RU2833524C1 - Heterocyclic compounds for tyk2 activity inhibition - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to compounds selected from:

or their pharmaceutically acceptable salts, which are selective binding agents for JH2 TYK2 and exhibit a significant inhibitory effect on the physiological function of TYK2.

EFFECT: obtaining novel compounds which selectively bind JH2 TYK2 and exhibit a significant inhibitory effect on the physiological function of TYK2, as well as having excellent pharmacokinetic properties in vivo.

4 cl, 2 dwg, 4 tbl, 14 ex

Description

Field of technology to which the present invention relates

The present invention relates to heterocyclic compounds useful for modulating TYK2 to cause inhibition of signal transduction. The compounds provide improved pharmacokinetic properties in animals.

Background of the present invention

Tyrosine kinase 2 (TYK2) is a non-receptor tyrosine protein kinase belonging to the Janus kinase (JAK) family and has been shown to be critical in regulating the signal transduction cascade downstream of receptors for IL-12, IL-23, and type I interferons.

Tandem kinase domains are a hallmark of JAKs. JH1 is the canonical protein tyrosine kinase domain, while JH2 is classified as a pseudokinase domain. The structure of the JAK family is shown in Fig. 1.

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

When cytokine receptors bind cytokines, phosphorylation of TYK2 and other members of its family JAK1 and/or JAK2 that are associated with intracellular regions is triggered, resulting in activation of signal transduction and transcription activating factors (STATs) by dimerization. Dimerized STATs then migrate into the nucleus and regulate the expression and transcription of related genes for complete signal transduction from the cell membrane to the nucleus. Thus, JAKs transmit cytokine-mediated signals through the JAK-STAT pathway and play an important role in many cellular functions, cytokine-dependent regulation of cell proliferation, differentiation, apoptosis, immune response, etc. TYK2-deficient mice are resistant to experimental models of colitis, psoriasis and multiple sclerosis, demonstrating the importance of TYK2-mediated signaling in autoimmunity and related disorders.

In humans, individuals expressing an inactive variant of TYK2 are protected from multiple sclerosis and possibly other autoimmune disorders. Genome-wide studies have found that other TYK2 variants are associated with autoimmune disorders such as Crohn's disease, psoriasis, systemic lupus erythematosus, and rheumatoid arthritis, further demonstrating the importance of TYK2 in autoimmunity.

TYK2 knockout mice have normal red blood cell counts and are able to survive. The absence of TYK2 expression is manifested by attenuated signaling of various proinflammatory cytokines and a severe imbalance in T helper cell differentiation. Genetic data support TYK2 as a common susceptibility gene for autoimmune diseases. Pathways regulated by TYK2 have been validated by antibody therapies for the treatment of diseases. 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 demonstrated significant efficacy in clinical trials.

TYK2 is associated with several cancers through a correlation between abnormal survival of acute lymphocytic leukemia (T-ALL) cells and TYK2 activation. Regarding the T-ALL oncogene, 88% of T-ALL cell lines and 63% of patient-derived T-ALL cells were dependent on TYK2 in gene knockout experiments (Sanda et al., Cancer Disc. 2013, 3, 564-77). The selective TYK2 inhibitor NDI-031301 induced apoptosis to inhibit the growth of human T-ALL cell lines and showed good safety and efficacy in KOPT-K1 T-ALL tumor mouse model (Akahane et. al., British J. Haematol. 2017, 177,271-82), demonstrating the promise of TYK2 selective inhibitors for the treatment of T-ALL. Thus, TYK2 is one of the hot targets for the treatment of inflammatory diseases, autoimmune diseases and cancer (Alicea-Velazquez et. al, Curr. Drug Targets 2011, 12, 546-55).

