CN116462688A - Aromatic fused ring derivatives, preparation method and use thereof - Google Patents

Abstract

The present invention relates to a substitutionAromatic condensed ring derivatives, a preparation method thereof and a pharmaceutical composition containing the derivatives are applied to medicine. In particular, the invention relates to a substituted aromatic condensed ring derivative shown in a general formula (I), a preparation method and pharmaceutically acceptable salts thereof, and application thereof as a therapeutic agent, in particular to an HPK1 inhibitor, wherein the definition of each substituent in the general formula (I) is the same as that in the specification.

Description

Aromatic fused ring derivatives and their preparation methods and uses

technical field

The invention relates to an aromatic condensed ring derivative, a preparation method thereof, a pharmaceutical composition containing the derivative and its use as a therapeutic agent, especially as an HPK1 inhibitor.

Background technique

Hematopoietic progenitor kinase (HPK1, also known as MAP4K1) is a mammalian Ste20-like serine/threonine kinase. As the expression product of gene MAP4K1, it is mainly found in hematopoietic stem cells and belongs to the mitogen-activated protein kinase family (MAP4K). It has been found that this family also includes five other members, MAP4K2, MAP4K3, MAP4K4, MAP4K5, and MAP4K6. HPK1 is a protein with a relative molecular weight of 97 kDa, which is mainly divided into three regions, the N-terminal Ste20-like kinase region, the C-terminal kinase region, and the middle part of four proline-rich regions (P1, P2, P3, and P4). The middle region usually interacts with the adapter protein containing the SH2/SH3 region to activate the transduction of a series of signal pathways.

When HPK1 is activated by various upstream signaling factors, including epidermal growth factor, prostaglandin E2, tumor growth factor, erythropoietin, T cell receptor, B cell receptor, etc., a series of biological cascade reactions will be triggered. HPK1 can also interact with the adapter proteins SLP-76 family, CARD11, GRB2 family, CRK family, etc. in the downstream signaling pathway to activate the JNK/SAPK signaling pathway of hematopoietic stem cells, thereby reversely regulating the T cell pathway. Therefore, HPK1 is involved in the regulation of cell proliferation and apoptosis, and also plays an important role in immunosuppression.

As a kinase that regulates immunosuppression, HPK1 is an inverse regulator of T cell receptors. When T cell receptors are activated, HPK1 in the cytoplasm recruits and gathers near the cell membrane. Activated HPK1 phosphorylates the adapter protein SLP76. Activated SLP76 acts as a docking site for T cell regulatory inhibitory protein 14-3-3. Various proteins combine to form a SLP76 complex, which triggers ubiquitination and degradation, eventually leading to the instability of the T cell receptor signaling complex, thereby downregulating T cells Signaling pathways and T cell proliferation.

However, T cells participate in the important process of anti-tumor immunity. They directly kill tumor cells through the antigen receptors of T cells to recognize specific antibodies on tumor cells; or indirectly kill tumor cells by activating macrophages and secreting other lymphokines. Therefore, the inhibition of HPK1 can make T cells proliferate and activate T cell signaling pathways, further enhance the killing effect on tumors, and inhibit tumor growth. Because HPK1 plays an important role in immune regulation, HPK1 can be used as a research direction for the treatment of inflammatory responses, autoimmune diseases (such as systemic lupus erythematosus, psoriasis) and malignant tumors (such as acute myeloid leukemia, bladder cancer, colon cancer, and pancreatic cancer).

Even for HPK1 target inhibitors, currently only two compounds have entered the clinic, namely Treadwell’s CFI-402411 (Phase II) and BeiGene’s BGB-15025 (Phase I). However, in the face of the fierce battle against tumors, medical workers are not far behind. Now more and more pharmaceutical companies have joined this battle, such as Nimbus Therapeutics and Arnold Medicine. As a relatively cutting-edge research direction, HPK1 inhibitors will inevitably bring about immune responses related to T cells while treating tumors. Therefore, it is necessary to find safer and more effective HPK1 inhibitors to reduce the risks during treatment and reduce the suffering of patients.

Contents of the invention

In view of the above technical problems, the present invention provides an aromatic fused ring derivative represented by general formula (I) or its stereoisomer, tautomer or pharmaceutically acceptable salt thereof:

in:

Ring A is selected from:

When it is a double bond, G is selected from CR ₁ ; X ₁ , X ₂ , X ₃ , and X ₄ are each independently selected from CR _x or N;

When it is a single bond, G is selected from C=O, X ₁ is selected from NR _x , X ₂ , X ₃ , and X ₄ are each independently selected from CR _x or N;

R _x is selected from hydrogen atom, hydroxyl, halogen, alkyl, cyano, alkoxy, cycloalkyl, heterocyclyl or -NR ₈ R ₉ , wherein said alkyl, alkoxy, cycloalkyl or heterocyclyl is optionally further substituted by one or more substituents selected from halogen, amino, hydroxyl, cyano, alkoxy or -NR ₈ R ₉ ;

R ₁ is selected from a hydrogen atom, hydroxyl, cyano, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, -S(O) _rR10 , -C(O) _OR10 , -C(O) _R10 , -S(O) _{rNR8R9 ,} -C(O) _NR8R9 or -NHC(O) _R10 , wherein the alkoxyl, cycloalkyl, _heterocyclyl , aryl or hetero Aryl is optionally further substituted by _one or _more substituents selected from halogen, amino, hydroxy, cyano or alkoxy;

R ₂ is selected from alkyl, -S(O) _{rR 10} , -S(O) _{rNR 8} R ₉ , -C(O)R ₁₀ , -C(O)NR ₈ R ₉ , -NHC(O)R ₁₀ or -P(O)R ₈ R ₉ , wherein the alkyl group is optionally further substituted by one or more substituents selected from halogen, amino, hydroxyl, cyano or alkoxy;

_R and _R are the same or different, each independently selected from a hydrogen atom, halogen, amino, hydroxyl, cyano, alkoxy, alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl, wherein the alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl or heteroaryl are optionally further substituted by one or more substituents selected from halogen, amino, hydroxyl, cyano or alkoxy;

_R is selected from a hydrogen atom, halogen, amino, hydroxyl, cyano, alkoxy, alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl, wherein said alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl or heteroaryl is optionally further substituted by one or more substituents selected from halogen, amino, hydroxyl, cyano or alkoxy;

R ₆ and R ₇ are the same or different, each independently selected from a hydrogen atom or an alkyl group;

R₈and R₉The same or different, each independently selected from a hydrogen atom, hydroxyl, halogen, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl or heteroaryl, wherein said alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl or heteroaryl is optionally further selected from one or more of hydroxyl, halogen, nitro, cyano, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, =O, -C(O)R₁₁, -C(O)OR₁₁, -OC(O)R₁₁, -NR₁₂R₁₃, -C(O)NR₁₂R₁₃、-SO₂NR₁₂R₁₃or -NR₁₂C(O)R₁₃replaced by substituents;

Alternatively, R ₈ and R ₉ form a 4-8 membered heterocyclic group together with the atoms they are connected to, wherein the 4-8 membered heterocyclic group contains one or more N, O or S(O) _r , and the 4-8 membered heterocyclic group is optionally further selected from one or more groups selected from hydroxyl, halogen, nitro, cyano, amino, alkyl, alkoxy, cycloalkyl, heterocyclic,

Aryl, heteroaryl, =O, -C(O)R ₁₁ , -C(O)OR ₁₁ , -OC(O)R ₁₁ , -NR ₁₂ R ₁₃ , -C(O)NR ₁₂ R ₁₃ , -SO ₂ NR ₁₂ R ₁₃ or -NR ₁₂ C(O)R ₁₃ are substituted by substituents;

Each R ₁₀ is independently selected from a hydrogen atom, alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl, wherein said alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl is optionally further replaced by one or more selected from hydroxyl, halogen, nitro, cyano, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, =O, -C(O)R ₁₁ , -C(O)OR ₁₁ , -OC(O)R _{11 , -NR Substituted by 12 R 13 , -C(O)NR 12 R 13 , -SO 2 NR 12 R 13 or -NR 12} C(O)R ₁₃ ;

R ₁₁ , R ₁₂ and R ₁₃ are each independently selected from a hydrogen atom, alkyl, amino, cycloalkyl, heterocyclyl, aryl or heteroaryl, wherein said alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl is optionally further substituted by one or more substituents selected from hydroxyl, halogen, nitro, amino, cyano, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, carboxyl or carboxylate;

n is 0, 1 or 2;

r is 0, 1 or 2.

