WO2022156765A1 - Pyrazolopyrazine-linked tricyclic compound and application thereof - Google Patents

Abstract

Disclosed is a pyrazolopyrazine-linked tricyclic compound represented by formula (I) or a pharmaceutically acceptable salt thereof. The compound has inhibitory activity on protein tyrosine phosphatase SHP2 and can be used to treat diseases related to SHP2, such as lung cancer.

Description

Pyrazolopyrazine tricyclic compound and its application

This application claims the following priority:

CN202110091198.9, application date: January 22, 2021;

CN202110264689.9, application date: March 11, 2021;

CN202110414584.7, application date: April 16, 2021.

technical field

The present invention relates to a class of pyrazolopyrazine tricyclic compounds and applications thereof, in particular to a compound represented by formula (I) or a pharmaceutically acceptable salt thereof.

Background technique

The phosphorylation of tyrosine kinases and the dephosphorylation of tyrosine phosphatases are ubiquitous signal transduction mechanisms in organisms, and they work together to regulate the level of tyrosine phosphorylation of intracellular proteins. SHP2 (SH2domain-containing protein-tyrosine phosphatase-2) is a non-transmembrane protein tyrosine phosphatase that plays a role in dephosphorylation and is one of the important members of the protein tyrosine phosphatase (PTP) family. The molecule is encoded by the PTPN11 gene, which can not only positively regulate the downstream signal transduction pathway through the catalytic activity of phosphatase, but also play a positive regulatory role as a phosphatase-independent adaptor protein, and can also play a negative role under specific conditions. Therefore, it is widely involved in the regulation of biological functions such as cell differentiation and migration 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 because of 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 overactivated; in lung cancer, SHP2, as an oncogene, promotes the occurrence and development of tumors by regulating various mechanisms. But in the process of hepatocarcinogenesis, SHP2 plays the role of tumor suppressor gene under the influence of specific environment. In conclusion, 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 (I) or a pharmaceutically acceptable salt thereof,

in,

T ₁ is CR ₄ or N;

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

When E ₁ is CH ₂ , R ₃ is H, Cl, Br, I or C _1-3 alkyl optionally substituted with ₁ , 2 or 3 R _a ;

R ₁ , R ₂ and R ₄ are each independently H, F, Cl, Br, I or C _1-3 alkyl optionally substituted with ₁ , 2 or 3 R _b ;

R ₅ is C _1-3 alkyl substituted by OH;

R ₆ is each independently H, F, Cl, Br, I, OH, NH ₂ or C _1-3 alkyl optionally substituted with ₁ , 2 or 3 R _c ;

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

m is 1, 2, 3 or 4.

In some embodiments of the present invention, when the above E ₁ is O, the above R ₃ is H or F, and other variables are as defined in the present invention.

In some aspects of the present invention, when the above E ₁ is CH ₂ , the above R ₃ is H, and other variables are as defined in the present invention.

In some embodiments of the present invention, the above R ₁ , R ₂ and R ₄ are each independently H, F, Cl, Br or I, and other variables are as defined in the present invention.

In some embodiments of the present invention, the above R ₅ is -CH ₂ -OH, and other variables are as defined in the present invention.

In some embodiments of the present invention, the above R ₆ are each independently Cl or NH ₂ , and other variables are as defined in the present invention.

为

In some aspects of the present invention, the above-mentioned structural units

for

In some aspects of the present invention, the above-mentioned compound is

in,

T ₁ , E ₁ , R ₁ , R ₂ , R ₃ , R ₅ , R ₆ and m are as defined in the present invention.

In some aspects of the present invention, the above-mentioned compound is

in,

T ₁ , E ₁ , R ₁ , R ₂ and R are as defined in the present invention.

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

In some aspects of the present invention, the above compound or a pharmaceutically acceptable salt thereof, wherein the compound is selected from,

The second aspect of the present invention also provides a pharmaceutical composition, which comprises the compound defined in any of the above technical solutions or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

The present invention also provides a method for treating a disease associated with SHP2 in a subject in need thereof, comprising providing the subject with an effective dose of a compound as defined in any of the above technical solutions or a pharmaceutically acceptable salt or pharmaceutical combination thereof thing.

The present invention also provides the use of the above compound or a pharmaceutically acceptable salt thereof in the preparation of a medicine for treating SHP2-related diseases; wherein the SHP2-related diseases refer to lung cancer, preferably non-small cell lung cancer.

technical effect

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

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

Use solid wedge keys unless otherwise specified

and wedge-dotted keys

Indicate the absolute configuration of a stereocenter, using a straight solid key

and straight dashed keys

Indicate the relative configuration of the stereocenter, with a wavy line

Represents a solid wedge key

or wedge-dotted key

or with wavy lines

Represents a straight solid key

and straight dashed keys

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 tautomers include interconversions by recombination of some 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).

The compounds of the present 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. 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.

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 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 term "C _1-3 alkyl" is used to denote a straight or branched chain saturated hydrocarbon group consisting 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 (eg methyl), divalent (eg methylene) or multivalent (eg methine) . Examples of _C1-3 alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), and the like.

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, 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, 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), 2-(trimethylsilyl)ethoxymethyl (SEM) and tert-butyldiphenyl Methylsilyl (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.

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 art. For example, single crystal X-ray diffraction method (SXRD), the cultured single crystal is collected by Bruker D8 venture diffractometer, the light source is CuKα radiation, and the scanning mode is:

After scanning and collecting relevant data, the crystal structure was further analyzed by the direct method (Shelxs97), and the absolute configuration could be confirmed.

The solvent used in the present invention is commercially available. The following abbreviations are used in the present invention: aq stands for water; DMF stands for N,N-dimethylformamide; EtOAc stands for ethyl acetate; EtOH stands for ethanol; MeOH stands for methanol; group; HOAc for acetic acid; rt for room temperature; THF for tetrahydrofuran; Boc ₂ O for di-tert-butyl dicarbonate; TFA for trifluoroacetic acid; DIPEA for diisopropylethylamine _; Sulfone; CS ₂ for carbon disulfide; TsOH for p-toluenesulfonic acid; mp for melting point; LDA for lithium diisopropylamide; EDTA for ethylenediaminetetraacetic acid; SEM for 2-trimethylsilylethoxymethyl ; PMB stands for p-methoxybenzyl; TBDPS stands for tert-butyldiphenylsilyl; TBS stands for tert-butyldimethylsilyl; OTf stands for trifluoromethanesulfonyl.

