Compounds for preparing HPK1 kinase inhibitors and synthesis methods thereof

Abstract

The present invention provides an intermediate compound Int-9 for preparing a compound having HPK1 kinase inhibitory activity, a method for preparing the intermediate compound, and its use in preparing compounds 33, 25, 70, 76 and 77 having HPK1 kinase inhibitory activity.

Description

[0001] This application is a divisional application of the invention application with application date of October 26, 2021, application number 202180070254.9, and invention name “A highly active HPK1 kinase inhibitor”. Technical Field

[0002] The present invention relates to a heterocyclic compound, in particular to a highly active HPK1 kinase inhibitor and use thereof. Background Art

[0003] HPK1, a member of the MAP4K family, is primarily expressed in hematopoietic cells and serves as an intracellular negative regulator of T cell proliferation and signaling. Antigen stimulation of T cells triggers the recruitment of the cytoplasmic adaptor protein SLP-76 to the lipid membrane TCR complex, providing binding sites for signal transduction kinases to facilitate TCR-mediated signaling and induce T cell activation. During this process, HPK1 is activated by phosphorylation by the tyrosine kinases Lck and Zap70, mediating T cell receptor protein interactions. HPK1 phosphorylates the adaptor protein SLP-76 at Ser376, enabling SLP-76 to bind to the scaffold protein 14-3-3ε and subsequently be degraded by the proteasome. This effect reduces SLP-76 binding to signal transduction kinases, blocking TCR signaling and subsequently inhibiting T cell activation and proliferation. HPK1 also regulates the maturation and activation of dendritic cells (DCs). Specifically, it inhibits the expression of T cell activation-related proteins such as CD80, CD86, and MHC complexes in DCs, thereby impairing DCs' ability to regulate T cell activation. The presentation of tumor antigens by activated DCs and the collaborative efforts between DCs and T cells are crucial components of the anti-tumor immune system. Furthermore, the tumor microenvironment is home to numerous immunosuppressive molecules, such as progesterone (PGE2) and transforming growth factor-β (TGF-β), whose immunosuppressive effects are also closely linked to HPK1. Overall, small molecule compounds that specifically inhibit HPK1 could potentially suppress tumor growth by enhancing anti-tumor immunity through multiple pathways, primarily by improving T cell function, enhancing DC function, and reversing the tumor's immunosuppressive microenvironment. Summary of the Invention

[0004] In one aspect, the present invention provides an intermediate for preparing a compound having HPK1 kinase inhibitory activity, having the following structure:

[0005]

[0006] In another aspect, the present invention provides a method for preparing the intermediate Int-9, characterized in that it comprises the following steps:

[0007]

[0008] Compound Int-6 (230 mg, 1.04 mmol) was dissolved in anhydrous DMF (10 mL) and NaH (42 mg, 60% content, 1.04 mmol) was added under ice bath. After the mixture was stirred at room temperature for 30 minutes, it was cooled to 0°C and a DMF (3 mL) solution of Int-8 (244 mg, 1.14 mmol) was added dropwise. After the addition was complete, the reaction was allowed to proceed at room temperature for 2 hours. LCMS detected that the reaction of the raw material was complete. 0.1N NaOH solution (1 mL) was added to the reaction solution and stirred at room temperature for 1 hour. The reaction solution was poured into water (40 mL), and a yellow solid precipitated. The solid was collected by filtration and dried to obtain Int-9.

[0009] Preferably, int-8 is prepared by the following method:

[0010]

[0011] Compound Int-8a (300 mg, 1.42 mmol) was dissolved in dichloromethane (10 mL). m-CPBA (604 mg, 85% content, 2.98 mmol) was added under ice-cooling. After completion of the addition, the reaction was continued under ice-cooling for 4 hours. LCMS confirmed complete reaction. The reaction solution was concentrated, and the residue was purified by silica gel column chromatography to yield Int-8 as a pale yellow solid.

