HPK1 kinase inhibitor compounds

Abstract

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

Description

[0001] This application is a divisional application of the invention application with application date of March 22, 2022, application number 202280006669.4, and invention name “HPK1 kinase inhibitor compounds”. Technical Field

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

[0003] 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 DC cells that assist T cell activation, thereby affecting the role of DC 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 improve T cell function, enhance DC cell function, and reverse the tumor immunosuppressive microenvironment at the same time, and enhance anti-tumor immune effects through multiple pathways, thereby achieving the effect of inhibiting tumor growth. However, there is currently a lack of effective inhibitors of HPK1 kinase activity.

[0004] Therefore, there is still an urgent need for effective HPK1 kinase activity inhibitors in the prior art to provide more effective options for anti-tumor treatment. Summary of the invention

[0005] The present invention unexpectedly discovered a compound of formula (I) having the ability to inhibit HPK1 kinase activity and a pharmaceutically acceptable salt, isotope derivative or stereoisomer thereof, having the following structure:

[0006]

[0007] It should be noted in particular that, in this text, when referring to a "compound" with a specific structural formula, it generally also encompasses its stereoisomers, diastereoisomers, enantiomers, racemic mixtures, and isotopic derivatives.

[0008] As is well known to those skilled in the art, a salt, solvate, or hydrate of a compound is an alternative form of existence of the compound, and they can all be converted into the said compound under certain conditions. Therefore, it should be noted in particular that in this text, when referring to a compound, it generally also includes its pharmaceutically acceptable salts, and further includes its solvates and hydrates.

[0009] Similarly, in this text, when referring to a compound, it generally also includes its prodrugs, metabolites, and N-oxides.

[0010] The pharmaceutically acceptable salts of 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 response, etc., and represent 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 functionality can be reacted 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, glucoheptanoates, glycerophosphates, glucuronates, 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.

[0011] 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 (e.g., 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.

[0012] As used herein, "solvate" means a physical association of a compound of the invention with one or more solvent molecules, whether organic or inorganic. This physical association includes hydrogen bonding. In certain cases, such as 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 may be present in a regular arrangement and / or a disordered arrangement. Solvates may contain stoichiometric or non-stoichiometric amounts of solvent molecules. "Solvate" encompasses both 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.

[0013] As used herein, "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 an organic molecule with a certain configuration has different arrangements of its atoms or groups in space due to the rotation or distortion of carbon-carbon single bonds. Common examples are the structures of alkanes and cycloalkanes, such as the chair and boat conformations in the structure of cyclohexane. "Stereoisomers" mean that when the compounds of the invention contain one or more asymmetric centers, they can exist as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures, and single diastereomers. The compounds of the invention have asymmetric centers, and each asymmetric center gives rise to two optical isomers. The scope of the invention includes all possible optical isomers and diastereomeric mixtures and pure or partially pure compounds. The compounds described herein may 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 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 invention.

[0014] As used herein, "isotope derivatives" of the invention mean molecules in which the compounds in this patent are isotopically labeled. Isotopes commonly used for isotope labeling are: hydrogen isotopes, 2 H and 3 H; carbon isotopes: 11 C, 13 C and 14 C; chlorine isotopes: 35 Cl and 37 Cl; fluorine isotope: 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 deuterium ( 2 H), can enhance metabolic stability, extend the half-life, and thus achieve the goal of reducing the dose and providing therapeutic advantages. 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.

[0015] 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.

[0016] 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.

[0017] 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.

[0018] 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.

[0019] 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.

[0020] In the process of describing exemplary embodiments of the present invention, other features of the present invention will become obvious. The described embodiments are used to illustrate the present invention and are not intended to be limiting thereof. The following examples are prepared, separated and characterized using the methods disclosed in the present invention.

