Pyrrolopyridine derivatives as hpk1 inhibitors, methods of preparation and uses thereof

Abstract

This invention discloses a compound of formula (I), its preparation method, and its application in pharmaceutical formulations. The compound of formula (I) exhibits excellent HPK1 and / or LCK kinase inhibitory activity and can significantly promote the release of immune-related cytokines, improve T cell dysfunction, and treat related diseases caused by HPK1 and / or LCK kinase abnormalities.

Description

A pyrrolopyridine derivative as an HPK1 inhibitor, its preparation method and its application

[0001] This application is a divisional application of the invention patent filed on February 20, 2023, with application number 202310137749.X and invention title "An HPK1 and / or LCK kinase modulator, preparation method and application thereof". Technical Field

[0002] This invention belongs to the pharmaceutical field and relates to a pyrrolopyridine derivative as an HPK1 inhibitor, its preparation method, and its application. Background Technology

[0003] The main treatments oncologists use to treat cancer are surgical resection, radiation therapy, and classic chemotherapy drugs. Unfortunately, surgical resection is not a viable option for many tumors or forms of cancer. Furthermore, radiation therapy and chemotherapy drugs not only target diseased cells but also ultimately damage healthy cells. More specific therapies targeting tumor cells have been developed by exploiting the expression of tumor-specific antigens or the inappropriate overexpression or activation of specific proteins within tumor cells; however, tumor cells are prone to mutation and can develop resistance to drugs that specifically target tumor cells.

[0004] Immunotherapy can reboot the body's immune system, enabling it to recognize and kill tumor cells. This novel anti-cancer strategy has become one of the most promising directions in the development of new anti-tumor drugs. However, currently only a small percentage of patients respond to immune checkpoint modulators. For example, only 5-15% of patients respond to ipilimumab, pembrolizumab, and nivolumab (Nat Rev Drug Discov, 2016, 15:235-247), while the proportion of patients responding to pembrolizumab and nivolumab is less than 40% (Immunity, 2016, 44:1255-1269). The proportion of patients with sustained responses to immunotherapy is even smaller. Most patients with malignant tumors not only do not benefit from it, but may also suffer additional harm from the toxic side effects of immunotherapy. How to lower the threshold for immunotherapy response and obtain sustained and effective responses is a hot topic worthy of attention in tumor immunotherapy.

[0005] Endogenous or adoptive cytotoxic T cells are important mediators of antitumor immunity. Sustained antigen exposure leads to the gradual loss of specific effector functions and proliferative capacity of T cells, as well as significant transcriptional, epigenetic, and metabolic changes, resulting in T cell dysfunction. T cell exhaustion is characterized by significant alterations in metabolic function, transcriptional programming, effector function (e.g., cytokine secretion, cytotoxicity), and the co-expression of multiple surface inhibitory receptors. The root cause of T cell exhaustion is sustained antigen exposure, leading to persistent TCR signaling. The prevention or reversal of T cell exhaustion has long been sought as a means to enhance T cell efficacy 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 monoclonal antibodies, CTLA-4 monoclonal antibodies, and CAR-T, and are expected to become key tools for solving the current difficulties in anti-tumor immunotherapy.

[0007] HPK1 is a negative regulator of T cell receptors, B cell receptors, and dendritic cells, and can target and 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, reducing the persistence of signal transduction microclusters. This leads to the recruitment of 14-3-3 proteins that bind to phosphorylated SLP76 and Gads, and the release of the 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 normally secreted by tumors, and this helps tumor cells escape from the immune system. Loss of HPK1 kinase function increases cytokine secretion, enhancing T cell signaling, viral clearance, and tumor growth inhibition. Therefore, HPK1 is considered a promising target for tumor immunotherapy.

[0008] More than a decade ago, researchers discovered that HPK1 might be a potential target for cancer immunotherapy, and several compounds, such as CFI-402411, BGB-15025, and PRJ1-3024, have entered clinical trials. However, no related drugs have yet been marketed. The main challenge in developing HPK1 modulators is the functional differences among HPK1 family members, making the design of highly selective inhibitory compounds difficult. Furthermore, 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 LCK and ZAP-70. LCK is a protein kinase specifically expressed by T lymphocytes, essential for T cell development and the activation and initiation of T cell antigen receptor (TCR)-mediated signal transduction pathways. Numerous studies have reported the use of LCK modulators, such as dasatinib, in the treatment of acute T-lymphoblastic leukemia. Dasatinib's transient blockade of CAR signaling not only prevents T cell exhaustion but also reverses the T cell exhaustion phenotype. This suggests that targeting LCK kinases holds promise as an important target for tumor immunotherapy.

[0010] There are currently no reports or patent applications for dual-target modulators that simultaneously target HPK1 and LCK kinases. Summary of the Invention

[0011] To address the shortcomings of existing technologies, one objective of this invention is to provide a pyrrolopyridine derivative as an HPK1 inhibitor and a method for its preparation; another objective of this invention is to provide the application of the pyrrolopyridine derivative in the preparation of drugs for the prevention and / or treatment of cancer; and yet another objective of this invention is to provide the application of the pyrrolopyridine derivative in combination with CAR-T and PD1-PDL1 in the preparation of drugs for the treatment of immune and cancer-related diseases.

