Pyrrolopyrazine derivatives targeting tumor immunokinases, methods of making and uses thereof

Abstract

The application discloses a compound as shown in formula (I) and a preparation method and application thereof in pharmaceutical preparations. The compound as shown in formula (I) has excellent HPK 1 and / or LCK kinase inhibiting activity and can significantly promote immune-related cytokine release, improve T cell dysfunction and treat related diseases caused by abnormal HPK 1 and / or LCK kinase.

Description

[0001] This application is a divisional application of the invention patent with the application date of February 20, 2023, the application number of 202310137749.X, and the invention name of "HPK1 and / or LCK kinase modulator, preparation method and application thereof". TECHNICAL FIELD

[0002] The present application belongs to the field of medicine, and relates to a pyrrolopyrazine derivative targeting tumor immune kinases, a preparation method and application thereof. BACKGROUND

[0003] The main treatment modalities used by oncologists to treat cancer are surgical resection, radiation therapy, and classic chemotherapeutic drugs. Unfortunately, surgical resection is not a viable option for many tumor or cancer forms. In addition, radiation therapy and chemotherapeutic drugs do not target only diseased cells, and thus end up damaging healthy cells. Therapies that more specifically target tumor cells have been developed by taking advantage of tumor-specific antigen expression or inappropriate overexpression or inappropriate activation of specific proteins within tumor cells, but tumor cells tend to mutate and can develop resistance to drugs that specifically target tumor cells.

[0004] Immunotherapy can restart the human immune system to enable it to recognize and kill tumor cells, and this new type of anti-cancer strategy has become the most promising development direction in the research and development of anti-tumor drugs. However, only a small part of patients can respond to the treatment of immune checkpoint modulators, for example, only 5-15% of patients respond to ipilimumab (Nat Rev Drug Discov, 2016, 15: 235-247), and the proportion of patients who can respond to pembrolizumab and nivolumab is less than 40% (Immunity, 2016, 44: 1255-1269). The proportion of patients who have a sustained response to immunotherapy is even rarer, and most malignant tumor patients not only cannot benefit from it, but also may suffer additional harm to the body due to the side effects of immunotherapy. How to reduce the threshold of immunotherapy response and obtain a sustained and effective response is a hot issue worthy of attention in current tumor immunotherapy.

[0005] Endogenous or adoptively transferred cytotoxic T cells are important mediators of anti-tumor immunity. Persistent antigen exposure leads to a progressive loss of specific effector functions and proliferative capacity of T cells and pronounced transcriptional, epigenetic and metabolic changes, resulting in T cell dysfunction. T cell exhaustion is characterized by marked changes in metabolic function, transcriptional programming, loss of effector functions (e.g., cytokine secretion, killing capacity) and co-expression of multiple surface inhibitory receptors. The root cause of T cell exhaustion is persistent antigen exposure, leading to persistent TCR signaling. Prevention or reversal of T cell exhaustion has long been sought as a means to enhance T cell effectiveness in patients with cancer or chronic infections.

[0006] Hematopoietic progenitor kinase 1 (HPK1) kinase modulators have significant synergistic anti-tumor effects with clinically investigated or marketed anti-tumor immune targets such as PD-1 / PD-L1 mAbs, CTLA-4 mAbs, CAR-Ts, etc., and are expected to become a key tool to solve the difficulties faced by current anti-tumor immunotherapy.

[0007] HPK1 is a negative regulator of T cell receptor, B cell receptor and dendritic cells, and can be targeted to enhance anti-tumor immunity. HPK1 is mainly expressed by hematopoietic cells, including early progenitor cells. In T cells, HPK1 negatively regulates T cell activation by phosphorylating SLP76 at Ser376 (J Exp Med, 2007, 204:681-691) and Gads at Thr254, which reduces the persistence of signaling microclusters, leading to the recruitment of 14-3-3 proteins bound to phosphorylated SLP76 and Gads, and the release of SLP76-Gads-14-3-3 complex from LAT-containing microclusters (J Cell Biol, 2011, 195(5):839-853). HPK1 can also be activated in response to prostaglandin E2, which is usually secreted by tumors, which helps tumor cells to escape from the immune system. Loss of HPK1 kinase function increases cytokine secretion, enhances T cell signaling, viral clearance and tumor growth inhibition. Therefore, HPK1 is considered a promising target for tumor immunotherapy.

[0008] Over a decade ago, researchers discovered that HPK1 could be a potential target for cancer immunotherapy. Several compounds, such as CFI-402411, BGB-15025, PRJ1-3024, etc., have entered clinical studies, but no related drugs have been marketed so far. The main challenge in the development of HPK1 modulators is that the functions of HPK1 family members are different, and it is difficult to design highly selective inhibitory compounds. In addition, it is difficult to avoid inhibiting other related kinases involved in TCR signaling, such as Src and other Ste20-like kinase families.

[0009] TCR signaling requires key protein kinases, including protein kinases LCK and ZAP-70. Among them, LCK is a protein kinase specifically expressed in T lymphocytes, which is essential for T cell development, activation and initiation of T cell antigen receptor (TCR) mediated signal transduction pathway. A large number of literatures have reported that LCK modulators represented by dasatinib can be used for the treatment of acute T lymphoblastic leukemia. The transient blockade of CAR signals by dasatinib can not only prevent the process of T cell exhaustion, but also reverse the phenotype of T cell exhaustion. It is shown that targeting LCK kinase is expected to become an important target for tumor immunity.