TYK2 and other JAK family members structurally share a JH1 (JAK Homology 1) kinase domain and an adjacent JH2 (JAK Homology 2) pseudokinase domain. JH2 can bind ATP but has no catalytic function and instead 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 JH1 kinase domain among the JAK family (JAK1, JAK2, JAK3, and TYK2), it is challenging to develop a JH1-selective TYK2 inhibitor without inhibiting JH1 by JAK1, JAK2, or JAK3. Most JAK inhibitors that bind to the kinase domain of JAK, including tofacitinib, ruxolitinib, baricitinib, upadacitinib, etc., are not very selective among JAK family members and exhibit dose-dependent clinical effects such as anemia. The development of highly selective TYK2 inhibitors remains attractive to pharmaceutical companies. Based on the structural differences between the ATP-binding pockets in JH1 and JH2 of TYK2, Bristol-Myers Squibb developed a highly selective JH2 binder BMS-986165, which inhibits only TYK2-mediated physiological functions without binding to the kinase domains (JH1) of JAK. BMS-986165 is currently in phase III clinical trials for autoimmune diseases (Wrobleski et al., J. Med. Chem. 2019, 62, 8973-95). The structure of BMS-986165 is shown below (WO 2014/074661):

There remains a need to develop new compounds that selectively bind to the pseudokinase domain (JH2) of TYK2 with minimal binding to the kinase domains of the JAK families, particularly JAK.2.

Brief description of the drawings

Figure 1 shows the general secondary structure of the JAK family (JAK.1, JAK2, JAK3, and TYK2).

Fig. 2 shows the efficacy in CD40 antibody-induced inflammatory bowel disease colitis in an in vivo animal model. Relative percentage changes in body weight of animals treated with vehicle, reference compound, and compound 3 at three different doses are plotted against the number of days after treatment.

Detailed description of the present invention

The inventors have discovered selective TYK.2 inhibitors that target not the catalytically active site of TYK.2, but the pseudokinase domain of TYK.2 (JH2). The present invention relates to compounds 1-8 and pharmaceutically acceptable salts or prodrugs thereof. Compounds 1-8 are selective binders for JH2 of TYK.2. By binding to the pseudokinase domain (JH2), compounds 1-8 inhibit the catalytic activity of TYK2 kinase, inhibit protein phosphorylation, and exhibit significant inhibitory effects on the physiological function of TYK2. Compounds 1-8 either weakly bind or do not bind to the kinase domain (JH1) of TYK2. Compounds 1-8 selectively inhibit the kinase activity of TYK2 by binding to JH2 and have low inhibitory activity against the kinase activity of other JAK family members. The selectivity of compounds 1-8 for inhibition of TYK2 over other JAK family members (JAK1, JAK2, and JAK3) minimizes effects such as anemia. Compounds 1-8 have been shown to have excellent pharmacokinetic properties in vivo in animals.

"Pharmaceutically acceptable salts" as used herein are salts that retain the desired biological activity of the parent compound and do not exhibit undesirable toxicological effects. Pharmaceutically acceptable salts include various crystalline polymorphs as well as amorphous forms of the various salts. Pharmaceutically acceptable salts of the present basic heterocyclic compounds can be prepared with inorganic acids or organic acids.

"Prodrug" as used herein refers to a compound that, when administered to an individual, undergoes chemical transformation via metabolic or chemical processes to form compound 1-8 and/or a salt thereof. Any compound that will be converted in vivo to a bioactive compound 1-8 is a prodrug within the scope of the invention. Various forms of prodrugs are well known in the art.

Compound 1 has a tri-deuterated methyl on the triazole ring and a tri-deuterated methylamide.

Compound 2 has a tri-deuterated methyl on the triazole ring.

Compound 3 has a trideuterated methoxy group on the benzene ring.

Compound 4 has a tri-deuterated methoxy group on the benzene ring and a tri-deuterated methylamide.

Compound 5 has (S)-6-(2,2-difluorocyclopropane-1-carboxamido) and tri-deuterated methylamide.

Compound 6 has (S)-6-(2,2-difluorocyclopropane-1-carboxamido) without any deuterium substitution.

Compound 7 has (R)-6-(2,2-difluorocyclopropane-1-carboxamido) and tri-deuterated methylamide.

Compound 8 has (R)-6-(2,2-difluorocyclopropane-1-carboxamido) without any deuterium substitution.

Compounds 1-5 and 7 have several deuterium substitutions on methyl to improve pharmacokinetic (PK) properties. Compounds 5-8 have a difluoro on cyclopropane. The compounds of the present invention have low binding activity to the kinase domains of JAKs and have high inhibitory activity on TYK2 cellular functions, for example, by inhibiting the secretion of γ-interferon and IL-23. The compounds of the present invention provide good bioavailability when administered orally. The compounds of the present invention are safe to use and effective in the treatment of inflammatory bowel disease (IBD), as demonstrated in a mouse model of CD40 antibody-induced colitis (IBD), which did not show significant body weight loss after treatment with compound 2, 3 and 5.

