CN115746026A

CN115746026A - Pyrrolopyrimidine derivatives, and preparation method and application thereof

Info

Publication number: CN115746026A
Application number: CN202111027020.4A
Authority: CN
Inventors: 江磊; 程战领; 冯志勇; 金贤; 刘胜洋; 聂士常; 石倩; 寿建勇; 汪兵; 许灵灵; 徐圆; 张建华; 张学标; 张毅; 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: 2021-09-02
Filing date: 2021-09-02
Publication date: 2023-03-07

Abstract

The invention provides a pyrrolopyrimidine derivative 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

Pyrrolopyrimidine derivatives, 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, 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. A complex and precise regulation and control system is arranged in a human body to ensure the normal operation of an 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 therapy 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 cancer treatment strategy is novel. 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. Hematopoietic progenitor cell kinase 1 (HPK 1), also known as MAP4K1 (a member of the MAP4K family), 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 negative regulator. 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 proteins, thereby reducing the persistence of signal nanoclusters and acting as a negative regulator of T cell activation. HPK1 can also be activated in response to prostaglandins (PGE 2), which are normally secreted by tumors, helping tumor cells to escape from the immune system. And HPK1 can also inhibit AP-1, and AP-1 plays a role in the processes of promoting cell proliferation, inhibiting differentiation, promoting invasion and metastasis of tumor cells and the like in the processes 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 knockout (HPK 1-/-) T cells is much faster than that of monomeric wild type, and mice transfected with HPK1-/-T cells can resist the growth of tumors, and Dendritic Cells (DCs) without HPK1 kinase have better antigen presentation capability and can better show anti-tumor immune response compared with wild type. In addition, animal experiment research shows that the inhibition of HPK1 and PD-1/PD-L1 antibody medicine have obvious synergistic antitumor activity. Thus, HPK1 kinase plays a key role in the treatment of diseases, in particular 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 small molecule HPK1 inhibitor with selectivity and high activity is expected to be developed through design, so that a novel oral medicine is provided for immune related diseases, particularly tumor treatment, and single medicine or combined chemotherapy, radiotherapy, tumor targeting medicine, other tumor immunotherapeutic agents (small molecule compounds and antibodies), tumor vaccines and the like are provided.

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 formula I below, or a pharmaceutically acceptable salt, an optical isomer or a hydrate thereof;

wherein,

m is 1,2,3 or 4;

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

p is 0, 1 or 2;

x is N or CH, when a substituent is attached to X, then X is C;

R ² selected from the group consisting of: halogen, CN, substituted or unsubstituted C1-C6 alkoxy, R ⁹ R ¹⁰ N-C(＝O)-、R ⁹ R ¹⁰ N-C(＝O)(CH ₂ ) _r O-、R ⁷ O-、R ⁸ C(＝O)N(R ⁹ ) -; wherein, R is ⁷ 、R ⁸ 、R ⁹ 、R ¹⁰ Each independently selected from the group consisting of: H. substituted or notSubstituted 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; or said R ⁸ And R ⁹ Or R ⁹ And R ¹⁰ Together 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; r is 0, 1 or 2;

each R is ¹ 、R ³ 、R ⁴ And R ⁵ Each independently selected from the group consisting of: H. halogen, OH, 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, R ⁷ -C(＝O)-、R ⁷ -C (= O) 3-12 membered heterocyclyl-, R having 1-3 heteroatoms selected from the group consisting of N, S and O ⁹ R ¹⁰ N-C(＝O)-、R ⁷ 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, -NH ₂ 、-NHS(O) ₂ CH ₃ C1-C6 amino group, carboxyl group, C1-C6 amido group (-C (= O) -N (Rc) ₂ or-NH-C (= O) (Rc), which is 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 substituent is selected from the group consisting of: halogen, C1-C6 alkyl, C1-C6 alkylene-OH, C1-C6 alkoxy, oxo, -S (O) ₂ CH ₃ CN, -OH, C6-C10 aryl, 3-10 membered heteroaryl having 1-3 heteroatoms selected from N, S and O, -C (O) CHNH ₂ -C (O) CHOH; with the proviso that said compound of formula I is a chemically stable structure;

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

and said compound is not a compound selected from the group consisting of:

in another preferred embodiment, m is 1 or 2; n is 0, 1 or 2; p is 0 or 1; r is ³ Is H.

In another preferred embodiment, R ² Selected from the group consisting of: halogen, CN, R ⁹ R ¹⁰ N-C(＝O)-、R ⁹ R ¹⁰ N-C(＝O)(CH ₂ ) _r O-、R ⁹ R ¹⁰ N-C(＝O)NR-、R ⁷ O-、R ⁸ C(＝O)N(R ⁹ ) -; wherein, R is ⁷ 、R ⁸ 、R ⁹ 、R ¹⁰ 、R ¹¹ 、R ¹² Each independently selected from the group consisting of: H. substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted 3-8 membered heterocyclyl having 1-3 heteroatoms selected from the group consisting of N, S and O; or said R ⁹ And R ¹⁰ Or R ¹¹ And R ¹² Together with the nitrogen atom to which they are attached form a substituted or unsubstituted 3-8 membered heterocyclic group having 1-3 heteroatoms selected from N, S and O.

In another preferred embodiment, R ² Selected from the group consisting of: halogen, CN, R ⁹ R ¹⁰ N-C(＝O)-、R ⁹ R ¹⁰ N-C(＝O)(CH ₂ ) _r O-、R ⁷ O-、R ⁸ C(＝O)N(R ⁹ ) -; wherein, R is ⁷ 、R ⁸ 、R ⁹ 、R ¹⁰ Each independently selected from the group consisting of: H. substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted C1-C3 heteroatom selected from the group consisting of N, S and O4-6 membered heterocyclic group of (a); or said R ⁸ And R ⁹ Or R ⁹ And R ¹⁰ Together with the nitrogen atom to which they are attached form a substituted or unsubstituted 4-6 membered heterocyclyl having 1-3 heteroatoms selected from N, S and O.