[0012] Preferably, int-6 is prepared by the following method:

[0013]

[0014] 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) under ice bath conditions at 0°C, and then heated to room temperature and stirred for 1 hour. The mixture was then recooled to 0°C and added dropwise to a solution of Int-2 (500 mg, 2.59 mmol) in tetrahydrofuran (10 mL) (0°C), and then heated to room temperature and stirred 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 Int-6 as a white solid.

[0015] More preferably, int-2 is prepared by the following method:

[0016]

[0017] Compound Int-1 (100 mg, 0.61 mmol) was dissolved in acetic acid (3 mL), and N-bromosuccinimide (109 mg, 0.61 mmol) was added. The reaction mixture was stirred at room temperature for 1 hour. The reaction was quenched by adding saturated aqueous sodium bicarbonate until bubbling ceased. The aqueous phase was extracted with methanol / dichloromethane (1 / 20, 50 mL x 2). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to yield compound Int-2a.

[0018] Compound Int-2a (37 mg, 0.15 mmol) was dissolved in methanol (1 mL), and cuprous iodide (3 mg, 0.015 mmol), 1,10-phenanthroline (3 mg, 0.03 mmol), and cesium carbonate (99 mg, 0.30 mmol) were added. The reaction mixture was purged with nitrogen and heated to 100°C in a microwave oven and stirred for 2 hours. The reaction mixture was cooled to room temperature, concentrated, and the residue was purified by preparative thin-layer chromatography (methanol / dichloromethane / triethylamine = 1 / 10 / 0.1) to afford Int-2 as a yellow solid.

[0019] More preferably, int-1 is prepared by the following method:

[0020]

[0021] 1-Methyl-3,5-dinitropyridin-2-one Int-1a (1.0 g, 5.02 mmol) was dissolved in methanol (50 mL). Ammonia methanol solution (7 mol / L, 8.61 mL, 60.27 mmol) and 1-methylpiperidin-4-one Int-1b (625 mg, 5.52 mmol) were added sequentially. The reaction mixture was heated to 50°C and stirred for 5 hours. After cooling to room temperature and allowing to stand for 48 hours, the reaction mixture was concentrated under reduced pressure. The residue was added with ethyl acetate (50 mL) and filtered. The filtrate was concentrated under reduced pressure to obtain Int-1c (1.0 g), a red solid, which was used directly in the next reaction.

[0022] Compound Int-1c (1.0 g) obtained in the previous step was dissolved in methanol (30 mL), and 10% Pd-C (400 mg) was added. The mixture was reacted at room temperature under a hydrogen atmosphere for 6 hours. The palladium-carbon was removed by filtration, and the filtrate was concentrated to yield Int-1.

[0023] In another aspect, the present invention provides the use of the intermediate Int-9 in the preparation of a compound having HPK1 kinase activity inhibition, wherein the compound having HPK1 kinase activity inhibition is selected from the following structures:

[0024]

[0025] Other features of the present invention will become apparent as the present invention describes exemplary embodiments, which are given to illustrate the present invention and are not intended to be limiting thereof. The following examples were prepared, isolated, and characterized using the methods disclosed herein.

[0026] 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 compounds of the present invention can be synthesized using the following methods and synthetic methods known in the field of organic synthetic chemistry or variations thereof known to those skilled in the art. Preferred methods include, but are not limited to, those described below. The reaction is carried out in a solvent or solvent mixture suitable for the kit materials used and for the desired transformation. It will be understood by those skilled in the art of organic synthesis that the functionality present on the molecule is consistent with the proposed transformation. This sometimes requires judgment to change the order of the synthesis steps or the raw materials to obtain the desired compounds of the present invention. DETAILED DESCRIPTION

[0027] Example

[0028] General Process

[0029] Unless a preparation route is provided, the raw materials and reagents used in the present invention are known products and can be synthesized according to methods known in the art or obtained by purchasing commercial products. No further purification is required for the commercially available reagents used. Room temperature refers to 20-30°C.