[0010] There is no report and patent application of a dual-targeting modulator targeting HPK 1 and LCK kinase. SUMMARY

[0011] In view of the deficiencies of the prior art, one object of the present application is to provide a pyrrolopyrazine derivative targeting tumor immunity kinase and a preparation method thereof. Another object of the present application is to provide the use of the pyrrolopyrazine derivative in the preparation of a drug for preventing and / or treating cancer. Another object of the present application is to provide the use of the pyrrolopyrazine derivative in combination with CAR-T and PD1-PDL1 in the preparation of a drug for treating immune and cancer related diseases.

[0012] In order to achieve the above objects, the present application provides a compound of formula (I) or a stereoisomer, tautomer thereof, or a pharmaceutically acceptable salt, hydrate, solvate thereof, or a PROTAC chimeric compound thereof;

[0013]

[0014] wherein:

[0015] X1 and X3 are C, and X2 is N;

[0016] X4 and X5 are each independently CH or N, and X4 and X5 are not simultaneously CH;

[0017] L1and L2are each independently selected from the group consisting of: null, NR, S, O, -NR-C(=O)R-, -NR-C(=O)NR-, -NR-C(=O)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-C6alkyl, substituted or unsubstituted C3-C8cycloalkyl, substituted or unsubstituted 3- to 12-membered heterocyclyl having from 1 to 3 heteroatoms selected from the group consisting of N, S, and O;

[0018] Ra is selected from the group consisting of: halogen, CN, CF3, substituted or unsubstituted C1-C6alkyl, or wherein said ring B is C3-C8cycloalkyl, 6- to 10-membered substituted or unsubstituted aryl, 5- to 10-membered substituted or unsubstituted heteroaryl, 3- to 10-membered substituted or unsubstituted heterocyclyl having from 1 to 3 heteroatoms selected from the group consisting of N, S, and O;

[0019] R1and R4are each independently selected from the group consisting of: H, halogen, CN, substituted or unsubstituted C1-C6alkyl, substituted or unsubstituted C1-C6alkoxy, substituted or unsubstituted C3-C8cycloalkyl, substituted or unsubstituted 3- to 12-membered heterocyclyl having from 1 to 3 heteroatoms selected from the group consisting of N, S, and O, substituted or unsubstituted C6-C10aryl;

[0020] R2is selected from the group consisting of: null, substituted or unsubstituted C1-C6alkyl, wherein L3is C1-C6alkyl, 3- to 10-membered heterocyclyl, C1-C6alkoxy, substituted amino wherein the substitution of the amino is C1-C6alkyl, 3- to 10-membered heterocyclyl, R5is NH2, C1-C6alkyl, 3- to 10-membered heterocyclyl, wherein said X is H, CN, halogen; R6is substituted or unsubstituted C1-C6alkyl, 3- to 10-membered heterocyclyl, C1-C6alkoxy, substituted amino wherein the substitution of the amino is C1-C6alkyl, 3- to 10-membered heterocyclyl;

[0021] t is 0, 1, 2, or 3;

[0022] s is 0, 1, 2, or 3;

[0023] R3is halogen, -C1-8alkyl, -C2-8alkenyl, -C2-8alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, oxo, -CN, -NO2, -OR 3a , -SO2R 3a , -SO2NR 3a R 3b , -COR 3a , -CO2R 3a , -CONR 3a R3b , -C(=NR 3a )NR 3b R 3c , -NR 3a R 3b , -NR 3a COR 3b , -NR 3a CONR 3b R 3c , -NR 3a CO2R 3b , -NR 3a SONR 3b R 3c , -NR 3a SO2NR 3b R 3c , or -NR 3a SO2R 3b , each of said -C1-8alkyl, -C2-8alkenyl, -C2-8alkynyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl being optionally substituted with at least one substituent R 3d ;

[0024] R 3a , R 3b , and R 3c are each independently hydrogen, -C1-8alkyl, -C2-8alkenyl, -C2-8alkynyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl, each of said -C1-8alkyl, -C2-8alkenyl, -C2-8alkynyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl being optionally substituted with at least one substituent R 3e ;

[0025] R 3d and R 3e are each independently hydrogen, halogen, -C1-8alkyl, -C2-8alkenyl, -C2-8alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, oxo, -CN, -NO2, -OR 3f , -SO2R 3f , -SO2NR 3f R 3g , -COR 3f , -CO2R 3f , -CONR 3f R 3g , -C(=NR 3f )NR 3g R 3h , -NR 3f R 3g , -NR 3f COR 3g , -NR 3f CONR 3gR 3h , -NR 3f CO2R 3f , -NR 3f SONR 3f R 3g , -NR 3f SO2NR 3g R 3h , or -NR 3f SO2R 3g , each of said -C1-8alkyl, -C2-8alkenyl, -C2-8alkynyl, cycloalkyl, heterocyclyl, aryl or heteroaryl is optionally substituted with at least one substituent selected from the group consisting of halogen, -C1-8alkyl, -OR 3i , -NR 3i R 3j , cycloalkyl, heterocyclyl, aryl or heteroaryl;

[0026] R 3f , R 3g , R 3h , R 3i , and R 3j are each independently hydrogen, -C1-8alkyl, C1-8alkoxy-C1-8alkyl-, -C2-8alkenyl, -C2-8alkynyl, cycloalkyl, heterocyclyl, aryl or heteroaryl;

[0027] Ring A is selected from the group consisting of: 6-10 membered substituted or unsubstituted aryl, 5-10 membered substituted or unsubstituted heteroaryl.

