound 005-6:

Compound 005-5 (3.6g, 11.75mmol, 1eq) was dissolved in methanol (100mL), hydrochloric acid methanol solution (4M, 293.81μL, 0.1eq) was added, and reacted at 50°C for 16 hours. Concentrate under reduced pressure to obtain compound 005-6. MS(ESI) m/z: 289.1 [M+H] ⁺ .

Step 6: Synthesis of compound 005-7:

Compound 005-6 (3g, 10.41mmol, 1eq) was dissolved in DMF (10mL), imidazole (2.13g, 31.22mmol, 3eq) was added, and tert-butyldiphenylchlorosilane (8.58g, 31.22mmol, 8.02mL, 3eq), gradually raised to 20°C and reacted for 1 hour. Add 50 mL of saturated sodium chloride solution dropwise to the reaction system, dilute with 100 mL of ethyl acetate, separate the layers, wash the aqueous phase with ethyl acetate (100 mL×3), combine the organic phases, dry with anhydrous sodium sulfate, filter, and depressurize the filtrate After concentration, the obtained crude product was purified by column chromatography (PE/EA=10:1-0:1) to obtain compound 005-7. MS (ESI) m/z: 527.2 [M+H] ⁺ .

Step 7: Synthesis of compound 005-8:

Compound 005-7 (4.2 g, 7.97 mmol, 1 eq) was dissolved in dioxane (50 mL), manganese dioxide (3.47 g, 39.87 mmol, 5 eq) was added, and reacted at 20°C for 1 hour. Suction filtration (diatomite filter aid), and the filtrate was concentrated under reduced pressure to obtain compound 005-8. MS (ESI) m/z: 525.2 [M+H] ⁺ .

Step 8: Synthesis of compound 005-9:

Compound 005-8 (30mg, 57.18μmol, 1eq) was dissolved in N,N-dimethylformamide (1mL), triethylamine (23.14mg, 228.72μmol, 31.83μL, 4eq) was added, and N- Phenyl bis(trifluoromethanesulfonyl)imide (40.86mg, 114.36μmol, 2eq) was gradually raised to 20°C and reacted for 20 minutes. 10mL saturated sodium chloride solution and 20mL ethyl acetate were diluted, separated, the organic phase was dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the crude product was separated by thin layer chromatography (PE/EA=10:1) to obtain Compound 005-9. MS(ESI)m/z:657.3[M+H] ⁺

Step 9: Synthesis of compound 005-10:

Compound 005-9 (17.74mg, 45.68μmol, 1eq, TFA) was dissolved in acetonitrile (3mL), and N,N-diisopropylethylamine (47.23mg, 365.44μmol, 63.65μL, 8eq) and compound were added at 20°C 001-4A (30mg, 45.68μmol, 1eq), reacted at 75°C for 16 hours. Dilute with 10mL ethyl acetate and 5mL saturated sodium chloride solution, separate the layers, wash the organic phase with saturated sodium chloride (15mL×3), dry the organic phase with anhydrous sodium sulfate, filter, concentrate the filtrate under reduced pressure, and subject the crude product to thin-layer chromatography The plates were separated (PE/EA=1:1) to obtain compound 005-10. MS (ESI) m/z: 781.5 [M+H] ⁺ .

Step 10: Synthesis of compound 005-11:

Compound 005-10 (100 mg, 128.03 μmol, 1 eq) was dissolved in trifluoroacetic acid (1 mL) and trifluoromethanesulfonic acid (0.1 mL), and reacted at 20°C for 7 hours. Add 7N ammonia methanol solution dropwise to pH>9, spin off the solvent under reduced pressure, dilute with 20mL ethyl acetate and 10mL saturated sodium chloride solution, separate the layers, wash the aqueous phase with ethyl acetate (20mL×3), combine the organic phases. Drying with sodium sulfate and filtering, the filtrate is concentrated under reduced pressure, and the crude product is purified by column chromatography (DCM/MeOH=100:1-20:1) to obtain compound 005-11. MS (ESI) m/z: 423.1 [M+H] ⁺ . ¹ H NMR (400MHz, CDCl3) δppm 1.17 (d, J = 6.27 Hz, 3H) 1.21 (s, 9H) 1.70 (br d, J = 15.06 Hz, 1H) 2.04 (br d, J = 8.28 Hz, 2H) 2.75-3.02(m,2H)3.22(br s,2H)3.37-3.53(m,2H)3.63(br d,J＝8.78Hz,1H)3.81(d,J＝9.54Hz,1H)4.07-4.37( m, 1H) 4.83 (br s, 2H) 8.08 (s, 1H).

