WO2023274418A1

WO2023274418A1 - Proteolysis-targeting chimeric compound

Info

Publication number: WO2023274418A1
Application number: PCT/CN2022/103762
Authority: WO
Inventors: 黄婧婕; 于涛; 谭冶; 李泽许; 吴成德; 陈曙辉
Original assignee: 南京明德新药研发有限公司
Priority date: 2021-07-02
Filing date: 2022-07-04
Publication date: 2023-01-05

Abstract

A novel proteolysis-targeting chimeric compound, and an application thereof in the preparation of a drug for treating related diseases. A compound represented by formula (I) and a pharmaceutically-acceptable salt thereof are specifically disclosed. PTM-L-ULM (I)

Description

Protein degradation targeting chimera-like compounds

This application claims the following priority:

CN202110750241.8, application date: July 02, 2021;

CN202110872914.7, application date: July 30, 2021.

technical field

The invention relates to a class of protein degradation targeting chimera compounds and their application in the preparation of drugs for treating related diseases, specifically disclosing the compounds represented by formula (I) and pharmaceutically acceptable salts thereof.

Background technique

BRD4 (Bromodomain-containing protein 4) is a protein encoded by the BRD4 gene in humans. BRD4 is a member of the BET family, which also includes BRD2, BRD3 and BRDT. Similar to other BET family members, BRD4 contains two bromodomains that recognize acetylated lysine residues. BRD4 also has an extended C-terminal domain with little sequence homology to other BET family members. BRD4 can bind to acetylated histones or non-histones, thereby regulating gene replication and transcription, affecting cell cycle, cell differentiation, signal transduction and other processes. The upregulation of BRD4 expression is closely related to the malignant development of various tumors. Inhibiting or degrading BRD4 can effectively control the malignant progression and distant metastasis of tumors. Therefore, BRD4 is a promising tumor epigenetic target.

PROTAC (Proteolysis Targeting Chimera) molecules are a class of bifunctional compounds that can simultaneously bind target proteins and E3 ubiquitin ligases. Such compounds can induce the intracellular proteasome system to recognize the target protein, thereby causing the degradation of the target protein, and can effectively reduce the content of the target protein in the cell. By introducing ligands that can bind different target proteins into PROTAC molecules, it is possible to apply PROTAC technology to the treatment of various diseases. This technology has attracted extensive attention from the pharmaceutical industry in recent years.

Contents of the invention

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

PTM-L-ULM

(I)

in,

PTM selected from

L is selected from single bond, *-Cy ₁ -Ak ₁ -Cy ₂ -Ak ₂ -Cy ₃ -Ak ₃ -(Cy ₄ -Ak ₄ ) _n -, *-Cy ₁ -(OCH ₂ CH ₂ ) _m -NH - and *-Cy ₁ -(OCH ₂ CH ₂ ) _m -O-, where * indicates connection with PTM;

Cy ₁ is selected from phenyl and 5-6 membered heteroaryl;

Ak ₁ is selected from single bonds and -O-;

Cy ₂ , Cy ₃ and Cy ₄ are independently selected from 3-6 membered heterocycloalkyl groups, and the 3-6 membered heterocycloalkyl groups are optionally substituted by 1, 2 or 3 R _a ;

Ak ₂ , Ak ₃ and Ak ₄ are independently selected from single bond, C _1-3 alkyl and -C _1-3 alkyl NH-, said C _1-3 alkyl and -C _1-3 alkyl NH - optionally substituted by 1, 2 or 3 R _b ;

n is 0 or 1;

m is 2, 3 or 4;

ULM is selected from the structures shown in formula (I-1), formula (I-2) and formula (I-3):

R ₁ , R ₂ , R ₃ and R ₄ are each independently selected from H;

Alternatively, R ₁ and R ₃ , R ₂ and R ₄ together with the carbon atoms they are connected to form =O and =NC _1-3 alkoxy;

R _a and R _b are independently selected from F, Cl, Br, I, oxo, C _1-3 alkyl and C _1-3 alkoxy;

requirement is:

When ULM is selected from formula (I-1), L is selected from single bond and *-Cy ₁ -Ak ₁ -Cy ₂ -Ak ₂ -Cy ₃ -Ak ₃ -(Cy ₄ -Ak ₄ ) _n -;

The "hetero" of the 3-6 membered heterocycloalkyl group and the 5-6 membered heteroaryl group represents a heteroatom independently selected from 1, 2, 3 or 4 O, S, N and C (=O) or heteroatom groups.

PTM-L-ULM

(I)

in,

PTM selected from

Cy ₁ is selected from phenyl, piperidinyl and 5-6 membered heteroaryl;

Ak ₁ is selected from single bonds and -O-;

n is 0 or 1;

m is 2, 3 or 4;

R ₁ , R ₂ , R ₃ and R ₄ are each independently selected from H;

R _a and R _b are independently selected from F, Cl, Br, oxo, C _1-3 alkyl and C _1-3 alkoxy;

The condition is: when ULM is selected from formula (I-1), L is selected from single bond and *-Cy ₁ -Ak ₁ -Cy ₂ -Ak ₂ -Cy ₃ -Ak ₃ -(Cy ₄ -Ak ₄ ) _n -;

The "hetero" of the 3-6 membered heterocycloalkyl and 5-6 membered heteroaryl is a heteroatom independently selected from O, S and N.

PTM-L-ULM

(I)

in,

PTM selected from

Cy ₁ is selected from phenyl, piperidinyl and 5-6 membered heteroaryl;

Ak ₁ is selected from single bonds and -O-;

n is 0 or 1;

m is 2, 3 or 4;

R ₁ , R ₂ , R ₃ and R ₄ are each independently selected from H;

In some solutions of the present invention, the above-mentioned R ₁ and R ₃ , R ₂ and R ₄ together with their connected carbon atoms constitute =O and =N-OCH ₃ , and other variables are as defined in the present invention.

In some solutions of the present invention, the above Cy ₁ is selected from phenyl and pyridyl, and other variables are as defined in the present invention.

In some schemes of the present invention, the above-mentioned Cy ₂ , Cy ₃ and Cy ₄ are independently selected from

said

Optionally substituted by 1, 2 or 3 R _a , other variables are as defined herein.

Other variables are as defined herein.

In some solutions of the present invention, the above-mentioned Ak ₂ , Ak ₃ and Ak ₄ are independently selected from single bonds, -CH ₂ - and -CH ₂ CH ₂ NH-, and the -CH ₂ - and -CH ₂ CH ₂ NH - optionally substituted by 1, 2 or 3 R _b , other variables are as defined in the present invention.

In some solutions of the present invention, the above-mentioned Ak ₂ , Ak ₃ and Ak ₄ are independently selected from single bonds, -CH ₂ - and -CH ₂ CH ₂ NH-, and other variables are as defined in the present invention.

In some solutions of the present invention, the above-mentioned L is selected from single bonds,

Other variables are as defined herein.

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

in,

L is as defined herein.

The present invention also provides a compound represented by the following formula or a pharmaceutically acceptable salt thereof,

In some schemes of the present invention, the following compounds or pharmaceutically acceptable salts thereof are also provided, which are selected from:

technical effect

The compound of the present invention has significant cell proliferation inhibitory activity on MDA-MB-231 cell line, and has weak or substantially inactive activity on GSPT1; the compound of the present invention can degrade BRD4 protein concentration-dependently in MDA-MB-231 cell line, And it has no obvious degradation activity on GSPT1; the compound of the present invention has excellent pharmacokinetic properties.

