CN113354648A

CN113354648A - Novel HPK1 inhibitor and preparation method and application thereof

Info

Publication number: CN113354648A
Application number: CN202010140893.5A
Authority: CN
Inventors: 江磊; 冯志勇; 金贤; 石倩; 寿建勇; 徐圆; 张建华; 赵海霞
Original assignee: Yinuo Zhejiang Pharmaceutical Co ltd; Shanghai Ennovabio Pharmaceuticals Co Ltd
Current assignee: Yinuo Zhejiang Pharmaceutical Co ltd; Shanghai Ennovabio Pharmaceuticals Co Ltd
Priority date: 2020-03-03
Filing date: 2020-03-03
Publication date: 2021-09-07
Also published as: WO2021175270A1; CN115279760A

Abstract

The invention provides an HPK1 kinase inhibitor and a preparation method and application thereof. Specifically, the invention provides a compound shown as a formula I, wherein the definition of each group is described in the specification. The compound has excellent HPK1 inhibition activity, so that the compound can be used for preparing a pharmaceutical composition for treating cancers and other diseases related to HPK1 activity.

Description

Novel HPK1 inhibitor and preparation method and application thereof

Technical Field

The invention relates to the field of small molecule drugs, in particular to a kinase inhibitor and preparation and application thereof.

Background

T cells and B cells and Dendritic Cells (DCs) are powerful weapons of the human immune system for combating foreign invaders such as viruses and bacteria, as well as self-infecting or abnormal cells such as cancerous cells. The human body has a set of complex and precise regulation and control system for ensuring the normal operation of the immune system. When cancerous cells are present in the body, these cancer cells that escape from the immune system proliferate abnormally to form tumors if they are not killed completely by the immune system. Traditional tumor therapy is mainly achieved by surgery, radiotherapy, chemotherapy and molecular targeted drugs. However, for many forms of tumors or cancers, surgical resection is often not a viable option. While radiotherapy and chemotherapy target tumor cells, they can also damage some healthy cells. Tumor cells are prone to mutation and may develop resistance to drugs that specifically target tumor cells, making cancer treatment difficult. In recent years, the immune system of a patient is utilized to overcome the immune escape strategy adopted by tumor cells, and the anti-tumor immunity of the body is enhanced, so that the novel cancer treatment strategy is provided. One strategy is to overcome the immune escape of tumor cells by recognizing tumor antigens as non-self antigens through the inhibition of negative regulators of the immune response that normally serve to maintain peripheral tolerance. The hematopoietic progenitor cell kinase 1(HPK1) is also called MAP4K1(MAP4K family member), is a negative regulator of Dendritic Cell (DCs), T cell and B cell activation response, and can be used for pertinently enhancing the anti-tumor immunity of the organism by inhibiting the activity of the hematopoietic progenitor cell kinase 1. HPK1 is expressed primarily by hematopoietic cells, including early hematopoietic progenitor cells. In T cells, HPK1 is thought to degrade downstream SLP76 protein at Ser376 and Gads protein at Thr254 by phosphorylating these proteins and recruiting 14-3-3 protein to reduce the persistence of signal micro-clusters, playing a role in negatively regulating T cell activation. HPK1 may also be activated in response to prostaglandins (PGE2) normally secreted by tumors, thereby facilitating escape of tumor cells from the immune system. In addition, HPK1 can also inhibit AP-1, and AP-1 plays a role in promoting cell proliferation, inhibiting differentiation, promoting invasion and metastasis of tumor cells and the like in the process of forming and developing tumors. Targeted disruption of the HPK1 kinase allele can allow T cells to increase production of Th1 cytokines (IL-2, IFN γ, etc.) in TCR responses. HPK1 has multiple roles in immunity and is involved in the pathogenesis of autoimmune diseases, cancer and inflammatory responses. The proliferation of HPK1 kinase-/-T cells is much faster than that of the monomeric wild type, and mice transfected with HPK1 kinase-/-T cells are resistant to the growth of cancer tumors, and Dendritic Cells (DCs) without HPK1 kinase have better antigen presenting capability than the wild type and can better show anti-tumor immune response. In addition, animal experiment research shows that the inhibition of HPK1 and the PD-1/PD-L1 antibody medicine have obvious synergistic antitumor activity. Thus, HPK1 kinase plays a key role in the treatment of disease, particularly cancer.

At present, no medicine exists on the market aiming at the target spot, and in order to meet the huge clinical needs in the future, a novel oral medicine is expected to be provided for immune related diseases, particularly tumor treatment by designing and developing a selective and high-activity small molecular HPK1 inhibitor, and the novel oral medicine is singly used or combined with tumor chemotherapy, radiotherapy, tumor targeting medicines, other tumor immunotherapeutic agents (small molecular compounds and antibodies), tumor vaccines and the like.

Disclosure of Invention

The invention aims to provide a small-molecule HPK1 inhibitor with selectivity and high activity.

In a first aspect of the present invention, there is provided a compound represented by the following formula I, or a pharmaceutically acceptable salt, optical isomer or hydrate thereof:

wherein the content of the first and second substances,

x and Y are each independently selected from the group consisting of: none, NR, S, O, -NR-C (═ O) R-, -C (═ O) NR-, -NR-C (═ S) NR-, -NR-C (═ O) NRCH₂-、-NR-C(＝S)NRCH₂-, wherein said R is selected from the group consisting of: H. substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted 3-12 membered heterocyclyl having 1-3 heteroatoms selected from the group consisting of N, S and O;

M¹、M²or M³Each independently is CH or N; when said M is¹Or M²When is CH, the

Can be located at said M¹Or M²Up (i.e. when M is present)¹Or M²Is C);

ring A is selected from the group consisting of: C3-C8 cycloalkyl, 5-12 membered heterocyclyl, 6-10 membered aromatic ring, or 5-10 membered heteroaromatic ring having 1-3 heteroatoms selected from N, S and O, or A is H;

m is 1,2,3 or 4;

n is 0, 1,2,3, 4 or 5;

p is 0, 1 or 2;

ra is selected from the group consisting of: halogen, CN, substituted or unsubstituted C1-C6 alkyl, or

Wherein said ring B is a C3-C8 cycloalkyl, 6-10 membered aryl, 5-10 membered heteroaryl, 3-10 membered heterocyclyl having 1-3 heteroatoms selected from the group consisting of N, S and O;

each R is¹、R²、R³And R⁴Each independently selected from the group consisting of: H. halogen, CN, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted 3-12 membered heterocyclyl having 1-3 heteroatoms selected from the group consisting of N, S and O, substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted 3-10 membered heteroaryl having 1-3 heteroatoms selected from the group consisting of N, S and O, R⁷-C(＝O)-、R⁷-C (═ O) 3-12 membered heterocyclyl-, R having 1-3 heteroatoms selected from the group consisting of N, S and O⁸-S(＝O)₂-、R⁹R¹⁰N-C(＝O)-、R⁹R¹⁰N-C(＝O)NR-、R¹¹R¹²N-S(＝O)₂-、R⁷O-、R⁸-S(O)₂NR-；

Each R is⁷、R⁸、R⁹、R¹⁰、R¹¹、R¹²Each independently selected fromGroup (2): H. substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted 3-12 membered heterocyclyl having 1-3 heteroatoms selected from the group consisting of N, S and O; or said R⁹And R¹⁰Or R¹¹And R¹²Together with the nitrogen atom to which they are attached form a substituted or unsubstituted 3-12 membered heterocyclyl having 1-3 heteroatoms selected from N, S and O;

unless otherwise specified, "substituted" means substituted with one or more (e.g., 2,3, 4, etc.) substituents selected from the group consisting of: halogen, C1-C6 alkoxy, halogenated C1-C6 alkoxy, C3-C8 cycloalkyl, halogenated C3-C8 cycloalkyl, methylsulfonyl, oxo (═ O), -CN, hydroxy, -NH₂、-NHS(O)₂CH₃C1-C6 amino, carboxyl, C1-C6 amido (-C (═ O) -n (rc)₂or-NH-C (═ O) (Rc), Rc being H or C1-C5 alkyl), or a substituted or unsubstituted group selected from: C1-C6 alkyl, C6-C10 aryl, 5-to 10-membered heteroaryl having 1-3 heteroatoms selected from N, S and O, - (CH)₂) -C6-C10 aryl, - (CH)₂) - (3-10 membered heteroaryl having 1-3 heteroatoms selected from N, S and O), - (5-10 membered heteroarylene having 1-3 heteroatoms selected from N, S and O) - (C1-C6 alkyl), 3-12 membered heterocyclyl (including monocyclic, spiro, bridged or fused rings) having 1-3 heteroatoms selected from N, S and O, and said substituents are selected from the group consisting of: halogen, C1-C6 alkyl, C1-C6 alkylene-OH, C1-C6 alkoxy, oxo, -S (O)₂CH₃-CN, -OH, C6-C10 aryl, 3-10 membered heteroaryl having 1-3 heteroatoms selected from N, S and O, -C (O) CHNH₂、-C(O)CHOH；

And in the compound of the formula I, each chiral center is in an R configuration or an S configuration.