[0012] To achieve the above objectives, the present invention provides a compound of formula (I) or its stereoisomers, tautomers, or pharmaceutically acceptable salts, hydrates, solvates, or PROTAC chimeric compounds thereof;

[0013]

[0014] in:

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

[0016] X4 and X5 are each independently CH or N, and X4 and X5 are not both CH at the same time;

[0017] L1 and L2 are each independently selected from: none, 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 R is selected from: H, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted 3-12 membered heterocyclic group having 1-3 heteroatoms selected from N, S and O;

[0018] Ra is selected from: halogens, CN, CF3, substituted or unsubstituted C1-C6 alkyl groups, or The ring B therein is a C3-C8 cycloalkyl, a 6-10 substituted or unsubstituted aryl, a 5-10 substituted or unsubstituted heteroaryl, or a 3-10 substituted or unsubstituted heterocyclic group having 1-3 heteroatoms selected from N, S and O.

[0019] R1 and R4 are each independently selected from: H, halogen, CN, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted 3-12 membered heterocyclic group having 1-3 heteroatoms selected from N, S and O, substituted or unsubstituted C6-C10 aryl;

[0020] R2 is selected from: unsubstituted, substituted, or non-substituted C1-C6 alkyl groups. Where L3 is a C1-C6 alkyl, 3-10 membered heterocyclic group, C1-C6 alkoxy group, or substituted amino group, wherein the amino group is substituted with a C1-C6 alkyl or 3-10 membered heterocyclic group, and R5 is NH2, C1-C6 alkyl, or 3-10 membered heterocyclic group. Wherein X is H, CN, or halogen; R6 is a substituted or unsubstituted C1-C6 alkyl, 3-10 heterocyclic group, C1-C6 alkoxy, or substituted amino group, wherein the amino group is substituted with a C1-C6 alkyl or a 3-10 heterocyclic group.

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

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

[0023] R3 represents halogens, -C1-8 alkyl groups, -C2-8 alkenyl groups, -C2-8 ynyl groups, cycloalkyl groups, heterocyclic groups, aryl groups, heteroaryl groups, oxoyl groups, -CN groups, -NO2 groups, and -OR groups. 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 The -C1-8 alkyl, -C2-8 alkenyl, -C2-8 ynyl, cycloalkyl, heterocyclic, aryl, or heteroaryl groups are each optionally substituented by at least one substituent R. 3d replace;

[0024] R 3a R 3b and R 3c Each of the following is independently hydrogen, -C1-8 alkyl, -C2-8 alkenyl, -C2-8 ynyl, cycloalkyl, heterocyclic, aryl, or heteroaryl, wherein the -C1-8 alkyl, -C2-8 alkenyl, -C2-8 ynyl, cycloalkyl, heterocyclic, aryl, or heteroaryl is optionally substituent for at least one substituent R. 3e replace;

[0025] R 3d and R 3e Each of these can be independently hydrogen, halogen, -C1-8 alkyl, -C2-8 alkenyl, -C2-8 ynyl, cycloalkyl, heterocyclic, 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 The -C1-8 alkyl, -C2-8 alkenyl, -C2-8 ynyl, cycloalkyl, heterocyclic, aryl, or heteroaryl groups are each optionally selected from at least one group selected from halogen, -C1-8 alkyl, -OR 3i -NR 3i R 3j Substitution with cycloalkyl, heterocyclic, aryl, or heteroaryl substituents;

[0026] R 3f R 3g R 3h R 3i and R 3j Each of them is independently hydrogen, -C1-8 alkyl, C1-8 alkoxy-C1-8 alkyl-, -C2-8 alkenyl, -C2-8 alkynyl, cycloalkyl, heterocyclic, aryl or heteroaryl;

[0027] Ring A is selected from: 6-10 substituted or unsubstituted aryl groups, 5-10 substituted or unsubstituted heteroaryl groups.

[0028] Furthermore, the compounds described in (I) are selected from the compounds in Table 1:

[0029] Table 1

[0030]

[0031]

[0032] The present invention also provides the use of compounds of formula (I) in the preparation of HPK1 and / or LCK kinase modulators.

[0033] The compounds of formula (I) of this invention can modulate HPK1 and / or LCK kinases and can be used to prepare dual-target modulators that target HPK1 and / or LCK kinases.

[0034] The present invention also provides a method for inhibiting HPK1 and / or LCK, the method comprising effectively contacting an effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition thereof, with HPK1 and / or LCK.

[0035] The present invention also provides a method for preparing compounds of formula (I), comprising the following steps:

[0036]

[0037] Pro1 and Pro2 are conventional protecting groups in organic synthesis, and coupling 1 and coupling 2 are conventional CC coupling or CN coupling in organic synthesis. Other variables are as described in formula (I).

[0038] For example, the compound of formula (I) can be synthesized as shown in Scheme I. Compound (1) is protected to obtain compound (2), and compound (2) is reacted with boric acid under palladium catalysis to give compound (3); if the Pro1 protecting group does not automatically deprotect, Pro2 protection is not required; if the Pro1 protecting group deprotects automatically, Pro2 protection is required to obtain compound (4); compound (4) can be used for a further coupling under a transition metal and with an L1 group, which may be aryl, heterocyclic, etc., to obtain compound of formula (I); if Pro2 deprotects itself, compound of formula (I) is obtained directly; otherwise, Pro2 needs to be deprotected to obtain compound of formula (I).

[0039] The pharmaceutically acceptable salts described in this invention include acid addition salts and base addition salts.

[0040] The acid addition salts include, but are not limited to, salts derived from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, and phosphonic acid, as well as salts derived from organic acids such as aliphatic monocarboxylic acids and dicarboxylic acids, phenyl-substituted alkanic acids, hydroxyalkanic acids, alkanedioic acids, aromatic acids, and aliphatic and aromatic sulfonic acids. Therefore, these salts include, but are not limited to, sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, nitrates, phosphoric acids, monohydrogen phosphates, dihydrogen phosphates, metaphosphates, pyrophosphates, hydrochlorides, hydrobromates, iodates, acetates, propionates, caprylates, isobutyrates, oxalates, malonates, succinates, octanoic acid, sebacic acid, fumarates, maleates, amygdalinates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, phthalates, benzenesulfonates, toluenesulfonates, phenylacetates, citrates, lactates, maleates, tartaric acid, and methanesulfonates, as well as salts of amino acids such as arginine salts, gluconates, galacturonic acids, etc. Acid addition salts can be prepared by contacting a free base in a sufficient quantity of the desired acid in a conventional manner to form a salt. The free base can be regenerated by contacting the salt with a base and then separated in a conventional manner.