The preferred version of the present invention, a compound described in general formula (I) or its stereoisomer, tautomer or its pharmaceutically acceptable salt, it is the compound described in general formula (II) or its stereoisomer, tautomer or its pharmaceutically acceptable salt:

Wherein: the definitions of G, X ₁ ~X ₄ , R ₂ ~R ₄ are as described in the general formula (I).

The preferred version of the present invention, a compound described in general formula (I) or its stereoisomer, tautomer or its pharmaceutically acceptable salt, it is the compound described in general formula (III) or its stereoisomer, tautomer or its pharmaceutically acceptable salt:

Wherein: R _x , R ₁ , R ₃ and R ₄ are as defined in the general formula (I).

A preferred version of the present invention, a compound described in general formula (II) or its stereoisomers, tautomers or pharmaceutically acceptable salts thereof, wherein:

selected from:

Wherein: the definitions of R _x and R ₁ to R ₄ are as described in the general formula (II).

In a preferred embodiment of the present invention, a compound described in general formula (III) or its stereoisomer, tautomer or pharmaceutically acceptable salt thereof, wherein:

R ₁ is selected from -C(O)OR ₁₀ ;

Rx is selected from an alkyl group or -NR ₈ R ₉ , wherein the alkyl group is optionally further substituted by -NR ₈ R ₉ ;

R ₈ and R ₉ are each independently selected from a hydrogen atom or an alkyl group;

R ₁₀ is selected from alkyl.

In a preferred embodiment of the present invention, a compound described in general formula (III) or its stereoisomer, tautomer or pharmaceutically acceptable salt thereof, wherein:

R ₁ is selected from -C(O)OR ₁₀ ;

Rx is selected from -NR ₈ R ₉ or

R ₈ is selected from a hydrogen atom;

_R9 is selected from alkyl;

R ₁₀ is selected from methyl, ethyl or isopropyl.

A preferred version of the present invention, a compound described in general formula (II) or its stereoisomers, tautomers or pharmaceutically acceptable salts thereof, wherein:

selected from:

R ₁ is selected from a hydrogen atom, an alkoxy group, a cyano group, -SR ₁₀ , -SO ₂ R ₁₀ , -C(O)R ₁₀ or -NHC(O)R ₁₀ ;

R ₁₀ is selected from alkyl or cycloalkyl;

The definitions of R _x and R ₂ are as described in general formula (II).

A preferred version of the present invention, a compound described in general formula (II) or its stereoisomers, tautomers or pharmaceutically acceptable salts thereof, wherein:

selected from:

R ₂ is selected from alkyl, -SO ₂ R ₁₀ , -SO ₂ NR ₈ R ₉ , -C(O)R ₁₀ , -C(O)NR ₈ R ₉ or -P(O)R ₈ R ₉ , wherein the alkyl group is optionally further substituted by -NR ₈ R ₉ ;

R ₈ and R ₉ are each independently selected from a hydrogen atom or an alkyl group;

R ₁₀ is selected from alkyl or cycloalkyl;

The definitions of R _x and R ₂ are as described in general formula (II).

A preferred version of the present invention, a compound described in general formula (II) or its stereoisomers, tautomers or pharmaceutically acceptable salts thereof, wherein:

selected from:

_R2 is selected from

R ₁ and R _x are defined as described in general formula (II).

A preferred version of the present invention, a compound described in general formula (II) or its stereoisomers, tautomers or pharmaceutically acceptable salts thereof, wherein:

selected from:

R ₂ is selected from -SO ₂ R ₁₀ , -SO ₂ NR ₈ R ₉ , -C(O)NR ₈ R ₉ , -C(O)R ₁₀ or -P(O)R ₈ R ₉ ;

R ₈ and R ₉ are each independently selected from a hydrogen atom, methyl, ethyl or isopropyl;

R ₁₀ is selected from methyl, ethyl, isopropyl or cyclopropyl;

The definitions of R _x and R ₁ are as described in general formula (II).

In a preferred embodiment of the present invention, the compound described in general formula (I) is selected from:

Or a stereoisomer, a tautomer, or a pharmaceutically acceptable salt thereof.

NOTE: If there is a discrepancy between the drawn structure and the given name for that structure, the drawn structure will be given greater weight.

The present invention provides a pharmaceutical composition, which contains an effective dose of the compound according to any one of the general formula (I), (II) or (III) or its stereoisomer, tautomer or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, excipient or their composition.

The present invention provides a compound described in general formula (I), (II) or (III) or its stereoisomer, tautomer or pharmaceutically acceptable salt thereof, or the use of its pharmaceutical composition in preparing HPK1 inhibitor.

The present invention also provides a compound described in general formula (I), (II) or (III) or its stereoisomer, tautomer or pharmaceutically acceptable salt thereof, or its pharmaceutical composition in the preparation of medicines for treating diseases mediated by HPK1, wherein the disease mediated by HPK1 is preferably inflammation, autoimmune disease or tumor, wherein the autoimmune disease is preferably systemic lupus erythematosus or psoriasis; wherein the tumor is selected from acute myeloid leukemia, bladder cancer, colon cancer, rectal cancer Cancer, pancreatic cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, lymphoma, blastoma, retinoblastoma, sarcoma, prostate cancer, cholangiocarcinoma, esophageal cancer, gastric cancer, liver cancer, glioma, cervical cancer, ovarian cancer, head and neck cancer, and multiple myeloma.

The present invention also provides a compound described in general formula (I), (II) or (III) or its stereoisomer, tautomer or pharmaceutically acceptable salt thereof, or its pharmaceutical composition in the preparation of medicines for treating inflammation, autoimmune disease or tumor, wherein said autoimmune disease is preferably systemic lupus erythematosus or psoriasis; wherein said tumor is selected from acute myeloid leukemia, urothelial carcinoma, colon cancer, rectal cancer, pancreatic cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, lymphoma, blastocystosis tumor, retinoblastoma, sarcoma, prostate cancer, cholangiocarcinoma, esophageal cancer, gastric cancer, liver cancer, glioma, cervical cancer, ovarian cancer, head and neck cancer, and multiple myeloma.

The pharmaceutical formulations of the present invention may be administered topically, orally, transdermally, rectally, vaginally, parenterally, intranasally, intrapulmonarily, intraocularly, intravenously, intramuscularly, intraarterially, intrathecally, intrathecally, intradermally, intraperitoneally, subcutaneously, subkeratally or by inhalation. The pharmaceutical composition containing the active ingredient may be in a form suitable for oral administration, such as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients suitable for the manufacture of tablets.