In another preferred embodiment, the term "substituted" refers to substitution with one or more (e.g., 2,3, 4, etc.) substituents selected from the group consisting of: halogen, C1-C6 alkoxy, halogenated C1-C6 alkoxy, C3-C8 cycloalkyl, halogenated C3-C8 cycloalkyl, methylsulfonyl, oxo (═ O), -CN, hydroxy, -NH₂C1-C6 amino, carboxyl, or substitutedOr unsubstituted groups selected from: C1-C6 alkyl, C6-C10 aryl, 5-10 membered heteroaryl having 1-3 heteroatoms selected from N, S and O, and said substituents are selected from the group consisting of: halogen, C1-C6 alkyl, C1-C6 alkylene-OH, C1-C6 alkoxy, oxo, -S (O)₂CH₃-CN, -OH, C6-C10 aryl, 3-10 membered heteroaryl having 1-3 heteroatoms selected from N, S and O, -C (O) CHNH₂、-C(O)CHOH。

In another preferred embodiment, the a ring is selected from the group consisting of:

wherein the content of the first and second substances,

z is selected from the group consisting of: NH, O or S;

q is 0, 1 or 2;

re and Rf are each independently selected from the group consisting of: H. CN, halogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, phenyl, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted 3-12 membered heterocyclyl having 1-3 heteroatoms selected from the group consisting of N, S and O; or said Re and Rf taken together with the atoms to which they are attached form a C3-C8 cycloalkyl, or a substituted or unsubstituted 3-12 membered heterocyclyl having 1-3 heteroatoms selected from N, S and O;

and said

May be further substituted by 1 or 2R²And (4) substitution.

In another preferred embodiment, the a ring is selected from the group consisting of: a C3-C8 cycloalkyl, a phenyl ring, a 5-6 membered heterocyclic ring, or a 5-10 membered heteroaromatic ring having 1-3 heteroatoms selected from N, S and O; and said R is²Selected from the group consisting of: H. halogen, CN, substituted or unsubstituted C1-C6 alkyl.

In another preferred embodiment, X is O.

In another preferred embodiment, Y is NH.

In another preferred embodiment, Y is-NH-C (O) NHCH₂-。

In another preferred embodiment, Y is-NH-C(S)NHCH₂-。

In another preferred embodiment, the B ring is selected from the group consisting of: a benzene ring, a 3-12 membered heterocyclic group, a 5-6 membered heteroaromatic ring, an 8-15 membered bicyclic fused ring containing a benzene ring structural unit, an 8-15 membered bicyclic fused ring containing a 5-6 membered heteroaromatic ring structural unit; wherein, R is⁴Selected from the group consisting of: H. halogen, substituted or unsubstituted C1-C6 alkyl, R⁹R¹⁰N-C(＝O)-、R⁹R¹⁰N-C(＝O)NR-、R¹¹R¹²N-S(＝O)₂-、R⁷O-、R⁸-S(O)₂NR-; wherein said substitution preferably comprises one or more groups selected from the group consisting of: -NH₂Substituted or unsubstituted C1-C6 alkyl.

In another preferred embodiment, the compound of formula I has a structure as shown in formula II-1 or II-2 below:

wherein the content of the first and second substances,

the Ra is independently selected from the group consisting of: halogen, CN, substituted or unsubstituted C1-C6 alkyl;

in another preferred embodiment, the compound of formula I has the structure shown in formula III below:

wherein the content of the first and second substances,

said R⁴Selected from the group consisting of: H. halogen, CN, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, R⁹R¹⁰N-C(＝O)-、R⁸-S(＝O)₂-、R⁹R¹⁰N-C(＝O)-、R⁹R¹⁰N-C(＝O)NR-、R¹¹R¹²N-S(＝O)₂-、R⁷O-、R⁸-S(O)₂NR-; wherein, R is⁷、R⁸、R⁹、R¹⁰、R¹¹、R¹²Each independently selected from the group consisting of: H. substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted 3-12 membered heterocyclyl having 1-3 heteroatoms selected from the group consisting of N, S and O; or said R⁹And R¹⁰Or R¹¹And R¹²Together with the nitrogen atom to which they are attached form a substituted or unsubstituted 3-12 membered heterocyclic group having 1-3 heteroatoms selected from N, S and O.

In another preferred embodiment, the compound of formula I is selected from the group consisting of:

in a second aspect of the present invention, there is provided a pharmaceutical composition comprising (1) a compound according to the first aspect of the present invention or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt, hydrate or solvate thereof; (2) a pharmaceutically acceptable carrier.

In a third aspect of the present invention, there is provided a use of a compound according to the first aspect of the present invention or a stereoisomer or a tautomer thereof, or a pharmaceutically acceptable salt, hydrate or solvate thereof, or a pharmaceutical composition according to the second aspect of the present invention for the preparation of a pharmaceutical composition for the prevention and/or treatment of a disease associated with an activity or an expression amount of HPK1 kinase.

In a fourth aspect of the present invention, there is provided a combined tumor chemotherapy regimen of the compound of the first aspect of the present invention and pharmaceutically acceptable salts, stereoisomers, esters, prodrugs, solvates and deuterated compounds thereof with other tumor immunotherapeutic agents selected from the group consisting of: small molecule compounds and antibodies (including but not limited to PD-1, PD-L1, CTLA-4, TIM-3, TGF- β and its receptors, LAG3 antagonists or TLR4, TLR7, TLR8, TLR9, STING agonists, etc.), radiation therapy regimens, tumor targeting drugs, tumor vaccines.

In another preferred embodiment, the additional tumor immunotherapeutic agent may be administered before, after, or simultaneously with the agent, or may be co-administered with other known therapies. Can also be used as vaccine adjuvant.

In a fifth aspect of the invention, there is provided the use of a compound according to the first aspect of the invention, and pharmaceutically acceptable salts, stereoisomers, esters, prodrugs, solvates and deuterated compounds thereof, in combination with CAR-T immunotherapy for the immunotherapy of cancer.

In another preferred embodiment, the disease includes, but is not limited to, cancer, metastatic cancer, inflammation and autoimmune related diseases.

In another preferred embodiment, the diseases include, but are not limited to: lymphoma, blastoma, medulloblastoma, retinoblastoma, sarcoma, liposarcoma, synovial cell sarcoma, neuroendocrine tumor, carcinoid tumor, gastrinoma, islet cell carcinoma, mesothelioma, schwannoma, acoustic neuroma, meningioma, adenocarcinoma, melanoma, leukemia or lymphoid malignancy, squamous cell carcinoma, epithelial squamous cell carcinoma, lung cancer, small cell lung cancer, non-small cell lung cancer, adenocarcinoma lung cancer, squamous lung cancer, peritoneal cancer, hepatocellular cancer, gastric cancer, intestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, metastatic breast cancer, colon cancer, rectal cancer, colorectal cancer, uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulval cancer, thyroid cancer, anal cancer, penile cancer, merkel cell cancer, esophageal cancer, biliary tract tumor, head and neck cancer, hematologic malignancies, nasopharyngeal carcinoma, multiple myeloma, large villous adenoma, non-hodgkin's lymphoma, bone cancer, testicular cancer, hodgkin's disease, seminoma, oral cancer, brain cancer, skin cancer, ductal breast cancer, renal pelvis cancer, wilms tumor, esophageal adenoma, retinoblastoma, glioma, neurofibroma, gastrointestinal stromal tumor, carcinoma in situ, endometrial carcinoma, myelodysplastic syndrome, and the like.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Detailed Description

The inventors have conducted long-term and intensive studies to design and synthesize a novel HPK1 kinase inhibitor. On this basis, the inventors have completed the present invention.

Term(s) for

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

As used herein, the term "about" when used in reference to a specifically recited value means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

As used herein, the term "comprising" or "includes" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of …," or "consisting of ….

Definition of

As used herein, the term "alkyl" includes straight or branched chain alkyl groups. E.g. C₁-C₈Alkyl represents a straight or branched chain alkyl group having 1 to 8 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, and the like.

As used herein, the term "alkenyl" includes straight or branched chain alkenyl groups. E.g. C₂-C₆Alkenyl means a straight or branched alkenyl group having 2 to 6 carbon atoms, such as vinyl, allyl, 1-propenyl, isopropenyl, 1-butenyl, 2-butenyl, or the like.

As used herein, the term "alkynyl" includesStraight or branched alkynyl. E.g. C₂-C₆Alkynyl means straight or branched chain alkynyl having 2 to 6 carbon atoms, such as ethynyl, propynyl, butynyl, or the like.

As used herein, the term "C₃-C₈Cycloalkyl "refers to cycloalkyl groups having 3 to 8 carbon atoms. It may be a single ring, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or the like. It may also be in the form of a double ring, for example a bridged or spiro ring.

As used herein, the term "C₁-C₈Alkoxy "means a straight or branched chain alkoxy group having 1 to 8 carbon atoms; for example, methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, tert-butoxy and the like.

As used herein, the term "3-12 membered heterocyclyl having 1-3 heteroatoms selected from the group consisting of N, S and O" refers to a saturated or partially saturated cyclic group having 3-12 atoms and wherein 1-3 atoms are heteroatoms selected from the group consisting of N, S and O. It may be monocyclic or may be in the form of a double ring, for example a bridged or spiro ring. Specific examples may be oxetane, azetidine, tetrahydro-2H-pyranyl, piperidinyl, tetrahydrofuranyl, morpholinyl, pyrrolidinyl, and the like.

As used herein, the term "C₆-C₁₀Aryl "means an aryl group having 6 to 10 carbon atoms, for example, phenyl or naphthyl and the like.

As used herein, the term "5-10 membered heteroaryl having 1-3 heteroatoms selected from the group consisting of N, S and O" refers to a cyclic aromatic group having 5-10 atoms and wherein 1-3 atoms are heteroatoms selected from the group consisting of N, S and O. It may be a single ring or a condensed ring form. Specific examples may be pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, pyrrolyl, pyrazolyl, imidazolyl, (1,2,3) -triazolyl, and (1,2,4) -triazolyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, and the like.