[0041] The base addition salts form with metals or amines, such as hydroxides of alkali metals and alkaline earth metals, or with organic amines. Examples of metals used as cations include, but are not limited to, sodium, potassium, magnesium, and calcium. Suitable amines include, but are not limited to, N,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine (ethane-1,2-diamine), N-methylglucosamine, and procaine. Base addition salts can be prepared by contacting the free acid form with a sufficient amount of the desired base in a conventional manner to form a salt. The free acid form can be regenerated by contacting the salt form with an acid, and the free acid can be separated in a conventional manner.

[0042] The stereoisomers described in this invention include enantiomers, diastereomers, and geometric isomers. Some compounds of this invention have cycloalkyl groups that can be substituted at more than one carbon atom; in this case, all their geometric forms, including cis and trans, and mixtures thereof, are within the scope of this invention.

[0043] The solvates described in this invention refer to the physical combination of the compounds of this invention with one or more solvent molecules. This physical combination includes various degrees of ionic and covalent bonding, including hydrogen bonding. In some cases, solvates can be isolated, for example when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid. "Solvate" includes both solution-phase and separable solvates. Representative solvates include ethanolides, methanolides, etc. A "hydrate" is a solvate in which one or more solvent molecules are H₂O.

[0044] The prodrugs described in this invention refer to compounds of formula (I) that are suitable for administration to patients without excessive toxicity, irritation, or allergic reactions, and are effective for their intended use, including acetals, esters, and zwitterionic forms. The prodrugs are converted in vivo (e.g., by hydrolysis in the blood) to yield the parent compound of the above formula.

[0045] The present invention also provides a pharmaceutical composition comprising a compound of formula (I) or a stereoisomer, tautomer, or pharmaceutically acceptable salt, hydrate, solvate, or PROTAC chimera thereof, and further comprising pharmaceutically acceptable excipients. The excipients are selected from: carriers, diluents, binders, lubricants, and wetting agents.

[0046] Preferably, the pharmaceutical composition further comprises a chemotherapeutic agent; wherein the chemotherapeutic agent is an immunotherapeutic agent.

[0047] Preferably, the pharmaceutical composition comprises a therapeutically effective amount of a compound of formula (I).

[0048] In some embodiments, these pharmaceutical compositions can be used to treat HPK1 and / or LCK kinase-mediated conditions or illnesses. The HPK1 and / or LCK kinase modulators of the present invention can also be incorporated into pharmaceutical compositions that further comprise compounds that can be used to treat cancer or other HPK1 and / or LCK kinase-mediated conditions.

[0049] The compounds of formula (I) of the present invention can be formulated into pharmaceutical compositions in the following forms: syrups, elixirs, suspensions, powders, granules, tablets, capsules, lozenges, aqueous solutions, creams, ointments, lotions, gels, emulsions, etc.

[0050] Pharmaceutical formulations are preferably unit dosage forms. In this form, the formulation is further divided into unit doses containing appropriate amounts of the active ingredient. A unit dosage form can be a packaged formulation containing discrete amounts of the formulation, such as tablets, capsules, and powders packaged in vials or ampoules. Alternatively, a unit dosage form can be a capsule, tablet, or any of these dosage forms in appropriate numbers in a packaged form.

[0051] The amount of active ingredient in a unit dose formulation can be varied or adjusted from 0.1 mg to 1000 mg, depending on the specific application and potency of the active ingredient. If desired, the composition may also contain other suitable therapeutic agents.

[0052] The pharmaceutical carrier may be determined in part according to the specific composition used and according to the specific method of administration of the composition. Therefore, the pharmaceutical compositions of the present invention exist in various suitable formulations.

[0053] The compounds of formula (I) of the present invention, alone or in combination with other suitable components, are formulated as aerosols (i.e., they can be “nebulized”) for administration via inhalation. The aerosols may be placed in an acceptable pressurized propellant such as dichlorodifluorohexane, propane, nitrogen, etc.

[0054] Formulations suitable for non-gastrointestinal administration, such as via intravenous, intramuscular, intradermal, and subcutaneous routes, include aqueous and non-aqueous isotonic sterile injections that may contain antioxidants, buffers, bacteriostatic agents, and solutes that make the formulation isotonic with the recipient's blood, as well as aqueous and non-aqueous sterile suspensions that may contain suspending agents, solubilizers, thickeners, stabilizers, and preservatives. In the practice of this invention, the compositions can be administered, for example, by intravenous infusion, oral, topical, intraperitoneal, intravesical, and intrathecal administration. Formulations of the compounds may be present in single-dose or multi-dose sealed containers such as ampoules and vials. Injectable solutions and suspensions may be prepared from sterile powders, granules, and tablets of the types previously described.

[0055] In the context of this invention, the dosage administered to the subject should be sufficient to produce a beneficial therapeutic response in the subject over time. The dosage is determined by the potency of the specific compound used and the condition of the subject, as well as the weight or body surface area of ​​the subject to be treated. The dosage will be determined based on the presence, nature, and extent of any adverse side effects accompanying the administration of the specific compound to the subject. In determining the effective amount of the compound to be administered in the treatment or prevention of a disease being treated, the physician may evaluate factors such as the circulating plasma level of the compound, the toxicity of the compound, and / or the disease progression.