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

Software naming, commercially available compounds use supplier catalog names.

Detailed ways

The present invention will be described in detail through 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.

Embodiment 1:

Step 1: Synthesis of Compound 001-3:

Under the protection of nitrogen, compound 001-1 (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. To the reaction mixture was slowly added dropwise a solution of lithium diisopropylamide (2.0 M, 10.69 mL, 1.1 eq) in tetrahydrofuran, the mixture was stirred at -78°C for 1 hour, and then the tetrahydrofuran of compound 001-2 was slowly added dropwise to the system The solution was reacted at -78°C for 30 minutes, and then the reaction system was slowly warmed to -25°C for 15 hours. After the reaction, it was quenched with saturated ammonium chloride solution (100 mL), extracted with ethyl acetate three times (50 mL×3), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was removed by rotary evaporation. Column chromatography: The crude product was separated by column chromatography (petroleum ether: ethyl acetate=0-10%) to obtain compound 001-3. MS (ESI) m/z: 388.2 [M+Na] ⁺ .

Step 2: Synthesis of Compound 001-4:

Under the protection of nitrogen, compound 001-3 (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. Then, the temperature was raised to 100°C and the reaction was refluxed for 15 hours. After the reaction was completed, it was cooled to room temperature, the organic solvent was removed under reduced pressure, and the pH was adjusted to 3-4 with dilute hydrochloric acid (1.0M), filtered, and the filter cake was washed with water. The washed filter cake was redissolved in ethyl acetate, and anhydrous sulfuric acid After drying with sodium, it was spin-dried to obtain compound 001-4, MS(ESI) m/z: 360.1 [M+Na] ⁺ .

Step 3: Synthesis of Compound 001-5:

Under the protection of nitrogen, compound 001-4 (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 to quench, and the pH was adjusted to 9 with aqueous sodium hydroxide solution (6M) slowly under ice bath. After extraction with ethyl acetate three times (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 the solvent was removed by rotary evaporation to obtain compound 001-5. MS (ESI) m/z: 342.1 [M+Na] ⁺ .

Step 4: Synthesis of Compound 001-7:

Under the protection of nitrogen, compound 001-5 (2g, 6.26mmol, 1.0eq) and compound 001-6 (2.28g, 18.79mol, 3.0eq) were added to a single-neck flask, followed by tetraethyl titanate (5mL) , 100 ℃ reflux reaction for 18 hours. After the reaction was completed, it was cooled to room temperature, the mixture was poured into ice water to quench, ethyl acetate (50 mL) was added, and the mixture was stirred for 1 hour. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was removed by rotary evaporation. Column chromatography: The crude product was separated by column chromatography (petroleum ether: ethyl acetate=0-20%) to obtain compound 001-7. MS (ESI) m/z: 445.1 [M+Na] ⁺ .

Step 5: Synthesis of Compound 001-8:

Under the protection of nitrogen, compound 001-7 (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 was gradually returned to 25°C for 12 hours. After the reaction, water was added under ice bath to quench the reaction, extracted 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 to obtain compound 001-8. MS (ESI) m/z: 447.1 [M+Na] ⁺ .

Step 6: Synthesis of Compound 001-9:

Compound 001-8 (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 solvent was spin-dried to obtain compound 001-9. MS(ESI) m/z: 325.1 [M+H] ⁺ .

Step 7: Synthesis of Compound 001-11:

Compound 001-10 (5g, 44.22mmol, 1eq) was dissolved in N,N-dimethylformamide (40mL), 4-methoxybenzyl chloride (6.93g, 44.22mmol, 6.02mL, 1eq) and potassium carbonate were added (6.11 g, 44.22 mmol, 1 eq), and reacted at 80° C. for 3 hours. The reaction solution was dropped dropwise into water (200 mL), diluted with ethyl acetate (300 mL), and the layers were separated. The aqueous phase was washed with ethyl acetate (200 mL), the organic phases were combined, washed with saturated sodium chloride (200 mL×4), The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain compound 001-11. 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 8: Synthesis of Compounds 001-12:

Compound 001-11 (5.2g, 22.30mmol, 1eq) was dissolved in tetrahydrofuran (150mL), bis(trimethylsilyl) lithium amide (1M, 26.76mL, 1.2eq) was added dropwise at -60°C, and the reaction was performed for 1 hour, -60 At °C, hexachloroethane (6.33 g, 26.76 mmol, 3.03 mL, 1.2 eq) dissolved in 30 mL of tetrahydrofuran was added dropwise, and the mixture was reacted at -60 °C for 1 hour. Add saturated ammonium chloride solution (100 mL), gradually increase to 20°C, dilute with ethyl acetate (300 mL), separate the layers, wash the aqueous phase with ethyl acetate (200 mL), combine the organic phases, and wash with saturated sodium chloride solution (100 mL) , dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the obtained crude product was purified by silica gel column chromatography (petroleum ether/ethyl acetate=100:1-10:1) to obtain compound 001-12. ¹ H NMR (400 MHz, CDCl ₃ ) δppm 8.08-8.47(m,1H)7.12-7.42(m,2H)6.77-7.01(m,2H)5.33(s,2H)3.82(s,3H).

Step 9: Synthesis of Compounds 001-14:

Compound 001-12 (5g, 18.68mmol, 1eq), compound 001-13 (9.50g, 56.04mmol, 3eq) were dissolved in ethanol (75mL) and water (75mL), and sodium bicarbonate (12.55g, 149.44mmol, 5.81mL) was added 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 (200 mL×2), concentrated hydrochloric acid was added dropwise to the aqueous phase until pH<4, diluted with ethyl acetate (200 mL), separated, and the aqueous phase was washed with ethyl acetate (200 mL). ), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain compound 001-14. ¹ H NMR (400MHz, DMSO-d ₆ ) δppm 8.07-8.20 (m, 1H) 7.31 (d, J=9.79Hz, 1H) 7.11 (d, J=8.53Hz, 2H) 6.91-6.94 (m, 1H) 5.22-5.36(m,2H)4.45(dt,J=9.79,3.39Hz,1H)3.82(br dd,J=11.04,3.51Hz,2H)3.73(s,3H)3.65(br d,J=3.26Hz , 1H) 3.18(s, 1H).