[0028] Further, the compound of formula (I) is selected from the group consisting of compounds in Table 1:

[0029] Table 1

[0030]

[0031] The present application also provides the use of a compound of formula (I) in the preparation of a HPK1 and / or LCK kinase modulator.

[0032] The compound of formula (I) of the present application can modulate HPK 1 and / or LCK kinase, and can be applied to prepare a dual-target modulator targeting HPK 1 and / or LCK kinase.

[0033] The present application also provides a method of inhibiting HPK1 and / or LCK, comprising contacting HPK1 and / or LCK with an effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition.

[0034] The present application also provides a method of preparing a compound of formula (I), comprising the following scheme:

[0035]

[0036] wherein Proi and Pro2 are conventional protecting groups in organic synthesis, couplingi and coupling2 are conventional C-C coupling or C-N coupling in organic synthesis, and other variables are as described for formula (I).

[0037] For example, a compound of formula (I) can be synthesized as shown in Scheme I. Compound (1) is protected to give compound (2), which is reacted with a boronic acid under palladium catalysis to give compound (3); if the Proi protecting group does not spontaneously fall off, no protection of Pro2 is needed; if the Proi protecting group spontaneously falls off, protection of Pro2 is needed to give compound (4); compound (4) can be used for the next coupling under transition metal and with a L1 group which can be aryl, heterocycle, etc. to give a compound of formula (I); if Pro2 spontaneously falls off, a compound of formula (I) is directly obtained, otherwise, deprotection of Pro2 is needed to give a compound of formula (I).

[0038] The pharmaceutically acceptable salts described herein include acid addition salts and base addition salts.

[0039] The acid addition salts include, but are not limited to, salts from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, and phosphonic acid, and salts from organic acids such as aliphatic monocarboxylic and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, alkanedioic acids, aromatic acids, and aliphatic and aromatic sulfonic acids. Thus, these salts include, but are not limited to, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, hydrochloride, hydrobromide, hydroiodide, acetate, propionate, caprylate, isobutyrate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, mandelate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, phthalate, benzenesulfonate, toluenesulfonate, phenylacetate, citrate, lactate, maleate, tartarate, and methanesulfonate, also salts with amino acids such as arginate, gluconate, galacturonate, and the like. The acid addition salts can be prepared by contacting the free base form with a sufficient amount of the desired acid to form the salt in conventional manner. The free base form can be regenerated by contacting the salt form with a base and isolating the free base in a conventional manner.

[0040] The base-addition salts are formed with metals or amines, such as the hydroxides of alkali and alkaline earth metals or with organic amines. Examples of metals useful as a cation include, but are not limited to sodium, potassium, magnesium, and calcium. Examples of appropriate amines include, but are not limited to, N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine (ethane- 1,2-diamine), N-methylglucamine and procaine. Base-addition salts can be prepared by contacting the free acid form with a sufficient amount of the desired base to provide the salt. The free acid form can be regenerated by contacting the salt form with an acid and isolating the free acid in the conventional manner.

[0041] The stereoisomers of the present application include enantiomers, diastereomers and geometric isomers. Some of the compounds of the present application have a cycloalkyl group which can be substituted at more than one carbon atom, in which case all geometric isomers, including cis- and trans-, and mixtures thereof, are within the scope of the present application.

[0042] The solvates of the present application refer to physical associations of the compounds of the present application with one or more solvent molecules. The physical associations include various degrees of ionization and covalency, including hydrogen bonding. In some cases, the solvates can be isolated, for example, when one or more solvent molecules are incorporated in the crystal lattice of the solid. "Solvate" includes both solution-phase and isolatable solvates. Representative solvates include ethanolate, methanolate, and the like. "Hydrate" is a solvate in which one or more solvent molecules are H2O.

[0043] The prodrugs of the present application refer to forms of the compounds of Formula (I) which are suitable for administration to a patient without undue toxicity, irritation, and allergic response, and which are effective for their intended use, and include esters, amides and zwitterions. The prodrugs are converted into the parent compounds of the above formula in vivo (e.g., by hydrolysis in blood).

[0044] The present application also provides a pharmaceutical composition comprising the compound of Formula (I) or a stereoisomer, tautomer, or a pharmaceutically acceptable salt, hydrate, solvate, or a PROTAC chimera thereof, and a pharmaceutically acceptable excipient. The excipient is selected from the group consisting of carriers, diluents, binders, lubricants, wetting agents.

[0045] Preferably, the pharmaceutical composition further comprises a chemotherapeutic agent; wherein the chemotherapeutic agent is an immunotherapeutic agent.

