Bridged bicyclic compounds as btk inhibitors

Abstract

The present invention relates to bridged bicyclic compounds A and B or a pharmaceutically acceptable salt thereof as inhibitors of Bruton's tyrosine kinase (BTK) and its C481 mutants. The present invention also relates to processes for preparing compounds A and B or a pharmaceutically acceptable salt thereof. Compounds A and B are useful for the treatment and / or prevention of relevant diseases mediated by BTK or its C481 mutants, especially cancer and autoimmune diseases.

Description

Technical Field

[0001] This invention relates to bridged bicyclic compounds or pharmaceutically acceptable salts thereof suitable for regulating or inhibiting the activity of Bruton's tyrosine kinase (BTK) and its C481 mutant. The invention also relates to methods for preparing these compounds or pharmaceutically acceptable salts thereof. Furthermore, the invention relates to the use and methods of using said compounds or pharmaceutically acceptable salts thereof in the treatment and / or prevention of cancer and autoimmune diseases. Background Technology

[0002] BTK is an important non-receptor tyrosine kinase that mediates cell signal transduction and is found in plasma cells, including B cells. B cells are activated via the B cell receptor (BCR), and BTK plays a crucial role in the BCR-mediated signaling pathway. Activation of the BCR on B cells leads to BTK activation, resulting in an increase in the concentration of downstream phospholipase C (PLC) and activation of the IP3 and DAG signaling pathways. This signaling pathway promotes cell proliferation, adhesion, and survival, playing a vital role in the development of B-cell lymphoma.

[0003] BTK inhibitors suppress the proliferation of B-cell lymphoma cells by inhibiting BTK activity, disrupting tumor cell adhesion, and promoting tumor cell apoptosis. This makes BTK a promising drug target in B-cell-related cancers, such as non-Hodgkin's lymphoma (NHL), chronic lymphocytic leukemia (CLL) / small lymphocytic lymphoma (SLL), mantle cell lymphoma (MCL), Waldenström macroglobulinemia (WM), marginal zone lymphoma (MZL), and central nervous system leukemia (CNSL). Several BTK inhibitors are currently on the market, including ibrutinib from Abbvie / JNJ, acalabrutinib from AstraZeneca, zanubrutinib from Beigene, and tirabrutinib from Gilead / Ono, with several others in clinical trials.

[0004] In addition to treating B-cell-associated lymphoid tissue (B-cell lymphoma), BTK inhibitors can also suppress the production of B-cell autoantibodies and cytokines. In autoimmune diseases, B cells present autoantigens, promote T-cell activation, secrete inflammatory factors that cause tissue damage, and simultaneously activate B cells to produce large amounts of antibodies, triggering an autoimmune response. The interaction between T cells and B cells forms a positive feedback regulatory chain, leading to uncontrolled autoimmune responses and exacerbating tissue pathological damage. Studies have shown that regulatory B cells exist in the body, negatively regulating immune responses and inhibiting immune-mediated inflammatory responses through mechanisms such as secreting interleukin-10 (IL-10) or transforming growth factor β1 (TGF-B1). Therefore, BTK can serve as a drug target for autoimmune diseases such as rheumatoid arthritis (RA), multiple sclerosis (MS), systemic lupus erythematosus (SLE), and pemphigus. For autoimmune indications, BTK inhibitors are still in the clinical research stage. Among them, Sanofi's rilzabrutinib and Merck Serono's evobrutinib have shown effective results in the treatment of pemphigus and multiple sclerosis, respectively.

[0005] Most marketed and investigational BTK inhibitors are irreversible inhibitors, inhibiting BTK activity by covalently binding to cysteine ​​residues at BTK protein 481. In some B-cell lymphoma patients treated with ibrutinib for a period of time, mutations occur at BTK C481, such as C481S, causing ibrutinib to lose its covalently binding site, leading to reduced ibrutinib activity and thus developing resistance (Quinquenel, et al. Blood 2019, 134, 641-644).

[0006] There is a need for an effective BTK inhibitor that suppresses the activity of BTK and its C481 mutant, thereby overcoming the drug resistance caused by the C481 mutation associated with irreversible BTK inhibitors. Summary of the Invention

[0007] definition

[0008] Unless otherwise stated, the following terms used in this application have the following meanings.