In another preferred embodiment, each R ¹ 、R ⁴ And R ⁵ Each independently selected from the group consisting of: H. halogen, CN, substituted or unsubstituted C1-C6 alkoxy.

In another preferred embodiment, the compound has the structure shown in formula II below:

in another preferred embodiment, the compound has the structure shown as follows:

in another preferred embodiment, the compound is selected from the group consisting of:

in another preferred embodiment, the compound is not a compound as found in PCT/CN 2021/078965. In a second aspect of the present invention, there is provided a pharmaceutical composition comprising (1) a compound according to claim 1, 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 the 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 the 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 level 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.), chemotherapy regimens, radiotherapy regimens, tumor targeting drugs, tumor vaccines, etc.

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 carcinoma, 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 carcinoma, kidney cancer, prostate cancer, vulval cancer, thyroid cancer, anal cancer, penile carcinoma, merkel cell carcinoma, esophageal cancer, biliary tract tumor, head and neck cancer, hematological malignancy, nasopharyngeal carcinoma, multiple myeloma, villous adenoma, non-hodgkin lymphoma, osteocarcinoma, testicular cancer, seminoma, carcinoma of the cell, oral carcinoma, squamous cell carcinoma, renal cell carcinoma, neuroblastoma, renal cell carcinoma in situ adenocarcinomas of the head and fibroadenoma.

In a fifth aspect of the invention, there is provided the use of a compound of 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.

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 repeated herein, depending on the space.

Detailed Description

The present inventors have conducted long and intensive studies to design and synthesize a novel class of HPK1 kinase inhibitors. 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 5% from the recited value. For example, as used herein, the expression "about 100" includes all values between 95 and 105 and (e.g., 95.1, 95.2, 95.3, 95.4, 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 823030A ".

Definition of

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

As used herein, the term "alkenyl" includes straight or branched chain alkenyl groups. E.g. C ₂ -C ₆ The alkenyl group means a straight-chain 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" includes straight or branched chain alkynyl groups. Such as C ₂ -C ₆ Alkynyl 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 "C ₃ -C ₈ Cycloalkyl "refers to cycloalkyl groups having 3 to 8 carbon atoms. It may be a monocyclic 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 "C ₁ -C ₈ Alkoxy "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 heterocyclic group having 1-3 heteroatoms selected from the following groups of N, S and O" refers to a saturated or partially saturated cyclic group having 3-12 atoms and in which 1-3 atoms are heteroatoms selected from the following groups 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 "C ₆ -C ₁₀ Aryl "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 group having 1-3 heteroatoms selected from the following groups N, S and O" refers to a cyclic aromatic group having 5-10 atoms, wherein 1-3 atoms are heteroatoms selected from the following groups 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 substituents selected from the group consisting of: halogen, nitrile group, nitro group, hydroxyl group, amino group, C ₁ -C ₆ Alkyl-amino, C ₁ -C ₆ Alkyl radical, C ₂ -C ₆ Alkenyl radical, C ₂ -C ₆ Alkynyl, C ₁ -C ₆ Alkoxy, halo C ₁ -C ₆ Alkyl, halo C ₂ -C ₆ Alkenyl, halo C ₂ -C ₆ Alkynyl, halo C ₁ -C ₆ Alkoxy, allyl, benzyl, C ₆ -C ₁₂ Aryl radical, C ₁ -C ₆ alkoxy-C ₁ -C ₆ Alkyl radical, C ₁ -C ₆ Alkoxy-carbonyl, phenoxycarbonyl, C ₂ -C ₆ Alkynyl-carbonyl, C ₂ -C ₆ Alkenyl-carbonyl, C ₃ -C ₆ Cycloalkyl-carbonyl, C ₁ -C ₆ Alkyl-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, the R and S configuration containing asymmetric center, the (Z) and (E) isomers of double bond, etc. Thus, individual stereochemical isomers of the compounds of the present invention or mixtures of enantiomers, diastereomers or geometric isomers (or configurational isomers) thereof are 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 shifts) include interconversion by proton shifts, 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 compound of the present invention coordinated to solvent molecules to form a complex in a specified ratio.

As used herein, the partial letter abbreviations are defined as follows:

"THF" refers to tetrahydroxyfurans.

"EtOAc" refers to ethyl acetate.

"MeOH" refers to methanol.

"DCM" refers to dichloromethane.

"DMF" refers to N, N-dimethylformamide.

"TFA" refers to trifluoroacetic acid.

"DMSO" refers to dimethyl sulfoxide.

"H ₂ O "means water.

"MeCN" refers to acetonitrile.

"DMA" refers to N, N-dimethylacetamide.

"DCE" refers to 1, 2-dichloroethane.

"DIPEA" refers to N, N-diisopropylethylamine.

"Pd ₂ (dba) ₃ "refers to tris (dibenzylideneacetone) dipalladium (0).

"HATU" refers to N, N, N ', N' -tetramethyl-O- (7-azabenzotriazol-1-yl) hexafluorophosphate urea.

A compound of formula I

The invention provides a compound shown as the following formula I, or pharmaceutically acceptable salt, optical isomer or hydrate thereof;

wherein,

m is 1,2,3 or 4;

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

p is 0, 1 or 2;

x is N or CH, when a substituent is attached to X, then X is C;

R ² selected from the group consisting of: halogen, CN, substituted or unsubstituted C1-C6 alkoxy, R ⁹ R ¹⁰ N-C(＝O)-、R ⁹ R ¹⁰ N-C(＝O)(CH ₂ ) _r O-、R ⁷ O-、R ⁸ C(＝O)N(R ⁹ ) -; wherein, R is ⁷ 、R ⁸ 、R ⁹ 、R ¹⁰ Each independently 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; or said R ⁸ And R ⁹ Or R ⁹ And R ¹⁰ Together 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; r is 0, 1 or 2;