The formulations of the invention are suitably presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form generally refers to that amount of the compound which produces a therapeutic effect.

Dosage forms for the topical or transdermal administration of a compound of this invention may include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, in admixture with any preservatives, buffers or propellants which may be required.

When the compound of the present invention is administered to humans and animals in the form of medicine, the compound may be provided alone or in the form of a pharmaceutical composition containing the active ingredient in combination with a pharmaceutically acceptable carrier, for example, 0.1% to 99.5% (more preferably, 0.5% to 90%) of the active ingredient.

Examples of pharmaceutically acceptable carriers include, but are not limited to: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as cornstarch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; Flower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) phosphate buffered saline; (21) cyclodextrins, such as targeting ligands attached to nanoparticles, such as AccurinsTM; and (22) other non-toxic compatible substances used in pharmaceutical formulations, such as polymer-based compositions.

Examples of pharmaceutically acceptable antioxidants include, but are not limited to: (1) water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as Citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid and their analogs. Solid dosage forms (such as capsules, troches pills, dragees, powders, granules, and the like) may include one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or bulking agents, such as starch, lactose, sucrose, glucose, mannitol and/or silicic acid; (2) binders, such as carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and/or acacia; (3) moisturizing (4) disintegrants, such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) dissolution retarders, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; Sodium Hydroxyl Sulfate and mixtures thereof; and (10) Colorants. Liquid dosage forms can include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. Liquid dosage forms may contain, in addition to the active ingredients, inert diluents commonly used in the art, such as water or other solvents; solubilizers and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butanediol, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerin, tetrahydrofuran methanol, polyethylene glycol, and anhydrous sorbitol Fatty acid esters of alcohols, and mixtures thereof.

Suspensions, in addition to the active compounds, may also contain suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum hydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

Ointments, pastes, creams and gels can contain, in addition to the active compounds, excipients such as animal and vegetable fats, oils, waxes, paraffins, starches, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to the active compounds, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. The sprays can contain other customary propellants, such as chlorofluorohydrocarbons, and volatile unsubstituted hydrocarbons, such as butane and propane.

Detailed Description of the Invention

Unless otherwise stated, some terms used in the present invention in the specification and claims are defined as follows:

"Alkyl" when used as a group or a part of a group refers to a C ₁ -C ₂₀ straight chain or branched aliphatic hydrocarbon group. It is preferably C ₁ -C ₁₀ alkyl, more preferably C ₁ -C ₆ alkyl. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethyl Butyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, etc. Alkyl groups can be substituted or unsubstituted.

"Cycloalkyl" refers to saturated or partially saturated monocyclic, fused, bridged and spiro carbocyclic rings. It is preferably a C ₃ -C ₁₂ cycloalkyl group, more preferably a C ₃ -C ₈ cycloalkyl group, and most preferably a C ₃ -C ₆ cycloalkyl group. Examples of monocyclic cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl, etc., preferably cyclopropyl and cyclohexenyl. Cycloalkyl groups can be optionally substituted or unsubstituted.

"Spirocycloalkyl" refers to a polycyclic group with 5 to 18 members, two or more ring structures, and one carbon atom (called spiro atom) shared between the single rings. The ring contains one or more double bonds, but none of the rings has an aromatic system with fully conjugated π electrons. Preferably it is 6 to 14 yuan, more preferably 7 to 10 yuan. According to the number of spiro atoms shared between the ring and the ring, the spiro cycloalkyl group is divided into single spiro, double spiro or polyspiro cycloalkyl, preferably single spiro and double spiro cycloalkyl, preferably 4/5, 4/6, 5/5 or 5/6. Non-limiting examples of "spirocycloalkyl" include, but are not limited to: spiro[4.5]decyl, spiro[4.4]nonyl, spiro[3.5]nonyl, spiro[2.4]heptyl.

"Fused cycloalkyl" refers to an aromatic system with 5 to 18 members, containing two or more ring structures sharing a pair of carbon atoms with each other, one or more rings may contain one or more double bonds, but none of the rings has a fully conjugated π electron, preferably 6 to 12 members, more preferably 7 to 10 members. According to the number of rings, it can be divided into bicyclic, tricyclic, tetracyclic or polycyclic condensed cycloalkyl groups, preferably bicyclic or tricyclic, more preferably 5-membered/5-membered or 5-membered/6-membered bicycloalkyl groups. Non-limiting examples of "fused cycloalkyl" include, but are not limited to, bicyclo[3.1.0]hexyl, bicyclo[3.2.0]hept-1-enyl, bicyclo[3.2.0]heptyl, decahydronaphthyl, or tetrahydrophenanthrenyl.

"Bridged cycloalkyl" refers to an aromatic system with 5 to 18 members, containing two or more ring structures, sharing two carbon atoms that are not directly connected to each other, one or more rings may contain one or more double bonds, but none of the rings has fully conjugated π electrons, preferably 6 to 12 members, more preferably 7 to 10 members. Preferably it is 6 to 14 yuan, more preferably 7 to 10 yuan. According to the number of constituent rings, it can be divided into bicyclic, tricyclic, tetracyclic or polycyclic bridged cycloalkyl groups, preferably bicyclic, tricyclic or tetracyclic, more preferably bicyclic or tricyclic. Non-limiting examples of "bridged cycloalkyl" include, but are not limited to: (1s,4s)-bicyclo[2.2.1]heptyl, bicyclo[3.2.1]octyl, (1s,5s)-bicyclo[3.3.1]nonyl, bicyclo[2.2.2]octyl, (1r,5r)-bicyclo[3.3.2]decyl.

"Heterocyclyl", "heterocycloalkyl", "heterocycle" or "heterocyclic" are used interchangeably in this application, and all refer to non-aromatic heterocyclic groups, wherein one or more ring-forming atoms are heteroatoms, such as oxygen, nitrogen, sulfur atoms, etc., including monocyclic heterocyclic groups, polycyclic heterocyclic groups, fused heterocyclic groups, bridged heterocyclic groups and spiroheterocyclic groups. It preferably has a 5 to 7 membered monocyclic ring or a 7 to 10 membered bicyclic or tricyclic ring, which may contain 1, 2 or 3 atoms selected from nitrogen, oxygen and/or sulfur.

Examples of "monocyclic heterocyclyl" include, but are not limited to, morpholinyl, oxetanyl, thiomorpholinyl, tetrahydrofuranyl, tetrahydropyranyl, 1,1-dioxo-thiomorpholinyl, piperidinyl, 2-oxo-piperidinyl, pyrrolidinyl, 2-oxo-pyrrolidinyl, piperazin-2-one, piperazinyl, hexahydropyrimidinyl,

Monocyclic heterocyclyl groups may be substituted or unsubstituted.

"Spiroheterocyclic group" refers to a polycyclic group with 5 to 18 members, two or more ring structures, and one atom shared between the single rings. The ring may contain one or more double bonds, but none of the rings has an aromatic system with fully conjugated π electrons. One or more ring atoms are selected from nitrogen, oxygen or S(O) _r (wherein r is selected from 0, 1 or 2) heteroatoms, and the remaining ring atoms are carbon. Preferably it is 6 to 14 yuan, more preferably 7 to 10 yuan. According to the number of spiro atoms shared between the rings, the spirocycloalkyl group can be divided into single spiroheterocyclyl, double spiroheterocyclyl or polyspiroheterocyclyl, preferably single spiroheterocyclyl and double spiroheterocyclyl. More preferably, it is a 4-membered/4-membered, 4-membered/5-membered, 4-membered/6-membered, 5-membered/5-membered or 5-membered/6-membered monospiro heterocyclic group. Non-limiting examples of "spiroheterocyclyl" include, but are not limited to: 1,7-dioxaspiro[4.5]decyl, 2-oxa-7-azaspiro[4.4]nonyl, 7-oxaspiro[3.5]nonyl, 5-oxaspiro[2.4]heptyl,

A spiroheterocyclyl can be substituted or unsubstituted.

