Bridged Bicyclic Compounds as BTK Inhibitors

Abstract

The present invention provides bridged bicyclic compounds A and B, or pharmaceutically acceptable salts thereof, as inhibitors of Bruton's tyrosine kinase (BTK) and its C481 mutant. A method for preparing compounds A and B, or pharmaceutically acceptable salts thereof, is also provided. Compounds A and B can be used to treat and / or prevent diseases mediated by BTK or its C481 mutant, particularly cancer and autoimmune diseases. [Formula 1] JPEG2023538090000008.jpg32170

Description

[Technical Field]

[0001] The present invention relates to a bridged bicyclic compound or a pharmaceutically acceptable salt thereof suitable for modulating or inhibiting the activity of Bruton's tyrosine kinase (BTK) and its C481 mutant. The present invention also relates to a method for producing the compound or a pharmaceutically acceptable salt thereof. Furthermore, the present invention relates to applications and methods of using the compound or a pharmaceutically acceptable salt thereof in the treatment and / or prevention of cancer and autoimmune diseases. [Background technology]

[0002] BTK is a key non-receptor tyrosine kinase that mediates cell signaling and is present in plasma cells, including B cells. B cells are activated via the B cell receptor (BCR), and BTK plays a key role in the BCR-mediated signaling pathway. Activation of the BCR on B cells activates BTK, leading to increased downstream phospholipase C (PLC) levels and activation of the IP3 and DAG signaling pathways. This signaling pathway can promote cell proliferation, adhesion, and survival and plays an important role in the development of B cell lymphoma.

[0003] By inhibiting BTK activity, BTK inhibitors inhibit B lymphoma cell proliferation, disrupt tumor cell adhesion, and promote tumor cell apoptosis, making BTK a compelling target for B cell-related cancers, such as non-Hodgkin lymphoma (NHL), chronic lymphocytic leukemia (CLL) / small lymphocytic lymphoma (SLL), mantle cell lymphoma (MCL), Waldenström's macroglobulinemia (WM), marginal zone lymphoma (MZL), and central nervous system leukemia (CNSL). Several BTK inhibitors are currently on the market, including Abbvie / JNJ's ibrutinib, AZ's acalabrutinib, Beigene's zanubrutinib, and Gilead / Ono's tirabrutinib, and many more are in clinical studies.

[0004] In addition to treating B cell-associated lymphomas, BTK inhibitors can also inhibit the production of B cell autoantibodies and cytokines. In autoimmune diseases, B cells present their own antigens, promote T cell activation, secrete inflammatory factors that cause tissue damage, and simultaneously produce numerous antibodies that activate B cells and induce autoimmune responses. T cells and B cells interact with each other to form a positive feedback regulatory chain that leads to uncontrolled autoimmune responses and exacerbates pathological tissue damage. Research has shown that the body contains regulatory B cells that can negatively regulate immune responses and inhibit immune-mediated inflammation through the secretion of interleukin-10 (IL-10) or transforming growth factor-β1 (TGF-β1) and other mechanisms. Therefore, BTK may be a potential target for autoimmune diseases such as rheumatoid arthritis (RA), multiple sclerosis (MS), systemic lupus erythematosus (SLE), and pemphigus. BTK inhibitors are still undergoing clinical investigation for the treatment of autoimmune diseases. Among them, Sanofi's rilzabrutinib and Merck Serono's evobrutinib have achieved effective results in the treatment of pemphigus and multiple sclerosis, respectively.

[0005] Most commercially available and research-based BTK inhibitors are irreversible inhibitors that inhibit BTK activity by covalently binding to the cysteine ​​residue at position 481 of the BTK protein. After a period of ibrutinib treatment, some B-cell lymphoma patients develop mutations at C481 in BTK, such as C481S, that cause ibrutinib to lose its covalent binding site to the protein, resulting in reduced ibrutinib activity and making the patients resistant to ibrutinib treatment (Quinquenel, et. al Blood 2019, 134, 641-644).

[0006] There is a need for BTK inhibitors that effectively inhibit the activity of BTK and its C481 mutant and overcome the drug resistance caused by the C481 mutation associated with irreversible BTK inhibitors. DETAILED DESCRIPTION OF THE INVENTION

[0007] Detailed Description of the Invention definition Unless otherwise stated, the following terms used in this application have the following meanings:

[0008] "CSF / plasma ratio (Kp,CSF)" refers to the ratio of the compound concentration in cerebrospinal fluid (CSF) to the compound concentration in plasma. The ability of a compound to cross the blood-brain barrier (BBB) ​​is assessed by measuring the compound's concentration in rodent CSF and plasma and determining the ratio (Kp,CSF).