[0056] The present invention also provides compounds of formula (I) or pharmaceutically acceptable salts, stereoisomers, esters, prodrugs, solvates or deuterated compounds thereof for the preparation of treatments, prevention and relief of diseases caused by overactivation of HPK1 and / or LCK kinases.

[0057] The present invention also provides the use of compounds of formula (I) or pharmaceutically acceptable salts, stereoisomers, esters, prodrugs, solvates or deuterated compounds thereof in the preparation of medicaments for the prevention and / or treatment of cancer.

[0058] The present invention also provides the use of compounds of formula (I) or pharmaceutically acceptable salts, stereoisomers, esters, prodrugs, solvates or deuterated compounds thereof in combination with PD-1, PD-L1, CTLA-4, TIM-3, TGF-β and their receptors, LAG3 antagonists or TLR4, TLR7, TLR8, TLR9, STING agonists in the preparation of medicaments for cancer immunotherapy.

[0059] The present invention 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 preparation of a medicament for cancer immunotherapy.

[0060] The present invention also provides the use of compounds of formula (I) or pharmaceutically acceptable salts, stereoisomers, esters, prodrugs, solvates or deuterated compounds thereof in combination with CAR-T immunotherapy in cancer immunotherapy.

[0061] The CAR-T immunotherapy mentioned refers to chimeric antigen receptor T-cell immunotherapy, which is one of the more effective treatments for malignant tumors. Its basic principle is to use the patient's own immune cells to eliminate cancer cells, and it belongs to a type of cell therapy.

[0062] The cancers described in this invention include lymphoma, blastoma, medulloblastoma, retinoblastoma, sarcoma, liposarcoma, synovial cell sarcoma, neuroendocrine tumors, carcinoid tumors, gastrinoma, islet cell carcinoma, mesothelioma, schwannoma, acoustic neuroma, meningioma, adenocarcinoma, melanoma, leukemia or lymphoid malignancies, squamous cell carcinoma, epithelial squamous cell carcinoma, lung cancer, small cell lung cancer, non-small cell lung cancer, adenocarcinoma, lung squamous cell carcinoma, peritoneal carcinoma, 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 cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, Merkel cell carcinoma, esophageal cancer, biliary tract tumors, head and neck cancers, and hematologic malignancies.

[0063] The hematologic malignancies described in this invention include, but are not limited to, acute T-lymphoblastic leukemia (T-ALL), chronic T-lymphoblastic leukemia, acute B-lymphoblastic leukemia, chronic B-lymphoblastic leukemia, plasma cell tumors, multiple myeloma, macroglobulinemia, Jekin's lymphoma, non-Hodgkin's lymphoma, essential thrombocytosis, and polycythemia vera.

[0064] Compared with the prior art, the present invention has the following advantages:

[0065] This invention combines immunotherapy with targeting HPK1 and / or LCK kinases, significantly promoting cytokine release and revitalizing T cell function. The HPK1 and / or LCK kinase-targeting compounds (such as those of formula (I)) provided by this invention exhibit good activity against immune factor release. Therefore, they can be used to prepare treatments, prevent and alleviate diseases caused by excessive activation of HPK1 and / or LCK kinases, or as lead compounds for designing candidate molecules with higher activity. Furthermore, the synthesis method of the HPK1 and / or LCK kinase modulators provided by this invention uses inexpensive and readily available raw materials, operates under mild conditions, is simple to operate, exhibits high regioselectivity, and achieves high yields, making it suitable for industrial production. Detailed Implementation

[0066] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0067] Example 1: Synthesis of Compound 1

[0068]

[0069] Step 1: Synthesis of Intermediate 1-1

[0070]

[0071] Triethylamine (5.05 g, 5.0 eq.) was added to 5-bromo-3-iodo-7-azaindole (3.21 g, 1.0 eq.) and di-tert-butyl dicarbonate (6.54 g, 3.0 eq.) in tetrahydrofuran (100 mL). 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 1-1 (3.4 g, 81%).

[0072] Step 2: Synthesis of intermediate 1-2

[0073]

[0074] K₂CO₃ (4.14 g, 3.0 eq.) and Pd(dppf)Cl₂ (365.5 mg, 0.015 eq.) were added to a mixture of compound 1-1 (3 g, 1.0 eq.) and 3,4-dimethoxyphenylboronic acid (1.30 g, 1.0 eq.) in dioxane (100 mL) and water (50 mL). 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 1-2 (1.8 mg, 78%).

[0075] Step 3: Synthesis of intermediates 1-3

[0076]

[0077] Compound 1-3 was prepared from compound 1-2 in a manner similar to that described in step 1 of Example 1.

[0078] Step 4: Synthesis of Compound 1

[0079] K₂CO₃ (245.04 mg, 3.0 eq.) and Pd(dppf)Cl₂ (21.95 mg, 0.015 eq.) were added to a mixture of compounds 1-3 (1.5 g, 1.0 eq.) and (4-(4-(tert-butoxycarbonyl)piperazin-1-yl)phenyl)boronic acid (1.38 g, 1.0 eq.) in dioxane (30 mL) and water (6 mL). 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 = 50:1) to give compound 1 (1.5 g, 65%).

[0080] ESI + -MS(m / z): 515.36.11 [M+H] + . 1 H NMR (400MHz, DMSO) δ11.85(s,1H),8.51(d,J=2.0Hz,1H),8.32(d,J=1.9Hz,1H),7.82(d,J=2.5Hz,1H),7.62(d,J=8.7Hz,2H), 7.35–7.26(m,2H),7.05(dd,J=8.4,6.9Hz,3H),3.86(s,3H),3.79(s,3H),3.54–3.44(m,4H),3.20–3.09(m,4H),1.43(s,9H).