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 indeterminate or unclear if it is not specifically defined, but should be understood according to its ordinary meaning. When a trade name appears herein, it is intended to refer to its corresponding trade name or its active ingredient.

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

The term "pharmaceutically acceptable salt" refers to a salt of a compound of the present invention, which is prepared from a compound having a specific substituent found in the present invention and a relatively non-toxic acid or base. 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, either neat solution or in a suitable inert solvent. When the compounds of the present invention contain relatively basic functional groups, acid addition salts of certain specific compounds of the present invention can be obtained by contacting such compounds with a sufficient amount of acid in neat solution or in a suitable inert solvent. Compounds contain 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 parent compound containing acid groups or bases by conventional chemical methods. In general, 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 both.

Unless otherwise stated, the term "isomer" is intended to include geometric isomers, cis-trans isomers, stereoisomers, enantiomers, optical isomers, diastereoisomers and interconversions isomer.

The compounds of the invention may exist in particular 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 and other mixtures, such as enantiomerically or diastereomerically enriched mixtures, all of which are subject to the present within the scope of the invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers, as well as mixtures thereof, are included within the scope of the present invention.

Unless otherwise stated, the terms "enantiomer" or "optical isomer" refer to stereoisomers that are mirror images of each other.

Unless otherwise stated, the terms "cis-trans isomers" or "geometric isomers" arise from the inability to rotate freely due to the double bond or the single bond of the carbon atoms forming the ring.

Unless otherwise indicated, the term "diastereoisomer" refers to stereoisomers whose molecules have two or more chiral centers and which are not mirror images of the molecules.

Unless otherwise specified, "(+)" means dextrorotation, "(-)" means levorotation, and "(±)" means racemization.

Unless otherwise noted, keys with wedge-shaped solid lines

and dotted wedge keys

Indicates the absolute configuration of a stereocenter, with a straight solid-line bond

and straight dashed keys

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

Indicates wedge-shaped solid-line bond

or dotted wedge key

or with tilde

Indicates a straight solid line key

or straight dotted key

Unless otherwise stated, the terms "enriched in an isomer", "enriched in an isomer", "enriched in an enantiomer" or "enantiomerically enriched" refer to one of the isomers or enantiomers 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 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 stated, the terms "isomer excess" or "enantiomeric excess" refer 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 other isomer or enantiomer is 10%, then 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 invention is desired, it can be prepared by asymmetric synthesis or derivatization with chiral auxiliary agents, wherein the resulting diastereomeric mixture 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 diastereoisomeric salt is formed with an appropriate optically active acid or base, and then a diastereomeric salt is formed by a conventional method known in the art. Diastereomeric resolution is performed and the pure enantiomers are recovered. Furthermore, the separation of enantiomers and diastereomers is usually accomplished by the use of chromatography using chiral stationary phases, optionally in combination with chemical derivatization methods (e.g. 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 compounds. For example, compounds may be labeled with radioactive isotopes such as tritium ( ³ H), iodine-125 ( ¹²⁵ I) or C-14 ( ¹⁴ C). For another example, heavy hydrogen can be used to replace 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 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 changes in isotopic composition of the compounds of the invention, whether radioactive or not, are included within the scope of the invention.

The term "optional" or "optionally" means that the subsequently described event or circumstance can but need not occur, and that the description includes instances where said event or circumstance occurs and instances where said event or circumstance does not occur .

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 a substituent is oxygen (ie =0), it means that two hydrogen atoms are replaced. 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 realizable basis.

When any variable (eg, R) occurs more than once in the composition or structure of a compound, its definition is independent at each occurrence. Thus, for example, if a group is substituted with 0-2 R, said group may optionally be substituted with up to two R, with independent options for each occurrence of R. Also, combinations of substituents and/or variations 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 the number of a substituent is 0, it means that the substituent does not exist, such as -A-(R) ₀ means that the structure is actually -A.

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

A substituent can be bonded to any atom on a ring when the bond of a substituent can cross-link two or more atoms on the ring, e.g., structural unit

It means that the substituent R can be substituted at any position on cyclohexyl or cyclohexadiene. When the enumerated substituent does not indicate which atom it is connected to the substituted group, this substituent can be bonded through any atom, for example, pyridyl as a substituent can be connected to any atom on the pyridine ring. The carbon atom is attached to the group being substituted.

When the linking group listed does not indicate its linking direction, its linking direction is arbitrary, for example,

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

It can also be formed by connecting loop A and loop B in the opposite direction to the reading order from left to right

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

Unless otherwise specified, when a group has one or more linkable sites, any one or more sites of the group can be linked to other groups through chemical bonds. When the connection method of the chemical bond is not positioned, and there is an H atom at the connectable site, when the chemical bond is connected, the number of H atoms at the site will decrease correspondingly with the number of chemical bonds connected to become the corresponding valence group. The chemical bonds that the site connects with other groups can use straight solid line bonds

Straight dotted key

or tilde

express. For example, the straight-shaped solid-line bond in _-OCH3 indicates that it is connected to other groups through the oxygen atom in the group;

The straight dotted line 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 1 and 2 carbon atoms in the phenyl group are connected to other groups;

Indicates that any connectable site on the piperidinyl group can be connected to other groups through a chemical bond, including at least

These 4 connection methods, even if the H atom is drawn on -N-, but

still include

For groups with this connection method, only when a chemical bond is connected, the H at this site will be reduced by one to become the corresponding monovalent piperidinyl group.

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

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

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

Unless otherwise specified, the terms "5-6-membered heteroaryl ring" and "5-6-membered heteroaryl" in the present invention can be used interchangeably, and the term "5-6-membered heteroaryl" means that there are 5 to 6 ring atoms A monocyclic group with a conjugated π-electron system, 1, 2, 3 or 4 ring atoms are heteroatoms independently selected from O, S and N, and the rest are carbon atoms. Where the nitrogen atom is optionally quaternized, the nitrogen and sulfur heteroatoms may be optionally oxidized (ie, NO and S(O) _p , where p is 1 or 2). The 5-6 membered heteroaryl can be attached to the rest of the molecule through a heteroatom or a carbon atom. The 5-6 membered heteroaryl includes 5 and 6 membered heteroaryl. Examples of the 5-6 membered heteroaryl groups include, but are not limited to, pyrrolyl (including N-pyrrolyl, 2-pyrrolyl and 3-pyrrolyl, etc.), pyrazolyl (including 2-pyrazolyl and 3-pyrrolyl Azolyl, etc.), imidazolyl (including N-imidazolyl, 2-imidazolyl, 4-imidazolyl and 5-imidazolyl, etc.), oxazolyl (including 2-oxazolyl, 4-oxazolyl and 5- Oxazolyl, etc.), triazolyl (1H-1,2,3-triazolyl, 2H-1,2,3-triazolyl, 1H-1,2,4-triazolyl and 4H-1, 2,4-triazolyl, etc.), tetrazolyl, isoxazolyl (3-isoxazolyl, 4-isoxazolyl and 5-isoxazolyl, etc.), thiazolyl (including 2-thiazolyl , 4-thiazolyl and 5-thiazolyl, etc.), furyl (including 2-furyl and 3-furyl, etc.), thienyl (including 2-thienyl and 3-thienyl, etc.), pyridyl (including 2 -pyridyl, 3-pyridyl and 4-pyridyl, etc.), pyrazinyl or pyrimidyl (including 2-pyrimidyl and 4-pyrimidyl, etc.).