[0009] "Isomers" refer to compounds that share a common molecular formula but differ in the bonding positions and spatial arrangement of their atoms. Isomers that differ in the spatial arrangement of their atoms are called "stereoisomers." Stereoisomers include optical isomers, geometric isomers, and conformational isomers.

[0010] The compounds of the present invention may exist as optical isomers. Optical isomers include enantiomers and diastereomers. An enantiomer is one of two stereoisomers that are mirror images of each other and are not superimposable. A racemic mixture or racemate is one that contains equal amounts of levorotatory and dextrorotatory enantiomers of a chiral molecule. Diastereomers are stereoisomers that are not mirror images of each other and are not superimposable. When a compound is a single isomer and the absolute configuration has been determined, it is referred to as the "R" or "S" isomer, depending on the arrangement of substituents around the chiral carbon atom. When the absolute configuration has not been determined, it is referred to as the (+) or (-) isomer, depending on the measured optical rotation. Methods for preparing and separating optical isomers are known to those skilled in the art.

[0011] The compounds of the present invention may have geometric isomers resulting from the distribution of substituents around a carbon-carbon double bond, a carbon-nitrogen double bond, a cycloalkyl group, or a heterocyclyl group. Substituents around a carbon-carbon double bond or a carbon-nitrogen bond are said to be in the Z or E configuration, and substituents around a cycloalkyl or heterocycle are said to be in the cis or trans configuration.

[0012] Compounds of the invention may also exhibit tautomerism, such as keto-enol tautomerism.

[0013] The present invention includes any tautomeric or stereoisomeric form and mixtures thereof and is not limited to the tautomeric or stereoisomeric forms specified in the compound nomenclature or chemical structure.

[0014] "Isotopes" include all stable isotopes of atoms in the compounds of the present invention. Isotopes include atoms with the same atomic number but different masses. Examples of isotopes suitable for incorporation into compounds of the present invention are isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, and chlorine. 2 H(D), 3 H, 13 C. 14 C. 15 N, 18 O. 17 O. 31 P, 32 P, 35 S, 18 F and 36 Examples include, but are not limited to, Cl. Isotopically labeled compounds of the present invention can generally be prepared by conventional techniques known to those skilled in the art, or by methods similar to those described in the embodiments, using an appropriate isotopically labeled reagent instead of a non-isotopically labeled reagent. Such compounds have a variety of potential uses, such as standards and reagents in determining biological activity. With stable isotopes, such compounds may advantageously alter biological, pharmacological, or pharmacokinetic properties. Deuterium 2 H(D) is a preferred isotope of the present invention. For example, the hydrogen of a methyl, methylene, or methine can be replaced with deuterium.

[0015] The compounds of the present invention can be administered in the form of prodrugs. A "prodrug" refers to a derivative that is converted into a biologically active compound under in vivo physiological conditions, for example, by oxidation, reduction, and hydrolysis (each occurring with or without the involvement of enzymes). Examples of prodrugs include compounds of the present invention in which the amino group is acylated, alkylated, or phosphorylated, such as eicosanoylamino, alanylamino, and pivaloyloxymethylamino; compounds of the present invention in which the hydroxyl group is acylated, alkylated, or phosphorylated or converted to a borate group, such as acetoxy, palmitoyloxy, pivaloyloxy, succinyloxy, fumaroyloxy, and alanyloxy; compounds of the present invention in which the carbonyl group is esterified or amidated; and compounds of the present invention in which the thiol group forms a disulfide bridge with a carrier molecule, such as a peptide, that selectively delivers drugs to a target and / or the cytosol of a cell. Prodrugs can be prepared from the compounds of the present invention by known methods.

[0016] "Pharmaceutically acceptable salt" refers to a salt made from a compound of the present invention and a pharmaceutically acceptable base or acid, including inorganic alkali or inorganic acids and organic bases or acids, provided that the compound of the present invention contains one or more acidic or basic groups. Compounds of the present invention containing an acidic group can exist in the form of a salt, such as an alkali metal salt, alkaline earth metal salt, or ammonium salt. For example, such salts include sodium salt, potassium salt, calcium salt, magnesium salt, or ammonia, or organic amine salts, such as salts of ethylamine, ethanolamine, triethanolamine, or amino acids. Compounds of the present invention containing a basic group can exist in the form of a salt, such as an inorganic acid salt or an organic acid salt. Examples of suitable acids include hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, nitric acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, oxalic acid, acetic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, formic acid, propanoic acid, pivalic acid, malonic acid, succinic acid, pimelic acid, fumaric acid, maleic acid, malic acid, sulfamic acid, phenylpropionic acid, gluconic acid, ascorbic acid, isonicotinic acid, citric acid, adipic acid, and other acids known to those skilled in the art. When the compounds of the present invention contain acidic and basic groups in the molecule, the present invention also includes internal salts in addition to the salt form. Each salt can be obtained by conventional methods known to those skilled in the art, for example, by mixing the compounds of the present invention with an organic or inorganic acid or base in a solvent or dispersant, or by anion or cation exchange with another salt.