Step 10: Synthesis of Compounds 001-15:

Compound 001-14 (4g, 11.89mmol, 1eq) was dissolved in methanol (100mL), Pd/C (0.4g, 10%) was added, hydrogen was replaced (400mg, 11.89mmol, 10% purity, 1eq), 50Psi at 30°C The reaction was carried out for 88 hours. The filtrate was subjected to suction filtration (diatomite-assisted filtration), and the solvent was removed from the filtrate under reduced pressure to obtain compound 001-15, and the crude product was directly subjected to the next reaction. MS (ESI) m/z: 307.1 [M+H] ⁺ .

Step 11: Synthesis of Compounds 001-16:

Compound 001-15 (3.6g, 11.75mmol, 1eq) was dissolved in methanol (100mL), methanol solution of hydrochloric acid (4M, 293.81μL, 0.1eq) was added, and the reaction was carried out at 50°C for 16 hours. Concentrate under reduced pressure to obtain compound 001-16. MS (ESI) m/z: 289.1 [M+H] ⁺ .

Step 12: Synthesis of Compounds 001-17:

Compound 001-16 (3g, 10.41mmol, 1eq) was dissolved in DMF (10mL), imidazole (2.13g, 31.22mmol, 3eq) was added, tert-butyldiphenylchlorosilane (8.58g, 31.22mmol, 8.02mL, 3eq), gradually raised to 20°C and reacted for 1 hour. Saturated sodium chloride solution (50 mL) was added dropwise to the reaction system, diluted with ethyl acetate (100 mL), the layers were separated, the aqueous phase was washed with ethyl acetate (100 mL×3), the organic phases were combined, dried over anhydrous sodium sulfate, and filtered. , the filtrate was concentrated under reduced pressure, and the obtained crude product was purified by silica gel column chromatography (PE/EA=10:1-0:1) to obtain compound 001-17. MS (ESI) m/z: 527.2 [M+H] ⁺ .

Step 13: Synthesis of Compounds 001-18:

Compound 001-17 (4.2g, 7.97mmol, 1eq) was dissolved in dioxane (50mL), manganese dioxide (3.47g, 39.87mmol, 5eq) was added, and the reaction was carried out at 20°C for 1 hour. Suction filtration (diatomite-assisted filtration), and the filtrate was concentrated under reduced pressure to obtain compound 001-18. MS (ESI) m/z: 525.2 [M+H] ⁺ .

Step 14: Synthesis of Compounds 001-19:

Compound 001-18 (30mg, 57.18μmol, 1eq) was dissolved in N,N-dimethylformamide (1mL), triethylamine (23.14mg, 228.72μmol, 31.83μL, 4eq) was added, and N- Phenylbis(trifluoromethanesulfonyl)imide (40.86mg, 114.36μmol, 2eq) was gradually raised to 20°C and reacted for 20 minutes. Saturated sodium chloride solution (10 mL) was diluted with ethyl acetate (20 mL), the layers were 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 001-19. MS(ESI) m/z: 657.3[M+H] ⁺

Step 15: Synthesis of Compounds 001-20:

001-19 (5 g, 9.39 mmol, 1 eq) was dissolved in dichloroethane (20 mL), aluminum trichloride (10.0 g, 75.1 mmol, 8 eq) was added in one portion at 25 °C, and the mixture was stirred at 40 °C for 1 hour. The reaction solution was dissolved in water (10 mL) and dichloromethane (10 mL), extracted and separated, and the aqueous phase was extracted three times with dichloromethane (100 mL, 100 mL, 100 mL). The organic phases were combined, washed once with saturated sodium chloride solution (100 mL), and finally the organic phases were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. Column chromatography: The crude product was separated by silica gel column chromatography (0%-10% methanol in dichloromethane) (TLC detection PE:EA=2:1-1:1) to obtain compound 001-20. MS (ESI) m/z: 299.0 [M+H] ⁺ .

Step 16: Synthesis of Compound 001-21:

001-20 (2.2g, 7.38mmol, 1eq) was dissolved in DMF (10mL), 2,6-lutidine (3.95g, 36.89mmol, 5.0eq) was added, and tert-butyldimethylsilyl was added at 0°C Trifluoromethanesulfonate (4.29g, 16.23mmol, 3.73mL, 2.2eq) was gradually raised to 20°C and reacted for 1 hour. Saturated sodium chloride solution (50 mL) was added dropwise to the reaction system, diluted with ethyl acetate (100 mL), the layers were separated, the aqueous phase was washed with ethyl acetate (100 mL×3), the organic phases were combined, dried over anhydrous sodium sulfate, and filtered. , the filtrate was spun under reduced pressure to remove the solvent. The crude product was subjected to silica gel column chromatography (PE/EA=5:1-2:1) to obtain compound 001-21. MS (ESI) m/z: 413.0 [M+H] ⁺ .

Step 17: Synthesis of Compounds 001-22:

001-21 (1.4g, 3.39mmol, 1eq) and 001-9 (1.10g, 3.39mmol, 1.0eq) were dissolved in acetonitrile (20mL), diisopropylethylamine (1.32g, 10.18mmol, 1.77mL, 3.0eq), then warmed 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 water (30 mL) and ethyl acetate (30 mL), and the layers were extracted while the aqueous phase was extracted three times with ethyl acetate (20 mL, 20 mL, 20 mL). The organic phases were combined, washed once with saturated sodium chloride solution (20 mL), and finally the organic phases were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. Column chromatography: The crude product was separated by column chromatography (10%-70% ethyl acetate in petroleum ether) (TLC detected petroleum ether:ethyl acetate=1:1) to obtain compound 001-22. MS (ESI) m/z: 587.2 [M+H] ⁺ .