"Fused heterocyclic group" refers to a polycyclic group containing two or more ring structures sharing a pair of atoms with each other. One or more rings may contain one or more double bonds, but none of the rings has a fully conjugated π-electron aromatic system, wherein one or more ring atoms are selected from nitrogen, oxygen or S(O) _r (where r is selected from 0, 1 or 2) heteroatoms, and the remaining ring atoms are carbon. Preferably it is 6 to 14 yuan, more preferably 7 to 10 yuan. According to the number of constituent rings, it can be divided into bicyclic, tricyclic, tetracyclic or polycyclic fused heterocyclic groups, preferably bicyclic or tricyclic, more preferably 5-membered/5-membered, 5-membered/6-membered or 5-membered/7-membered bicyclic fused heterocyclic groups. Non-limiting examples of "fused heterocyclyl" include, but are not limited to: octahydropyrrolo[3,4-c]pyrrolyl, octahydro-1H-isoindolyl, 3-azabicyclo[3.1.0]hexyl, octahydrobenzo[b][1,4]dioxine,

A fused heterocyclic group can be substituted or unsubstituted.

"Bridged heterocyclic group" refers to a polycyclic group with 5 to 14 members, 5 to 18 members, containing two or more ring structures, sharing two atoms not directly connected to each other, one or more rings may contain one or more double bonds, but none of the rings has an aromatic system with fully conjugated π electrons, wherein one or more ring atoms are selected from nitrogen, oxygen or S(O) _r (wherein r is selected from 0, 1 or 2) heteroatoms, and the remaining ring atoms are carbon. Preferably it is 6 to 14 yuan, more preferably 7 to 10 yuan. According to the number of constituent rings, it can be divided into bicyclic, tricyclic, tetracyclic or polycyclic bridged heterocyclic groups, preferably bicyclic, tricyclic or tetracyclic, more preferably bicyclic or tricyclic. Non-limiting examples of "bridged heterocyclyl" include, but are not limited to: 2-azabicyclo[2.2.1]heptyl, 2-azabicyclo[2.2.2]octyl, 8-oxa-3-aza-bicyclo[3.2.1]octyl, 2-azabicyclo[3.3.2]decyl.

Bridged heterocyclyl groups may be substituted or unsubstituted.

"Aryl" means a carbocyclic aromatic system containing one or two rings, wherein the rings may be joined together in a fused fashion. The term "aryl" includes monocyclic or bicyclic aryl groups, such as phenyl, naphthyl, tetrahydronaphthyl aromatic groups. Preferred aryl groups are C ₆ -C ₁₀ aryl groups, more preferred aryl groups are phenyl and naphthyl, most preferably naphthyl. Aryl groups can be substituted or unsubstituted.

"Heteroaryl" refers to an aromatic 5 to 6 membered monocyclic ring or 8 to 10 membered bicyclic ring, which may contain 1 to 4 atoms selected from nitrogen, oxygen and/or sulfur. Examples of "heteroaryl" include, but are not limited to, furyl, pyridyl, 2-oxo-1,2-dihydropyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, thienyl, isoxazolyl, oxazolyl, oxadiazolyl, imidazolyl, pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, thiazolyl, isothiazolyl, 1,2,3-thiadiazolyl, benzodioxolyl, benzothienyl , benzimidazolyl, indolyl, isoindolyl, 1,3-dioxo-isoindolyl, quinolinyl, indazolyl, benzisothiazolyl, benzoxazolyl, benzisoxazolyl,

Heteroaryl groups can be substituted or unsubstituted.

"Alkoxy" refers to a group of (alkyl-O-). Wherein, alkyl refers to relevant definitions herein. C ₁ -C ₆ alkoxy is preferred. Examples include, but are not limited to: methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, and the like.

"Hydroxy" means an -OH group.

"Halogen" refers to fluorine, chlorine, bromine and iodine.

"Amino" refers to _-NH2 .

"Cyano" refers to -CN.

"Nitro" refers to _-NO2 .

"Benzyl" means _-CH2 -phenyl.

"Carboxy" refers to -C(O)OH.

"Carboxylate group" refers to -C(O)O-alkyl or -C(O)O-cycloalkyl, wherein the definitions of alkyl and cycloalkyl are as above.

"DMSO" means dimethylsulfoxide.

"TFA" means trifluoroacetic acid.

"Ts" refers to p-toluenesulfonyl.

"Tf" refers to p-trifluoromethylbenzenesulfonyl.

"Leaving group", or leaving group, an atom or functional group that is separated from a larger molecule in a chemical reaction, is a term used in nucleophilic substitution reactions and elimination reactions. In a nucleophilic substitution reaction, the reactant attacked by the nucleophile is called the substrate, and the atom or atomic group that breaks off with a pair of electrons from the substrate molecule is called the leaving group. Groups that are easy to accept electrons and have strong ability to bear negative charges are good leaving groups. The smaller the pKa of the conjugate acid of the leaving group, the easier it is for the leaving group to detach from other molecules. The reason is that when the pKa of its conjugate acid is smaller, the corresponding leaving group does not need to combine with other atoms, and the tendency to exist in the form of anion (or electrically neutral leaving group) is also enhanced. Common leaving groups include, but are not limited to, halogen, methanesulfonyl, -OTs, -OTf, or -OH.

"Substituted" means that one or more hydrogen atoms in the group, preferably 1 to 3 hydrogen atoms are independently substituted by the corresponding number of substituents. It goes without saying that substituents are only in their possible chemical positions and that a person skilled in the art can determine (by experiment or theory) possible or impossible substitutions without undue effort. For example, an amino or hydroxyl group with free hydrogen may be unstable when bonded to a carbon atom with an unsaturated (eg, ethylenic) bond.

"Substituted" or "substituted" described in this specification, unless otherwise specified, means that the group can be substituted by one or more groups selected from the following groups: alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, amino, haloalkyl, hydroxyalkyl, carboxyl, carboxylate, =O, -C( O)R₁₁, -C(O)OR₁₁, -OC(O)R₁₁, -NR₁₂R₁₃, -C(O)NR₁₂R₁₃、-SO₂NR₁₂R₁₃or -NR₁₂C(O)R₁₃replaced by substituents;

R ₁₁ , R ₁₂ and R ₁₃ are each independently selected from a hydrogen atom, alkyl, amino, cycloalkyl, heterocyclyl, aryl or heteroaryl, wherein said alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl is optionally further substituted by one or more substituents selected from hydroxyl, halogen, nitro, amino, cyano, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, carboxyl or carboxylate.

The compounds of the present invention may contain asymmetric centers or chiral centers and thus exist in different stereoisomeric forms. It is contemplated that all stereoisomeric forms of the compounds of the invention, including but not limited to, diastereoisomers, enantiomers and atropisomers and geometric (conformational) isomers and mixtures thereof, such as racemic mixtures, are within the scope of the present invention.