[0017] A "pharmaceutical composition" refers to a composition comprising one or more compounds of the present invention or pharmaceutically acceptable salts, stable isotopic derivatives, isomers, prodrugs, and mixtures thereof, and other components such as pharmaceutically acceptable carriers and excipients.

[0018] "Cancer, lymphoma, or leukemia" includes, but is not limited to, B-cell malignancies, B-cell lymphoma, diffuse large B-cell lymphoma, chronic lymphocytic leukemia, small lymphocytic lymphoma, non-Hodgkin's lymphoma (e.g., ABC-DLBCL), mantle cell lymphoma, follicular lymphoma, Waldenstrom's macroglobulinemia, marginal zone lymphoma, central nervous system lymphoma, chronic lymphocytic lymphoma, B-cell prolymphocytic leukemia, plasma cell lymphoma, multiple myeloma, various solid tumors (e.g., melanoma, bone cancer, brain cancer, colon cancer, liver cancer, skin cancer, kidney cancer, lung cancer, muscle cancer, bladder cancer, gastrointestinal / gastrointestinal cancer, breast cancer, ovarian cancer, head and neck cancer, prostate cancer), etc.

[0019] "Autoimmune or inflammatory diseases" include, but are not limited to, arthritis, multiple sclerosis, osteoporosis, inflammatory bowel disease, colitis, Crohn's disease, lupus, rheumatoid arthritis, psoriatic arthritis, lupus nephritis, Sjogren's syndrome, IgG4-related disease, idiopathic thrombocytopenic purpura, immune thrombocytopenia, Wright's syndrome, psoriasis, Behcet's disease, asthma, pemphigus, diabetes, myasthenia gravis, Guillain-Barré syndrome, Graves' disease, Hashimoto's thyroiditis, vasculitis, autoimmune vasculitis, granulomatosis with polyangiitis, autoimmune hepatitis, and the like.

[0020] A "therapeutically effective amount" refers to an amount of a compound of the present invention that can effectively inhibit the activity of BTK and its C481 mutant and / or treat or prevent a disease mediated by BTK and its C481 mutant.

[0021] "Patient" refers to a mammal, preferably a human.

[0022] The present invention relates to two bridged bicyclic compounds, A and B (having the structures shown below), as reversible BTK inhibitors. These compounds effectively inhibit the activity of BTK and its C481 mutant, overcoming drug resistance caused by the C481 mutation associated with irreversible BTK inhibitors.

[0023] The present invention is directed to Compound A or B, or its pharmaceutically acceptable salts, prodrugs, stable isotopic derivatives, isomers, and mixtures thereof.

[0024] [ka]

[0025] Compounds A and B effectively inhibited the activity of BTK and its C481 mutant, with IC 50 Compounds A and B are non-brain penetrants, with a Kp,CSF of less than 0.1.

[0026] The present invention also relates to pharmaceutical compositions comprising Compound A or B, or pharmaceutically acceptable salts, stable isotope derivatives, isomers and prodrugs thereof, and one or more pharmaceutically acceptable carriers or excipients.

[0027] The present invention further relates to pharmaceutical compositions comprising a compound of formula (I) or a pharmaceutically acceptable salt, stable isotope derivative, isomer, prodrug, or mixture thereof, and at least one additional therapeutic agent, which may be a small molecule chemotherapeutic agent (e.g., an anti-inflammatory steroid, a kinase-targeting agent, an apoptosis inhibitor, an inflammation modulator, a cytotoxic agent, or a DNA damage-related agent) or a macromolecular immune and / or inflammation modulator (e.g., a CD20 antibody, a CD19 antibody, or a PD antibody).

[0028] Compound A or B and the other therapeutic agent may be present in the same pharmaceutical composition or in separate pharmaceutical compositions. The compound of formula (I) and the other therapeutic agent may be administered simultaneously or sequentially, in the same or different forms.

