A highly active HPK1 kinase inhibitor

Abstract

The present invention provides compounds having inhibitory activity against HPK1 kinase and pharmaceutical compositions comprising the compounds. The present invention also provides the use of the compounds in the prevention and / or treatment of cancer, tumor, inflammatory disease, autoimmune disease or immune-mediated disease.

Description

Technical Field

[0001] The present invention relates to a heterocyclic compound, in particular to a highly active HPK1 kinase inhibitor and application thereof. Background Art

[0002] HPK1 is a member of the MAP4K family. It is mainly expressed in hematopoietic cells and acts as an intracellular negative regulator of T cell proliferation and signal transduction. Antigen stimulation of T cells causes the cytoplasmic adaptor protein SLP-76 to be recruited to the lipid membrane TCR complex, providing binding sites for signal transduction-related kinases to achieve TCR-mediated signal transduction and induce T cell activation. In this process, HPK1 is activated by phosphorylation of tyrosine kinases Lck and Zap70, and participates in regulating T cell receptor protein interactions. HPK1 phosphorylates the Ser376 site of the adaptor protein SLP-76, allowing SLP-76 to bind to the scaffold protein 14-3-3ε and then be degraded by the proteasome. This effect reduces the binding of SLP-76 to signal transduction-related kinases and blocks TCR signal transduction, thereby inhibiting T cell activation and proliferation. On the other hand, HPK1 is also involved in regulating the maturation and activation of dendritic cells (DCs), especially inhibiting the expression of proteins such as CD80, CD86 and MHC complexes in DCs that assist T cell activation, thereby affecting the role of DCs in regulating T cell activation; and the presentation of tumor antigens by activated DCs and the cooperation between DCs and T cells are one of the most important links in the anti-tumor immune system. In addition, there are a large number of immunosuppressive molecules such as PGE2 and TGF-β in the tumor microenvironment, and the immunosuppressive effects mediated by these factors are also closely related to HPK1. In general, small molecule compounds that specifically target and inhibit HPK1 can inhibit tumor growth by enhancing anti-tumor immune effects through multiple pathways such as improving T cell function, enhancing DCs cell function and reversing the tumor immunosuppressive microenvironment. Summary of the invention

[0003] The present invention provides a compound capable of inhibiting HPK1 kinase activity and a pharmaceutically acceptable salt, isotope derivative and stereoisomer.

[0004]

[0005]

[0006] It is specifically noted that, herein, when referring to a "compound" having a specific structural formula, stereoisomers, diastereomers, enantiomers, racemic mixtures and isotopic derivatives thereof are generally also encompassed.

[0007] It is well-known to those skilled in the art that salts, solvates, and hydrates of a compound are alternative forms of existence of the compound, and they can all be converted into the compound under certain conditions. Therefore, it should be particularly noted that when referring to a compound in this article, generally its pharmaceutically acceptable salts are also included, and further its solvates and hydrates are included.

[0008] Similarly, when referring to a compound in this article, generally its prodrugs, metabolites, and N-oxides are also included.