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, -NH ₂ 、-NHS(O) ₂ CH ₃ C1-C6 amino group, carboxyl group, C1-C6 amido group (-C (= O) -N (Rc) ₂ or-NH-C (= O) (Rc), rc being H or C1-C5Alkyl), 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 substituent is selected from the group consisting of: halogen, C1-C6 alkyl, C1-C6 alkylene-OH, C1-C6 alkoxy, oxo, -S (O) ₂ CH ₃ -CN, -OH, C6-C10 aryl, 3-10 membered heteroaryl having 1-3 heteroatoms selected from N, S and O, -C (O) CHNH ₂ -C (O) CHOH; with the proviso that said compound of formula I is a chemically stable structure;

and said compound is not a compound selected from the group consisting of:

preferably, m, n, p, X, R are ¹ 、R ² 、R ³ 、R ⁴ 、R ⁷ 、R ⁸ 、R ⁹ 、R ¹⁰ 、R ¹¹ 、R ¹² Each 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 preferred embodiments 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 by using a five-membered and six-membered compound, and modifying by using a substituent group to obtain the compound shown in the formula I.

The method 2 comprises the following steps:

the method 3 comprises the following steps:

pharmaceutical compositions and methods of administration

Since the compound of the present invention has excellent inhibitory activity against HPK1 kinase, the compound of the present invention and various crystalline 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 HPK1 kinase activity or expression level.

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 to 2000 mg of a compound of the invention per dose, more preferably, 10 to 200 mg 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. hard gelatinFatty acid, magnesium stearate), calcium sulfate, vegetable oil (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyol (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifier (such as propylene glycol, glycerol, 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) injection, and the like.

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 a composition 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, especially 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.

In the case of pharmaceutical compositions, a safe and effective amount of a compound of the present invention is administered to a mammal (e.g., a human) in need of treatment, wherein the administration is a pharmaceutically acceptable and effective dose, and the daily dose for a human of 60kg body weight is usually 1 to 2000 mg, 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. The percentage and parts of each column chromatography in the purification method are calculated by volume. The percentages and parts are by weight unless otherwise indicated.

In each example: analytical method I

LCMS instrument: waters Acquity UPLC-MS, UV detector: acquity UPLC

And (3) chromatographic column: acquity UPLC HSS T3.8 uM, column temperature 40 DEG C

Mobile phase: a: H2O (0.1% tfa or 0.1% ammonium bicarbonate), B: acetonitrile, gradient elution.

The test materials and reagents used in the following examples are commercially available without specific reference.

Synthesis of an intermediate A1:

synthesis of Compound A1-1:

to a solution of 4-chloro-7H-pyrrolo [2,3-d ] pyrimidine (100g, 650mmol) in N, N-dimethylformamide (500 mL) was added N-iodosuccinimide (160g, 716mmol) in portions at room temperature. The reaction was stirred at 15 ℃ for 10 min and tested by LCMS for complete conversion of starting material. Water (1.2L) was slowly added to the reaction solution, and the mixture was slurried and filtered to obtain a crude product. Dissolving the crude product in ethyl acetate, drying with anhydrous sodium sulfate, filtering, and concentrating the filtrate under reduced pressure to obtain residue. To the residue was added petroleum ether (500 mL), slurried and purified, and filtered to give a brown solid product A1-1 (206 g, yield: 100%).

MS(ESI):m/z＝280.0,282.0[M+H] ⁺ .

¹ H NMR(400MHz,CD ₃ OD)δ8.57-8.53(m,1H),7.72(s,1H)

Synthesis of Compound A1-2

A solution of Compound A1-1 (151g, 540 mmol) in THF (1.5L) was cooled to 0 deg.C in an ice-salt bath. Under nitrogen protection, 60% sodium hydride solid (32g, 810mmol) was added in portions to the reaction solution, stirred for 10 minutes, and then 2- (trimethylsilyl) ethoxymethyl chloride (99g, 594mmol) was slowly added dropwise. After the addition was complete, the reaction mixture was stirred at 0 ℃ for 2 hours and the conversion of starting material was complete by LCMS. The reaction mixture was quenched by pouring it into 1.5L of water, and extracted with ethyl acetate (500 mL. Times.3). The combined organic phases were washed with saturated brine (1L), dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated under reduced pressure to give the crude product. To the crude product was added absolute ethanol (500 mL), and the mixture was heated to 70 ℃ in an oil bath and stirred for 30 minutes until the solid was completely dissolved, cooled, and recrystallized to give the title compound A1-2 (100 g, yield: 45.2%) as a white solid.

MS(ESI):m/z＝410.1,412.1[M+H] ⁺ .

¹ H NMR(400MHz,CDCl ₃ )δ8.65(s,1H),7.54(s,1H),5.62(s,2H),3.54-3.50(m,2H),0.94-0.90(m,2H),-0.04(s,9H).

Synthesis of Compound A1

To compound A1-2 (5.0 g,12.2 mmol) in DMSO (40 mL) at room temperature was added K in this order ₂ CO ₃ (6.74g, 48.8mmol) and Compound B1 (2.12g, 14.6mmol) were replaced with nitrogen three times, and then the reaction mixture was heated to 90 ℃ and stirred for 2 hours, and the reaction was examined by LCMS. After the reaction mixture was cooled to room temperature, the reaction mixture was added to water (400 mL) and extracted with ethyl acetate (100 mL. Times.2). The combined organic phases were washed successively with water (100 mL. Times.2) and saturated brine (100 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 and purified by silica gel column (PE: EA = 3) to give a brown oily product (3.34 g, yield: 52.8%).

ESI-MS m/z＝519.0[M+H] ⁺ .

¹ H NMR(400MHz,CDCL ₃ )δ8.42(s,1H),7.40(s,1H),6.36-6.32(m,2H),5.60(s,2H),3.86-.62(m,2H),3.56-3.52(m,2H),0.94-0.90(m,2H),-0.04(s,9H).