[0030] Unless otherwise specified in the reaction examples, all reactions were carried out under a nitrogen atmosphere, which means that the reaction flask was connected to a nitrogen balloon of approximately 1 L.

[0031] The hydrogenation reaction is usually carried out by evacuating the flask and filling it with hydrogen, and this operation is repeated three times. The hydrogen atmosphere means that the reaction flask is connected to a hydrogen balloon of about 1L.

[0032] Microwave reaction use Initiator + microwave reactor.

[0033] The structures of the compounds of the present invention were determined by nuclear magnetic resonance (NMR) and mass spectrometry (MS). -6 The unit of ppm is given. NMR measurements were performed using a Bruker Ascend TM The NMR spectra were obtained using a 500 nm NMR spectrometer. The solvents used were deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), and deuterated methanol (CD3OD), with tetramethylsilane (TMS) as the internal standard. The following abbreviations are used for NMR signal multiplicities: s = singlet, brs = broad, d = doublet, t = triplet, and m = multiplet. Coupling constants are listed as J values and are measured in Hz.

[0034] LC-MS analysis was performed using a Thermo LC-MS / MS instrument (UltiMate 3000+MSQ PLUS). HPLC analysis was performed using a Thermo HPLC instrument (UltiMate 3000). Reverse-phase preparative chromatography was performed using a Thermo HPLC instrument (UltiMate 3000). Flash column chromatography was performed using an Agilent FS-9200T automatic column analyzer, and silica gel prepacked columns were performed using a Santai HPLC instrument. Pre-packed columns. Thin layer chromatography silica gel plates use Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plates. The specifications used for thin layer chromatography separation and purification products are 0.4mm to 0.5mm.

[0035] The synthetic methods of some intermediates in the present invention are as follows:

[0036] Intermediate 1

[0037]

[0038] Intermediate 1 was prepared by the following steps:

[0039]

[0040] Step 1: Dissolve 1-methyl-3,5-dinitropyridine-2-one Int-1a (1.0 g, 5.02 mmol) in methanol (50 mL), and 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) in sequence. The reaction mixture was heated to 50 ° C and stirred for 5 hours. After cooling to room temperature and standing for 48 hours, the reaction solution was concentrated under reduced pressure, and the residue was added with ethyl acetate (50 mL) and filtered. The filtrate was concentrated under reduced pressure to obtain a red solid Int-1c (1.0 g), which was used directly in the next reaction. ESI-MS (m / z): 194.4 [M+H] + ; 1 HNMR (500MHz, DMSO-d6) δ9.14 (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).

[0041] 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 under a hydrogen atmosphere for 6 hours. Filter to remove the palladium-carbon, and concentrate the filtrate to obtain a yellow solid Int-1 (800 mg, 94.70% yield). ESI-MS (m / z): 164.2 [M+H] +.

[0042] Intermediate 2

[0043]

[0044] Intermediate 2 was prepared by the following steps:

[0045]

[0046] Step 1: Compound Int-1 (100 mg, 0.61 mmol) was dissolved in acetic acid (3 mL), and N-bromosuccinimide (109 mg, 0.61 mmol) was added. The reaction mixture was stirred at room temperature for 1 hour. Saturated aqueous sodium bicarbonate was added to quench the reaction until bubbles disappeared. The aqueous phase was extracted with methanol / dichloromethane (1 / 20, 50 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to afford compound Int-2a (38 mg, 25% yield). ESI-MS (m / z): 242.3 [M+H] + ; 1 HNMR (500MHz, DMSO-d6) δ6.77(s,1H),5.25(s,2H),3.37(s,2H),2.69(t,J=6.0Hz,2H),2.60(t,J=6.0Hz,2H),2.32(s,3H).