Step 18: Synthesis of Compounds 001-24:

Dissolve 001-22 (500mg, 852.0μmol, 1eq), 001-23 (150mg, 852.0μmol, 1.0eq) in dimethyl sulfoxide (5mL), add potassium carbonate (141mg, 846.42μmol, 1.2eq) at one time , 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, extracted and separated, and 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 phases were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude product of compound 001-24 was obtained, which was directly used in the next reaction without further purification. MS (ESI) m/z: 743.2, 744.2 [M+H] ⁺ .

Step 19: Synthesis of Compounds 001-25:

001-24 (240mg, 381.49μμmol, 1eq) was dissolved in ethyl acetate (8mL), then saturated aqueous ammonium chloride solution (8mL) was added, iron powder (170.44mg, 3.05mmol, 8eq) was added at one time, and then the temperature was raised 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 spin-dried to obtain crude product. The crude product of compound 001-25 was obtained, which was directly used in the next reaction without further purification. MS (ESI) m/z: 713.2, 714.2 [M+H] ⁺ .

Step 20: Synthesis of Compound 001:

001-25 (200 mg, 280.3 μmol, 1.0 eq) was dissolved in hydrochloric acid/methanol (4 M, 3.13 mL, 25 eq), and the mixture was stirred at 25° C. for 1 hour. The reaction solution was directly spin-dried to obtain crude product. The obtained crude product was dissolved in water (5 mL), the insoluble matter was filtered off, and the insoluble matter was filtered off, and the resulting crude product was subjected to preparative high performance liquid chromatography (chromatographic column: Welch Xtimate C18 100*40mm*3 μm; mobile phase: [water (0.075% trifluoroacetic acid)-acetonitrile]) ; Acetonitrile%: 17%-47%, 8min) separation and purification. The trifluoroacetate salt of compound 001 was obtained. MS (ESI) m/z: 495.1 [M+H] ⁺ . ¹ H NMR (400MHz, CD ₃ OD) δppm 8.41(s, 1H), 8.08(d, J=6.02Hz, 1H), 7.48-7.36(m, 2H), 7.20-7.18(m, 1H), 7.08( m, 1H), 4.87(s, 2H), 4.55(s, 1H), 3.66-3.63(m, 1H), 3.57-3.54(m, 1H), 3.27-3.14(m, 4H), 2.10-2.08( m, 1H), 2.05–1.99 (m, 1H), 1.99–1.84 (m, 1H), 1.75–1.72 (m, 1H).

Embodiment 2:

Step 1: Synthesis of Compound 002-2:

Compound 002-1 (1.5 g, 3.68 mmol, 1 eq) was dissolved in dichloromethane (10 mL), and trifluoroacetic acid (5 mL) 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 solvent was spin-dried to obtain compound 002-2. MS (ESI) m/z: 308.1 [M+H] ⁺ .

Step 2: Synthesis of Compound 002-3:

002-2 (1.1g, 3.58mmol, 1eq) and 001-21 (1.48g, 3.58mmol, 1.0eq) were dissolved in acetonitrile (20mL), diisopropylethylamine (1.39g, 10.73mmol, 3.0eq), then warmed 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 water (30 mL) and ethyl acetate (30 mL), and the layers were extracted while the aqueous phase was extracted three times with ethyl acetate (20 mL, 20 mL, 20 mL). The organic phases were combined, washed once with saturated sodium chloride solution (20 mL), and finally the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain compound 002-3. MS (ESI) m/z: 570.2, 571.2 [M+H] ⁺ .

Step 3: Synthesis of Compound 002-4:

Dissolve 002-3 (700mg, 1.23mmol, 1eq), 001-23 (217mg, 1.23mmol, 1.0eq) in dimethyl sulfoxide (5mL), add potassium carbonate (339mg, 2.46μmol, 2eq) at one time, The mixture was stirred at 25°C for 3 hours. The reaction solution was dissolved in water (20 mL) and ethyl acetate (20 mL), the layers were extracted, and 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 phases were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The obtained crude product was directly used in the next reaction without further purification. Compound 002-4. MS (ESI) m/z: 726.2, 727.2 [M+H] ⁺ .

Step 4: Synthesis of Compound 002-5:

Dissolve 002-4 (500mg, 688μmol, 1eq) in ethyl acetate (8mL), add saturated aqueous ammonium chloride solution (8mL), add iron powder (114.4mg, 2.04mmol, 3eq) at one time, and then heat up to 50 °C was stirred 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 spin-dried to obtain crude product. The obtained crude product was directly used in the next reaction without further purification. Compound 002-5. MS (ESI) m/z: 696.2, 697.2 [M+H] ⁺ .

Step 5: Synthesis of Compound 002:

002-5 (350 mg, 502.6 μmol, 1.0 eq) was dissolved in hydrochloric acid/methanol (4 M, 3.13 mL, 25 eq), and the mixture was stirred at 25° C. for 1 hour. The reaction solution was directly spin-dried to obtain crude product. The crude product was separated by preparative high performance liquid chromatography (chromatographic column: Welch Xtimate C18 100*40mm*3μm; mobile phase: [water (0.075% trifluoroacetic acid)-acetonitrile]; acetonitrile%: 2%-32%, 8min) purification. The trifluoroacetate salt of compound 002 was obtained. MS (ESI) m/z: 478.2, 479.0 [M+H] ⁺ . ¹ H NMR (400MHz, CD ₃ OD) δppm 8.62(d, J=4.4Hz, 1H), 8.46(s, 1H), 8.11-8.06(m, 2H), 7.52-7.49(m, 1H), 7.28( d, J＝6.4Hz, 1H), 4.89-4.85(s, 2H), 4.65(s, 1H), 3.73-3.65(m, 1H), 3.63-3.55(m, 1H), 3.31-3.20(m, 4H), 2.20-2.00 (m, 2H), 1.90–1.84 (m, 1H), 1.75–1.72 (m, 1H).