[0021] 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 following methods, as well as synthetic methods known in the field of organic synthetic chemistry or variations thereof understood by those skilled in the art, can be used to synthesize the compounds of the present invention. Preferred methods include, but are not limited to, those described below. The reactions are carried out in solvents or solvent mixtures that are suitable for the materials used in the kits and 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 the starting materials to obtain the desired compounds of the present invention.

[0022] Specific test methods Examples

[0023] General procedure

[0024] 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.

[0025] Room temperature refers to 20 - 30 °C.

[0026] 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.

[0027] For hydrogenation reactions, the system is usually evacuated, filled with hydrogen, and this operation is repeated 3 times. A hydrogen atmosphere means that the reaction flask is connected to a hydrogen balloon of about 1 L.

[0028] Microwave reactions use Initiator + microwave reactor.

[0029] 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 spectrometer, and the solvents used for the measurements are deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), deuterated methanol (CD3OD), 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 constants are listed as J values and measured in Hz.

[0030] The determination by LC-MS was performed using a Thermo liquid chromatography-mass spectrometry instrument (UltiMate 3000 + MSQ PLUS). The determination by HPLC was performed using a Thermo high-performance liquid chromatography instrument (UltiMate 3000). Reverse-phase preparative chromatography was carried out using a Thermo (UltiMate 3000) reverse-phase preparative chromatograph. Flash column chromatography was performed using an Agela (FS-9200T) automatic column chromatography machine, and the silica gel pre-packed column was used with Santai pre-packed column. The thin-layer chromatography silica gel plate was the Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plate, and the specifications used for the thin-layer chromatography separation and purification of the product were 0.4 mm to 0.5 mm.

[0031] The synthesis methods of some intermediates in the present invention are as follows:

[0032] Intermediate 1

[0033]

[0034] Intermediate 1 was prepared by the following steps:

[0035]

[0036] 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 standing 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 1H NMR (500 MHz, DMSO-d6) 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).

[0037] Second step: 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. The palladium carbon was filtered off, and the filtrate was concentrated to obtain a yellow solid Int-1d (800 mg, yield 94.70%). ESI-MS (m / z): 164.2 [M+H]+ 。

[0038] Step 3: Dissolve compound Int-1d (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 compound Int-1e (38 mg, yield 25%). ESI-MS (m / z): 242.3 [M+H] + ; 1 H NMR (500 MHz, DMSO-d6) 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).

[0039] Step 4: Dissolve compound Int-1e (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 purging 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-1 (20 mg, yield 67%). ESI-MS (m / z): 194.5 [M+H] + ; 1 H NMR (500 MHz, DMSO-d6) 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).

[0040] Example 10

[0041] 4-((5-Chloro-2-((2-methoxy-6-methyl-5,6,7,8-tetrahydro-1,6-naphthyridin-3-yl)amino)pyrimidin-4-

[0042] yl)amino)-3,3-dimethylbutanoic acid

[0043]

[0044] Compound 10 was prepared by the following steps:

[0045]

[0046] Step 1: Dissolve compound 10a (200 mg, 1.02 mmol) and 2,4,5-trichloropyrimidine (43 mg, 1.33 mmol) 1d in isopropanol (8 mL). Add DIPEA (264 mg, 2.04 mmol) to the above reaction solution, and stir the reaction solution at room temperature overnight. LCMS detection shows that the raw materials are completely converted. Concentrate the reaction solution to obtain a crude product, and purify it by silica gel column chromatography (petroleum ether / ethyl acetate = 3 / 1) to obtain colorless oily compound 10b (312 mg, yield 100%). ESI-MS (m / z): 306.3 [M+H] + 。

[0047] Step 2: Dissolve compound 10b (360 mg, 1.18 mmol) and Int-1 (227 mg, 1.18 mmol) in dioxane (10 mL). Sequentially add cesium carbonate (766 mg, 2.35 mmol), Brettphos Pd G3 (213 mg, 0.23 mmol) and Brettphos (126 mg, 0.23 mmol). Stir the reaction solution at 110 °C under a nitrogen atmosphere overnight. LCMS detection shows that the raw materials are completely converted. Concentrate the reaction solution to obtain a crude product, and purify it by silica gel column chromatography (methylene chloride / methanol = 10 / 1) to obtain brown oil 10c (280 mg, yield 51%). ESI-MS (m / z): 463.5 [M+H] + 。