Pharmaceutical compositions

The present invention relates to pharmaceutical compositions comprising one or more pharmaceutically acceptable carriers and an active compound of compounds 1-8 or a pharmaceutically acceptable salt thereof. The active compound or its pharmaceutically acceptable salt in the pharmaceutical compositions is generally present in an amount of about 0.01-20%, or 0.05-20%, or 0.1-20%, or 0.2-15%, or 0.5-10% or 1-5% (w/w) for a topical formulation; about 0.1-5% for an injection formulation, 0.1-5% for a patch, about 1-90% for a tablet formulation and 1-100% for a capsule formulation.

In one embodiment, the active compound is incorporated into any suitable carrier, including creams, gels, lotions or other types of suspensions that can stabilize the active compound and deliver it to the affected area for topical application. In another embodiment, the pharmaceutical composition may have a dosage form such as tablets, capsules, granules, fine granules, powders, textures, suppositories, injection solutions, patches or the like. The above-mentioned pharmaceutical composition can be prepared by conventional methods.

Pharmaceutically acceptable carriers, which are inactive ingredients, can be selected by those skilled in the art using generally accepted criteria. Pharmaceutically acceptable carriers include, but are not limited to, non-aqueous based solutions, suspensions, emulsions, microemulsions, micellar solutions, gels, and ointments. Pharmaceutically acceptable carriers may also contain ingredients that include, but are not limited to, saline and aqueous electrolyte solutions; ionic and non-ionic osmotic agents such as sodium chloride, potassium chloride, glycerol, and dextrose; pH adjusters and buffers such as hydroxide, phosphate, citrate, acetate, borate salts; and trolamine; antioxidants such as salts, acids and/or bases of bisulfite, sulfite, metabisulfite, thiosulfite, ascorbic acid, acetylcysteine, cysteine, glutathione, butylated hydroxyanisole, butylated hydroxytoluene, tocopherol, and ascorbyl palmitate; surfactants such as lecithin, phospholipids including, but not limited to, phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol; poloxamers and poloxamines, polysorbates such as polysorbate 80, polysorbate 60 and polysorbate 20, polyethers such as polyethylene glycols and polypropylene glycols; polyvinyls such as polyvinyl alcohol and povidone; Cellulose derivatives such as methylcellulose, hydroxypropylcellulose, hydroxyethylcellulose, carboxymethylcellulose and hydroxypropylcellulose and their salts; petroleum derivatives such as mineral oil and white petrolatum; fats such as lanolin, peanut oil, palm oil, soybean oil; mono-, di- and triglycerides; acrylic acid polymers such as carboxypolymethylene gel and hydrophobically modified cross-linked acrylate copolymer; polysaccharides such as dextrans and glycosaminoglycans such as sodium hyaluronate. Such pharmaceutically acceptable carriers may be protected from bacterial contamination by well known preservatives which include, but are not limited to, benzalkonium chloride, ethylenediaminetetraacetic acid and its salts, benzethonium chloride, chlorhexidine, chlorobutanol, methylparaben, thimerosal and phenylethyl alcohol, or may be formulated as a preservative-free single or multiple use formulation.

For example, a tablet or capsule formulation of the active compound may contain other excipients that do not have biological activity and do not react with the active compound. Excipients for a tablet or capsule may include fillers, binders, lubricants and glidants, disintegrants, humectants, and release rate modifiers. Binders promote aggregation of the particles of the formulation and are essential to the formulation of tablets. Examples of tablet or capsule excipients include, but are not limited to, carboxymethylcellulose, cellulose, ethylcellulose, hydroxypropylmethylcellulose, methylcellulose, gum karaya, starch, gum tragacanth, gelatin, magnesium stearate, titanium dioxide, poly(acrylic acid), and polyvinylpyrrolidone. For example, a tablet formulation may contain inactive ingredients such as colloidal silicon dioxide, crospovidone, hypromellose, magnesium stearate, microcrystalline cellulose, polyethylene glycol, sodium starch glycolate, and/or titanium dioxide. A capsule formulation may contain inactive ingredients such as gelatin, magnesium stearate, and/or titanium dioxide.