Synthesis of an intermediate A2:

synthesis of Compound A2-1:

to a solution of the compound 3,4, 5-trifluoronitrobenzene (100g, 565mmol) in N, N-dimethylformamide (0.5L) was added potassium carbonate (156g, 1.13mmol) in portions with stirring, and then to the reaction mixture was added 4-methoxybenzyl alcohol (93.5g, 678mmol). After nitrogen displacement, the reaction mixture was stirred at 40 ℃ for 13 hours and the reaction was checked by HPLC. The reaction solution was filtered, and the cake was washed with ethyl acetate (1L), to which water (1L) was added for extraction. The organic phase was washed with saturated brine (800 mL. Times.4), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the crude product. The crude product was slurried with petroleum ether (200 mL) and filtered to give the title compound A2-1 (109 g, purity: 90%, yield: 65%) as a white solid.

¹ H NMR(400MHz,CDCl ₃ )δ7.88–7.73(m,2H),7.37–7.31(m,2H),6.90–6.85(m,2H),5.29(s,2H),3.80(s,3H).

Synthesis of Compound A2-2:

in a 500mL three-necked flask equipped with a mechanical stirrer, compound A2-1 (40g, 135.5 mmol), iron powder (38g, 677.4 mmol) and ammonium chloride (36.2g, 677.4 mmol) were dissolved in ethanol/water (200 mL/65 mL), and after nitrogen substitution, the reaction mixture was stirred at 65 ℃ for 3 hours, and the reaction was examined by LC-MS. After the raw materials are completely converted, the reaction solution is cooled to room temperature and then filtered, a filter cake is washed by ethanol (500 mL), and a crude product is obtained by decompressing and concentrating the filtrate. The crude product was purified via column chromatography (petroleum ether: ethyl acetate = 3) to give a white solid A2-2 (33 g, purity: 90%, yield: 92%).

MS(ESI):m/z＝266.1[M+H] ⁺ .

¹ H NMR(400MHz,DMSO-d ₆ )δ7.28(d,J＝8.0Hz,2H),6.93–6.88(m,2H),6.17(d,J＝8.0Hz,2H),5.38(s,2H),4.83(s,2H),3.75(s,3H).

Synthesis of Compound A2-3:

intermediate A2-2 (20g, 75.47mmol) was dissolved in 100mL of dichloromethane solution, and the raw material 1,1' -thiocarbonylbis (pyridin-2 (1H) -one (19.28g, 83mmol) was added, and the mixture was stirred at 40 ℃ for 1 hour, the reaction was detected by a TLC plate, after the conversion of the raw material was completed, the reaction solution was cooled to room temperature, then DIEA (19.5 g, 151mmol) was slowly dropped into the reaction solution, followed by (1- (aminomethyl) cyclopropyl) methanol (9.16g, 90.6 mmol) being slowly dropped, after the dropping, the mixture was stirred at 30 ℃ for 13 hours, and the reaction solution was detected by LC-MS, and the reaction solution was concentrated under reduced pressure to obtain a crude product, which was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 1) to obtain red oily compound A2-3 (19 g, purity: 90%, yield: 62%).

MS(ESI):m/z＝409.3[M+H] ⁺ .

¹ H NMR(400MHz,DMSO-d ₆ )δ9.76(s,1H),7.94(s,1H),7.33(m,4H),6.93(d,J＝8.0Hz,2H),5.02(s,2H),4.77(s,1H),3.75(s,3H),3.48(s,2H),3.31(d,J＝5.2Hz,2H),0.44(m,4H).

Synthesis of Compounds A2-4:

to the compound A2-3 (1- (3, 5-difluoro-4- ((4-methoxybenzyl) oxy) phenyl) -3-

To a solution of ((1- (hydroxymethyl) cyclopropyl) methyl) thiourea (16.0 g,39.2 mmol) and N, N-diisopropylethylamine (16.2 mL,97.9 mmol) in dichloromethane (200 mL) was added methyl iodide (19.5 mL, 313mmol) slowly. The reaction mixture was stirred at 40 ℃ for 16 hours and the reaction was checked by LC-MS. After the reaction mixture was cooled to room temperature, it was diluted with dichloromethane (200 mL) and washed with saturated brine (100 mL. Times.2). The organic phase is passed through anhydrous Na ₂ SO ₄ Drying, filtration and concentration of the filtrate under reduced pressure gave crude A2-4 (16.0 g) which was used directly in the next step.

MS(ESI):m/z＝423.0[M+H] ⁺ .

Synthesis of Compounds A2-5:

is provided with magnetismIn a 500mL reaction flask with a mechanical stirrer, compound A2-4 (16.0 g,37.9 mmol) and tetrahydrofuran (180 mL) were added in this order, and the stirrer was turned on and ground sodium hydroxide solid (6.06g, 151mmol) was added to the reaction solution. The mixture was stirred at room temperature for 3 hours. After the LC-MS detection reaction is completed, reducing pressure to remove tetrahydrofuran in the reaction liquid to obtain a residue. Ethyl acetate (200 mL) was added to the residue, which was washed with brine (100 mL) and anhydrous Na ₂ SO ₄ Drying, filtering, and concentrating the filtrate under reduced pressure to obtain crude product. The crude product was isolated and purified by column chromatography (dichloromethane: methanol = 0-25) to give the title compound A2-5 (12.0 g, yield: 84.6%).

MS(ESI):m/z＝375.0[M+H] ⁺ .

¹ H NMR(400MHz,CDCl ₃ )δ7.34(d,J＝8.4Hz,2H),6.94-6.79(m,4H),5.01(s,2H),4.07(s,2H),3.80(s,3H),3.30(s,2H),0.78-0.64(m,4H).

Synthesis of compound A2:

to a solution of compound A2-5 (11.8g, 31.5 mmol) in dichloromethane (100 mL) was added trifluoroacetic acid (9.4mL, 126mmol). The reaction mixture was stirred at room temperature for 3 hours and the reaction was checked by LC-MS. After the reaction was complete, the mixture was concentrated under reduced pressure to give a residue, which was then redissolved by the addition of dichloromethane (100 mL) which was saturated NaHCO ₃ Neutralizing with water solution. The mixture was concentrated under reduced pressure to give a residue, which was purified by column chromatography (dichloromethane: methanol = 0-6) to give the title compound A2 (18.5 g, crude containing CF3CO2 Na).