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 the methods well known to those skilled in the art Equivalent alternatives, preferred embodiments include but are not limited to the examples of the present invention.

The structure of the compounds of the present invention can be confirmed by conventional methods known to those skilled in the art. If the present invention involves the absolute configuration of the compound, the absolute configuration can be confirmed by conventional technical means in the art. For example, in single crystal X-ray diffraction (SXRD), the cultured single crystal is collected with a Bruker D8 venture diffractometer to collect diffraction intensity data, the light source is CuKα radiation, and the scanning method is:

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

The solvent used in the present invention is commercially available.

The present invention adopts the following abbreviations: aq stands for water; eq stands for equivalent, equivalent; DCM stands for dichloromethane; PE stands for petroleum ether; DMSO stands for dimethyl sulfoxide; EtOAc stands for ethyl acetate; EtOH stands for ethanol; MeOH stands for Methanol; Cbz stands for benzyloxycarbonyl, which is an amine protecting group; BOC stands for tert-butoxycarbonyl, which is a kind of amine protecting group; rt stands for room temperature; O/N stands for overnight; THF stands for tetrahydrofuran; Boc ₂ O stands for di-tert-tert Butyl dicarbonate; TFA stands for trifluoroacetic acid; DIEA stands for diisopropylethylamine; iPrOH stands for 2-propanol; mp stands for melting point; DIAD stands for diisopropyl azodicarboxylate; HATU stands for 2-( 7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate.

Compounds are named according to the conventional naming principles in this field or using

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

Description of drawings

Fig. 1 is the test result of the degradation activity of the compound of the present invention on BRD4 and GSPT1 in MDA-MB-231 cell line.

detailed description

The present invention will be described in detail through examples below, but it does not imply any unfavorable limitation to 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

synthetic route:

Step 1: Synthesis of Compound 01-3

Compound 01-1 (20g, 143.77mmol, 1eq) was dissolved in tetrahydrofuran (500mL), triphenylphosphine (45.22g, 172.40mmol, 1.20eq) and DIAD (36.17g, 178.89mmol, 34.78mL, 1.24eq ), the mixture was stirred at 0°C for 10 minutes, then 01-2 (30.38g, 150.96mmol, 1.05eq) was added, and the mixture was stirred and reacted at 20°C for 16 hours. After the reaction was completed, saturated aqueous sodium bicarbonate (100 mL) was added to the reaction solution to quench the reaction, extracted with ethyl acetate (3×100 mL), and the organic phases were combined. Compound 01-3 was obtained after purification by column chromatography (petroleum ether:ethyl acetate=4:1). LCMS m/z = 223.1 [M-100+1] ⁺ .

Step 2: Synthesis of Compound 01-4

Compound 01-3 was dissolved in dichloromethane (50mL), hydrochloric acid methanol solution (4M, 50.00mL, 6.45eq) was added at 0°C, and the mixture was stirred and reacted at 15°C for 1 hour. After the reaction was completed, it was concentrated under reduced pressure, dissolved in water (50 mL), and extracted with dichloromethane (3×100 mL). Take the aqueous phase, neutralize with saturated aqueous sodium bicarbonate solution (100mL), extract with dichloromethane (3×100mL), combine the organic phases, and concentrate to obtain compound 01-4; After soaking and filtering, the filtrate was collected and concentrated to obtain compound 01-4; it was directly used in the next reaction.

Step 3: Synthesis of Compound 01-6

Add dichloroethane (150mL) to 01-4 (6.0g, 23.19mmol, 1eq), then add 01-5 (15.88g, 92.77mmol, 4eq) and acetic acid (6.96g, 115.96mmol, 6.63mL, 5eq ), sodium acetate borohydride (29.49g, 139.16mmol, 6eq) was added at 20°C, and the mixture was stirred and reacted at 20°C for 2 hours. After the reaction was completed, an aqueous solution (10 mL) was added to the reaction solution to quench the reaction, extracted with dichloromethane (3×10 mL), the organic phases were combined, dried over anhydrous sodium sulfate and then spin-dried. Compound 01-6 was obtained by preparative thin-layer chromatography on a silica gel plate (dichloromethane:methanol=20:1) and column chromatography (dichloromethane:methanol:triethylamine=20:1:0.2). LCMS m/z = 378.1 [M+1] ⁺ .

Step 4: Synthesis of compound 01-7

Compound 01-6 (4.8g, 9.69mmol, 76.2% purity, 1eq) was dissolved in dichloromethane (50mL), hydrochloric acid methanol solution (4M, 60.96mL, 25.16eq) was added at 0°C, and the mixture was stirred at 0°C React for 1 hour. After the reaction was completed, the solvent was spin-dried, then dissolved in methanol (50 mL), added with sodium bicarbonate solution, filtered to collect the filtrate, and concentrated to obtain compound 01-7, which was directly used in the next reaction.

Step 5: Synthesis of compound 01-8

Add dichloroethane (150mL) to compound 01-7 (3.8g, 13.70mmol, 1eq), then add 01-5 (9.38g, 54.81mmol, 4eq) and acetic acid (4.11g, 68.51mmol, 3.92mL, 5eq), sodium acetate borohydride (17.42g, 82.22mmol, 6eq) was added at 20°C, and the mixture was stirred and reacted at 20°C for 2 hours. After the reaction was completed, an aqueous solution (10 mL) was added to the reaction solution to quench the reaction, extracted with dichloromethane (3×10 mL), and the organic phases were combined. The organic phase was concentrated under reduced pressure to obtain compound 01-8, which was directly used in the next reaction.

Step 6: Synthesis of compound 01-9

Add ethanol (40mL) and water (10mL) to compound 01-8 (2.0g, 4.62mmol, 1eq), then add ammonium chloride (2.47g, 46.24mmol, 10eq) and iron powder (2.58g, 46.24mmol, 10eq), the mixture was stirred and reacted at 20°C for 1 hour. After the reaction is complete, filter the reaction solution, wash the filter cake with ethanol, collect the filtrate, concentrate and then add methanol/dichloromethane (1:1, 50mL) to soak, filter, collect the filtrate, concentrate to obtain compound 01-9, and directly use Next reaction.

Step 7: Synthesis of Compound 01-11

Add DMF (10mL) to compound 01-9 (0.8g, 1.73mmol, 87% purity, 1eq), then add compound 01-10 (693.15mg, 1.73mmol, 1eq) and DIEA (670.39mg, 5.19mmol, 903.50 μL, 3eq), HATU (1.31g, 3.46mmol, 2eq) was added at 20°C, and the mixture was stirred and reacted at 20°C for 2 hours. After the reaction was completed, an aqueous solution (10 mL) was added to the reaction solution to quench the reaction, extracted with dichloromethane (3×10 mL), and the organic phases were combined. The crude product was separated by preparative silica gel column (dichloromethane:methanol=10:1) to obtain compound 01-11. LCMS m/z = 785.3 [M+1] ⁺ .