Step 11: Synthesis of compound 005-13:

Compound 005-12 (1g, 7.04mmol, 1eq) was dissolved in tetrahydrofuran (20mL), replaced with nitrogen, added dropwise lithium bis(trimethylsilyl)amide (1M, 8.45mL, 1.2eq) at -60℃ and reacted at -60℃ For 1 hour, hexachloroethane (2.00 g, 8.45 mmol, 956.64 μL, 1.2 eq) dissolved in 5 mL of tetrahydrofuran was added dropwise at -60°C, and reacted at -60°C for 1 hour. The reaction solution was added dropwise with 20 mL of saturated ammonium chloride solution, diluted with 40 mL of ethyl acetate, separated, the aqueous phase was washed with ethyl acetate (40 mL), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the residue The compound was purified by column chromatography (PE/EA=100:1-10:1) to obtain compound 005-13, ¹ H NMR (400MHz, CDCl ₃ ) δppm7.44 (dd, J=7.40, 5.40Hz, 1H) 8.45 (t, J = 5.90 Hz, 1H).

Step 12: Synthesis of compound 005-14:

Compound 005-11 (35mg, 82.83μmol, 1eq) and compound 005-13 (17.55mg, 99.40μmol, 1.2eq) were dissolved in dimethyl sulfoxide (1.5mL), replaced with nitrogen, added potassium carbonate (13.74mg, 99.40 μmol, 1.2eq) react at 25°C for 3 hours. Dilute with 20 mL of water and 20 mL of ethyl acetate, separate the layers, wash the aqueous phase with ethyl acetate (20 mL×3), combine the organic phases, dry with anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure. The crude product is separated by a thin layer chromatography plate ( DCM/MeOH=20:1) to give compound 005-14, MS(ESI) m/z: 579.2[M+H] ⁺ , ¹ H NMR(400MHz, CDCl ₃ )δppm 1.28(s,12H)1.81(br d,J=13.05Hz,3H)2.12-2.24(m,1H)2.89(s,1H)2.95-3.09(m,3H)3.51-3.61(m,2H)3.72(br d,J=9.03Hz,2H )3.90(br d,J＝9.03Hz,1H)4.22-4.29(m,1H)4.89(s,2H)7.89(d,J＝5.02Hz,1H)8.41(s,1H)8.57(d,J＝ 5.27Hz, 1H).

Step 13: Synthesis of compound 005-15:

Compound 005-14 (40mg, 69.08μmol, 1eq) was dissolved in ethyl acetate (1mL) and saturated ammonium chloride solution (1mL), reduced iron powder (23.15mg, 414.46μmol, 6eq) was added, and reacted at 50°C for 2 hours. Reduce to 25°C, filter with suction (diatomaceous earth filter aid), wash the filter cake with methanol, spin off the solvent under reduced pressure of the filtrate, dilute with 20 mL of ethyl acetate, add saturated sodium bicarbonate solution to pH>9, separate the aqueous phase with acetic acid Wash with ethyl ester (20 mL×3), combine the organic phases, dry with anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure to obtain compound 005-15. MS (ESI) m/z: 549.2 [M+H] ⁺ .

Step 14: Synthesis of compound 005:

Compound 005-15 (35 mg, 63.74 μmol, 1 eq) was dissolved in a methanol solution of hydrochloric acid (1.5 mL, 4M) and methanol (1.5 mL), and reacted at 25° C. for 30 minutes. Concentrated under reduced pressure, and the crude product was separated by reverse column: separation column: Welch Xtimate C18 100*40mm*3μm; mobile phase: [water (0.075% trifluoroacetic acid)-acetonitrile]; B%: 5%-35%, 8min, The trifluoroacetate salt of compound 005 was isolated. MS(ESI)m/z:445.2[M+H] ⁺ , ¹ H NMR(400MHz,CD ₃ OD)δppm 1.35(d,J=6.53Hz,3H)1.33-1.37(m,1H)1.80(br d ,J=13.05Hz,1H)1.91-2.12(m,3H)2.95-3.18(m,2H)3.46-3.67(m,3H)3.84-3.93(m,1H)3.94-4.07(m,1H)4.26- 4.43 (m, 1H) 4.81 (br d, J = 5.52 Hz, 1H) 4.92 (br s, 2H) 7.09 (d, J = 6.02 Hz, 1H) 8.07 (d, J = 5.77 Hz, 1H) 8.40 (s ,1H).

Example 6:

Step 1: Synthesis of compound 006-2:

Under the protection of nitrogen, compound 006-1 (5.0 g, 19.43 mmol, 1 eq) was dissolved in anhydrous tetrahydrofuran (60 mL), replaced with nitrogen three times and then cooled to -78°C. The tetrahydrofuran solution of lithium diisopropylamide (2.0M, 10.69mL, 1.1 eq) was slowly added dropwise to the reaction mixture. The mixture was stirred at -78°C for 1 hour, and then the compound 006-2A tetrahydrofuran was slowly added dropwise to the system The solution was reacted at -78°C for 30 minutes, and then the reaction system was slowly heated to -25°C for 15 hours. After the reaction, it was quenched with 100mL saturated ammonium chloride solution, extracted with ethyl acetate three times (50mL×3), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was subjected to column chromatography (petroleum ether). : Ethyl acetate = 0-10%) isolated to obtain compound 006-2. MS(ESI) m/z: 388.2 [M+Na] ⁺ .