MS(ESI):m/z＝255.2[M+H] ⁺ .

¹ H NMR(400MHz,DMSO-d ₆ )δ10.62(br s,2H),7.15-7.01(m,2H),4.39(s,2H),3.27(s,2H),0.75(brd,J＝3.2Hz,4H).

Example 1: (4- (4- (4- ((5-oxa-7-azaspiro [2.5] oct-6-en-6-yl) amino) -2, 6-difluorophenoxy) -7H-pyrrolo [2,3-d ] pyrimidin-5-yl) phenyl) (3, 3-difluoropyrrolidin-1-yl) methanone

Synthesis of Compound 1-1

1, 4-dioxane (8 mL) and water (2 mL) were added in this order to a mixture of A1 (1036mg, 2.00mmol), (3, 3-difluoropyrrolidin-1-yl) (4- (4, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) phenyl) methanone (876 mg, 2.60mmol), anhydrous potassium phosphate (1065 mg, 5.00mmol) and 1,1' -bis (diphenylphosphino) ferrocene ] dichloropalladium (II) (164mg, 0.20mmol), and after three times of replacement with nitrogen, the reaction was heated to 90 ℃ and stirred for 3 hours. After the reaction solution is cooled to room temperature, the residue is obtained by decompression and concentration. The residue was isolated and purified by silica gel column chromatography (petroleum ether: ethyl acetate = 1) to give the title compound 1-1 (1140 mg, yield: 95%) as a pale yellow solid.

MS(ESI):m/z＝602.2[M+H] ⁺ .

Synthesis of Compound 1-2

To a solution of compound 1-1 (1140mg, 1.89mmol) in dichloromethane (8 mL) at room temperature was added 1,1' -thiocarbonylbis (pyridin-2 (1H) -one (483mg, 2.08mmol), the reaction was stirred at 40 ℃ for 1 hour, the reaction was checked by LC-MS, after completion of the conversion of the raw material, (1- (aminomethyl) cyclopropyl) methanol (382mg, 3.78mmol) was added to the reaction solution, and the reaction mixture was further stirred at room temperature for 1 hour, the solvent was concentrated under reduced pressure to give a residue, and the residue was isolated and purified by reverse phase column chromatography (acetonitrile: aqueous solution of ammonium hydrogencarbonate = 0-4) to give the title compound 1-2 (1020 mg, yield: 72%) as a white solid.

MS(ESI):m/z＝745.2[M+H] ⁺ .

Synthesis of Compounds 1-3

To a solution of compound 1-2 (950mg, 1.28mmol) and triethylamine (89mg, 3.84mmol) in dichloromethane (5 mL) was added 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (491mg, 2.56mmol) in portions under an ice-water bath. After the addition was complete, the reaction was stirred at room temperature for 48 hours. The reaction was checked by LC-MS. After the reaction is finished, the solvent is distilled off under reduced pressure to obtain a crude product. The crude product was isolated and purified by silica gel column chromatography (methanol: dichloromethane = 6) to give the title compound 1-3 (410 mg, yield: 45%) as a white solid.

MS(ESI):m/z＝711.2[M+H] ⁺ .

¹ H NMR(400MHz,CD ₃ OD)δ8.37(s,1H),7.90(s,1H),7.87(s,1H),7.80(s,1H),7.62(s,1H),7.60(s,1H),7.01(s,1H),6.98(s,1H),5.74(s,2H),4.08(s,2H),4.01–3.88(m,2H),3.88–3.78(m,2H),3.65(t,J＝8.0Hz,2H),3.23(s,2H),2.44(s,2H),0.91(t,J＝7.9Hz,2H),0.77–0.62(m,4H),-0.06(s,9H).

Synthesis of example 1

To a solution of compound 1-3 (410mg, 0.58mmol) in dichloromethane (2 mL) at room temperature was added trifluoroacetic acid (2 mL). The reaction was stirred at room temperature for 2 hours and the reaction was checked by LC-MS. The residue was concentrated under reduced pressure, and then dissolved by adding methanol (3 mL). The solution was cooled to 0 ℃ in an ice bath, anhydrous potassium carbonate (321mg, 2.32mmol) was added, and the reaction solution was stirred at room temperature for 2 hours. After the reaction is completed, the reaction solution is concentrated under reduced pressure to obtain a residue. The residue was purified by reverse phase column chromatography (55% acetonitrile in aqueous ammonium bicarbonate) to give example 1 (248 mg, yield: 74%) as a white solid.

MS(ESI):m/z＝581.1[M+H] ⁺ .

¹ H NMR(400MHz,CD ₃ OD)δ8.30(s,1H),7.89(s,1H),7.87(s,1H),7.66(s,1H),7.60(s,1H),7.58(s,1H),7.00(s,1H),6.99(d,J＝10.5Hz,2H),6.97(s,1H),4.06(s,2H),4.01–3.90(m,2H),3.87–3.78(m,2H),3.23(s,2H),2.54–2.36(m,2H),0.73–0.64(m,4H).

Example 2: (4- (4- (4- ((5-oxa-7-azaspiro [2.5] oct-6-en-6-yl) amino) -2, 6-difluorophenoxy) -7H-pyrrolo [2,3-d ] pyrimidin-5-yl) phenyl) (azetidin-1-yl) methanone

Synthesis of Compound 2-1

1, 4-dioxane (40 mL) and water (10 mL) were added in this order to a mixture of A1 (5.18g, 10.00mmol), methyl 4- (4, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) benzoate (2.88g, 11.00mmol), anhydrous potassium phosphate (4.24g, 20.00mmol) and 1,1' -bis (diphenylphosphino) ferrocene ] dichloropalladium (II) (818mg, 1.00mmol), and the reaction was stirred at 90 ℃ for 3 hours under nitrogen. After the reaction was cooled to room temperature, the reaction mixture was concentrated to a residue. The residue was isolated and purified by silica gel column chromatography (petroleum ether: ethyl acetate =2 1) to give the title compound 2-1 (5.26 g, yield: 99%) as a pale yellow solid.