Step 8: Synthesis of Compound 01-12

Compound 01-11 (0.3g, 381.97μmol, 1eq) was dissolved in dichloromethane (6mL), hydrochloric acid methanol (4M, 18.00mL, 188.49eq) was added dropwise at 0°C, and the mixture was stirred at 0°C for 1 hour. After the reaction was completed, it was concentrated under reduced pressure to obtain compound 01-12 hydrochloride, which was directly used in the next reaction.

Step 9: Synthesis of compound 01

Dissolve 01-12 hydrochloride (0.16g, 221.69μmol, 1eq) in DMSO (5mL), add 01-13 (61.23mg, 221.69μmol, 1eq) and DIEA (229.21mg, 1.77mmol, 308.90μL, 8eq ), the mixture was heated to 80°C and stirred for 2 hours. After the reaction was completed, an aqueous solution (50 mL) was added to the reaction solution to quench the reaction, extracted with dichloromethane (3 × 10 mL), the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product, which was prepared by preparing a thin-layer chromatography silica gel plate (2 Chloromethane: methanol: triethylamine=10:1:0.1) separation and purification, and then through the preparation of high performance liquid chromatography column (chromatographic column: Boston Prime C18 150*30mm*5 μm; mobile phase: [water (NH ₃ H ₂ O +NH ₄ HCO ₃ )-ACN]; Acetonitrile: 45%-75%, 7min) separation, and then normal phase chiral chromatography (column: DAICEL CHIRALPAK ID (250mm*30mm, 10μm); mobile phase: [MeOH-ACN ]; ACN:50%-50%, 60min) purification to obtain compound 01. ¹ H NMR (400MHz, CDCl ₃ )δppm 8.60(br s,1H),8.19(br s,1H),7.43-7.32(m,5H),7.30-7.23(m,2H),7.08(d,J= 7.0Hz, 1H), 6.76(br d, J=8.8Hz, 2H), 6.50(d, J=8.5Hz, 1H), 4.84(br dd, J=5.4, 11.9Hz, 1H), 4.56(dd, J=5.6,8.2Hz,1H),4.25-4.12(m,3H),4.01(br s,2H),3.69(br dd,J=8.5,13.8Hz,1H),3.54-3.36(m,4H) ,2.98(br s,2H),2.85-2.65(m,3H),2.60(s,3H),2.50(br s,2H),2.33(s,3H),2.19-1.57(m,11H); LCMS m/z = 942.0 [M+1] ⁺ ; ee% = 97.6%.

Example 2

synthetic route:

Step 1: Synthesis of compound 02

Compound 01-12 hydrochloride (0.07g, 96.99μmol, 1eq) was dissolved in DMSO (2mL), and 02-1 (26.79mg, 96.99μmol, 1eq) and DIEA (100.28mg, 775.90μmol, 135.15μL, 8eq), the mixture was heated to 80°C and stirred for 3 hours. After the reaction was completed, an aqueous solution (50 mL) was added to the reaction solution to quench the reaction, extracted with dichloromethane (3 × 10 mL), the organic phases were combined, dried over anhydrous sodium sulfate and concentrated, and prepared a thin-layer chromatography silica gel plate (dichloromethane : methyl alcohol: triethylamine=10:1:0.1) separation; then through the preparation of high performance liquid chromatography column (chromatographic column: Boston Prime C18 150*30mm*5 μ m; mobile phase: [water (NH ₃ H ₂ O+NH ₄ HCO ₃ )-ACN]; Acetonitrile: 45%-75%, 7min) separation, and then through normal phase chiral chromatography (column: DAICEL CHIRALPAK ID (250mm*30mm, 10μm); Mobile phase: [MeOH-ACN]; ACN : 50%-50%, 60min) purified to obtain compound 02. ¹ H NMR (400MHz, CDCl ₃ ) δppm 8.64(br s,1H),8.03(br s,1H),7.57(d,J=8.3Hz,1H),7.44-7.24(m,6H),6.87-6.67 (m,3H),6.47(br d,J=8.4Hz,1H),4.85(br dd,J=5.6,12.4Hz,1H),4.62-4.51(m,1H),4.01-3.94(m,2H ),3.86(br s,2H),3.69(br dd,J＝8.4,14.0Hz,2H),3.56(br s,2H),3.44-3.36(m,1H),2.85-2.63(m,5H) , 2.61 (s, 3H), 2.34 (s, 3H), 2.15-1.94 (m, 4H), 1.61 (m, 10H); LCMS m/z = 941.4 [M+1] ⁺ ; ee% = 100%.

Example 3

synthetic route:

Step 1: Synthesis of compound 03-3

Compound 03-1 (2.0g, 7.19mmol, 1eq), 03-2 (1.58g, 7.19mmol, 1.39mL, 1eq) was dissolved in DMF (20mL), and potassium carbonate (1.99g, 14.38mmol, 2.0eq ), the mixture was heated to 80°C and stirred for 10 hours. After the reaction was completed, an aqueous solution (50mL) was added to the reaction solution to quench the reaction, extracted with dichloromethane (3×50mL), the organic phases were combined, dried over anhydrous sodium sulfate, concentrated, and subjected to column chromatography (PE:EA:TEA= 1:1:0.01) to obtain compound 03-3. ¹ H NMR (400MHz, CDCl3) δppm 7.32-7.22 (m, 5H), 5.11-5.04 (m, 2H), 4.06-3.91 (m, 2H), 3.46-3.39 (m, 4H), 2.61 (br t, J＝11.9Hz, 2H), 2.28(br s, 4H), 2.10(d, J＝7.0Hz, 2H), 1.65(br d, J＝13.3Hz, 2H), 1.59-1.50(m, 1H), 1.38 (s, 9H), 1.06-0.92 (m, 2H); LCMS m/z = 418.1 [M+1] ⁺ .

Step 2: Synthesis of compound 03-4

Compound 03-3 (1.8g, 4.31mmol, 1eq) was dissolved in methanol (10mL), palladium carbon (0.5g, 10% content) was added under nitrogen protection, and hydrogen gas (8.71mg, 4.31mmol, 1eq) was introduced, The mixture was stirred and reacted at 25°C for 10 hours. After the reaction was completed, it was filtered, the filter cake was washed with methanol, and concentrated to obtain compound 03-4. The crude product was directly used in the next reaction. LCMS m/z = 284.2 [M+1] ⁺ .

Step 3: Synthesis of compound 03-5

Compound 03-4 (0.5g, 1.76mmol, 1eq), 02-1 (487.32mg, 1.76mmol, 1eq) was dissolved in DMSO (5mL), DIEA (1.82g, 14.11mmol, 2.46mL, 8eq) was added, The mixture was heated to 80°C and stirred for 3 hours. After the reaction was completed, water (100 mL) was added to the reaction solution, and a solid precipitated out. The filter cake was collected by filtration and concentrated to obtain compound 03-5.