Step 2: Synthesis of compound 006-3:

Under the protection of nitrogen, compound 006-2 (6.91g, 18.91mmol, 1eq) was dissolved in dioxane (80mL) and methanol (32mL), and then sodium hydroxide aqueous solution (6M, 16mL, 5.08eq) was added After that, the temperature was raised to 100° C. and the reaction was refluxed for 15 hours. After the reaction, cool to room temperature, remove the organic solvent under reduced pressure, adjust the pH to 3-4 with dilute hydrochloric acid (1.0M), filter, wash the filter cake with water, and redissolve the washed filter cake in ethyl acetate, anhydrous sulfuric acid The sodium was dried and concentrated under reduced pressure to obtain compound 006-3, MS (ESI) m/z: 360.1 [M+Na] ⁺ .

Step 3: Synthesis of compound 006-4:

Under the protection of nitrogen, compound 006-3 (6.10 g, 18.08 mmol, 1 eq) and polyphosphoric acid (40 mL, 1.0 eq) were added to a single-neck flask and reacted at 120° C. for 1 hour. After the reaction was completed, it was cooled to room temperature, the reaction mixture was poured into ice water for quenching, and the pH was slowly adjusted to 9 with an aqueous sodium hydroxide solution (6M) under an ice bath. It was extracted three times with ethyl acetate (50 mL×3), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was removed by rotary evaporation, and the crude product was dissolved in dichloromethane (100 mL). After adding di-tert-butyl dicarbonate (6.12g, 28.05mmol, 6.44mL, 3.0eq) and triethylamine (5.68g, 56.10mmol, 7.81mL, 6.0eq), the reaction mixture was reacted at 25°C for 2 hours. After the reaction was completed, water was added to separate the layers, the aqueous phase was extracted with ethyl acetate (50 mL×3), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain compound 006-4. MS (ESI) m/z: 342.1 [M+Na]+.

Step 4: Synthesis of compound 006-5:

Under the protection of nitrogen, compound 006-4 (2g, 6.26mmol, 1eq) and compound 006-5A (2.28g, 18.79mol, 3.0eq) were added to a single-necked flask, followed by tetraethyl titanate (5mL) 100 The reaction was refluxed at °C for 18 hours. After the reaction was completed, the mixture was cooled to room temperature, the mixture was poured into ice water for quenching, ethyl acetate (50 mL) was added, and the mixture was stirred for 1 hour, and the layers were separated. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was separated by column chromatography (petroleum ether: ethyl acetate=0-20%) to obtain compound 006-5. MS(ESI) m/z: 445.1 [M+Na] ⁺ .

Step 5: Synthesis of compound 006-6:

Under the protection of nitrogen, compound 006-5 (2.54 g, 6.01 mmol, 1 eq) was dissolved in tetrahydrofuran (25 mL), cooled to -20° C., and sodium borohydride (455 mg, 12.02 mmol, 2.0 eq) was added. The reaction system gradually returned to 25°C and reacted for 12 hours. After the reaction, the reaction was quenched by adding water under an ice bath, and the organic phases were combined after extraction with ethyl acetate (50 mL×3), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain compound 006-6. MS(ESI) m/z: 447.1 [M+Na] ⁺ .

Step 6: Synthesis of compound 006-7:

Compound 006-6 (34 mg, 80 μmol, 1 eq) was dissolved in dichloromethane (5 mL), and trifluoroacetic acid (91.2 mg, 800 μmol, 59.23 μL, 10 eq) was added. The reaction mixture was reacted at 25° C. for 2 hours, potassium carbonate was added to neutralize the reaction system to neutrality, and the reaction system was concentrated under reduced pressure to obtain compound 006-7. MS(ESI) m/z: 325.1 [M+H] ⁺ .

Step 7: Synthesis of compound 006-8:

Compound 005-6 (3g, 10.41mmol, 1eq) was dissolved in DMF (10mL), 2,6-lutidine (2.13g, 31.22mmol, 3eq) was added, and tert-butyldimethylsilyl three was added at 0°C. The fluoromethanesulfonate (8.58g, 31.22mmol, 8.02mL, 3eq) was gradually raised to 20°C and reacted for 1 hour. Add 50 mL of saturated sodium chloride solution dropwise to the reaction system, dilute with 100 mL of ethyl acetate, separate the layers, wash the aqueous phase with ethyl acetate (100 mL×3), combine the organic phases, dry with anhydrous sodium sulfate, filter, and depressurize the filtrate After concentration, the crude product was subjected to column chromatography (PE/EA=10:1-0:1) to obtain compound 006-8. MS (ESI) m/z: 403.1 [M+H] ⁺ .

