A highly active HPK1 kinase inhibitor

Abstract

The present invention provides a compound capable of inhibiting HPK1 kinase activity and a pharmaceutical composition comprising the compound. The present invention also provides the use of the compound in preventing and / or treating cancer, tumors, inflammatory diseases, autoimmune diseases or immune-mediated diseases.

Description

Technical Field

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

[0002] 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. On the other hand, HPK1 is also involved in regulating the maturation and activation of dendritic cells (DCs). In particular, it inhibits the expression of proteins related to T cell activation in DCs, such as CD80, CD86, and MHC complexes, thereby affecting the role of DCs in regulating T cell activation. The presentation of tumor antigens by activated DCs and the collaboration between DCs and T cells are one of the most important links in the anti-tumor immune system. In addition, the tumor microenvironment contains a large number of immunosuppressive molecules such as PGE2 and TGF-β, and the immunosuppressive effects mediated by these factors are also closely related to HPK1. Overall, small molecule compounds that specifically target and inhibit HPK1 can inhibit tumor growth by enhancing anti-tumor immunity through multiple pathways, such as improving T cell function, enhancing DC cell function, and reversing the tumor's immunosuppressive microenvironment. Summary of the Invention

[0003] The present invention provides a compound capable of inhibiting HPK1 kinase activity and pharmaceutically acceptable salts, isotope derivatives and stereoisomers.

[0004]

[0005] It is important to note that, herein, when reference is made to a "compound" having a particular structural formula, it generally also encompasses stereoisomers, diastereomers, enantiomers, racemic mixtures and isotopic derivatives thereof.

[0006] It is well known to those skilled in the art that the 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 is particularly noted that when a compound is mentioned in this article, it generally also includes its pharmaceutically acceptable salts, and further includes its solvates and hydrates.

[0007] Similarly, reference herein to a compound generally also includes prodrugs, metabolites, and N-oxides thereof.

[0008] Pharmaceutically acceptable salts of the present invention may be formed using, for example, the following inorganic or organic acids: "Pharmaceutically acceptable salts" refers to salts that, within the scope of sound medical judgment, are suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, or the like, commensurate with a reasonable benefit / risk ratio. Such salts may be prepared in situ during the final isolation and purification of the compounds of the invention, or separately by reacting the free base or free acid with a suitable reagent, as summarized below. For example, the free base function may be reacted with a suitable acid. Furthermore, when the compounds of the invention possess an acidic moiety, suitable pharmaceutically acceptable salts thereof may 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 of an amino group formed with inorganic acids (e.g., hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid) or organic acids (e.g., acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid), or salts formed using other methods known in the art, such as ion exchange. Other pharmaceutically acceptable salts include adipate, sodium alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hernisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, and the like. 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, for example, halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates, and aryl sulfonates.

[0009] The pharmaceutically acceptable salts of the present invention can be prepared by conventional methods, for example, by dissolving the compound of the present invention in a water-miscible organic solvent (e.g., acetone, methanol, ethanol and acetonitrile), adding an excess of an organic acid or an aqueous inorganic acid solution thereto to precipitate the salt from the resulting mixture, removing the solvent and the remaining free acid therefrom, and then isolating the precipitated salt.

[0010] As used herein, "solvate" refers to 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 some cases, for example, when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid, the solvate will be capable of isolation. The solvent molecules in the solvate may exist in a regular and / or disordered arrangement. The solvate may contain stoichiometric or non-stoichiometric amounts of solvent molecules. "Solvate" encompasses both solution-phase and isolatable solvates. Exemplary solvates include, but are not limited to, hydrates, ethanolates, methanolates, and isopropanolates. Solvation methods are well known in the art.

[0011] "Stereoisomerism" as used herein is divided into conformational isomerism and configurational isomerism. Configurational isomerism can be further divided into cis-trans isomerism and optical isomerism (i.e., optical isomerism). Conformational isomerism refers to the stereoisomerism phenomenon in which the atoms or atomic groups of an organic molecule with a certain configuration have different spatial arrangements 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 that occur in the structure of cyclohexane. "Stereoisomers" refer to compounds of the present invention that contain one or more asymmetric centers and can therefore 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 can produce two optical isomers. The scope of the present invention includes all possible optical isomers and diastereomeric mixtures, as well as pure or partially purified compounds. The compounds of the present invention may exist as tautomers, 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. Each tautomer and mixtures thereof are included in the compounds of the present invention. Enantiomers, diastereomers, racemates, mesomorphs, cis-trans isomers, tautomers, geometric isomers, epimers and mixtures thereof of all compounds of formula (I) are included within the scope of the present invention.