[0009] The "CSF / plasma ratio (Kp, CSF)" refers to the ratio of the concentration of a compound in cerebrospinal fluid (CSF) to the concentration of the compound in plasma. The ability of a compound to cross the blood-brain barrier (BBB) ​​is assessed by measuring its concentration in the CSF and plasma of rodents and determining the ratio (Kp, CSF).

[0010] "Isomers" are compounds with the same molecular formula but different atomic bonding positions or spatial arrangements. Isomers with different spatial arrangements of atoms are called "stereoisomers." Stereoisomers include optical isomers, geometric isomers, and conformational isomers.

[0011] The compounds of this invention can exist as optical isomers. Optical isomers include enantiomers and diastereomers. Enantiomers are two stereoisomers that are mirror images of each other but cannot overlap. Racemic mixtures or racemates are mixtures in which the number of left- and right-handed enantiomers of a chiral molecule is equal. Diastereomers are two stereoisomers that are not mirror images of each other and cannot overlap. When an optical isomer is a single isomer and its absolute configuration is determined, it is an "R" or "S" absolute configuration based on the configuration of the substituents on the chiral carbon atom; when the absolute configuration of an optical isomer is not determined, it is (+) or (-) based on the measured optical rotation value. Methods for preparing and separating optical isomers are known in the art.

[0012] The compounds of the present invention may also have geometric isomers, resulting from the distribution of substituents around carbon-carbon double bonds, carbon-nitrogen double bonds, cycloalkyl groups, or heterocyclic groups. Substituents around carbon-carbon double bonds or carbon-nitrogen bonds are designated as Z or E configurations, and substituents around cycloalkyl groups or heterocyclic groups are designated as cis or trans configurations.

[0013] The compounds of the present invention may also exhibit tautomerism, such as keto-enol tautomerism.

[0014] This invention includes any tautomer or stereoisomer and mixtures thereof, and is not limited to any tautomer or stereoisomer used in compound nomenclature or chemical structural formulas.

[0015] "Isotope" refers to all stable isotopes of atoms appearing in the compounds of this invention. Isotopes include those atoms having the same atomic number but different mass numbers. Examples of isotopes suitable for inclusion in the compounds of this invention are hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, and chlorine, respectively, for example, but not limited to, [examples of isotopes]. 2 H(D), 3 H, 13 C 14 C 15 N、 18 O、 17 O、 31 P, 32 P, 35 S, 18 F and 36Cl. The isotope-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 appended examples, using a suitable isotope-labeled reagent instead of a non-isotope-labeled reagent. Such compounds have a variety of potential uses, for example, as standards and reagents for determining biological activity. In the case of stable isotopes, such compounds have the potential to advantageously alter biological, pharmacological, or pharmacokinetic properties. Deuterium 2 H(D) is a preferred isotope of the present invention. For example, hydrogen in methyl, methylene or methine can be replaced by deuterium.

[0016] The compounds of this invention can be administered in the form of prodrugs. A "prodrug" is a derivative which, under physiological conditions in vivo, is converted into a biologically active compound through oxidation, reduction, and hydrolysis (each process with or without enzymatic involvement). Examples of prodrugs include compounds in which the amino group is acylated, alkylated, or phosphorylated, such as eicosanoylamino, alanylamino, and neopentanoyloxymethylamino; the hydroxyl group is acylated, alkylated, or phosphorylated, or converted to a borate ester, such as acetoxy, palmitoyloxy, neopentanoyloxy, succinoxy, fumaroyloxy, and alanyloxy; the carbonyl group is esterified or amidated; and the thiol forms a disulfide bond with a carrier molecule, selectively delivering the drug to the target and / or the cytosol of cells, such as a peptide. Prodrugs can be prepared from the compounds of this invention according to well-known methods.