[0046] Preferably, the pharmaceutical composition comprises a therapeutically effective amount of the compound of Formula (I).

[0047] In certain embodiments, these pharmaceutical compositions are useful for treating HPK1 and / or LCK kinase-mediated disorders or conditions. The HPK 1 and / or LCK kinase modulators of the present application can also be incorporated into pharmaceutical compositions further comprising a compound useful for treating cancer or other HPK1 and / or LCK kinase-mediated disorders.

[0048] The compounds of the present application, as Formula (I), can be formulated into pharmaceutical compositions in the form of syrups, elixirs, suspensions, powders, granules, tablets, capsules, lozenges, aqueous or oily solutions, creams, ointments, lotions, gels, emulsions, and the like.

[0049] The pharmaceutical preparations are preferably in unit dosage forms. In such form, the preparation is subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or ampules. Also, the unit dosage form can be a capsule, tablet, or it can be the appropriate number of any of these in packaged form.

[0050] The quantity of active component in a unit dose preparation can be varied or adjusted from 0.1 mg to 1000 mg, depending on the particular application and the potency of the active component. The composition, if desired, can also contain other suitable therapeutic agents.

[0051] The pharmaceutically-acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method of administration. Thus, if desired, the pharmaceutical compositions of the application can be formulated with various materials having various degrees of acidity, alkalinity, and the like. Thus, the pharmaceutical compositions of the present application can be present in a variety of formulations.

[0052] The compounds of the present application, as Formula (I), alone or in combination with other suitable components, are formulated into aerosol formulations (i.e., they can be "nebulized") to be administered via inhalation. The aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen and the like.

[0053] Formulations suitable for parenteral administration, such as, for example, by intra venous, intramuscular, intradermal, and subcutaneous routes, include aqueous and nonaqueous sterile injection solutions which can contain anti-oxidants, buffers, bacteriostats, and solutes which render the formulation isotonic with the blood of the recipient, and aqueous and nonaqueous sterile suspensions which can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. In the practice of this application, compositions can be administered, for example, intravenously, orally, topically, intraperitoneally, intravesically, and intrathecally. Formulations for injection can be presented in unit dosage form, e.g., in ampules or in multidose containers. Solutions and suspensions for injection can be prepared from sterile powders, granules, and tablets of the kind previously described.

[0054] In the context of the present application, the subject should be dosed sufficiently to result in a beneficial therapeutic response in the subject over time. The dose will depend on the potency of the particular compound employed, and the condition of the subject, as well as the body weight or surface area of the subject to be treated. The size of the dose will depend on the ability of the particular compound to elicit the desired therapeutic response in the particular subject over the course of the treatment, in the presence or absence of any adverse side effects of the compound being administered. In determining the effective amount of the compound to be administered, the physician can evaluate such factors as the subject's circulation plasma levels of the compound, compound toxicity, and / or the progression of the disease.

[0055] The present application also provides the use of a compound of formula (I) or a pharmaceutically acceptable salt, stereoisomer, ester, prodrug, solvate or deuterated compound thereof in the manufacture of a medicament for treating, preventing and alleviating diseases caused by over-activation of HPK 1 and / or LCK kinase.

[0056] The present application also provides the use of a compound of formula (I) or a pharmaceutically acceptable salt, stereoisomer, ester, prodrug, solvate or deuterated compound thereof in the manufacture of a medicament for treating, preventing and alleviating diseases caused by over-activation of HPK 1 and / or LCK kinase.

[0057] The present application also provides the use of a compound of formula (I) or a pharmaceutically acceptable salt, stereoisomer, ester, prodrug, solvate or deuterated compound thereof in combination with a PD-1, PD-L1, CTLA-4, TIM-3, TGF-β and its receptor, LAG3 antagonist or TLR4, TLR7, TLR8, TLR9, STING agonist in the manufacture of a medicament for cancer immunotherapy.

[0058] The present application also provides the use of a compound of formula (I) or a pharmaceutically acceptable salt, stereoisomer, ester, prodrug, solvate or deuterated compound thereof in combination with CAR-T in the manufacture of a medicament for cancer immunotherapy.

[0059] The present application also provides the use of a compound of formula (I) or a pharmaceutically acceptable salt, stereoisomer, ester, prodrug, solvate or deuterated compound thereof in combination with CAR-T immunotherapy in cancer immunotherapy.

[0060] The CAR-T immunotherapy refers to chimeric antigen receptor T cell immunotherapy, which is one of the more effective treatments for malignant tumors at present, and its basic principle is to use the patient's own immune cells to eliminate cancer cells, which belongs to a kind of cell therapy.

[0061] The cancer according to the present application includes 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, lung squamous carcinoma, peritoneal cancer, hepatocellular carcinoma, gastric cancer, intestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver cancer, breast cancer, metastatic breast cancer, colon cancer, rectal cancer, colorectal cancer, uterine cancer, salivary gland cancer, renal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, Merkel cell carcinoma, esophageal cancer, biliary tract tumor, head and neck cancer and hematological malignancy.

[0062] The hematological malignancy according to the present application includes, but is not limited to, acute T lymphoblastic leukemia (T-ALL), chronic T lymphoblastic leukemia, acute B lymphoblastic leukemia, chronic B lymphoblastic leukemia, plasma cell neoplasm, multiple myeloma, macroglobulinemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, primary thrombocytosis, polycythemia vera.