[0048] Step 3: Dissolve compound 10c (260 mg, 0.56 mmol) in a mixed solution of tetrahydrofuran (5 mL) and water (5 mL). Add lithium hydroxide (23 mg, 0.56 mmol) to the above reaction solution, and stir the reaction solution at room temperature for four hours. Acidify the reaction solution to pH = 3 with 1N hydrochloric acid aqueous solution, and then extract with ethyl acetate. Concentrate the extracted aqueous phase to obtain a crude product. Purify the crude product by reverse-phase preparative HPLC to obtain compound 10 (3.59 mg, yield 1.47%). ESI-MS (m / z): 435.2 [M+H] + ; 1 H NMR (500 MHz, DMSO-d6) δ 8.13 (s, 1H), 7.95 (s, 1H), 7.62 (s, 1H), 7.25 (s, 1H), 3.88 (s, 2H), 3.45 (s, 3H), 3.38 (s, 2H), 2.75 (d, J = 6.0 Hz, 2H), 2.67 (d, J = 6.0 Hz, 2H), 2.35 (s, 3H), 2.15 (s, 2H), 0.96 (s, 6H).

[0049] Example 13

[0050] 1 - ((((5 - chloro - 2 - ((2 - methoxy - 6 - methyl - 5,6,7,8 - tetrahydro - 1,6 - naphthyridin - 3 - yl)amino)pyrimidin

[0051] -4 - yl)amino)methyl)cyclobutane - 1 - carboxylic acid

[0052]

[0053] Replace ethyl 3,3 - dimethyl - 4 - aminobutyrate hydrochloride in the first step of Example 10 with methyl 4 - (aminomethyl)cyclobutane carboxylate hydrochloride, and compound 13 can be obtained by a similar method and reaction steps. ESI - MS(m / z): 433.3[M + H] + ; 1 H NMR(500MHz, DMSO - d6)δ8.15(s, 1H), 7.97(s, 1H), 7.63(s, 1H), 7.01(t, J = 6.1Hz, 1H), 3.88(s, 3H), 3.75(d, J = 6.0Hz, 2H), 3.44(s, 2H), 2.75(t, J = 5.8Hz, 2H), 2.66(t, J = 5.8Hz, 2H), 2.35(s, 3H), 2.28 - 2.21(m, 2H), 2.06 - 1.99(m, 2H), 1.84 - 1.76(m, 2H).

[0054] Biological screening and results of HPK1 inhibitors

[0055] Test Example 1: Detection of the inhibitory ability of compounds on HPK1 kinase activity (Method 1)

[0056] The reagents required for use are as follows

[0057]

[0058]

[0059] Experimental procedure

[0060] The specific operations are as follows: Configure 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 tested compound (a 1 mM compound stock solution in DMSO) with the buffer to a maximum concentration of 60 μM (including 6% DMSO), and configure a gradient concentration of the compound with 8 points by 5-fold dilution starting from a 60 μM concentration using the buffer containing 6% DMSO; Subsequently, dilute the HPK1 kinase with the buffer to 30 nM. Add 2 μl of the HPK1 kinase dilution 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 it in a 25°C constant temperature incubator (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 it in a 25°C constant temperature incubator 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.

[0061]

[0062] The above results indicate that the compounds of the present invention have excellent inhibitory ability against HPK1 kinase activity.