[0017] "Pharmaceutically acceptable salt" refers to a salt formed by the compound of the present invention with a pharmaceutically acceptable base or acid, including inorganic bases or acids and organic bases or acids, under conditions where the compound contains one or more acidic or basic groups. Compounds of the present invention containing acidic groups may exist in the form of salts, such as alkali metal salts, alkaline earth metal salts, or ammonium salts. For example, such salts include sodium salts, potassium salts, calcium salts, magnesium salts, or ammonia or organic amine salts, such as ethylamine, ethanolamine, triethanolamine, or salts of amino acids. Compounds of the present invention containing basic groups may exist in the form of salts, such as inorganic acid salts or organic acid salts. 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, propionic acid, pentanoic 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 well known to those skilled in the art. If the compounds of the present invention contain both acidic and basic groups in the molecule, the present invention also includes internal salts in addition to the salt forms described above. 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.

[0018] "Pharmaceutical composition" refers to a composition containing one or more of the compounds of the present invention or their pharmaceutically acceptable salts, stable isotope derivatives, isomers, prodrugs and mixtures thereof, as well as other components such as pharmaceutically acceptable carriers and excipients.

[0019] "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, Waldenström macroglobulinemia, marginal zone lymphoma, central nervous system lymphoma, chronic lymphocytic lymphoma, B-cell pre-lymphocytic leukemia, plasma cell lymphoma, multiple myeloma, and various solid tumors (e.g., melanoma, bone cancer, brain cancer, colon cancer, liver cancer, skin cancer, kidney cancer, lung cancer, muscle cancer, bladder cancer, digestive tract / gastrointestinal cancer, breast cancer, ovarian cancer, head and neck cancer, prostate cancer), etc.

[0020] "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, Sjögren's syndrome, IgG4-related disease, idiopathic thrombocytopenic purpura, immune thrombocytopenic purpura, Reiter's syndrome, psoriasis, Behcet's disease, asthma, pemphigus, diabetes, myasthenia gravis, Guillain-Barré syndrome, Graves' disease, Hashimoto's thyroiditis, vasculitis, autoimmune vasculitis, granulomatous polyangiitis, and autoimmune hepatitis.

[0021] "Therapeutic effective amount" refers to the amount of the compound of the present invention that can effectively inhibit the activity of BTK and its C481 mutant, and / or treat or prevent diseases mediated by BTK and its C481 mutant.

[0022] "Patient" refers to mammals, especially humans.

[0023] This invention relates to reversible BTK inhibitors of two bridging bicyclic compounds A and B (structures shown below), which can effectively inhibit the activity of BTK and its C481 mutant, thereby overcoming drug resistance caused by the C481 mutation associated with irreversible BTK inhibitors.

[0024] This invention relates to compound A or B, or pharmaceutically acceptable salts, prodrugs, stable isotope derivatives, isomers, and mixtures thereof.

[0025]

[0026] Compounds A and B effectively inhibited the activity of BTK and its C481 mutant, IC50 50Less than 10 nM. Compounds A and B are non-brain permeabilizers, with Kp and CSF less than 0.1.

[0027] The present invention also relates to pharmaceutical compositions comprising compound A or B or a pharmaceutically acceptable salt thereof, a stable isotope derivative, an isomer and a prodrug, and one or more pharmaceutically acceptable carriers or excipients.

[0028] The present invention also relates to pharmaceutical compositions comprising a compound of general formula (I) or a pharmaceutically acceptable salt thereof, a stable isotope derivative, an isomer, a prodrug, or a mixture thereof, and at least one additional therapeutic agent, wherein the pharmaceutical agent may be a small molecule chemotherapeutic agent (such as 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 large molecule immunomodulator and / or inflammation modulator (such as a CD-20 antibody, a CD19 antibody, or a PD-1 antibody).

[0029] Compound A or B and another therapeutic agent may be present in the same pharmaceutical composition or in different pharmaceutical compositions. The compound of formula (I) and another pharmaceutical agent may be administered simultaneously or sequentially in the same or different forms.

