WO2023160572A1 - Pyrazole derivative, pharmaceutical composition, and use - Google Patents

Abstract

Disclosed are a pyrazole derivative, a pharmaceutical composition, and use. The pyrazole derivative has a structure represented by formula (I). The pyrazole derivative has a good selectivity for inhibiting the activity of CDK2.

Description

Pyrazole derivatives, pharmaceutical composition and application technical field

The invention relates to the technical field of medicine, in particular to a pyrazole derivative, a pharmaceutical composition and its application.

Background technique

Cyclin-dependent kinases (CDKs) belong to the serine/threonine kinase family. They are involved in cell proliferation, transcription and other physiological processes. According to the different functions of CDKs, they can be divided into two categories: 1) one type of CDK participates in cell cycle regulation, mainly including CDK1, CDK2, CDK4, CDK6, etc.; 2) another type of CDK participates in transcriptional regulation, mainly including CDK7, CDK8, CDK9, CDK12, CDK13, etc.

CDK2 activity is of interest to researchers because dysregulation of CDK2 activity occurs frequently in a variety of human cancers. CDK2 plays a key role in promoting G1/S transition and S phase progression. CDK2 forms a complex with cyclin E (Cyclin E), phosphorylates members of the retinoblastoma family (pRb, etc.), leads to the release and activation of E2F transcription factors, and promotes the transition of the cell cycle from G1 phase to S phase, thereby making CDK2/ Cyclin A is activated to promote cell cycle DNA synthesis, replication and other processes.

Cyclin E1 copy number gain and overexpression have been identified in ovarian cancer, gastric cancer, endometrial cancer, breast cancer and other cancers, and are positively correlated with poor prognosis of the corresponding tumors. In ER+ breast cancer cells, the high expression of Cyclin E2 is often accompanied by hormone therapy resistance (Mol. Cancer Ther., 2012, 11, 1488-1499), and the amplification or overexpression of Cyclin E is associated with poor prognosis of breast cancer are closely related (N. Engl. J. Med, 2002, 347, 1566-1575). In HER2+ breast cancer, the amplification of Cyclin E has also been reported to have a certain contribution to the drug resistance of trastuzumab (Proc. Natl. Acad. Sci., 2011, 108, 3671-3676). It has been reported that the overexpression of Cyclin E also plays an important role in the progression of triple-negative breast cancer (Breast Care, 2011, 6, 273-278) or inflammatory breast cancer (Oncotarget, 2017, 8, 14897-14911). Therefore, CDK2 may become an important anti-tumor target.

Currently, there are a small number of small molecule inhibitors with CDK2 activity in the clinical research stage, such as Pfizer's PF-3600, the structural formula is: It can inhibit CDK2, CDK4 and CDK6, but it also has strong inhibitory activity on other CDK subtypes, which inevitably brings some off-target side effects. In addition, so far, no CDK2 inhibitors have been approved, so there is an urgent need to develop novel, safe and effective CDK2 inhibitors that can treat a variety of cancers, especially small molecule inhibitors that selectively target CDK2, which may With better security.

Contents of the invention

The object of the present invention is to provide a pyrazole derivative with good selectivity for CDK2 activity inhibition.

The first aspect of the present invention provides a compound represented by formula (I), or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof:

in,

R ₁ is selected from H, halogen, C ₁ - ₆ alkyl, halogenated C ₁ - ₆ alkyl, C _3-7 cycloalkyl, 3 to 7 membered heterocycloalkyl, nitro, isocyano, -( CH ₂ ) _n R ₅ ;

R ₅ is selected from -CN, -OR _6, -NR ₆ R ₇ , -C(O)OR ₆ , -C(O)R ₆ , -S(O) ₂ R ₆ , -P(O)(OR ₆ ) ₂ , -O-alicyclic group, -O-aliphatic heterocyclic group, -O-aryl group, -O-heteroaryl group or -C(O)-heteroaryl group, wherein the alicyclic group, aliphatic Cyclic, aryl, heteroaryl are unsubstituted or substituted by one or more R ₀ ;

_R is selected from halogen, C _1-6 alkyl, halogenated C _1-6 alkyl, _{C 3-7} cycloalkyl, 3-7 membered heterocycloalkyl _, cyano, amino, nitro or hydroxyl;

R ₆ and R ₇ are each independently _{selected from H, C 1-6 alkyl, halogenated C 1-6 alkyl ,} C _3-7 cycloalkyl, _3-7 membered heterocycloalkyl, aryl or heteroaryl base;

n is 0, 1, 2, 3 or 4;

Ring A is selected from

X is each independently N or _CR6 ;

R _is selected from H, hydroxyl _, C _1-6 alkyl, halogenated C _1-6 alkyl, C _3-7 cycloalkyl or 3-7 membered heterocycloalkyl _;

R ₂ and R ₃ are each independently _selected from H, C _1-6 alkyl, halogenated C _1-6 alkyl, C _{3-7 cycloalkyl} or 3-7 membered heterocycloalkyl, wherein the C _{3 -7} cycloalkyl and 3-7 membered heterocycloalkyl are unsubstituted or substituted by one or more _R9 ;

R ₉ is selected from halogen, C _{1-6 alkyl, halogenated C 1-6 alkyl} , cyano, amino, nitro or hydroxyl _;

R ₄ is selected from H, halogen, amino, hydroxy or C ₁ -C ₆ alkyl.

In some preferred embodiments, the compound has a structure shown in any one of formulas (I-1) to (I-3):

Further preferably, the compound has a structure shown in formula (I-4):

In some preferred embodiments, R ₁ is selected from H, halogen, C _3-7 cycloalkyl, 3 to 7 membered heterocycloalkyl, nitro, isocyano, -(CH ₂ ) _n R ₅ ;

Further preferably, n is 1, and the R ₁ is -CH ₂ R ₅ , at this time, the compound has the structure shown in formula (I-5):

Further preferably, the R ₅ is selected from -CN, -OR ₆ or -NR ₆ R ₇ ;

_The R ₆ and R _{7 are} each independently selected from H, C _1-6 alkyl or halogenated C _1-6 alkyl.

In some embodiments, R ₅ is selected from -CN, -OC _{1-3 alkyl , -N(H)C 1-3 alkyl , -N} (C _1-3 alkyl)(C _1-3 alkyl ).

In some preferred embodiments, the R ₂ is selected from H, C ₁ -C ₆ alkyl or halogenated C ₁ -C ₆ alkyl;

The R ₃ is selected from H, C ₁ -C ₆ alkyl, halogenated C ₁ -C ₆ alkyl, C _3-7 cycloalkyl or 3-7 membered heterocycloalkyl, wherein the C _3-7 Cycloalkyl and 3-7 membered heterocycloalkyl are unsubstituted or substituted with one or more R ₉ .

Further preferably, said R ₂ is selected from H;

The R ₃ is selected from H, C ₁ -C ₆ alkyl, halogenated C ₁ -C ₆ alkyl, unsubstituted or 5-membered heterocycloalkyl substituted by one R ₉ .

Further preferably, the R ₉ is _{selected from halogen, C 1-6 alkyl} or halogenated C _1-6 alkyl; the heteroatom of the 5-membered heterocycloalkyl is _an oxygen atom.

Further preferably, in the formula (I) One of the following structures is selected:

In some preferred embodiments, _R4 is selected from H, F or methyl.

In some preferred embodiments, R _is selected from C _3-7 cycloalkyl, 3-7 membered heterocycloalkyl, cyano, amino, nitro or hydroxyl.

In some preferred embodiments, R ₈ is _{selected from H, C 1-6 alkyl ,} halogenated C _1-6 alkyl, C _3-7 cycloalkyl or 3-7 membered heterocycloalkyl.

In some embodiments, the compounds include but are not limited to the following structures:

The second aspect of the present invention provides a pharmaceutical composition, which comprises the compound described in the first aspect of the present invention, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, and a pharmaceutically acceptable carrier.

The third aspect of the present invention provides the compound described in the first aspect of the present invention, or its pharmaceutically acceptable salt, or its stereoisomer and the pharmaceutical composition described in the third aspect of the present invention in the preparation of treatment or prevention and CDK2 activity related or by Drug applications for diseases mediated by CDK2 activity.

In certain embodiments, the disease associated with or mediated by CDK2 activity is cancer.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (such as embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, we will not repeat them here.

After long-term and in-depth research, the present inventor unexpectedly found a compound as shown in formula (I). The compound has unexpected inhibitory activity on CDK2, can be used to treat various cancers with high expression of Cyclin E, and especially has excellent therapeutic effect on cancer patients resistant to CDK4/6 inhibitors. Based on the above findings, the inventors have accomplished the present invention.