[0009] The pharmaceutically acceptable salts described in the present invention can be formed using, for example, the following inorganic or organic acids: "Pharmaceutically acceptable salts" refer to salts that, within the scope of reasonable medical judgment, are suitable for contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic reaction, etc., and have a reasonable benefit / risk ratio. The salts can be prepared in situ during the final isolation and purification of the compounds of the present invention, or separately by reacting the free base or free acid with a suitable reagent, as outlined below. For example, the free base function can react with a suitable acid. In addition, when the compounds of the present invention carry an acidic moiety, suitable pharmaceutically acceptable salts can include metal salts, such as alkali metal salts (e.g., sodium or potassium salts); and alkaline earth metal salts (e.g., calcium or magnesium salts). Examples of pharmaceutically acceptable non-toxic acid addition salts are salts formed by the reaction of an amino group with an inorganic acid (e.g., hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid) or an organic acid (e.g., acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid), or salts formed by using other methods in the prior art such as ion exchange. Other pharmaceutically acceptable salts include adipates, alginates, ascorbates, aspartates, benzenesulfonates, benzoates, bisulfates, borates, butyrates, camphorates, camphorsulfonates, citrates, cyclopentanepropionates, digluconates, dodecyl sulfates, ethanesulfonates, formates, fumarates, glucoheptonates, glycerophosphates, gluconates, hernisulfates, heptanoates, hexanoates, hydroiodides, 2-hydroxyethanesulfonates, lactates, lactobionates, laurates, lauryl sulfates, malates, maleates, malonates, methanesulfonates, 2-naphthalenesulfonates, nicotinates, nitrates, oleates, oxalates, palmitates, pamoates, pectates, persulfates, 3-phenylpropionates, phosphates, picrates, pivalates, propionates, stearates, succinates, sulfates, tartrates, thiocyanates, p-toluenesulfonates, undecanoates, valerates, etc. Representative alkali metal or alkaline earth metal salts include salts of sodium, lithium, potassium, calcium, magnesium, etc. Other pharmaceutically acceptable salts include (where appropriate) non-toxic ammonium salts, quaternary ammonium salts, and amine cations formed with counterions, such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates, and aryl sulfonates.

[0010] The pharmaceutically acceptable salts of the present invention can be prepared by conventional methods, for example, by dissolving the compounds of the present invention in a water-miscible organic solvent (such as acetone, methanol, ethanol, and acetonitrile), adding an aqueous solution of an excess of an organic or inorganic acid thereto such that the salt precipitates from the resulting mixture, removing the solvent and the remaining free acid therefrom, and then isolating the precipitated salt.

[0011] As used herein, the term "solvate" means a physical association of a compound of the present invention with one or more solvent molecules, whether organic or inorganic. This physical association includes hydrogen bonding. In some cases, for example, when one or more solvent molecules are incorporated into the lattice of a crystalline solid, the solvate will be capable of being isolated. The solvent molecules in the solvate can be present in a regular or disordered arrangement. Solvates can contain stoichiometric or non-stoichiometric amounts of solvent molecules. The term "solvate" encompasses both the solution phase and isolable solvates. Exemplary solvates include, but are not limited to, hydrates, ethanolates, methanolates, and isopropanolates. Solvation methods are well known in the art.

[0012] As used herein, the term "stereoisomerism" is divided into conformational isomerism and configurational isomerism. Configurational isomerism can be further divided into cis-trans isomerism and optical isomerism (i.e., enantiomerism). Conformational isomerism refers to a stereoisomeric phenomenon in which organic molecules with a certain configuration have different arrangements of atoms or groups in space due to the rotation or distortion of carbon-carbon single bonds. Common examples include the structures of alkanes and cycloalkanes, such as the chair and boat conformations in the structure of cyclohexane. "Stereoisomers" refer to compounds of the present invention that contain one or more asymmetric centers and can thus exist as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures, and single diastereomers. The compounds of the present invention have asymmetric centers, and each asymmetric center gives rise to two optical isomers. The scope of the present invention includes all possible optical isomers and diastereomeric mixtures and pure or partially pure compounds. The compounds described herein can exist in tautomeric forms, which have different hydrogen attachment points due to the displacement of one or more double bonds. For example, a ketone and its enol form are keto-enol tautomers. All tautomers and their mixtures are included in the compounds of the present invention. All enantiomers, diastereomers, racemates, meso forms, cis-trans isomers, tautomers, geometric isomers, epimers, and mixtures thereof of the compounds of formula (I) are included within the scope of the present invention.

[0013] The "isotope derivatives" of the present invention refer to molecules in which the compounds in this patent are isotope-labeled. Isotopes commonly used for isotope labeling are: hydrogen isotopes, 2 H and 3 H; carbon isotopes: 11 C, 13 C and 14C; chlorine isotopes: 35 Cl and 37 Cl; fluorine isotopes: 18 F; iodine isotopes: 123 I and 125 I; nitrogen isotopes: 13 N and 15 N; oxygen isotopes: 15 O, 17 O and 18 O and sulfur isotopes 35 S. These isotope-labeled compounds can be used to study the distribution of pharmaceutical molecules in tissues. In particular, deuterium 3 H and carbon 13 C are more widely used because they are easy to label and convenient to detect. Substitution of certain heavy isotopes, such as heavy hydrogen ( 2 H), can enhance metabolic stability, extend the half-life, and thus achieve the goal of reducing the dose and providing a therapeutic advantage. Isotope-labeled compounds generally start from labeled starting materials and are synthesized using known synthetic techniques in the same way as non-isotope-labeled compounds.