[0081] Example 2: Synthesis of Compound 2

[0082]

[0083] 100 mL of ethyl acetate was added to compound 1 (200 mg, 1.0 eq.), followed by 20 mL of ethyl acetate solution saturated with hydrogen chloride. The mixture was stirred at room temperature for 4 hours. The reaction solution was concentrated under reduced pressure, and 100 mL of methanol and 10 mL of triethylamine were added. The mixture was stirred for 1 hour and then concentrated under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane:MeOH = 5:1) to give compound 2 (150 mg, 93%).

[0084] ESI + -MS(m / z): 415.36 [M+H] + . 1 H NMR (400MHz, DMSO) δ11.86(s,1H),8.51(d,J=1.7Hz,1H),8.31(d,J=1.6Hz,1H),7.82(s,1H),7.62(d,J=8.6H z,2H),7.34–7.24(m,2H),7.10–7.00(m,3H),3.86(s,3H),3.79(s,3H),3.26–3.15(m,4H),3.03–2.90(m,4H).

[0085] Example 3: Synthesis of Compound 3

[0086]

[0087] Compound 3 (160 mg, 80%) was prepared from compounds 1-3 (200 mg, 1.0 eq.) and 4-(4-methyl-1-piperazinyl)phenylboronic acid (101.85 mg, 1.0 eq.) in a manner similar to that described in step 4 of Example 1. ESI+-MS (m / z): 429.34 [M+H] + . 1 H NMR (400MHz, DMSO) δ11.84(s,1H),8.50(d,J=1.3Hz,1H),8.31(d,J=1.0Hz,1H),7.82(d,J=2.0Hz,1H),7.60(d,J=8.5Hz,2H),7.34 –7.25(m,2H),7.04(dd,J=8.1,4.9Hz,3H),3.86(s,3H),3.79(s,3H),3.21–3.13(m,4H),2.49–2.41(m,J=3.9Hz,4H),2.23(s,3H).

[0088] Example 4: Synthesis of Compound 4

[0089]

[0090] Compound 4 (170 mg, 85%) was prepared from compounds 1-3 (200 mg, 1.0 eq.) and (6-(4-methylpiperazin-1-yl)pyridin-3-yl)boronic acid (102.31 mg, 1.0 eq.) in a manner similar to that described in step 4 of Example 1. ESI+-MS (m / z): 430.36 [M+H] + . 1 H NMR (400MHz, DMSO) δ11.93(s,1H),8.51(dd,J=3.6,2.5Hz,2H),8.34(d,J=1.9Hz,1H),7.94(dd,J=8.8,2.5Hz,1H),7.83(d,J=2.4Hz,1H),7. 37–7.25(m,2H),7.03(d,J=8.3Hz,1H),6.93(d,J=8.9Hz,1H),3.86(s,3H),3.79(s,3H),3.58–3.50(m,4H),2.47–2.36(m,4H),2.22(s,3H).

[0091] Example 5: Synthesis of Compound 5

[0092]

[0093] Compound 5 (200 mg, 74%) was prepared from compounds 1-3 (200 mg, 1.0 eq.) and 1-methyl-4-((4-(4,4,5,5-tetramethyl-1,3,2-dioxoboronyl-2-yl)phenyl)sulfonyl)piperazine (169.44 mg, 1.0 eq.) in a manner similar to that described in step 4 of Example 1. + -MS(m / z): 493.25 [M+H] + . 1 H NMR (400MHz, DMSO) δ12.05(s,1H),8.66(s,1H),8.52(s,1H),8.06(d,J=7.8Hz,2H),7.95–7.87(m,1H),7.81(d,J=7.8Hz,2 H),7.43–7.25(m,2H),7.05(d,J=8.0Hz,1H),3.87(s,3H),3.80(s,3H),3.02–2.84(m,4H),2.44–2.32(m,4H),2.14(s,3H).

[0094] Example 6: Synthesis of Compound 6

[0095]

[0096] Compound 6 (150 mg, 65%) was prepared from compounds 1-3 (200 mg, 1.0 eq.) and 4-(4-tetrahydropyranyl)phenylboronic acid pinacol ester (133.33 mg, 1.0 eq.) in a manner similar to that described in step 4 of Example 1. ESI+-MS (m / z): 415.35 [M+H] + . 1 H NMR (400MHz, DMSO) δ11.91(s,1H),8.55(d,J=1.2Hz,1H),8.38(s,1H),7.85(d,J=2.0Hz,1H),7.68(d,J=8.0Hz,2H),7.41–7.27(m,4H),7.04( d,J=8.2Hz,1H),4.01–3.93(m,J=10.7Hz,2H),3.86(s,3H),3.80(s,3H),3.45(td,J=11.0,5.4Hz,2H),2.88–2.74(m,1H),1.77–1.64(m,4H).

[0097] Example 7: Synthesis of Compound 7

[0098]

[0099] Compound 7 (160 mg, 70%) was prepared from compounds 1-3 (200 mg, 1.0 eq.) and (6-morpholinopyridin-3-yl)boronic acid (96.23 mg, 1.0 eq.) in a manner similar to that described in step 4 of Example 1. ESI+-MS (m / z): 417.21 [M+H] + . 1 H NMR (400MHz, DMSO) δ11.90(s,1H),8.54(dd,J=11.0,2.1Hz,2H),8.36(d,J=1.8Hz,1H),7.98(dd,J=8.8,2.5Hz,1H),7.84(d,J=2.5Hz, 1H),7.37–7.26(m,2H),7.04(d,J=8.3Hz,1H),6.94(d,J=8.8Hz,1H),3.87(s,3H),3.80(s,3H),3.77–3.68(m,4H),3.55–3.45(m,4H).