For example, the active compound patch formulation may contain certain inactive ingredients such as 1,3-butylene glycol, dihydroxyaluminum aminoacetate, sodium diethate, D-sorbitol, gelatin, kaolin, methylparaben, polysorbate 80, povidone, propylene glycol, propylparaben, sodium carboxymethylcellulose, sodium polyacrylate, tartaric acid, titanium dioxide, and purified water. The patch formulation may also contain a skin penetration enhancer such as lactate esters (e.g., lauryl lactate) or diethylene glycol monoethyl ether.

Topical formulations containing the active compound may be in the form of a gel, cream, lotion, liquid, emulsion, ointment, spray, solvent, and suspension. Inactive ingredients in topical formulations include, for example, but are not limited to, diethylene glycol monoethyl ether (emollient/permeation enhancer), DMSO (solubility enhancer), silicone elastomer (rheology/texture modifier), caprylic/capric triglyceride (emollient), octisalate (emollient/UV filter), silicone fluid (emollient/diluent), squalene (emollient), sunflower oil (emollient), and silicon dioxide (thickener).

Directions for use

The inventor demonstrated that the present compounds specifically bind to the pseudokinase domain of TYK2 (JH2) and significantly inhibit the physiological function of TYK2 in NK92 cells. The compounds also exhibit excellent pharmacokinetic properties in rats.

The present invention relates to a method for the prevention or treatment of TYK2-mediated diseases, including but not limited to autoimmune diseases, inflammatory diseases (including inflammatory bowel disease and inflammatory bowel disease), various types of cancer, skin diseases, diabetes mellitus, eye diseases, neurodegenerative diseases, allergic reactions, asthma, other obstructive pulmonary diseases and transplant rejection, etc. The method is particularly suitable for the treatment of inflammatory bowel disease, psoriasis and systemic lupus erythematosus (SLE). The method comprises administering to a patient in need thereof an effective amount of a compound of the present invention or a prodrug thereof, a pharmaceutically acceptable salt thereof. An "effective amount" as used herein is an amount effective for treating a disease by improving the pathological condition or reducing the symptoms of the diseased.

The pharmaceutical composition of the present invention can be used by topical administration and systemic administration. Topical administration includes topical application. Systemic administration includes oral (including buccal or sublingual), parenteral (such as intravenous, intramuscular, subcutaneous or rectal) and other systemic routes of administration. In systemic administration, the active compound first reaches the plasma and then is distributed to the target tissues. Topical administration and oral administration are preferred routes of administration in the present invention.

The dosage of the composition may vary depending on the degree of damage and the individual response of each patient. When administered systemically, the concentrations of the delivered active compound in plasma may vary, but are generally 1×10 ^-10 -1×10 ^-4 mol/liter, and preferably 1×10 ^-8 -1×10 ^-5 mol/liter.

In one embodiment, the composition is applied topically to the affected area and rubbed into it. The composition is applied topically at least 1 or 2 times a day, or 3-4 times a day, depending on the health condition and whether the pathology is chronic or acute. Basically, the composition for topical application contains about 0.01-20%, or 0.05-20%, or 0.1-20%, or 0.2-15%, 0.5-10 or 1-5% (w/w) of the active compound. The active compound passes through the skin and is delivered to the site of discomfort.

In one embodiment, the pharmaceutical composition is administered to an individual orally. The dosage for oral administration is generally at least 0.1 mg/kg/day and less than 1000 mg/kg/day. For example, the dosage for oral administration is from 0.5 mg to 1 g, from 1 mg to 700 mg, or from 5 mg to 300 mg of the compound per day.

Those skilled in the art will appreciate that a wide range of delivery mechanisms are also suitable for the present invention.

The present invention is intended for the treatment of such mammalian individuals as humans, horses and dogs. The present invention is particularly suitable for the treatment of humans.

The following examples further illustrate the present invention. These examples are intended to illustrate the present invention only and should not be considered as limiting.

EXAMPLES

Examples 1-8 illustrate the synthesis of the present compounds. The product at each reaction step is obtained by separation methods 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 conventionally from literature data (searchable in SciFinder) or purchased.