[0063] Compared with the prior art, the present application has the following advantages:

[0064] The present application combines immunotherapy, targets HPK 1 and / or LCK kinase, can significantly promote the release of cytokines, and significantly revitalize the function of T cells. The HPK 1 and / or LCK kinase targeting compound (such as the compound of formula (I)) provided by the present application has good immune factor release activity, and thus can be used for designing candidate molecules with higher activity as a lead compound for the treatment, prevention and alleviation of diseases caused by excessive activation of HPK 1 and / or LCK kinase. Moreover, the synthesis method of the HPK 1 and / or LCK kinase modulator provided by the present application has the advantages of cheap raw materials, mild reaction conditions, simple operation, high regioselectivity, high yield and facilitation of industrial production. DETAILED DESCRIPTION

[0065] The present application will be further described by way of examples, but the present application is not limited to the scope of the examples. The experimental methods in the following examples are not specified, and are selected according to conventional methods and conditions, or according to the instructions of the commercial product.

[0066] Example 1: Synthesis of compound 1

[0067]

[0068] Step 1: Synthesis of intermediate 1-1

[0069]

[0070] To a mixture of compound 2-bromo-7-iodo-5H-pyrrolo[2,3-b]pyrazine (3 g, 1.0 eq.) and 3,4-dimethoxyphenylboronic acid (1.69 g, 1.0 eq.) in dioxane (100 mL) and water (50 mL) was added K2CO3(4.14 g, 3.0 eq.) and Pd(dppf)Cl2(365.5 mg, 0.015 eq.). The reaction mixture was stirred at 110 °C for 12 h under nitrogen. The mixture was cooled to room temperature and concentrated under reduced pressure, the residue was purified by silica gel column chromatography (dichloromethane:MeOH = 20:1) to give compound 1-1 (1.2 g, 40%). ESI+-MS (m / z): 355.90 [M+Na] + .

[0071] Step 2: Synthesis of intermediate 1-2

[0072]

[0073] To a mixture of compound 1-1 and di-tert-butyl dicarbonate (6.54 g, 3.0 eq.) in tetrahydrofuran (100 mL) was added triethylamine (5.05 g, 5.0 eq.). The reaction mixture was stirred at 50 °C for 6 h. The mixture was cooled to room temperature and concentrated under reduced pressure, the residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 20:1) to give compound 1-2. ESI+-MS (m / z): 456.08 [M+H]+.1H NMR (400 MHz, DMSO) δ 8.60 (s, 1H), 8.58 (s, 1H), 7.75 (dd, J = 8.3, 2.0 Hz, 1H), 7.66 (d, J = 2.0 Hz, 1H), 7.05 (d, J = 8.5 Hz, 1H), 3.86 (s, 3H), 3.81 (s, 3H), 1.66 (s, 9H).

[0074] Step 3: Synthesis of compound 1

[0075] To a mixture of compound 1-2 (200 mg, 1.0 eq.) and (6-(4-methylpiperazin-1- yl)pyridin-3-yl)boronic acid (102.78 mg, 1.0 eq.) in dioxane (30 mL) and water (6 mL) was added K2CO3 (245.04 mg, 3.0 eq.) and Pd(dppf)Cl2 (21.95 mg, 0.015 eq.). The reaction mixture was stirred at 110 °C for 12 h under nitrogen. The mixture was cooled to room temperature and concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane:MeOH = 50:1) to give compound 1 (150 mg, 75%). ESI+-MS (m / z): 431.31 [M+H]+.1H NMR (400 MHz, DMSO) δ 12.19 (s, 1H), 8.97 (d, J = 2.3 Hz, 1H), 8.84 (s, 1H), 8.41 - 8.30 (m, 2H), 8.01 (d, J = 1.7 Hz, 1H), 7.88 (dd, J = 8.3, 1.8 Hz, 1H), 7.06 (d, J = 8.4 Hz, 1H), 6.98 (d, J = 9.0 Hz, 1H), 3.91 (s, 3H), 3.81 (s, 3H), 3.63 - 3.56 (m, 4H), 2.47 - 2.38 (m, 4H), 2.23 (s, 3H).

[0076] Example 2: Synthesis of compound 2

[0077]

[0078] Compound 2 (160 mg, 81%) was prepared in a similar manner as described in Example 1, step 3, from compound 1-2 (200 mg, 1.0 eq.) and 4-(1-methyl-4-piperidinyl)benzeneboronic acid pinacol ester (139.03 mg, 1.0 eq.). ESI + -MS (m / z): 429.34 [M+H]+. + . 1 H NMR (400 MHz, DMSO) δ 12.25 (s, 1H), 8.88 (s, 1H), 8.40 (s, 1H), 8.15 (d, J = 7.7 Hz, 2H), 8.02 (s, 1H), 7.90 (d, J = 8.2 Hz, 1H), 7.41 (d, J = 7.8 Hz, 2H), 7.06 (d, J = 8.4 Hz, 1H), 3.91 (s, 3H), 3.81 (s, 3H), 3.69 - 3.47 (m, 4H), 2.69 - 2.59 (m, J = 9.4, 4.0 Hz, 1H), 2.39 (s, 3H), 1.95 - 1.76 (m, J = 20.7, 1.5 Hz, 4H).