Unless otherwise indicated, structures depicted herein also include all isomeric (e.g., diastereoisomers, enantiomers, and atropisomers) and geometric (conformational) isomeric forms of such structures; for example, the R and S configurations of each asymmetric center, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers. Thus individual stereoisomers as well as enantiomeric mixtures, diastereomeric mixtures, and geometric (conformational) isomers of compounds of the present invention Body mixtures are within the scope of the present invention.

"Pharmaceutically acceptable salt" refers to certain salts of the above compounds that can maintain their original biological activity and are suitable for medical use. The pharmaceutically acceptable salt of the compound represented by the general formula (I) may be a metal salt or an amine salt with a suitable acid.

"Pharmaceutical composition" means a mixture containing one or more compounds described herein or a physiologically acceptable salt or prodrug thereof and other chemical components, as well as other components such as physiologically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to promote the administration to the organism, facilitate the absorption of the active ingredient and thus exert biological activity.

The synthetic method of compound of the present invention

In order to accomplish the purpose of the present invention, the present invention adopts following technical scheme:

The present invention provides a preparation method of a compound of general formula (I) or its stereoisomer, tautomer or pharmaceutically acceptable salt thereof, said method comprising:

The compound of the general formula (IA) and the compound of (IB) undergo a coupling reaction under a catalyst condition and under basic conditions to obtain the compound of the general formula (I);

in:

X is selected from a leaving group, and the leaving group is selected from halogen or OTf;

Ring A, R ₃ -R ₇ and n are as defined in general formula (I).

Described catalyst is preferably tridibenzylidene acetone dipalladium;

The reagent providing basicity is preferably cesium carbonate.

Detailed ways

The following examples are used to further describe the present invention, but these examples do not limit the scope of the present invention.

Example

The examples give the preparation of representative compounds represented by formula (I) and relevant structural identification data. It must be noted that the following examples are used to illustrate the present invention rather than limit the present invention. The ¹ H NMR spectrum was measured with a Bruker instrument (400 MHz), and the chemical shifts are expressed in ppm. Tetramethylsilane internal standard (0.00 ppm) was used. Notation in ¹ H NMR: s = singlet, d = doublet, t = triplet, m = multiplet, br = broadened, dd = doublet of doublet, dt = doublet of triplet. If the coupling constant is provided, its unit is Hz.

The mass spectrum is measured by LC/MS instrument, and the ionization method can be ESI or APCI.

Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plates are used for thin-layer chromatography silica gel plates. The specifications of silica gel plates used in thin-layer chromatography (TLC) are 0.15mm-0.2mm, and the specifications of thin-layer chromatography separation and purification products are 0.4mm-0.5mm.

Column chromatography generally uses Yantai Huanghai silica gel 200-300 mesh silica gel as the carrier.

In the following examples, unless otherwise specified, all temperatures are in degrees Celsius. Unless otherwise specified, various starting materials and reagents were purchased from commercial sources or synthesized according to known methods. Commercially available raw materials and reagents were used directly without further purification.

CD ₃ OD: deuterated methanol.

CDCl ₃ : deuterated chloroform.

DMSO-d ₆ : deuterated dimethylsulfoxide.

The nitrogen atmosphere means that the reaction bottle is connected to a nitrogen balloon with a volume of about 1 L.

Unless otherwise specified in the examples, the solution in the reaction refers to an aqueous solution.

The compound is purified by silica gel column chromatography eluent system and thin-layer chromatography, wherein the eluent system is selected from: A: petroleum ether and ethyl acetate system; B: methylene chloride and methanol system; C: methylene chloride: ethyl acetate; wherein the volume ratio of the solvent varies according to the polarity of the compound, and can also be adjusted by adding a small amount of acidic or alkaline reagents, such as acetic acid or triethylamine.

Example 1

2-((5-(2-aminopropan-2-yl)-8-(methylthio)-2,7-naphthyridin-3-yl)amino)-7,7-dimethyl-7,8-

dihydro-5H-pyrano[4,3-b]pyridin-5-one

2-((5-(2-aminoisopropyl-2-yl)-8-(methylthio)-2,7-naphthyridin-3-yl)amino)-7,7-dimethyl-7,8-dihydro-5H-pyran

[4,3-b]pyridin-5-one

first step

4-bromo-6-chloro-2,7-naphthyridin-1(2H)-one

4-Bromo-6-chloro-2,7-naphthyridin-1(2H)-one

6-Chloro-2,7-naphthyridin-1(2H)-one 1a (3g, 16.61mmol), N-bromosuccinimide (3.55g, 19.93mmol) were sequentially added into N,N-dimethylformamide (30mL), and stirring was continued at 20°C for 2 hours. LC-MS detected that the reaction of raw materials was complete. The reaction solution was poured into ice water, a large amount of khaki solid precipitated, filtered, the filter cake was washed twice with water, and dried to obtain 4-bromo-6-chloro-2,7-naphthyridin-1(2H)-one 1b (3.7g, 14.26mmol, 85.83% yield).

MS m/z(ESI): 260.3[M+H] ⁺

second step

4-bromo-1,6-dichloro-2,7-naphthyridine

4-Bromo-1,6-dichloro-2,7-naphthyridine

4-Bromo-6-chloro-2,7-naphthyridin-1(2H)-one 1b (500mg, 1.93mmol) was added to phosphorus oxychloride (3.40g, 22.16mmol, 5mL), and stirring was continued at 110°C for 3 hours under nitrogen protection. LC-MS monitoring, the raw material reaction is completed, the reaction liquid is cooled to room temperature, and the decompression is concentrated. The ice cubes are quenched to the residue. The 50ml saturated sodium bicarbonate solution is added to adjust the pH to the weak alkali. To 4-bromine-1,6-dichlori-2,7-piomididine 1c (500 mg, 1.80 mmol, 93.36 % yield).

MS m/z(ESI): 281.1[M+H] ⁺

third step

4-bromo-6-chloro-1-(methylthio)-2,7-naphthyridine

4-Bromo-6-chloro-1-(methylthio)-2,7-naphthyridine

4-Bromo-1,6-dichloro-2,7-naphthyridine 1c (1g, 3.60mmol), sodium methyl mercaptide (1.26g, 3.60mmol, 20% purity) were sequentially added to N,N-dimethylformamide (10mL), and stirred at 25°C for 12 hours under nitrogen protection. LC-MS monitored the completion of the reaction of the raw materials. The reaction solution was poured into 200 mL of ice water, and a large amount of khaki solid was precipitated. After filtration, the filter cake was washed twice with water and dried to obtain 4-bromo-6-chloro-1-(methylthio)-2,7-naphthyridine 1d (900 mg, 3.11 mmol, 86.38% yield).

MS m/z(ESI): 290.3[M+H] ⁺

the fourth step

6-chloro-4-(1-ethoxyvinyl)-1-(methylthio)-2,7-naphthyridine

6-Chloro-4-(1-ethoxyvinyl)-1-(methylthio)-2,7-naphthyridine

4-Bromo-6-chloro-1-(methylthio)-2,7-naphthyridine 1d (1g, 3.45mmol), tributyl(1-ethoxyethylene)tin (2.49g, 6.91mmol), tetrakis(triphenylphosphine)palladium (199.52mg, 172.66μmol) were sequentially added to toluene (10mL), and stirring was continued for 3 hours at 80°C under nitrogen protection. LC-MS monitored the completion of the raw material reaction, added water, extracted with ethyl acetate, and concentrated under reduced pressure. The obtained residue was further separated and purified by silica gel column chromatography (eluent: system A) to obtain 6-chloro-4-(1-ethoxyvinyl)-1-(methylthio)-2,7-naphthyridine 1e (660 mg, 2.35 mmol, 68.07% yield).