[0047] Step 2: Compound Int-2a (37 mg, 0.15 mmol) was dissolved in methanol (1 mL), and cuprous iodide (3 mg, 0.015 mmol), 1,10-phenanthroline (3 mg, 0.03 mmol), and cesium carbonate (99 mg, 0.30 mmol) were added. The reaction mixture was purged with nitrogen and heated to 100°C in a microwave oven and stirred for 2 hours. The reaction mixture was cooled to room temperature, concentrated, and the residue was purified by preparative thin-layer chromatography (methanol / dichloromethane / triethylamine = 1 / 10 / 0.1) to give Int-2 (20 mg, 67% yield) as a yellow solid. ESI-MS (m / z): 194.5 [M+H] + ; 1 HNMR (500MHz, DMSO-d6) δ6.54 (s, 1H), 4.68 (s, 2H), 3.80 (s, 3H), 3.30 (s, 2H), 2.64 (t, J = 5.6Hz, 2H), 2.59 (t, J = 5.7Hz, 2H), 2.31 (s, 3H).

[0048] Intermediate 6

[0049]

[0050] Intermediate 6 was prepared by the following steps:

[0051]

[0052] Step 1: 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) in an ice bath at 0°C, then allowed to warm to room temperature and stirred for 1 hour. The mixture was then recooled to 0°C and added dropwise to a solution of Int-2 (500 mg, 2.59 mmol) in tetrahydrofuran (10 mL) at 0°C, then allowed to warm to room temperature and stirred for 30 minutes. The reaction mixture 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 afford Int-6 (550 mg, 96% yield) as a white solid. ESI-MS (m / z): 222.5 [M+H] + .

[0053] Intermediate 8

[0054]

[0055] Intermediate 8 was prepared by the following steps:

[0056]

[0057] Step 1: Dissolve compound Int-8a (300 mg, 1.42 mmol) in dichloromethane (10 mL). Add m-CPBA (604 mg, 85% content, 2.98 mmol) under ice-cooling. Continue the reaction under ice-cooling for 4 hours. LCMS analysis indicates complete reaction. The reaction solution is concentrated, and the residue is purified by silica gel column chromatography to afford Int-8 (300 mg, 86% yield) as a pale yellow solid. ESI-MS (m / z): 244.3 [M+H] + .

[0058] Intermediate 9

[0059]

[0060] Intermediate 9 was prepared by the following steps:

[0061]

[0062] Step 1: 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 stirring the mixture at room temperature for 30 minutes, cool to 0°C and add a solution of Int-8 (244 mg, 1.14 mmol) in DMF (3 mL) dropwise. After the addition is complete, react at room temperature for 2 hours. LCMS shows that the reaction of the raw material is complete. 0.1N NaOH solution (1 mL) is added to the reaction solution and stirred at room temperature for 1 hour. The reaction solution is poured into water (40 mL), and a yellow solid precipitates. The solid is collected by filtration and dried to obtain Int-9 (230 mg, 62% yield). ESI-MS (m / z): 357.2 [M+H] + .

[0063] The synthesis method of the embodiment compounds of the present invention is as follows:

[0064] Example 25

[0065] N-(2-methoxy-6-methyl-5,6,7,8-tetrahydro-1,6-naphthyridin-3-yl)-8-(2-methoxyphenyl)pyrido[3,4-d]

[0066] Pyrimidine-2-amine

[0067]

[0068] Compound 25 was prepared by the following steps:

[0069]

[0070] Step 1: Int-9 (50 mg, 0.14 mmol) and 2-methoxyphenylboronic acid (32 mg, 0.21 mmol) were dissolved in a mixed solution of THF (10 mL) and water (2 mL), and [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane complex (11 mg, 14 umol) and sodium carbonate (29 mg, 0.28 mmol) were added. The reaction system was replaced with nitrogen and heated to 60°C and stirred overnight. LCMS detection of product formation was performed. The reaction solution was concentrated and the residue was purified by Prep-HPLC to obtain a yellow solid 25 (24 mg, 41% yield). ESI-MS (m / z): 429.1 [M+H] + ; 1HNMR(500MHz,DMSO-d6)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=7.4,1.6H z,1H),7.23(d,J=8.3Hz,1H),7.14(t,J=7.4Hz,1H),3.90(s,3H),3.59(s,3 H), 3.08 (s, 2H), 2.71 (t, J = 5.9Hz, 2H), 2.61 (t, J = 5.9Hz, 2H), 2.40 (s, 3H).