[0100] Example 8: Synthesis of Compound 8

[0101]

[0102] Compound 8 (200 mg, 83%) was prepared from compounds 1-3 (200 mg, 1.0 eq.) and 6-(4-Boc-1-piperazinyl)pyridine-3-boronic acid pinacol ester (180.09 mg, 1.0 eq.) in a manner similar to that described in step 4 of Example 1. ESI+-MS (m / z): 516.32 [M+H] + . 1 H NMR (400MHz, DMSO) δ11.88(s,1H),8.52(dd,J=9.4,1.4Hz,2H),8.35(s,1H),7.97(dd,J=8.8,1.9Hz,1H),7.83(d,J=2.0Hz,1H),7.37–7.25( m,2H),7.03(d,J=8.2Hz,1H),6.96(d,J=8.8Hz,1H),3.86(s,3H),3.79(s,3H),3.59–3.51(m,J=5.0Hz,4H),3.50–3.41(m,4H),1.44(s,9H).

[0103] Example 9: Synthesis of Compound 9

[0104]

[0105] Compound 9 (140 mg, 87%) was prepared from compound 8 (200 mg, 1.0 eq.) in a manner similar to that described in Example 2. ESI+-MS (m / z): 416.37 [M+H] + . 1H NMR (400MHz, DMSO) δ11.90(s,1H),8.52(d,J=4.6Hz,2H),8.35(s,1H),7.96(dd,J=8.5,1.7Hz,1H),7.83(s,1H),7.38–7. 22(m,2H),7.04(d,J=8.1Hz,1H),6.94(d,J=8.8Hz,1H),3.87(s,3H),3.80(s,3H),3.57–3.52(m,4H),2.97–2.81(m,4H).

[0106] Example 10: Synthesis of Compound 10

[0107]

[0108] Compound 10 (120 mg, 64%) was prepared from compounds 1-3 (200 mg, 1.0 eq.) and 4-(dimethylcarbamoyl)phenylboronic acid (89.35 mg, 1.0 eq.) in a manner similar to that described in step 4 of Example 1. ESI+-MS (m / z): 402.27 [M+H] + . 1 H NMR (400MHz, DMSO) δ11.97(s,1H),8.61(d,J=1.9Hz,1H),8.45(d,J=1.7Hz,1H),7.85(dd,J=13.9,5.3Hz,3H),7.52(d,J=8. 2Hz,2H),7.34(dd,J=8.2,1.8Hz,1H),7.30(d,J=1.6Hz,1H),7.05(d,J=8.3Hz,1H),3.87(s,3H),3.80(s,3H),3.00(s,6H).

[0109] Example 11: Synthesis of Compound 11

[0110]

[0111] Compound 11 (150 mg, 77%) was prepared from compounds 1-3 (200 mg, 1.0 eq.) and 1-methyl-4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxoboronyl-2-yl)-1H-pyrazol-1-yl)piperidine (134.72 mg, 1.0 eq.) in a manner similar to that described in step 4 of Example 1. ESI+-MS (m / z): 418.34 [M+H] + . 1 H NMR (400MHz, DMSO) δ11.80(s,1H),8.55(d,J=1.8Hz,1H),8.36(s,2H),8.00(s,1H),7.78(d,J=2.4Hz,1H),7.32–7.24(m,2H),7.04(d,J=8. 3Hz,1H),4.23–4.13(m,1H),3.88(s,3H),3.80(s,3H),3.01–2.94(m,J=11.5Hz,2H),2.32(s,3H),2.25(t,J=9.3Hz,2H),2.11–2.02(m,4H).

[0112] Example 12: Synthesis of Compound 12

[0113]

[0114] Compound 12 (120 mg, 62%) was prepared from compounds 1-3 (200 mg, 1.0 eq.) and 4-(4-morpholino)phenylboronic acid (95.83 mg, 1.0 eq.) in a manner similar to that described in step 4 of Example 1. ESI+-MS (m / z): 416.31 [M+H] + . 1 H NMR (400MHz, DMSO) δ11.85(s,1H),8.52(d,J=2.0Hz,1H),8.32(d,J=1.8Hz,1H),7.82(d,J=2.5Hz,1H),7.62(d,J= 8.7Hz,2H),7.35–7.26(m,2H),7.10–7.00(m,3H),3.86(s,3H),3.80(s,3H),3.78–3.71(m,4H),3.18–3.10(m,4H).

[0115] Example 13: Synthesis of Compound 13

[0116]

[0117] Compound 13 (135 mg, 73%) was prepared from compounds 1-3 (200 mg, 1.0 eq.) and 4-(2H-tetrazol-5-yl)phenylboronic acid pinacol ester (125.92 mg, 1.0 eq.) in a manner similar to that described in step 4 of Example 1. ESI+-MS (m / z): 399.28 [M+H] + . 1 H NMR (400MHz, DMSO) δ12.01(s,1H),8.69(d,J=1.1Hz,1H),8.53(s,1H),8.17(d,J=8.1Hz,2H),8.05(d,J=8.1Hz,2H) ,7.89(d,J=1.3Hz,1H),7.36(dd,J=8.2,1.2Hz,1H),7.32(s,1H),7.06(d,J=8.3Hz,1H),3.89(s,3H),3.81(s,3H).