Detailed ways

Definition of Terms

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" refers to those compounds, materials, compositions and/or dosage forms which, within the scope of sound medical judgment, are suitable for use in contact with human and animal tissues without excessive 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. 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 functionalities, acid addition salts can be obtained by contacting such compounds with a sufficient amount of the acid, either neat solution or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include salts of inorganic acids including, for example, hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogenphosphate, dihydrogenphosphate, sulfuric acid, Hydrogen sulfate, hydriodic acid, phosphorous acid, etc.; and organic acid salts, such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, Fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, methanesulfonic acid and similar acids; also salts of amino acids such as arginine and the like , and salts of organic acids such as glucuronic acid. Certain specific compounds of the present invention contain basic and acidic functional groups and can thus 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 reaction with the free acid or base in water or an organic solvent or a mixture of both. Forms of these compounds are prepared by reacting stoichiometric amounts of the appropriate base or acid.

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 and straight dashed keys

Unless otherwise stated, when there is a double bond structure in the compound, such as carbon-carbon double bond, carbon-nitrogen double bond and nitrogen-nitrogen double bond, and each atom on the double bond is connected with two different substituents (including nitrogen atom In the double bond of the nitrogen atom, a lone pair of electrons on the nitrogen atom is regarded as a substituent connected to it), if there is a wavy line between the atom on the double bond and its substituent in the compound Linked means the (Z) isomer, (E) isomer or a mixture of the two isomers of the compound. For example, the following formula (A) means that the compound exists as a single isomer of formula (A-1) or formula (A-2) or as two isomers of formula (A-1) and formula (A-2). The following formula (B) means that the compound exists in the form of a single isomer of formula (B-1) or formula (B-2) or in the form of both formula (B-1) and formula (B-2) It exists as a mixture of isomers. The following formula (C) represents that the compound exists in the form of a single isomer of formula (C-1) or formula (C-2) or in the form of two isomers of formula (C-1) and formula (C-2). It exists in the form of a mixture.

Unless otherwise stated, the term "tautomer" or "tautomeric form" means that isomers with different functional groups are in dynamic equilibrium at room temperature and are rapidly interconvertible. For example in the present invention, For tautomers. If tautomerism is possible (eg, in solution), then chemical equilibrium of the tautomers can be achieved. For example, proton tautomers (also called prototropic tautomers) include interconversions via migration of a proton, such as keto-enol isomerization and imine-enol Amine isomerization. Valence isomers (valence tautomers) include interconversions by recombination of some bonding electrons. A specific example of keto-enol tautomerization is the interconversion between two tautomers of pentane-2,4-dione and 4-hydroxypent-3-en-2-one.

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. white. 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.

"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 it does not.

The term "substituted" means that any one or more hydrogen atoms on a specific atom are replaced by a substituent, which may include deuterium and hydrogen variants, as long as the valence of the specific atom is normal and the substituted compound is stable of. The kind and number of substituents can be arbitrary on the basis of chemically achievable. When a substituent is oxygen (ie =0), it means that two hydrogen atoms are replaced. Oxygen substitution does not occur on aromatic groups.

When any variable (eg R ₁ ) 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 ₁ , said group may optionally be substituted with up to two R ₁ , with independent options for each case of R ₁ . 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 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.

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 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 is connected 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, "3-7 membered ring" means a "ring" with 3-7 atoms arranged around it.

Unless otherwise specified, the term "C _1-6 alkyl" is used to denote a straight or branched chain saturated hydrocarbon group consisting of 1 to 6 carbon atoms. It is preferably C _1-4 alkyl, which may be monovalent (such as methyl), divalent (such as methylene) or multivalent (such as methine). Examples of C _1-3 alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), butyl (including n-butyl, isobutyl, tert-butyl and sec-butyl).

Unless otherwise specified, the term "C _1-6 alkoxy" denotes those alkyl groups containing 1 to 6 carbon atoms attached to the rest of the molecule through an oxygen atom. It is preferably C _1-3 alkoxy. 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 "C _1-6 alkylamino" denotes those alkyl groups containing 1 to 6 carbon atoms attached to the rest of the molecule through an amino group. Preferably it is C _1-3 alkylamino. Examples of C _1-3 alkylamino include, but are not limited to, -NHCH ₃ , -N(CH ₃ ) ₂ , -NHCH ₂ CH ₃ , -N(CH ₃ )CH 2 CH 3 , -NHCH ₂ CH 2 CH ₃ , -NHCH ₂ CH ₂ CH ₃ , - NHCH ₂ (CH ₃ ) ₂ etc.

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

Unless otherwise specified, the terms "5-membered heteroaryl" and "5-membered heteroaryl" in the present invention can be used interchangeably, and the term "5-membered heteroaryl" means a single ring consisting of 5 ring atoms with a conjugated π-electron system. Cyclic group whose 1, 2, 3 or 4 ring atoms are independently selected Heteroatoms from O, S and N, the remainder being 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). A 5-membered heteroaryl can be attached to the rest of the molecule through a heteroatom or a carbon atom. Examples of the 5-membered heteroaryl group include, but are not limited to, pyrrolyl (including N-pyrrolyl, 2-pyrrolyl and 3-pyrrolyl, etc.), pyrazolyl (including 2-pyrazolyl and 3-pyrazolyl etc.), imidazolyl (including N-imidazolyl, 2-imidazolyl, 4-imidazolyl and 5-imidazolyl, etc.), oxazolyl (including 2-oxazolyl, 4-oxazolyl and 5-oxazole base, 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.).

Unless otherwise specified, C _n-n+m or C _n -C _n+m includes any specific instance of n to n+m carbons, for example C _1-12 includes C ₁ , C ₂ , C ₃ , C ₄ , C ₅ , C ₆ , C ₇ , C ₈ , C ₉ , C ₁₀ , C ₁₁ , and C ₁₂ , also including any range from n to n+m, for example, C _1-12 includes C _1-3 , C _1-6 , C _1-9 , C _3-6 , C _3-9 , C _3-12 , C _6-9 , C _6-12 , and C _9-12 etc.; similarly, n to n +m means that the number of atoms on the ring is from n to n+m, for example, a 3-12-membered ring includes a 3-membered ring, a 4-membered ring, a 5-membered ring, a 6-membered ring, a 7-membered ring, an 8-membered ring, and a 9-membered ring , 10-membered rings, 11-membered rings, and 12-membered rings, also including any range from n to n+m, for example, 3-12-membered rings include 3-6-membered rings, 3-9-membered rings, 5-6-membered rings ring, 5-7-membered ring, 6-7-membered ring, 6-8-membered ring, and 6-10-membered ring, etc.

Unless otherwise specified, "C _3-7 cycloalkyl" means a saturated cyclic hydrocarbon group composed of 3 to 7 carbon atoms, which includes monocyclic and bicyclic systems, wherein bicyclic systems include spiro rings, fused rings and bridge ring. The C _3-7 cycloalkyl group includes C _3-6 , C _4-6 , C _4-5 , C _5-7 or C _5-6 cycloalkyl group; it can be monovalent, divalent or multivalent . Examples of C _3-7 cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and the like.

Unless otherwise specified, the term "3-7 membered heterocycloalkyl" by itself or in combination with other terms denotes a saturated cyclic group consisting of 3 to 7 ring atoms, respectively, whose 1, 2, 3 or 4 ring atoms is a heteroatom independently selected from O, S, and N, and the remainder is carbon atoms, wherein the nitrogen atom is optionally quaternized, and the nitrogen and sulfur heteroatoms may be optionally oxidized (i.e., NO and S(O) _p , 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 "3-7 membered heterocycloalkyl", a heteroatom may occupy the attachment position of the heterocycloalkyl to the rest of the molecule. The 3-7-membered heterocycloalkyl group includes 5-7-membered, 3-membered, 4-membered, 5-membered, 6-membered and 7-membered heterocycloalkyl groups and the like. Examples of 3-7 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 or hexahydropyridazinyl, etc.

Unless otherwise specified, the term "3-7 membered nitrogen-containing heterocycloalkyl" means a 3-7 membered heterocycloalkyl group containing at least one N atom.

Cycloaliphatic refers to a saturated or partially unsaturated all-carbocyclic ring system. Where "partially unsaturated" refers to ring moieties that include at least one double or triple bond, "partially unsaturated" is intended to encompass rings having multiple sites of unsaturation, but is not intended to include aryl or heteroaryl moiety. Non-limiting examples include cyclopropyl rings, cyclobutyl rings, cyclopentyl rings, cyclopentenyl rings, cyclohexyl rings, cyclohexenyl rings, cyclohexadienyl rings, cycloheptyl rings, cycloheptyl rings Alkenyl ring, cyclopentanone ring, cyclopentane-1,3-dione ring, etc.