[0071] Example 33

[0072] N-(2-Methoxy-6-methyl-5,6,7,8-tetrahydro-1,6-naphthyridin-3-yl)-8-(piperidin-1-yl)pyrido[3,4-d]pyrimidine

[0073] -2-amine

[0074]

[0075] Compound 33 was prepared by the following steps:

[0076]

[0077] Step 1: Dissolve Int-9 (50 mg, 140 μmol) in N-methylpyrrolidone (5 mL) and add piperidine (178 mg, 2.1 mmol). Heat the reaction mixture to 120°C and stir for 2 hours. LCMS confirms the reaction is complete. The reaction mixture is concentrated and the residue is purified by Prep-HPLC to afford 33 (31 mg, 55% yield) as a yellow solid. ESI-MS (m / z): 406.5 [M+H] + ; 1 HNMR(500MHz,DMSO-d6)δ9.23(s,1H),8.59(s,1H),8.01-7.90(m,2H),7.12(d,J=5.4Hz,1H),3.87(s,3H),3.69(br s, 4H), 3.48 (s, 2H), 2.80 (t, J = 6.0Hz, 2H), 2.68 (t, J = 6.0Hz, 2H), 2.38 (s, 3H), 1.59 (br s, 6H).

[0078] Example 76

[0079] (R)-6-methyl-3-((8-(2-methylpiperidin-1-yl)pyrido[3,4-d]piperidin-2-yl)amino)-5,6,7,8-tetrahydro

[0080] -1,6-naphthyridin-2(1H)-one

[0081]

[0082] By replacing 3-amino-2,2-dimethyl-1-propanol in the first step of Example 70 with R-2-methylpiperidine, compound 76 can be obtained using a similar method and reaction steps. ESI-MS (m / z): 405.4 [M+H] + ; 1 H NMR (500MHz, DMSO-d6) δ11.97(s,1H),9.30(s,1H),8.41(s,1H),8.08(s,1H),8.03(d,J=5.3Hz,1H),7.19(d,J=5.3Hz,1H),5.13(br s,1H),4.15-4.06(m,1H),3.28-3.22(m,2H),2.65-2.55(m,4H),2.35(s,3H),2 .02-1.94(m,1H),1.80-1.75(m,2H),1.70-1.57(m,3H),1.10(d,J=6.8Hz,3H).

[0083] Example 77

[0084] 6-Methyl-3-((8-(((1-methylcyclopropyl)methyl)amino)pyrido[3,4-d]pyrimidin-2-yl)amino)-5,6,7,8-tetrahydro-1,6-naphthyridine-2(1H)-one

[0085]

[0086] By replacing 3-amino-2,2-dimethyl-1-propanol in the first step of Example 70 with 1-methylcyclopropylethylamine, compound 77 can be obtained using a similar method and reaction steps. ESI-MS (m / z): 392.2 [M+H] + ; 1H NMR (500MHz, DMSO-d6) δ11.95(s,1H),9.23(s,1H),8.42(s,1H),8.23(s,1H),7.83(d,J=5.6Hz,1H),6.90(d,J=5.7Hz,1H),6.74(br s,1H),3.45(d,J=5.7Hz,2H),3.34(s,2H),2.64-2.56(m,4H),2.34(s,3H),1.17(s,3H),0.63-0.57(m,2H),0.36-0.32(m,2H).