[0029] The present invention provides a method for treating or preventing a disease mediated by BTK or its C481 mutant. The method comprises administering to a patient in need thereof a therapeutically effective amount of Compound A or B, or a pharmaceutically acceptable salt, stable isotope derivative, isomer, prodrug, or mixture thereof. Diseases include, for example, B-cell malignancies, B-cell lymphoma, diffuse large B-cell lymphoma, chronic lymphocytic leukemia, small lymphocytic lymphoma, non-Hodgkin's lymphoma (e.g., ABC-DLBCL), mantle cell lymphoma, follicular lymphoma, Waldenstrom's macroglobulinemia, marginal zone lymphoma, central nervous system lymphoma, chronic lymphocytic lymphoma, B-cell prolymphocytic leukemia, plasma cell lymphoma, multiple myeloma, and various solid tumors ( For example, lung cancer, prostate cancer, head and neck cancer, breast cancer, ovarian cancer, uterine cancer, pancreatic cancer, colon cancer, rectal cancer, stomach cancer, esophageal cancer, brain cancer, liver cancer, kidney cancer, skin cancer, muscle cancer, epithelial cancer, bladder cancer, neuroblastoma, melanoma, bone cancer, melanoma), arthritis, multiple sclerosis, osteoporosis, inflammatory bowel disease, colitis, Crohn's disease, lupus, rheumatoid arthritis, psoriatic arthritis, lupus nephritis, Sjogren's syndrome, IgG4-related disease, idiopathic Cancer, lymphoma, leukemia, autoimmune or inflammatory diseases, especially B-cell lymphoma, diffuse large B-cell lymphoma, chronic lymphocytic leukemia, small lymphocytic lymphoma, such as thrombocytopenic purpura, immune thrombocytopenia, Wright's syndrome, psoriasis, Behçet's disease, asthma, pemphigus, diabetes, myasthenia gravis, Guillain-Barré syndrome, Graves' disease, Hashimoto's thyroiditis, vasculitis, autoimmune vasculitis, granuloma with polyangiitis, autoimmune hepatitis These conditions include, but are not limited to, lymphoma, non-Hodgkin's lymphoma (e.g., ABC-DLBCL), mantle cell lymphoma, follicular lymphoma, Waldenstrom's macroglobulinemia, marginal zone lymphoma, central nervous system lymphoma, chronic lymphocytic lymphoma, rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, lupus nephritis, sicca syndrome, IgG4-related disease, idiopathic thrombocytopenic purpura, immune thrombocytopenia, pemphigus, and urticaria.

[0030] In the present invention, the pharmaceutical composition may be in any dosage form including, but not limited to, tablets, capsules, liquids, lyophilized preparations and injectables.

[0031] The pharmaceutical preparation of the present invention may be administered in the form of a dosage unit containing a predetermined amount of the active ingredient. Such a unit may contain 1 mg to 1 g, preferably 5 mg to 700 mg, particularly preferably 10 mg to 500 mg of the compound of the present invention, depending on the disease to be treated, the method of administration, and the age, weight, and condition of the patient. Pharmaceutical preparations may be produced by methods well known in the pharmaceutical art, for example, by formulating the active ingredient with one or more additives or one or more adjuvants.

[0032] The pharmaceutical formulations of the present invention are suitable for administration in any suitable manner, for example orally (including orally or sublingually) or parenterally (including subcutaneously, intramuscularly, intravenously or intradermally).

[0033] The present invention also provides methods for preparing compounds A and B. The compounds of the present invention may be prepared by the following representative methods and examples, which should not be construed as limiting the scope of the present invention in any way. The compounds of the present invention may be synthesized by synthetic techniques known to those skilled in the art or by a combination of methods known in the art with the methods of the present invention. The products obtained at each step of the reaction are isolated by separation techniques known in the art, including, but not limited to, extraction, filtration, distillation, crystallization, and chromatographic separation. The starting materials and reagents used in the synthesis may be prepared in a conventional manner based on literature (e.g., SciFinder) or may be purchased commercially. [Example]

[0034] Starting materials in the present invention were synthesized by methods known in the art or purchased from ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, Accela ChemBio Inc., Beijing Ouhe, etc.

[0035] The structures of the compounds were determined by nuclear magnetic resonance (NMR) or mass spectrometry (MS). NMR measurements were performed using a Bruker ASCEND-400 NMR spectrometer. Deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), or deuterated methanol (CD3OD) was used as the solvent. Tetramethylsilane (TMS) was used as the internal standard, and the chemical shifts were measured at 10 -6 The values ​​are shown in units of ppm. MS measurements were performed using an Agilent SQD (ESI) mass spectrometer (Agilent 6120).