The structure of the compound was determined using nuclear magnetic resonance (NMR) or mass spectrometry (MS). NMR was measured on a Bruker ASCEND-400 NMR spectrometer. Solvents were deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform ( _CDCl3 ) or deuterated methanol ( _CD3OD ). The internal standard was tetramethylsilane (TMS). Chemical shift is given in units of 10 ^-6 (ppm).

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

HPLC was measured using an Agilent 1260 DAD high-performance liquid chromatograph (Poroshell120 EC-C18 column, 50×3.0 mm, 2.7 μm) or a Waters Arc high-performance liquid chromatograph (Sunfire C18, 150×4.6 mm, 5 μm column).

Thin layer chromatography (TLC) was performed using Qingdao Ocean GF254 silica gel plate. The specifications of TLC for reaction monitoring and product separation/purification are 0.15-0.2 mm and 0.4-0.5 mm thickness, respectively.

Column chromatography was carried out mainly using Qingdao Ocean 200-(300 mesh) silica gel as a carrier.

Known starting materials used at the present scale can be synthesized according to methods known in the art or can be purchased from ABCR GmbH & Co, Acros Organics, Sigma-Aldrich Chemical Company, Accela ChemBio Inc., Beijing Ouhe chemicals and other companies.

In the examples below, unless otherwise stated, all reactions were carried out under argon or nitrogen atmosphere.

The hydrogenation reaction was typically started in a reactor that was evacuated, filled with hydrogen, and the cycle was repeated three times.

The microwave reaction was carried out using a CEM Discover-SP microwave reactor.

In the examples below, unless otherwise stated, the reaction temperature was room temperature from 20°C to 30°C.

The reaction progress was monitored using an Agilent LCMS (1260/6120). TLC monitoring can also be performed. The solvent system for TLC was A: dichloromethane and methanol system; B: petroleum ether and ethyl acetate system; C: the system shown in the examples. The volume ratio of the solvents was adjusted according to the polarity of the compound.

The eluent system for column chromatography and TLC used in the purification process of the compounds included A: dichloromethane and methanol system; B: petroleum ether and ethyl acetate system; C: the system shown in the examples. The volume ratio of the solvents was adjusted according to the polarity of the compound, and a small amount of triethylamine and an acidic or basic reagent could be added for adjustment.

Purification of the compound can also be accomplished using an automated preparative mass spectrometry-oriented Waters system (prep-HPLC with SQD2 mass detector). Depending on the polarity of the compound, an appropriate elution profile of acetonitrile/water (containing 0.1% trifluoroacetic acid or formic acid) or acetonitrile/water (containing 0.05% ammonium hydroxide) was used to wash the HPLC column (XBridge-C18, 19×150 mm, 5 μm) at a flow rate of 20 mL/min.

Example 1. 6-(cyclopropanecarboxamido)-4-((2-methoxy-3-(1-(methyl-d ₃ )-1H-1,2,4-triazol-3-yl)phenyl)amino)-N- (methyl-d ₃ )pyridazine-3-carboxamide (1)

Stage 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 h, the solution was diluted with water (200 mL) and then extracted with ethyl acetate (3 x 60 mL). The organic phases were combined, washed with brine (2 x 100 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated to dryness under reduced pressure to afford the title compound 1b (10 g, solid) in 98% yield.

MS mass/charge (IEC): 212 [M+1]

Stage 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 h, the solvent was removed under reduced pressure to afford the title compound 1c (crude, 10 g, solid). The crude product was used in the next step without further purification. MS m/z (ESI): 197 [M+1]

Stage 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 give solution A. Hydrazine hydrate (25 mL) was slowly added to a mixture of acetic acid (35 mL) and ethanol (150 mL) at 0 °C, followed by the addition of solution A. After gradually warming to room temperature and stirring for 12 h, 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 give the title compound 1d (6 g, solid) in 55% yield.

MS mass/charge (IEC): 221 [M+1]

Stage 4

3-(2-methoxy-3-nitrophenyl)-1-(methyl-d ₃ )-1H-1,2,4-triazole (1e)

To a mixture of 3-(2-methoxy-3-nitrophenyl)-1-(methyl-d ₃ )-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 reversed-phase preparative HPLC to afford the title compound 1e (530 mg, solid) in 42% yield.