[0030] This invention provides a method for treating or preventing diseases mediated by BTK or its C481 mutant. The method comprises administering to a patient in need a therapeutically effective amount of compound A or B, or a pharmaceutically acceptable salt, stable isotope derivative, isomer, prodrug, or mixture thereof. The diseases include, but are not limited to, cancer, lymphoma, leukemia, autoimmune diseases, or inflammatory diseases, such as 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, Waldenström macroglobulinemia, marginal zone lymphoma, central nervous system lymphoma, chronic lymphocytic lymphoma, and B-cell prelymphocytic lymphoma. Leukemia, plasma cell lymphoma, multiple myeloma, various solid tumors (e.g., 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), arthritis, multiple sclerosis, osteoporosis, inflammatory bowel disease, colitis, Crohn's disease, lupus, rheumatoid arthritis, psoriasis Arthritis, lupus nephritis, Sjögren's syndrome, IgG4-related disease, idiopathic thrombocytopenic purpura, immune thrombocytopenic purpura, Reiter's syndrome, psoriasis, Behcet's disease, asthma, pemphigus, diabetes, myasthenia gravis, Guillain-Barré syndrome, Graves' disease, Hashimoto's thyroiditis, vasculitis, autoimmune vasculitis, granulomatous polyangiitis, autoimmune hepatitis, especially B-cell lymphoma, diffuse large B-cell lymphoma, chronic lymphocytic leukemia. Leukemia, small lymphocytic lymphoma, non-Hodgkin's lymphoma (e.g., ABC-DLBCL), mantle cell lymphoma, follicular lymphoma, Waldenström macroglobulinemia, marginal zone lymphoma, central nervous system lymphoma, chronic lymphocytic lymphoma, rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, lupus nephritis, Sjögren's syndrome, IgG4-related disease, idiopathic thrombocytopenic purpura, immune thrombocytopenia, pemphigus, urticaria, etc.

[0031] According to the present invention, the pharmaceutical composition may be in any dosage form, including but not limited to tablets, capsules, solutions, lyophilized preparations and injections.

[0032] The pharmaceutical formulations of the present invention can be administered in dose units containing a predetermined amount of the active ingredient. Such units may contain 1 mg to 1 g, preferably 5 mg to 700 mg, more preferably 10 mg to 500 mg of the compound of the present invention, depending on the disease being treated, the method of administration, and the patient's age, weight, and physical condition. The pharmaceutical formulations can be prepared using methods well-known in the pharmaceutical industry, for example, by formulating the active ingredient together with one or more excipients or one or more adjuvants.

[0033] The pharmaceutical formulations of the present invention are suitable for administration by any suitable method, such as oral (including oral or sublingual) or parenteral (including subcutaneous, intramuscular, intravenous or intradermal).

[0034] This invention also provides methods for preparing compounds A and B. The preparation of the compounds of this invention can be accomplished through the following exemplary methods and examples, but these methods and examples should not be considered in any way as limiting the scope of this invention. The compounds described in this invention can also be synthesized using synthetic techniques known to those skilled in the art, or by combining methods known in the art with the methods described in this invention. The products obtained from each reaction step are obtained using separation techniques known in the art, including but not limited to extraction, filtration, distillation, crystallization, and chromatographic separation. The starting materials and chemical reagents required for synthesis can be conventionally synthesized or purchased according to literature (available from SciFinder). Example

[0035] The starting materials of this invention can be synthesized according to methods known in the art, or can be purchased from chemical companies such as ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, Accela ChemBio Inc., and Beijing Coupling Chemical Co.

[0036] The structures of the compounds of this invention were determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). NMR measurements were performed using a Bruker Aschend-400 NMR spectrometer, with solvents such as deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), or deuterated methanol (CD3OD), and tetramethylsilane (TMS) as the internal standard. Chemical shifts were expressed as 10⁻⁶. -6 (ppm) are given as the unit. MS determinations were performed using an Agilent SQD (ESI) mass spectrometer (Agilent 6120).

[0037] HPLC was performed using an Agilent 1260DAD high-performance liquid chromatograph (Poroshell 120 EC-C18, 50×3.0 mm, 2.7 μm column) or a WatersArc high-performance liquid chromatograph (Sunfirc C18, 150×4.6 mm, 5 μm column).

[0038] Thin-layer chromatography (TLC) uses GF254 silica gel plates from Qingdao Haiyang Chemical Co., Ltd., with a thickness of 0.15-0.2 mm. For TLC separation / purification of products, silica gel plates with a thickness of 0.4-0.5 mm are used.

[0039] Column chromatography typically uses 200-300 mesh silica gel from Qingdao Haiyang Chemical Co., Ltd.