Step 8: Synthesis of compound 006-9:

Compound 006-8 (4.2 g, 7.97 mmol, 1 eq) was dissolved in dioxane (50 mL), manganese dioxide (3.47 g, 39.87 mmol, 5 eq) was added, and reacted at 20°C for 1 hour. Suction filtration (diatomite filtration aid), and the filtrate was concentrated under reduced pressure to obtain compound 006-9. MS (ESI) m/z: 401.0 [M+H] ⁺ .

Step 9: Synthesis of compound 006-10:

Compound 006-9 (30mg, 57.18μmol, 1eq) was dissolved in N,N-dimethylformamide (1mL), triethylamine (23.14mg, 228.72μmol, 31.83μL, 4eq) was added, and N- Phenyl bis(trifluoromethanesulfonyl)imide (40.86mg, 114.36μmol, 2eq) was gradually raised to 20°C and reacted for 20 minutes. 10mL saturated sodium chloride solution and 20mL ethyl acetate were diluted, separated, the organic phase was dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the crude product was separated by thin layer chromatography (PE/EA=10:1) to obtain Compound 006-10. MS(ESI)m/z:533.1[M+H] ⁺

Step 10: Synthesis of compound 006-11:

Dissolve 006-10 (1g, 1.88mmol, 1eq) and 006-7 (609.18mg, 1.88mmol, 1eq) in acetonitrile (20mL), add diisopropylethylamine (242.66mg, 1.88mmol, 327.04μL at a time) ,1eq), then heated to 75°C and stirred for 16 hours. The reaction liquid was cooled to 25°C and concentrated under reduced pressure at 43°C. The concentrate was dissolved in 30 mL of water and 30 mL of ethyl acetate, and the extraction layer was separated, while the aqueous phase was extracted three times with ethyl acetate (20 mL, 20 mL, 20 mL). The organic phases were combined and washed once with saturated sodium chloride solution (20 mL). Finally, the organic phase was dried with anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude product was subjected to column chromatography (10%-70% ethyl acetate in petroleum ether). ) (Petroleum ether detected by TLC: ethyl acetate = 1:1) to obtain compound 006-11. MS (ESI) m/z: 707.4 [M+H] ⁺ .

Step 11: Synthesis of compound 006-12:

Dissolve 006-11 (450mg, 636.50μmol, 1eq) in dichloroethane (20mL), add aluminum trichloride (678.97mg, 5.09mmol, 278.27μL, 8eq) for the next 2520, and stir the mixture in 25 1 hour. The reaction solution was dissolved in 10 mL of water and 10 mL of dichloromethane, and the extraction layer was separated, while the aqueous phase was extracted three times with dichloromethane (10 mL, 10 mL, 10 mL). The organic phases were combined, washed once with saturated sodium chloride solution (10 mL), and finally the organic phase was dried with anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude product was subjected to column chromatography (0%-10% methanol in dichloromethane) (TLC detection DCM: MeOH=10:1) separated to obtain compound 006-12. MS (ESI) m/z: 473.2 [M+H] ⁺ .

Step 12: Synthesis of compound 006-13:

Dissolve 006-12 (200mg, 423.21μmol, 1eq), 005-13 (89.65mg, 507.85μmol, 1.2eq) in dimethyl sulfoxide (5mL), add potassium carbonate (116.98mg, 846.42μmol, 2eq) at once ), the mixture was stirred at 25°C for 3 hours. The reaction solution was dissolved in 20 mL of water and 20 mL of ethyl acetate, and the extraction layer was separated, while the aqueous phase was extracted three times with ethyl acetate (10 mL, 10 mL, 10 mL). The organic phases were combined, washed once with saturated sodium chloride solution (15 mL), and finally the organic phase was dried with anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The obtained crude product was directly used in the next reaction without further purification. The obtained crude product was directly used in the next reaction without further purification. Compound 006-13 was obtained. MS(ESI) m/z: 629.1 [M+H] ⁺ .

Step 13: Synthesis of compound 006-14:

Dissolve 006-13 (240mg, 381.49μmol, 1eq) in ethyl acetate (8mL), add saturated aqueous ammonium chloride solution (8mL), add iron powder (170.44mg, 3.05mmol, 8eq) in one go, and then heat to Stir at 50°C for 3 hours. The reaction solution was cooled to 25°C, filtered with Celite, and the filtrate was concentrated under reduced pressure. The reaction solution was directly concentrated under reduced pressure to obtain a crude product. The obtained crude product was directly used in the next reaction without further purification. Compound 006-14 was obtained. MS (ESI) m/z: 599.1 [M+H] ⁺ .