Embodiment 3:

Step 1: Synthesis of Compound 003-2:

Compound 003-1 (1.1 g, 2.45 mmol, 1 eq) was dissolved in dichloromethane (10 mL), and trifluoroacetic acid (5 mL) 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 solvent was spin-dried to obtain compound 003-2. MS (ESI) m/z: 327.1 [M+H] ⁺ .

Step 2: Synthesis of Compound 003-3:

003-2 (520mg, 1.33mmol, 1.2eq) and 001-21 (548mg, 1.33mmol, 1.0eq) were dissolved in acetonitrile (20mL), and diisopropylethylamine (0.97g, 7.2mmol, 3.0ml) was added at one time eq), then warmed 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 water (30 mL) and ethyl acetate (30 mL), and the layers were extracted while the aqueous phase was extracted three times with ethyl acetate (20 mL, 20 mL, 20 mL). The organic phases were combined, washed once with saturated sodium chloride solution (20 mL), and finally the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain compound 003-3. MS (ESI) m/z: 589.2 [M+H] ⁺ .

Step 3: Synthesis of Compound 003-4:

Dissolve 003-3 (300mg, 645.3μmol, 1eq), 001-23 (217mg, 1.23μmol, 1.0eq) in dimethyl sulfoxide (5mL), add potassium carbonate (133.8mg, 968.1μmol, 2eq) at one time , the mixture was stirred at 25°C for 3 hours. The reaction solution was dissolved in water (20 mL) and ethyl acetate (20 mL), the layers were extracted, and 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. Compound 003-4. MS (ESI) m/z: 745.2 [M+H] ⁺ .

Step 4: Synthesis of Compound 003-5:

Dissolve 003-4 (470mg, 630.6μmol, 1eq) in ethyl acetate (8mL), add saturated aqueous ammonium chloride solution (8mL), add iron powder (281.7mg, 5.04mmol, 8eq) at one time, and then warm 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 spin-dried to obtain crude product. The obtained crude product was directly used in the next reaction without further purification. Compound 003-5. MS (ESI) m/z: 715.2 [M+H] ⁺ .

Step 5: Synthesis of Compound 003:

003-5 (448 mg, 626.26 μmol, 1.0 eq) was dissolved in hydrochloric acid/methanol (4 M, 1.0 mL, 25 eq), and the mixture was stirred at 25° C. for 1 hour. The reaction solution was directly spin-dried to obtain crude product. The crude product was separated by preparative high performance liquid chromatography (chromatographic column: Welch Xtimate C18 100*40mm*3um; mobile phase: [water (0.075% trifluoroacetic acid)-acetonitrile]; acetonitrile%: 16%-46%, 8min) purification. The trifluoroacetate salt of compound 003 was obtained. MS (ESI) m/z: 497.0 [M+H] ⁺ . ¹ H NMR (400 MHz, CD ₃ OD) δppm 8.43 (s, 1H), 8.08 (d, J=6.02 Hz, 1H), 7.56 (dd, J=8.16, 5.65 Hz, 1H), 7.12 (d, J= 5.77Hz, 1H), 6.83(m, 2H), 4.87(s, 2H), 3.80-3.75(m, 1H), 3.64-3.36(m, 3H), 2.42-2.32(m, 1H), 2.18-1.97 (m, 4H).

Embodiment 4:

Step 1: Synthesis of Compound 004-3:

Compound 004-1 (7.13 g, 33.93 mmol, 1 eq) was dissolved in tetrahydrofuran (160 mL), and a solution of lithium bisisopropylamide in tetrahydrofuran (2M, 22.06 mL, 1.3 eq) was added when the temperature was lowered to -78 °C, The reaction system was reacted at -78 °C for 1 hour, 004-2 (10 g, 37.32 mmol, 1.1 eq) was added, the system was reacted at -78 °C for another 1 hour, and then the temperature was slowly raised to 25 °C with stirring. After the reaction was completed, it was quenched with saturated ammonium chloride solution (25 mL), extracted three times with ethyl acetate (150 mL×3), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was removed by rotary evaporation. Column chromatography: The crude product was separated by column chromatography (petroleum ether: ethyl acetate=0-10%) to obtain compound 004-3. MS(ESI) m/z: 296.8&298.8[M-100+H] ⁺ .

Step 2: Synthesis of Compound 004-4:

Under the protection of nitrogen, compound 004-3 (12.00 g, 30.21 mmol, 1 eq) was dissolved in a mixed solution of NN dimethylacetamide (100 mL) and water (10 mL), and dichlorobis[di-tert-butyl] was added. -(4-Dimethylaminophenyl)phosphine]palladium (2.14g, 3.02mmol, 2.14mL, 0.1eq) and triethylamine (12.23g, 120.82mmol, 16.82mL, 4eq), the system was purged with nitrogen three times, The temperature was raised to 130°C for 5 hours. The reaction solution was cooled to room temperature, 150 ml of water was added, extracted with ethyl acetate (200 mL×3), the organic phases were combined, concentrated under reduced pressure to a concentrated solution volume of about 150 ml, washed 4 times with water, twice with saturated brine, and anhydrous Dry over sodium sulfate and concentrate under reduced pressure to obtain crude product. The crude product was isolated by column chromatography (30%-35% ethyl acetate in petroleum ether) to give 004-4, MS (ESI) m/z: 263.9 [M-56+H] ⁺ .

Step 3: Synthesis of Compound 004-5:

004-4 (8.83g, 27.65mmol, 1eq) was dissolved in tetraethyl titanate (85ml), 001-6 (10.05g, 82.94mmol, 3eq) was added, the system was purged with nitrogen three times, and then heated to 130°C The reaction was carried out for 3 hours. After the reaction was completed, the reaction solution was cooled to room temperature, the reaction solution was added to ice water, stirred for 40 minutes, the supernatant was added to a separatory funnel, and then ethyl acetate was added for extraction (200 mL×3), and the organic phases were combined and used Washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude product. The crude product was isolated by column chromatography (25%-35% ethyl acetate in petroleum ether) to give compound 004-5. MS (ESI) m/z: 322.9 [M-100+H] ⁺ .