[0040] Unless otherwise specified in the examples, all reactions were carried out at room temperature (20℃-30℃) under an argon or nitrogen atmosphere. An argon or nitrogen atmosphere refers to a reaction flask connected to an approximately 1L volume argon or nitrogen balloon.

[0041] The hydrogenation reaction is carried out under a hydrogen atmosphere. A hydrogen atmosphere means that after the reaction flask is evacuated and then refilled with hydrogen (repeated 3 times), a hydrogen balloon with a volume of about 1L is connected to it.

[0042] The reaction progress in the examples was monitored using an Agilent LCMS (1260 / 6120) or thin-layer chromatography. Solvent elution systems used for column chromatography and TLC included a) dichloromethane and methanol, b) petroleum ether and ethyl acetate, or as shown in the examples. The solvent ratios were adjusted according to the polarity of the compound, and further adjustments were made by adding small amounts of triethylamine, or acidic or basic reagents as needed. Alternatively, compound purification was performed using a Waters MS-guided automated prep-HPLC system with an MS detector (SQD2), eluting at a flow rate of 20 mL / min under appropriate acetonitrile / water (containing 0.1% TFA or formic acid) or acetonitrile / water (containing 0.05% 25-28% ammonium hydroxide) gradients (XBridge-C18, 19 × 150 mm, 5 μm). Some compounds, after purification by prep-HPLC, could be prepared as HCl salts by adding 1 N HCl to the collected fraction and then drying under reduced pressure.

[0043] The abbreviation DMF stands for N,N-dimethylformamide.

[0044] The abbreviation DIPEA refers to N,N-diisopropylethylamine.

[0045] The abbreviation DBU refers to 1,8-diazabicycloundec-7-ene.

[0046] The abbreviation NIS stands for N-iodosuccinimide.

[0047] Pd(dppf)Cl2 refers to [1,1′-bis(diphenylphosphino)ferrocene]palladium dichloride.

[0048] The abbreviation HATU refers to 2-(7-azabenzotriazole)-N,N,N′,N′-tetramethylurea hexafluorophosphate.

[0049] Example 1

[0050] 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)

[0051]

[0052] first step

[0053] (4-((5-fluoro-2-methoxybenzamido)methyl)phenyl)boronic acid (1b)

[0054] Oxaloyl chloride (279 mg, 2.2 mmol) was added 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. The mixture was gradually heated to room temperature and stirred for 1 hour, then cooled again to 0 °C, and a suspension of (4-(aminomethyl)phenyl)borate (374 mg, 2 mmol) and DIPEA (516 mg, 4 mmol) in tetrahydrofuran (20 mL) was added. After stirring at room temperature for 15 hours, the solvent was removed under reduced pressure, and the residue was dissolved in ethyl acetate (100 mL), followed by washing with saturated ammonium chloride (50 mL) and brine (50 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was subjected to reduced pressure to remove the solvent, yielding target compound 1b (460 mg, 76%).

[0055] MS m / z (ESI): 304 [M+H]

[0056] Step 2

[0057] 1-(2,5-dioxopyrrolidone-1-yl)4-methylbicyclo[2.2.2]octane-1,4-dicarboxylic acid ester (1d)

[0058] DCC (1.07 g, 5.2 mmol) was added to a THF (50 mL) solution of (methoxycarbonyl)bicyclo[2.2.2]octane-1-carboxylic acid 1c (850 mg, 4 mmol) and 1-hydroxypyrrolidine-2,5-dione (552 mg, 4.8 mmol) at 0 °C. The mixture was heated to room temperature and stirred for 15 hours, then cooled to 0 °C and filtered. The filtrate was concentrated to dryness to give the target compound 1d (1.4 g, crude product).

[0059] MS m / z (ESI): 310 [M+H]

[0060] Step 3

[0061] 4-(((5-oxo-4,5-dihydro-1,2,4-triazin-6-yl)methyl)carbamoyl)bicyclo[2.2.2]octane-1-carboxylic acid methyl ester (1e)

[0062] A solution of 1d (1.4 g, crude) in THF (5 mL) was added dropwise to a DMF (5 mL) solution of 6-aminomethyl-4H-[1,2,4]triazine-5-one (252 mg, 2 mmol) and DIPEA (1.03 g, 8 mmol). The mixture was stirred for 3 hours and then concentrated to dryness. The residue was purified by silica gel column chromatography (dichloromethane / methanol = 50 / 1 to 10 / 1) to give the target compound 1e (150 mg, 23% in two steps).