[0087] HPK1 inhibitor biological screening and results

[0088] Test Example 1: Detection of the Inhibitory Ability of Compounds on HPK1 Kinase Activity (Method 1)

[0089] The required reagents are as follows

[0090]

[0091] Experimental procedures

[0092] The specific operation is as follows: prepare the enzymatic reaction system buffer (10mM MOPS, pH 7.2, 5mMβ-glycerol-phosphate, 10mM MgCl2, 0.8mM EDTA, 2mM EGTA, 0.1mM DTT); dilute the test compound (1mM compound stock solution in DMSO) with buffer to a maximum concentration of 60uM (containing 6% DMSO), and prepare a gradient concentration of the compound starting from 60μM concentration and 5-fold dilution with buffer containing 6% DMSO for a total of 8 points; then use buffer to dilute HPK1 kinase to 30nM. In a Greiner 384-well microplate (Cat. No. 784075), 2 μl of HPK1 kinase dilution buffer was added to each well, and 2 μl of buffer was added to control wells. After brief centrifugation, 1 μl of diluted compound was added to the reaction wells, and 1 μl of buffer containing 6% DMSO was added to the control wells. After brief centrifugation, the plates were incubated in a 25°C incubator (Shanghai Yiheng Scientific Instrument Co., Ltd., Cat. No. LRH-150) for 20 minutes. 3 μl of substrate (10 μM MBP and 20 μM ATP dissolved in distilled water) was added to each well. After brief centrifugation, the plates were incubated in a 25°C incubator for 60 minutes. Enzyme activity was measured using the ADP-Glo Kinase Assay Kit, performed according to the kit's instructions. Data are expressed as the half-maximal inhibitory concentration (IC50) of the compounds.

[0093] Compound number IC50(nM) 25 0.16 33 0.52 70 0.29 76 <0.1 77 <0.1

[0094] Test Example 2: Detection of the ability of compounds to stimulate the secretion of cytokine interleukin-2 (IL-2) by Jurkat cells (Method 2)

[0095] The reagents and cells required are as follows

[0096] Experimental reagents:

[0097]

[0098] Experimental cells:

[0099]

[0100] Experimental procedures

[0101] The specific operation is as follows: Dissolve the compound powder in DMSO to 10mM, take 2μl of the compound and add it to 998μl of RPMI1640 medium (containing 10% FBS in this experiment), and vortex to mix thoroughly to obtain the highest concentration point. Dilute the compound solution 3-fold with 0.2% DMSO medium, for a total of 8 concentration points. Treat with RPMI 1640 medium containing 0.1% DMSO as a control. Add 1×10 cells / well to each well of a Corning 96-well cell culture plate (Cat. No. 3599). 5 Jurkat E6-1 cells were then treated with an equal volume of compound dilutions. A control group was treated with RPMI 1640 medium containing 0.2% DMSO and incubated at 37°C for 1 hour. Anti-human CD3 Antibody and Anti-human CD28 Antibody were then added at a final concentration of 1 μg / ml and incubated at 37°C for 24 hours. IL-2 levels in cell supernatants were measured using the Human IL-2 DuoSet ELISA Kit according to the kit's instructions. Data are presented as the highest fold ratio of the compound-stimulated signal to the 0.1% DMSO signal.

[0102]

[0103] NA: indicates that no enhancement of IL-2 release was detected.

[0104] Test Example 3: Detection of the ability of compounds to stimulate the secretion of cytokine interleukin-2 (IL-2) by mouse spleen cells (Method 3)

[0105] The reagents and cells required are as follows

[0106] Experimental reagents:

[0107]

[0108] Experimental animals:

[0109]

[0110] Experimental procedures

[0111] The specific operation is as follows: Compound powder is dissolved to 10mM in DMSO, 2μl of compound is added to 998μl of RPMI1640 medium (containing 10% FBS in this experiment), and vortexed to mix thoroughly as the highest concentration point. The compound solution is gradually diluted 3-fold with 0.2% DMSO medium, for a total of 8 concentration points. A solution containing 0.1% DMSO in RPMI 1640 medium is used as a control. 10 mice are added to each well of a Corning 96-well cell culture plate (Cat. No. 3599). 5 Spleen cells were then treated with an equal volume of compound dilutions. The control group was treated with RPMI 1640 medium containing 0.2% DMSO and incubated at 37°C in a cell culture incubator (Thermo Fisher Scientific, model: 3111) for 1 hour. Concanavalin A Cells were incubated at 37°C for 24 hours. IL-2 levels in cell supernatants were measured using the Mouse IL-2 DuoSet ELISA Kit according to the kit's instructions. Data are presented as the highest fold ratio of the compound-stimulated signal to the 0.1% DMSO-induced signal.