[0036] HPLC measurements were performed using an Agilent 1260 DAD high pressure liquid chromatograph (column: Poroshell 120 EC-C18, 50×3.0 mm, 2.7 μm) or a Waters Arc high pressure liquid chromatograph (column: Sunfire C18, 150×4.6 mm, 5 μm).

[0037] Thin layer chromatography (TLC) was performed using GF254 silica gel plates with a thickness of 0.15 to 0.2 mm manufactured by Qingdao Haiyang Chemical Co., Ltd., and separation / purification of products by thin layer chromatography was performed using silica plates with a thickness of 0.4 to 0.5 mm.

[0038] For column chromatography, 200-300 mesh silica gel manufactured by Qingdao Haiyang Chemical Co., Ltd. was usually used.

[0039] Unless otherwise stated in the examples, reactions were carried out at room temperature (20-30° C.) in an argon or nitrogen atmosphere using a balloon with a volume of approximately 1 L.

[0040] The hydrogenation was carried out three times in a hydrogen atmosphere using a balloon with a capacity of about 1 L attached to the reaction vessel after evacuating and then filling with hydrogen.

[0041] Reactions were monitored using an Agilent LCMS (1260 / 6120) or thin-layer chromatography. The elution solvent systems for column chromatography and TLC were a) dichloromethane / methanol, b) petroleum ether / ethyl acetate, or other systems as indicated. The solvent ratio was adjusted according to the polarity of the compounds and, if necessary, further adjusted by adding small amounts of TEA or acidic or alkaline reagents. Compounds were purified using a Waters MS-guided autopreparation system (referred to as preparative HPLC) equipped with an MS detector (SQD2) and eluted with the appropriate acetonitrile / water (containing 0.1% TFA or formic acid) or acetonitrile / water (25–28% ammonium hydroxide, 0.05%) gradient (XBridge-C18, 19 × 150 mm, 5 μm) at a flow rate of 20 mL / min. Some compounds were prepared as hydrochloride salts by adding 1 N hydrochloric acid to the fractions obtained after preparative HPLC purification, followed by drying under reduced pressure.

[0042] DMF refers to N,N-dimethylformamide. DIPEA refers to N,N-diisopropylethylamine. DBU refers to 1,8-diazabicycloundec-7-ene. NIS refers to N-iodosuccinimide. Pd(dppf)Cl2 refers to [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium. HATU refers to 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate.

[0043] Example 1 Preparation of 4-(4-amino-5-(4-((5-fluoro-2-methoxybenzamido)methyl)phenyl)imidazo[5,1-f][1,2,4]triazin-7-yl)bicyclo[2.2.2]octane-1-carboxylic acid (Compound A)

[0044] [ka]

[0045] Step 1 (4-((5-fluoro-2-methoxybenzamido)methyl)phenyl)boronic acid (1b) To a solution of 5-fluoro-2-methoxybenzoic acid 1a (340 mg, 2 mmol) and DMF (0.05 mL) in dichloromethane (10 mL) at 0 °C was added oxalyl chloride (279 mg, 2.2 mmol). The mixture was gradually warmed to room temperature and stirred for 1 h. Then, it was cooled again to 0 °C, and a suspension of (4-(aminomethyl)phenyl)boronic acid hydrochloride (374 mg, 2 mmol) and DIPEA (516 mg, 4 mmol) in THF (20 mL) was added. After stirring at room temperature for 15 h, the solvent was removed under reduced pressure. The residue was dissolved in ethyl acetate (100 mL), washed with saturated ammonium chloride (50 mL) and brine (50 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to give the title compound 1b (460 mg, 76%). MS m / z (ESI): 304 [M+1]

[0046] Step 2: 1-(2,5-dioxopyrrolidin-1-yl)4-methylbicyclo[2.2.2]octane-1,4-dicarboxylate (1d) To a solution of (methoxycarbonyl)bicyclo[2.2.2]octane-1-carboxylic acid 1c (850 mg, 4 mmol) and 1-hydroxy-pyrrolidine-2,5-dione (552 mg, 4.8 mmol) in THF (50 mL) at 0 °C was added DCC (1.07 g, 5.2 mmol). After warming to room temperature and stirring for 15 h, the mixture was cooled to 0 °C and filtered. The filtrate was concentrated to dryness to give the title compound 1d (1.4 g, crude). MS m / z (ESI): 310 [M+H] +