Heteroalicyclic group means that 1, 2 or 3 ring carbon atoms in a saturated or partially unsaturated alicyclic group are replaced by a heteroatom selected from nitrogen, oxygen or S(O) _t (wherein t is an integer from 0 to 2) Substituted, but not including -OO-, -OS- or -SS- ring moieties, the remaining ring atoms are carbon. Non-limiting examples include propylene oxide rings, azetidine rings, oxetane rings, tetrahydrofuran rings, tetrahydrothiophene rings, tetrahydropyrrole rings, piperidine rings, pyrroline rings, oxazolidine rings , piperazine ring, dioxolane, dioxane, morpholine ring, thiomorpholine ring, thiomorpholine-1,1-dioxide, tetrahydropyran ring, azetidine- 2-keto ring, oxetane-2-one ring, pyrrolidin-2-one ring, pyrrolidin-2,5-dione ring, piperidin-2-one ring, dihydrofuran-2(3H )-keto ring, dihydrofuran-2,5-dione ring, tetrahydro-2H-pyran-2-one ring, piperazin-2-one ring, morpholin-3-one ring. Non-limiting examples of partially unsaturated monoheterocyclic rings include 1,2-dihydroazetidinium rings, 1,2-dihydrooxetidine rings, 2,5-dihydro-1H- Pyrrole ring, 2,5-dihydrofuran ring, 2,3-dihydrofuran ring, 2,3-dihydro-1H-pyrrole ring, 3,4-dihydro-2H-pyran ring, 1,2, 3,4-tetrahydropyridine ring, 3,6-dihydro-2H-pyran ring, 1,2,3,6-tetrahydropyridine ring, 4,5-dihydro-1H-imidazole ring, 1,4 ,5,6-tetrahydropyrimidine ring, 3,4,7,8-tetrahydro-2H-1,4,6-oxadiazosin ring, 1,6-dihydropyrimidine ring, 4,5,6, 7-tetrahydro-1H-1,3-diazepine Cyclo, 2,5,6,7-tetrahydro-1,3,5-oxadiazepine Ring etc.

"Aryl" and "aromatic ring" are used interchangeably and both refer to an all-carbon monocyclic or fused polycyclic (that is, rings sharing adjacent pairs of carbon atoms) group having a conjugated pi-electron system, which Can be fused to a cycloalkyl ring, heterocycloalkyl ring, cycloalkenyl ring, heterocycloalkenyl ring or heteroaryl. "C _6-10 aryl" refers to a monocyclic or bicyclic aryl group having 6 to 10 carbon atoms, and non-limiting examples of the aryl group include phenyl, naphthyl, and the like.

"Heteroaryl" and "heteroaryl ring" are used interchangeably and both refer to a monocyclic, bicyclic or polycyclic 4n+2 aromatic ring system having ring carbon atoms and ring heteroatoms (e.g., having A group of shared 6 or 10 π electrons) where each heteroatom is independently selected from nitrogen, oxygen and sulfur. In the present invention, heteroaryl also includes ring systems in which the aforementioned heteroaryl ring is fused to one or more cycloalkyl rings, heterocycloalkyl rings, cycloalkenyl rings, heterocycloalkenyl rings or aromatic rings. Heteroaryl rings can be optionally substituted. "5 to 10 membered heteroaryl" refers to a monocyclic or bicyclic heteroaryl group having 5 to 10 ring atoms, of which 1, 2, 3 or 4 ring atoms are heteroatoms. "5 to 6 membered heteroaryl" means a monocyclic heteroaryl group having 5 to 6 ring atoms, of which 1, 2, 3 or 4 ring atoms are heteroatoms, non-limiting examples include thienyl, furan base, thiazolyl, isothiazolyl, imidazolyl, oxazolyl, pyrrolyl, pyrazolyl, triazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2 ,5-triazolyl, 1,3,4-triazolyl, tetrazolyl, isoxazolyl, oxadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadi Azolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, tetrazine base. "8 to 10 membered heteroaryl" refers to a bicyclic heteroaryl group having 8 to 10 ring atoms, of which 1, 2, 3 or 4 ring atoms are heteroatoms, non-limiting examples include indolyl, isoindolyl, indazolyl, benzene Triazolyl, benzothienyl, isobenzothienyl, benzofuryl, benzisofuryl, benzimidazole, benzoxazolyl, benzisoxazolyl, benzoxadiazole Base, benzothiazolyl, benzisothiazolyl, benzothiadiazolyl, indenazinyl, purinyl, pyrido[3,2-d]pyrimidinyl, pyrido[2,3-d]pyrimidinyl , pyrido[3,4-d]pyrimidinyl, pyrido[4,3-d]pyrimidinyl, 1,8-naphthyridinyl, 1,7-naphthyridinyl, 1,6-naphthyridinyl, 1 , 5-naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl and quinazolinyl. "Heteroatom" means nitrogen, oxygen or sulfur. In heteroaryl groups containing one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valence permits. Heteroaryl bicyclic ring systems can include one or more heteroatoms in one or both rings.

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, brosylate, tosylate Esters, etc.; acyloxy groups, such as acetoxy, trifluoroacetoxy, etc.

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

Unless otherwise defined, the "substituents independently selected from ..." in the present invention means that when more than one hydrogen on the group is replaced by a substituent, the types of the substituents may be the same or different, so The selected substituents are each independent species.

Usually, the compound of the present invention or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof can be administered in a suitable dosage form with one or more pharmaceutically acceptable carriers. These dosage forms are suitable for oral, rectal, topical, buccal and other parenteral administration (eg, subcutaneous, intramuscular, intravenous, etc.). For example, dosage forms suitable for oral administration include capsules, tablets, granules, syrups and the like. The compounds of the present invention contained in these formulations may be solid powders or granules; solutions or suspensions in aqueous or non-aqueous liquids; water-in-oil or oil-in-water emulsions and the like. The above-mentioned dosage forms can be made from the active compound and one or more carriers or excipients through common pharmaceutical methods. The aforementioned carriers need to be compatible with the active compound or other excipients. For solid formulations, commonly used non-toxic carriers include, but are not limited to, mannitol, lactose, starch, magnesium stearate, cellulose, grape Sugar, sucrose, etc. Carriers for liquid preparations include water, physiological saline, aqueous dextrose, ethylene glycol, polyethylene glycol, and the like. The active compounds can form solutions or suspensions with the above-mentioned carriers.

The compositions of the present invention are formulated, dosed and administered in a manner consistent with medical practice. The "therapeutically effective amount" of a compound to be administered is determined by factors such as the particular condition to be treated, the individual being treated, the cause of the condition, the target of the drug, and the mode of administration.

"Therapeutically effective amount" refers to the amount of a compound of the present invention that will elicit a biological or medical response in an individual, such as reducing or inhibiting enzyme or protein activity or improving symptoms, alleviating symptoms, slowing or delaying disease progression, or preventing disease, etc.

The therapeutically effective amount of the compound of the present invention or its pharmaceutically acceptable salt or its stereoisomer contained in the said pharmaceutical composition of the present invention or said pharmaceutical composition is preferably 0.1mg-5g/kg (body weight ).

"Patient" means an animal, preferably a mammal, more preferably a human. The term "mammal" refers to warm-blooded vertebrate mammals including, for example, cats, dogs, rabbits, bears, foxes, wolves, monkeys, deer, mice, pigs and humans.

"Treating" means alleviating, delaying progression, attenuating, preventing, or maintaining an existing disease or condition (eg, cancer). Treatment also includes curing, preventing its development, or alleviating to some extent one or more symptoms of a disease or disorder.

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 following abbreviations are used in the present invention: Pd/C stands for palladium on carbon; _H2 stands for hydrogen; _N2 stands for nitrogen; mL stands for milliliter; _MeNH2 stands for dimethylamine; _BBr3 stands for boron tribromide.

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.

The present invention will be described in detail through the following examples, but it does not mean 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

step 1:

Compound 1-1 was dissolved in tetrahydrofuran (2 mL), and the air was replaced with nitrogen 3 times. The system was cooled to 0-10°C, and diisobutylaluminum hydride (1M, 4.43mL, 5.00eq) was added slowly under nitrogen atmosphere. Monitor after reacting at 20-30°C for 3 hours. LCMS showed that compound 1-1 was completely consumed, and a target product peak (m/z=183.9) was monitored. Water (0.70 mL), 15% aqueous sodium hydroxide solution (0.70 mL) and water (2.00 mL) were thus added to the reaction solution under ice-water bath conditions. After the reaction was stirred for 10 minutes, anhydrous sodium sulfate was added to dry, filtered, and the filtrate was concentrated to obtain the crude product compound 1-2. LCMS: m/z (M+H) ⁺ = 183.9. ¹ H NMR: (400MHz, DMSO-d6) δ8.78(d, J=4.6Hz, 1H), 7.72(d, J=4.8Hz, 1H), 6.92(s, 1H), 5.51(t, J= 5.8Hz, 1H), 4.70 (d, J = 5.8Hz, 2H).