[0014] The present invention also provides the use of the compounds of the present invention in the preparation of drugs for preventing and / or treating cancer, tumors, inflammatory diseases, autoimmune diseases or immune-mediated diseases.

[0015] In addition, the present invention provides a pharmaceutical composition for preventing and / or treating cancer, tumors, inflammatory diseases, autoimmune diseases, neurodegenerative diseases, attention-related diseases or immune-mediated diseases, which comprises the compounds of the present invention as an active ingredient.

[0016] In addition, the present invention provides a method for preventing and / or treating cancer, tumors, inflammatory diseases, autoimmune diseases, neurodegenerative diseases, attention-related diseases or immune-mediated diseases, which comprises administering the compounds of the present invention to a mammal in need thereof.

[0017] When the compounds of the present invention or their pharmaceutically acceptable salts are administered in combination with another anti-cancer agent or immune checkpoint inhibitor for treating cancer or tumors, the compounds of the present invention or their pharmaceutically acceptable salts can provide enhanced anti-cancer effects.

[0018] When the compounds of the present invention or their pharmaceutically acceptable salts are administered in combination with another therapeutic agent for treating inflammatory diseases, autoimmune diseases and immune-mediated diseases, the compounds of the present invention or their pharmaceutically acceptable salts can provide enhanced therapeutic effects.

[0019] In the process of describing the exemplary embodiments of the present invention, other features of the present invention will become apparent. The described embodiments are for illustrative purposes of the present invention and are not intended to be limiting. The following examples are prepared, separated, and characterized using the methods disclosed in the present invention.

[0020] The compounds of the present invention can be prepared in a variety of ways known to those skilled in the art of organic synthesis. The present invention compounds can be synthesized using the following methods, as well as synthetic methods known in the field of organic synthetic chemistry or through variations thereof known to those skilled in the art. Preferred methods include but are not limited to those described below. The reactions are carried out in solvents or solvent mixtures suitable for the materials used in the kits and suitable for the transformations to be achieved. Those skilled in the art of organic synthesis will understand that the functionality present on the molecule is consistent with the proposed transformation. This sometimes requires making judgments to change the order of synthetic steps or starting materials to obtain the desired compounds of the present invention. Detailed Description

[0021] Examples

[0022] General Procedure

[0023] When the preparation routes are not included, the starting materials and reagents used in the present invention are all known products, which can be synthesized according to methods known in the art or obtained by purchasing commercially available products. Commercially available reagents used do not require further purification. Room temperature refers to 20 - 30 °C.

[0024] Unless otherwise specified in the reaction examples, the reactions are carried out under a nitrogen atmosphere. A nitrogen atmosphere means that the reaction flask is connected to a nitrogen balloon of about 1 L.

[0025] For hydrogenation reactions, usually vacuum is pumped, hydrogen is filled, and the operation is repeated 3 times. A hydrogen atmosphere means that the reaction flask is connected to a hydrogen balloon of about 1 L.

[0026] Microwave reactions use Initiator + microwave reactor.

[0027] The structures of the compounds of the present invention are determined by nuclear magnetic resonance (NMR) and mass spectrometry (MS). The NMR chemical shift (δ) is given in units of 10 -6 (ppm). The NMR measurements are performed using a (Bruker Ascend TM 500 type) nuclear magnetic resonance instrument, and the solvents for measurement are deuterated dimethyl sulfoxide (DMSO - d6), deuterated chloroform (CDCl 3 ), deuterated methanol (CD 3 OD), and the internal standard is tetramethylsilane (TMS). The following abbreviations are used for the multiplicity of NMR signals: s = singlet, brs = broad singlet, d = doublet, t = triplet, m = multiplet. The coupling constant is listed as the J value and measured in Hz.