Unless otherwise specified, the groups of the present invention may be substituted with a substituent selected from the group consisting of: halogen, nitrile group, nitro group, hydroxyl group, amino group, C₁-C₆Alkyl-amino, C₁-C₆Alkyl radical, C₂-C₆Alkenyl radical, C₂-C₆Alkynyl, C₁-C₆Alkoxy, halo C₁-C₆Alkyl, halo C₂-C₆Alkenyl, halo C₂-C₆Alkynyl, halo C₁-C₆Alkoxy, allyl, benzyl, C₆-C₁₂Aryl radical, C₁-C₆alkoxy-C₁-C₆Alkyl radical, C₁-C₆Alkoxy-carbonyl, phenoxycarbonyl, C₂-C₆Alkynyl-carbonyl, C₂-C₆Alkenyl-carbonyl, C₃-C₆Cycloalkyl-carbonyl, C₁-C₆Alkyl-sulfonyl, and the like.

As used herein, "halogen" or "halogen atom" refers to F, Cl, Br, and I. More preferably, the halogen or halogen atom is selected from F, Cl and Br. "halogenated" means substituted with an atom selected from F, Cl, Br, and I.

Unless otherwise specified, the structural formulae depicted herein are intended to include all isomeric forms (e.g., enantiomers, diastereomers and geometric isomers (or configurational isomers)): for example, R, S configuration containing an asymmetric center, (Z), (E) isomers of double bonds, and the like. Thus, individual stereochemical isomers of the compounds of the present invention or mixtures of enantiomers, diastereomers or geometric isomers (or configurational isomers) thereof are intended to be within the scope of the present invention.

As used herein, the term "tautomer" means that structural isomers having different energies may exceed the low energy barrier, thereby converting with each other. For example, proton tautomers (i.e., proton transmutations) include interconversion by proton shift, such as 1H-indazoles and 2H-indazoles. Valence tautomers include interconversion by recombination of some of the bonding electrons.

As used herein, the term "solvate" refers to a complex of a compound of the present invention coordinated to solvent molecules in a specific ratio.

A compound of formula I

The invention provides a compound shown as the following formula I:

wherein the content of the first and second substances,

x and Y are each independently selected from the group consisting of: none, NR, S, O, -NR-C (═ O) R-, -NR-C (═ O) NR-, -NR-C (═ O) NR-, -NR-C (═ S) NR-, -NR-C (═ O) NRCH₂-、-NR-C(＝S)NRCH₂-, wherein said R is selected from the group consisting of: H. substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted 3-12 membered heterocyclyl having 1-3 heteroatoms selected from the group consisting of N, S and O;

M¹、M²or M³Each independently is CH or N; when said M is¹Or M²When is C, the

Can be located at said M¹Or M²The above step (1);

m is 1,2,3 or 4;

n is 0, 1,2,3, 4 or 5;

p is 0, 1 or 2;

each R is¹、R²、R³And R⁴Each independently selected from the group consisting of: H. halogen, CN, substituted or unsubstituted C1-C6 alkyl, or a salt thereofSubstituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted 3-12 membered heterocyclyl having 1-3 heteroatoms selected from the group consisting of N, S and O, substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted 3-10 membered heteroaryl having 1-3 heteroatoms selected from the group consisting of N, S and O, R⁷-C(＝O)-、R⁷-C (═ O) 3-12 membered heterocyclyl-, R having 1-3 heteroatoms selected from the group consisting of N, S and O⁸-S(＝O)₂-、R⁹R¹⁰N-C(＝O)-、R⁹R¹⁰N-C(＝O)NR-、R¹¹R¹²N-S(＝O)₂-、R⁷O-、R⁸-S(O)₂NR-；

Each R is⁷、R⁸、R⁹、R¹⁰、R¹¹、R¹²Each independently selected from the group consisting of: H. substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted 3-12 membered heterocyclyl having 1-3 heteroatoms selected from the group consisting of N, S and O; or said R⁹And R¹⁰Or R¹¹And R¹²Together with the nitrogen atom to which they are attached form a substituted or unsubstituted 3-12 membered heterocyclyl having 1-3 heteroatoms selected from N, S and O;

unless otherwise specified, "substituted" means substituted with one or more (e.g., 2,3, 4, etc.) substituents selected from the group consisting of: halogen, C1-C6 alkoxy, halogenated C1-C6 alkoxy, C3-C8 cycloalkyl, halogenated C3-C8 cycloalkyl, methylsulfonyl, oxo (═ O), -CN, hydroxy, -NH₂、-NHS(O)₂CH₃C1-C6 amino, carboxyl, C1-C6 amido (-C (═ O) -n (rc)₂or-NH-C (═ O) (Rc), Rc being H or C1-C5 alkyl), or a substituted or unsubstituted group selected from: C1-C6 alkyl, C6-C10 aryl, 5-to 10-membered heteroaryl having 1-3 heteroatoms selected from N, S and O, - (CH)₂) -C6-C10 aryl, - (CH)₂) - (3-10 membered heteroaryl having 1-3 heteroatoms selected from N, S and O), - (5-10 membered heteroarylene having 1-3 heteroatoms selected from N, S and O) - (C1-C6 alkyl), 3-12 membered heteroaryl having 1-3 heteroatoms selected from the group N, S and OA heterocyclic radical (including monocyclic, spiro, bridged or fused rings) and said substituents are selected from the group consisting of: halogen, C1-C6 alkyl, C1-C6 alkylene-OH, C1-C6 alkoxy, oxo, -S (O)₂CH₃-CN, -OH, C6-C10 aryl, 3-10 membered heteroaryl having 1-3 heteroatoms selected from N, S and O, -C (O) CHNH₂、-C(O)CHOH；

In another preferred embodiment, the term "substituted" refers to substitution with one or more (e.g., 2,3, 4, etc.) substituents selected from the group consisting of: halogen, C1-C6 alkoxy, halogenated C1-C6 alkoxy, C3-C8 cycloalkyl, halogenated C3-C8 cycloalkyl, methylsulfonyl, oxo (═ O), -CN, hydroxy, -NH₂C1-C6 amine groups, carboxyl groups, or substituted or unsubstituted groups selected from: C1-C6 alkyl, C6-C10 aryl, 5-10 membered heteroaryl having 1-3 heteroatoms selected from N, S and O, and said substituents are selected from the group consisting of: halogen, C1-C6 alkyl, C1-C6 alkylene-OH, C1-C6 alkoxy, oxo, -S (O)₂CH₃-CN, -OH, C6-C10 aryl, 3-10 membered heteroaryl having 1-3 heteroatoms selected from N, S and O, -C (O) CHNH₂、-C(O)CHOH。

Preferably, said X, Y, A, B, M¹、M²、M³、m、n、p、q、Ra、Re、Rf、R¹、R²、R³、R⁴、R⁷、R⁸、R⁹、R¹⁰、R¹¹、R¹²Each independently is the corresponding group in the specific compound in each example.

The compounds of the invention may be useful as inhibitors of HPK1 kinase, and in a preferred embodiment, are selective inhibitors of HPK1 kinase.

Preparation of Compounds of formula I

The compounds of formula I of the present invention may be prepared by the following exemplary methods:

the method comprises the following steps:

and carrying out substitution and coupling reaction on the quinary-hexabasic compound, and modifying by a substituent group to obtain the compound shown in the formula I.

Compounds in which a is an unsaturated ring can be prepared by method 2:

and (3) carrying out cyclization reaction by using the Ia compound to obtain the compound shown in the formula I'.

Pharmaceutical compositions and methods of administration

The compound of the present invention and various crystal forms, pharmaceutically acceptable inorganic or organic salts, hydrates or solvates thereof, and pharmaceutical compositions containing the compound of the present invention as a main active ingredient are useful for the prevention and/or treatment of diseases (e.g., cancer) associated with the activity or expression level of HPK1 kinase, since the compound of the present invention has excellent inhibitory activity against HPK1 kinase.

The pharmaceutical compositions of the present invention comprise a safe and effective amount of a compound of the present invention in combination with a pharmaceutically acceptable excipient or carrier. Wherein "safe and effective amount" means: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical composition contains 1-2000mg of a compound of the invention per dose, more preferably, 10-200mg of a compound of the invention per dose. Preferably, said "dose" is a capsule or tablet.

"pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances which are suitable for human use and must be of sufficient purity and sufficiently low toxicity. By "compatible" is meant herein that the components of the composition are capable of intermixing with and with the compounds of the present invention without significantly diminishing the efficacy of the compounds. Examples of pharmaceutically acceptable carrier moieties are cellulose and its derivatives (e.g. sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate etc.), gelatin, talc, solid lubricants (e.g. sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate etc.), gelatin, talc, and the likeStearic acid, magnesium stearate), calcium sulfate, vegetable oil (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyol (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifier (such as propylene glycol, glycerin, mannitol, sorbitol, etc.)

) Wetting agents (e.g., sodium lauryl sulfate), coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, and the like.

The mode of administration of the compounds or pharmaceutical compositions of the present invention is not particularly limited, and representative modes of administration include (but are not limited to): oral, parenteral (intravenous, intramuscular or subcutaneous).

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) fillers or extenders, for example, starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants, for example, glycerol; (d) disintegrating agents, for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) slow solvents, such as paraffin; (f) absorption accelerators, e.g., quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glycerol monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage forms may also comprise buffering agents.

Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared using coatings and shells such as enteric coatings and other materials well known in the art. They may contain opacifying agents and the release of the active compound or compounds in such compositions may be delayed in release in a certain part of the digestive tract. Examples of embedding components which can be used are polymeric substances and wax-like substances. If desired, the active compound may also be in microencapsulated form with one or more of the above-mentioned excipients.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly employed in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1, 3-butylene glycol, dimethylformamide and oils, in particular, cottonseed, groundnut, corn germ, olive, castor and sesame oils or mixtures of such materials and the like.

In addition to these inert diluents, the compositions can also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances, and the like.

Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols and suitable mixtures thereof.

The compounds of the present invention may be administered alone or in combination with other pharmaceutically acceptable compounds.