MS m/z(ESI): 281.5[M+H] ⁺

the fifth step

1-(6-chloro-1-(methylthio)-2,7-naphthyridin-4-yl)ethan-1-one

1-(6-Chloro-1-(methylthio)-2,7-naphthyridin-4-yl)ethan-1-one

6-Chloro-4-(1-ethoxyvinyl)-1-(methylthio)-2,7-naphthyridine 1e (660 mg, 2.35 mmol) and concentrated hydrochloric acid (0.7 mL) were sequentially added to tetrahydrofuran (7 mL), and stirring was continued for 3 hours under nitrogen protection at 0°C. LC-MS monitored the completion of the raw material reaction, poured the reaction solution into 50 mL of water, added saturated sodium bicarbonate solution to adjust the pH to weakly alkaline, extracted with ethyl acetate (25 mL×2), dried, and concentrated under reduced pressure to obtain 1-(6-chloro-1-(methylthio)-2,7-naphthyridin-4-yl)ethan-1-one 1f (500 mg, 1.98 mmol, 84.17% yield).

MS m/z(ESI): 253.1[M+H] ⁺

step six

2-(6-chloro-1-(methylthio)-2,7-naphthyridin-4-yl)propan-2-ol

2-(6-Chloro-1-(methylthio)-2,7-naphthyridin-4-yl)propan-2-ol

1-(6-Chloro-1-(methylthio)-2,7-naphthyridin-4-yl)ethan-1-one 1f (500mg, 1.98mmol) and methylmagnesium bromide (5mL, 1mol/L, THF) were sequentially added to tetrahydrofuran (5mL), and stirred continuously for 5 hours under nitrogen protection at 0°C. LC-MS monitored the completion of the raw material reaction, added water, extracted with ethyl acetate, concentrated under reduced pressure, and the obtained residue was further separated and purified by silica gel column chromatography (eluent: system A) to obtain 1 g (200 mg, 744.15 μmol, 37.61% yield) of 2-(6-chloro-1-(methylthio)-2,7-naphthyridin-4-yl)propan-2-ol.

MS m/z(ESI): 269.1[M+H] ⁺

step seven

4-(2-azidopropan-2-yl)-6-chloro-1-(methylthio)-2,7-naphthyridine

4-(2-Azidopropan-2-yl)-6-chloro-1-(methylthio)-2,7-naphthyridine

Add 1 g (100 mg, 372.08 μmol) of 2-(6-chloro-1-(methylthio)-2,7-naphthyridin-4-yl) propan-2-ol into dichloromethane (3 mL), slowly add azidotrimethylsilane (111.25 mg, 967.40 μmol) and boron trifluoride diethyl ether (110.90 mg, 781.36 μmol), at room temperature, under nitrogen protection conditions Stirring was continued for 12 hours. LC-MS monitored the completion of the raw material reaction, added water, and then extracted with ethyl acetate. The combined organic phases were concentrated under reduced pressure, and the obtained residue was further separated and purified by silica gel column chromatography (eluent: system A) to obtain 4-(2-azidopropan-2-yl)-6-chloro-1-(methylthio)-2,7-naphthyridine 1h (80 mg, 272.32 μmol, 73.19% yield).

MS m/z(ESI): 293.1[M+H] ⁺

eighth step

2-((5-(2-azidopropan-2-yl)-8-(methylthio)-2,7-naphthyridin-3-yl)amino)-7,7-dimethyl-7,8-

dihydro-5H-pyrano[4,3-b]pyridin-5-one

2-((5-(2-azidopropyl-2-yl)-8-(methylthio)-2,7-naphthyridin-3-yl)amino)-7,7-dimethyl-7,8-dihydro-5H-pyran[4,3-

b] pyridin-5-one

4-(2-azidopropan-2-yl)-6-chloro-1-(methylthio)-2,7-naphthyridine 1h (80.00 mg, 272.32 μmol), 2-amino-7,7-dimethyl-8H-pyrano[4,3-b]pyridin-5-one 1j (52.34 mg, 272.32 μmol, prepared by referring to WO2021146370A1) Trisdibenzylideneacetone dipalladium (24.94 mg, 27.23 μmol), 4,5-bisdiphenylphosphine-9,9-dimethylxanthene (31.51 mg, 54.46 μmol), and cesium carbonate (266.18 mg, 816.95 μmol) were sequentially added to 1,4-dioxane (3 mL), and stirring was continued for 1 hour at 125° C. under nitrogen protection under microwave conditions. LC-MS monitored the completion of the raw material reaction, added water, extracted with ethyl acetate, and concentrated the combined organic phases under reduced pressure. The obtained residue was further separated and purified by silica gel column chromatography (eluent: system B) to obtain 2-((5-(2-azidopropyl-2-yl)-8-(methylthio)-2,7-naphthyridin-3-yl)amino)-7,7-dimethyl-7,8-dihydro-5H-pyran[4,3-b]pyridin-5-one 1i (75 mg, 166.84 μmol, 61.27% yield).

MS m/z(ESI):450.2[M+1]

Ninth step

2-((5-(2-aminopropan-2-yl)-8-(methylthio)-2,7-naphthyridin-3-yl)amino)-7,7-dimethyl-7,8-dihydro-5H-pyrano[4,3-b]pyridin-5-one

2-((5-(2-aminoisopropyl-2-yl)-8-(methylthio)-2,7-naphthyridin-3-yl)amino)-7,7-dimethyl-7,8-dihydro-5H-pyran[4,3-b]pyridin-5-one

2-((5-(2-azidopropyl-2-yl)-8-(methylthio)-2,7-naphthyridin-3-yl)amino)-7,7-dimethyl-7,8-dihydro-5H-pyran[4,3-b]pyridin-5-one 1i (70mg, 155.72μmol), zinc powder (50.91mg, 778.59μmol), glacial acetic acid (46.76mg , 778.59 μmol) were sequentially added into tetrahydrofuran (3 mL), and stirring was continued for 2 hours at 25° C. under hydrogen protection. LC-MS monitored the completion of the reaction, filtered the reaction solution, concentrated the filtrate under reduced pressure, added saturated sodium bicarbonate solution (50 mL) to the residue, extracted with ethyl acetate (25 mL×2) twice, combined the organic phases, washed with saturated brine (25 mL), dried over anhydrous sodium sulfate, and concentrated by filtration. , 7-Dimethyl-7,8-dihydro-5H-pyrano[4,3-b]pyridin-5-one 1 (15 mg, 33.65 μmol, 21.61% yield).

MS m/z(ESI):424.2[M+1]

¹ H NMR (400MHz, Chloroform-d) δ9.47(s,1H),9.42(s,1H),8.52(s,1H),8.27(d,J=8.6Hz,1H),8.08(s,1H),7.20(d,J=8.6Hz,1H),3.18(s,2H),2.74(s,3H),1 .82(s,6H).

biological evaluation

Test example 1, the test of compound of the present invention to HPK1 kinase inhibitory activity

The following method is used to determine the degree of inhibition of the compound of the present invention on the kinase activity of recombinant human HPK1 under in vitro conditions. This method uses the ADP-Glo ^TM Kinase Assay kit (product number V9102) from Promega. The above kit is a luminescent kinase detection kit, which is used to detect the ADP content produced by the kinase reaction. The ADP content is positively correlated with the kinase activity. By measuring the ADP content, it reflects the inhibitory strength of the compound on the HPK1 kinase activity. For detailed experimental operation, please refer to the kit instruction manual.