[0028] For LC-MS determination, a Thermo liquid chromatography-mass spectrometry instrument (UltiMate 3000 + MSQ PLUS) was used. For HPLC determination, a Thermo high-performance liquid chromatography instrument (UltiMate 3000) was used. For reverse-phase preparative chromatography, a Thermo (UltiMate 3000) reverse-phase preparative chromatography instrument was used. For flash column chromatography, an Agela (FS-9200T) automatic column chromatography machine was used, and the silica gel pre-packed column used was Santai pre-packed column. For thin-layer chromatography silica gel plates, Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plates were used, and the specifications for thin-layer chromatography separation and purification of products were 0.4 mm to 0.5 mm.

[0029] The synthetic methods of some intermediates in the present invention are as follows:

[0030] Intermediate 1

[0031]

[0032] Intermediate 1 was prepared by the following steps:

[0033]

[0034] First step: Dissolve 1-methyl-3,5-dinitropyridin-2-one Int-1a (1.0 g, 5.02 mmol) in methanol (50 mL), and successively add ammonia methanol solution (7 mol / L, 8.61 mL, 60.27 mmol) and 1-methylpiperidin-4-one Int-1b (625 mg, 5.52 mmol). The reaction mixture was heated to 50 °C and stirred for 5 hours. After cooling to room temperature, it was left to stand for 48 hours, the reaction solution was concentrated under reduced pressure, and the residue was filtered after adding ethyl acetate (50 mL). The filtrate was concentrated under reduced pressure to obtain a red solid Int-1c (1.0 g), which was directly used for the next step of the reaction. ESI-MS (m / z): 194.4 [M+H] + ; 1 HNMR (500 MHz, DMSO-d 6 ) δ 9.14 (d, J = 2.5 Hz, 1H), 8.36 (d, J = 2.5 Hz, 1H), 3.64 (s, 2H), 3.02 (t, J = 6.0 Hz, 2H), 2.74 (t, J = 6.0 Hz, 2H), 2.39 (s, 3H).

[0035] Step 2: Dissolve the compound Int-1c (1.0 g) obtained in the previous step in methanol (30 mL), add 10% Pd-C (400 mg), and react at room temperature for 6 hours under a hydrogen atmosphere. Filter off the palladium carbon, and concentrate the filtrate to obtain a yellow solid Int-1 (800 mg, yield 94.70%). ESI-MS (m / z): 164.2 [M+H] + 。

[0036] Intermediate 2

[0037]

[0038] Intermediate 2 is prepared by the following steps:

[0039]

[0040] Step 1: Dissolve the compound Int-1 (100 mg, 0.61 mmol) in acetic acid (3 mL), add N-bromosuccinimide (109 mg, 0.61 mmol), and stir the reaction mixture at room temperature for 1 hour. Quench the reaction by adding saturated aqueous sodium bicarbonate until no more bubbles are produced. Extract the aqueous phase with methanol / dichloromethane (1 / 20, 50 mL × 2), combine the organic phases, dry over anhydrous sodium sulfate, filter, and concentrate to obtain the compound Int-2a (38 mg, yield 25%). ESI-MS (m / z): 242.3 [M+H] + ; 1 HNMR (500 MHz, DMSO-d 6 ) δ 6.77 (s, 1H), 5.25 (s, 2H), 3.37 (s, 2H), 2.69 (t, J = 6.0 Hz, 2H), 2.60 (t, J = 6.0 Hz, 2H), 2.32 (s, 3H).

[0041] Step 2: Dissolve the compound Int-2a (37 mg, 0.15 mmol) in methanol (1 mL), add copper(I) iodide (3 mg, 0.015 mmol), 1,10-phenanthroline (3 mg, 0.03 mmol), and cesium carbonate (99 mg, 0.30 mmol). After displacing the reaction mixture with nitrogen, heat it to 100 °C with microwave irradiation and stir for 2 hours. Cool the reaction to room temperature, concentrate the reaction solution, and purify the residue by preparative thin-layer chromatography (methanol / dichloromethane / triethylamine = 1 / 10 / 0.1) to obtain a yellow solid Int-2 (20 mg, yield 67%). ESI-MS (m / z): 194.5 [M+H] + ; 1 HNMR (500 MHz, DMSO-d 6)δ 6.54 (s, 1H), 4.68 (s, 2H), 3.80 (s, 3H), 3.30 (s, 2H), 2.64 (t, J = 5.6 Hz, 2H), 2.59 (t, J = 5.7 Hz, 2H), 2.31 (s, 3H).