Step 2:

Compound 1-2 was dissolved in tetrahydrofuran (2.00 mL), the air was replaced with nitrogen three times, and the temperature was lowered to 0-10°C. Under nitrogen atmosphere, sodium hydride (28.3mg, 708umol, 60% purity, 1.30eq) was added to the reaction solution; after 30 minutes of reaction, methyl iodide (116mg, 817umol, 50.9uL, 1.50eq) was added. Monitor after 1 hour of reaction at 10-20°C. LCMS showed that compound 1-2 was completely consumed, and a target product peak (m/z=198.5) was monitored. The reaction solution was slowly poured into saturated ammonium chloride solution (3.00 mL), Ethyl acetate (3.00 mL*3) extracted three times. The organic phases were combined, washed once with saturated brine (3.00 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain a crude product. The crude product was purified by preparative HPLC (preparative column: 3_Phenomenex Luna C18 75*30mm*3um; mobile phase: [water(TFA)-ACN]; B%: 22%-52%, 7mins). Sodium carbonate was used to adjust the pH value of the preparation solution to 7-8, extracted three times with ethyl acetate (10.0 mL*2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain compound 1-3. LCMS: m/z (M+H) ⁺ = 198.5. ¹ H NMR: (400MHz, DMSO-d6) δ8.81 (dd, J = 4.71, 0.92Hz, 1H), 7.76 (d, J = 4.77Hz, 1H), 6.99(s,1H), 4.64(s,2H), 3.34(s,3H).

Step 3:

Compound 1-4 (300mg, 839umol, 1.00eq) and p-nitrobenzoyl chloride (219mg, 1.09mmol, 1.30eq) were dissolved in dichloromethane (3.00mL) under nitrogen protection. The reaction system was cooled to 0°C, and pyridine (199mg, 2.52mmol, 203uL, 3.00eq) and 4-dimethylaminopyridine (10.3mg, 83.9umol, 0.10eq) were added sequentially. Monitoring after 2 hours of reaction, TLC (petroleum ether:ethyl acetate=2:1) showed that compound 1-4 (Rf=0.20) was completely consumed and a major point (Rf=0.70) was formed. The reaction solution was concentrated to obtain compound 1-6. LCMS: m/z (M+H) ⁺ = 523.3.

Step 4:

Compound 1-6 (600mg, 1.15mmol, 1.00eq) was dissolved in dichloromethane (6.00mL), and N,N-diisopropylethylamine (594mg, 4.59mmol, 800uL, 4.00eq) and isopropylamine were added successively (204mg, 3.44mmol, 296uL, 3.00eq). After reacting at 25°C for 1 hour, TLC (petroleum ether:ethyl acetate=2:1) showed that compound 1-6 (Rf=0.70) was completely consumed, and a major new spot (Rf=0.30) was formed. The reaction solution was slowly introduced into water (3.00 mL), and extracted twice with dichloromethane (3.00 mL*2). The organic phases were combined, washed twice with saturated brine (3.00 mL*2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain a crude product. The crude product was purified by column chromatography (300-400 mesh silica gel, petroleum ether:ethyl acetate=100:1 to 2:1, product: Rf=0.3) to obtain compound 1-7. LCMS: m/z (M+H) ⁺ = 443.3.

Step 5:

Compound 1-7 (300mg, 678umol, 1.00eq) was dissolved in anhydrous tetrahydrofuran (3.00mL), the air was replaced with nitrogen three times, and wet palladium on carbon (30.0mg, 50% purity) was added. The nitrogen was replaced three times with hydrogen (15 psi). Monitored after 2 hours of reaction at 25°C under hydrogen (15 psi). TLC (petroleum ether: ethyl acetate = 1:1) showed that compound 1-7 (Rf = 0.70) was completely consumed and a major new spot (Rf = 0.40) was formed. The reaction solution was filtered, and the filtrate was concentrated to obtain compound 1-8. LCMS: m/z (M+H) ⁺ = 309.3.

Step 6:

Compound 1-8 (312.mg, 1.01mmol, 2.50eq), compound 1-3 (0.08g, 404umol, 1.00eq) were dissolved in dioxane (2.00mL), and tris(dibenzylideneacetone was added successively) ) Dipalladium (37.1mg, 40.5umol, 0.10eq), sodium carbonate (85.8mg, 810umol, 2.00eq) and 4,5-bisdiphenylphosphine-9,9-dimethyloxaoxanthene (46.9mg, 81.0umol, 0.20eq). Replace the air with nitrogen for 3 times, and monitor after reacting at 50-60°C for 12 hours. LCMS showed compound 1-8 The consumption was complete and a target product peak (m/z=470.3) was monitored. The reaction solution was filtered, and the filtrate was concentrated to obtain a crude product. The crude product was purified by column chromatography (300-400 mesh silica gel, petroleum ether:ethyl acetate=10:1 to 1:1) to obtain compound 1-9. LCMS: m/z (M+H) ⁺ = 470.4.

Step 7:

Compound 1-9 (60.0 mg, 128 umol, 1.00 eq) was dissolved in anhydrous formic acid (3.00 mL). React at 100°C for 16 hours and monitor the thickness. TLC (petroleum ether:ethyl acetate=1:2) showed that compound 1-9 (Rf=0.60) was completely consumed and a new spot (Rf=0.10) was formed. The reaction solution was concentrated to remove formic acid, diluted with dichloromethane (10.0 mL), washed twice with saturated aqueous sodium bicarbonate and once with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain a crude product. The crude product was purified by column chromatography (300-400 mesh silica gel, petroleum ether: ethyl acetate = 5:1 to 0:1, product: petroleum ether: ethyl acetate = 1:2, R _f = 0.10) to obtain compound 1 . LCMS: m/z (M+H) ⁺ = 414.3. ¹ H NMR: (400MHz, CDCl3) δ7.86 (m, 1H) 7.36-7.51 (m, 1H) 6.71-6.87 (m, 1H) 6.48-6.69 (m, 1H) 5.09-5.29 (m, 1H) 4.71 (s,1H)4.65(s,2H)3.66-3.90(m,1H)3.44(s,3H)3.13-3.28(m,1H)2.43-2.61(m,1H)2.11-2.22(m,1H)1.87 -1.98(m,4H)1.09-1.16(m,6H).

Example 2

step 1:

Compound 1-3 (5.00g, 25.3mmol, 100% purity, 1.00eq) and potassium fluoride (7.35g, 127mmol, 2.96mL, 5.00eq) were dissolved in N,N-dimethylformamide ( 50.0 mL). The air was replaced with N ₂ for 3 times, under N ₂ conditions, 140°C was monitored after 16 hours of reaction, LCMS showed that compounds 1-3 were completely consumed and a product peak (m/z=182.0 (M+H)+) was generated. Water (50.0 mL) was added to the reaction liquid, and extracted three times with ethyl acetate (100 mL*3). The organic phases were combined, washed three times with saturated aqueous sodium chloride solution (100 mL*3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain a crude product. Purification by column chromatography (SiO2, petroleum ether:ethyl acetate=5:1 to 2:1, product: Rf=0.60) gave compound 2-1. LCMS: m/z = 181.9 (M+H) ⁺ .

Step 2:

At room temperature, compound 1-4 (8.00g, 22.4mmol, 1.00eq) and imidazole (2.29g, 33.6mmol, 1.50eq) were dissolved in N,N-dimethylformamide (80.0mL), and dimethyl tert-butylchlorosilane (5.06 g, 33.6 mmol, 4.11 mL, 1.50 eq). After replacing N ₂ three times, the reaction was monitored under N ₂ at 20°C for 6 hours. LCMS showed that compound 1-4 was completely consumed and a target product peak (m/z=472.1 (M+H)+) was detected. The reaction solution was slowly poured into water (80.0 mL), and extracted 3 times with ethyl acetate (80.0 mL*3). The organic phases were combined, washed 3 times with saturated aqueous sodium chloride solution (50.0 mL*3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain a crude product. The crude product was purified by column chromatography (SiO2, petroleum ether:ethyl acetate=5:1 to 2:1, product: Rf=0.50) to obtain compound 2-2. LCMS: m/z = 472.3 (M+H) ⁺ .

Step 3:

At room temperature, tetrahydrofuran (100mL) and ethyl acetate (100mL) were added to a 500ml three-necked flask, and wet palladium on carbon (1.89g, 1.78mmol, 1.91mL, 10.0%purity) and compound 2-2 were added sequentially under the protection of _N2 (9.50 g, 20.0 mmol, 99.5% purity, 1.00 eq). The hydrogen was replaced three times. Under the condition of hydrogen (15.0Psi), 20°C was monitored after 3 hours of reaction. LCMS showed that compound 2-2 was completely consumed and a target product peak (m/z=338.0(M+H)+) was detected. After filtration, the filtrate was concentrated to obtain compound 2-3. LCMS: m/z = 338.0 (M+H) ⁺ .