[0012] The "isotope derivative" of the present invention refers to a molecule in which the compound in this patent is isotopically labeled. The isotopes commonly used as isotope labels are: hydrogen isotopes, 2 H and 3 H; Carbon isotope: 11 C, 13 C and 14C; Chlorine isotope: 35 Cl and 37 Cl; Fluorine isotope: 18 F; Iodine isotope: 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 These isotope-labeled compounds can be used to study the distribution of pharmaceutical molecules in tissues. 3 H and carbon 13 C, because they are easy to label and detect, they are more widely used. Some heavy isotopes, such as deuterium ( 2 H), substitution can enhance metabolic stability and prolong half-life, thereby achieving a reduction in dosage and providing therapeutic advantages. Isotope-labeled compounds are generally synthesized from labeled starting materials using known synthetic techniques similar to those used for synthesizing non-isotope-labeled compounds.

[0013] The present invention also provides use of the compound of the present invention in preparing a medicament for preventing and / or treating cancer, tumor, inflammatory disease, autoimmune disease or immune-mediated disease.

[0014] Furthermore, 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, comprising the compound of the present invention as an active ingredient.

[0015] Furthermore, 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 a compound of the present invention to a mammal in need thereof.

[0016] When the compound of the present invention or a pharmaceutically acceptable salt thereof is administered in combination with another anticancer agent or immune checkpoint inhibitor for treating cancer or tumors, the compound of the present invention or a pharmaceutically acceptable salt thereof may provide enhanced anticancer effects.

[0017] When the compounds of the present invention, or pharmaceutically acceptable salts thereof, 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 pharmaceutically acceptable salts thereof, may provide enhanced therapeutic effects.

[0018] 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.

[0019] 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

[0020] Example

[0021] General Process

[0022] 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.

[0023] 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.

[0024] 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.

[0025] Microwave reaction use Initiator + microwave reactor.

[0026] 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.

[0027] 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.

[0028] The synthetic methods of some intermediates in the present invention are as follows:

[0029] Intermediate 1

[0030]

[0031] Intermediate 1 was prepared by the following steps:

[0032]

[0033] 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).

[0034] 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] +.

[0035] Intermediate 2

[0036]

[0037] Intermediate 2 was prepared by the following steps:

[0038]

[0039] 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).

[0040] 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).

[0041] Intermediate 6

[0042]

[0043] Intermediate 6 was prepared by the following steps:

[0044]

[0045] 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] + .

[0046] Intermediate 8

[0047]

[0048] Intermediate 8 was prepared by the following steps:

[0049]

[0050] 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] + .

[0051] Intermediate 9

[0052]

[0053] Intermediate 9 was prepared by the following steps:

[0054]

[0055] 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] + .

[0056] The synthesis method of the embodiment compounds of the present invention is as follows:

[0057] Example 33

[0058] N-(2-Methoxy-6-methyl-5,6,7,8-tetrahydro-1,6-naphthyridin-3-yl)-8-(piperidin-1-yl)pyrido[3,4-d]pyrimidine

[0059] -2-amine

[0060]

[0061] Compound 33 was prepared by the following steps:

[0062]

[0063] 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).

[0064] HPK1 inhibitor biological screening and results

[0065] Test Example 1: Detection of the Inhibitory Ability of Compounds on HPK1 Kinase Activity (Method 1)

[0066] The required reagents are as follows

[0067]

[0068] Experimental procedures

[0069] 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 at 60μM concentration and diluted 5-fold 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 plate was 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 plate was 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 compound.

[0070] Compound number IC50(nM) 33 0.52

[0071] Test Example 2: Detection of the ability of compounds to stimulate the secretion of cytokine interleukin-2 (IL-2) by Jurkat cells (Method 2)

[0072] The reagents and cells required are as follows

[0073] Experimental reagents:

[0074]

[0075] Experimental cells:

[0076]

[0077] Experimental procedures

[0078] 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 1×10 cells per well in 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 in a 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 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, 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.

[0079]

[0080] NA: indicates that no enhancement of IL-2 release 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 or a pharmaceutically acceptable salt thereof 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.

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