Step 4: Synthesis of Compound 004-6:

Dissolve 004-5 (7.34g, 17.37mmol, 1eq) in tetrahydrofuran (70mL), cool to 0°C and add sodium borohydride (1.31g, 34.74mmol, 2eq) with stirring, the reaction system gradually returns to 25°C for reaction 16 hours. Water was added to the reaction solution to quench (150 ml) unreacted sodium borohydride, extracted with ethyl acetate (200 mL×3), the combined organic phases were dried over anhydrous sodium sulfate, and the crude product was concentrated under reduced pressure. The crude product was purified by SFC to give compound 004-6. MS(ESI)m/z:325.0[M-100+H] ⁺

Step 5: Synthesis of Compound 004-7:

004-6 (1.10g, 2.59mmol, 1eq) was dissolved in dichloromethane (10mL), trifluoroacetic acid (3.84g, 33.68mmol, 2.49mL, 13eq) was added, and the mixture was reacted at 25°C for 50 minutes. After the reaction, part of the trifluoroacetic acid was removed by concentration under reduced pressure, water (30 ml) was added to the concentrated solution, potassium carbonate (3 g) was added to remove the excess trifluoroacetic acid, and the organic phase was extracted with ethyl acetate (50 mL×3). After the combination, it was dried with anhydrous sodium sulfate, filtered, and the solvent was removed by rotary evaporation to obtain compound 004-7. MS(ESI) m/z: 325.1 [M+H] ⁺ .

Step 6: Synthesis of Compound 004-8:

004-7 (783.67mg, 1.90mmol, 1eq) and 001-21 (740.00mg, 2.28mmol, 1.2eq) were dissolved in acetonitrile (12mL), diisopropylethylamine (736.69mg, 5.70mmol, 992.84uL, 3eq), the system was purged with nitrogen three times, then heated to 75°C and stirred for 16 hours. The reaction solution was cooled to room temperature and concentrated under reduced pressure. The concentrate was dissolved in water (50 mL) and ethyl acetate (50 mL), and extracted three times with ethyl acetate (50 mL×3). The organic phases were combined, washed once with saturated sodium chloride solution (50 mL), and finally the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain crude compound 004-8. MS (ESI) m/z: 587.3 [M+H] ⁺ .

Step 7: Synthesis of Compound 004-9:

Dissolve 004-8 (970mg, 1.65mmol, 1eq) and 001-23 (291.80mg, 1.65mmol, 1eq) in dimethyl sulfoxide (2ml), add potassium carbonate (456.89mg, 3.31mmol, 2eq), the mixture is in The reaction was carried out at 25°C for 1.5 hours. After the reaction, 50 ml of water was added to the reaction solution, extracted three times with ethyl acetate (50 mL × 3), the organic phases were combined, the organic phases were washed three times with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain crude product 004 -9, MS (ESI) m/z: 743.2 [M+H] ⁺ .

Step 8: Synthesis of Compound 004-10:

Dissolve 004-9 (1.20g, 1.62mmol, 1eq) in ethyl acetate (8mL), then add saturated aqueous ammonium chloride solution (8mL), add iron powder (723.12mg, 12.95mmol, 8eq) at one time, then warm up Stir to 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 spin-dried to obtain crude product. The obtained crude product was directly used in the next reaction without further purification. Compound 004-10. MS (ESI) m/z: 713.2 [M+H] ⁺ .

Step 9: Synthesis of Compound 004:

004-10 (1.06g, 1.48mmol, 1eq) was dissolved in hydrochloric acid/methanol (4M, 1.0mL, 25eq), and the mixture was stirred at 25°C for 1 hour. The reaction solution was directly spin-dried to obtain crude product.

The crude product was separated by preparative high performance liquid chromatography (chromatographic column: Phenomenex Genimi NX C18 150*40mm*5μm; mobile phase: [water (0.225% trifluoroacetic acid)-acetonitrile]; acetonitrile %: 1%-30%, 10min) Isolation and Purification. The trifluoroacetate salt of compound 004 was obtained. MS (ESI) m/z: 495.1 [M+H] ⁺ . ¹ H NMR (400MHz, CD ₃ OD) δppm 8.41(s, 1H), 8.08(d, J=6.02Hz, 1H), 7.48-7.36(m, 2H), 7.20-7.18(m, 1H), 7.08( m, 1H), 4.87(s, 2H), 4.55(s, 1H), 3.66-3.63(m, 1H), 3.57-3.54(m, 1H), 3.27-3.14(m, 4H), 2.10-2.08( m, 1H), 2.05–1.99 (m, 1H), 1.99–1.84 (m, 1H), 1.75–1.72 (m, 1H).

The crude product was separated and purified by preparative high performance liquid chromatography (chromatographic column: Welch Xtimate C18 150*25mm*5μm; mobile phase: [water (0.225% formic acid)-acetonitrile]; acetonitrile%: 1%-30%, 10min). The formate salt of compound 004 was obtained. MS (ESI) m/z: 495.1 [M+H] ⁺ . ¹ H NMR (400MHz, CD ₃ OD) δppm 8.41(s, 1H), 8.08(d, J=6.02Hz, 1H), 7.40-7.30(m, 1H), 7.27-7.18(m, 1H), 7.08( m, 1H), 6.94(d, J=6.02Hz, 1H), 4.87(s, 2H), 4.31(s, 1H), 3.62-3.45(m, 2H), 3.27-3.14(m, 3H), 3.05 -2.92(m, 1H), 2.15-1.99(m, 2H), 1.81-1.65(m, 2H).

biological test

Experimental Example 1: In vitro evaluation

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

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

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

Isoform 1 (canonical)) was expressed in E. coli with an N-terminal StrepII-TEV, C-terminal histidine tag. Mw = 71.93 kDa.