Step 4:

Dissolve compound 2-3 (6.20g, 18.4mmol, 1.00eq) in tetrahydrofuran (100mL) at room temperature, cool down to -20°C, add lithium bis(trimethylsilyl)amide, 1M n-hexane solution under _N2 protection (1.00M, 55.1mL, 3.00eq), stirred at -20°C for 1 hour, compound 2-1 (3.43g, 17.8mmol, 94.1% purity, 0.970eq) was dissolved in tetrahydrofuran (20.0mL) and added. The air was replaced with N ₂ for 3 times, under N ₂ , the reaction was monitored at -20°C for 1 hour. LCMS showed that compound 3 was completely consumed and a target product peak (m/z=499.2(M+H) ⁺ ) was detected. The reaction solution was slowly poured into saturated aqueous ammonium chloride solution (300 mL), and extracted three times with ethyl acetate (120 mL*3). The organic phases were combined, washed twice with a saturated aqueous sodium chloride solution (120 mL*2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain a crude product. The crude product was purified by column chromatography (SiO2, petroleum ether:ethyl acetate=5:1 to 1:1, product: Rf=0.50) to obtain compound 2-4. LCMS: m/z=499.2(M+H) ⁺¹ H NMR: (400MHz,DMSO-d6)δ9.00(s,1H),8.04(d,J=4.4Hz,1H),7.30(d,J ＝4.4Hz,1H),6.85-6.77(m,1H),6.01(s,1H),4.56(s,2H),4.37-4.25(m,1H),3.33-3.31(m,3H),3.04- 2.91(m,1H),2.31-2.21(m,1H),1.96-1.86(m,1H),1.85-1.72(m,2H),1.65-1.59(m,1H),1.57-1.52(m,1H ), 1.49(s,9H), 0.83(s,9H), 0.05-0.02(m,6H).

Step 5:

Compound 2-4 (9.00 g, 16.4 mmol, 91.0% purity, 1.00 eq) was dissolved in formic acid (200 g, 4.34 mol, 164 mL, 264 eq) at room temperature. The air was replaced with N ₂ for 3 times, and stirred at 20°C for 2 hours under N ₂ . The mixture was concentrated to remove most of the formic acid, methanol (160 mL) and water (40.0 mL) were added to the residue and stirred to dissolve, and lithium hydroxide monohydrate (6.89 g, 164 mmol, 10.0 eq) was added under N ₂ protection. The air was replaced with _N2 3 times, and monitored after 1 hour of reaction at 20 °C. LCMS showed that compound 2-4 was consumed completely, and a target product peak (m/z=385.1 (M+H) ⁺ ) was monitored. The reaction solution was slowly poured into water (450 mL), extracted 3 times with ethyl acetate (160 mL*3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain a crude product. Purification using column chromatography (SiO 2 , petroleum ether:ethyl acetate=50:1 to 0:1, product: Rf=0.50) gave compound 2-5. LCMS: m/z = 385.1 (M+H) ⁺ .

Step 6:

At room temperature, compound 2-5 (101mg, 260umol, 98.8% purity, 1.00eq) was dissolved in 2-methyltetrahydrofuran (1.00mL), and 1,1-carbonyldiimidazole (127mg, 780umol, 3.00eq) and 4-Dimethylaminopyridine (6.36 mg, 52.0 umol, 0.200 eq). The air was replaced with N ₂ 3 times, and stirred at 20 °C for 1 h under N ₂ . Compound 2-6 (237mg, 2.34mmol, 9.00eq) and triethylamine (263mg, 2.60mmol, 362uL, 10.0eq) were added sequentially. Replace empty with _N2 Gas three times, under N ₂ conditions, 20 ° C after 24 hours of reaction monitoring, LCMS showed complete consumption of compound 2-5 and monitored a target product peak (m/z = 512.1 (M+H) ⁺ ). The reaction solution was slowly poured into water (5.00 mL), and extracted 3 times with ethyl acetate (10.0 mL*3). The organic phases were combined, washed once with saturated aqueous sodium chloride (10.0 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain compound 2-7. LCMS: m/z = 512.1 (M+H) ⁺ .

Step 7:

Compound 2-7 (159 mg, 311 umol, 1.00 eq) was dissolved in formic acid (3.55 g, 77.1 mmol, 2.91 mL, 248 eq) at room temperature. Use N ₂ to replace the air 3 times, under N ₂ conditions, monitor after 1 hour of reaction at 80°C, LCMS shows that compound 2-7 is completely consumed, and a target product peak (m/z=456.1(M+H)+) is detected . Concentrate the mixture to remove most of the formic acid, add dichloromethane (10.0mL) to the residue to dissolve it completely, and wash with saturated aqueous sodium bicarbonate (20.0mL*3) and saturated aqueous sodium chloride (20.0mL*3) respectively. Extracted 3 times, dried over anhydrous sodium sulfate, filtered, and concentrated the filtrate to obtain a crude product. The crude product was purified and lyophilized by HPLC (preparative column: Phenomenex luna C18 150*25mm*10um; mobile phase: [water(FA)-ACN]; B%: 9%-39%, 10min) to obtain compound 2. LCMS: m/z=456.1(M+H) ⁺ . ¹ H NMR: (400MHz, MeOD) δ7.93(d, J=4.4Hz, 1H), 7.39(s, 1H), 7.07(s, 1H) ,6.30(s,1H),5.09(s,1H),4.64(s,2H),3.98-3.91(m,1H),3.90-3.82(m,2H),3.57(d,J＝8.8Hz,1H ),3.43(s,3H),3.24-3.12(m,1H),2.61-2.50(m,1H),2.27-2.08(m,2H),2.01-1.81(m,5H),1.42(s,3H ).

Example 3

step 1:

Compound 2-5 (101 mg, 260 umol, 98.8% purity, 1.00 eq) was dissolved in 2-methyltetrahydrofuran (1.00 mL) at room temperature. 1,1-Carbonyldiimidazole (127mg, 780umol, 3.00eq) and 4-dimethylaminopyridine (6.36mg, 52.0umol, 0.200eq) were added sequentially. The air was replaced with N ₂ for 3 times, and the reaction was carried out at 20° C. for 1 hour under N ₂ condition. Triethylamine (79.0mg, 780umol, 109uL, 3.00eq), N,N-dimethylformamide (1.00mL) and compound 3-1 (193mg, 1.56mmol, 6.00eq) were added in sequence. Use N ₂ to replace the air 3 times, under N ₂ conditions, 80 ° C reaction for 16 hours monitoring, TLC (petroleum ether: ethyl acetate=0:1) showed that compound 2-5 (Rf=0.50) was completely consumed and there was a major A point (Rf = 0.70) was generated. The reaction solution was poured into water (6.00 mL), and extracted 3 times with ethyl acetate (10.0 mL*3). Combine the organic phases, saturated aqueous sodium chloride (10.0 mL) was washed once, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain compound 3-2.

Step 2:

Compound 3-2 (180mg, 362umol, 1.00eq) was dissolved in formic acid (4.13g, 89.7mmol, 3.38mL, 248eq) at room temperature. The air was replaced with N ₂ for 3 times, under N ₂ , the reaction was monitored at 80°C for 1 hour, LCMS showed that compound 9 was completely consumed, and a target product peak (m/z=442.1(M+H) ⁺ ) was detected. Concentrate the mixture to remove most of the formic acid, add dichloromethane (10.0mL) to the residue to dissolve it completely, and wash with saturated aqueous sodium bicarbonate (20.0mL*3) and saturated aqueous sodium chloride (20.0mL*3) respectively. Extracted 3 times, dried over anhydrous sodium sulfate, filtered, and concentrated the filtrate to obtain a crude product. The crude product was purified and lyophilized by HPLC (preparative column: Phenomenex luna C18 150*25mm*10um; mobile phase: [water(FA)-ACN]; B%: 6%-36%, 10min) to obtain compound 3. LCMS: m/z=442.2(M+H) ⁺ . ¹ H NMR: (400MHz, DMSO) δ12.26-12.03(m, 1H), 10.07-9.89(m, 1H), 8.01(d, J=4.8 Hz,1H),7.41-7.32(m,2H),7.27(s,1H),6.70-6.46(m,1H),5.12-4.96(m,1H),4.56(s,2H),4.10-3.95( m,1H),3.73(dd,J=6.0,8.8Hz,2H),3.64(d,J=7.2Hz,1H),3.42(dd,J=4.0,8.4Hz,1H),3.32(s,3H ), 3.19-3.02(m,1H), 2.12-1.60(m,8H).