[0047] Step 3: Methyl 4-(((5-oxo-4,5-dihydro-1,2,4-triazin-6-yl)methyl)carbamoyl)bicyclo[2.2.2]octane-1-carboxylate (1e) To a solution of 6-aminomethyl-4H-[1,2,4]triazin-5-one (252 mg, 2 mmol) and DIPEA (1.03 g, 8 mmol) in DMF (5 mL) was added dropwise a solution of 1d (1.4 g, crude product) in THF (5 mL). The mixture was stirred for 3 h and then concentrated to dryness. The residue was purified by silica gel column chromatography (dichloromethane / methanol = 50 / 1 to 10 / 1) to give the title compound 1e (150 mg, 23% over two steps). MS m / z (ESI): 321 [M+H] +

[0048] Step 4: Methyl 4-(4-oxo-3,4-dihydroimidazo[5,1-f][1,2,4]triazin-7-yl)bicyclo[2.2.2]octane-1-carboxylate (1f) To a solution of 1e (150 mg, 0.47 mmol) in MeCN (10 mL) was added POCl (2 mL). The mixture was heated to 80 °C and stirred for 5 h. The mixture was then concentrated to dryness to give the title compound 1f (150 mg, 100%). MS m / z (ESI): 303 [M+H] +

[0049] Step 5 Methyl 4-(5-iodo-4-oxo-3,4-dihydroimidazo[5,1-f][1,2,4]triazin-7-yl)bicyclo[2.2.2]octane-1-carboxylate (1g). To a solution of 1f (150 mg, crude, 0.47 mmol) in DMF (5 mL) was added NIS (529 mg, 2.35 mmol). The mixture was heated to 55° C. and stirred for 5 h. After cooling to room temperature, the mixture was purified by preparative HPLC to give the title compound 1g (80 mg, 40%). MS m / z (ESI): 429 [M+H] +

[0050] Step 6: Methyl 4-(4-amino-5-iodoimidazo[5,1-f][1,2,4]triazin-7-yl)bicyclo[2.2.2]octane-1-carboxylate (1h) To a solution of 1H-[1,2,4]triazole (131 mg, 1.9 mmol) in pyridine (2 mL) was added POCl (86 mg, 0.56 mmol). The mixture was stirred for 10 min, and a solution of 1g (80 mg, 0.19 mmol) in pyridine (2 mL) was added. After stirring for an additional 1 h, a solution of NH (2 M in isopropanol, 10 mL) was added to the mixture and stirred for 0.5 h. The mixture was then concentrated to dryness, and the residue was purified by preparative HPLC to give the title compound 1h (60 mg, 74%). MS m / z (ESI): 428 [M+H] +

[0051] Step 7 Methyl 4-(4-amino-5-(4-((5-fluoro-2-methoxybenzamido)methyl)phenyl)imidazo[5,1-f][1,2,4]triazin-7-yl)bicyclo[2.2.2]octane-1-carboxylate (1i).

[0052] A mixture of 1h (60 mg, 0.14 mmol), 1b (64 mg, 0.21 mmol), K2CO3 (39 mg, 0.28 mmol), Pd(dppf)Cl2 (11 mg, 0.014 mmol), 1,4-dioxane (4 mL), and water (1 mL) was heated to 100 °C under a nitrogen atmosphere and stirred for 2 h. After cooling to room temperature, the mixture was concentrated to dryness, and the residue was purified by silica gel column chromatography (petroleum ether / EtOAc = 20:1 to 1:2) to give the title compound 1i (60 mg, 77%). MS m / z (ESI): 559 [M+H] + . 1H NMR (400 MHz, CD3OD) δ 7.80 (s, 1H), 7.64 - 7.57 (m, 3H), 7.52 (d, J = 8.3 Hz, 2H), 7.25 (ddd, J = 9.1, 7.6, 3.3 Hz, 1H), 7.17 (dd, J = 9.1, 4.2 Hz, 1H), 4.69 (s, 2H), 3.96 (s, 3H), 3.66 (s, 3H), 2.26 (dd, J = 9.7, 6.2 Hz, 6H), 1.93 (dd, J = 9.7, 6.3 Hz, 6H).