Step 14: Synthesis of compound 006:

006-14 (300 mg, 500.73 μmol, 1 eq) was dissolved in hydrochloric acid/methanol (4M, 3.13 mL, 25 eq), and the mixture was stirred at 25 compound for 1 hour. The reaction solution was directly concentrated under reduced pressure to obtain a crude product. Pre-HPLC: Dissolve the obtained crude product with water (5mL), filter out the insoluble matter, and use high performance liquid chromatography (column: Welch Xtimate C18 100*40mm*3μm; mobile phase: [water (0.075% trifluoroacetic acid)) -Acetonitrile]; Acetonitrile%: 17%-47%, 8min) separation and purification. The trifluoroacetate salt of compound 006 was obtained. MS (ESI) m/z: 495.1 [M+H] ⁺ . 1H NMR (400MHz, METHANOL-d4) δppm 8.43 (s, 1H) 8.07 (d, J = 6.02 Hz, 1H) 7.55 (dd, J = 8.28, 5.02 Hz, 1H) 7.10-7.20 (m, 3H) 4.87 ( s, 2H) 4.47 (s, 1H) 3.64 (br d, J = 12.30 Hz, 1H) 3.54 (br d, J = 13.55 Hz, 1H) 3.19-3.28 (m, 4H) 2.07-2.16 (m, 1H) 1.93-2.03 (m, 1H) 1.86 (br d, J = 12.55 Hz, 1H) 1.71 (br d, J = 12.80 Hz, 1H).

Biological test

Experimental example 1: In vitro evaluation

Reaction buffer:

60mM hydroxyethylpiperazine ethanesulfonic acid (HEPES) (pH 7.4), 1mM ethylenediaminetetraacetic acid (EDTA), 75mM KCl, 75mM NaCl, 0.01% Brij-35, 5mM dithiothreitol (DTT) and 10 %DMSO (final).

Enzyme: PTPN11/SHP2-FL (FL stands for full-length enzyme) (produced by RBC, no CAS number)

Recombinant human PTPN11 full length (Genbank accession number #NM_002834; aa 2-597,

Isotype 1 (canonical)) is expressed in E. coli, with N-terminal StrepII-TEV and C-terminal histidine tags. Mw=71.93kDa.

Activating peptide: H ₂ N-LN(pY)IDLDLV(dPEG8)LST(pY)ASINFQK-amide (based on publication)

Substrate: DiFMUP[6,8-difluoro-7-hydroxy-4-methylcoumarin]

The final concentration in the determination:

0.35μM activation peptide

100μM DiFMUP

step:

1. Prepare the specified enzyme/peptide and substrate in the newly prepared reaction buffer

2. Add the enzyme/peptide solution to the reaction well

3. Provide the compound to the enzyme solution (Echo550; nanoliter range) in 100% DMSO by acoustic technology, and incubate at room temperature for 30 minutes

4. Add the substrate solution to the reaction well to initiate the reaction

5. Monitoring enzyme activity (Ex/Em 355/460), as a time course measurement of the fluorescence signal increase of the fluorescent substrate at room temperature for 60 minutes.

Data analysis: take the slope of the linear part of the time course measurement × (signal/min), and calculate the% enzyme activity relative to the DMSO control. Subtract the background slope of the basal enzyme activity (no peptide).

Table 1. In vitro screening test results of the compounds of the present invention

Compound PTPN11/SHP2-FL (IC ₅₀ nM) The formate of compound 001 105.3 Formate of compound 002 899.8 Compound 003 9.03 The formate of compound 004 140.4 Trifluoroacetate of compound 005 <10 Trifluoroacetate of compound 006 3.78

Conclusion: The compound has a good inhibitory activity on PTPN11/SHP2-FL.

Experimental Example 2: Evaluation of Compound H358 Cell Activity

Purpose:

This experiment aims to verify the inhibitory effect of the compound of the present invention on the proliferation of KRAS G12C mutant NCI-H358 human non-small cell lung cancer cells.