Example 4

step 1:

Compound 2-5 (170mg, 437umol, 98.8% purity, 1.00eq) was dissolved in dichloromethane (2.00mL) at room temperature and cooled to 0°C. Pyridine (104mg, 1.31mmol, 106uL, 3.00eq), 4-dimethylaminopyridine (5.34mg, 43.7umol, 0.100eq) and 4-nitrophenol chloroformate (114mg, 568umol, 1.30eq) were added sequentially. The air was replaced with N ₂ for 3 times, and the reaction was carried out at 20° C. for 0.5 h under the condition of N ₂ . Then add compound 4-1 (639mg, 8.74mmol, 918uL, 20.0eq), replace the air with _N2 for 3 times, under N2 conditions, monitor after 2 hours at 20°C, LCMS shows that compound 5 still remains (m/z =385.0 (M+H) ⁺ ), and one product peak (m/z = 484.2 (M+H) ⁺ ) was formed. The reaction solution was poured into water (5.00 mL), and extracted 3 times with ethyl acetate (10.0 mL*3). The organic phases were combined, washed once with saturated aqueous sodium chloride (10.0 mL), anhydrous Dry over sodium sulfate, filter, and concentrate the filtrate to obtain crude product. Purification by column chromatography (SiO2, petroleum ether: ethyl acetate = 5:1 to 1:1, product: Rf = 0.50) gave compound 4-2. LCMS: m/z=484.2(M+H) ⁺ .

Step 2:

Compound 4-2 (180mg, 372umol, 100% purity, 1.00eq) was dissolved in formic acid (4.20g, 91.2mmol, 3.44mL, 245eq) at room temperature. Use N ₂ to replace the air for 3 times, under N ₂ condition, monitor after reacting at 80°C for 1 hour, LCMS shows that compound 4-2 is completely consumed, and a target product peak (m/z=428.0(M+H) ⁺ ) is detected . Concentrate the mixture to remove most of the formic acid, add dichloromethane (10.0mL) to the residue to dissolve it completely, and wash with saturated aqueous sodium bicarbonate (20.0mL*3) and saturated aqueous sodium chloride (20.0mL*3) respectively. Extracted 3 times, dried over anhydrous sodium sulfate, filtered, and concentrated the filtrate to obtain the crude product compound 4-3. LCMS: m/z=428.0(M+H) ⁺ .

Step 3:

Compound 4-3 (100mg, 234umol, 1.00eq) was dissolved in dichloromethane (1mL) at room temperature, cooled to -78°C, and BBr ₃ (117mg, 468umol, 45.1uL, 2.00eq) was added dropwise. Use N ₂ to replace the air 3 times, under N ₂ conditions, monitor after 1 hour of reaction at 0°C, LCMS shows that compound 4-3 still remains (m/z=428.1(M+H) ⁺ ), and there is a target product peak (m/z = 414.1 (M+H) ⁺ ). The reaction solution was poured into 0-5°C ice water (15.0 mL), adjusted to pH 6-8 with saturated aqueous sodium bicarbonate solution, and extracted three times with dichloromethane (20.0 mL*3). The organic phases were combined, washed twice with a saturated aqueous sodium chloride solution (30.0 mL*2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain a crude product. The crude product was purified and lyophilized by HPLC (preparative column: Phenomenex luna C18 150*25mm*10um; mobile phase: [water(FA)-ACN]; B%: 10%-40%, 10min) to obtain compound 4. LCMS: m/z=414.1(M+H) ⁺ . ¹ H NMR: (400MHz, MeOD) δ7.95(d, J=4.8Hz, 1H), 7.39(d, J=4.8Hz, 1H), 7.13 (s,1H),6.29(s,1H),5.07(d,J=3.2Hz,1H),4.80(s,2H),3.26-3.12(m,1H),2.64-2.50(m,1H), 2.22-2.08 (m, 1H), 2.06-1.76 (m, 5H), 1.29 (s, 9H).

Example 5

step 1:

At room temperature, compound 1 (700mg, 1.69mmol, 1.00eq) was dissolved in dichloromethane (7mL), cooled to -70°C, and boron tribromide (5.20g, 20.8mmol, 2.00mL, 12.3eq) was added dropwise . Use _N2 to replace air 3 times, under _N2 condition, After reacting at -70～-50°C for 1 hour, LCMS showed that compound 1 was completely consumed, and the target product peak (m/z=463.9 (M+H) ⁺ ) was monitored. The reaction solution was poured into 0-5°C ice water (50.0 mL), adjusted to pH 6-8 with saturated aqueous sodium bicarbonate solution, and extracted three times with dichloromethane (50.0 mL*3). The organic phases were combined, washed twice with saturated aqueous sodium chloride solution (50.0 mL*2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain compound 5-1. LCMS: m/z=463.9(M+H) ⁺ .

Step 2:

Compound 5-1 (100 mg, 216 mmol, 1.00 eq) was dissolved in tetrahydrofuran (7 mL) at room temperature. Use N ₂ to replace the air 3 times, under N ₂ conditions, slowly add MeNH ₂ (2M, 541uL, 5.00eq) solution dropwise, monitor after 1 hour of reaction at 20°C, LCMS shows that compound 5-1 is completely consumed, and the target product is detected Peak (m/z = 413.2 (M+H)+). The reaction solution was slowly poured into 1M hydrochloric acid aqueous solution (keep the solution pH=7-8), and extracted with dichloromethane (15.0 mL*3) for 3 times. The organic phases were combined, washed twice with saturated aqueous sodium chloride solution (15.0 mL*2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain a crude product. The crude product was purified and lyophilized by HPLC (preparative column: column: Phenomenex luna C18 150*25mm*10um; mobile phase: [water(FA)-ACN]; B%: 1%-31%, 10min) to obtain compound 5 of formate. LCMS: m/z=413.2(M+H) ⁺ . ¹ H NMR: (400MHz, MeOD) δ8.50(s, 1H), 7.97(d, J=4.8Hz, 1H), 7.44(d, J= 4.4Hz,1H),7.11(s,1H),6.41-6.38(m,1H),5.13-5.08(m,1H),4.38(s,2H),3.73-3.67(m,1H),3.19-3.13 (m,1H), 2.77(s,3H), 2.60-2.54(m,1H), 2.15-1.83(m,5H), 1.13-1.10(m,6H).

Example 6

At room temperature, compound 1 (700mg, 1.69mmol, 1.00eq) was dissolved in dichloromethane (7mL), cooled to -70°C, and boron tribromide (5.20g, 20.8mmol, 2.00mL, 12.3eq) was added dropwise . Use N ₂ to replace the air 3 times, under N ₂ conditions, monitor at -70～-50°C for 1 hour, LCMS shows that compound 1 is completely consumed, and the target product peak (m/z=400.1(M+H)+ ). The reaction solution was poured into 0-5°C ice water (50.0 mL), adjusted to pH 6-8 with saturated aqueous sodium bicarbonate solution, and extracted three times with dichloromethane (50.0 mL*3). The organic phases were combined, washed twice with a saturated aqueous sodium chloride solution (50.0 mL*2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain a crude product. The crude product was purified and lyophilized using HPLC (preparative column: column: YMC Triart C18 250*50mm*7um; mobile phase: [water(FA)-ACN]; B%: 30%-60%, 20min) to obtain compound 6 . LCMS: m/z=400.1(M+H) ⁺ . ¹ H NMR: (400MHz, MeOD) δ7.91(s, 1H), 7.37(d, J=4.8Hz, 1H), 7.05(s, 1H) ,6.38-6.25(m,1H),5.16-5.09(m,1H),4.79(s,2H),3.72-3.63(m,1H),3.13(d,J＝0.8Hz,1H),2.59-2.52 (m, 1H), 2.14-1.82 (m, 5H), 1.11 (d, J=6.4Hz, 6H).

Example 7

At room temperature, compound 5-1 (0.24g, 519umol, 1.00eq) was dissolved in tetrahydrofuran (3mL), potassium cyanide (70.0mg, 1.08mmol, 46.1uL, 2.07eq), dimethyl sulfoxide (0.5mL ), after reacting at room temperature for 2 hours, water (0.5mL) and octadecadecron-6 (206mg, 779umol, 1.50eq) were added. After monitoring at 70°C for 16 hours, LCMS showed that compound 5-1 was completely consumed, and the target product peak (m/z=409.2 (M+H)+) was detected. The reaction solution was slowly poured into water (10 mL), extracted 3 times with ethyl acetate (10 mL*3), washed 3 times with saturated aqueous sodium chloride solution (10 mL*3), combined the organic phases, dried over anhydrous sodium sulfate, filtered, Concentration of the filtrate gave crude product. The crude product was purified and lyophilized by HPLC (preparative column: column: Phenomenex luna C18 150*25mm*10um; mobile phase: [water(HCl)-ACN]; B%: 11%-41%, 10min) to obtain compound 7 . LCMS: m/z=409.2(M+H) ⁺ .1H NMR: (400MHz,MeOD)δ8.23(d,J=6.0Hz,1H),7.53-7.50(m,2H),6.20(s,1H ),5.11(d,J=2.8Hz,1H),4.25(s,2H),3.73-3.68(m,1H),3.26-3.24(m,1H),2.66-2.61(m,1H),2.19- 2.03 (m, 1H), 2.02-1.79 (m, 4H), 1.12 (d, J=6.4Hz, 6H).