The experimental procedure is briefly described as follows: the test compound was first dissolved in DMSO to prepare a stock solution, and then the buffer solution (20mM MgCl ₂ , 50μM DTT, 0.1mg/ml BSA, 40mMTris, pH7.4) was prepared according to the buffer solution provided in the reagent instruction manual, and the buffer solution was used for serial dilution. The final concentration of the test compound in the reaction system ranged from 1000nM to 0.02nM. The reaction was carried out in a 384-well microplate. First, the compound and recombinant human HPK1 protein (final concentration 1 ng/μL, purchased from Signalchem, Cat. No. M23-11G-10) were added to the wells, and incubated at room temperature for 5 minutes, and then ATP solution (from ADP-Glo ^TM Kinase Assay kit component V915A, final concentration 10 μM) and substrate MBP (final concentration 0.2 μg/μM) were added to the reaction solution. μL, purchased from Signalchem, Cat. No. M42-51N), and incubated at room temperature for 60 minutes with shaking. Then add 5 μL of ADP-Glo Reagent to the reaction system, and continue to shake and incubate at room temperature for 40 minutes. Then add 10 μL of Kinase Detection Reagent to the reaction system, and continue to shake and incubate at room temperature for 30 minutes. After the incubation, the chemiluminescence intensity value of each well was measured on a microplate reader in Luminescence mode. The percentage inhibition rate of the compound at each concentration was calculated by comparing the luminescence intensity ratio of the control group (0.1% DMSO), and the compound concentration-inhibition rate was used for nonlinear regression analysis by GraphPad Prism 5 software to obtain the _IC50 value of the compound, as shown in Table 1.

Table 1 The compound of the present invention inhibits the IC50 data of HPK1 kinase activity

It can be seen from Table 1 that the compound of the present invention has a good inhibitory effect on HPK1 kinase.

Test example 2, the assay of compound of the present invention to p-SLP76 S376 inhibitory activity in Jurkat cell

The following method is used to determine the inhibitory activity of the compounds of the present invention on p-SLP76 S376 in Jurkat cells. This method uses the Phospho-SLP-76 (Ser376) cellular kit (Catalog No. 63ADK076PEG) from Cisbio Company, and the detailed experimental operation can refer to the kit instruction manual. Jurkat cells were purchased from the Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

The experimental procedure is briefly described as follows: Jurkat cells were cultured in RPMI 1640 complete medium containing 10% fetal bovine serum, 100 U penicillin, 100 μg/mL streptomycin and 1 mM Sodium Pyruvate. 8 uL of Jurkat cells (100,000 per well) were spread in a 384-well plate, the medium was complete medium, and cultured at 37° C. in a 5% CO2 incubator. The test compound was dissolved in DMSO to prepare a 10mM stock solution, then diluted with RPMI 1640 complete medium, and 2uL of RPMI 1640 complete medium containing the corresponding concentration of the test compound was added to each well. The uL lysate was lysed for 45 minutes, and finally 4uL of Phospho-SLP-76Cryptate antibody and Phospho-SLP-76d2 antibody were added and incubated overnight at room temperature. Under the excitation wavelength of 304nM, the fluorescence intensity of each well with emission wavelengths of 620nM and 665nM was measured on a microplate reader in TF-FRET mode, and the ratio of the fluorescence intensity of each well 665/620 was calculated. By comparing with the fluorescence intensity ratio of the control group (0.1% DMSO), the percentage inhibition rate of the test compound at each concentration was calculated, and the non-linear regression analysis was performed with the concentration of the test compound on the value-inhibition rate by GraphPad Prism 5 software to obtain the _IC50 value of the compound, as shown in Table 2.

Table 2 _IC50 data of compounds of the present invention on p-SLP76 S376 inhibitory activity in Jurkat cells

Example number IC ₅₀ (nM) 1 77.99

It can be concluded from Table 2 that the compound of the present invention has a better inhibitory effect on p-SLP76 S376 in Jurkat cells.

Test example 3, pharmacokinetic study of compound of the present invention ICR mice

1. Purpose of the experiment

Taking ICR mice as the experimental animals, the LC/MS/MS method was used to determine the compound 1 administered orally, and the drug concentration in plasma at different times was measured to study the pharmacokinetic characteristics of the compound of the present invention in mice.

2. Experimental plan

2.1 Experimental drugs and animals;

Compounds of the invention

ICR mice, male, 29.7-34.6 g, were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.

2.2 Drug preparation

Weigh an appropriate amount of drug, add DMA, Cremophor EL and 5% GS in sequence, and mix by ultrasonic vortexing to prepare a 1 mg/mL administration preparation. Wherein DMA:Cremophor EL:5%GS=5:10:85 (v/v/v).

2.3 Administration

ICR mice, the test compound group (nine rats), were fasted overnight and administered intragastrically (PO, dosage 10 mg/kg, volume 10 mL/kg), and fed 4 hours after administration.

3. Operation

About 0.1 mL of blood was collected from the orbital vein before and 0.25 hours, 0.5 hours, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours and 24 hours after the administration, and anticoagulated with heparin sodium. Blood samples were placed on ice after collection, and plasma was separated by centrifugation (centrifugation conditions: 1500g, 10 minutes). The collected plasma was stored at –40–20°C before analysis.

LC-MS/MS was used to determine the content of the compound to be tested in mouse plasma after intragastric administration.

4. Results of pharmacokinetic parameters

The pharmacokinetic parameters of the compounds of the present invention are shown in the table below.

Table 3 Pharmacokinetic Results

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

Claims (17)