MS mass/charge (IEC): 238 [M+1]

Stage 5

2-methoxy-3-(1-(methyl-d ₃ )-1H-1,2,4-triazol-3-yl)aniline (1f)

To a solution of 3-(2-methoxy-3-nitrophenyl)-1-(methyl-d ₃ )-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 hydrogen atmosphere for 12 h and then filtered. The filtrate was concentrated to dryness under reduced pressure to give the desired product 1f (430 mg, solid). The product was used in the next reaction without further purification.

MS mass/charge (IEC): 208 [M+1]

Stage 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 (3.5 mL) was added lithium bromide (6.3 g, 72.5 mmol). The resulting mixture was stirred at room temperature for 12 h and filtered. The resulting solid was washed with acetonitrile (8 mL) and dried in vacuo to give the title compound 1h (4.53 g, solid) in 90% yield.

MS mass/charge (IER): 193 [M+1]

Stage 7

Zinc 6-chloro-4-((2-methoxy-3-(1-(methyl-d ₃ )-1H-1,2,4-triazol-3-yl)phenyl)amino)pyridazine-3-carboxylate (1i )

To a mixture of lithium 4,6-dichloropyridazine-3-carboxylate 1h (380 mg, 1.9 mmol), 2-methoxy-3-(1-(methyl-d ₃ )-1H-1,2,4-triazol-3-yl)aniline 1f (471 mg, 2.27 mmol), isopropanol (0.5 mL) and water (5 mL) was added zinc acetate (350 mg, 1.9 mmol) 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 under vacuum to give the title compound 1i (490 mg, solid) in 71% yield.

MS mass/charge (IEC): 364 [M+1]

Stage 8

Methyl 6-(cyclopropanecarboxamido)-4-((2-methoxy-3-(1-(methyl-d ₃ )-1H-1,2,4-triazol-3-yl)phenyl)amino)pyridazine-3-carboxylate (1j)

To a mixture of zinc 6-chloro-4-((2-methoxy-3-(1-(methyl-d ₃ )-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) were added successively potassium carbonate (320 mg, 7.8 mmol) and 1,8-Diazabicycloundec-7-ene (180 mg, 1.5 mmol) was added sequentially. The resulting mixture was stirred at 80 °C for 72 h under nitrogen atmosphere. After cooling to room temperature, the solvent was removed under reduced pressure and the residue was purified by reversed-phase preparative HPLC to afford the title compound 1j (560 mg, solid) in 99% yield.

MS mass/charge (IEC): 413 [M+1]

Stage 9

6-(cyclopropanecarboxamido)-4-((2-methoxy-3-(1-(methyl-d ₃ )-1H-1,2,4-triazol-3-yl)phenyl)amino)-N-(methyl- d ₃ )pyridazine-3-carboxamide (1)

A mixture of methyl 6-(cyclopropanecarboxamido)-4-((2-methoxy-3-(1-(methyl-d ₃ )-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 hour. After cooling to room temperature, the solvent was removed under reduced pressure and the residue was purified by reversed-phase preparative HPLC to give the title compound 1 (44 mg, solid) in 15% yield.

MS mass/charge (IEC): 429 [M+1]

^1H NMR (400 MHz, DMSO- _d6 ) δ 11.32 (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).

Example 2. 6-(cyclopropanecarboxamido)-4-((2-methoxy-3-(1-(methyl-d ₃ )-1H-1,2,4-triazol-3-yl)phenyl)amino)-N- methylpyridazine-3-carboxamide (2)

Compound 2 was synthesized according to the methods of Example 1, except that methylamine hydrochloride (CH ₃ NH ₂ ⋅HCl) was used in step 9 instead of deuterated methylamine hydrochloride (CD ₃ NH ₂ ⋅HCl).

MS mass/charge (IEC): 426 [M+1]

¹ H NMR (400 MHz, DMSO-d ₆ ) 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).

Example 3. 6-(cyclopropanecarboxamido)-4-((2-(methoxy-d ₃ )-3-(1-methyl-1H-1,2,4-triazol-3-yl)phenyl)amino)-N- methylpyridazine-3-carboxamide (3)

Stage 1

N-methylformaldehyde

To a solution of methyl hydrazine 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 h and filtered. Then methyl formate (17 g, 277 mmol) was added to the filtrate and stirred at room temperature for 18 h. The solvent was removed under reduced pressure to give the title compound 5b (22 g, crude). The crude product was used directly in the next step without further purification.