Example 8

At room temperature, compound 5-1 (100mg, 216umol, 1.00eq) was dissolved in tetrahydrofuran (1mL), and dimethylamine tetrahydrofuran solution (1M, 1.08mL, 5.00eq) was slowly added dropwise _. Under the condition of N ₂ , after reacting at room temperature for 1 hour, LCMS showed that the compound 5-1 was completely consumed, and the target product peak (RT=0.385min, m/z=427.1(M+H) ⁺ ) was detected. The reaction solution was filtered, and the filtrate was concentrated to obtain a crude product. The crude product was purified and lyophilized by HPLC (preparative column: column: Phenomenex luna C18 150*25mm*10um; mobile phase: [water(HCl)-ACN]; B%: 0%-30%, 10min) to obtain compound 8 . LCMS: m/z=427.1(M+H) ⁺ . ¹ H NMR: (400MHz, MeOD) δ8.32(d, J=5.6Hz, 1H), 7.91(s, 1H), 7.60(d, J= 5.6Hz,1H),6.32(s,1H),5.13(s,1H),4.69(s,2H),3.63-3.58(m,1H),3.29-3.22(m,1H),2.99(s,6H ),2.70-2.55(m,1H),2.20-2.18(m,4H),2.04-1.90(m,4H),1.20-1.12(m,6H).

In Vitro Activity Test

Experimental example 1: CDK2/CyclinE enzyme activity test in vitro

Experimental Materials:

CDK2/CyclinE 1 was purchased from Xingnokang. Ulight-4E-BP1 polypeptide, Eu-anti-phospho-tyrosine antibody, and 1X detection buffer were purchased from PerkinElmer. High-purity ATP was purchased from Promega. EDTA was purchased from Sigma. Nivo Multilabel Analyzer (PerkinElmer).

experimental method:

Kinase buffer preparation: Kinase buffer contains 50mM HEPES, 1mM EDTA, 10mM MgCl ₂ , 0.01% Brij-35, PH7.4 Add 2.38g HEPES, 58mg EDTA, 406mg MgCl ₂ , 20mg Brij-35 to 200ml buffer, adjust pH to 7.4.

Preparation of stop solution:

Use 100 μL of 1 MEDTA stock solution plus 0.625uL of 1X detection buffer and mix with 1725uL of distilled water to prepare a stop solution.

Dilute enzyme, Ulight-4E-BP1 polypeptide, ATP and inhibitors in Kinase Buffer. Dilute the Eu-anti-phospho-tyrosine antibody to a concentration of 8nM/L with detection buffer. The compound to be tested was diluted 5 times to the 8th concentration, that is, diluted from 4 μM to 0.0512 nM with a row gun, and the final concentration of DMSO was 4%, and a double-well experiment was set up. Add 2.5 μL inhibitor concentration gradients, 5 μL CDK2/CyclinE 1 enzyme (10 ng), 2.5 μL substrate and ATP mixture (4 mMATP, 100 nM Ulight-4E-BP1 polypeptide) to the microwell plate, and the final concentration gradient of the compound is now 1μM was diluted to 0.0128Nm, and the final concentrations of ATP and substrate were 1mM and 25nM. The reaction system was placed at 25°C for 120 minutes. After the reaction is over, add 5 μL of stop solution to each well, and continue to react at 25 degrees for 5 minutes. After the reaction is completed, add 5uL of Eu-anti-phospho-tyrosine antibody dilution solution to each well, and react at 25 degrees for 60 minutes with a PerkinElmer Nivo multi-label analyzer TR - Data acquisition in FRET mode (excitation at 320 nm and emission at 615 nm and 665 nm).

data analysis:

Use the equation (Sample-Min)/(Max-Min)*100% to convert the original data into an inhibition rate, and the value of IC ₅₀ can be obtained by curve fitting with four parameters (log(inhibitor)vs.response in GraphPad Prism --Variable slope mode derived). Table 1 provides the CDK2/CyclinE 1 enzymatic inhibitory activity of the compounds of the present invention.

Experimental example 2: CDK1/CyclinB1 enzyme activity test in vitro

Experimental Materials:

CDK1/CyclinB1 was purchased from CARNA. Ulight-4E-BP1 polypeptide, Eu-anti-phospho-tyrosine antibody, and 1X detection buffer were purchased from PerkinElmer. High-purity ATP was purchased from Promega. EDTA was purchased from Sigma. Nivo Multilabel Analyzer (PerkinElmer).

experimental method:

Kinase buffer preparation: Kinase buffer contains 50mM HEPES, 1mM EDTA, 10mM MgCl2, 0.01% Brij-35, PH7.4 Add 2.38g HEPES, 58mg EDTA, 406mg MgCl2, 20mg Brij-35, Adjust pH to 7.4.

Preparation of stop solution:

Use 100 μL of 1 MEDTA stock solution plus 0.625uL of 1X detection buffer and mix with 1725uL of distilled water to prepare a stop solution.

Dilute enzyme, Ulight-4E-BP1 polypeptide, ATP and inhibitors in Kinase Buffer.

Dilute the Eu-anti-phospho-tyrosine antibody to a concentration of 8nM/L with detection buffer.

The compound to be tested was diluted 5 times to the 8th concentration, that is, diluted from 4 μM to 0.0512 nM with a row gun, and the final concentration of DMSO was 4%, and a double-well experiment was set up. Add 2.5 μL inhibitor concentration gradients, 5 μL CDK1/CyclinB1 enzyme (0.5 ng), 2.5 μL substrate and ATP mixture (4mMATP, 200nM Ulight-4E-BP1 polypeptide) to the microwell plate, and the final concentration gradient of the compound is now 1 μM diluted to 0.0128 nM, ATP and substrate at final concentrations of 1 mM and 50 nM. The reaction system was placed at 25°C for 60 minutes. After the reaction is over, add 5 μL of stop solution to each well, and continue to react at 25 degrees for 5 minutes. After the reaction is completed, add 5uL of Eu-anti-phospho-tyrosine antibody dilution solution to each well, and react at 25 degrees for 60 minutes with a PerkinElmer Nivo multi-label analyzer TR - Data acquisition in FRET mode (excitation at 320 nm and emission at 615 nm and 665 nm).

data analysis:

Use the equation (Sample-Min)/(Max-Min)*100% to convert the original data into an inhibition rate, and the value of IC ₅₀ can be obtained by curve fitting with four parameters (log(inhibitor)vs.response in GraphPad Prism --Variable slope mode derived). Table 1 provides the CDK1/CyclinB 1 enzymatic inhibitory activity of the compounds of the present invention.

Experimental example 3: GSK3β enzyme activity test in vitro

Experimental Materials:

GSK3βActive was purchased from SignalChem; GSK3Substrate was purchased from SignalChem; ADP-Glo Kinase Assay was purchased from Promega; Kinase assay buffer III was purchased from SignalChem; Nivo multi-label analyzer (PerkinElmer).

experimental method:

The compound to be tested was diluted to 100 μM with 100% DMSO as the first concentration, and then diluted 5 times to the eighth concentration with a row gun, that is, diluted from 100 μM to 0.0013 μM. Each concentration point of the compound was diluted 20 times with 1X kinase buffer to prepare a compound working solution containing 5% DMSO, and 1 μL of the compound concentration gradient working solution was added to the microplate to set up duplicate wells. Add 2 μl of GSK3β enzyme (1ng), 2 μl of the mixture of substrate and ATP (62.5 μM ATP, 0.5 μg/μl GSK3Substrate) to the microwell plate, at this time the final concentration gradient of the compound is 1 μ M and dilute to 0.013 nM, and the final concentration of ATP and substrate is 25 μM and 0.2 μg/μl. The reaction system was placed at 25°C for 60 minutes. After the reaction is over, add 5 μl of ADP-Glo reagent to each well, and continue to react at 25 degrees for 40 minutes. After the reaction is completed, add 10 uL of kinase detection reagent to each well. After reacting at 25 degrees for 30 minutes, use a PerkinElmer Nivo multi-label analyzer to read chemiluminescence, and the integration time is 0.5 Second.

data analysis:

Use the equation (Sample-Min)/(Max-Min)*100% to convert the original data into an inhibition rate, and the value of IC50 can be obtained by curve fitting with four parameters (log(inhibitor)vs.response- in GraphPad Prism -Variable slope mode derived). Table 1 provides the enzymatic inhibitory activity of the compounds of the present invention on GSK3β.