[0079] Example 3: Synthesis of compound 3

[0080]

[0081] Compound 3 (200 mg, 87%) was prepared in a similar manner as described in example 1 step 3 from compound 1-2 (200 mg, 1.0 eq.) and 1-methyl-4-((4-(4,4,5,5- tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)sulfonyl)piperazine (169.05 mg, 1.0 eq.). ESI + -MS (m / z): 494.26 [M+H] + . 1 H NMR (400 MHz, DMSO) δ 12.40 (s, 1H), 9.04 (s, 1H), 8.48 (dd, J = 10.2, 5.0 Hz, 3H), 8.00 (s, 1H), 7.89 (d, J = 8.3 Hz, 3H), 7.07 (d, J = 8.4 Hz, 1H), 3.93 (s, 3H), 3.81 (s, 3H), 3.06 - 2.89 (m, 4H), 2.44 - 2.29 (m, 4H), 2.14 (s, 3H).

[0082] Example 4: Synthesis of compound 4

[0083]

[0084] Compound 4 (150 mg, 78%) was prepared in a similar manner as described in example 1 step 3 from compound 1-2 (200 mg, 1.0 eq.) and 4-(4-tetrahydropyranyl)benzeneboronic acid pinacol ester (133.02 mg, 1.0 eq.). ESI + -MS (m / z): 438.31 [M+Na] + . 1 H NMR (400 MHz, DMSO) δ 12.24 (s, 1H), 8.88 (s, 1H), 8.40 (d, J = 2.4 Hz, 1H), 8.15 (d, J = 8.2 Hz, 2H), 8.03 (d, J = 1.7 Hz, 1H), 7.90 (dd, J = 8.3, 1.8 Hz, 1H), 7.41 (d, J = 8.2 Hz, 2H), 7.07 (d, J = 8.4 Hz, 1H), 4.03 - 3.94 (m, J = 10.8 Hz, 2H), 3.92 (s, 3H), 3.81 (s, 3H), 3.51 - 3.40 (m, 2H), 2.90 - 2.77 (m, 1H), 1.72 (dd, J = 10.9, 7.7 Hz, 4H).

[0085] Example 5: Synthesis of compound 5

[0086]

[0087] Compound 5 (150 mg, 77%) was prepared from compound 1-2 (200 mg, 1.0 eq.) and (6-morpholinopyridin-3-yl)boronic acid (96.07 mg, 1.0 eq.) in a similar manner as described in Example 1, step 3. ESI + -MS (m / z): 418.29 [M+H] + . 1 H NMR (400 MHz, DMSO) δ 12.19 (s, 1H), 8.99 (d, J = 2.3 Hz, 1H), 8.85 (s, 1H), 8.37 (dd, J = 8.7, 2.5 Hz, 2H), 8.00 (d, J = 1.8 Hz, 1H), 7.88 (dd, J = 8.3, 1.9 Hz, 1H), 7.06 (d, J = 8.4 Hz, 1H), 7.00 (d, J = 9.0 Hz, 1H), 3.91 (s, 3H), 3.80 (s, 3H), 3.77 - 3.70 (m, 4H), 3.60 - 3.50 (m, 4H).

[0088] Example 6: Synthesis of compound 6

[0089]

[0090] Compound 6 was prepared from compound 1-2 and 6-(4-Boc-l-piperazinyl)pyridine-3- boronic acid pinacol ester in a similar manner as described in Example 1, step 3. ESI + -MS (m / z): 417.33 [M+H] + . 1 H NMR (400 MHz, DMSO) δ 12.16 (s, 1H), 8.97 (d, J = 1.9 Hz, 1H), 8.84 (s, 1H), 8.42 - 8.29 (m, 2H), 8.00 (s, 1H), 7.88 (d, J = 8.3 Hz, 1H), 7.06 (d, J = 8.4 Hz, 1H), 6.97 (d, J = 9.0 Hz, 1H), 3.91 (s, 3H), 3.80 (s, 3H), 3.60 - 3.50 (m, 4H), 2.92 - 2.75 (m, 4H).

[0091] Example 7: Synthesis of compound 7

[0092]

[0093] Compound 7 (120 mg, 62%) was prepared in a similar manner as described in Example 1, step 3 from compound 1-2 (200 mg, 1.0 eq.) and l-methyl-4-(4-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)-lH-pyrazol-l- yl)piperidine (134.41 mg, 1.0 eq.). ESI + -MS (m / z): 419.30 [M+H] + . 1 H NMR (400 MHz, DMSO) δ 12.12 (s, 1H), 8.67 (s, 1H), 8.47 (s, 1H), 8.32 (d, J = 2.5 Hz, 1H), 8.15 (s, 1H), 8.03 (d, J = 1.8 Hz, 1H), 7.84 (dd, J = 8.3, 1.9 Hz, 1H), 7.04 (d, J = 8.4 Hz, 1H), 4.25 (ddd, J = 15.4, 10.2, 5.3 Hz, 1H), 3.92 (s, 3H), 3.80 (s, 3H), 3.06 - 2.92 (m, J = 11.4 Hz, 2H), 2.32 (s, 3H), 2.25 (t, J = 9.7 Hz, 2H), 2.14 - 2.01 (m, 4H).