[0042] Intermediate 6

[0043]

[0044] Intermediate 6 is prepared by the following steps:

[0045]

[0046] The first step: Under the condition of an ice bath at 0 °C, formic acid (2.14 g, 46.57 mmol, 1.76 mL) was added dropwise to acetic anhydride (3.17 g, 31.05 mmol, 2.93 mL), and then the mixture was stirred at room temperature for 1 hour. Then the mixture was cooled to 0 °C again and added dropwise to a solution of Int-2 (500 mg, 2.59 mmol) in tetrahydrofuran (10 mL) (at 0 °C), and then stirred at room temperature for 30 minutes. The reaction solution was diluted with dichloromethane and washed three times with saturated sodium bicarbonate solution. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (dichloromethane / methanol = 10 / 1) to obtain a white solid Int-6 (550 mg, yield 96%). ESI-MS (m / z): 222.5 [M + H] + .

[0047] Intermediate 8

[0048]

[0049] Intermediate 8 is prepared by the following steps:

[0050]

[0051] The first step: Compound Int-8a (300 mg, 1.42 mmol) was dissolved in dichloromethane (10 mL), and m-CPBA (604 mg, 85% content, 2.98 mmol) was added under an ice bath. After the addition was complete, the reaction was continued under an ice bath for 4 hours, and the raw material reaction was detected to be complete by LCMS. The reaction solution was concentrated, and the residue was purified by silica gel column chromatography to obtain a pale yellow solid Int-8 (300 mg, yield 86%). ESI-MS (m / z): 244.3 [M + H] + .

[0052] Intermediate 9

[0053]

[0054] Intermediate 9 was prepared by the following steps:

[0055]

[0056] The first step: Dissolve compound Int-6 (230 mg, 1.04 mmol) in anhydrous DMF (10 mL), and add NaH (42 mg, 60% content, 1.04 mmol) under ice bath. After the mixture was stirred at room temperature for 30 minutes, it was cooled to 0 °C, and a solution of Int-8 (244 mg, 1.14 mmol) in DMF (3 mL) was added dropwise. After the dropwise addition, the reaction was carried out at room temperature for 2 hours, and the raw material reaction was detected to be complete by LCMS. Add 0.1 N NaOH solution (1 mL) to the reaction solution and stir at room temperature for 1 hour. Pour the reaction solution into water (40 mL), yellow solid precipitated, filter and collect the solid, and dry to obtain Int-9 (230 mg, yield 62%). ESI-MS (m / z): 357.2 [M+H] + 。

[0057] Example 17

[0058] 1-(4-(2-((2-Methoxy-6-methyl-5,6,7,8-tetrahydro-1,6-naphthyridin-3-yl)amino)quinazolin-8-yl)-3,6-dihydropyridin-1(2H)-yl)ethan-1-one

[0059]

[0060] Compound 17 was prepared by the following steps:

[0061]

[0062] The first step: Dissolve 2-chloro-8-bromoquinazoline 1a (220 mg, 0.90 mmol) and compound 17a (307 mg, 0.99 mmol) in a mixed solvent of 1,4-dioxane (4 mL) and water (0.4 mL), add sodium carbonate (191 mg, 1.81 mmol) and Pd(dppf)Cl 2 (66 mg, 90 umol), after replacing the nitrogen in the reaction system, heat to 90 °C and stir for 16 hours. After the reaction solution was cooled to room temperature, the reaction solution was filtered through diatomaceous earth, and the filtrate was concentrated. The residue was separated by column chromatography (petroleum ether / ethyl acetate = 5 / 1) to obtain compound 17b (260 mg, yield 83%). ESI-MS (m / z): 346.3 [M+H] + 。

[0063] Step 2: Dissolve compound 17b (200 mg, 0.57 mmol) in dichloromethane (2 mL), add dioxane hydrochloride solution (4 N, 0.72 mL), and stir the reaction mixture at room temperature for 16 h. Concentrate the reaction solution to obtain compound 17c (160 mg, crude product), which is directly used in the next step of the reaction.