Step 4: Synthesis of compound 03-6

Compound 03-5 (0.3g, 555.94μmol, 1eq) was dissolved in dichloromethane (5mL), and methanol hydrochloride (4M, 138.99μL, 1eq) was added at 0°C, and the mixture was gradually warmed to 25°C and stirred for 1 hour. After the reaction was completed, it was concentrated under reduced pressure to obtain compound 03-6 hydrochloride, and the crude product was directly used in the next reaction. LCMS m/z = 440.1 [M+1] ⁺ .

Step 5: Synthesis of compound 03-8

Compound 03-6 hydrochloride (0.25g, 525.25μmol, 1eq) and 03-7 (74.11mg, 525.25μmol, 55.72μL, 1eq) were dissolved in DMSO (10mL), and DIEA (203.65mg, 1.58mmol, 274.46μL, 3eq), the mixture was heated to 100°C and stirred for 6 hours. After the reaction was completed, the reaction solution was poured into (100 mL) water, solids were precipitated, filtered, and the filter cake was collected to obtain compound 03-8, which was directly used in the next reaction. LCMS m/z = 561.2 [M+1] ⁺ .

Step 6: Synthesis of compound 03-9

Compound 03-8 (0.1g, 178.38μmol, 1eq) was dissolved in a mixture solvent of ethanol (10mL) and water (2mL), ammonium chloride (95.42mg, 1.78mmol, 10eq) and iron powder (99.63mg, 1.78mmol, 10eq). The mixture was stirred and reacted at 25°C for 1 hour. After the reaction was completed, the solvent was concentrated under reduced pressure and spin-dried, added dichloromethane (50 mL) for soaking, filtered, and the filtrate was collected, concentrated under reduced pressure and added dichloromethane (10 mL) for extraction. The organic phase was dried over anhydrous sodium sulfate and concentrated to obtain compound 03-9. LCMS m/z = 531.1 [M+1] ⁺ .

Step 7: Synthesis of compound 03

Compound 03-9 (50 mg, 94.23 μmol, 1 eq) was dissolved in DMF (5 mL), and 01-10 (37.78 mg, 94.23 μmol, 1 eq), DIEA (36.54 mg, 282.69 μmol, 49.24 μL, 3eq) and HATU (71.66mg, 188.46μmol, 2eq), the mixture was stirred at 25°C for 3 hours. After the reaction was completed, an aqueous solution (10 mL) was added to the reaction solution to quench the reaction, extracted with dichloromethane (3×10 mL), the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was separated and purified by preparative thin-layer chromatography silica gel plate (dichloromethane:methanol:triethylamine=10:1:0.1), and then purified by preparative high-performance liquid chromatography column (column: Boston Prime C18 150*25mm*5 μm; flow Phase: [water (NH ₃ H ₂ O+NH ₄ HCO ₃ )-ACN]; acetonitrile: 60%-90%, 7min) separation and purification to obtain compound 03. ¹ H NMR (400MHz, CDCl ₃ ) δppm 8.49(s, 1H), 8.06(br d, J=11.0Hz, 1H), 7.69(d, J=8.5Hz, 1H), 7.42(dd, J=8.8, 12.0Hz, 4H), 7.38-7.32(m, 2H), 7.28(s, 1H), 7.05(dd, J=2.1, 8.7Hz, 1H), 6.90(d, J=9.0Hz, 2H), 4.96- 4.89(m,1H),4.63(dd,J=6.0,8.0Hz,1H),3.74(dd,J=8.3,14.1Hz,1H),3.61(br d,J=12.3Hz,2H),3.49- 3.40(m,5H),2.92-2.55(m,12H),2.40(s,3H),2.28(br d,J＝7.3Hz,2H),2.18-2.09(m,1H),1.88(br d, J = 11.5 Hz, 2H), 1.72-1.68 (m, 3H), 1.45-1.25 (m, 3H); LCMS m/z = 913.3 [M+1] ⁺ .

Example 4

synthetic route:

Step 1: Synthesis of compound 04-1

Compound 03-4 (0.5g, 1.76mmol, 1eq), 01-13 (487.32mg, 1.76mmol, 1eq) was dissolved in DMSO (10mL), DIEA (1.82g, 14.11mmol, 2.46mL, 8eq) was added, The mixture was heated to 80°C and stirred for 3 hours. After the reaction was completed, water (100 mL) was added to the reaction solution, and a solid precipitated out. The filter cake was collected by filtration and dried to obtain compound 04-1, which was directly used in the next reaction. LCMS m/z = 540.1 [M+1] ⁺ .

Step 2: Synthesis of compound 04-2

Compound 04-1 (0.3g, 555.94μmol, 1eq) was dissolved in dichloromethane (10mL), hydrochloric acid methanol (4M, 6.01mL, 43.25eq) was added dropwise at 0°C, and the mixture was gradually warmed to 25°C and stirred for reaction 1 Hour. After the reaction was completed, the reaction solution was concentrated under reduced pressure to obtain compound 04-2 hydrochloride, which was directly used in the next reaction. LCMS m/z = 440.1 [M+1] ⁺ .

Step 3: Synthesis of compound 04-3

Compound 04-2 hydrochloride (0.25g, 525.25μmol, 1eq) and 03-7 (74.11mg, 525.25μmol, 55.72μL, 1eq) were dissolved in DMSO (10mL), and DIEA (203.65mg, 1.58mmol, 274.46μL, 3eq), the mixture was heated to 100°C and stirred for 6 hours. After the reaction was completed, water (100 mL) was poured into the reaction liquid, stirred, filtered, and the filter cake was collected and dried to obtain compound 04-3. LCMS m/z = 561.1 [M+1] ⁺ .

Step 4: Synthesis of Compound 04-4

Compound 04-3 (0.1g, 178.38μmol, 1eq) was dissolved in ethanol (50mL) and water (10mL), ammonium chloride (95.42mg, 1.78mmol, 10eq) and iron powder (99.62mg, 1.78mmol, 10eq), the mixture was stirred and reacted at 25°C for 1 hour. After the reaction was complete, the solvent was spin-dried, soaked in dichloromethane (50 mL), filtered, and the filtrate was collected, concentrated under reduced pressure and extracted by adding dichloromethane (10 mL); the organic phase was dried over anhydrous sodium sulfate and concentrated to obtain compound 04-4 . LCMS m/z = 531.2 [M+1] ⁺ .

Step 5: Synthesis of Compound 04

Compound 04-4 (0.05g, 94.23μmol, 1eq) was dissolved in DMF (5mL), 01-10 (37.78mg, 94.23μmol, 1eq), DIEA (24.36mg, 188.46μmol, 32.83μL, 2eq) were added, HATU (35.83mg, 94.23μmol, 1eq) was added at 0°C, and the mixture was gradually heated to 25°C and stirred for 1 hour. After the reaction was completed, an aqueous solution (10mL) was added to the reaction solution to quench the reaction, extracted three times with dichloromethane (3×10mL), the organic phases were combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the crude product was prepared on a thin-layer chromatography silica gel plate (dichloromethane:methanol:triethylamine=20:1:0.2) separation and purification, and then through preparative thin-layer chromatography silica gel plate (dichloromethane:methanol:triethylamine=10:1:0.1) secondary separation and purification, Finally, compound 04 was obtained by normal-phase chiral chromatography (column: DAICEL CHIRALPAK IA (250mm*30mm, 10μm); mobile phase: [DCM-MeOH]; MeOH: 80%-80%, 60min). ¹ H NMR (400MHz, CDCl ₃ ) δppm 8.43(s, 1H), 7.98(br s, 1H), 7.53(t, J=7.8Hz, 1H), 7.42-7.31(m, 5H), 7.29-7.23( m,2H),7.11(d,J=8.0Hz,1H),6.83(br d,J=8.8Hz,2H),4.89(br dd,J=5.3,12.0Hz,1H),4.57(br t, J=6.9Hz,1H),3.68(br dd,J=8.2,14.2Hz,1H),3.54(br d,J=11.5Hz,2H),3.44-3.27(m,5H),3.05(q,J ＝7.4Hz, 2H), 2.88-2.55(m, 12H), 2.38-2.22(m, 5H), 2.04(br d, J＝5.8Hz, 1H), 1.82(br d, J＝11.0Hz, 3H) , 1.60-1.58 (m, 3H); LCMS Rt = 0.666 min, m/z = 913.3 [M+1] ⁺ .