Experimental Materials:

The cell line NCI-H358 (purchased from Pronox), RPMI1640 medium, penicillin/streptomycin antibiotics were purchased from Vicente, and fetal bovine serum was purchased from Biosera. CellTiter-Glo (cell viability chemiluminescence detection reagent) reagent was purchased from Promega.

experimental method:

NCI-H358 cell anti-proliferation experiment:

Plant NCI-H358 cells in a white 96-well plate, 80μL of cell suspension per well, which contains 4000 NCI-H358 cells. The cell plate was placed in a carbon dioxide incubator for overnight culture. The compound to be tested was diluted 5-fold to the 9th concentration with a discharge gun, that is, diluted from 2000 μM to 5.12 nM, and a double-well experiment was set up. Add 78 μL of culture medium to the middle plate, and then transfer 2 μL of each well of the serially diluted compound to the middle plate according to the corresponding position. After mixing, transfer 20 μL of each well to the cell plate. The concentration of the compound transferred to the cell plate ranges from 10 μM to 0.026 nM. The cell plate was placed in a carbon dioxide incubator for 5 days. Another cell plate is prepared, and the signal value is read as the maximum value (Max value in the following equation) on the day of drug addition to participate in data analysis. Add 25μL of cell viability chemiluminescence detection reagent to each well of this cell plate, and incubate for 10 minutes at room temperature to stabilize the luminescence signal. Use multi-marker analyzer to read. After the incubation of the cell plate with the compound added, 25 μL of cell viability chemiluminescence detection reagent per well was added to the cell plate, and the luminescence signal was stabilized by incubating at room temperature for 10 minutes. Use multi-marker analyzer to read.

data analysis:

Using the equation (Sample-Min)/(Max-Min)*100% to convert the original data into the inhibition rate, the IC ₅₀ value can be obtained by four-parameter curve fitting ("log(inhibitor) vs. GraphPad Prism" response--Variable slope" mode).

Table 2. In vitro screening test results of the compounds of the present invention

Compound H358(IC ₅₀ nM)

Trifluoroacetate of compound 005 427 Trifluoroacetate of compound 006 19.1

Conclusion: The compound has a good inhibitory activity on H358 cells.

Experimental example 3: Evaluation of compound pharmacokinetics

Experimental purpose: To test the pharmacokinetics of the compound in CD-1 mice

Experimental Materials:

CD-1 mouse (male, 32-33g)

Experimental operation:

The rodent pharmacokinetic characteristics of the compound after intravenous injection and oral administration were tested by standard protocols. In the experiment, the candidate compound was prepared as a clear solution and given to mice by a single intravenous injection and oral administration. The vehicle for intravenous injection and oral administration is a certain proportion of hydroxypropyl β cyclodextrin aqueous solution or physiological saline solution. Collect whole blood samples within 24 hours, centrifuge at 3000g for 15 minutes, separate the supernatant to obtain a plasma sample, add 4 times volume of acetonitrile solution containing internal standard to precipitate the protein, centrifuge to take the supernatant, add equal volume of water, and centrifuge to take the supernatant Sample, quantitative analysis of blood drug concentration by LC-MS/MS analysis method, and calculation of pharmacokinetic parameters, such as peak concentration, peak time, clearance rate, half-life, area under the drug-time curve, bioavailability, etc.

Experimental results:

Table 3 Pharmacokinetic test results

Conclusion: The compound of the present invention can significantly improve single or partial indexes of rat pharmacokinetics.

Experimental example 4: CYP inhibition experiment of human liver microsomes

The purpose of the research project is to use a 5-in-1 probe substrate of CYP isoenzymes to evaluate the inhibitory properties of the test product on human liver microsomal cytochrome P450 isoenzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4).

Mixed human liver microsomes (HLM) were purchased from Corning Inc. (Steuben, New York, USA) or XenoTech, LLC. (Lenexa, KS, USA) or other suppliers, and stored at less than -70°C before use Down.

Add the diluted working solution of the test substance to the incubation system containing human liver microsomes, the probe substrate and the cofactors of the circulating system. The control without the test substance but containing the solvent is used as the enzyme activity control ( 100%). The concentration of metabolites generated by the probe substrate in the sample was determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Use SigmaPlot (V.11) to make a non-linear regression analysis of the average percentage activity versus concentration of the test product. ^{The IC 50} value is calculated by a three-parameter or four-parameter inverse logarithmic equation. The test results are shown in Table 4:

Table 4

Conclusion: The compound of the present invention has a weak degree of inhibition of the five CYP isoenzymes.

Experimental Example 5: Inhibition test of hERG potassium ion channel

1. The purpose of the experiment:

The automatic patch clamp method was used to detect the effect of Example 1 to be tested on the hERG potassium ion channel.

2. Experimental method

2.1. Cell culture

The cells stably expressing the hERG potassium channel used in the experiment were derived from CHO-hERE of Aviva Biosciences, and the CHO-hERG was cultured in _{an environment of 5% CO 2} and 37°C. CHO hERG culture medium is shown in Table 5.

Table 5 CHO hERG culture medium

Reagent Supplier Catalog Number Volume(mL) F12Hams Invitrogen 31765-092 500 FBS Invitrogen 10099-141 50 G418/Geneticin Invitrogen 10131-027 1 Hygromycin B Invitrogen 10687-010 1

2.2. Preliminary preparation of cells

The CHO-hERG cells to be used in the experiment should be cultured for at least two days, and the cell density should reach 75% or more. Before the experiment, the cells were digested with TrypLE, and then resuspended in extracellular fluid to collect the cells.