When administered in combination, the pharmaceutical composition further comprises one or more other pharmaceutically acceptable compounds. One or more of the other pharmaceutically acceptable compounds may be administered simultaneously, separately or sequentially with a compound of the invention.

When the pharmaceutical composition is used, a safe and effective amount of the compound of the present invention is suitable for mammals (such as human beings) to be treated, wherein the administration dose is a pharmaceutically-considered effective administration dose, and for a human body with a weight of 60kg, the daily administration dose is usually 1 to 2000mg, preferably 20 to 500 mg. Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are by weight.

Intermediate a1 synthesis:

5-iodo-4-chloro-7H-pyrrolo [2,3-d]Pyrimidines

To 4-chloro-7H-pyrrolo [2,3-d ] at room temperature]To a solution of pyrimidine (10 g, 65.12 mmol) in N, N-dimethylformamide (400 ml) was added N-iodosuccinimide (15.7 g, 69.77 mmol) in portions. The reaction was stirred at rt overnight and the reaction was checked by LCMS. Concentrating the reaction solution to dryness, suspending the residue in 250 ml of 10% sodium sulfite aqueous solution, filtering, recrystallizing the filter cake with ethanol to obtain white solid 5-iodo-4-chloro-7H-pyrrolo [2, 3-d%]Pyrimidine (12.6 g, purity: 90%, yield: 69.3%). MS (ESI) M/z 280.0, 282.0[ M + H ]]⁺.

4-chloro-5-iodo-7- (2-trimethylsilyl-ethoxymethyl) -7H-pyrrolo [2,3-d]Pyrimidines

60% sodium hydride (2.53 g) was added under ice-water bath6.22 mmol) were added portionwise to 5-iodo-4-chloro-7H-pyrrolo [2, 3-d)]Pyrimidine (12.6 g, 45.16 mmol) in N, N-dimethylformamide (120 ml) and the reaction mixture was stirred at 0 ℃ for a further 30 minutes. 2- (trimethylsilyl) ethoxymethyl chloride (9.77 g, 58.71 mmol) in N, N-dimethylformamide (10 ml) was then added dropwise to the reaction mixture, and after addition, the reaction mixture was stirred at room temperature for 1.5 h and the reaction was monitored by LCMS. Pouring the reaction solution into 500 ml of water, extracting with 500 ml of diethyl ether, drying the organic phase with anhydrous sodium sulfate, filtering, concentrating the filtrate, passing the residue through a normal phase silica gel chromatographic column, eluting with a solvent (petroleum ether: ethyl acetate ═ 5:1) to obtain a white solid intermediate 4-chloro-5-iodo-7- (2-trimethylsilyl-ethoxymethyl) -7H-pyrrolo [2,3-d]Pyrimidine (14.0 g, purity: 90%, yield: 75.7%). MS (ESI) M/z 410.1,412.1[ M + H ]]⁺.

Tert-butyl (4- ((5-iodo-7- ((2- (trimethylsilyl) ethoxy) methyl) -7H pyrrolo [2, 3-d)]Pyrimidin-4-yl) Oxy) -3, 5-difluorophenyl) carbamate

To 4-chloro-5-iodo-7- (2-trimethylsilyl-ethoxymethyl) -7H-pyrrolo [2,3-d]To a solution of pyrimidine (2.00 g, 4.88 mmol) and tert-butyl (3, 5-difluoro-4-hydroxyphenyl) carbamate (intermediate B1, 1.26 g, 5.13 mmol) in dimethyl sulfoxide (20 ml) was added potassium carbonate (1.35 g, 9.76 mmol). The mixture was then heated to 110 degrees and stirred for 2 hours. The reaction was checked by LCMS and cooled to room temperature after the reaction was complete. The reaction solution was poured into 60 ml of water, and the mixture was extracted with ethyl acetate (20 ml. times.3). The combined organic phases were washed successively with water (20 ml. times.3), saturated brine (20 ml), dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated under reduced pressure to give the crude product. The crude product was isolated on flash silica gel (petroleum ether: ethyl acetate ═ 5:1) to give tert-butyl (4- ((5-iodo-7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2, 3-d) as a white solid]Pyrimidin-4-yl) oxy) -3,5-difluorophenyl) carbamate (2.43 g, yield: 80.5%). MS (ESI) M/z 619.0[ M + H ]]⁺.

3, 5-difluoro-4- ((5-iodo-7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2, 3-d)] Pyrimidin-4-yl) oxo) anilines

Coupling (4- ((5-iodo-7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2, 3-d)]Pyrimidin-4-yl) oxy) -3, 5-difluorophenyl) carbamate (2.27 g, 3.67 mmol) in 1,1,1,3,3, 3-hexafluoropropane-2-ol solution (30 mL) was added to a 150mL sealed tube. The reaction solution was heated to 110 ℃ and stirred for 1 hour. The reaction was checked by LCMS and after the reaction was complete, the reaction was cooled to room temperature. Concentrating under reduced pressure to obtain crude product, separating with rapid silica gel column (petroleum ether: ethyl acetate: 3:1) to obtain pale yellow 3, 5-difluoro-4- ((5-iodo-7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2, 3-d)]Pyrimidin-4-yl) oxo) aniline (1.6 g, yield: 84.1%). MS (ESI) M/z 618.9[ M + H ]]⁺.

¹H NMR(400MHz,CDCl₃)δ:8.42(s,1H),7.40(s,1H),6.36–6.28(m,2H),5.60(s,2H),3.85(s,2H),3.59–3.50(m,2H),1.26(t,J＝7.1Hz,1H),0.97–0.88(m,2H),-0.04(s,9H).

Intermediate a2 synthesis:

4-chloro-5- (trifluoromethyl) -7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2,3-d] Pyrimidines

Under the protection of nitrogen, the nitrogen is used for protecting the air,to 4-chloro-5-iodo-7- (2-trimethylsilyl-ethoxymethyl) -7H-pyrrolo [2,3-d]To a solution of pyrimidine (1.10 g, 2.68 mmol), methyl 2, 2-difluoro-2- (fluorosulfonyl) acetate in anhydrous N, N-dimethylformamide (10 ml) was added cuprous iodide (1.02 g, 5.37 mmol) as a solid. The reaction solution was heated to 100 ℃ and stirred for 2 hours. The reaction was checked by LCMS and, after the reaction was complete, the reaction was cooled to room temperature. The reaction solution was poured into 30 ml of water, and the mixture was extracted with ethyl acetate (15 ml. times.3). The combined organic phases were washed successively with water (20 ml. times.3), saturated brine (20 ml), dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated under reduced pressure to give the crude product. The crude product was isolated on flash silica gel (petroleum ether: ethyl acetate ═ 6:1) to give 4-chloro-5- (trifluoromethyl) -7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2,3-d ] as a white solid]Pyrimidine (670 mg, purity: 86%, yield: 61%). MS (ESI) 352.0[ M + H ] M/z]⁺.

Tert-butyl (3, 5-difluoro-4- ((5- (trifluoromethyl) -7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pir Pyrrole [2,3-d]Pyrimidin-4-yl) oxy) phenyl) carbamates

To 4-chloro-5- (trifluoromethyl) -7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2,3-d]To a solution of pyrimidine (450 mg, 1.28 mmol) and tert-butyl (3, 5-difluoro-4-hydroxyphenyl) carbamate (intermediate B1, 345 mg, 1.41 mmol) in dimethyl sulfoxide (5.0 ml) was added anhydrous potassium carbonate (353 mg, 2.56 mmol). The mixture was then heated to 110 degrees and stirred for 2 hours. The reaction was checked by LCMS and cooled to room temperature after the reaction was complete. The reaction solution was poured into 20 ml of water, and the mixture was extracted with ethyl acetate (10 ml. times.3). The combined organic phases are dried over anhydrous sodium sulfate, filtered and the filtrate is concentrated under reduced pressure to obtain a crude product. The crude product was isolated on flash silica gel (petroleum ether: ethyl acetate ═ 3:1) to give tert-butyl (3, 5-difluoro-4- ((5- (trifluoromethyl) -7- ((2- (tris) -1) as a white solidMethylsilyl) ethoxy) methyl) -7H-pyrrolo [2,3-d]Pyrimidin-4-yl) oxy) phenyl) carbamate (430 mg, yield: 60%). MS (ESI) M/z 561.1[ M + H ]]⁺.

3, 5-difluoro-4- ((5- (trifluoromethyl) -7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrole And [2,3-d ]]Pyrimidin-4-yl) oxo) anilines

((3, 5-difluoro-4- ((5- (trifluoromethyl) -7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2, 3-d)]Pyrimidin-4-yl) oxy) phenyl) carbamate (330 mg, 0.59 mmol) in 1,1,1,3,3, 3-hexafluoropropane-2-ol (8 ml) was added to 20 ml of microwave. The reaction solution was heated to 110 ℃ and stirred for 1 hour. The reaction was checked by LCMS and after the reaction was complete, the reaction was cooled to room temperature. Concentrating under reduced pressure to obtain crude product, separating with rapid silica gel column (petroleum ether: ethyl acetate: 3:2) to obtain light yellow solid 3, 5-difluoro-4- ((5- (trifluoromethyl) -7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2, 3-d)]Pyrimidin-4-yl) oxo) aniline (252 mg, yield: 92.9%). MS (ESI) M/z 461.0.[ M + H ]]⁺.

¹H NMR(400MHz,CDCl₃)δ:8.52(s,1H),7.67(s,1H),6.33(d,J＝9.1Hz,2H),5.66(s,2H),3.64–3.56(m,2H),0.99–0.90(m,2H),-0.01–-0.04(m,9H).