MS(ESI):m/z＝527.2[M+H] ⁺ .

Synthesis of Compound 2-2

To a solution of compound 2-1 (5.26g, 10.00mmol) in dichloromethane (50 mL), 1' -thiocarbonylbis (pyridin-2 (1H) -one (2.55g, 11.0 mmol) was added, the reaction mixture was heated to 40 ℃ and stirred for 1 hour, TLC detection reaction, after complete conversion of the starting material, (1- (aminomethyl) cyclopropyl) methanol (2.02g, 20.0 mmol) was then added to the reaction solution, stirring was continued at room temperature for 1 hour, the reaction was concentrated under reduced pressure to give a residue, which was purified by reverse phase column chromatography (acetonitrile: aqueous ammonium bicarbonate solution = 0-5), to give the title compound 2-2 (6.20 g, yield: 93%) as a white solid.

MS(ESI):m/z＝670.2[M+H] ⁺ .

Synthesis of Compounds 2-3

To a mixture of compound 2-2 (3.45g, 5.15mmol) and triethylamine (1.56g, 15.4 mmol) in dichloromethane (50 mL) was added 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (1.97g, 10.3mmol) in portions under an ice-water bath. The reaction mixture was then stirred at room temperature for 48 hours. After the reaction, the reaction solution was concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography (SiO) ₂ Methanol: dichloromethane =0-7:93 To afford title compound 2-3 (1.45 g, yield: 45%) as a white solid.

MS(ESI):m/z＝636.2[M+H] ⁺ .

Synthesis of Compounds 2-4

Compound methyl 2-3 (1.45g, 2.28mmol) and lithium hydroxide monohydrate (382mg, 9.12mmol) were added to a mixed solution of methanol (20 mL) and water (4 mL), and the reaction solution was stirred at room temperature for 4 hours. The reaction was concentrated under reduced pressure to give the title compound 2-4 (1.49 g, crude) as a white solid.

MS(ESI):m/z＝622.3[M+H] ⁺ 。

Synthesis of Compounds 2 to 5

To a mixture of the compound 2-4 (80mg, 0.13mmol) in dichloromethane (3 mL) were added 2- (7-azobenzotriazol) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (99mg, 0.26mmol) and triethylamine (39mg, 0.39mmol), and the reaction mixture was stirred at room temperature for 0.5 hour. Then, azetidine (15mg, 0.26mmol) was added to the reaction solution, and the reaction solution was stirred at room temperature for 2 hours. After the reaction is finished, the solvent is decompressed and concentrated to obtain a residue. The residue was isolated and purified by silica gel column chromatography (dichloro: methanolic = 0-6) to give the title compound 2-5 (50 mg, yield: 58%) as a pale yellow solid.

MS(ESI):m/z＝661.1[M+H] ⁺ .

Synthesis of example 2

To a solution of compound 2-5 (50mg, 0.076mmol) in dichloromethane (2 mL) was added trifluoroacetic acid (2 mL). The reaction mixture was stirred at room temperature for 3 hours, concentrated under reduced pressure to give a residue, and methanol (3 mL) was added to dissolve the residue. The solution was then cooled to 0 ℃ in an ice bath, and anhydrous potassium carbonate (53mg, 0.38mmol) was added and stirred at room temperature for 1 hour. After the reaction is completed, the reaction solution is concentrated under reduced pressure. The crude product was purified by reverse phase preparative column to give the title compound, example 2 (20mg, 50%) as a white solid.

MS(ESI):m/z＝531.3[M+H] ⁺ .

¹ H NMR(400MHz,CD ₃ OD)δ8.32(s,1H),7.90(s,1H),7.88(s,1H),7.77–7.63(m,3H),7.06-6.95(m,2H),4.45(t,J＝7.4Hz,2H),4.22(t,J＝7.5Hz,2H),4.09(s,2H),3.25(s,2H),2.51–2.29(m,2H),0.86–0.61(m,4H).

Example 3:4- (4- (4- ((5-oxa-7-azaspiro [2.5] oct-6-en-6-yl) amino) -2, 6-difluorophenoxy) -7H-pyrrolo [2,3-d ] pyrimidin-5-yl) -N-cyclopropylbenzamide

Synthesis of Compound 3-1

To a mixture of compound 2-4 (300mg, 0.48mmol) in dichloromethane (8 mL) was added 2- (7-azobenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate (365mg, 0.96mmol) and triethylamine (146mg, 1.44mmol), and the reaction mixture was stirred at room temperature for 0.5 hour. Cyclopropylamine (55mg, 0.96mmol) was then added and the reaction stirred at room temperature for an additional 2 hours. And (5) detecting by LC-MS, and after the reaction is finished, concentrating under reduced pressure to obtain a crude product. The crude product was isolated and purified by silica gel column chromatography (methanol: dichloromethane =6 94) to give the title compound 3-1 (120mg, 38%) as a pale yellow solid.

MS(ESI):m/z＝661.2[M+H] ⁺ .

Synthesis of example 3

To a solution of compound 3-1 (120mg, 0.18mmol) in dichloromethane (2 mL) was added trifluoroacetic acid (2 mL). The reaction was stirred at room temperature for 3 hours, LCMS checked for complete conversion of the starting material, and dichloromethane was evaporated under reduced pressure to give a residue. Methanol (5 mL) was then added to the residue to redissolve, the solution was cooled to 0 ℃ in an ice bath, and anhydrous potassium carbonate (249mg, 1.80mmol) was then added and the reaction stirred at room temperature for 1 hour. And (5) detecting by LC-MS, and after the reaction is finished, concentrating under reduced pressure to obtain a crude product. The crude product was isolated and purified by reverse phase preparative column to give the title compound 3 (45mg, 47%) as a white solid.

MS(ESI):m/z＝531.1[M+H] ⁺ .