Max well: Positive control well reading value No enzyme blank well

Min well: Negative control wells read as wells containing 1% DMSO solvent

Experimental example 4: In vitro OVCAR3 cell activity test

Experimental Materials:

1640 medium, fetal bovine serum, and penicillin/streptomycin antibiotics were purchased from Vicente. CellTiter-Glo (Cell Viability Chemiluminescence Detection Reagent) reagent was purchased from Promega. OVCAR3 cell line was purchased from Nanjing Kebai Biotechnology Co., Ltd. Envision Multilabel Analyzer (PerkinElmer).

experimental method:

The OVCAR3 cells were seeded in a white 384-well plate, with 40 μL of cell suspension per well, which contained 300 OVCAR3 cells. Cell plates were cultured overnight in a carbon dioxide incubator.

The compound to be tested was diluted 5 times to the 8th concentration, that is, diluted from 2000 μM to 0.00512 μM with a row gun, 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 gradient dilution compound to the middle plate according to the corresponding position, transfer 10 μL of each well to the cell plate after mixing. Compound concentrations ranged from 10 [mu]M to 0.026 nM were transferred to the cell plate. Cell plates were cultured in a carbon dioxide incubator for 7 days. Prepare another cell plate, and read the signal value on the day of drug addition as the maximum value (Max value in the following equation) to participate in data analysis. Add 10 μL of cell viability chemiluminescent detection reagent to each well of the cell plate, and incubate at room temperature for 10 minutes to stabilize the luminescent signal. Read using a multi-label analyzer.

Add 10 μL per well of cell viability chemiluminescent detection reagent to the cell plate, and incubate at room temperature for 10 minutes to stabilize the luminescent signal. Read using a multi-label analyzer.

data analysis:

Use the equation (Sample-Min)/(Max-Min)*100% to convert the original data into an inhibition rate, and the value of _IC50 can be obtained by curve fitting with four parameters ("log(inhibitor) vs. response--Variable slope" mode). Table 1 provides the inhibitory activity of the compounds of the present invention on the proliferation of OVCAR3 cells.

Experimental example 5: In vitro T47D cell activity test

Experimental Materials:

1640 medium, fetal bovine serum, and penicillin/streptomycin antibiotics were purchased from Vicente. CellTiter-Glo (Cell Viability Chemiluminescence Detection Reagent) reagent was purchased from Promega. T-47D cell line was purchased from Nanjing Kebai Biotechnology Co., Ltd. Envision Multilabel Analyzer (PerkinElmer).

experimental method:

Sow T-47D cells in white 384-well plates, 40 μL of cell suspension per well, which contains 300 T-47D cells. Cell plates were cultured overnight in a carbon dioxide incubator.

The compound to be tested was diluted 5 times to the 8th concentration, that is, diluted from 2000 μM to 0.00512 μM with a row gun, 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 gradient dilution compound to the middle plate according to the corresponding position, transfer 10 μL of each well to the cell plate after mixing. Compound concentrations ranged from 10 [mu]M to 0.026 nM were transferred to the cell plate. Cell plates were cultured in a carbon dioxide incubator for 7 days. Prepare another cell plate, and read the signal value on the day of drug addition as the maximum value (Max value in the following equation) to participate in data analysis. Add 10 μL of cell viability chemiluminescent detection reagent to each well of the cell plate, and incubate at room temperature for 10 minutes to stabilize the luminescent signal. Read using a multi-label analyzer.

Add 10 μL per well of cell viability chemiluminescent detection reagent to the cell plate, and incubate at room temperature for 10 minutes to stabilize the luminescent signal. Read using a multi-label analyzer.

data analysis:

Use the equation (Sample-Min)/(Max-Min)*100% to convert the original data into an inhibition rate, and the value of _IC50 can be obtained by curve fitting with four parameters ("log(inhibitor) vs. response--Variable slope" mode). Table 1 provides the inhibitory activity of the compounds of the present invention on the proliferation of T-47D cells.

Table 1

Experimental results:

It can be seen from Table 1 that the compound of the present invention has good activity on CDK2 kinase. Inhibitory activity against CDK1 kinase is not strong, and inhibitory activity against GSK3β is not strong. Good selectivity for CDK1 and GSK3β. And it has good cell anti-proliferation activity on OVCAR3 and T47D.

The technical features of the above-mentioned embodiments can be combined arbitrarily. To make the description concise, all possible combinations of the technical features in the above-mentioned embodiments are not described. However, as long as there is no contradiction in the combination of these technical features, should be considered as within the scope of this specification.

The above-mentioned embodiments only express several implementation modes of the present invention, and the descriptions thereof are relatively specific and detailed, but should not be construed as limiting the patent scope of the invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent for the present invention should be based on the appended claims.

Claims (12)

1. A compound having a structure shown in formula (I), or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof:
   

   in,

   _R is selected from H, halogen, C _1-6 alkyl, halogenated C _1-6 alkyl, C _3-7 cycloalkyl, 3 to 7 membered heterocycloalkyl, nitro, isocyano or -( CH ₂ ) _n R ₅ ;

   R ₅ is selected from -CN, -OR ₆ , -NR ₆ R ₇ , -C(O)OR ₆ , -C(O)R ₆ , -S(O) ₂ R ₆ , -P(O)(OR ₆ ) ₂ , -O-alicyclic group, -O-aliphatic heterocyclic group, -O-aryl group, -O-heteroaryl group or -C(O)-heteroaryl group, wherein the alicyclic group, aliphatic Cyclic, aryl, heteroaryl are unsubstituted or substituted by one or more R ₀ ;

   _R is selected from halogen, C _1-6 alkyl, halogenated C _1-6 alkyl, C _3-7 cycloalkyl, 3-7 membered heterocycloalkyl, cyano, amino, nitro or hydroxyl;

   R and R are _each independently selected from H, C _1-6 alkyl, _halogenated C _1-6 alkyl, C _3-7 cycloalkyl, 3-7 membered heterocycloalkyl, aryl or heteroaryl base;

   n is 0, 1, 2, 3 or 4;

   Ring A is selected from

   X is each independently N or _CR6 ;

   R _is selected from H, hydroxyl, C _1-6 alkyl, halogenated C _1-6 alkyl, C _3-7 cycloalkyl or 3-7 membered heterocycloalkyl;

   R ₂ and R ₃ are each independently selected from H, C _1-6 alkyl, halogenated C _1-6 alkyl, C _3-7 cycloalkyl or 3-7 membered heterocycloalkyl, wherein the C _{3 -7} cycloalkyl and 3-7 membered heterocycloalkyl are unsubstituted or substituted by one or more _R9 ;

   _R is selected from halogen, C _1-6 alkyl, halogenated C _1-6 alkyl, cyano, amino, nitro or hydroxyl;

   R ₄ is selected from H, halogen, amino, hydroxy or C ₁ -C ₆ alkyl.
2. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein the compound has any of formulas (I-1) to (I-3) structure:
   
   
3. The compound according to claim 2, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein the compound has a structure shown in formula (I-4):
4. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein said R ₁ is -CH ₂ R ₅ .
5. The compound according to claim 4, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein said R ₅ is selected from -CN, -OR ₆ or -NR ₆ R ₇ ;

   The R ₆ and R ₇ are each independently selected from H, C _1-6 alkyl or halogenated C _1-6 alkyl.
6. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein said R ₂ is selected from H, C ₁ -C ₆ alkyl or halogenated C ₁ - _C6 alkyl;

   The R ₃ is selected from H, C ₁ -C ₆ alkyl, halogenated C ₁ -C ₆ alkyl, C _3-7 cycloalkyl or 3-7 membered heterocycloalkyl, wherein the C _3-7 Cycloalkyl and 3-7 membered heterocycloalkyl are unsubstituted or substituted with one or more R ₉ .
7. The compound as claimed in claim 6, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein said _R is selected from H;

   The R ₃ is selected from H, C ₁ -C ₆ alkyl, halogenated C ₁ -C ₆ alkyl, unsubstituted or 5-membered heterocycloalkyl substituted by one R ₉ .
8. The compound according to claim 7, or a pharmaceutically acceptable salt _thereof , or a stereoisomer thereof, wherein said R is selected from halogen, C _1-6 alkyl or halogenated C _1-6 Alkyl group; the heteroatom of the 5-membered heterocycloalkyl group is an oxygen atom.
9. The compound according to claim 8, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein in the formula (I) One of the following structures is selected:
   
10. The compound according to any one of claims 1-9, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein the compound has one of the following structures:
    
11. A pharmaceutical composition, characterized by comprising the compound according to any one of claims 1-10, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof; and a pharmaceutically acceptable carrier.
12. According to the compound described in any one of claims 1-10, or its pharmaceutically acceptable salt, or its stereoisomer, or the pharmaceutical composition described in claim 11 in the preparation of treatment or prevention related to CDK2 activity or Drug application for diseases mediated by CDK2 activity.