2.3. Preparation of intracellular and extracellular fluids

Extracellular fluid needs to be prepared once a month. The intracellular fluid must be frozen in aliquots and stored at -20°C. See Table 6 for the composition of the intracellular and extracellular fluid.

Table 6 Cell fluid composition

Composition Extracellular fluid (mM) Intracellular fluid (mM) NaCl 145 - KCl 4 120 KOH - 31.25 CaCl ₂ 2 5.374 MgCl ₂ 1 1.75 Glucose 10 - Na ₂ ATP - 4 HEPES 10 10 EGTA - 10 pH 7.4with NaOH 7.2with KOH Osmotic pressure 295mOsm 285mOsm

2.4. Compound preparation

The test compound and the positive control Amitriptyline are dissolved in DMSO into a stock solution of a certain concentration, and then diluted according to different gradients, and finally added to the extracellular fluid according to a certain ratio, and diluted to the test concentration. Check with the naked eye for precipitation before the start of the experiment. Finally, the concentration of DMSO in the test solution and the positive control Amitriptyline should not exceed 0.3%.

2.5. Voltage stimulation program

Keep the clamping potential at -80mv, first give -50mv voltage stimulation, last 80ms to record the cell leakage current value, then depolarize to +20mv for 4800ms, open the hERG channel, and then repolarize to -50mv for 5000ms , Draw out the hERG tail current and record it. Finally, the voltage is restored to the clamping potential -80mv and maintained for 3100ms. The above voltage stimulation is repeated every 15000ms.

2.6. QPatch ^HTX whole cell patch clamp recording

The hERG QPatch ^HTX experiment was performed at room temperature. The whole cell protocol, voltage stimulation protocol and compound detection protocol were established on the software of QPatch Assay Software 5.2 (Sophion Bioscience).

First, perform 30 repetitive voltage stimulations, which is the baseline area for subsequent analysis, and then add 5 μl of extracellular fluid, which is repeated three times. The concentration of each compound is added sequentially, and the addition volume is still 5 μl and repeated three times. Incubate the cells at each test concentration for at least 5mins. During the entire recording process, all indicators need to meet the data analysis acceptance standard. If the standard is not met, the cell will not be included in the analysis range, and the compound will be tested again. The above recording process is automated by the Qpatch analysis software. The test concentration of each compound was 0.24μM, 1.20μM, 6.00μM, 30.00μM, and each concentration was repeated for at least two cells.

2.7. Data analysis

In each complete current record, based on the percentage of peak current in the negative control, the percentage inhibition of the concentration of each compound can be calculated. The standard Greek equation is used to fit the dose-effect relationship curve. The specific equation is as follows:

I _(C) =I _b +(I _fr -I _b )*c ⁿ /(IC ₅₀ ⁿ +c ⁿ )

C is the test concentration of the compound, n is the slope

Curve fitting and inhibition rate calculation are all analyzed by Qpatch analysis software. If the inhibition rate at the lowest concentration exceeds half inhibition or the inhibition rate at the highest concentration does not reach half inhibition, the corresponding IC _{50 of} the compound is lower than the lowest concentration or IC ₅₀ value Greater than the highest concentration.

2.8. Test results

Table 7 shows the _{hERG IC 50} value results of the compounds of the examples.

Table 7 hERG IC ₅₀ value results of the example compounds

Test sample hERG IC ₅₀ (nM) Example 5 >20

Conclusion: The compound of the present invention does not significantly inhibit hERG.

Experimental Example 6: In vivo drug efficacy study on H358 transplanted tumor model

On the basis of the above-mentioned in vitro evaluation, some of the compounds with high activity and representative structure were selected for in vivo research.

Experimental purpose: Balb/c nude mice were used as test animals, and human lung cancer H358 xenograft tumor model was used to conduct in vivo drug efficacy tests to evaluate the anti-tumor effects of the test compounds.

Experimental instruments: ultra-clean workbench, CO ₂ incubator (Thermo-311, Thermo), centrifuge (Centrifuge 5720R, Eppendorf), automatic cell counter (Countess II, Life Technologies), pipette (10-20μL) , Microscope (Ts2, Nikon), vernier caliper, cell culture flask (T25/T75/T225, Corning), constant temperature water tank (HWS12).

Experimental reagents: DMEM (11995-065, Gibo), fetal bovine serum (FBS) (10091-148, Gibco), 0.25% trypsin (25200-056, Gibco), penicillin double antibody (P/S) ( SV30010, GE), phosphate buffered saline (PBS) (10010-023, Gibco), Matrigel (356234, Corning), Gln (25030-081, Gibco).