¹ H NMR(400MHz,CD ₃ OD)δ8.30(s,1H),7.85–7.81(m,4H),7.68(s,1H),7.26–7.19(m,2H),4.50(s,2H),3.39(s,2H),2.89–2.81(m,1H),0.95–0.86(m,4H),0.84–0.77(m,2H),0.66–0.61(m,2H).

The following compounds were obtained by a method similar to that of example 3, substituting the corresponding starting materials.

Example 13N- (4- ((5- (2, 3-difluoro-4-methoxyphenyl) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) oxo) -3, 5-difluorophenyl) -5-oxa-7-azaspiro [2.5] oct-6-en-6-amine

Synthesis of Compound 13-1

To a solution of the compounds A1-2 (5g, 12.2mmol) and A2 (6g, 24.5mmol) in dimethyl sulfoxide (40 mL) was added cesium carbonate (11.9g, 36.6 mmol), and the reaction was stirred at 90 ℃ for 2 hours under nitrogen protection. The reaction is detected by LC-MS, and after the reaction is finished, the reaction liquid is cooled to room temperature. Then, a saturated ammonium chloride solution (150 mL) was added, and extraction was performed with dichloromethane (50 mL. Times.2). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was separated and purified by flash normal phase column chromatography (petroleum ether: ethyl acetate = 2) to give the title compound 13-1 (2.9 g, yield: 31%) as a yellow solid.

MS(ESI):m/z＝628.1[M+H] ⁺ .

Synthesis of Compound 13-2

To a mixture of compound 13-1 (120mg, 0.19mmol), (2, 3-difluoro-4-methoxyphenyl) boronic acid (54mg, 0.29mmol) and potassium carbonate (122mg, 0.57mmol) in 1, 4-dioxane (1.0 ml) and water (0.3 ml) was added chlorine (2-dicyclohexylphosphino-2 ',4',6 '-triisopropyl-1, 1' -biphenyl) [2- (2 '-amino-1, 1' -biphenyl ] under argon protection]Palladium (II) (15mg, 0.019mmol). The reaction solution was heated to 90 ℃ and stirred for 4 hours. The reaction was checked by LC-MS. After the reaction mixture was cooled to room temperature, ethyl acetate (5 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, and subjecting the crude product to reverse C-18 column chromatography (NH) ₄ HCO ₃ ) Isolation and purification afforded the title compound 13-2 (74 mg, yield: 57 percent) As a white solid.

MS(ESI):m/z＝644.2[M+H] ⁺ .

Synthesis of example 13

To compound 12-2 (74mg, 0.114mmol) in anhydrous dichloromethane (2.0 mL) at 0 deg.C 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, anhydrous methanol (3.0 mL) was added, and after cooling to 0 degree with ice bath, anhydrous potassium carbonate was added to the reaction solution to adjust pH =10-11. Starting a stirrer, stirring for 30 minutes, filtering, and concentrating the filtrate under reduced pressure to obtain a crude product. The crude product is subjected to reverse C-18 column chromatography (NH) ₄ HCO ₃ ) Title compound was isolated as title compound example 13 (25 mg, yield: 42%).

MS(ESI):m/z＝514.1[M+H] ⁺ .

¹ H NMR(400MHz,CD ₃ OD)δ8.28(s,1H),7.53(s,1H),7.38–7.31(m,2H),7.19(m,2H),6.94(s,2H),4.49(s,2H),3.91(s,3H),3.37(s,2H),0.89(m,4H).

The following compounds were obtained by a method similar to that of example 13, 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 Cl2;0.1 mg/ml BSA;50 μ M DTT. The compound was dissolved in 100% DMSO, the concentration of the stock solution was 10mM. The DMSO solution of the compound was serially diluted in a three-fold gradient from 100uM for eleven concentrations, and after the diluted compound was diluted with the enzyme activity test buffer at 1. Negative and positive control wells were added 1uL of 1. 2.5 Xsubstrate/ATP working solution is prepared as enzyme activity test buffer solution containing 0.25ug/uLMBP protein and 45uM ATP, and 2uL 2.5 Xsubstrate/ATP working solution is added into each working hole. 2.5 Xenzyme reaction working solution is prepared to be enzyme activity test buffer solution containing 0.5ng/uL HPK1 recombinant protein (Signalchem, the product number is M23-11G-10), each working well is added with 2uL 2.5 Xenzyme reaction working solution, and negative control wells are only added with 2uL enzyme activity test buffer solution. After a sealing plate film is pasted, the reaction solution is placed at room temperature for 30 minutes after simple centrifugation, 5uL ADP-Glo reagent (Promega, product number V1901) is added into each hole after the reaction is finished, the reaction solution is reacted at room temperature for 40 minutes, then 10uL kinase detection reagent (Promega, product number V1901) is added, and the final luminescence signal is measured after the reaction at room temperature for 20 minutes.

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 Inhibition rate = (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 calculated ₅₀ . The results of the experiment are shown in the following table:

TABLE 1HPK1 enzyme Activity test results

Wherein A represents IC ₅₀ The value is less than or equal to 50nM.

Biological test example 2SLP76 phosphorylation cytology test

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

TIB-152 ^TM ) Cells were tested, and the first day of the experiment cells were diluted to 10% with medium (RPMI 1640+0.5% FBS) ⁶ Per ml, 100uL per well ⁵ The amount of cells was plated in 96-well cell culture plates and starved for 4 hours of culture. Compounds were dissolved in 100% DMSO, the stock concentration was 4mM. Four-fold gradient dilutions of compound in DMSO were performed sequentially from 10mM for 9 concentrations, and 4uL of diluted compound was diluted to 196uL of 37 pre-warmed RPMI 1640 and mixed well. 50uL of the final diluted compound was added to the cells, incubated at 37 ℃ for 20 minutes, and 50uL of the diluted human CD3/CD 28T cell activator (Stemcell, cat # 10971) was added to the cells to give a final concentration of 1/40 of the total system 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, 150uL of Cell lysate (supplied from ELISA kit, cell Signaling, cat. No. 30794C) 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 5 minutes, and 50uL of the supernatant was added to FastScan ^TM Phospho-SLP-76 (Ser 376) ELISA Kit (Cell Signaling, cat # 30794C) tested the level of cellular SLP76 phosphorylation.