Example 5

synthetic route:

Step 1: Synthesis of compound 05-3

Dissolve triphenylphosphine (11.78g, 44.93mmol, 1.25eq) in tetrahydrofuran (100mL) at 0°C. After nitrogen protection, slowly add DIAD (9.09g, 44.93mmol, 8.74mL, 1.25eq) dropwise, at 0°C Stir from a clear solution until a precipitate forms. Then a tetrahydrofuran (30 mL) solution of 05-1 (5 g, 35.94 mmol, 1 eq) and 05-2 (6.85 g, 39.54 mmol, 1.1 eq) was added, and the temperature was raised to 60° C. and stirred for 16 hours. After the reaction was completed, the reaction solution was cooled to room temperature, water (100 mL) was added to the reaction solution, and then extracted with ethyl acetate (200 mL×2), the organic phases were collected and combined, washed with brine (100 mL), anhydrous sodium sulfate dry. After filtration and spin-drying, compound 05-3 was obtained by column chromatography (petroleum ether: ethyl acetate = 1:0-10:1) purification.

Step 2: Synthesis of compound 05-4

Compound 05-3 (10g, 33.98mmol, 1eq) was dissolved in dichloromethane (20mL), added with methanol hydrochloride (4M, 66.67mL, 7.85eq), and stirred at 25°C for 2 hours. After the reaction was complete, the reaction solution was spin-dried, then water (30 mL) was added, ethyl acetate (50 mL×2) was added for extraction, the aqueous phase was collected, and the pH was adjusted to 7 with saturated aqueous sodium bicarbonate solution. Then it was extracted with dichloromethane (500 mL×2), and the organic phase was collected and dried over anhydrous sodium sulfate. Filter and concentrate for later use. At the same time, the aqueous phase was spin-dried, dissolved with dichloromethane:methanol=(4:1, 200 mL), filtered, and the organic phase was collected and concentrated under reduced pressure. The two batches of products were combined to obtain compound 05-4, which was directly used in the next reaction. LCMS m/z = 194.8 [M+1] ⁺ .

Step 3: Synthesis of compound 05-6

05-4 (6.6g, 28.62mmol, 1eq) and 05-5 (8.68g, 28.62mmol, 1eq) were dissolved in dichloroethane (100mL), and acetic acid (1.72g, 28.62mmol, 1.64mL, 1eq) was added And sodium acetate borohydride (9.10g, 42.92mmol, 1.5eq), stirred at 25°C for 3 hours. After the reaction was completed, saturated ammonium chloride aqueous solution (50 mL) was added to the reaction solution, stirred for 30 minutes, extracted with ethyl acetate (200 mL×2), the organic phase was collected, dried with anhydrous sodium sulfate, filtered and spin-dried. The crude product was purified by column chromatography (petroleum ether: ethyl acetate = 10:1-1:1) to obtain compound 05-6. LCMS m/z = 382.5 [M-100+1] ⁺ .

Step 4: Synthesis of Compound 05-7

Compound 05-6 (8 g, 16.61 mmol, 1 eq) was dissolved in DCM (25 mL), and methanolic hydrochloric acid (4M, 25 mL, 6.02 eq) was added. Stir at 25°C for 2 hours. The reaction solution was directly spin-dried, and then rotary-evaporated under reduced pressure with ethyl acetate (30 mL×3). Ethyl acetate (30 mL) was added to the crude product for slurry filtration, and the filter cake was collected to obtain compound 05-7 hydrochloride. ¹ H NMR (400MHz, CD ₃ OD) δppm 3.22-3.30(m,3H)3.31-3.36(m,1H)3.78-4.01(m,3H)4.38-4.53(m,2H)5.24-5.37(m,1H ) 7.01-7.14 (m, 2H) 8.18-8.32 (m, 2H).

Step 5: Synthesis of Compound 05-8

05-7 hydrochloride (2g, 6.58mmol, 1eq) and 01-5 (1.13g, 6.58mmol, 1eq) were dissolved in dichloroethane (50mL), and acetic acid (395.40mg, 6.58mmol, 376.57μL, 1eq) and sodium acetate borohydride (5.58g, 26.34mmol, 4eq), react at 25°C for 12 hours. After the reaction was completed, saturated ammonium chloride aqueous solution (30 mL) was added to the reaction liquid, and after stirring for 30 min, it was extracted with ethyl acetate (100 mL×2), and the organic phase was collected, dried with anhydrous sodium sulfate, filtered and spin-dried. Purification by column chromatography (dichloromethane:methanol=1:0-10:1) gave compound 05-8. ¹ H NMR (400MHz, CDCl ₃ ) δppm 1.40-1.51(m,9H)2.63-2.85(m,3H)3.26-3.42(m,2H)3.50-3.85(m,5H)3.94-4.15(m,4H) 4.85-5.01 (m, 1H) 6.77-6.94 (m, 2H) 8.13-8.32 (m, 2H). LCMS m/z = 423.4 [M+1] ⁺ .

Step 6: Synthesis of compound 05-9

Compound 05-8 (1.00g, 2.37mmol, 1eq) was dissolved in tetrahydrofuran (20mL), triethylamine (239.52mg, 2.37mmol, 329.46μL, 1eq) was added, then cooled to 0°C, Boc ₂ O was slowly added dropwise (516.59mg, 2.37mmol, 543.78μL, 1eq), stirred at 0°C for 10 minutes, then slowly raised the temperature to 25°C and stirred for 12 hours. Pour ice water (20 mL) into the reaction solution to quench, then extract with ethyl acetate (200 mL×2), collect the organic phase, dry over anhydrous sodium sulfate, filter and spin dry. Purification by column chromatography (petroleum ether:ethyl acetate=10:1-0:1) gave compound 05-9. LCMS m/z = 523.6 [M+1] ⁺ .

Step 7: Synthesis of Compound 05-10

Compound 05-9 (0.6g, 1.15mmol, 1eq) was dissolved in dichloromethane (10mL), triethylamine (697.07mg, 6.89mmol, 958.84μL, 6eq) was added, then the temperature was lowered to 0°C and methane was slowly added dropwise Sulfonyl chloride (0.37g, 3.23mmol, 250.00μL, 2.81eq), stirred at 25°C for 2 hours. After the reaction was completed, the reaction solution was slowly poured into ice water (20 mL) to quench. Then it was extracted with ethyl acetate (100 mL×2), the organic phases were collected and combined, dried over anhydrous sodium sulfate, filtered, concentrated to dryness and left for two days to obtain compound 05-10. LCMS m/z = 471.0 [M+23] ⁺ .