[0118] Example 14: Synthesis of Compound 14

[0119]

[0120] Compound 14 (170 mg, 83%) was prepared from compounds 1-3 (200 mg, 1.0 eq.) and pinacol 4-(4-methyl-1-piperazinmethyl)phenylboronic acid (146.41 mg, 1.0 eq.) in a manner similar to that described in step 4 of Example 1. ESI + -MS(m / z): 443.39 [M+H] + . 1 H NMR (400MHz, DMSO) δ11.92(s,1H),8.56(d,J=1.6Hz,1H),8.39(d,J=1.3Hz,1H),7.85(d,J=2.1Hz,1H),7.70(d,J=8.0Hz,2H),7.39(d,J=7.9 Hz,2H),7.35–7.26(m,2H),7.04(d,J=8.2Hz,1H),3.86(s,3H),3.80(s ,3H),3.49(s,2H),3.44–3.34(m,4H),2.41–2.32(m,4H),2.16(s,3H).

[0121] Example 15: Synthesis of Compound 15

[0122]

[0123] Compound 15 (155 mg, 78%) was prepared from compounds 1-3 (200 mg, 1.0 eq.) and 4-morpholine methylphenylboronic acid (102.31 mg, 1.0 eq.) in a manner similar to that described in step 4 of Example 1. ESI + -MS(m / z): 430.30 [M+H] + . 1 H NMR (400MHz, DMSO) δ11.92(s,1H),8.56(d,J=2.0Hz,1H),8.39(d,J=2.0Hz,1H),7.85(s,1H),7.71(d,J=8.1Hz,2H),7.41(d,J=8. 1Hz,2H),7.36–7.23(m,2H),7.04(d,J=8.3Hz,1H),3.86(s,3H),3.80(s,3H),3.65–3.54(m,4H),3.50(s,2H),2.45–2.30(m,4H).

[0124] Example 16: Synthesis of Compound 16

[0125]

[0126] Compound 16 (160 mg, 80%) was prepared from compounds 1-3 (200 mg, 1.0 eq.) and 4-(1-methyl-4-piperidinyl)phenylboronic acid pinacol ester (139.35 mg, 1.0 eq.) in a manner similar to that described in step 4 of Example 1. ESI + -MS(m / z): 428.33 [M+H] + . 1 H NMR (400MHz, DMSO) δ11.92(s,1H),8.54(s,1H),8.37(s,1H),7.84(s,1H),7.67(d,J=5.5Hz,2H),7.31(dd,J=19.0,5.5Hz,4H),7.04(d,J=7.0H z,1H),3.86(s,3H),3.79(s,3H),3.09–2.93(m,J=7.1Hz,2H),2.62–2. 54(m,J=0.7Hz,1H),2.33(s,3H),2.27–2.14(m,2H),1.87–1.68(m,4H).

[0127] Example 17: Synthesis of Compound 17

[0128]

[0129] Compound 17 (145 mg, 72%) was prepared from compounds 1-3 (200 mg, 1.0 eq.) and (2-(4-methylpiperazin-1-yl)pyrimidin-5-yl)boronic acid (102.78 mg, 1.0 eq.) in a manner similar to that described in step 4 of Example 1. ESI + -MS(m / z): 431.26 [M+H] + . 1 H NMR (400MHz, DMSO) δ11.93(s,1H),8.78(s,2H),8.51(d,J=1.7Hz,1H),8.40(s,1H),7.84(d,J=2.2Hz,1H),7.32 (dd,J=12.2,3.9Hz,2H),7.02(d,J=8.3Hz,1H),3.87(s,3H),3.83–3.75(m,7H),2.45–2.34(m,4H),2.23(s,3H).

[0130] Example 18: Synthesis of Compound 18

[0131]

[0132] Compound 18 (150 mg, 81%) was prepared from compounds 1-3 (200 mg, 1.0 eq.) and 2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborhecyclopentan-2-yl)phenyl)-1,3,4-oxadiazole (125.92 mg, 1.0 eq.) in a manner similar to that described in step 4 of Example 1. + -MS(m / z): 399.36 [M+H] + . 1 H NMR (400MHz, DMSO) δ12.01(s,1H),9.37(s,1H),8.66(s,1H),8.51(s,1H),8.12(d,J=8.0Hz,2H),8.0 2(d,J=8.1Hz,2H),7.88(s,1H),7.37–7.26(m,2H),7.05(d,J=8.2Hz,1H),3.87(s,3H),3.80(s,3H).

[0133] Example 19: Synthesis of Compound 19

[0134]

[0135] Compound 9 (200 mg, 1.0 eq.) and 1-iodo-2-methoxyethane (44.82 mg, 0.5 eq.) were added to acetonitrile (30 mL) with K₂CO₃ (245.04 mg, 3.0 eq.). The reaction mixture was stirred at 60 °C for 4 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 = 10:1) to give compound 19 (80 mg, 70%). ESI + -MS(m / z): 443.39 [M+H] + . 1 H NMR (400MHz, DMSO) δ11.88(d,J=1.7Hz,1H),8.51(dd,J=3.6,2.4Hz,2H),8.34(d,J=1.8Hz,1H),7.94(dd,J=8.8,2.5Hz,1H),7.83(d,J=2.4Hz,1H),7.37– 7.26(m,2H),7.03(d,J=8.3Hz,1H),6.93(d,J=8.9Hz,1H),3.86(s,3H),3.79 (s,3H),3.53(s,4H),3.40–3.28(m,6H),3.25(s,3H),1.17(t,J=7.1Hz,2H).