The average of the positive and negative wells was calculated as a positive control (Signal) _pos ) And negative control value (Signal) _neg ). The working well Signal value (Signal) _test ) According to the formula Inhibition rate = (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 calculated ₅₀ . The results of the experiment are shown in the following table:

TABLE 2SLP76 phosphorylation inhibition assay results

Wherein A represents IC ₅₀ The value is less than or equal to 1000nM.

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,

m is 1,2,3 or 4;

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

p is 0, 1 or 2;

x is N or CH, when a substituent is attached to X, then X is C;

R ² selected from the group consisting of: halogen, CN, substituted or unsubstituted C1-C6 alkoxy, R ⁹ R ¹⁰ N-C(＝O)-、R ⁹ R ¹⁰ N-C(＝O)(CH ₂ ) _r O-、R ⁷ O-、R ⁸ C(＝O)N(R ⁹ ) -; wherein, R is ⁷ 、R ⁸ 、R ⁹ 、R ¹⁰ Each independently 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; or said R ⁸ And R ⁹ Or R ⁹ And R ¹⁰ Together 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;r is 0, 1 or 2;

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, -NH ₂ 、-NHS(O) ₂ CH ₃ C1-C6 amino, carboxyl, C1-C6 amide (- = O) -N (Rc) ₂ or-NH-C (= O) (Rc), which is H or C1-C5 alkyl), or a substituted or unsubstituted group selected from: C1-C6 alkyl, C6-C10 aryl, 5-10 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 substituent is selected from the group consisting of: halogen, C1-C6 alkyl, C1-C6 alkylene-OH, C1-C6 alkoxy, oxo, -S (O) ₂ CH ₃ CN, -OH, C6-C10 aryl, 3-10 membered heteroaryl having 1-3 heteroatoms selected from N, S and O, -C (O) CHNH ₂ -C (O) CHOH; with the proviso that said compound of formula I is a chemically stable structure;

and said compound is not a compound selected from the group consisting of:

2. the compound of claim 1, or a pharmaceutically acceptable salt, optical isomer, or hydrate thereof, wherein m is 1 or 2; n is 0, 1 or 2; p is 0 or 1; r is ³ Is H.

3. The compound of claim 1, or a pharmaceutically acceptable salt, optical isomer, or hydrate thereof, wherein R is ² Selected from the group consisting of: halogen, CN, R ⁹ R ¹⁰ N-C(＝O)-、R ⁹ R ¹⁰ N-C(＝O)(CH ₂ ) _r O-、R ⁹ R ¹⁰ N-C(＝O)NR-、R ⁷ O-、R ⁸ C(＝O)N(R ⁹ ) -; wherein, R is ⁷ 、R ⁸ 、R ⁹ 、R ¹⁰ Each independently selected from the group consisting of: H. substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted 3-8 membered heterocyclyl having 1-3 heteroatoms selected from the group consisting of N, S and O; or said R ⁹ And R ¹⁰ Together with the nitrogen atom to which they are attached form a substituted or unsubstituted 3-8 membered heterocyclic group having 1-3 heteroatoms selected from N, S and O.

4. The compound of claim 1, or a pharmaceutically acceptable salt, optical isomer, or hydrate thereof, wherein R is ² Selected from the group consisting of: halogen, CN, R ⁹ R ¹⁰ N-C(＝O)-、R ⁹ R ¹⁰ N-C(＝O)(CH ₂ ) _r O-、R ⁷ O-、R ⁸ C(＝O)N(R ⁹ ) -; wherein, R is ⁷ 、R ⁸ 、R ⁹ 、R ¹⁰ Each independently selected from the group consisting of: H. substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted C1-C3 cycloalkyl, or a substituted or unsubstituted C3 cycloalkyl groupA 4-6 membered heterocyclic group of a heteroatom selected from the group consisting of N, S and O; or said R ⁸ And R ⁹ Or R ⁹ And R ¹⁰ Together with the nitrogen atom to which they are attached form a substituted or unsubstituted 4-6 membered heterocyclic group having 1-3 heteroatoms selected from N, S and O.

5. The compound of claim 1, or a pharmaceutically acceptable salt, optical isomer, or hydrate thereof, wherein each R is ¹ 、R ⁴ And R ⁵ Each independently selected from the group consisting of: H. halogen, CN, substituted or unsubstituted C1-C6 alkoxy.

6. The compound of claim 1, or a pharmaceutically acceptable salt, optical isomer, or hydrate thereof, wherein the compound has the structure shown in formula II:

7. the compound of claim 1, or a pharmaceutically acceptable salt, optical isomer, or hydrate thereof, wherein the compound has the structure shown in formula:

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

9. 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.

10. 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 of a pharmaceutical composition according to claim 8, for the preparation of a pharmaceutical composition for the prophylaxis and/or treatment of diseases which are associated with the activity or expression of HPK1 kinase.

11. The compounds of claim 1 and pharmaceutically acceptable salts, stereoisomers, esters, prodrugs, solvates and deuterated compounds thereof, alone or in combination with tumor chemotherapy regimens, other tumor immunotherapeutic agents (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, etc., can be administered before, after, or concurrently with the agents, or can be co-administered with other known therapies. Can also be used as vaccine adjuvant.

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.

13. The use of any one of claims 10 to 12, wherein the disease includes, but is 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 carcinoma, esophageal cancer, biliary tract tumors, head and neck cancer, hematological malignancies, nasopharyngeal cancer, multiple myeloma, villous adenoma in the large field, non-hodgkin's lymphoma, bone cancer, testicular cancer, hodgkin's disease, seminoma, oral cancer, brain cancer, skin cancer, ductal breast cancer, renal pelvis cancer, nephroblastoma, esophageal adenoma, retinoblastoma, glioma, neurofibroma, gastrointestinal stromal tumors, carcinoma in situ, endometrial cancer, and myelodysplastic syndrome.