Step 8: Synthesis of Compound 05-11

Compound 05-10 (0.57g, 1.27mmol, 1eq) was dissolved in dichloromethane (5mL), and hydrochloric acid methanol solution (4M, 5mL, 15.74eq) was added, and stirred at 25°C for 0.5 hours. The reaction solution was spin-dried to obtain compound 05-11 hydrochloride, and the crude product was directly used in the next reaction. LCMS m/z = 349.1 [M+1] ⁺ .

Step 9: Synthesis of Compound 05-12

Compound 05-11 hydrochloride (280 mg, 803.78 μmol, 1 eq) and 01-13 (222.02 mg, 803.78 μmol, 1 eq) were dissolved in DMF (10 mL), and DIEA (207.77 mg, 1.61 mmol, 280.01 μL, 2 eq) was added , heated to 90°C and stirred for 2 hours. After the reaction was completed, the reaction solution was directly spin-dried, and then purified by preparative thin-layer chromatography on silica gel plate (dichloromethane:methanol=10:1) to obtain compound 05-12. LCMS m/z = 605.1 [M+1] ⁺ .

Step 10: Synthesis of Compound 05-13

05-12 (100 mg, 165.41 μmol, 1 eq) was dissolved in ethanol (4 mL), iron powder (92.37 mg, 1.65 mmol, 10 eq) was added, then ammonium chloride (86.71 mg, 1.62 mmol, 9.8 eq) and H2O ( 1 mL), heated to 80°C and stirred for 1 hour. After the reaction was completed, the reaction solution was filtered, and the filtrate was collected. The filter cake was then rinsed with ethyl acetate (50 mL). The filtrate was collected, water (20 mL) was added, and extracted with ethyl acetate (100 mL×2). The organic phase was collected, dried over anhydrous sodium sulfate, filtered and spin-dried to obtain compound 05-13, which was directly used in the next reaction. LCMS m/z = 575.2 [M+1] ⁺ .

Step 11: Synthesis of

Compounds

05 and 06

Dissolve 05-13 (70mg, 121.83μmol, 1eq) and 01-10 (48.84mg, 121.83μmol, 1eq) in DMF (6mL), cool to 0°C, add DIEA (47.24mg, 365.48μmol, 63.66μL, 3eq ) and HATU (92.64mg, 243.65μmol, 2eq), stirred at 25°C for 1 hour. After the reaction was completed, water (20 mL) was added to the reaction liquid to quench, and then extracted with ethyl acetate (100 mL×2), the organic phase was collected, dried with anhydrous sodium sulfate, filtered and spin-dried. By preparing a high-performance liquid chromatography column (chromatographic column: Boston Prime C18 150*30mm*5μm; mobile phase: [water (NH ₃ H ₂ O+NH ₄ HCO ₃ )]; acetonitrile: 45%-75%, 7min) Separation and purification, and then resolution by normal-phase chiral chromatography column (column: DAICEL CHIRALPAK ID (250mm*30mm, 10μm); mobile phase: [MeOH-ACN]; ACN: 50%-50%, 80min) to obtain

compound

05 and 06.

Compound 05: retention time: 5.512min; ¹ H NMR (400MHz, CDCl ₃ ) δppm 1.68(s,3H)2.04-2.17(m,1H)2.41(s,3H)2.68(s,3H)2.70-2.95(m ,5H)3.31(br s,2H)3.48(br dd,J＝14.18,5.65Hz,1H)3.57-3.68(m,1H)3.73-3.84(m,2H)3.90(br d,J＝6.78Hz, 2H)4.28-4.43(m,2H)4.48(br s,2H)4.59-4.69(m,2H)4.74(br d,J＝5.27Hz,1H)4.83(br s,1H)4.92(br d,J =9.29Hz,1H)6.53-6.76(m,3H)7.24(d,J=7.03Hz,1H)7.30-7.37(m,2H)7.38-7.54(m,5H)8.31-8.61(m,1H)8.93 (br s, 1H); LCMS m/z = 957.3 [M+1] ⁺ ; ee% = 97.6%. SFC detection method: chromatographic column: Boston Prime C18 150*30mm*5um; mobile phase: [water (NH ₃ H ₂ O+NH ₄ HCO ₃ )-ACN]; B%: 45%-75%, 7min).

Compound 06: retention time: 9.322min; ¹ H NMR (400MHz, CDCl ₃ ) δppm 1.68(s,3H)2.04-2.17(m,1H)2.41(s,3H)2.68(s,3H)2.70-2.95(m ,5H)3.31(br s,2H)3.48(br dd,J＝14.18,5.65Hz,1H)3.57-3.68(m,1H)3.73-3.84(m,2H)3.90(br d,J＝6.78Hz, 2H)4.28-4.43(m,2H)4.48(br s,2H)4.59-4.69(m,2H)4.74(br d,J＝5.27Hz,1H)4.83(br s,1H)4.92(br d,J =9.29Hz,1H)6.53-6.76(m,3H)7.24(d,J=7.03Hz,1H)7.30-7.37(m,2H)7.38-7.54(m,5H)8.31-8.61(m,1H)8.93 (br s, 1H); LCMS m/z=957.2[M+1] ⁺ ; ee%=96.6%. SFC detection method: chromatographic column: Boston Prime C18 150*30mm*5um; mobile phase: [water (NH ₃ H ₂ O+NH ₄ HCO ₃ )-ACN]; B%: 45%-75%, 7min).

Experimental Example 1: Research on Antiproliferative Activity of Compounds on Tumor Cells

The effect of the test compound on the anti-proliferation activity of the cells was determined in the MDA-MB-231 cell line by means of CTG.

Cell culture medium: complete cell culture medium (RPMI 1640+10% serum+1% L-glutamine+1% double antibody)

The specific operation steps are as follows:

(1) Inoculate MDA-MB-231 cells into a 96-well plate with 100 μL per well (3000 cells per well/MDA-MB-231) cell complete medium, and incubate the cells at 37°C and 5% CO ₂ 24 hours

(2) Replace the complete medium of the cells with 100 μL of serum-free medium and starve overnight

(3) Prepare the compound (the initial concentration of the compound is 10 μM, three-fold dilution of 9 concentrations. Then each concentration of the compound is diluted 100 times with serum-free medium), and add 25 μL of the diluted compound to the well plate containing the cells middle

(4) Incubate for 72 hours at 37°C and 5% CO ₂

(5) Subsequent operations refer to the instructions of the Promega CTG kit.

The results are shown in Table 1.

Experimental Example 2: Study on off-target activity of compounds on GSPT1

The effect of the test compound on the degradation activity of GSPT1 was determined in HEK293 cell line by HiBiT method.