[0094] Example 8: Synthesis of compound 8

[0095]

[0096] Compound 8 (150 mg, 78%) was prepared in a similar manner as described in Example 1, step 3 from compound 1-2 (200 mg, 1.0 eq.) and 4-(4-morpholinyl)benzeneboronic acid (95.61 mg, 1.0 eq.). ESI + -MS (m / z): 417.27 [M+H] + . 1 H NMR (400 MHz, DMSO) δ 12.15 (s, 1H), 8.83 (s, 1H), 8.35 (d, J = 2.5 Hz, 1H), 8.11 (d, J = 8.7 Hz, 2H), 8.07 (d, J = 1.2 Hz, 1H), 7.88 (dd, J = 8.3, 1.4 Hz, 1H), 7.07 (t, J = 9.4 Hz, 3H), 3.92 (s, 3H), 3.81 (s, 3H), 3.79 - 3.73 (m, 4H), 3.25 - 3.16 (m, 4H).

[0097] Example 9: Synthesis of compound 9

[0098]

[0099] Compound 9 (120 mg, 65%) was prepared from compound 1-2 (200 mg, 1.0 eq.) and 4-(2H-tetrazol-5-yl)benzeneboronic acid (87.29 mg, 1.0 eq.) in a similar manner as described in Example 1, Step 3. ESI + -MS (m / z): 400.24 [M+H] + . 1 H NMR (400 MHz, DMSO) δ 12.35 (s, 1H), 9.04 (s, 1H), 8.47 (dd, J = 11.0, 5.4 Hz, 3H), 8.23 (d, J = 8.2 Hz, 2H), 8.05 (s, 1H), 7.90 (d, J = 8.3 Hz, 1H), 7.62 (d, J = 5.8 Hz, 1H), 7.09 (d, J = 8.3 Hz, 1H), 3.94 (s, 3H), 3.82 (s, 3H).

[0100] Example 10: Synthesis of compound 10

[0101]

[0102] Compound 10 (180 mg, 87%) was prepared from compound 1-2 (200 mg, 1.0 eq.) and (4-((4-methylpiperazin-1-yl)methyl)phenyl)boronic acid (108.08 mg, 1.0 eq.) in a similar manner as described in Example 1, Step 3. ESI + -MS (m / z): 444.29 [M+H] + . 1 H NMR (400 MHz, DMSO) δ 12.26 (s, 1H), 8.90 (s, 1H), 8.40 (d, J = 2.1 Hz, 1H), 8.18 (d, J = 8.1 Hz, 2H), 8.03 (d, J = 1.7 Hz, 1H), 7.89 (dd, J = 8.3, 1.8 Hz, 1H), 7.46 (d, J = 8.1 Hz, 2H), 7.06 (d, J = 8.4 Hz, 1H), 3.92 (s, 3H), 3.81 (s, 3H), 3.55 (s, 2H), 3.54 - 3.40 (m, 4H), 2.50 - 2.41 (m, 4H), 2.29 (s, 3H).

[0103] Example 11: Synthesis of compound 11

[0104]

[0105] Compound 11 (120 mg, 65%) was prepared in a similar manner as described in Example 1, step 3 from compound 1-2 (200 mg, 1.0 eq.) and 2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-1,3,4-oxadiazole (125.63 mg, 1.0 eq.). ESI + -MS (m / z): 422.18 [M+Na] + . 1 H NMR (400 MHz, DMSO) δ 12.32 (d, J = 2.5 Hz, 1H), 9.38 (s, 1H), 9.02 (s, 1H), 8.44 (dd, J = 12.8, 5.6 Hz, 3H), 8.18 (d, J = 8.4 Hz, 2H), 8.03 (d, J = 1.9 Hz, 1H), 7.87 (dd, J = 8.3, 1.9 Hz, 1H), 7.07 (d, J = 8.4 Hz, 1H), 3.93 (s, 3H), 3.82 (s, 3H).

[0106] Example 12: Synthesis of compound 12

[0107]

[0108] Compound 12 (135 mg, 68%) was prepared in a similar manner as described in Example 1, step 3 from compound 1-2 (200 mg, 1.0 eq.) and 4-(4-methyl-1-piperazinyl)benzeneboronic acid (101.62 mg, 1.0 eq.). ESI + -MS (m / z): 430.24 [M+H] + . 1 H NMR (400 MHz, DMSO) δ 12.14 (s, 1H), 8.82 (s, 1H), 8.34 (s, 1H), 8.09 (d, J = 9.3 Hz, 3H), 7.87 (d, J = 8.0 Hz, 1H), 7.06 (t, J = 7.3 Hz, 3H), 3.92 (s, 3H), 3.81 (s, 3H), 3.30 - 3.15 (m, 4H), 2.50 - 2.40 (m, 4H), 2.23 (s, 3H).