Intermediate B1 synthesis:

2, 6-difluoro-4-nitrophenol

2, 6-difluorophenol (1.7 g, 13mmol) was dissolved in acetic acid (20 ml) and concentrated nitric acid (1.9 g, 20 mmol) was added dropwise. The reaction solution was reacted at 70 ℃ for 2 hours. The reaction was complete by TLC. The reaction solution was poured into ice water (100 ml), and extracted with ethyl acetate (100 ml × 2). The combined organic phases are washed with saturated salt water, dried over anhydrous sodium sulfate, filtered and the filtrate is concentrated under reduced pressure to obtain a crude product. The crude product was isolated and purified by silica gel column (petroleum ether: ethyl acetate 4:1) to give 2, 6-difluoro-4-nitrophenol (1.57 g, yield: 69%) as a yellow solid.

¹H NMR(400MHz,DMSO-d₆)δ:7.95(dd,J＝6.9,2.2Hz,2H).

Tert-butyl (3, 5-difluoro-4-hydroxyphenyl) carbamate

To a solution of 2, 6-difluoro-4-nitrophenol (1.57 g, 9 mmol) and di-tert-butyl dicarbonate (2.9 g, 13mmol) in methanol (30 ml) was added Pd/C (500 mg) under argon. The reaction solution was replaced with hydrogen three times, and then stirred at room temperature under a hydrogen (15psi) atmosphere for 16 hours. The reaction was complete by TLC. The reaction solution is filtered, and the filtrate is decompressed and concentrated to obtain coarse product. The crude product was isolated and purified by silica gel column (oleyl ether: ethyl acetate ═ 4:1) to give a grey solid (1.3 g, yield: 59%).

¹HNMR(400MHz,DMSO-d₆)δ:9.64(s,1H),9.40(s,1H),7.12(d,J＝9.6Hz,2H),1.46(s,9H).

Example 1: (2- ((3, 5-difluoro-4- ((5- (2-fluoro-4-methoxyphenyl) -7H-pyrrolo [2, 3-d)]Pyrimidine-4- Yl) oxo) phenyl) amino) -5-methyl-5, 6-dihydro-4H-1, 3-oxazin-5-yl) methanol

3, 5-difluoro-4- ((5- (2-fluoro-4-methoxyphenyl) -7- ((2- (trimethylsilyl) ethoxy)) Methyl) -7H-pyrrolo [2,3-d]Pyrimidin-4-yl) oxo) anilines

To 3, 5-difluoro-4- ((5-iodo-7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2, 3-d) under an argon blanket]Pyrimidin-4-yl) oxo) aniline (a1, 400 mg, 0.945 mmol), (2-fluoro-4-methoxyphenyl) boronic acid (178 mg, 1.04 mmol), potassium phosphate (401 mg, 1.89 mmol), and 1, 4-dioxane (6.0 ml)/water (2.0 ml) was added to a mixture of [1,1' -bis (diphenylphosphino) ferrocene]Palladium dichloride dichloromethane complex (76 mg, 0.094 mmol). The reaction solution was heated to 100 ℃ and stirred for 2 hours. The reaction was checked by LCMS. After the reaction solution was cooled to room temperature, ethyl acetate (10 ml. times.2) was added and extracted. Drying the combined organic phase with anhydrous sodium sulfate, filtering, concentrating the filtrate under reduced pressure to obtain crude product, separating the crude product with flash silica gel column (petroleum ether: ethyl acetate ═ 2:1) to obtain 3, 5-difluoro-4- ((5- (2-fluoro-4-methoxyphenyl) -7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2,3-d ] pyrrole]Pyrimidin-4-yl) oxo) aniline (439 mg, yield: 83.9%). MS (ESI) M/z 517.1[ M + H ]]⁺.

1- (3, 5-difluoro-4- ((5- (2-fluoro-4-methoxyphenyl) -7- ((2- (trimethylsilyl) ethoxy) methyl) Yl) -7H-pyrrolo [2,3-d]Pyrimidin-4-yl) oxo) phenyl) -3- ((3-methyloxetan-3-yl) methyl) urea

To a 50ml round bottom flask equipped with a magnetic stirrer was added 3, 5-difluoro-4- ((5- (2-fluoro-4-methoxyphenyl) -7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2, 3-d)]Pyrimidin-4-yl) oxo) aniline (102 mg, 0.197 mmol) and triethylamine (80 mg, 0.79 mmol) in dry dichloromethane (3.0 ml) was replaced with argon three timesThen, the reaction solution was cooled in an ice bath. Triphosgene (41 mg, 0.138 mmol) was then added to the reaction solution, and the reaction solution was stirred at 0 ℃ for 20 minutes. Then, (3-methyloxetan-3-yl) methylamine (120 mg, 1.18 mmol) was added to the reaction solution, the ice bath was removed, and the reaction solution was stirred at room temperature for 30 minutes. Reaction was checked by LCMS. Concentrated under reduced pressure to give an oil. Separating the oily substance with reverse C-18 silica gel column (HCOOH) to obtain white solid 1- (3, 5-difluoro-4- ((5- (2-fluoro-4-methoxyphenyl) -7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2, 3-d)]Pyrimidin-4-yl) oxo) phenyl) -3- ((3-methyloxetan-3-yl) methyl) urea (78 mg, 61.4% yield). MS (ESI) M/z 644.2[ M + H ]]⁺.

(2- ((3, 5-difluoro-4- ((5- (2-fluoro-4-methoxyphenyl) -7H-pyrrolo [2, 3-d)]Pyrimidin-4-yl) oxo) Phenyl) amino) -5-methyl-5, 6-dihydro-4H-1, 3-oxazin-5-yl) methanol

To 1- (3, 5-difluoro-4- ((5- (2-fluoro-4-methoxyphenyl) -7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2, 3-d) at 0 degrees]Pyrimidin-4-yl) oxo) phenyl) -3- ((3-methyloxetan-3-yl) methyl) urea (78 mg, 0.12 mmol) in anhydrous dichloromethane (3.0 ml) was added trifluoroacetic acid (1.0 ml). The ice bath was removed and the reaction was stirred at room temperature for 4 hours. The oily residue of the solution was concentrated under reduced pressure, and after adding anhydrous methanol (4.0 ml) and cooling to 0 degree with an ice bath, anhydrous potassium carbonate was added to the reaction solution to adjust pH to 10-11. Starting a stirrer, stirring for 30 minutes, filtering, and concentrating the filtrate under reduced pressure to obtain a crude product. Separating the crude product with reverse C-18 silica gel column (NH4HCO3) to obtain white solid (2- ((3, 5-difluoro-4- ((5- (2-fluoro-4-methoxyphenyl) -7H-pyrrolo [2, 3-d)]Pyrimidin-4-yl) oxo) phenyl) amino) -5-methyl-5, 6-dihydro-4H-1, 3-oxazin-5-yl) methanol (51 mg, yield: 82%). MS (ESI) M/z 514.0.[ M + H ]]⁺.

¹H NMR(400MHz,CD₃OD)δ8.25(s,1H),7.57(t,J＝8.8Hz,1H),7.45(d,J＝1.6Hz,1H),6.93(s,2H),6.80–6.74(m,2H),4.16(dd,J＝10.6,1.6Hz,1H),3.94(d,J＝10.6Hz,1H),3.81(s,3H),3.52(d,J＝11.2Hz,1H),3.43(d,J＝11.2Hz,1H),3.25(d,J＝13.6Hz,1H),3.04(d,J＝13.6Hz,1H),1.01(s,3H).

Example 2- ((3, 5-difluoro-4- ((5- (2-fluoro-4-methoxyphenyl) -7H-pyrrolo [2, 3-d)]Pyrimidine-4- Yl) oxo) phenyl) amino) -5-fluoro-5, 6-dihydro-4H-1, 3-oxazin-5-yl) methanol

1- (3, 5-difluoro-4- ((5- (2-fluoro-4-methoxyphenyl) -7- ((2- (trimethylsilyl) ethoxy) methyl) Yl) -7H-pyrrolo [2,3-d]Pyrimidin-4-yl) oxo) phenyl) -3- ((3-fluorooxetan-3-yl) methyl) urea

To a 50ml round bottom flask equipped with a magnetic stirrer was added 3, 5-difluoro-4- ((5- (2-fluoro-4-methoxyphenyl) -7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2, 3-d)]Pyrimidin-4-yl) oxo) aniline (100 mg, 0.193 mmol) and triethylamine (79 mg, 0.77 mmol) in dry dichloromethane (3.0 ml) were replaced three times with argon, and the reaction was then cooled in an ice bath. Triphosgene (40 mg, 0.135 mmol) was then added to the reaction and the reaction was stirred at 0 ℃ for 20 minutes. Then, (3-fluorooxetan-3-yl) methylamine (102 mg, 0.969 mmol) was added to the reaction solution, the ice bath was removed, and the reaction solution was stirred at room temperature for 30 minutes. Reaction was checked by LCMS. Concentrated under reduced pressure to give an oil. Separating the oily substance with reverse C-18 silica gel column (HCOOH) to obtain white solid 1- (3, 5-difluoro-4- ((5- (2-fluoro-4-methoxyphenyl) -7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2, 3-d)]Pyrimidin-4-yl) oxo) phenyl) -3- ((3-fluorooxetan-3-yl) methyl) urea (80 mg, yield: 63.8％)。MS(ESI):m/z＝648.1[M+H]⁺.