[0064] Step 3: Dissolve the compound 17c (160 mg) obtained in the previous step in tetrahydrofuran (5 mL), and sequentially add N,N - diisopropylethylamine (219 mg, 1.70 mmol, 0.29 mL) and acetyl chloride (67 mg, 0.85 mmol) at 0 °C. Stir the reaction mixture at 0 °C for 1 h. Dilute the reaction solution with ethyl acetate, wash it with water, dry the organic phase with anhydrous sodium sulfate, filter and concentrate. Purify the residue by silica gel column chromatography (dichloromethane / methanol = 10 / 1) to obtain compound 17d (120 mg, yield 72% for two - step conversion). ESI - MS (m / z): 288.3 [M + H] + 。

[0065] Step 4: Dissolve compound 17d (44 mg, 0.15 mmol) and Int - 2 (20 mg, 0.10 mmol) in 1,4 - dioxane (2 mL), add BrettPhos Pd G3 (9 mg, 10 μmol), BrettPhos (11 mg, 20 μmol) and cesium carbonate (67 mg, 0.20 mmol). After replacing the nitrogen in the reaction system, heat it to 100 °C and stir for 16 h. After the reaction solution is cooled to room temperature, filter the reaction solution through diatomaceous earth, and concentrate the filtrate. Purify the residue by Prep - TLC, and then purify the crude product by Prep - HPLC to obtain compound 17 (1.45 mg, yield 3%). ESI - MS (m / z): 445.4 [M + H] + ; 1 HNMR(500MHz,DMSO - d 6)δ9.29(d, J = 1.7 Hz, 1H), 8.40(s, 0.5H), 8.35(s, 0.5H), 8.18(s, 0.5H), 8.14(s, 0.5H), 7.91 - 7.81(m, 1H), 7.65(t, J = 6.1 Hz, 1H), 7.37(dd, J = 7.7, 3.4 Hz, 1H), 5.96(d, J = 3.4 Hz, 1H), 4.17(brs, 1H), 4.09(br s, 1H), 3.89(s, 3H), 3.62(t, J = 5.7 Hz, 1H), 3.58(t, J = 5.6 Hz, 1H), 3.40(s, 2H), 2.84 - 2.74(m, 2H), 2.70 - 2.55(m, 4H), 2.36(s, 3H), 2.08(s, 3H).

[0066] Example 25

[0067] N-(2-Methoxy-6-methyl-5,6,7,8-tetrahydro-1,6-naphthyridin-3-yl)-8-(2-methoxyphenyl)pyrido[3,4-d]

[0068] pyrimidin-2-amine

[0069]

[0070] Compound 25 was prepared by the following steps:

[0071]

[0072] First step: Dissolve Int-9 (50 mg, 0.14 mmol) and 2-methoxyphenylboronic acid (32 mg, 0.21 mmol) in a mixed solution of THF (10 mL) and water (2 mL), add [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane complex (11 mg, 14 umol), and sodium carbonate (29 mg, 0.28 mmol). After replacing the reaction system with nitrogen, heat it to 60 °C and stir overnight, and detect the formation of the product by LCMS. Concentrate the reaction solution, and purify the residue by Prep-HPLC to obtain yellow solid 25 (24 mg, yield 41%). ESI-MS (m / z): 429.1 [M + H] + ; 1 HNMR(500MHz, DMSO-d 6)δ9.49(s,1H),8.55(d,J=5.3Hz,1H),8.33(s,1H),8.05(s,1H),7.85(d,J=5.4Hz,1H),7.58-7.50(m,1H),7.34(dd,J=7.4,1.6Hz,1H),7.23(d,J=8.3Hz,1H),7.14(t,J=7.4Hz,1H),3.90(s,3H),3.59(s,3H),3.08(s,2H),2.71(t,J=5.9Hz,2H),2.61(t,J=5.9Hz,2H),2.40(s,3H).