Activating peptide: H2N _- LN(pY)IDLDLV(dPEG8)LST(pY)ASINFQK-amide (based on publication)

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

Final concentrations in assay: 0.35 μM activating peptide; 100 μM DiFMUP

step:

1. Prepare the indicated enzymes/peptides and substrates in freshly prepared reaction buffer

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

3. Provide compounds in 100% DMSO into enzyme solution (Echo550; nanoliter range) by acoustic technique and incubate for 30 minutes at room temperature

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

5. Monitor the enzymatic activity (Ex/Em 355/460) as a time course measurement of the 60 min increase in the fluorescence signal of the fluorogenic substrate at room temperature.

Data Analysis: Take the slope of the linear portion of the time course measurement x (signal/min) and calculate the % enzyme activity relative to the DMSO control. Subtract the background slope of enzyme basal activity (no peptide). The results of the in vitro screening test of the compounds of the present invention are shown in Table 1:

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

compound PTPN11/SHP2-FL (IC ₅₀ nM) Trifluoroacetate salt of compound 001 1.61 Trifluoroacetate salt of compound 002 23.4 Trifluoroacetate salt of compound 003 4.22 Trifluoroacetate salt of compound 004 2.60

Conclusion: The compounds of the present invention have good inhibitory activity on PTPN11/SHP2-FL.

Experimental Example 2: Evaluation of Cell Activity of Compound H358

Purpose:

The purpose of this experiment is to verify the inhibitory effect of the compounds of the present invention on the proliferation of KRAS G12C-mutated NCI-H358 human non-small cell lung cancer cells.

Experimental Materials:

Cell line NCI-H358 (purchased from Proceedings), 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 cells were seeded in a white 96-well plate, 80 μL of cell suspension per well, which contained 4000 NCI-H358 cells. Cell plates were incubated overnight in a carbon dioxide incubator. The compounds to be tested were diluted 5-fold to the ninth concentration, that is, from 2000 μM to 5.12 nM, and a double-well experiment was set up. Add 78 μL of medium to the middle plate, and then transfer 2 μL of each well of the compound to the middle plate according to the corresponding position. After mixing, transfer 20 μL of each well to the cell plate. Compound concentrations transferred to cell plates ranged from 10 [mu]M to 0.026 nM. The cell plates were placed in a carbon dioxide incubator for 5 days. Another cell plate was prepared, and the signal value was read on the day of drug addition as the maximum value (Max value in the following equation) to participate in data analysis. Add 25 μL of chemiluminescence detection reagent for cell viability to each well of this cell plate, incubate at room temperature for 10 minutes to stabilize the luminescence signal, and use a multi-label analyzer to read.

data analysis:

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

Table 2 shows the results of the cell activity screening test of the compound H358 of the present invention.

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

Compound number H358 (IC ₅₀ nM)

Trifluoroacetate salt of compound 001 46.5 Trifluoroacetate salt of compound 003 136.0 Formate salt of compound 004 16.0

Conclusion: The compounds of the present invention have good inhibitory activity on H358 cells.

Experimental Example 3: Compound Pharmacokinetic Evaluation

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

Experimental material: CD-1 mice (male, 32-33g)

Experimental operation:

The rodent pharmacokinetic characteristics of compounds after intravenous injection and oral administration were tested by standard protocols. In the experiment, candidate compounds were formulated into clear solutions and administered to mice by single intravenous injection and oral administration. Intravenous and oral vehicles are 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 plasma samples, add 4 times the volume of acetonitrile solution containing the internal standard to precipitate proteins, centrifuge the supernatant, add equal volume of water, mix well, and then centrifuge to get the samples. The supernatant was injected, and the plasma drug concentration was quantitatively analyzed by LC-MS/MS analysis method, and the pharmacokinetic parameters, such as peak concentration, peak time, clearance rate, half-life, area under the drug-time curve, bioavailability, etc., were calculated.

The pharmacokinetic test results of the compounds of the present invention are shown in Table 3.

Table 3. Compound pharmacokinetic test results

Conclusion: The compounds of the present invention can significantly improve the pharmacokinetics single or partial indexes in mice.

Experimental Example 4: Inhibition test of hERG potassium channel

1. Experimental purpose:

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

2. Experimental method

2.1. Cell Culture

The cells stably expressing the hERG potassium channel used in the experiment were obtained from CHO-hERE of Aviva Biosciences, and CHO-hERG was cultured in 5% CO ₂ at 37°C. CHO hERG medium is shown in Table 4.

Table 4. CHO hERG broth

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

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

2.3. Preparation of intracellular and extracellular fluids

Extracellular fluid needs to be prepared once a month. The intracellular fluid must be aliquoted and stored at -20°C. The composition of intracellular and extracellular fluids is shown in Table 5.

Table 5. Components of intracellular and extracellular fluids

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 formulation

The compound to be tested and the positive control Amitriptyline were dissolved in DMSO into a stock solution of a certain concentration, then diluted according to different gradients, and finally added to the extracellular fluid according to a certain proportion to be diluted to the concentration to be tested. Visually inspect for the presence or absence of precipitation prior to the start of the experiment. Finally, in the test solution and the positive control Amitriptyline, the maximum concentration of DMSO should not exceed 0.3%.

2.5. Voltage stimulation protocol

Hold the clamping potential at -80mv, first give -50mv voltage stimulation for 80ms to record the cell leakage current value, then depolarize to +20mv, maintain 4800ms, open the hERG channel, and then repolarize to -50mv for 5000ms , elicited the hERG tail current and recorded, and finally, the voltage returned to the clamping potential -80mv and maintained for 3100ms. The above voltage stimulation was repeated every 15000ms.

2.6. QPatch ^HTX Whole Cell Patch Clamp Recording

hERG QPatch ^HTX experiments were performed at room temperature. Whole cell protocols, voltage stimulation protocols and compound detection protocols were established on the software of QPatch Assay Software 5.2 (Sophion Bioscience).

30 repetitions of voltage stimulation were performed first, and this segment was the baseline region for subsequent analysis, followed by the addition of 5 μL of extracellular fluid, which was repeated three times. The effect concentrations of each compound were added sequentially, again in a 5 μL addition volume and repeated three times. Incubate cells for at least 5 mins at each test concentration. During the entire recording process, each index must meet the acceptance criteria for data analysis. If the criteria are not met, the cell will not be included in the analysis range, and the compound will be re-tested. The above recording process is automated by the Qpatch analysis software. Each compound was tested at concentrations of 0.24 μM, 1.20 μM, 6.00 μM, and 30.00 μM in sequence, and each concentration was replicated in at least two cells.