(2- ((3, 5-difluoro-4- ((5- (2-fluoro-4-methoxyphenyl) -7H-pyrrolo [2, 3-d)]Pyrimidin-4-yl) oxo) Phenyl) amino) -5-fluoro-5, 6-dihydro-4H-1, 3-oxazin-5-yl) methanol

With 1- (3, 5-difluoro-4- ((5- (2-fluoro-4-methoxyphenyl) -7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2, 3-d)]Pyrimidin-4-yl) oxo) phenyl) -3- ((3-fluorooxetan-3-yl) methyl) urea (80 mg, 0.123 mmol) as starting material with reference to (2- ((3, 5-difluoro-4- ((5- (2-fluoro-4-methoxyphenyl) -7H-pyrrolo [2, 3-d)]Synthesis of pyrimidin-4-yl) oxo) phenyl) amino) -5-methyl-5, 6-dihydro-4H-1, 3-oxazin-5-yl methanol (example ENN0885) (2- ((3, 5-difluoro-4- ((5- (2-fluoro-4-methoxyphenyl) -7H-pyrrolo [2, 3-d)]Pyrimidin-4-yl) oxo) phenyl) amino) -5-fluoro-5, 6-dihydro-4H-1, 3-oxazin-5-yl) methanol (54 mg, yield: 84.5%). MS (ESI) M/z 518.2[ M + H ]]⁺.

¹H NMR(400MHz,CD₃OD)δ:8.26(d,J＝5.0Hz,1H),7.56(dd,J＝11.1,6.6Hz,1H),7.44(d,J＝1.4Hz,1H),7.14(s,2H),6.79–6.72(m,2H),4.34–4.21(m,2H),3.79(d,J＝5.7Hz,3H),3.76–3.44(m,5H).

Example 3N- (3, 5-difluoro-4- ((3- (2-fluoro-4-methoxyphenyl) -1H-pyrrolo [2, 3-b)]Pyridine-4- Yl) oxo) phenyl) -5-oxa-7-azaspiro [2.5]Oct-6-en-6-amines

1- (3, 5-difluoro-4- ((5- (2-fluoro-4-methoxyphenyl) -7- ((2- (trimethylsilyl) ethoxy) methyl) Yl) -7H-pyrrolo [2,3-d]Pyrimidin-4-yl) oxo) phenyl) -3- ((1- (hydroxymethyl) cyclopropyl) methyl) thiourea

To a 10 ml round bottom flask equipped with a magnetic stirrer was added 3, 5-difluoro-4- ((5- (2-fluoro-4-methoxyphenyl) -7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2,3-d ] at room temperature]Pyrimidin-4-yl) oxo) aniline (150 mg, 0.29 mmol) was dissolved in anhydrous dichloromethane (3.0 ml) and 1,1' -thiocarbonylbis (pyridin-2 (1H) -one) (74 mg, 0.32 mmol) was added, argon was substituted three times, and the reaction was stirred in a 40-degree oil bath for 1 hour. After complete conversion of the starting material, the reaction was cooled to room temperature and then (1- (aminomethyl) cyclopropyl) methanol (59 mg, 0.58 mmol) was added. The reaction was stirred at room temperature for 3 hours. The reaction was checked by LCMS. Concentrating the solvent under reduced pressure to obtain oily crude product, and separating with reverse C-18 silica gel column (HCOOH) to obtain white solid 1- (3, 5-difluoro-4- ((5- (2-fluoro-4-methoxyphenyl) -7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2, 3-d)]Pyrimidin-4-yl) oxo) phenyl) -3- ((1- (hydroxymethyl) cyclopropyl) methyl) thiourea (180 mg, yield: 94%). MS (ESI) M/z 660.1[ M + H ]]⁺.

N- (3, 5-difluoro-4- ((3- (2-fluoro-4-methoxyphenyl) -1- ((2- (trimethylsilyl) ethoxy) methyl) 1H-pyrrolo [2,3-b ] yl]Pyridin-4-yl) oxo) phenyl) -5-oxa-7-azaspiro [2.5]Oct-6-en-6-amines

To a 10 ml flask equipped with a magnetic stirrer was added in sequence 1- (3, 5-difluoro-4- ((5- (2-fluoro-4-methoxyphenyl) -7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2, 3-d)]Pyrimidin-4-yl) oxo) phenyl) -3- ((1- (hydroxymethyl) cyclopropyl) methyl) thiourea (80 mg, 0.12 mmol) and triethylamine (37 mg, 0.36 mmol) and anhydrous dichloromethane (1.5 ml) were replaced with argon three times, and the reaction was cooled in an ice-water bath 10After a minute, 3- (((ethylimino) methylene) amino) -N, N-dimethylpropan-1-amine hydrochloride (47 mg, 0.24 mmol) was then added to the reaction. The ice bath was removed and the reaction was stirred at room temperature for 24 hours. Reaction was checked by LCMS. Concentrating the solvent under reduced pressure to obtain oily crude product, and separating with reverse C-18 silica gel column (HCOOH) to obtain white solid N- (3, 5-difluoro-4- ((3- (2-fluoro-4-methoxyphenyl) -1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrrolo [2, 3-b)]Pyridin-4-yl) oxo) phenyl) -5-oxa-7-azaspiro [2.5]Oct-6-en-6-amine (24 mg, yield: 32%). MS (ESI) M/z 626.1[ M + H ]]⁺.

N- (3, 5-difluoro-4- ((3- (2-fluoro-4-methoxyphenyl) -1H-pyrrolo [2, 3-b)]Pyridin-4-yl) oxy) benzene Yl) -5-oxa-7-azaspiro [2.5]Oct-6-en-6-amines

With N- (3, 5-difluoro-4- ((3- (2-fluoro-4-methoxyphenyl) -1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrrolo [2, 3-b)]Pyridin-4-yl) oxo) phenyl) -5-oxa-7-azaspiro [2.5]Oct-6-en-6-amine (24 mg, 0.038 mmol) as raw material reference (2- ((3, 5-difluoro-4- ((5- (2-fluoro-4-methoxyphenyl) -7H-pyrrolo [2, 3-d)]Synthesis of pyrimidin-4-yl) oxo) phenyl) amino) -5-methyl-5, 6-dihydro-4H-1, 3-oxazin-5-yl methanol (example ENN0885) N- (3, 5-difluoro-4- ((3- (2-fluoro-4-methoxyphenyl) -1H-pyrrolo [2, 3-b)]Pyridin-4-yl) oxo) phenyl) -5-oxa-7-azaspiro [2.5]Oct-6-en-6-amine (3.6 mg, yield: 19%). MS (ESI) M/z 496.1[ M + H ]]⁺.

¹H NMR(400MHz,CD₃OD)δ:8.25(s,1H),7.57(t,J＝8.8Hz,1H),7.45(d,J＝1.4Hz,1H),6.96(d,J＝10.4Hz,2H),6.80–6.73(m,2H),4.08(s,2H),3.81(s,3H),3.22(s,2H),0.74–0.64(m,4H).

Example 4- (3, 5-difluoro-4- ((5- (2-fluoro-4-methoxyphenyl) -7H-pyrrolo [2, 3-d)]Pyrimidine-4- Oxy) phenyl) -3- ((1- (hydroxymethyl) cyclopropyl) methyl) thiourea

With 1- (3, 5-difluoro-4- ((5- (2-fluoro-4-methoxyphenyl) -7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2, 3-d)]Pyrimidin-4-yl) oxo) phenyl) -3- ((1- (hydroxymethyl) cyclopropyl) methyl) thiourea (20 mg, 0.030 mmol) as starting material reference (2- ((3, 5-difluoro-4- ((5- (2-fluoro-4-methoxyphenyl) -7H-pyrrolo [2, 3-d)]Synthesis of pyrimidin-4-yl) oxo) phenyl) amino) -5-methyl-5, 6-dihydro-4H-1, 3-oxazin-5-yl methanol (example ENN0885) 1- (3, 5-difluoro-4- ((5- (2-fluoro-4-methoxyphenyl) -7H-pyrrolo [2, 3-d)]Pyrimidin-4-yl) oxo) phenyl) -3- ((1- (hydroxymethyl) cyclopropyl) methyl) thiourea (2.5 mg, yield: 15.6%). MS (ESI) 530.0[ M + H ]]⁺.

¹H NMR(400MHz,CD₃OD)δ8.27(s,1H),7.57(t,J＝8.8Hz,1H),7.47(s,1H),7.32(d,J＝9.5Hz,2H),6.82–6.73(m,2H),3.81(s,3H),3.65(s,2H),3.45(s,2H),0.59(t,J＝5.1Hz,2H),0.51(t,J＝5.0Hz,2H).

Example 5 (2- ((3, 5-difluoro-4- ((5- (trifluoromethyl) -7H-pyrrolo [2, 3-d)]Pyrimidin-4-yl) oxy Substituted) phenyl) amino) -substituted ketone5-fluoro-5, 6-dihydro-4H-1, 3-oxazin-5-yl) methanol

1- (3, 5-difluoro-4- ((5- (trifluoromethyl) -7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyri-dine Pyrrolo [2,3-d]Pyrimidin-4-yl) oxo) phenyl) -3- ((3-fluorooxetan-3-yl) methyl) urea

With 3, 5-difluoro-4- ((5- (trifluoromethyl) -7- ((2- (trimethylsilyl) ethoxy)Methyl) -7H-pyrrolo [2,3-d]Pyrimidin-4-yl) oxo) aniline (77 mg, 0.167 mmol) and (3-fluorooxetan-3-yl) methylamine (88 mg, 0.836 mmol) as starting materials reference compound 1- (3, 5-difluoro-4- ((5- (2-fluoro-4-methoxyphenyl) -7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2, 3-d)]METHOD FOR THE SYNTHESIS OF 1- (3, 5-DIFLUORO-4- ((5- (TRIFLUOROMETHYL) -7- ((2- (TRIFLUOROMETHYL) ETHOXY) METHYL) -7H-PYRROLO [2, 3-D) UREA (COMPOUND 2-1)]Pyrimidin-4-yl) oxo) phenyl) -3- ((3-fluorooxetan-3-yl) methyl) urea (65 mg, yield: 65.7%) MS (ESI) M/z 592.1[ M + H ]]⁺.