Experimental operation: Take out H358 cells from the cell bank, add DMEM medium after resuscitation and place them in a CO2 incubator (incubator temperature is 37°C, CO2 concentration is 5%). After the cells are filled with the bottom of the culture flask for 80-90 passages, the cells will continue to be cultured in a CO2 incubator after the tape is worn. Repeat the process until the number of cells meets the inoculation requirements, and start to collect the logarithmic growth phase cells, using an automatic cell counter Count, and resuspend the cells with PBS and Matrigel (volume ratio 1:1) according to the results to prepare a cell suspension (8*10 ⁷ /mL) and place in an ice box for later use. Use female Balb/c nude mice, 6-8 weeks old, weighing about 18-22 grams. Keep the mice in an environment free of special pathogens. A single ventilated cage has 5 mice per cage. All cages and bedding are disinfected before use. All animals can freely obtain standard certified laboratory diets. Before the experiment, the nude mice were labeled with a disposable mouse universal ear tag, and 75% medical alcohol was used to disinfect the skin of the inoculation site before inoculation. Each mouse was subcutaneously inoculated with 0.1mL H358 tumor cells on the right back. When the tumor volume reached 100-200mm Start group administration at ^{3 o'clock.} Test compound The test compound was formulated as a 1 mg/mL 5% DMSO/95% (10% HP-β-cyclodextrin aqueous solution) solution for oral administration. The dosage and efficacy are shown in Table 8. The tumor volume was measured with a vernier caliper twice a week, and the calculation formula was V=0.5*D*d*d, where D and d are the long diameter and short diameter of the tumor, respectively. The anti-tumor effect was determined by dividing the average tumor increase volume of animals treated with the compound by the average tumor increase volume of untreated animals. The calculation formula of the inhibition rate is TGL(%)=1-[(Vt-V0) administration group/(Vt-V0) control group]*100%. After the experiment, all animals were euthanized.

Table 8. Results of in vivo efficacy test

Conclusion: The compound of the present invention can significantly inhibit the growth of transplanted tumors in H358 nude mice.

Claims (18)

The compound of formula (III) or a pharmaceutically acceptable salt thereof,

in, Structural units

for

Structural units

for

T ₃ is CR1 or N; T4 is CR4 or N; T5 is N or NH; R1 is H, F, Cl, Br, I, OH, NH2 or C1-3 alkyl; R2 is H, F, Cl, Br, I, OH, NH2 or C1-3 alkyl; R3 is F, Cl, Br, I or C _1-3 alkyl, said C _1-3 alkyl optionally substituted with 1, 2 or 3 R _a; R ₄ is H, F, Cl, Br, I, OH, NH2 or C1-3 alkyl; R5 is each independently H, F, Cl, Br, I, OH, NH2 or C1-3 alkyl, and the C1-3 alkyl is optionally substituted with 1, 2 or 3 Rb; R6 is H, F, Cl, Br, I or C1-3 alkyl, and the C1-3 alkyl is optionally substituted with 1, 2 or 3 Rc; RT1 is OH, C1-3 alkyl or C1-3 alkoxy; RT2 is H or C1-3 alkyl substituted by OH; Ra, Rb and Rc are each independently F, Cl, Br, I, OH or NH2; m is 1, 2 or 3; n is 1, 2 or 3. The compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein R1 is H, F, Cl, Br, I, OH or NH2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein R ₂ is NH ₂ . The compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein R3 is F, Cl, Br, I or CH3, and the CH3 alkyl group is optionally substituted with 1, 2, or 3 Ra. The compound or a pharmaceutically acceptable salt thereof according to claim 4, wherein R3 is -CH3. The compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein R4 is H, F, Cl, Br, I, OH or NH2. The compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein R5 is each independently H, F, Cl, Br, I or CH3, and the CH3 alkyl group is optionally substituted by 1, 2 or 3 Replaced by Rb. The compound or a pharmaceutically acceptable salt thereof according to claim 7, wherein R5 is each independently H, F, Cl, Br, I, CH3 or CF3. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein R ₆ is H, F, Cl, Br, or I, respectively. The compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein RT1 is OH or -OCH3. The compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein RT2 is H or -CH2-OH. The compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein the structural unit

for

The compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein the structural unit

for

The compound according to claim 1 or a pharmaceutically acceptable salt thereof, which has the structure (II-1)

in, n, T ₃ , T ₄ , R5 and structural units

As defined in claim 1. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, which has the structure (I-1)

in, R1 and structural unit

As defined in claim 1. The following compounds or their pharmaceutically acceptable salts,

The compound according to claim 16 or a pharmaceutically acceptable salt thereof,

The use of the compound according to any one of claims 1-17 or a pharmaceutically acceptable salt thereof in the preparation of a medicine for treating diseases related to SHP2 inhibitors.