[0112]

[0113]

[0114] NA: indicates that no enhancement of IL-2 release was detected.

[0115] Test Example 4: Detection of the ability of compounds to stimulate the secretion of cytokine interleukin-6 (IL-6) by DC2.4 cells (Method 3)

[0116] The reagents and cells required are as follows

[0117] Experimental reagents:

[0118]

[0119] Experimental cells:

[0120]

[0121] Experimental procedures

[0122] The specific operation is as follows: Dissolve the compound powder in DMSO to 10mM, take 2μl of the compound and add it to 998μl of RPMI1640 medium (containing 10% FBS in this experiment), and vortex to mix it to obtain the highest concentration point. Dilute the compound solution 3 times with 0.2% DMSO medium, for a total of 8 concentration points. Use RPMI 1640 medium solution containing 0.1% DMSO as a control. Add 10 cells / well to each well of a Corning 96-well cell culture plate (Cat. No. 3599). 5 DC2.4 cells were then added with an equal volume of compound dilutions. The control group was added with RPMI 1640 medium containing 0.2% DMSO and incubated at 37°C in a cell culture incubator (Thermo Fisher Scientific, model: 3111) for 1 hour. LPS Cells were incubated at 37°C for 24 hours. IL-2 levels in cell supernatants were measured using the Mouse IL-6 DuoSet ELISA Kit according to the kit's instructions. Data are presented as the highest fold ratio of the compound-stimulated signal to the 0.1% DMSO-stimulated signal.

[0123]

[0124] NA: indicates that no enhancement of IL-6 release was detected.

[0125] Test Example 5: Detection of the ability of compounds to stimulate PBMC secretion of cytokine interleukin-2 (IL-2) (Method 5)

[0126] The reagents and cells required are as follows

[0127] Experimental reagents:

[0128]

[0129]

[0130] Experimental cells:

[0131]

[0132] Experimental procedures

[0133] The specific operation is as follows: Dissolve the compound powder in DMSO to 10mM, take 2μl of the compound and add it to 998μl of RPMI1640 medium (containing 10% FBS in this experiment), and vortex to mix thoroughly to obtain the highest concentration point. Dilute the compound solution 3-fold with 0.2% DMSO medium, for a total of 8 concentration points. Treat with RPMI 1640 medium containing 0.1% DMSO as a control. Add 1×10 cells / well to each well of a Corning 96-well cell culture plate (Cat. No. 3599). 5 PBMC cells were then treated with an equal volume of compound dilutions. A control group was treated with RPMI 1640 medium containing 0.2% DMSO and incubated in a 37°C cell culture incubator (Thermo Fisher Scientific, Model: 3111) for 1 hour. Anti-human CD3 Antibody and Anti-human CD28 Antibody were then added at a final concentration of 0.1 μg / ml and incubated at 37°C for 24 hours. IL-2 levels in cell supernatants were measured using the Human IL-2 DuoSet ELISA Kit. The Human IL-2 DuoSet ELISA assay was performed according to the kit's instructions. Data are presented as the highest fold ratio of the compound-stimulated signal to the 0.1% DMSO signal.

[0134]

[0135]

[0136] NA: indicates that no enhancement of IL-2 release was detected.

Claims

1. For preparing an intermediate compound Int-9 having HPK1 kinase inhibitory activity, having the following structure: Int-9.

2. Use of the intermediate compound Int-9 according to claim 1 in the preparation of a compound having HPK1 kinase activity inhibition, wherein the compound having HPK1 kinase activity inhibition is selected from the following structures: 25 33 76 77 。

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