2.7. Data Analysis

In each full current recording, based on the percentage of peak current in the negative control, the percent inhibition for each compound acting concentration can be calculated. The dose-response curve is obtained by fitting the standard Hexian equation, and the specific equation is as follows:

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

C is the compound test concentration, n is the slope

Curve fitting and inhibition rate calculation are both completed 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 6 shows the hERG IC ₅₀ values of the compounds of the examples.

Table 6. Example compound hERG _IC50 value results

Test sample hERG _IC50 (nM) Trifluoroacetate salt of compound 001 18.3 Trifluoroacetate salt of compound 003 6.4 Formate salt of compound 004 9.8

Conclusion: The compounds of the present invention do not significantly inhibit hERG.

Experimental Example 5: In Vitro Microsomal Stability Experiment

1. Experimental materials

Human and animal microsomes were purchased from Corning or Xenotech and stored in a -80°C freezer.

Nicotinamide Adenine Dinucleotide Phosphate, Reduced (NADPH), Supplier: Chem-impex international, Cat. No. 00616

Control compounds: testosterone, diclofenac, propafenone

2. Experimental steps

2.1 Preparation of working solution

Stock solution: 10mM DMSO solution

Working concentration preparation: 100% acetonitrile diluted to 100 μM (organic phase content: 99% ACN, 1% DMSO)

2.2 Experimental steps

Prepare two 96-well incubation plates, named T60 incubation plate and NCF60 incubation plate respectively.

Add 445 μL of microsomal working solution (the concentration of liver microsomal protein is 0.56 mg/mL) to the T60 incubation plate and NCF60 incubation plate respectively, and then place the above incubation plate in a 37°C water bath for pre-incubation for about 10 minutes.

After the pre-incubation, add 5 μL of the test substance or control compound working solution to the T60 incubation plate and NCF60 incubation plate respectively, and mix well. Add 50 μL of potassium phosphate buffer to each well of the NCF60 incubation plate to start the reaction; add 180 μL of stop solution (200 ng/mL tolbutamide and 200 ng/mL labetalol in acetonitrile) and 6 μL of NADPH regeneration system working solution to the T0 stop plate , remove 54 μL of sample from the T60 incubation plate to the T0 stop plate (T0 sample generation). Add 44 μL of NADPH regeneration system working solution to each well of the T60 incubation plate to start the reaction. Add only 54 μL of microsomal working solution, 6 μL of NADPH regeneration system working solution and 180 μL of stop solution to the Blank plate. Therefore, in the test or control compound samples, the final reaction concentration of compound, testosterone, diclofenac and propafenone was 1 μM, the final concentration of liver microsomes was 0.5 mg/mL, and the final concentration of DMSO and acetonitrile in the reaction system was The concentrations were 0.01% (v/v) and 0.99% (v/v), respectively.

After an appropriate incubation time (eg 5, 15, 30, 45 and 60 minutes), add 180 μL of stop solution (200ng/mL tolbutamide and 200ng/mL labetalol in acetonitrile) to the sample wells of each stop plate, respectively. Remove 60 μL of sample from the T60 incubation plate to stop the reaction.

All sample plates were shaken and centrifuged at 3220×g for 20 minutes, then 80 μL of supernatant per well was diluted into 240 μL of purified water for liquid chromatography tandem mass spectrometry analysis.

The results of the MMS results of the compounds of the present invention are shown in Table 7.

Table 7. MMS results of compounds of the present invention

Test sample MMS (mL/min/kg), H (human), M (mouse) Trifluoroacetate salt of compound 001 11.9, 57.5 Formate salt of compound 004 11.9, 64.1

Conclusion: The compounds of the present invention have better stability of liver microsomes.

Claims (13)

A compound of formula (I) or a pharmaceutically acceptable salt thereof,

in, T ₁ is CR ₄ or N; When E ₁ is O, R ₃ is H, F, Cl, Br, I or C _1-3 alkyl optionally substituted with ₁ , 2 or 3 R _a ; When E ₁ is CH ₂ , R ₃ is H, Cl, Br, I or C _1-3 alkyl optionally substituted with ₁ , 2 or 3 R _a ; R ₁ , R ₂ and R ₄ are each independently H, F, Cl, Br, I or C _1-3 alkyl optionally substituted with ₁ , 2 or 3 R _b ; R ₅ is C _1-3 alkyl substituted by OH; R ₆ is each independently H, F, Cl, Br, I, OH, NH ₂ or C _1-3 alkyl optionally substituted with ₁ , 2 or 3 R _c ; R _a , R _b and R _c are each independently F, Cl, Br, I, OH or NH ₂ ; m is 1, 2, 3 or 4. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein when E ₁ is O, R ₃ is H or F. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein when E ₁ is CH ₂ , R ₃ is H. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein R ₁ , R ₂ and R ₄ are each independently H, F, Cl, Br or I. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein R ₅ is -CH ₂ -OH. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein R ₆ is each independently Cl or NH ₂ . The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein the structural unit

for

The compound according to claim 1 or a pharmaceutically acceptable salt thereof, which is

in, T ₁ , E ₁ , R ₁ , R ₂ , R ₃ , R ₅ , R ₆ and m are as defined in claims 1-7. The compound according to claim 8 or a pharmaceutically acceptable salt thereof, which is

in, T ₁ , E ₁ , R ₁ , R ₂ and R are as defined in claim 8 . The following compounds or their pharmaceutically acceptable salts,

The compound according to claim 10, or a pharmaceutically acceptable salt thereof, selected from the group consisting of:

A pharmaceutical composition comprising the compound of any one of claims 1-11 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. Use of the compound according to any one of claims 1 to 11 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition according to claim 12 in the preparation of a medicament for the treatment of SHP2-related diseases, wherein the SHP2-related disease The disease refers to lung cancer, preferably non-small cell lung cancer.