[0053] Step 8 4-(4-amino-5-(4-((5-fluoro-2-methoxybenzamido)methyl)phenyl)imidazo[5,1-f][1,2,4]triazin-7-yl)bicyclo[2.2.2]octane-1-carboxylic acid (A) To a solution of 1i (55 mg, 0.098 mmol) in THF (10 mL) was added lithium hydroxide solution (1N, 4 mL). The mixture was heated to 40 °C and stirred for 48 h. After cooling to room temperature, glacial acetic acid (1 mL) was added to the reaction mixture and extracted with ethyl acetate (2 × 50 mL). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by preparative HPLC to give the title compound A (40 mg, solid, 75%). MS m / z (ESI): 545 [M+1] 1 H NMR (400 MHz, CD3OD) δ 7.80 (s, 1H), 7.64 - 7.56 (m, 3H), 7.53 (d, J = 8.1 Hz, 2H), 7.28 - 7.22 (m, 1H), 7.17 (dd, J = 9.1, 4.2 Hz, 1H), 4.69 (s, 2H), 3.96 (d, J = 7.6 Hz, 3H), 2.30 - 2.22 (m, 6H), 1.98 - 1.90 (m, 6H).

[0054] Example 2 Preparation of N-(4-(4-amino-7-(4-(morpholine-4-carbonyl)bicyclo[2.2.2]octan-1-yl)imidazo[5,1-f][1,2,4]triazin-5-yl)benzyl)-5-fluoro-2-methoxybenzamide (Compound B)

[0055] [ka]

[0056] To a solution of A (75 mg, 0.137 mmol), morpholine (12 mg, 0.137 mmol), and DIPEA (53 mg, 0.409 mmol) in DMF (5 mL) was added HATU (58 mg, 0.151 mmol). The resulting mixture was stirred for 5 min and then purified by preparative HPLC to give the title compound B (40 mg, solid, 48%). MS m / z (ESI): 614.2 [M+H]+ 1 H NMR (400 MHz, DMSO-d6) δ 8.84 (t, J = 6.0 Hz, 1H), 7.88 (s, 1H), 7.58 (d, J = 8.2 Hz, 2H), 7.52 (dd, J = 9.2, 3.3 Hz, 1H), 7.46 (d, J = 8.2 Hz, 2H), 7.39 - 7.29 (m, 1H), 7.19 (dd, J = 9.1, 4.3 Hz, 1H), 4.57 (d, J = 6.1 Hz, 2H), 3.90 (s, 3H), 3.57 (d, J = 9.6 Hz, 8H), 2.21 - 2.09 (m, 6H), 1.97 - 1.84 (m, 6H).

[0057] Example 3 BTK Activity Inhibition Assay The effects of the compounds of the present invention on BTK activity were evaluated by an in vitro kinase assay (Table 1).

[0058] The experimental method is summarized below: The enzyme activity of BTK was determined by detecting the level of substrate phosphorylation in the kinase reaction using a homogeneous time-resolved fluorescence (HTRF) kinase detection kit (Cisbio, catalog number 62TK0PEC). The reaction buffer contained the enzyme reaction buffer (1x) in the kit, 5 mM MgCl, 1 mM DTT, 10 nM SEB, and 0.01% Tween® 20. The kinase reaction solution contained human recombinant BTK protein (Carna Biosciences, catalog number 08-180) diluted to 0.2 ng / μL in the reaction buffer. The substrate reaction solution contained biotin-labeled tyrosine kinase substrate diluted to 0.5 μM in the reaction buffer and 40 μM ATP. The detection buffer contained Eu diluted to 0.05 ng / μL in the reaction buffer. 3+ The compounds were diluted to 100 μM in DMSO, followed by four serial dilutions in DMSO down to a minimum concentration of 6.1 nM, and a final 40-fold dilution in reaction buffer. 50 If the value is too low, reduce the initial concentration of the compound.

[0059] Add 4 μL of test compound solution and 2 μL of kinase reaction solution to a 384-well detection plate (Corning, catalog number 3674), mix thoroughly, and incubate at room temperature for 15 minutes; add 4 μL of substrate reaction solution and incubate for 50 minutes; add 10 μL of detection buffer, mix thoroughly, and leave for 60 minutes; and detect signals at 620 nm and 665 nm using an Envision plate reader (Perkin Elmer). The signal value (absorbance at 665 nm / absorbance at 620 nm) positively correlates with the degree of substrate phosphorylation, thereby detecting the kinase activity of BTK. In this experiment, the group without BTK served as a negative control (100% inhibition), and the group containing BTK but no compound served as a positive control (0% inhibition). The inhibition curve was plotted, and the corresponding IC of the test compound was determined. 50Values ​​are calculated with XLfit software (ID Business Solutions Ltd., UK).

[0060] Example 4 BTK C481S Activity Inhibition Assay The effects of compounds of the present invention on BTK C481S activity were evaluated using an in vitro kinase assay (Table 1).