(2- ((3, 5-difluoro-4- ((5- (trifluoromethyl) -7H-pyrrolo [2, 3-d)]Pyrimidin-4-yl) oxo) phenyl) amino Yl) -5-fluoro-5, 6-dihydro-4H-1, 3-oxazin-5-yl) methanol

To 1- (3, 5-difluoro-4- ((5- (trifluoromethyl) -7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2, 3-d)]Pyrimidin-4-yl) oxo) phenyl) -3- ((3-fluorooxetan-3-yl) methyl) urea (65 mg, 0.11 mmol) as starting material reference (2- ((3, 5-difluoro-4- ((5- (2-fluoro-4-methoxyphenyl) -7H-pyrrolo [2, 3-d)]Synthesis of pyrimidin-4-yl) oxo) phenyl) amino) -5-methyl-5, 6-dihydro-4H-1, 3-oxazin-5-yl methanol (example ENN0885) (2- ((3, 5-difluoro-4- ((5- (trifluoromethyl) -7H-pyrrolo [2, 3-d)]Pyrimidin-4-yl) oxo) phenyl) amino) -5-fluoro-5, 6-dihydro-4H-1, 3-oxazin-5-yl) methanol (29 mg, yield: 57.2%). MS (ESI) M/z 462.0[ M + H ]]⁺.

¹H NMR(400MHz,CD₃OD)δ:8.35(s,1H),7.87(d,J＝1.2Hz,1H),7.25–7.10(m,2H),4.29(d,J＝29.6Hz,2H),3.74–3.48(m,4H).

Example 6: n- (3, 5-difluoro-4- ((5- (trifluoromethyl) -7H-pyrrolo [2, 3-d)]Pyrimidin-4-yl) oxo) Phenyl) -5-oxa-7-azaspiro [2.5]Oct-6-en-6-amines

1- (3, 5-difluoro-4- ((5- (trifluoromethyl) -7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyri-dine Pyrrolo [2,3-d]Pyrimidin-4-yl) oxo) phenyl) -3- ((1- (hydroxymethyl) cyclopropyl) methyl) thiourea

To a 10 ml round bottom flask equipped with a magnetic stirrer was added 3, 5-difluoro-4- ((5- (trifluoromethyl) -7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2,3-d ] at room temperature]Pyrimidin-4-yl) oxo) aniline (252 mg, 0.55 mmol) was dissolved in anhydrous dichloromethane (4.0 ml) and 1,1' -thiocarbonylbis (pyridin-2 (1H) -one) (140 mg, 0.60 mmol) was added, argon was substituted three times, and the reaction was stirred in a 40-degree oil bath for 1 hour. After complete conversion of the starting material, the reaction was cooled to room temperature and then (1- (aminomethyl) cyclopropyl) methanol (111 mg, 1.09 mmol) was added. The reaction was stirred at room temperature for 1 hour. The reaction was checked by LCMS. Concentrating the solvent under reduced pressure to obtain oily crude product, and separating with reverse C-18 silica gel column (HCOOH) to obtain white solid 1- (3, 5-difluoro-4- ((5- (trifluoromethyl) -7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2, 3-d)]Pyrimidin-4-yl) oxo) phenyl) -3- ((1- (hydroxymethyl) cyclopropyl) methyl) thiourea (292 mg, yield: 88.4%). MS (ESI) M/z 604.0[ M + H ]]⁺.

N- (3, 5-difluoro-4- ((3- (trifluoromethyl) -1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyri-dine Pyrrolo [2,3-b]Pyridin-4-yl) oxo) phenyl) -5-oxa-7-azaspiro [2.5]Oct-6-en-6-amines

To a 10 ml flask equipped with a magnetic stirrer was added in sequence 1- (3, 5-difluoro-4- ((5- (trifluoromethyl) -7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2, 3-d)]Pyrimidin-4-yl) oxo) phenyl) -3- ((1- (hydroxymethyl) cyclopropyl) methyl) thiourea (292 mg, 0.483 mmol) and triethylamine (196 mg, 1.93 mmol) and anhydrous dichloromethane (5.0 ml), after three times replacement with argon, the reaction was cooled in an ice-water bath for 10 minutes, and then 3- (((ethylimino) methylene) amino) -N, N-dimethylpropan-1-amine hydrochloric acid (185 mg, 0.967 mmol) was added to the reaction. The ice bath was removed and the reaction was stirred at room temperature for 24 hours. Reaction was checked by LCMS. Concentrating the solvent under reduced pressure to obtain oily crude product, and separating with reverse C-18 silica gel column (HCOOH) to obtain white solid N- (3, 5-difluoro-4- ((3- (trifluoromethyl) -1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrrolo [2, 3-b)]Pyridin-4-yl) oxo) phenyl) -5-oxa-7-azaspiro [2.5]Oct-6-en-6-amine (150 mg, yield: 54.4%). MS (ESI) M/z 570.0[ M + H ]]⁺.

N- (3, 5-difluoro-4- ((3- (trifluoromethyl) -1H-pyrrolo [2, 3-b)]Pyridin-4-yl) oxo) phenyl) -5- Oxa-7-azaspiro [2.5 ]]Oct-6-en-6-amines

With N- (3, 5-difluoro-4- ((3- (trifluoromethyl) -1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrrolo [2, 3-b)]Pyridin-4-yl) oxo) phenyl) -5-oxa-7-azaspiro [2.5]Oct-6-en-6-amine (150 mg, 0.263 mmol) as raw material reference (2- ((3, 5-difluoro-4- ((5- (2-fluoro-4-methoxyphenyl) -7H-pyrrolo [2, 3-d)]Synthesis of pyrimidin-4-yl) oxo) phenyl) amino) -5-methyl-5, 6-dihydro-4H-1, 3-oxazin-5-yl methanol (example ENN0885) Synthesis of N- (3, 5-difluoro-4- ((3- (trifluoromethyl) -1H-pyrrolo [2, 3-b)]Pyridin-4-yl) oxo) phenyl) -5-oxa-7-azaspiro [2.5]Oct-6-en-6-amine (45 mg, yield: 38.9%). MS (ESI) 440.0[ M + H ] M/z]⁺.

¹H NMR(400MHz,CD₃OD)δ:8.29(s,1H),7.86(d,J＝1.2Hz,1H),7.16(d,J＝8.3Hz,2H),4.43(s,2H),3.31(s,2H),0.82(d,J＝14.8Hz,4H).

The following compounds were obtained in a similar manner to example 1, substituting the corresponding starting materials.

Biological test example 1HPK1 ADP-Glo enzymatic Activity test

Preparing enzyme activity test buffer containing 40mM Tris, pH7.5; 20mM MgCl 2; 0.1mg/ml BSA; 50 μ M DTT. Compounds were dissolved in 100% DMSO and the stock concentration was 10 mM. The DMSO solution of the compound is continuously diluted in a gradient manner by three times from 100uM to eleven concentrations, after the diluted compound is diluted by enzyme activity test buffer solution in a ratio of 1:20, 1ul of the diluted compound is added into a working hole, and each concentration is two times of the working hole. Negative control wells and positive control wells were added 1ul of 1:20 diluted DMSO solution. 2.5 Xsubstrate/ATP working solution was prepared as enzyme activity assay buffer containing 0.25ug/ul MBP protein and 45uM ATP, 2ul of 2.5 Xsubstrate/ATP working solution was added to each well. 2.5 Xenzyme reaction working solution is prepared as enzyme activity test buffer solution containing 0.5ng/ul HPK1 recombinant protein (Signalchem, cat # M23-11G-10), 2ul 2.5 Xenzyme reaction working solution is added into each working well, and only 2ul enzyme activity test buffer solution is added into a negative control well. After a sealing plate film is pasted, the reaction solution is placed at room temperature for 30 minutes after simple centrifugation, 5ul of ADP-Glo reagent (Promega, product number V1901) is added into each hole after the reaction is finished, the reaction solution is reacted for 40 minutes at room temperature, 10ul of kinase detection reagent (Promega, product number V1901) is added subsequently, and the final luminescence signal is measured after the reaction is carried out for 20 minutes at room temperature.

The average values of the positive and negative wells were calculated as positive control values (Signal)_pos) And negative control value (Signal)_neg). The working well Signal value (Signal)_test) According to the formula (Signal)_pos-Signal_test)/(Signal_pos–Signal_neg) X 100% the inhibition was calculated. The obtained inhibition rate is subjected to nonlinear fitting in GraphPad Prism software to draw a concentration-inhibition rate curve, and IC is calculated₅₀。

Biological test example 2GLK ADP-Glo enzymatic Activity test

Preparing enzyme activity test buffer containing 40mM Tris, pH7.5; 20mM MgCl 2; 0.1mg/ml BSA; 50 μ M DTT. Compounds were dissolved in 100% DMSO and the stock concentration was 10 mM. The DMSO solution of the compound is continuously diluted by three times of gradient from 1mM to eleven concentrations, after the diluted compound is diluted by enzyme activity test buffer solution in a ratio of 1:20, 1ul of the diluted compound is added into a working hole, and each concentration is two times of the working hole. Negative control wells and positive control wells were added 1ul of 1:20 diluted DMSO solution. 2.5 Xsubstrate/ATP working solution was prepared as enzyme activity assay buffer containing 0.5ug/ul PKA substrate polypeptide and 105uM ATP, 2ul of 2.5 Xsubstrate/ATP working solution was added to each well. 2.5 Xenzyme reaction working solution is prepared as enzyme activity test buffer solution containing 2.5ng/ul GLK recombinant protein (Signalchem, product number M25-11G-10), 2ul 2.5 Xenzyme reaction working solution is added into each working well, and only 2ul enzyme activity test buffer solution is added into a negative control well. After a sealing plate film is pasted, the reaction solution is placed at room temperature for 1 hour after simple centrifugation, 5ul of ADP-Glo reagent (Promega, product number V1901) is added into each hole after the reaction is finished, the reaction solution is reacted for 40 minutes at room temperature, 10ul of kinase detection reagent (Promega, product number V1901) is added subsequently, and the final luminescence signal is measured after the reaction for 20 minutes at room temperature.