[0063] MS m / z (ESI): 321 [M+H]

[0064] Step 4

[0065] 4-(4-oxo-3,4-dihydroimidazo[5,1-f][1,2,4]triazin-7-yl)bicyclo[2.2.2]octane-1-carboxylic acid methyl ester (1f)

[0066] POCl3 (2 mL) was added to a MeCN (10 mL) solution of 1e (150 mg, 0.47 mmol). The mixture was heated to 80 °C and stirred for 5 hours. The mixture was then concentrated to dryness to give the target compound 1f (150 mg, 100%).

[0067] MS m / z (ESI): 303 [M+H]

[0068] Step 5

[0069] 4-(5-iodo-4-oxo-3,4-dihydroimidazo[5,1-f][1,2,4]triazin-7-yl)bicyclo[2.2.2]octane-1-carboxylic acid methyl ester (1g)

[0070] NIS (529 mg, 2.35 mmol) was added to 1 f (150 mg, crude, 0.47 mmol) of DMF (5 mL). The mixture was heated to 55 °C and stirred for 5 hours. After cooling to room temperature, the mixture was purified by prep-HPLC to give 1 g (80 mg, 40%) of the target compound.

[0071] MS m / z (ESI): 429 [M+H]

[0072] Step 6

[0073] 4-(4-amino-5-iodoimidazole[5,1-f][1,2,4]triazin-7-yl)bicyclo[2.2.2]octane-1-carboxylic acid methyl ester (1h)

[0074] POCl3 (86 mg, 0.56 mmol) was added to a pyridine (2 mL) solution of 1H-[1,2,4]triazole (131 mg, 1.9 mmol). After stirring the mixture for 10 minutes, 1 g (80 mg, 0.19 mmol) of pyridine (2 mL) solution was added. After stirring again for 1 hour, NH3 solution (2 M isopropanol solution, 10 mL) was added to the mixture and stirred for 0.5 hours. The mixture was then concentrated to dryness, and the residue was purified by prep-HPLC to give the target compound 1h (60 mg, 74%).

[0075] MS m / z (ESI): 428 [M+H]

[0076] Step 7

[0077] 4-(4-amino-5-(4-((5-fluoro-2-methoxybenzoylamino)methyl)phenyl)imidazo[5,1-f][1,2,4]triazin-7-yl)bicyclo[2.2.2]octane-1-carboxylic acid methyl ester (li)

[0078] The 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 and stirred for 2 hours under a nitrogen atmosphere. 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 target compound 1i (60 mg, 77%).

[0079] MS m / z (ESI): 559 [M+H]

[0080] Step 8

[0081] 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)

[0082] Lithium hydroxide solution (1N, 4 mL) was added to a 1i (55 mg, 0.098 mmol) solution of THF (10 mL). The mixture was heated to 40 °C and stirred for 48 hours. 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 prep-HPLC to give target compound A (40 mg, solid, 75%).

[0083] MS m / z (ESI): 545 [M+1]

[0084] 1 H NMR (400MHz, CD3OD) δ7.80 (s, 1H), 7.64-7.56 (m, 3H), 7.53 (d, J=8.1Hz, 2H), 7.28-7.22 (m, 1H), 7.1 7 (dd, J=9.1, 4.2Hz, 1H), 4.69 (s, 2H), 3.96 (d, J=7.6Hz, 3H), 2.30-2.22 (m, 6H), 1.98-1.90 (m, 6H).

[0085] Example 2

[0086] N-(4-(4-amino-7-(4-(morpholin-4-carbonyl)bicyclo[2.2.2]oct-1-yl)imidazolium[5,1-f][1,2,4]triazine-5-yl)benzyl)-5-fluoro-2-methoxybenzamide (Compound B)

[0087]

[0088] HATU (58 mg, 0.151 mmol) was added to a DMF (5 mL) solution of A (75 mg, 0.137 mmol), morpholine (12 mg, 0.137 mmol), and DIPEA (53 mg, 0.409 mmol). The resulting mixture was stirred at room temperature for 5 minutes and then purified by prep-HPLC to give target compound B (40 mg, solid, 48%).

[0089] MS m / z (ESI): 614.2 [M+1]

[0090] 1 H NMR (400MHz, DMSO-d6) δ8.84 (t, J=6.0Hz, 1H), 7.88 (s, 1H), 7.58 (d, J=8.2Hz, 2H), 7.52 (dd, J=9.2, 3.3Hz, 1H), 7.46 (d, J=8.2Hz, 2H), 7.39 -7.29 (m, 1H), 7.19 (dd, J=9.1, 4.3Hz, 1H), 4.57 (d, J=6.1Hz, 2H), 3.90 (s, 3H), 3.57 (d, J=9.6Hz, 8H), 2.21-2.09 (m, 6H), 1.97-1.84 (m, 6H).

[0091] Example 3. BTK activity inhibition test

[0092] The effect of the compounds of the present invention on BTK activity was evaluated using in vitro kinase assays (Table 1).

[0093] The experimental methods are summarized as follows:

[0094] The enzyme activity of BTK was determined by detecting the phosphorylation level of the substrate in the kinase reaction using a homogeneous time-resolved fluorescence (HTRF) kinase assay kit (Cisbio, catalog number 62TK0PEC). The reaction buffer contained the kit's own enzyme reaction buffer (1×), 5 mM MgCl2, 1 mM DTT, 10 nM SEB, and 0.01% Tween-20; human recombinant BTK protein (CarnaBiosciences, catalog number 08-180) was diluted with the reaction buffer to a kinase reaction solution of 0.2 ng / μL; the substrate reaction solution contained biotin-labeled tyrosine kinase substrate diluted with the reaction buffer to 0.5 μM and 40 μM ATP; the assay buffer contained Eu diluted with the reaction buffer to 0.05 ng / μL. 3+ Labeled cage-like antibodies and 31.25 nM streptavidin-labeled XL665 antibody were used. The compounds were dissolved and diluted to 100 μM in DMSO, then serially diluted 4-fold with DMSO to a minimum concentration of 6.1 nM. Each concentration point was then diluted 40-fold with reaction buffer. If the compound IC50... 50 The value is very low, which can reduce the initial concentration of the compound.

[0095] Add 4 μL of compound solution of varying concentration 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. Then add 4 μL of substrate reaction solution and incubate the reaction mixture at room temperature for 50 minutes. Next, add 10 μL of detection buffer, mix thoroughly, and let stand at room temperature for 60 minutes. Read the signal values ​​at 620 nm and 665 nm wavelengths using an Envision plate reader (Perkin Elmer). Signal value (absorbance) 665nm Absorbance 620nm The kinase activity of BTK was positively correlated with the phosphorylation level of the substrate, thus allowing for the detection of BTK kinase activity. In this experiment, the group without BTK served as a negative control (100% inhibition), while the group with BTK but without the compound served as a positive control (0% inhibition). Inhibition curves were plotted using XLfit software (IDBusiness Solutions Ltd., UK), and their IC50 values ​​were calculated. 50 value.

[0096] Example 4. Activity Inhibition Test of BTK C481S

[0097] The effect of the compounds of the present invention on BTK C481S activity was evaluated using in vitro kinase assays (Table 1).

[0098] The experimental methods are summarized as follows:

[0099] The enzyme activity of BTK C481S was determined by detecting the phosphorylation level of the substrate in the kinase reaction using a homogeneous time-resolved fluorescence (HTRF) kinase assay kit (Cisbio, catalog number 62TKOPEC). The reaction buffer contained the kit's own enzyme reaction buffer (1×), 5 mM MgCl2, 1 mM DTT, 10 nM SEB, and 0.01% Tween-20; human recombinant BTKC481S protein (purified in-house) was diluted with the reaction buffer to a kinase reaction solution of 1.5 ng / μL; the substrate reaction solution contained biotin-labeled tyrosine kinase substrate diluted with the reaction buffer to 0.4 μM and 35 μM ATP; the assay buffer contained Eu diluted with the reaction buffer to 0.05 ng / μL. 3+ Labeled cage-like antibodies and 31.25 nM streptavidin-labeled XL665 antibody were used. The compounds were dissolved and diluted to 100 μM in DMSO, then serially diluted 4-fold with DMSO to a minimum concentration of 6.1 nM. Each concentration point was then diluted 40-fold with reaction buffer. If the compound IC50... 50 The value is very low, which can reduce the initial concentration of the compound.