[0061] The experimental method is summarized below: The enzyme activity of BTK C481S was determined by detecting the level of substrate phosphorylation in the kinase reaction using a homogeneous time-resolved fluorescence (HTRF) kinase detection kit (Cisbio, catalog number 62TK0PEC). The reaction buffer contained the enzyme reaction buffer (1x) in the kit, 5 mM MgCl, 1 mM DTT, 10 nM SEB, and 0.01% Tween® 20; the kinase reaction solution contained human recombinant BTK C481S protein (purified in-house) diluted to 1.5 ng / μL in the reaction buffer; the substrate reaction solution contained biotin-labeled tyrosine kinase substrate diluted to 0.5 μM in the reaction buffer and 35 μM ATP; and the detection buffer contained Eu diluted to 0.05 ng / μL in the reaction buffer. 3+ The compounds were dissolved in DMSO and diluted to 100 μM, followed by four serial dilutions in DMSO down to a minimum concentration of 6.1 nM, and a final 40-fold dilution in reaction buffer. 50 If the value is too low, reduce the initial concentration of the compound.

[0062] Add 4 μL of test compound solution and 2 μL of kinase reaction solution to a 384-well detection plate (Corning, catalog number 3674), mix thoroughly, and incubate at room temperature for 15 minutes; add 4 μL of substrate reaction solution and incubate for 50 minutes; add 10 μL of detection buffer, mix thoroughly, and leave for 60 minutes; and detect signals at 620 nm and 665 nm using an Envision plate reader (Perkin Elmer). The signal value (absorbance at 665 nm / absorbance at 620 nm) positively correlates with the degree of substrate phosphorylation, thereby detecting the kinase activity of BTK C481S. In this experiment, the group without BTK served as a negative control (100% inhibition), and the group containing BTK C481S but no compound served as a positive control (0% inhibition). The inhibition curve was plotted, and the corresponding IC of the test compound was determined. 50 Values ​​are calculated with XLfit software (ID Business Solutions Ltd., UK).

[0063] Table 1 shows the IC of BTK for compounds A and B. 50 and BTK C481S IC 50 Shows.

[0064] [Table 1]

[0065] Example 5 Brain penetration activity test Compound A or B was orally administered at a dose of 5 mg / kg to 12 male Sprague Dawley rats in a 0.5 mg / mL solution of 5% N,N-dimethylacetamide, 10% solutol, and 85% saline. Plasma, brain tissue homogenate (to measure Kp, brain), and cerebrospinal fluid (to measure Kp, CSF) samples were collected (from three animals per time point) at 1, 2, 4, and 8 hours after administration.

[0066] Compound concentrations in plasma, brain tissue homogenate, and CSF were quantified by LC-MS / MS on an API-4500 mass spectrometer. The analytical limit of quantitation (LOQ) was 1 ng / mL. Pharmacokinetic (PK) parameters were calculated using WinNonlin with a non-compartmental approach. Kp, brain and Kp, CSF were calculated as AUC brain / AUC plasma and AUC CSF / AUC plasma, respectively. The results are shown in Table 2 and indicate that both Compounds A and B are non-brain-penetrating agents.

[0067] [Table 2]

[0068] The invention, and the manner and process of making and using it, are described in this specification in such full, clear, concise and exact terms as to enable any person skilled in the art to which the invention pertains to make and use the same. It is understood that the specification sets forth preferred embodiments of the invention and that modifications may be made therein without departing from the scope of the invention as set forth in the claims. To particularly point out and distinctly claim the subject matter regarded as invention, the following claims conclude this specification.

Claims

1. Compound A or B, or a pharma- ceutically acceptable salt, stable isotope derivative or stereoisomer thereof. 【Chemistry 1】

2. A pharmaceutical composition comprising compound A or B, or a pharma- ceutically acceptable salt, stable isotope derivative or stereoisomer thereof, and a pharma- ceutically acceptable carrier thereof.

3. 1. A pharmaceutical composition for preventing or treating a disease mediated by BTK or its C481 mutant, comprising a therapeutically effective amount of compound A or B, or a pharma- ceutical acceptable salt, stable isotope derivative, or stereoisomer thereof, wherein the disease is selected from the group consisting of cancer, lymphoma, leukemia, autoimmune disease, and inflammatory disease.

4. 4. The pharmaceutical composition according to claim 3, wherein the disease is B-cell lymphoma, chronic lymphocytic leukemia, small lymphocytic lymphoma, mantle cell lymphoma, Waldenstrom's macroglobulinemia, marginal zone lymphoma, follicular lymphoma, central nervous system lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, multiple myeloma, rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, lupus nephritis, Sjogren's syndrome, IgG4-related disease, idiopathic thrombocytopenic purpura, immune thrombocytopenia, or pemphigus.

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