Cell culture medium: DMEM+10% serum+2mM L-glutamine+1mM sodium pyruvate+double antibody

The specific operation steps are as follows:

1. Compound Preparation

(1) Take 9 μL of 10mM compound mother solution and add it to the LDV plate

(2) Add 6 μL DMSO to each compound well

(3) The compound was diluted 3 times with DMSO

(4) Transfer 25nL of the above compound solution to the test plate

2. Experimental operation

(1) Digest the cells with trypsin, count the cells, and resuspend the cells at 4*10 ⁵ cell/mL

(2) Add the above cell suspension to the test plate, 25 μL/well

(3) Put the test plate into the incubator and incubate overnight

(4) Add NanoGlo lysis buffer + furazine and LgBit to the test plate, 25 μL/well, and shake for 10 minutes

(5) Centrifuge the test plate at 4000g for 5 minutes to remove foam

(6) Read the RLU value on the Envision instrument.

The results are shown in Table 1.

Table 1 Compound of the present invention in vitro screening test results

Conclusion: the compound of the present invention has significant cell proliferation inhibitory activity on MDA-MB-231 cell line, and has weak or almost no activity on GSPT1.

Experimental example 3: Research on the degradation activity of compounds on BRD4

The effect of the test compound on the degradation activity of BRD4 was determined in the MDA-MB-231 cell line by Western Blot method.

Cell culture medium: RPMI 1640+10% serum

Experimental steps:

(1) MDA-MB-231 cells were recovered and cultured to a suitable state;

(2) MDA-MB-231 cells were seeded in a 12-well plate at 2×10 ⁵ cells per well, and treated with a certain concentration of the test compound after adhering overnight;

(3) After 16 hours of treatment, the supernatant of the cultured cell samples was discarded, washed twice with DPBS (Duchener's phosphate buffered saline, 21-031-CVR, CORNING), and then washed with a certain amount of preheated 100°C containing 1 Lyse the cells with 2% SDS lysate (SDS Lysis Buffer, P0013G, Biyuntian) of loading buffer and 1× reducing reagent, and denature at 100°C for 15 minutes after collection;

(4) The above samples were separated by SDS-PAGE and transferred to PVDF membrane (Biorad);

(5) Cut the band according to the molecular weight of the target protein, block it with blocking solution (3% bovine serum albumin TBS–T solution, where TBS–T solution is Tris–HCl buffer containing 0.2% Tween–20) for 1 hour, and then Prepare with primary antibodies BRD4 (#13440S, CST), GSPT1 (ab49878, Abcam) and anti–β-actin (#4970, CST) in blocking solution at 1:1000, 1:1000 and 1:2000 respectively 4°C Incubate overnight;

(6) Finally, incubate with HRP-linked secondary antibody (anti-rabbit IgG (#7074, CST), prepared by diluting 1:2000 in blocking solution) at room temperature for 1 hour, and then detect with chemiluminescence substrate (Clarity ECL, Biorad) bands on the membrane.

The results are shown in Figure 1.

Conclusion: The compound of the present invention can degrade BRD4 protein in a concentration-dependent manner in MDA-MB-231 cell line, and has no obvious degradation activity on GSPT1.

Experimental Example 4: Pharmacokinetic properties of compounds in mice

Purpose of the experiment: Female Balb/c nude mice were used as test animals to measure the blood drug concentration of the compound and evaluate the pharmacokinetic behavior after a single administration. Experimental operation: Select 4 healthy adult female Balb/c nude mice, 2 for the intravenous injection group and 2 for the oral administration group. The compound to be tested was mixed with an appropriate amount of solvent (10% DMSO, 40% PEG300, 5% Tween-80plus 45% saline), vortexed and sonicated to prepare a 1.0 mg/mL clear solution, which was filtered through a microporous membrane for use. After intravenous administration of 1 mg/kg or oral administration of 5 mg/kg to mice, the whole blood was collected for a certain period of time to prepare plasma, and the drug concentration was analyzed by LC-MS/MS method, and the drug concentration was calculated by Phoenix WinNonlin software (Pharsight, USA) generation parameters. The results are shown in Table 2.

Table 2 The results of pharmacokinetic properties of compounds of the present invention in mice

Conclusion: The compound of the present invention has excellent pharmacokinetic properties.

Claims

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

PTM-L-ULM

(I)

in,

PTM selected from

L is selected from single bond, *-Cy 1 -Ak 1 -Cy 2 -Ak 2 -Cy 3 -Ak 3 -(Cy 4 -Ak 4 ) n -, *-Cy 1 -(OCH 2 CH 2 ) m -NH - and *-Cy 1 -(OCH 2 CH 2 ) m -O-, where * indicates connection with PTM;

Cy 1 is selected from phenyl and 5-6 membered heteroaryl;

Ak 1 is selected from single bonds and -O-;

Cy 2 , Cy 3 and Cy 4 are independently selected from 3-6 membered heterocycloalkyl groups, and the 3-6 membered heterocycloalkyl groups are optionally substituted by 1, 2 or 3 R a ;

Ak 2 , Ak 3 and Ak 4 are independently selected from single bond, C 1-3 alkyl and -C 1-3 alkyl NH-, said C 1-3 alkyl and -C 1-3 alkyl NH - optionally substituted by 1, 2 or 3 R b ;

n is 0 or 1;

m is 2, 3 or 4;

ULM is selected from the structures shown in formula (I-1), formula (I-2) and formula (I-3):

R 1 , R 2 , R 3 and R 4 are each independently selected from H;

Alternatively, R 1 and R 3 , R 2 and R 4 together with the carbon atoms they are connected to form =O and =NC 1-3 alkoxy;

R a and R b are independently selected from F, Cl, Br, I, oxo, C 1-3 alkyl and C 1-3 alkoxy;

requirement is:

When ULM is selected from formula (I-1), L is selected from single bond and *-Cy 1 -Ak 1 -Cy 2 -Ak 2 -Cy 3 -Ak 3 -(Cy 4 -Ak 4 ) n -;

The "hetero" of the 3-6 membered heterocycloalkyl group and the 5-6 membered heteroaryl group represents a heteroatom independently selected from 1, 2, 3 or 4 O, S, N and C (=O) or heteroatom groups.
The compound or the pharmaceutically acceptable salt thereof according to claim 1, wherein R 1 and R 3 , R 2 and R 4 together with their connected carbon atoms form =O and =N-OCH 3 .
The compound or a pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein Cy 1 is selected from phenyl and pyridyl.
The compound or pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein Cy 2 , Cy 3 and Cy 4 are independently selected from

said

Optionally substituted with 1, 2 or 3 R a .
The compound according to claim 4 or a pharmaceutically acceptable salt thereof, wherein Cy 2 , Cy 3 and Cy 4 are independently selected from
The compound or pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein Ak 2 , Ak 3 and Ak 4 are independently selected from single bonds, -CH 2 - and -CH 2 CH 2 NH-, so The -CH 2 - and -CH 2 CH 2 NH- are optionally substituted by 1, 2 or 3 R b .
The compound or a pharmaceutically acceptable salt thereof according to claim 6, wherein Ak 2 , Ak 3 and Ak 4 are independently selected from single bonds, -CH 2 - and -CH 2 CH 2 NH-.
The compound or pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein L is selected from single bonds,
The compound or pharmaceutically acceptable salt thereof according to any one of claims 1-8, which is selected from:

in,

L is as defined in any one of claims 1-8.
A compound represented by the following formula or a pharmaceutically acceptable salt thereof,
The compound or pharmaceutically acceptable salt thereof according to claim 10, which is selected from: