Heterocyclic compounds that inhibit TYK2 activity

Abstract

The present invention relates to compounds 1 to 8 and their pharmaceutically acceptable salts or prodrugs. Compounds 1 to 8 are selective binding agents for JH2 of TYK2, significantly inhibit the physiological function of TYK2, and have excellent in vivo pharmacokinetics. Compounds 1 to 5 and 7 have some deuterium substitutions in the methyl groups to improve pharmacokinetics (PK).

Description

[Technical Field]

[0001] The present invention relates to heterocyclic compounds useful for modulating TYK2 to block signal transduction, which compounds have improved pharmacokinetics in animals. [Background technology]

[0002] Tyrosine kinase 2 (TYK2) is a non-receptor tyrosine protein kinase belonging to the Janus kinase (JAK) family and has been shown to be important in regulating signaling cascades downstream of IL-12, IL-23, and type I interferon receptors.

[0003] JAKs are characterized by tandem kinase domains. JH1 is a canonical protein tyrosine kinase domain, and JH2 is classified as a pseudokinase domain. The structure of the JAK family is shown in Figure 1.

[0004] Recent biochemical and structural data suggest that the pseudokinase domain of TYK2 has low catalytic activity and negatively regulates the activity of the kinase domain.

[0005] Cytokine binding to cytokine receptors results in phosphorylation of TYK2 and its family members JAK1 and / or JAK2, which bind to the intracellular domain, leading to dimerization and activation of signal transducers and activators of transcription (STATs). The dimerized STATs then translocate to the nucleus, where they regulate the expression and transcription of associated genes, completing the signal transduction pathway from the plasma membrane to the nucleus. Thus, JAKs transduce cytokine-mediated signals via the JAK-STAT pathway, playing an important role in cytokine-dependent regulation of many cellular functions, including cell proliferation, differentiation, apoptosis, and immune responses. TYK2-deficient mice are resistant to experimental models of colitis, psoriasis, and multiple sclerosis, demonstrating the importance of TYK2-mediated signaling in autoimmune and related diseases.

[0006] In humans, individuals expressing inactive variants of TYK2 may be protected from multiple sclerosis and other autoimmune diseases. Genome-wide studies have shown that other variants of TYK2 are associated with autoimmune diseases such as Crohn's disease, psoriasis, systemic lupus erythematosus, and rheumatoid arthritis, further demonstrating the importance of TYK2 in autoimmunity.

[0007] TYK2 knockout mice have normal red blood cell counts and are viable. Lack of TYK2 expression is manifested by impaired signaling of various proinflammatory cytokines and severe imbalance in T helper cell differentiation. Evidence from genetic association studies supports TYK2 as a common susceptibility gene for autoimmune diseases. TYK2-regulated pathways have been identified in antibody therapies for disease treatment. For example, ustekinumab, which targets IL-12 / IL-23 for the treatment of psoriasis, and anifrolumab, which targets the type I interferon receptor for the treatment of systemic lupus erythematosus (SLE), have shown remarkable efficacy in clinical trials.

[0008] TYK2 has been implicated in some cancers due to the correlation between TYK2 activation and abnormal cell survival in acute lymphoblastic leukemia (T-ALL). As a T-ALL oncogene, gene knockout experiments demonstrated that 88% of T-ALL cell lines and 63% of patient-derived T-ALL cells were dependent on TYK2 (Sanda et al., Cancer Disc. 2013, 3, 564-77). The TYK2-selective inhibitor NDI-031301 induced apoptosis and inhibited proliferation of human T-ALL cell lines and showed favorable safety and efficacy in a mouse model using KOPT-K1 T-ALL tumor cells (Akahane et al., British J. Haematol. 2017, 177, 271-82), demonstrating the potential of selective TYK2 inhibitors for the treatment of T-ALL. Therefore, TYK2 is one of the hot targets for treating inflammatory diseases, autoimmune diseases and cancer (Alicea-Velazquez et. al, Curr. Drug Targets 2011, 12, 546-55).

[0009] TYK2 and other members of the JAK family structurally possess a pseudokinase domain JH2 (JAK homology 2) adjacent to the kinase domain JH1 (JAK homology 1). JH2 can bind ATP but does not have catalytic function; instead, it negatively regulates the kinase activity of JH1 (Staerk et al., J. Biol. Chem. 2015, 280, 41893-99). Due to the high sequence similarity of the kinase domain JH1 among the JAK family (JAK1, JAK2, JAK3, and TYK2), it is difficult to develop a selective inhibitor of TYK2 JH1 without inhibiting JH1 of JAK1, JAK2, or JAK3. Most JAK inhibitors that bind to the kinase domain of JAKs, such as tofacitinib, ruxolitinib, baricitinib, and upadacitinib, have poor selectivity among JAKs and exhibit clinically dose-dependent side effects, such as anemia. The development of highly selective TYK2 inhibitors continues to be attractive to pharmaceutical companies. Based on the structural differences between the ATP-binding pockets of TYK2 JH1 and JH2, Bristol-Myers Squibb Company has developed a highly selective JH2 binder, BMS-986165, which inhibits only the physiological functions mediated by TYK2 without binding to the JAK kinase domain (JH1). BMS-986165 is currently in Phase III clinical trials for autoimmune diseases (Wrobleski et al., J. Med. Chem. 2019, 62, 8973-95).

[0010] The structure of BMS-986165 is shown below (WO 2014 / 074661):

[0011] [ka]

[0012] There is a continuing need to develop novel compounds that selectively bind to the pseudokinase domain (JH2) of TYK2 and have minimal binding to the kinase domain of the JAK family, particularly JAK2. [Brief explanation of the drawings]

[0013] [Figure 1] FIG. 1 shows the general secondary structure of the JAK family (JAK1, JAK2, JAK3 and TYK2). [Figure 2] Figure 2 shows the in vivo efficacy in an animal model of anti-CD40 antibody-induced IBD colitis. The relative body weight change (%) of animals treated with vehicle, reference compound, and three different doses of Compound 3 is plotted against the number of days after treatment. DETAILED DESCRIPTION OF THE INVENTION

[0014] The present inventors have discovered selective TYK2 inhibitors that target the TYK2 pseudokinase domain (JH2) rather than the catalytically active site of TYK2. The present invention relates to compounds 1-8 and pharmaceutically acceptable salts or prodrugs thereof. Compounds 1-8 are selective binders for TYK2 JH2. By binding to the pseudokinase domain (JH2), compounds 1-8 inhibit the kinase catalytic activity of TYK2, inhibiting protein phosphorylation and exhibiting significant inhibitory effects on the physiological function of TYK2. Compounds 1-8 bind weakly or not at all to the kinase domain (JH1) of TYK2. By binding to JH2, compounds 1-8 selectively inhibit the kinase activity of TYK2 and have low inhibitory activity against the kinase activity of other JAK family members. The selectivity of compounds 1-8 in inhibiting TYK2 over other JAK family members (JAK1, JAK2, and JAK3) minimizes side effects such as anemia. Compounds 1-8 have been shown to have excellent in vivo pharmacokinetics in animals.

[0015] [Table A-1]

[0016] [Table A-2]

[0017] As used herein, a "pharmaceutically acceptable salt" is a salt that retains the desired biological activity of the parent compound and does not impart undesired toxicity. Pharmaceutically acceptable salt forms include various crystalline polymorphs as well as amorphous forms of different salts. Pharmaceutically acceptable salts of the present basic heterocyclic compounds can be formed with inorganic or organic acids.

[0018] As used herein, "prodrug" refers to a compound that, upon administration to a subject, undergoes conversion by metabolic or chemical processes to yield a compound of Compounds 1-8 and / or a salt thereof. Compounds that are converted in vivo to provide a bioactive agent of Compounds 1-8 are prodrugs within the scope of the present invention. Various forms of prodrugs are well known in the art.

[0019] Compound 1 has a trideuterated methyl group attached to the triazole ring and a trideuterated methyl amide group.

[0020] Compound 2 has a trideuterated methyl group attached to the triazole ring.

[0021] Compound 3 has a trideuterated methoxy group attached to the benzene ring.

[0022] Compound 4 has a trideuterated methoxy group attached to the benzene ring and a trideuterated methylamide group.

[0023] Compound 5 has an (S)-6-(2,2-difluorocyclopropane-1-carboxamide) group and a trideuterated methylamide group.

[0024] Compound 6 has an (S)-6-(2,2-difluorocyclopropane-1-carboxamide) group but no deuterated substituents.

[0025] Compound 7 has an (R)-6-(2,2-difluorocyclopropane-1-carboxamide) group and a trideuterated methylamide group.

[0026] Compound 8 has an (R)-6-(2,2-difluorocyclopropane-1-carboxamide) group but no deuterated substituents.

[0027] Compounds 1-5 and 7 have several deuterium substitutions on the methyl group to improve pharmacokinetic (PK) properties. Compounds 5-8 have difluoro groups on the cyclopropane ring. The compounds of the present invention have low binding activity to the kinase domain of JAK and high inhibitory activity against TYK2 cellular functions, such as inhibition of gamma interferon and IL-23 secretion. The compounds of the present invention have good bioavailability when administered orally. The compounds of the present invention are safe to use and have been shown to be effective in treating inflammatory bowel disease (IBD) in anti-CD40 colitis (IBD) model mice, and treatment with compounds 2, 3, and 5 did not result in significant weight loss.

[0028] Pharmaceutical Composition The present invention provides pharmaceutical compositions comprising one or more pharmaceutically acceptable carriers and an active compound or a pharmaceutically acceptable salt thereof of Compounds 1 to 8. Generally, the active compound or a pharmaceutically acceptable salt thereof in the pharmaceutical composition is present in an amount of about 0.01 to 20%, 0.05 to 20%, 0.1 to 20%, 0.2 to 15%, 0.5 to 10%, or 1 to 5% (w / w) for topical preparations, about 0.1 to 5% for injections, 0.1 to 5% for patches, about 1 to 90% for tablets, and 1 to 100% for capsules.

[0029] In one embodiment, the active compound is formulated in an acceptable carrier, including a cream, gel, lotion, or other suspension that stabilizes the active compound and can be delivered to the affected area by topical application. In another embodiment, the pharmaceutical composition can be in the form of a tablet, capsule, granule, fine granule, powder, syrup, suppository, injection, patch, etc. The pharmaceutical composition can be prepared by conventional methods.

[0030] Pharmaceutically acceptable carriers for the inactive ingredients can be selected by those skilled in the art according to conventional criteria, including, but not limited to, non-aqueous solutions, suspensions, emulsions, microemulsions, micellar solutions, gels, and ointments. Pharmaceutically acceptable carriers include saline and aqueous electrolyte solutions; ionic and non-ionic osmotic agents such as sodium chloride, potassium chloride, glycerin, and glucose; pH adjusters and buffers such as hydroxides, phosphates, citrates, acetates, and borates; trolamine; antioxidants such as bisulfites, sulfites, metabisulfites, thiosulfites, ascorbic acid, acetylcysteine, cysteine, glutathione, butylated hydroxyanisole, butylated hydroxytoluene, tocopherols, salts, acids, and / or bases of ascorbyl palmitate; surfactants such as lecithin, phospholipids, including, but not limited to, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinosithiol; poloxamers and poloxamines, polysorbate 80, polysorbate 60, and the like. Other ingredients may include, but are not limited to, polysorbates such as polysorbate 20, polyethers such as polyethylene glycol and polypropylene glycol; polyvinyls such as polyvinyl alcohol and povidone; cellulose derivatives such as methylcellulose, hydroxypropylcellulose, hydroxyethylcellulose, carboxymethylcellulose, and hydroxypropylmethylcellulose and their salts; petroleum derivatives such as mineral oil and white petrolatum; fats such as lanolin, peanut oil, palm oil, and soybean oil; mono-, di-, and triglycerides; polymers of acrylic acid such as carboxypolymethylene gel and hydrophobically modified crosslinked acrylate copolymers; polysaccharides such as dextran, and glycosaminoglycans such as sodium hyaluronate.Such pharmaceutically acceptable carriers may be preserved against microbial contamination through the use of well-known preservatives, including, but not limited to, benzalkonium chloride, ethylenediaminetetraacetic acid and its salts, benzethonium chloride, chlorhexidine, chlorobutanol, methylparaben, thimerosal, and phenylethyl alcohol, and may be formulated as single-use or multi-use unpreserved preparations.

[0031] For example, tablets or capsules containing active compounds may contain excipients that are not biologically active and do not react with the active compound. Tablet or capsule excipients may include fillers, binders, lubricants and glidants, disintegrants, wetting agents, and release rate modifiers. Binders promote adhesion of formulation particles and are important in tablets. Examples of tablet or capsule excipients include, but are not limited to, carboxymethylcellulose, cellulose, ethylcellulose, hydroxypropylmethylcellulose, methylcellulose, karaya gum, starch, tragacanth gum, gelatin, magnesium stearate, titanium dioxide, poly(acrylic acid), and polyvinylpyrrolidone. For example, tablets may contain inactive ingredients such as colloidal silicon dioxide, crospovidone, hypromellose, magnesium stearate, microcrystalline cellulose, polyethylene glycol, sodium starch glycolate, and / or titanium dioxide. Capsules may contain inactive ingredients such as gelatin, magnesium stearate, and / or titanium dioxide.

[0032] For example, an active compound patch may contain inactive ingredients such as 1,3-butylene glycol, dihydroxyaluminum aminoacetate, disodium edetate, D-sorbitol, gelatin, kaolin, methylparaben, polysorbate 80, povidone, propylene glycol, propylparaben, sodium carboxymethylcellulose, sodium polyacrylate, tartaric acid, titanium dioxide, and purified water. The patch may also contain a skin permeation enhancer such as a lactate ester (e.g., lauryl lactate) or diethylene glycol monoethyl ether.

[0033] Topical formulations containing the active compound may be in the form of gels, creams, lotions, liquids, emulsions, ointments, sprays, solutions, and suspensions. Inactive ingredients in topical formulations include, but are not limited to, diethylene glycol monoethyl ether (emollient / penetration enhancer), DMSO (solubility enhancer), silicone elastomer (rheology / texture modifier), caprylic / capric triglyceride (emollient), octisalate (emollient / UV filter), silicone oil (emollient / thinner), squalene (emollient), sunflower oil (emollient), and silicon dioxide (thickener).

[0034] How to use The present inventors demonstrated that the present compounds specifically bind to the pseudokinase domain (JH2) of TYK2 and significantly inhibit the physiological function of TYK2 in NK92 cells. These compounds also show excellent pharmacokinetics in rats.

[0035] The present invention relates to a method for preventing or treating TYK2-mediated diseases, including, but not limited to, autoimmune diseases, inflammatory diseases (including intestinal inflammation), cancer, skin diseases, diabetes, eye diseases, neurodegenerative diseases, allergic reactions, asthma, other obstructive airway diseases, and transplant rejection. The method is particularly useful for treating inflammatory bowel disease, psoriasis, and systemic lupus erythematosus (SLE). The method comprises administering an effective amount of a compound of the present invention, or a prodrug thereof, or a pharmaceutically acceptable salt thereof, to a patient in need thereof. As used herein, an "effective amount" refers to an amount effective to treat a disease by ameliorating a pathological condition or alleviating symptoms.

[0036] The pharmaceutical composition of the present invention can be applied by local administration and systemic administration. Local administration includes administration to a local area. Systemic administration includes oral (including buccal or sublingual), parenteral (intravenous, intramuscular, subcutaneous, or rectal, etc.), and other systemic administration routes. In systemic administration, the active compound first enters the plasma and then distributes to the target tissue. Topical administration and oral administration are preferred administration routes of the present invention.

[0037] The dosage of the composition varies based on the extent of the injury and the individual response of each patient. When administered systemically, the plasma concentration of the active compound delivered varies; however, typically, it is greater than 1×10 -10 ~1×10 -4 mol / L, preferably 1×10 -8 ~1×10 -5 mol / L.

[0038] In some embodiments, the composition is applied topically to the affected area and rubbed in. The composition is applied topically at least once or twice daily, or three to four times daily, depending on the medical event and whether the condition is chronic or acute. Typically, topical compositions contain about 0.01-20%, 0.05-20%, 0.1-20%, 0.2-15%, 0.5-10%, or 1-5% (w / w) of the active compound. The active compound penetrates the skin and is delivered to the area of ​​concern.

[0039] In some embodiments, the pharmaceutical composition is orally administered to a subject. The oral dosage is typically at least 0.1 mg / kg / day and less than 1000 mg / kg / day. For example, the oral dosage is 0.5 mg to 1 g, preferably 1 mg to 700 mg or 5 mg to 300 mg of the compound per day.

[0040] Those skilled in the art will recognize that a wide variety of delivery mechanisms are suitable for the present invention.

[0041] The present invention is useful for treating mammals such as humans, horses and dogs. The present invention is particularly useful for treating humans.

[0042] The following examples further illustrate the present invention. These examples are intended to be merely illustrative of the invention and should not be construed as limiting. [Example]

[0043] Examples 1-8 describe the synthesis of compounds of the present invention. The products of each reaction step can be obtained by 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 the synthesis can be synthesized by conventional methods according to the literature (searchable in SciFinder) or purchased commercially.

[0044] The structure of the compound is determined by nuclear magnetic resonance (NMR) or mass spectrometry (MS). NMR was measured on a Bruker ASCEND-400 NMR spectrometer. The solvent used was deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), or deuterated methanol (CD3OD). Tetramethylsilane (TMS) was used as the internal standard. The unit of chemical shift is 10 -6 (ppm).

[0045] MS was measured using an Agilent SQD (ESI) mass spectrometer (manufacturer: Agilent, model: 6120).

[0046] HPLC was performed using an Agilent 1260 DAD high pressure liquid chromatograph (Poroshell 120 EC-C18, 50 × 3.0 mm, 2.7 μm column) or a Waters Arc high pressure liquid chromatograph (Sunfire C18, 150 × 4.6 mm, 5 μm column).

[0047] Thin-layer chromatography (TLC) was performed using Qingdao Ocean GF254 silica gel plates, with thicknesses of 0.15–0.2 mm for reaction monitoring and 0.4–0.5 mm for product separation and purification, respectively.

[0048] Most of the column chromatography was performed using Qingdao Ocean silica gel 200~ (300 mesh) as the carrier.

[0049] Known starting materials used in the present invention can be synthesized by methods known in the art or can be purchased commercially from ABCR GmbH & Co. KG, Acros Organics, Sigma-Aldrich Chemical Company, Accela ChemBio Inc., Beijing Ouhe Chemicals, and other companies.

[0050] In the following examples, all reactions were carried out under an argon or nitrogen atmosphere unless otherwise specified.

[0051] The hydrogenation reaction was typically carried out in a reactor that was evacuated and filled with hydrogen three times.

[0052] Microwave reactions were carried out using a CEM Discover-SP microwave reactor.

[0053] In the following examples, the reaction temperature is room temperature, 20°C to 30°C, unless otherwise specified.

[0054] The progress of the reaction was monitored by Agilent LCMS (1260 / 6120). In some cases, it was also monitored by TLC. The solvents used for TLC were: A: dichloromethane and methanol system; B: petroleum ether and ethyl acetate system; and C: the system shown in the examples. The volume ratio of the solvents was adjusted depending on the polarity of the compounds.

[0055] The column chromatography and TLC used in the purification process of the compounds were eluted with A: dichloromethane and methanol system, B: petroleum ether and ethyl acetate system, and C: the system shown in the examples. The volume ratio of the solvents was adjusted according to the polarity of the compounds, and a small amount of triethylamine and an acidic or basic reagent was added.

[0056] Compounds were also purified using Waters' mass spectrometry-oriented automated preparation system (prep-HPLC with a mass detector of SQD2). Depending on the polarity of the compounds, an appropriate acetonitrile / water (containing 0.1% trifluoroacetic acid or formic acid) or acetonitrile / water (containing 0.05% ammonium hydroxide) elution profile was used, and the purification was performed on a reversed-phase high-pressure column (XBridge-C18, 19 × 150 mm, 5 μm) at a flow rate of 20 mL / min.

[0057] Example 1 6-(cyclopropanecarboxamido)-4-((2-methoxy-3-(1-(methyl-d3)-1H-1,2,4-triazol-3-yl)phenyl)amino)-N-(methyl-d3)pyridazine-3-carboxamide (1)

[0058] [ka]

[0059] Process 1 Methyl 2-methoxy-3-nitrobenzoate (1b) To a solution of methyl 2-fluoro-3-nitrobenzoate 1a (10 g, 50 mmol) in methanol (50 mL) was added sodium methoxide (12.6 g, 70 mmol) at room temperature. After stirring at room temperature for 4 hours, the solution was diluted with water (200 mL) and then extracted with ethyl acetate (3 × 60 mL). The organic phases were combined, washed with saturated brine (2 × 100 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated to dryness under reduced pressure to give the target compound 1b (10 g, solid) in 98% yield.

[0060] MS m / z (ESI): 212 [M+1]

[0061] Process 2 2-Methoxy-3-nitrobenzamide (1c) To a solution of methyl 2-methoxy-3-nitrobenzoate 1b (10 g, 47 mmol) in methanol (40 mL) was added ammonium hydroxide (20 mL) at room temperature. After stirring at room temperature for 48 hours, the solvent was removed under reduced pressure to give the desired compound 1c (crude, 10 g, solid). The crude product was used in the next step without further purification.

[0062] MS m / z (ESI): 197 [M+1]

[0063] Process 3 3-(2-Methoxy-3-nitrophenyl)-1H-1,2,4-triazole (1d) A solution of 2-methoxy-3-nitrobenzamide 1c (10 g, 51 mmol) in N,N-dimethylformamide dimethyl acetal (50 mL) was heated to 95 °C and stirred for 2 h. After cooling to room temperature, the solvent was removed under reduced pressure, and the residue was dissolved in ethanol (30 mL) to obtain solution A. To a mixture of acetic acid (35 mL) and ethanol (150 mL) was slowly added hydrazine hydrate (25 mL) at 0 °C, followed by solution A. The mixture was gradually warmed to room temperature and stirred for 12 h, after which the solvent was removed under reduced pressure. The residue was dispersed in water (400 mL) and filtered. The resulting solid was washed with water and dried to obtain the target compound 1d (6 g, solid) in 55% yield.

[0064] MS m / z (ESI): 221 [M+1]

[0065] Process 4 3-(2-Methoxy-3-nitrophenyl)-1-(methyl-d3)-1H-1,2,4-triazole (1e) To a mixture of 3-(2-methoxy-3-nitrophenyl)-1-(methyl-d3)-1H-1,2,4-triazole 1d (1.2 g, 5.3 mmol), potassium carbonate (2.2 g, 16 mmol), and N,N-dimethylformamide (10 mL) was added deuterated iodomethane (1 g, 6.9 mmol). After stirring at room temperature for 12 h, the resulting solution was purified by reverse-phase preparative HPLC to give the target compound 1e (530 mg, solid) in 42% yield.

[0066] MS m / z (ESI): 238 [M+1]

[0067] Process 5 2-Methoxy-3-(1-(methyl-d3)-1H-1,2,4-triazol-3-yl)aniline (1f) To a solution of 3-(2-methoxy-3-nitrophenyl)-1-(methyl-d3)-1H-1,2,4-triazole 1e (530 mg, 1.61 mmol) in methanol (10 mL) was added 10% palladium on carbon (50 mg). The reaction mixture was stirred under a hydrogen atmosphere for 12 hours and then filtered. The filtrate was concentrated to dryness under reduced pressure to give the target compound 1f (430 mg, solid). The product was used in the next reaction without further purification.

[0068] MS m / z (ESI): 208 [M+1]

[0069] Process 6 Lithium 4,6-dichloropyridazine-3-carboxylate (1h) To a mixture of methyl 4,6-dichloropyridazine-3-carboxylate 1g (5 g, 24.15 mmol), diisopropylethylamine (9.4 g, 72.5 mmol), acetonitrile (13.5 mL), and water (3.25 mL) was added lithium bromide (6.3 g, 72.5 mmol). The resulting mixture was stirred at room temperature for 12 hours and then filtered. The resulting solid was washed with acetonitrile (8 mL) and dried in vacuo to give the target compound 1h (4.53 g, solid) in 90% yield.

[0070] MS m / z (ESI): 193 [M+1]

[0071] Process 7 6-chloro-4-((2-methoxy-3-(1-(methyl-d3)-1H-1,2,4-triazol-3-yl)phenyl)amino)pyridazine-3-carboxylate zinc salt (1i) To a mixture of lithium 4,6-dichloropyridazine-3-carboxylate 1h (380 mg, 1.9 mmol), 2-methoxy-3-(1-(methyl-d3)-1H-1,2,4-triazol-3-yl)aniline 1f (471 mg, 2.27 mmol), isopropanol (0.5 mL), and water (5 mL), zinc acetate (350 mg, 1.9 mmol) was added at room temperature. The mixture was heated to 65 °C and stirred for 12 h. After cooling to room temperature, the reaction mixture was diluted with water (30 mL), stirred for 30 min, and filtered. The solid was washed with water (2 × 30 mL) and tetrahydrofuran (2 × 30 mL) and dried in vacuo to give the target compound 1i (490 mg, solid) in 71% yield.

[0072] MS m / z (ESI): 364 [M+1]

[0073] Process 8 6-(cyclopropanecarboxamido)-4-((2-methoxy-3-(1-(methyl-d3)-1H-1,2,4-triazol-3-yl)phenyl)amino)pyridazine-3-carboxylate methyl (1j) To a mixture of zinc 6-chloro-4-((2-methoxy-3-(1-(methyl-d3)-1H-1,2,4-triazol-3-yl)phenyl)amino)pyridazine-3-carboxylate 1i (490 mg, 1.15 mmol), cyclopropanecarboxamide (300 mg, 3.45 mmol), (2R)-1-[(1R)-1-[bis(1,1-dimethylethyl)phosphino]ethyl]-2-(dicyclohexylphosphino)ferrocene (63 mg, 0.115 mmol), palladium acetate (25 mg, 0.0575 mmol), toluene (9 mL), and acetonitrile (5 mL) was added potassium carbonate (320 mg, 7.8 mmol) and 1,8-diazabicycloundec-7-ene (180 mg, 1.5 mmol), sequentially. The resulting mixture was stirred under nitrogen at 80° C. for 72 h. After cooling to room temperature, the solvent was removed under reduced pressure, and the residue was purified by reverse-phase preparative HPLC to give the target compound 1j (560 mg, solid) in 99% yield.

[0074] MS m / z (ESI): 413 [M+1]

[0075] Process 9 6-(Cyclopropanecarboxamido)-4-((2-methoxy-3-(1-(methyl-d3)-1H-1,2,4-triazol-3-yl)phenyl)amino)-N-(methyl-d3)pyridazine-3-carboxamide (1) A mixture of methyl 6-(cyclopropanecarboxamido)-4-((2-methoxy-3-(1-(methyl-d3)-1H-1,2,4-triazol-3-yl)phenyl)amino)pyridazine-3-carboxylate 1j (280 mg, 0.68 mmol), deuterated methylamine hydrochloride (60 mg, 0.81 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (181 mg, 0.95 mmol), 1-hydroxybenzotriazole (53 mg, 0.34 mmol), acetonitrile (3 mL), N-methylpyrrolidone, and N-methylimidazole (41 mg, 0.5 mmol) was heated to 65 °C and stirred for 1 h. After cooling to room temperature, the solvent was removed under reduced pressure, and the residue was purified by reverse-phase preparative HPLC to give the target compound 1 (44 mg, solid) in 15% yield.

[0076] MS m / z (ESI): 429 [M+1] 1 H NMR (400 MHz, DMSO-d6) δ11.32 (s, 1H), 10.97 (s, 1H), 9.13 (s, 1H), 8.56 (s, 1H), 8.15 (s, 1H), 7.65 (dd, J = 7.8, 1.5 Hz, 1H), 7.54 - 7.46 (m, 1H), 7.32 - 7.22 (m, 1H), 3.72 (s, 3H), 2.12 - 2.03 (m, 1H), 0.88 - 0.73 (m, 4H).

[0077] Example 2 6-(cyclopropanecarboxamido)-4-((2-methoxy-3-(1-(methyl-d3)-1H-1,2,4-triazol-3-yl)phenyl)amino)-N-methylpyridazine-3-carboxamide (2) Compound 2 was synthesized by the method of Example 1, except that in step 9, methylamine hydrochloride (CH3NH2·HCl) was used instead of deuterated methylamine hydrochloride (CD3NH2·HCl).

[0078] MS m / z (ESI): 426 [M+1] 1 H NMR (400 MHz, DMSO-d6) δ 11.32 (s, 1H), 10.97 (s, 1H), 9.22 - 9.11 (m, 1H), 8.56 (s, 1H), 8.15 (s, 1H), 7.66 (dd, J = 7.8, 1.5 Hz, 1H), 7.51 (dd, J = 8.0, 1.5 Hz, 1H), 7.31 - 7.22 (m, 1H), 3.72 (s, 3H), 2.86 (d, J = 4.8 Hz, 3H), 2.15 - 2.01 (m, 1H), 0.87 - 0.75 (m, 4H).

[0079] Example 3 6-(cyclopropanecarboxamido)-4-((2-(methoxy-d3)-3-(1-methyl-1H-1,2,4-triazol-3-yl)phenyl)amino)-N-methylpyridazine-3-carboxamide (3)

[0080] [ka]

[0081] Process 1 N-Methylformohydrazide To a solution of methylhydrazine sulfate 3a (40 g, 277 mmol) in methanol (250 mL) was added sodium methoxide (100 g, 554 mmol) at room temperature. The resulting mixture was stirred for 24 hours and filtered. Methyl formate (17 g, 277 mmol) was then added to the filtrate, and the mixture was stirred at room temperature for 18 hours. The solvent was removed under reduced pressure to give the target compound 5b (22 g, crude). The crude product was used directly in the next step without further purification.

[0082] MS m / z (ESI): 75 [M+1]

[0083] Process 2 5-chloro-2-(methoxy-d3)benzonitrile (3d) To a mixture of 5-chloro-2-hydroxybenzonitrile 3c (4 g, 26 mmol), potassium carbonate (7.3 g, 53 mmol), and N,N-dimethylformamide (30 mL) was added deuterated methyl iodide (10 g, 78 mmol) at room temperature. The resulting mixture was heated to 70 °C and stirred for 12 h. After cooling to room temperature, the reaction mixture was diluted with water (200 mL) and extracted with ethyl acetate (2 × 100 mL). The combined organic phase was washed with saturated brine (2 × 100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to dryness under reduced pressure to give the target compound 3d (4.3 g, solid) in 97% yield.

[0084] MS m / z (ESI): 171 [M+1]

[0085] Process 3 3-(5-chloro-2-(methoxy-d3)phenyl)-1-methyl-1H-1,2,4-triazole sulfate (3e) A solution of 5-chloro-2-(methoxy-d3)benzonitrile 3d (4.3 g, 25.3 mmol) and N-methylformylhydrazine (4.1 g, 58 mmol) in tetrahydrofuran (20 mL) was added sequentially to a solution of potassium tert-butoxide (11.3 g, 101 mmol) in tetrahydrofuran (30 mL) at 0 °C. After stirring at room temperature for 12 h, water (50 mL) was added to the mixture, which was then heated to 40 °C and stirred for 40 min. After cooling to room temperature, the organic phase was separated, washed with saturated brine (40 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was dissolved in ethyl acetate (40 mL). Concentrated sulfuric acid (5 g) was slowly added to the resulting solution at room temperature, and the mixture was stirred for 12 h. The mixture was then filtered and dried to give the target compound 3e (5.6 g, solid) in 83% yield.

[0086] MS m / z (ESI): 227 [M+1]

[0087] Process 4 3-(5-chloro-2-(methoxy-d3)-3-nitrophenyl)-1-methyl-1H-1,2,4-triazole (3f) To a solution of 3-(5-chloro-2-(methoxy-d3)phenyl)-1-methyl-1H-1,2,4-triazole sulfate 3e (5.6 g, 24.7 mmol) in sulfuric acid (25 g) was added nitric acid (2 g) at 0°C. The resulting solution was gradually warmed to room temperature, stirred for 12 hours, and cooled to 0°C again. Water (67 mL) and methanol (47 mL) were added to the solution at 0°C, followed by warming to room temperature and stirring for 1 hour. The solution was heated to 40°C, and ammonium hydroxide (42 mL) was added. The solution was cooled to 20°C, stirred for 2 hours, and filtered. The solid was washed with water (2 × 30 mL) and dried in vacuo to give the target compound 3f (3.37 g, solid) in 50% yield.

[0088] MS m / z (ESI): 272 [M+1]

[0089] Process 5 2-(Methoxy-d3)-3-(1-methyl-1H-1,2,4-triazol-3-yl)aniline (3g) To a solution of 3-(5-chloro-2-(methoxy-d3)-3-nitrophenyl)-1-methyl-1H-1,2,4-triazole 3f (3.37 g, 12.25 mmol) in methanol (10 mL) was added 10% palladium on carbon (400 mg) and sodium bicarbonate (1.6 g, 25 mmol). The resulting mixture was stirred under a hydrogen atmosphere for 12 hours and then filtered. The filtrate was concentrated to dryness under reduced pressure, and the residue was dissolved in dichloromethane (25 mL). The resulting mixture was filtered, and the filtrate was concentrated to dryness under reduced pressure to give the target compound 3g (2.35 g, solid) in a 92% yield.

[0090] MS m / z (ESI): 208 [M+1]

[0091] Process 6 6-chloro-4-((2-(methoxy-d3)-3-(1-methyl-1H-1,2,4-triazol-3-yl)phenyl)amino)pyridazine-3-carboxylate zinc(3h) To a mixture of lithium 4,6-dichloropyridazine-3-carboxylate 1h (3 g, 15.1 mmol), 2-(methoxy-d3)-3-(1-methyl-1H-1,2,4-triazol-3-yl)aniline 3g (2.35 g, 11.3 mmol), isopropanol (2.5 mL), and water (18 mL) was added zinc acetate (2.5 g, 13.6 mmol) at room temperature. The mixture was heated to 65 °C and stirred for 12 h. After cooling to room temperature, the mixture was diluted with water (20 mL), stirred for 30 min, and filtered. The solid was washed with water (2 × 30 mL) and tetrahydrofuran (2 × 30 mL) and dried in vacuo to give the target compound 3h (4.3 g, solid) in 100% yield.

[0092] MS m / z (ESI): 364 [M+1]

[0093] Process 7 Methyl 6-(cyclopropanecarboxamido)-4-((2-(methoxy-d3)-3-(1-methyl-1H-1,2,4-triazol-3-yl)phenyl)amino)pyridazine-3-carboxylate (3i) A mixture of 6-chloro-4-((2-(methoxy-d3)-3-(1-methyl-1H-1,2,4-triazol-3-yl)phenyl)amino)pyridazine-3-zinc carboxylate 3h (4.3 g, 11 mmol), cyclopropanecarboxamide (2.4 g, 27.56 mmol), (2R)-1-[(1R)-1-[bis(1,1-dimethylethyl)phosphino]ethyl]-2-(dicyclohexylphosphino)ferrocene (600 mg, 1.1 mmol), palladium acetate (125 mg, 0.55 mmol), toluene (34 mL), acetonitrile (17 mL), potassium carbonate (3.1 g, 22 mmol), and 1,8-diazabicycloundec-7-ene (1.7 g, 11 mmol) was heated to 80° C. under a nitrogen atmosphere and stirred for 12 hours. After cooling to room temperature, aqueous acetic acid (50%, 17 mL) and glacial acetic acid (40 mL) were added sequentially to the mixture. After stirring at room temperature for 1 hour, the resulting homogeneous mixture was washed with petroleum ether (2 × 20 mL). Water (50 mL) was added, and the mixture was aged at room temperature for 4 hours and filtered. The solid was washed sequentially with aqueous acetonitrile (50%, 20 mL) and acetonitrile (20 mL), and then dried under vacuum at 65 °C for 30 minutes to give the target compound 3i (3 g, solid) in 66% yield.

[0094] MS m / z (ESI): 413 [M+1]

[0095] Process 8 6-(cyclopropanecarboxamido)-4-((2-(methoxy-d3)-3-(1-methyl-1H-1,2,4-triazol-3-yl)phenyl)amino)-N-methylpyridazine-3-carboxamide A mixture of methyl 6-(cyclopropanecarboxamido)-4-((2-(methoxy-d3)-3-(1-methyl-1H-1,2,4-triazol-3-yl)phenyl)amino)pyridazine-3-carboxylate 3i (1.5 g, 3.38 mmol), methylamine hydrochloride (280 mg, 4.0 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (900 mg, 4.73 mmol), 1-hydroxybenzotriazole (230 mg, 1.7 mmol), acetonitrile (3 mL), N-methylpyrrolidone (3 mL), and N-methylimidazole (200 mg, 2.4 mmol) was heated to 65 °C and stirred for 12 hours. After completion of the reaction, the reaction was quenched with water (1.5 mL) and acetonitrile (4.5 mL). The resulting mixture was aged at 65°C for 1 hour and at 0°C for 3 hours, and then filtered. The solid was washed successively with aqueous acetonitrile (33%, 4.5 mL) and acetonitrile (4.5 mL) and dried under vacuum at 65°C for 8 hours to give the target compound 3 (811 mg, solid) in a 56% yield.

[0096] MS m / z (ESI): 426 [M+1] 1 H NMR (400 MHz, DMSO-d6) δ 11.32 (s, 1H), 10.97 (s, 1H), 9.24 - 9.08 (m, 1H), 8.57 (s, 1H), 8.15 (s, 1H), 7.66 (dd, J = 7.8, 1.5 Hz, 1H), 7.52 (dd, J = 7.9, 1.5 Hz, 1H), 7.33 - 7.20 (m, 1H), 3.96 (s, 3H), 2.87 (d, J = 4.8 Hz, 3H), 2.14 - 2.01 (m, 1H), 0.91 - 0.73 (m, 4H).

[0097] Compound 3 could be converted to the hydrochloride salt by the following procedure: A reaction flask was charged with 3 (5.00 g, 11.752 mmol) and DMSO (27 mL). The resulting mixture was heated to 50–55 °C with stirring until the solid became a completely homogeneous solution. Concentrated hydrochloric acid (36%–38%, 1.18 g) was then added to the mixture, followed by water (3 mL) and seed crystals (25 mg). The resulting mixture was stirred at 50–55 °C for 0.5 h, cooled to 35–40 °C, and isopropanol (60 mL) was added dropwise over 0.5–1.0 h. The mixture was stirred at 35–40 °C for 0.5 h. The mixture was slowly cooled to 20–25 °C over 1 h, stirred overnight, and filtered. The filter cake was washed with isopropanol (2 × 15 mL) and dried under reduced pressure at 65 °C overnight to give the monohydrochloride salt of 3 (4.5 g, solid) in 83% yield.

[0098] MS m / z (ESI): 426 [M+1] 1 H NMR (400 MHz, DMSO-d6) δ 13.72 (brs, 1H), 12.13 (s, 1H), 11.40 (s, 1H), 9.22 (q, J = 4.5 Hz, 1H), 8.87 (s, 1H), 8.00 (s, 1H), 7.78 (dd, J = 7.9, 1.5 Hz, 1H), 7.61 (dd, J = 8.0, 1.4 Hz, 1H), 7.35 (t, J = 7.9 Hz, 1H), 4.01 (s, 3H), 2.89 (d, J = 4.8 Hz, 3H), 2.14 - 2.00 (m, 1H), 1.00 - 0.84 (m, 4H).

[0099] Example 4 6-(cyclopropanecarboxamido)-4-((2-(methoxy-d3)-3-(1-methyl-1H-1,2,4-triazol-3-yl)phenyl)amino)-N-(methyl-d3)pyridazine-3-carboxamide (4) Compound 4 was synthesized by the method of Example 3, except that in step 8, deuterated methylamine hydrochloride (CD3NH2·HCl) was used instead of methylamine hydrochloride (CH3NH2·HCl).

[0100] MS m / z (ESI): 429 [M+1] 1 H NMR (400 MHz, DMSO-d6) δ11.32 (s, 1H), 10.98 (s, 1H), 9.14 (s, 1H), 8.57 (s, 1H), 8.15 (s, 1H), 7.66 (dd, J = 7.8, 1.6 Hz, 1H), 7.52 (dd, J = 7.9, 1.5 Hz, 1H), 7.32 - 7.21 (m, 1H), 3.96 (s, 3H), 2.14 - 2.03 (m, 1H), 0.89 - 0.75 (m, 4H).

[0101] Example 5 (S)-6-(2,2-difluorocyclopropane-1-carboxamide)-4-((2-methoxy-3-(1-methyl-1H-1,2,4-triazol-3-yl)phenyl)amino)-N-(methyl-d3)pyridazine-3-carboxamide (5)

[0102] [ka]

[0103] Process 1 (S)-N-(2,4-Dimethoxybenzyl)-2,2-difluorocyclopropane-1-carboxamide (5b) To a mixture of (S)-2,2-difluorocyclopropane-1-carboxylic acid 5a (1.5 g, 12.3 mmol), HATU (5.7 g, 15 mmol), diisopropylethylamine (4.8 g, 37 mmol), and N,N-dimethylformamide (15 mL) was added 2,4-dimethoxybenzylamine (4.0 g, 24.4 mmol). After stirring at room temperature for 3 h, the solvent was removed under reduced pressure, and the residue was purified by reverse-phase preparative HPLC to give the target compound 5b (4.4 g, solid).

[0104] MS m / z (ESI): 272 [M+1]

[0105] Process 2 (S)-2,2-Difluorocyclopropane-1-carboxamide (5c) A solution of (S)-N-(2,4-dimethoxybenzyl)-2,2-difluorocyclopropane-1-carboxamide 5b in trifluoroacetic acid (10 mL) was heated to 70 °C and stirred for 1 h. After cooling to room temperature, the mixture was concentrated to dryness, and the residue was purified by silica gel column chromatography (dichloromethane / methanol, 100 / 0 to 9 / 1) to give the target compound 5d (1.4 g, solid) in a 93% yield over two steps.

[0106] MS m / z (ESI): 122 [M+1]

[0107] Process 3 3-(5-chloro-2-methoxyphenyl)-1-methyl-1H-1,2,4-triazole (5d) To a solution of potassium tert-butoxide (34 g, 290 mmol) in tetrahydrofuran (200 mL) was added 5-chloro-2-methoxybenzonitrile (20 g, 120 mmol) and methyl formylhydrazide 3b (22 g, crude) sequentially at 0 °C. After stirring at room temperature for 72 h, water (500 mL) was added, and the mixture was extracted with ethyl acetate (3 × 300 mL). The combined organic phases were washed with saturated brine (2 × 300 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to dryness under reduced pressure to give the desired compound 5d (17.1 g, solid) in 88% yield.

[0108] MS m / z (ESI): 224 [M+1]

[0109] Process 4 3-(5-chloro-2-methoxy-3-nitrophenyl)-1-methyl-1H-1,2,4-triazole (5e) To a solution of 3-(5-chloro-2-methoxyphenyl)-1-methyl-1H-1,2,4-triazole 5d (16.13 g, 72 mmol) in concentrated sulfuric acid (72 g) was added concentrated nitric acid (8.5 g, 87 mmol) at 0 °C. After stirring for 2 h, a mixture of water (250 g) and methanol (150 g) was added to the resulting solution at 0 °C. The mixture was then adjusted to pH > 7 with ammonium hydroxide and filtered. The solid was washed with water (2 × 100 mL) and purified by silica gel column chromatography (100 / 0 to 3 / 7 petroleum ether / ethyl acetate) to give the target compound 5e (17.1 g, solid) in 88% yield.

[0110] MS m / z (ESI): 269 [M+1]

[0111] Process 5 2-Methoxy-3-(1-methyl-1H-1,2,4-triazol-3-yl)aniline (5f) To a solution of 3-(5-chloro-2-methoxyphenyl)-1-methyl-1H-1,2,4-triazole 5e (17 g, 63 mmol) in methanol was added 10% palladium on carbon (3 g) and sodium bicarbonate (10.5 g, 126 mmol). The mixture was stirred under a hydrogen atmosphere for 5 hours and then filtered. The filtrate was concentrated to dryness under reduced pressure, and the residue was purified by reverse-phase preparative HPLC to give the target compound 5f (8.8 g, solid) in 68% yield.

[0112] MS m / z (ESI): 205 [M+1]

[0113] Process 6 6-chloro-4-((2-methoxy-3-(1-methyl-1H-1,2,4-triazol-3-yl)phenyl)amino)pyridazine-3-carboxylate zinc salt (5g) To a mixture of lithium 4,6-dichloropyridazine-3-carboxylate 1h (4.53 g, 22.87 mmol), 2-methoxy-3-(1-methyl-1H-1,2,4-triazol-3-yl)aniline 5f (5.6 g, 27.44 mmol), isopropanol (4.5 mL), and water (34 mL) was added zinc acetate (4.2 g, 22.87 mmol). The resulting mixture was heated to 65 °C and stirred for 12 h. After cooling to room temperature, the mixture was diluted with water (30 mL), aged for 30 min, and filtered. The solid was washed with water (2 × 30 mL) and tetrahydrofuran (2 × 30 mL) and dried in vacuo to give the target compound 5g (7.6 g, solid) in 93% yield.

[0114] MS m / z (ESI): 361 [M+1]

[0115] Process 7 (S)-6-(2,2-difluorocyclopropane-1-carboxamido)-4-((2-methoxy-3-(1-methyl-1H-1,2,4-triazol-3-yl)phenyl)amino)pyridazine-3-carboxylate zinc(5h) 6-chloro-4-((2-methoxy-3-(1-methyl-1H-1,2,4-triazol-3-yl)phenyl)amino)pyridazine-3-carboxylate zinc 5g (1.6 g, 3.93 mmol), (S)-2,2-difluorocyclopropane-1-carboxamide 5c (1.2 g, 9.8 mmol), (2R)-1-[(1R)-1-[bis(1,1-dimethylethyl)phosphino]ethyl] A mixture of [[(2-(dicyclohexylphosphino)-2-(dicyclohexylphosphino)ferrocene] (220 mg, 0.393 mmol), palladium acetate (44 mg, 0.196 mmol), toluene (18 mL), acetonitrile (11 mL), potassium carbonate (1.1 g, 7.8 mmol), and 1,8-diazabicycloundec-7-ene (600 mg, 3.93 mmol) was stirred at 80 °C for 72 h under a nitrogen atmosphere. After cooling to room temperature, the mixture was diluted with acetic acid (27 mL) and water (9 mL), and the resulting solution was washed with petroleum ether (2 × 30 mL). Water (50 mL) was then added and the mixture was allowed to stand for 3 h. The mixture was filtered, and the solid was dried in vacuo to give the target compound 5h (1.1 g, solid) in 62% yield.

[0116] MS m / z (ESI): 446 [M+1]

[0117] Process 8 (S)-6-(2,2-difluorocyclopropane-1-carboxamide)-4-((2-methoxy-3-(1-methyl-1H-1,2,4-triazol-3-yl)phenyl)amino)-N-(methyl-d3)pyridazine-3-carboxamide To a mixture of (S)-zinc 6-(2,2-difluorocyclopropane-1-carboxamido)-4-((2-methoxy-3-(1-methyl-1H-1,2,4-triazol-3-yl)phenyl)amino)pyridazine-3-carboxylate 5h (1.1 g, 2.46 mmol), deuterated methylamine hydrochloride (210 mg, 2.95 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (660 mg, 3.44 mmol), 1-hydroxybenzotriazole (190 mg, 1.23 mmol), acetonitrile (6 mL), and N-methylpyrrolidone (6 mL) was added N-methylimidazole (141 mg, 1.72 mmol). The reaction mixture was heated to 65° C. and stirred for 1 hour. After cooling to room temperature, the mixture was concentrated to dryness under reduced pressure, and the residue was purified by reverse-phase preparative HPLC to give target compound 5 (420 mg, solid) in 37% yield.

[0118] MS m / z (ESI): 462 [M+1] 1 H NMR (400 MHz, DMSO-d6) δ 11.52 (s, 1H), 11.01 (s, 1H), 9.18 (s, 1H), 8.58 (s, 1H), 8.09 (s, 1H), 7.67 (dd, J = 7.8, 1.6 Hz, 1H), 7.53 (dd, J = 7.9, 1.5 Hz, 1H), 7.33 - 7.23 (m, 1H), 3.95 (s, 3H), 3.73 (s, 3H), 3.13 - 2.97 (m, 1H), 2.10 - 1.95 (m, 2H).

[0119] Example 6 (S)-6-(2,2-difluorocyclopropane-1-carboxamide)-4-((2-methoxy-3-(1-methyl-1H-1,2,4-triazol-3-yl)phenyl)amino)-N-methylpyridazine-3-carboxamide (6) Compound 6 was synthesized by the procedure of Example 5, except that in step 8, deuterated methylamine hydrochloride (CD3NH2·HCl) was replaced with methylamine hydrochloride (CH3NH2·HCl).

[0120] MS m / z (ESI): 459 [M+1] 1 H NMR (400 MHz, DMSO-d6) δ 11.52 (s, 1H), 11.01 (s, 1H), 9.20 (d, J = 4.8 Hz, 1H), 8.56 (s, 1H), 8.09 (s, 1H), 7.67 (dd, J = 7.8, 1.6 Hz, 1H), 7.52 (dd, J = 8.0, 1.5 Hz, 1H), 7.33 - 7.23 (m, 1H), 3.95 (s, 3H), 3.73 (s, 3H), 3.11 - 2.98 (m, 1H), 2.86 (d, J = 4.8 Hz, 3H), 2.10 - 1.94 (m, 2H).

[0121] Example 7 (R)-6-(2,2-difluorocyclopropane-1-carboxamido)-4-((2-methoxy-3-(1-methyl-1H-1,2,4-triazol-3-yl)phenyl)amino)-N-(methyl-d3)pyridazine-3-carboxamide (7) Compound 7 was synthesized by the procedure of Example 5, except that in step 1, (S)-2,2-difluorocyclopropane-1-carboxylic acid (5a) was replaced with (R)-2,2-difluorocyclopropane-1-carboxylic acid.

[0122] MS m / z (ESI): 462 [M+1] 1 H NMR (400 MHz, DMSO-d6) δ 11.52 (s, 1H), 11.01 (s, 1H), 9.18 (s, 1H), 8.56 (s, 1H), 8.09 (s, 1H), 7.67 (dd, J = 7.8, 1.6 Hz, 1H), 7.53 (dd, J = 7.9, 1.5 Hz, 1H), 7.28 (m, 1H), 3.95 (s, 2H), 3.73 (s, 3H), 3.11 - 2.99 (m, 1H), 2.10 - 1.95 (m, 2H).

[0123] Example 8 (R)-6-(2,2-difluorocyclopropane-1-carboxamide)-4-((2-methoxy-3-(1-methyl-1H-1,2,4-triazol-3-yl)phenyl)amino)-N-methylpyridazine-3-carboxamide (8) Compound 8 was synthesized by the procedure described in Example 5, except that (i) in step 1, (S)-2,2-difluorocyclopropane-1-carboxylic acid (5a) was replaced with (R)-2,2-difluorocyclopropane-1-carboxylic acid, and (ii) in step 8, deuterated methylamine hydrochloride (CDNH·HCl) was replaced with methylamine hydrochloride (CHNH·HCl).

[0124] MS m / z (ESI): 459 [M+1] 1 H NMR (400 MHz, DMSO-d6) δ 11.52 (s, 1H), 11.01 (s, 1H), 9.27 - 9.16 (m, 1H), 8.56 (s, 1H), 8.09 (s, 1H), 7.67 (dd, J = 7.8, 1.6 Hz, 1H), 7.52 (dd, J = 8.0, 1.5 Hz, 1H), 7.33 - 7.24 (m, 1H), 3.95 (s, 2H), 3.73 (s, 2H), 3.11 - 2.99 (m, 1H), 2.86 (d, J = 4.8 Hz, 3H), 2.08 - 1.95 (m, 2H).

[0125] Example 9 Enzyme Activity Assay of JAK2 Kinase Domain The effect of the compounds of the present invention on the enzymatic activity of recombinant JAK2 kinase domain (JH1) is evaluated by detecting the amount of substrate phosphorylation in the kinase reaction using an HTRF kinase assay detection kit (Cisbio, Cat. No. 62TK0PEC) (Table 1).

[0126] The experimental method is outlined below: A reaction buffer containing the following components: enzyme buffer (1x), 5 mM MgCl2, 1 mM DTT, and 0.01% Brij35 (included in the kit); human recombinant JAK2 kinase domain protein (Carna Biosciences, Cat. No. 08-045) diluted to 0.15 ng / μL in reaction buffer; substrate reaction solution containing 2.5 μM ATP and biotinylated tyrosine kinase substrate diluted to 0.25 μM in reaction buffer; and 0.1 ng / μL Eu 3+ Detection solution containing labeled caged antibody (Cisbio, Cat. No. 61T66KLB) and 12.5 nM streptavidin-labeled XL665.

[0127] Test compounds are dissolved in DMSO to 1 mM and serially diluted four times with DMSO to a minimum concentration of 61 nM. Each concentration is further diluted 40-fold with reaction buffer.

[0128] 4 μL of compound solution and 2 μL of JAK2 kinase solution were added to a 384-well assay plate (Corning, Cat. No. 3674). The mixture was incubated at room temperature for 15 minutes, and then 4 μL of substrate reaction solution was added. After further incubation at room temperature for 30 minutes, 10 μL of detection solution was added to the reaction mixture and left at room temperature for 30 minutes. The progress of the reaction was measured at 620 nm and 665 nm using an Envision plate reader (Perkin Elmer). The ratio of absorbance at 620 nm and 665 nm positively correlates with the degree of substrate phosphorylation, thereby detecting JAK2 kinase activity. In this experiment, the group without JAK2 kinase protein represented 100% inhibition, and the group containing JAK2 kinase protein but without test compound represented 0% inhibition. The inhibition rate of JAK2 kinase activity by the test compound was calculated using the following formula: Inhibition rate = 100 - 100 × (ratio 化合物 -ratio 100%阻害 ) / (ratio 0%阻害 -ratio 100%阻害 )

[0129] IC of test compound 50is calculated from the eight concentrations using the following formula using XLfit software (ID Business Solutions Ltd., UK): Y=Bottom+(Top-Bottom) / (1+10^((logIC 50 -X) × gradient coefficient) (wherein Y is the inhibition rate, X is the logarithm of the concentration of the test compound, Bottom is the lower plateau value of the S-shaped curve, Top is the upper plateau value of the S-shaped curve, and the slope coefficient is the slope coefficient of the curve.)

[0130] Example 10 Enzyme Activity Assay of TYK2 Kinase Domain The effect of the compounds of the present invention on the enzymatic activity of recombinant TYK2 kinase domain (JH1) is evaluated by detecting the amount of substrate phosphorylation in the kinase reaction using an HTRF kinase assay detection kit (Cisbio, Cat. No. 62TK0PEC) (Table 1).

[0131] The experimental method is outlined below: A reaction buffer containing the following components: enzyme buffer (1x), 5 mM MgCl2, 1 mM DTT, 10 nM SEB (Cisbio, Cat. No. 61SEBALB), 0.625 mM EGTA, and 0.01% Brij35 (included in the kit); human recombinant TYK2 kinase (JH1) domain protein (Carna Biosciences, Cat. No. 08-147) diluted to 0.25 ng / μL in reaction buffer; a substrate reaction solution containing 11.25 μM ATP and biotinylated tyrosine kinase substrate diluted to 0.5 μM in reaction buffer; and 0.1 ng / μL Eu 3+ Detection solution containing labeled caged antibody (Cisbio, Cat. No. 61T66KLB) and 25 nM streptavidin-labeled XL665.

[0132] Test compounds are dissolved in DMSO to 1 mM and serially diluted four times with DMSO to a minimum concentration of 61 nM. Each concentration is further diluted 40-fold with reaction buffer.

[0133] Four microliters of compound solution and two microliters of TYK2 kinase solution were added to a 384-well assay plate (Corning, Cat. No. 3674). The mixture was incubated at room temperature for 15 minutes, followed by the addition of 4 microliters of substrate reaction solution. After further incubation at room temperature for 40 minutes, 10 microliters of detection solution was added to the reaction mixture, which was then left at room temperature for 30 minutes. The progress of the reaction was measured at 620 nm and 665 nm using an Envision plate reader (Perkin Elmer). The ratio of absorbance at 620 nm and 665 nm positively correlates with the degree of substrate phosphorylation, thus detecting TYK2 kinase activity. In this experiment, the group containing no TYK2 kinase protein represented 100% inhibition, while the group containing TYK2 kinase protein but no test compound represented 0% inhibition. The inhibition rate of TYK2 kinase activity by the test compound was calculated using the following formula: Inhibition rate = 100 - 100 × (ratio 化合物 -ratio 100%阻害 ) / (ratio 0%阻害 -ratio 100%阻害 )

[0134] IC of test compound 50 is calculated from the eight concentrations using the following formula using XLfit software (ID Business Solutions Ltd., UK): Y=Bottom+(Top-Bottom) / (1+10^((logIC 50 -X) × gradient coefficient) (wherein Y is the inhibition rate, X is the logarithm of the concentration of the test compound, Bottom is the lower plateau value of the S-shaped curve, Top is the upper plateau value of the S-shaped curve, and the slope coefficient is the slope coefficient of the curve.)

[0135] Example 11 TYK2 Pseudokinase Domain Binding Assay The binding of the compounds of the present invention to the TYK2 pseudokinase domain (JH2) is determined by using a time-resolved fluorescence energy transfer (TR-FRET) biochemical assay by competition with a commercially available fluorescein-labeled probe (Alexa-Fluor 647-conjugated kinase tracer 178) (Table 1).

[0136] The experimental method is outlined below: The binding buffer contained 20 mM Hepes pH 7.5, 150 mM NaCl, 10 mM MgCl2, 0.015% Brij35, 2 mM DTT, 0.625 mM EGTA, and 100 mM KF. The JH2 domain of TYK2 (amino acids 556–871 of the full-length protein) was expressed and purified at the Protein Purification and Identification Platform of Tsinghua University. Test compounds were dissolved in DMSO to 0.1 mM and serially diluted four times with DMSO to a minimum concentration of 61 nM. Each concentration of the sample was further diluted 40-fold with the reaction buffer.

[0137] Five μL of compound solution and five μL of TYK2 JH2 domain solution (160 nM) were added to a 384-well assay plate (Corning, Cat. No. 4512). The mixture was incubated at room temperature for 30 minutes, followed by the addition of 10 μL of a mixture of a fluorescein-labeled probe (ThermoFisher, Cat. No. PV5593) (20 nM) and a GST-europium (Eu)-labeled antibody (Cisbio, Cat. No. 61GSTKLA) (40 ng / mL). After a further 30 minutes of incubation at room temperature, the HTRF signal (the ratio of the fluorescence intensity at the emission wavelength of the fluorescein acceptor at 615 nm to the fluorescence intensity at the emission wavelength of the europium donor at 665 nm) was measured using an Envision plate reader (Perkin Elmer). The percent inhibition was calculated by comparing the positive control without test compound and the negative control without protein using the following formula: Inhibition rate (%) = 100 - 100 × (signal 化合物 -signal 陰性対照 ) / (signal 陽性対照 -signal陰性対照 )

[0138] IC of test compound 50 is calculated from the eight concentrations using the following formula using XLfit software (ID Business Solutions Ltd., UK): Y=Bottom+(Top-Bottom) / (1+10^((logIC 50 -X) × gradient coefficient) (wherein Y is the inhibition rate, X is the logarithm of the concentration of the test compound, Bottom is the lower plateau value of the S-shaped curve, Top is the upper plateau value of the S-shaped curve, and the slope coefficient is the slope coefficient of the curve.)

[0139] [Table 1]

[0140] The inhibitory activity of the compounds of the present invention against the kinase domain of JAK2 or TYK2 is weak or slight. Table 1 shows the IC values ​​of compounds 2, 3, 4, 7, and 8 that directly inhibit the kinase activity. 50 is greater than 10 μM, whereas the IC of the reference compound 50 All test and reference compounds bound strongly to TYK2 JH2 (IC in the nanomolar range). 50 ).

[0141] Example 12 Inhibition of IL-12-induced IFN-γ secretion in NK92 cells The effect of compounds of the invention on TYK2-induced IFN-γ secretion in NK92 cells is assessed by enzyme-linked immunosorbent assay (ELISA) (Table 2).

[0142] IL-12 receptors are mainly expressed on activated T cells, NK cells (NK92 is a NK cell line), DCs, and B cells. Binding of IL-12 activates the JAK2 / TYK2 signaling pathway in NK cells and T lymphocytes, inducing IFN-γ secretion.

[0143] The experimental method is outlined below: Test compounds were dissolved in DMSO to a concentration of 2.5 mM and serially diluted four times with DMSO to a minimum concentration of 0.31 nM. Each concentration was further diluted 50-fold with FBS-free MEM alpha medium (Gibco, Cat. No. 12561-056). NK92 cells (Nanjing Cobioer, Cat. No. CBP60980) were cultured in complete MEMα medium containing 12.5% ​​FBS (Ausbian, Cat. No. VS500T), 12.5% ​​horse serum (Gibco, Cat. No. 16050-122), 0.02 mM folic acid (Sigma, Cat. No. F8758), 0.2 mM inositol (Sigma, Cat. No. 17850), 0.55 mM β-mercaptoethanol (Gibco, Cat. No. 21985-023), 200 U / mL IL-2 (R&D Systems, Cat. No. 202-1L), and 100 U / mL penicillin (ThermoFisher, Cat. No. 15140122). Once 80-90% of the culture vessel surface is covered, disperse the cells and seed 100,000 cells per well (80 µL of complete MEMα medium without IL-2) into a 96-well plate (ThermoFisher, Cat No. 167425). Then, incubate the 96-well plate overnight at 37 °C / 5% CO.

[0144] After overnight incubation, 10 μL of test compound and 10 μL of 50 ng / mL IL-12 (R&D Systems, Cat. No. 219-1L) were added to each well and mixed gently. The 96-well plate was then further incubated at 37°C / 5% CO2 for 24 hours. The plate was centrifuged at 800 rpm for 10 minutes at room temperature, and 50 μL of the supernatant from each well was transferred to another 96-well plate coated with anti-IFN-γ antibody (Sigma, Cat. No. CLS3695). IFN secretion was detected according to the instructions of the Human IFN-γ DuoSet ELISA Kit (R&D Systems, Cat. No. DY285B). In the experiment, the group in which IL-12 and test compound were replaced with MEMα medium served as the unstimulated control group (100% inhibition), and the group containing IL-12 and 0.2% DMSO served as the stimulated group (0% inhibition). The inhibition rate of IL-12-induced IFN-γ secretion in NK-92 cells by the test compound is calculated by the following formula: Inhibition rate = 100 - 100 × (signal 化合物 -signal 非刺激対象 ) / (signal 刺激対象 -Signal 非刺激対象 )

[0145] IC of test compound 50 is calculated from the eight concentrations using the following formula using XLfit software (ID Business Solutions Ltd., UK): Y=Bottom+(Top-Bottom) / (1+10^((logIC 50 -X) × gradient coefficient) (wherein Y is the inhibition rate, X is the logarithm of the concentration of the test compound, Bottom is the lower plateau value of the S-shaped curve, Top is the upper plateau value of the S-shaped curve, and the slope coefficient is the slope coefficient of the curve.)

[0146] [Table 2]

[0147] The compounds of the present invention had a significant inhibitory effect on TYK2-induced IFN-γ secretion in NK92 cells.

[0148] Example 13 In vivo PK measurements in rats The pharmacokinetics of compound 3 of the present invention and the reference compound BMS-986165 were evaluated. Compound 3 has an OCD3 on the benzene ring, while the reference compound has a CD3 in the amide moiety. Methyl groups are generally unstable in vivo, undergoing hydrolysis by amidase in the case of methylamides and oxidative demethylation by CYPs in the case of methoxy groups and methyltriazoles. Replacing the methyl with a trideuterated methyl improves the compound's bioavailability and in vivo exposure, increasing the compound's efficacy at the same dose.

[0149] Compound 3 and the reference compound were orally administered at a dose of 5 mg / kg to three male Sprague-Dawley rats at 0.5 mg / mL in a solution containing 5% N,N-dimethylacetamide, 20% solutol, and 75% saline. Blood samples were collected at 0.25, 0.5, 1, 2, 4, 8, and 24 hours post-dose. Plasma compound concentrations were quantified by LC-MS / MS using an API-4500 mass spectrometer. The analytical limit of quantification (LOQ) was 1 ng / mL. Pharmacokinetic (PK) parameters were calculated using non-compartmental methods using WinNonlin and are shown in Table 3. Compound 3 of the present invention demonstrated superior in vivo exposure compared to the reference compound.

[0150] [Table 3]

[0151] Example 14 Evaluation of in vivo efficacy using an animal model of anti-CD40 antibody-induced colitis Female CB17-Scid mice (8–10 weeks old, 18–20 g) obtained from the Vital River laboratory in Beijing were randomly divided into five groups (n = 8 per group). On day 0, colitis was induced in the mice by a single intraperitoneal injection of 100 μg of FGK4.5 anti-CD40 mAb (BioXCell, Cat. No. EB0016-2) in PBS. Starting from day 0 to day 7, mice in the treatment groups were orally administered 0, 1.5, 5, or 15 mg / kg of compound 3 or 5 mg / kg of BMS-986165 dissolved in the vehicle DMSO / Solutol / PEG-400 (10:5:30) twice daily. Mice in the vehicle group were orally administered the same vehicle. Mice were weighed daily and monitored for signs of colitis, including weight loss, loose stools, and diarrhea. All animals were euthanized on day 8. Spleen tissues were collected and weighed. The groups treated with Compound 3 at doses of 1.5 mg / kg, 5 mg / kg, and 15 mg / kg, as well as the group treated with the reference compound at 5 mg / kg, showed significant protection from colitis, preventing weight loss (Figure 2, Table 4) and spleen enlargement (Table 4) compared with the vehicle group.

[0152] [Table 4]

[0153] It will be understood that this specification describes preferred embodiments of the invention and that modifications can be made without departing from the scope of the invention as set forth in the claims.

Claims

1. Compound 3 represented by the following chemical structural formula or a pharmaceutically acceptable salt thereof. 【Chemical 1】

2. Compound 2 represented by the following chemical structural formula or a pharmaceutically acceptable salt thereof. 【Chemistry 2】

3. Compound 4 represented by the following chemical structural formula or a pharmaceutically acceptable salt thereof. 【Chemistry 3】

Images