[0100] Add 4 μL of compound solution of varying concentration 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. Then add 4 μL of substrate reaction solution and incubate the reaction mixture at room temperature for 50 minutes. Next, add 10 μL of detection buffer, mix thoroughly, and let stand at room temperature for 60 minutes. Read the signal values ​​at 620 nm and 665 nm using an Envision plate reader (Perkin Elmer). Signal value (absorbance) 665nm Absorbance 620nm The activity of BTK C481S was positively correlated with the degree of phosphorylation of the substrate, thus allowing the detection of kinase activity. In this experiment, the group without BTK C481S served as a negative control (100% inhibition), while the group with BTK C481S but without the compound served as a positive control (0% inhibition). XLfit software (ID Business Solutions Ltd., UK) was used to plot the compound inhibition curves and calculate their IC50 values. 50 value.

[0101] Table 1 shows the BTK IC50 of compounds A and B. 50 and BTK C481S IC 50 The result.

[0102] Table 1

[0103] Compound numbering <![CDATA[BTK IC 50 (nM)]]> <![CDATA[BTK C481S IC 50 (nM)]]> A 0.7 1.3 B 1.0 1.2

[0104] Example 5. Brain permeability test

[0105] Compound A or B was orally administered at a dose of 5 mg / kg to 12 male Sprague Dawley rats in a solution containing 5% N,N-dimethylacetamide, 10% solubilol, and 85% physiological saline at a concentration of 0.5 mg / mL. Plasma, brain tissue homogenate (for Kp, brain measurement), and cerebrospinal fluid (for Kp, CSF measurement) samples were collected at 1, 2, 4, and 8 hours post-administration (samples were taken from three animals at each time point).

[0106] The concentrations of compounds in plasma, brain tissue homogenate, and CSF were quantified by LC-MS / MS using an API-4500 mass spectrometer. The limit of quantitation (LOQ) was 1 ng / mL. Pharmacokinetic (PK) parameters were calculated using WinNonlin non-compartmental methods. Kp, brain and Kp, CSF were calculated using AUCbrain / AUCplasma and AUCCSF / AUCplasma, respectively. The results are shown in Table 2. Compounds A and B are non-brain permeabilizers.

[0107] Table 2

[0108] Compound numbering <![CDATA[K p ,brain]]> <![CDATA[K p ,CSF]]> A 0.0355 0.0054 B 0.0315 0.0043

[0109] The present invention, as well as the manner and process of making and using it, are now described in full, clear, concise and precise terminology to enable any person skilled in the art to make and use them. It should be understood that preferred embodiments of the invention have been described above, and modifications may be made thereto without departing from the scope of the invention as defined in the claims. The following claims summarize the specification in order to particularly point out and explicitly claim the subject matter considered inventive.

Claims

1. Compound A or B, or a pharmaceutically acceptable salt, stable isotopic derivative, or stereoisomer thereof: AB, The stable isotope derivatives mentioned above are selected from the following group: 2 H(D), 13 C, 15 N, 18 O, 17 O, 31 P.

2. A pharmaceutical composition comprising compound A or compound B according to claim 1, or a pharmaceutically acceptable salt thereof, a stable isotope derivative, or a stereoisomer thereof, and a pharmaceutically acceptable carrier thereof, wherein the stable isotope derivative is selected from the group consisting of: 2 H(D), 13 C, 15 N, 18 O, 17 O, 31 P.

3. Use of compound A or B of claim 1, or a pharmaceutically acceptable salt, stable isotope derivative, or stereoisomer thereof, in the preparation of a medicament for the prevention or treatment of BTK-mediated related diseases, wherein a therapeutically effective amount of compound A or B, or a pharmaceutically acceptable salt, stable isotope derivative, or stereoisomer thereof, is administered to a patient in need, and wherein the BTK-mediated diseases are selected from cancer, autoimmune diseases, and inflammatory diseases.

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

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