[0136] Example 20: Synthesis of Compound 20

[0137]

[0138] Compound 20 (95 mg, 81%) was prepared from compound 9 (200 mg, 1.0 eq.) and dimethylcarbamoyl chloride (25.78 mg, 0.5 eq.) in a manner similar to that described in Example 19. (ESI) + -MS(m / z): 487.35 [M+H] + . 1 H NMR (400MHz, DMSO) δ11.89(s,1H),8.52(d,J=8.9Hz,2H),8.35(s,1H),7.97(d,J=8.7Hz,1H),7.83(s,1H),7.43–7.2 3(m,2H),6.99(dd,J=33.6,8.3Hz,2H),3.86(s,3H),3.79(s,3H),3.60–3.51(m,4H),3.28–3.19(m,4H),2.79(s,6H).

[0139] Example 21: Synthesis of Compound 21

[0140]

[0141] Compound 21 (90 mg, 84%) was prepared from compound 9 (200 mg, 1.0 eq.) and acryloyl chloride (21.69 mg, 0.5 eq.) in a manner similar to that described in Example 19. (ESI) + -MS(m / z): 443.39 [M+H] + . 1 H NMR (400MHz, DMSO)

[0142] Example 22: Synthesis of Compound 22

[0143]

[0144] Compound 22 (80 mg, 71%) was prepared from compound 2 (200 mg, 1.0 eq.) and dimethylcarbamoyl chloride (24.71 mg, 0.5 eq.) in a manner similar to that described in Example 19. (ESI) + -MS(m / z): 486.25 [M+H] + . 1 H NMR (400MHz, DMSO) δ11.85(d,J=1.7Hz,1H),8.51(d,J=2.0Hz,1H),8.32(d,J=1.8Hz,1H),7.82(d,J=2.4Hz,1H),7.62(d,J=8.7Hz,2H), 7.37–7.25(m,2H),7.05(dd,J=8.5,2.5Hz,3H),3.86(s,3H),3.79(s,3H),3.30–3.23(m,4H),3.21–3.13(m,J=4.9Hz,4H),2.78(s,6H).

[0145] Other compounds described in this invention can be synthesized and prepared by referring to the examples above.

[0146] Biological activity assay

[0147] Experimental Example 1: Inhibition assay of HPK1 and LCK kinases

[0148] 1. Dilute the compound 3-fold with DMSO in a dilution plate, with an initial concentration of 1-10 μM.

[0149] 2. Dilute the compound 50-fold in 1x kinase reaction buffer and shake on a shaker for 20 minutes.

[0150] 3. Prepare 2x kinase using 1x enzyme reaction buffer.

[0151] 4. Add 2 μL of kinase (prepared in step 3) to each well of the reaction plate.

[0152] 5. Add 1 μL of the compound diluted in buffer to each well, seal the plate with sealing film, centrifuge at 1000g for 30 seconds, and let stand at room temperature for 10 minutes.

[0153] 6. Prepare a 4x MBP Protein and ATP (final ATP concentration 10 μM) mixture using 1x enzyme reaction buffer, and add 1 μL of the 4x MBP Protein / ATP mixture to the reaction plate.

[0154] 7. Seal the plate with sealing film, centrifuge at 1000g for 30 seconds, and react at room temperature for 60 minutes.

[0155] 8. Transfer 4 μL of ADP-Glo ​​to a 384 reaction plate, centrifuge at 1000 rpm / min for 1 min, and incubate at 25 °C for 40 min.

[0156] 9. Transfer 8 μL of Detection solution to a 384 reaction plate, centrifuge at 1000 rpm for 1 min, and incubate at 25 °C for 40 min.

[0157] 10. Use a Biotek multi-function plate reader to read the RLU (Relative Luminescence Unit) signal. The signal intensity is used to characterize the activity level of the kinase.

[0158] (3) Data processing

[0159] The inhibition rate for each well is calculated using the following formula:

[0160]

[0161] 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 DMSOs.

[0162] Computing IC 50 And plot the inhibition curve of the compound:

[0163] The ICso (half-maximal inhibitory concentration) of the compound was obtained using the following nonlinear fitting formula: data analysis was performed using Graphpad 9.3 software.

[0164] Y=Bottom+(Top-Bottom) / (1+10^((L ogIC 50-X)*Hill Slope))

[0165] X: Log value of compound concentration; Y: Inhibition rate (% inhibition).

[0166] (4) Experimental Results

[0167] The inhibitory activity of the compounds of this invention on HPK1 / LCK enzymes and their IL-2 stimulatory effect:

[0168] Experimental results:

[0169] Table 2

[0170]

[0171]

[0172] IC 50 In the IL-2 stimulation factor, A = <100 nM; B = 100–500 nM; C = 500–1000 nM; D = >1000 nM, A = >2; B = 1–2; C = <1

[0173] Some of the compounds in this invention have good inhibitory effects on HPK1, some have good inhibitory effects on LCK, and some can inhibit both HPK1 and LCK. In addition, some compounds show significant stimulatory effects on the cytokine IL-2, which can improve tumor immunity. This indicates that the compounds in this invention have good application potential for diseases caused by HPK1 and / or LCK kinases.

[0174] The above provides a detailed description of the HPK1 and / or LCK modifiers provided by this invention, their preparation, and their applications.

[0175] This document uses specific embodiments to illustrate the principles and implementation methods of the present invention. The descriptions of the embodiments above are merely for the purpose of helping to understand the method and central idea of ​​the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall under the protection of the claims of the present invention.

[0176] Although the foregoing invention has been described in detail by way of illustration and examples for purposes of clarity, it will be apparent to those skilled in the art that certain minor changes and modifications may be made. Therefore, the description and examples should not be construed as limiting the scope of the invention.

Claims

1. A compound or a pharmaceutically acceptable salt thereof, said compound having the structure shown below; 2. A pharmaceutical composition, characterized in that, It comprises the compound of claim 1 or a pharmaceutically acceptable salt thereof, and further 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 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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