The average values of the positive and negative wells were calculated as positive control values (Signal)_pos) And negative control value (Signal)_neg). The working well Signal value (Signal)_test) According to the formula (Signal)_pos-Signal_test)/(Signal_pos–Signal_neg) X 100% the inhibition was calculated. The obtained inhibition rate is subjected to nonlinear fitting in GraphPad Prism software to draw a concentration-inhibition rate curve, and IC is calculated₅₀. The results of the experiment are shown in the following table:

TABLE 1HPK1, GLK enzyme activity test results

Wherein A represents IC₅₀The value is less than or equal to 50 nM; b denotes 50nM < IC₅₀The value is less than or equal to 500 nM; c denotes 500nM < IC₅₀The value is less than or equal to 10 uM.

Biological test example 3SLP76 Phosphorylcytology test

Inhibition of HPK1 inhibited phosphorylation of SLP76 downstream thereof. Phosphorylation of SLP76 protein Jurkat (ATCC, Clone E6-1) was used

TIB-152^TM) Cells were tested by diluting the cells to 10 with culture medium (RPMI 1640+ 0.5% FBS) the first day of the experiment⁶Per ml, 100ul per well, 10⁵The amount of cells was plated in 96-well cell culture plates and starved overnight. Compounds were dissolved in 100% DMSO and the stock concentration was 10 mM. The DMSO solution of the compound is diluted by four times of gradient from 10mM to eight concentrations, 10ul of the diluted compound is diluted into 40ul of RPMI 1640 and mixed evenly, 10ul of the diluted compound is added into 240ul of RPMI 1640 preheated at 37 ℃ and mixed evenly. 50ul of the final diluted compound was added to the cells and incubated at 37 ℃ for 20 minutes, and 50ul of the diluted human CD3/CD 28T cell activator (Stemcell, cat # 10971) was added to make the final concentration volume of activator 1/40 and incubated at 37 ℃ for 30 minutes. After the reaction, the cells were centrifuged at 1200rpm in a 4-degree centrifuge for 5 minutes, the medium was aspirated off, 120. mu.l of Cell lysate (supplied from ELISA kit, Cell Signaling, cat # 25424) was added and left on ice for 30 minutes to lyse the cells sufficiently, the lysed cells were spun down uniformly and then centrifuged at 4000rpm in a 4-degree centrifuge for 10 minutes, and 50. mu.l of the supernatant was added to FastScan^TMPhospho-SLP-76(Ser376) ELISA Kit (Cell Signaling, cat # 25424) tested the phosphorylation level of cellular SLP 76. In addition, 25ul of the supernatant was added to Pierce BCA Protein Assay Kit (Pierce, cat # 23227) to test for total Protein concentration instead of total SLP-76 Protein level. Dividing the reading value of phosphorylated SLP-76 by the reading value of total protein concentration to obtain the Ratio of phosphorylated SLP-76 to total protein concentration Ratio_testAnd calculating a positive control Ratio_max(does not containTest compound plus cell activator only) and negative control Ratio_min(without test compound and without cell activator) and the reading was converted to Inhibition rate (%) (Ratio) by the following equation_max-Ratio_test)/(Ratio_max-Ratio_min) X 100%. The obtained inhibition rate is subjected to nonlinear fitting in GraphPad Prism software to draw a concentration-inhibition rate curve, and IC is calculated₅₀. The results of the experiment are shown in the following table:

TABLE 2 SLP76 phosphorylation inhibition assay results

Example numbering	IC50(nM)
		1	A
2	A
		3	A
4	B
		5	B
6	B
		7	A
8	A
		9	B
11	B

Wherein A represents IC₅₀The value is less than or equal to 1000 nM; b denotes 1000nM < IC₅₀The value is less than or equal to 20000nM

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims

1. A compound represented by formula I below, or a pharmaceutically acceptable salt, optical isomer, or hydrate thereof;

wherein the content of the first and second substances,

Can be located at said M¹Or M²Upper (i.e., M)¹Or M²Is C);

m is 1,2,3 or 4;

n is 0, 1,2,3, 4 or 5;

p is 0, 1 or 2;

Each R is⁷、R⁸、R⁹、R¹⁰、R¹¹、R¹²Each independently selected from the group consisting of: H. substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C3-C8 cycloalkaneA substituted or unsubstituted 3-12 membered heterocyclic group having 1-3 heteroatoms selected from the group consisting of N, S and O; or said R⁹And R¹⁰Or R¹¹And R¹²Together with the nitrogen atom to which they are attached form a substituted or unsubstituted 3-12 membered heterocyclyl having 1-3 heteroatoms selected from N, S and O;

2. The compound of claim 1, or a pharmaceutically acceptable salt, optical isomer, or hydrate thereof, wherein ring a is selected from the group consisting of:

wherein the content of the first and second substances,

z is selected from the group consisting of: NH, O or S;

q is 0, 1 or 2;

and said

May be further substituted by 1 or 2R²And (4) substitution.

3. The compound of claim 1, or a pharmaceutically acceptable salt, optical isomer, or hydrate thereof, wherein ring a is selected from the group consisting of: a C3-C8 cycloalkyl, a phenyl ring, a 5-6 membered heterocyclic ring, or a 5-10 membered heteroaromatic ring having 1-3 heteroatoms selected from N, S and O; and said R is²Selected from the group consisting of: H. halogen, CN, substituted or unsubstituted C1-C6 alkyl.

4. The compound of claim 1, or a pharmaceutically acceptable salt, optical isomer, or hydrate thereof, wherein ring B is selected from the group consisting of: a benzene ring, a 3-12 membered heterocyclic group, a 5-6 membered heteroaromatic ring, an 8-15 membered bicyclic fused ring containing a benzene ring structural unit, an 8-15 membered bicyclic fused ring containing a 5-6 membered heteroaromatic ring structural unit; wherein, R is⁴Selected from the group consisting of: H. halogen, substituted or unsubstituted C1-C6 alkyl, R⁹R¹⁰N-C(＝O)-、R⁹R¹⁰N-C(＝O)NR-、R¹¹R¹²N-S(＝O)₂-、R⁷O-、R⁸-S(O)₂NR-; wherein said substitution preferably comprises one or more groups selected from the group consisting of: -NH₂Substituted or unsubstituted C1-C6 alkyl.

5. The compound of claim 1, or a pharmaceutically acceptable salt, optical isomer, or hydrate thereof, wherein the compound of formula I has a structure as shown in formula II-1 or II-2 below:

wherein the content of the first and second substances,

each Ra is independently selected from the group consisting of: halogen, CN, substituted or unsubstituted C1-C6 alkyl.

6. The compound of claim 5, or a pharmaceutically acceptable salt, optical isomer, or hydrate thereof, wherein the compound of formula I has the structure shown in formula III below:

wherein the content of the first and second substances,

7. The compound of claim 1, or a pharmaceutically acceptable salt, optical isomer, or hydrate thereof, wherein the compound of formula I is selected from the group consisting of:

8. a pharmaceutical composition comprising (1) a compound of claim 1 or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt, hydrate, or solvate thereof; (2) a pharmaceutically acceptable carrier.

9. Use of a compound according to claim 1 or a stereoisomer or a tautomer thereof, or a pharmaceutically acceptable salt, hydrate or solvate thereof, or a pharmaceutical composition according to claim 8, for the preparation of a pharmaceutical composition for the prophylaxis and/or treatment of a disease associated with the activity or expression of HPK1 kinase.

10. The use according to claim 9, wherein the disease is selected from the group consisting of: lymphoma, blastoma, medulloblastoma, retinoblastoma, sarcoma, liposarcoma, synovial cell sarcoma, neuroendocrine tumor, carcinoid tumor, gastrinoma, islet cell carcinoma, mesothelioma, schwannoma, acoustic neuroma, meningioma, adenocarcinoma, melanoma, leukemia or lymphoid malignancy, squamous cell carcinoma, epithelial squamous cell carcinoma, lung cancer, small cell lung cancer, non-small cell lung cancer, adenocarcinoma lung cancer, squamous lung cancer, peritoneal cancer, hepatocellular cancer, gastric cancer, intestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, metastatic breast cancer, colon cancer, rectal cancer, colorectal cancer, uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulval cancer, thyroid cancer, anal cancer, penile cancer, merkel cell cancer, esophageal cancer, biliary tract tumor, head and neck cancer, hematologic malignancies, nasopharyngeal carcinoma, multiple myeloma, large villous adenoma, non-hodgkin's lymphoma, bone cancer, testicular cancer, hodgkin's disease, seminoma, oral cancer, brain cancer, skin cancer, ductal breast cancer, renal pelvis cancer, wilms tumor, esophageal adenoma, retinoblastoma, glioma, neurofibroma, gastrointestinal stromal tumor, carcinoma in situ, endometrial carcinoma, myelodysplastic syndrome, and the like.

11. The compound of claim 1, and pharmaceutically acceptable salts, stereoisomers, esters, prodrugs, solvates and deuterated compounds thereof, in combination with a tumor chemotherapeutic regimen of another tumor immunotherapeutic agent selected from the group consisting of: small molecule compounds and antibodies (including but not limited to PD-1, PD-L1, CTLA-4, TIM-3, TGF- β and its receptors, LAG3 antagonists or TLR4, TLR7, TLR8, TLR9, STING agonists, etc.), radiation therapy regimens, tumor targeting drugs, tumor vaccines.

12. The use of a compound of claim 1, and pharmaceutically acceptable salts, stereoisomers, esters, prodrugs, solvates, and deuterated compounds thereof, in combination with CAR-T immunotherapy in cancer immunotherapy.