[0063] Test Example 2: Detection of the ability of the compound to stimulate the secretion of cytokine interleukin-2 (IL-2) by Jurkat cells and the effect of the compound on the viability of Jurkat cells (Method 2)

[0064] The reagents and cells required for use are as follows:

[0065] Experimental reagents:

[0066]

[0067]

[0068] Experimental cells:

[0069] Cell Cell type Brand Jurkat E6-1 Human T lymphocyte leukemia cells Cell Bank of the Chinese Academy of Sciences

[0070] Experimental steps

[0071] 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 (both 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 1×105 Jurkat E6-1 cells to each well of a Corning 96-well cell culture plate (product number: 3599), and then add an equal volume of the compound dilution. The control group is added with RPMI 1640 medium containing 0.2% DMSO, and it is incubated in a 37°C cell culture incubator (Thermo Fisher Scientific, model: 3111) for 1 h. Subsequently, add Anti-human CD3 Antibody with a final concentration of 1 μg / ml and Anti-human CD28 Antibody with a final concentration of 1 μg / ml, and incubate in a 37°C cell culture incubator for 24 h. Collect the culture supernatant, and use the Human IL-2 DuoSet ELISA KIT to detect the IL-2 content in the cell supernatant. The detection of Human IL-2 DuoSet ELISA is carried out according to the operation instructions of the kit. The IL-2 secretion data is described by the highest multiple ratio of the stimulation signal of the compound to the signal of 0.1% DMSO; Collect the cells and use the Luminescent Cell Viability Assay kit to detect cell viability. The cell viability data is described by the half-maximal inhibitory concentration IC50 of the compound.

[0072]

[0073] NA: Indicates that no enhanced release of IL-2 was detected.

[0074] The above results show that compared with the control, the compound of the present invention significantly increases the level of the cytokine interleukin-2 secreted by Jurkat cells, and at the same time has no adverse effect on the cell viability of Jurkat.

[0075] Test Example 3: Detection of the ability of the compound to stimulate the secretion of the cytokine interleukin-2 (IL-2) by human PBMC cells. Effect of the compound on the viability of human PBMC cells (Method 3)

[0076] The reagents required for use are as follows

[0077]

[0078]

[0079] Experimental cell source information:

[0080]

[0081] Experimental procedures

[0082] The specific operations are as follows: After human PBMCs are taken out from liquid nitrogen according to the standard operation, they are thawed and recovered in a 37 °C water bath. The cells are resuspended with RPMI 1640 medium (containing 10% FBS in this experiment) and centrifuged and washed twice; subsequently, human PBMC cells are resuspended in RPMI 1640 medium for standby. The compound powder is dissolved in DMSO to 10 mM, and 2 μl of the compound is added to 998 μl of RPMI 1640 medium. After vortex mixing, it is the highest concentration point. The compound solution is gradually diluted 3-fold with 0.2% DMSO medium, with a total of 8 concentration points. Treatment with RPMI 1640 medium solution containing 0.1% DMSO is used as a control. 1×105 human PBMC cells are added to each well of a Corning 96-well cell culture plate (product number: 3599), and then an equal volume of the compound dilution is added. 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. Subsequently, Anti-human CD3 Antibody with a final concentration of 0.01 μg / ml and Anti-human CD28 Antibody with a concentration of 1 μg / ml are added and incubated in a 37 °C cell culture incubator for 24 h. The content of IL-2 in the cell supernatant is detected using the Human IL-2 DuoSet ELISA KIT, and the detection is carried out according to the operation instructions of the kit. The data are described by the highest multiple ratio of the stimulation signal of the compound to the signal of 0.1% DMSO. The cells are collected and the Luminescent CellViability Assay kit is used to detect cell viability, and the cell viability data are described by the half-maximal inhibitory concentration IC50 of the compound.

[0083]

[0084] NA: It indicates that no enhanced release of IL-2 is detected.

[0085] The above results show that compared with the control, the compound of the present invention significantly increases the level of the cytokine interleukin-2 secreted by PBMC cells, and at the same time has no adverse effect on the cell viability of PBMC.

Claims

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

3. Use of the compound or the pharmaceutically acceptable salt thereof according to claim 1 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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