1. A compound represented by general formula (I) or its stereoisomers, tautomers or pharmaceutically acceptable salts thereof: in: Ring A is selected from: When it is a double bond, G is selected from CR ₁ ; X ₁ , X ₂ , X ₃ , and X ₄ are each independently selected from CR _x or N; When it is a single bond, G is selected from C=O, X ₁ is selected from NR _x , X ₂ , X ₃ , and X ₄ are each independently selected from CR _x or N; R _x is selected from hydrogen atom, hydroxyl, halogen, alkyl, cyano, alkoxy, cycloalkyl, heterocyclyl or -NR ₈ R ₉ , wherein said alkyl, alkoxy, cycloalkyl or heterocyclyl is optionally further substituted by one or more substituents selected from halogen, amino, hydroxyl, cyano, alkoxy or -NR ₈ R ₉ ; R ₁ is selected from a hydrogen atom, hydroxyl, cyano, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, -S(O) _rR10 , -C(O) _OR10 , -C(O) _R10 , -S(O) _{rNR8R9 ,} -C(O) _NR8R9 or -NHC(O) _R10 , wherein the alkoxyl, cycloalkyl, _heterocyclyl , aryl or hetero Aryl is optionally further substituted by _one or _more substituents selected from halogen, amino, hydroxy, cyano or alkoxy; R ₂ is selected from alkyl, -S(O) _{rR 10} , -S(O) _{rNR 8} R ₉ , -C(O)R ₁₀ , -C(O)NR ₈ R ₉ , -NHC(O)R ₁₀ or -P(O)R ₈ R ₉ , wherein the alkyl group is optionally further substituted by one or more substituents selected from halogen, amino, hydroxyl, cyano or alkoxy; _R and _R are the same or different, each independently selected from a hydrogen atom, halogen, amino, hydroxyl, cyano, alkoxy, alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl, wherein the alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl or heteroaryl are optionally further substituted by one or more substituents selected from halogen, amino, hydroxyl, cyano or alkoxy; _R is selected from a hydrogen atom, halogen, amino, hydroxyl, cyano, alkoxy, alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl, wherein said alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl or heteroaryl is optionally further substituted by one or more substituents selected from halogen, amino, hydroxyl, cyano or alkoxy; R ₆ and R ₇ are the same or different, each independently selected from a hydrogen atom or an alkyl group; R₈and R₉The same or different, each independently selected from a hydrogen atom, hydroxyl, halogen, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl or heteroaryl, wherein said alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl or heteroaryl is optionally further selected from one or more of hydroxyl, halogen, nitro, cyano, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, =O, -C(O)R₁₁, -C(O)OR₁₁, -OC(O)R₁₁, -NR₁₂R₁₃, -C(O)NR₁₂R₁₃、-SO₂NR₁₂R₁₃or -NR₁₂C(O)R₁₃replaced by substituents; or, R₈and R₉Together with the atoms they are connected to form a 4-8 membered heterocyclic group, wherein the 4-8 membered heterocyclic group contains one or more N, O or S(O)_r, and the 4-8 membered heterocyclic group is optionally further replaced by one or more selected from hydroxyl, halogen, nitro, cyano, amino, alkyl, alkoxy, cycloalkyl, heterocyclic, aryl, heteroaryl, =O, -C(O)R₁₁, -C(O)OR₁₁, -OC(O)R₁₁, -NR₁₂R₁₃, -C(O)NR₁₂R₁₃、-SO₂NR₁₂R₁₃or -NR₁₂C(O)R₁₃replaced by substituents; Each R ₁₀ is independently selected from a hydrogen atom, alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl, wherein said alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl is optionally further replaced by one or more selected from hydroxyl, halogen, nitro, cyano, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, =O, -C(O)R ₁₁ , -C(O)OR ₁₁ , -OC(O)R _{11 , -NR Substituted by 12 R 13 , -C(O)NR 12 R 13 , -SO 2 NR 12 R 13 or -NR 12} C(O)R ₁₃ ; R ₁₁ , R ₁₂ and R ₁₃ are each independently selected from a hydrogen atom, alkyl, amino, cycloalkyl, heterocyclyl, aryl or heteroaryl, wherein said alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl is optionally further substituted by one or more substituents selected from hydroxyl, halogen, nitro, amino, cyano, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, carboxyl or carboxylate; n is 0, 1 or 2; r is 0, 1 or 2. 2. compound according to claim 1 or its stereoisomer, tautomer or its pharmaceutically acceptable salt, it is the compound described in general formula (II) or its stereoisomer, tautomer or its pharmaceutically acceptable salt: Wherein: G, X ₁ ~ X ₄ , R ₂ ~ R ₄ are as defined in claim 1. 3. The compound according to claim 1 or its stereoisomer, tautomer or its pharmaceutically acceptable salt, which is the compound described in general formula (III) or its stereoisomer, tautomer or its pharmaceutically acceptable salt: Wherein: the definitions of R _x , R ₁ , R ₃ and R ₄ are as described in claim 1. 4. The compound according to claim 2 or its stereoisomer, tautomer or pharmaceutically acceptable salt thereof, wherein: selected from: Wherein: the definitions of R _x and R ₁ to R ₄ are as described in claim 2. 5. The compound or its stereoisomer, tautomer or pharmaceutically acceptable salt thereof according to claim 3, wherein: R ₁ is selected from -C(O)OR ₁₀ ; Rx is selected from an alkyl group or -NR ₈ R ₉ , wherein the alkyl group is optionally further substituted by -NR ₈ R ₉ ; R ₈ and R ₉ are each independently selected from a hydrogen atom or an alkyl group; R ₁₀ is selected from alkyl. 6. The compound according to claim 5 or its stereoisomer, tautomer or pharmaceutically acceptable salt thereof, wherein: Rx is selected from -NR ₈ R ₉ or R ₈ is selected from a hydrogen atom; _R9 is selected from alkyl; R ₁₀ is selected from methyl, ethyl or isopropyl. 7. The compound or its stereoisomer, tautomer or pharmaceutically acceptable salt thereof according to claim 4, wherein: R ₁ is selected from a hydrogen atom, an alkoxy group, a cyano group, -SR ₁₀ , -SO ₂ R ₁₀ , -C(O)R ₁₀ or -NHC(O)R ₁₀ ; R ₁₀ is selected from alkyl or cycloalkyl. 8. The compound or its stereoisomer, tautomer or pharmaceutically acceptable salt thereof according to claim 4, wherein: R ₂ is selected from alkyl, -SO ₂ R ₁₀ , -SO ₂ NR ₈ R ₉ , -C(O)R ₁₀ , -C(O)NR ₈ R ₉ or -P(O)R ₈ R ₉ , wherein the alkyl group is optionally further substituted by -NR ₈ R ₉ ; R ₈ and R ₉ are each independently selected from a hydrogen atom or an alkyl group; R ₁₀ is selected from alkyl or cycloalkyl. 9. The compound according to claim 8 or its stereoisomer, tautomer or pharmaceutically acceptable salt thereof, wherein: _R2 is selected from 10. The compound according to claim 8 or its stereoisomer, tautomer or pharmaceutically acceptable salt thereof, wherein: R ₂ is selected from -SO ₂ R ₁₀ , -SO ₂ NR ₈ R ₉ , -C(O)NR ₈ R ₉ , -C(O)R ₁₀ or -P(O)R ₈ R ₉ ; R ₈ and R ₉ are each independently selected from a hydrogen atom, methyl, ethyl or isopropyl; R ₁₀ is selected from methyl, ethyl, isopropyl or cyclopropyl. 11. The compound according to claim 1 or its stereoisomer, tautomer or pharmaceutically acceptable salt thereof, wherein said compound is: 12. A pharmaceutical composition, which contains an effective dose of the compound according to any one of claims 1 to 11 or its stereoisomer, tautomer or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, excipient or their composition. 13. Use of the compound according to any one of claims 1-11 or its stereoisomer, tautomer or pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 12 in the preparation of an HPK1 inhibitor. 14. The compound according to any one of claims 1 to 11 or its stereoisomer, tautomer or pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 12 in the preparation of medicines for treating diseases mediated by HPK1, wherein said diseases mediated by HPK1 are preferably inflammation, autoimmune diseases or tumors, wherein said autoimmune diseases are preferably systemic lupus erythematosus or psoriasis. 15. The use according to claim 14, wherein the tumor is selected from acute myeloid leukemia, bladder cancer, colon cancer, rectal cancer, pancreatic cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, lymphoma, blastoma, retinoblastoma, sarcoma, prostate cancer, bile duct cancer, esophageal cancer, gastric cancer, liver cancer, glioma, cervical cancer, ovarian cancer, head and neck cancer and multiple myeloma. 16. The compound according to any one of claims 1 to 11 or its stereoisomer, tautomer or pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 12 in the preparation of medicines for treating inflammation, autoimmune disease or tumor, wherein said autoimmune disease is preferably systemic lupus erythematosus or psoriasis. 17. The use according to claim 16, wherein the tumor is selected from acute myeloid leukemia, bladder cancer, colon cancer, rectal cancer, pancreatic cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, lymphoma, blastoma, retinoblastoma, sarcoma, prostate cancer, bile duct cancer, esophageal cancer, gastric cancer, liver cancer, glioma, cervical cancer, ovarian cancer, head and neck cancer and multiple myeloma.