[0109] Example 13: Synthesis of compound 13

[0110]

[0111] Compound 13 (160 mg, 80%) was prepared from compound 1-2 (200 mg, 1.0 eq.) and 2-(4-methylpiperazino)pyridine-5-boronic acid tetramethylpropanol ester (140.50 mg, 1.0 eq.) in a similar manner as described in Example 1, step 3. ESI + -MS (m / z): 432.27 [M+H] + .

[0112] Other compounds of the present application can be prepared by reference to the example synthesis preparation methods shown above.

[0113] Biological activity assay

[0114] Experimental Example 1: Inhibition assay for HPK 1 and LCK kinases

[0115] 1. Dilute the compounds 3-fold in DMSO with a starting concentration of 1-10 μΜ in dilution plate.

[0116] 2. Dilute the compounds 50-fold into lx kinase reaction buffer and shake on a shaker for 20 minutes.

[0117] 3. Prepare 2x kinase with lx enzyme reaction buffer.

[0118] 4. Add 2 μΐ^of kinase (prepared in step 3) to each well of the reaction plate.

[0119] 5. Add 1 μΐ^of the compound diluted in buffer to each well, seal the plate with a plate sealer and centrifuge at 1000 g for 30 seconds and incubate at room temperature for 10 minutes.

[0120] 6. Prepare 4x MBP Protein and ATP (ATP final concentration 10 μΜ) mix with lx enzyme reaction buffer and add 1 μΐ^of 4x MBP Protein / ATP mix to the reaction plate.

[0121] 7. Seal the plate with a plate sealer and centrifuge at 1000 g for 30 seconds and incubate at room temperature for 60 minutes.

[0122] 8. Transfer 4 μΐ^of ADP-Glo to the 384 reaction plate and centrifuge at 1000 rpm / min for 1 min and incubate at 25 °C for 40 min.

[0123] 9. Transfer 8 μΐ^of Detection solution to the 384 reaction plate and centrifuge at 1000 rpm / min for 1 min and incubate at 25 °C for 40 min.

[0124] 10. Read the RLU (Relative luminescence unit) signal using Biotek Multifunctional plate reader. The signal intensity is used to characterize the degree of kinase activity.

[0125] (3) Data processing

[0126] Calculate the inhibition rate of each well, and the inhibition rate is calculated according to the following formula:

[0127]

[0128] The Lum positive control is the average of the RLU readings of all positive controls, and the Lum negative control is the average of the RLU readings of all negative control empty DMSO.

[0129] Calculate IC 50 and draw the inhibition curve of the compound:

[0130] The ICso (half inhibitory concentration) of the compound is obtained by using the following nonlinear fitting formula: data analysis is performed using Graphpad 9.3 software.

[0131] Y = Bottom + (Top-Bottom) / (1 + 10^((LogIC 50 -X) * Hill Slope))

[0132] X: compound concentration log value; Y: inhibition rate (% inhibition).

[0133] (4) Experimental results

[0134] The inhibitory activity of the compound of the present application on HPK1 enzyme / LCK enzyme and the stimulation of IL-2:

[0135] Experimental results:

[0136] Table 2

[0137] Compound No. HPK 1 IC 50 ]]> LCK IC 50 ]]> Stimulation fold of IL-2 1 2.2 2 2.2 3 2.2 4 1 A A B 2 B A B 3 C C C 4 B B C 5 B C B 6 A B C 7 A A C 8 A A C 9 C C C 10 C C C 11 C C C 12 B B B 13 B A B

[0138] Where IC 50 A = <100 nM; B = 100-500 nM; C = 500-1000 nM; D = >1000 nM of IL-2 stimulation fold A = >2; B = 1-2; C = <1

[0139] Some of the compounds of the present application have better inhibitory effect on HPK1, some have better inhibitory effect on LCK, and some can simultaneously inhibit HPK1 and LCK, and part of the compounds show obvious stimulation effect on the cytokine IL-2, which can improve tumor immunity, indicating that the compounds of the present application have better application potential for diseases caused by HPK1 and / or LCK kinase.

[0140] The HPK1 and / or LCK modulator provided by the present application, preparation and application thereof are described in detail above.

[0141] The principles and implementation modes of the present application are described by using specific examples in this paper, and the above examples are only used to help understand the method and the central idea of the present application. It should be pointed out that for ordinary skilled persons in the art, some improvements and modifications can be made without departing from the principles of the present application, and these improvements and modifications also fall within the protection scope of the claims of the present application.

[0142] Although the foregoing application has been described in detail by means of illustrations and examples for the purpose of clarity and understanding, it is clear to those skilled in the art that certain minor changes and modifications can be made. Therefore, the description and examples should not be interpreted as limiting the scope of the present application.

Claims

1. A compound or a pharmaceutically acceptable salt thereof, said compound being selected from the compounds in Table 1; Table 1 2. A pharmaceutical composition, characterized in that, It comprises the compound of claim 1 or a pharmaceutically acceptable salt thereof, and also comprises pharmaceutically acceptable excipients.

3. An HPK1 and / or LCK kinase modulator, characterized in that, It comprises the compound of claim 1 or a pharmaceutically acceptable salt thereof.

4. The use of a compound as described in claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating, preventing and alleviating diseases caused by overactivation of HPK1 and / or LCK kinases.

5. Use of a compound as described in claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention and / or treatment of cancer.

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