[0073] Biological Screening and Results of HPK1 Inhibitors

[0074] Test Example 1: Detection of the Inhibitory Ability of Compounds on HPK1 Kinase Activity (Method 1)

[0075] The reagents required are as follows

[0076]

[0077] Experimental Procedures

[0078] The specific operations are as follows: Prepare the buffer for the enzymatic reaction system (10 mM MOPS, pH 7.2, 5 mM β-glycerol-phosphate, 10 mM MgCl2, 0.8 mM EDTA, 2 mM EGTA, 0.1 mM DTT); Dilute the test compound (a 1 mM compound stock solution in DMSO) with the buffer to a maximum concentration of 60 μM (including 6% DMSO), and prepare a gradient concentration of the compound with 5-fold dilutions starting from a 60 μM concentration using the buffer containing 6% DMSO for a total of 8 points; Subsequently, dilute HPK1 kinase with the buffer to 30 nM. Add 2 μl of the diluted HPK1 kinase solution to each well in a Greiner 384-well microplate (product number: 784075), and supplement 2 μl of the buffer in the control wells; After brief centrifugation, add 1 μl of the diluted compound to the reaction wells and 1 μl of the buffer containing 6% DMSO to the control wells; After brief centrifugation, place them in an incubator at 25 °C (Shanghai Yiheng Scientific Instrument Co., Ltd., product number: LRH-150) and incubate for 20 min. Add 3 μl of the reaction substrate (10 μM MBP and 20 μM ATP dissolved in distilled water) to each well, after brief centrifugation, place them in an incubator at 25 °C and incubate for 60 min, and use the ADP-Glo Kinase Assay Kit to detect the enzymatic reaction activity. The detection using the ADP-Glo Kinase Assay Kit is carried out according to the operation instructions of the kit. The data is described by the half-maximal inhibitory concentration IC50 of the compound.

[0079] Compound number IC50 (nM) 17 <0.1 25 0.16

[0080] Experimental Example 2: Detection of the ability of a compound to stimulate the secretion of the cytokine interleukin-2 (IL-2) by Jurkat cells (Method 2)

[0081] The reagents and cells required are as follows

[0082] Experimental reagents:

[0083]

[0084] Experimental cells:

[0085]

[0086]

[0087] Experimental procedure

[0088] The specific operations are as follows: Dissolve the compound powder in DMSO to 10 mM. Take 2 μl of the compound and add it to 998 μl of RPMI 1640 medium (containing 10% FBS in this experiment). After vortexing and mixing, it is the highest concentration point. Gradually dilute the compound solution 3-fold with 0.2% DMSO medium, with a total of 8 concentration points. Use the RPMI 1640 medium solution containing 0.1% DMSO as a control. Add 1×10 5 Jurkat E6-1 cells, and then add an equal volume of the compound dilution. The control group is added with RPMI 1640 medium containing 0.2% DMSO, and incubated in a 37°C cell culture incubator (Thermo Fisher Scientific, model: 3111) for 1 h. Then add Anti-human CD3 Antibody and Anti-human CD28 Antibody at a final concentration of 1 μg / ml, and incubate in a 37°C cell culture incubator for 24 h. Use the Human IL-2 DuoSet ELISA KIT to detect the IL-2 content in the cell supernatant, and the Human IL-2 DuoSet ELISA detection is carried out according to the operation instructions of the kit. The data is described by the highest multiple ratio of the stimulation signal of the compound to the signal of 0.1% DMSO.

[0089]

[0090] NA: Indicates that no enhanced release of IL-2 was detected.

Claims

1. A compound having the following structure or a pharmaceutically acceptable salt thereof: 。 2. A pharmaceutical composition comprising the compound according to claim 1 and a pharmaceutically acceptable carrier.

3. Use of the compound according to claim 1 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition according to claim 2 in the preparation of a medicament for preventing and / or treating cancer, tumor, inflammatory disease, autoimmune disease or immune-mediated disease.

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