MS mass/charge (IEC): 75 [M+1]

Stage 2

5-chloro-2-(methoxy-d ₃ )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 x 100 mL). The combined organic phases were washed with brine (2 x 100 mL), dried over anhydrous sodium sulfate, filtered and concentrated to dryness under reduced pressure to give the title compound 3d (4.3 g, solid) in 97% yield.

MS mass/charge (IEC): 171 [M+1]

Stage 3

3-(5-chloro-2-(methoxy-d ₃ )phenyl)-1-methyl-1H-1,2,4-triazole sulfate (3e)

To a solution of potassium tert-butoxide (11.3 g, 101 mmol) in tetrahydrofuran (30 mL) at 0 °C were added sequentially 5-chloro-2-(methoxy-d ₃ )benzonitrile 3d (4.3 g, 25.3 mmol) and a solution of N-methylformylhydrazide (4.1 g, 58 mmol) in tetrahydrofuran (20 mL). After stirring at room temperature for 12 h, water (50 mL) was added, 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 stirred for 12 hours. The mixture was then filtered and dried to give the target compound 3e (5.6 g, solid) in 83% yield.

MS mass/charge (IEC): 227 [M+1]

Stage 4

3-(5-chloro-2-(methoxy-d ₃ )-3-nitrophenyl)-1-methyl-1H-1,2,4-triazole (3f)

To a solution of 3-(5-chloro-2-(methoxy-d ₃ ) 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 h, and then cooled again to 0 °C. Water (67 mL) and methanol (47 mL) were added to the solution at 0 °C, then warmed to room temperature and stirred for 1 h. The solution was heated to 40 °C, and ammonium hydroxide (42 mL) was added to it. The solution was cooled to 20 °C, stirred for 2 h, and then filtered. The solid was washed with water (2×30 mL) and dried under vacuum to give the title compound 3f (3.37 g, solid) in 50% yield.

MS mass/charge (IEC): 272 [M+1]

Stage 5

2-(methoxy-d ₃ )-3-(1-methyl-1H-1,2,4-triazol-3-yl)aniline (3g)

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

MS mass/charge (IEC): 208 [M+1]

Stage 6

Zinc 6-chloro-4-((2-(methoxy-d ₃ )-3-(1-methyl-1H-1,2,4-triazol-3-yl)phenyl)amino)pyridazine-3-carboxylate (3h )

To a mixture of lithium 4,6-dichloropyridazine-3-carboxylate 1h (3 g, 15.1 mmol), 2-(methoxy-d ₃ )-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 under vacuum to give the title compound 3h (4.3 g, solid) in 100% yield.

MS mass/charge (IEC): 364 [M+1]

Stage 7

Methyl 6-(cyclopropanecarboxamido)-4-((2-(methoxy-d ₃ )-3-(1-methyl-1H-1,2,4-triazol-3-yl)phenyl)amino)pyridazine-3-carboxylate (3i)

A mixture of zinc 6-chloro-4-((2-(methoxy-d ₃ )-3-(1-methyl-1H-1,2,4-triazol-3-yl)phenyl)amino)pyridazine-3-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 nitrogen atmosphere and stirred for 12 h. After cooling to room temperature, aqueous acetic acid (50%, 17 mL) and glacial acetic acid (40 mL) were added successively. After stirring at room temperature for 1 h, the resulting homogeneous mixture was washed with petroleum ether (2 x 20 mL). Water (50 mL) was added and the mixture was kept at room temperature for 4 h and then filtered. The solid was washed successively with aqueous acetonitrile (50%, 20 mL) and acetonitrile (20 mL) and then dried in vacuo at 65 °C for 30 min to give the desired product 3i (3 g, solid) in 66% yield.

MS mass/charge (IEC): 413 [M+1]

Stage 8

6-(cyclopropanecarboxamido)-4-((2-(methoxy-d ₃ )-3-(1-methyl-1H-1,2,4-triazol-3-yl)phenyl)amino)-N-methylpyridazin-3 -carboxamide

A mixture of methyl 6-(cyclopropanecarboxamido)-4-((2-(methoxy-d ₃ )-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 h. After completion of the reaction, it was quenched with water (1.5 mL) and acetonitrile (4.5 mL). The resulting mixture was heated at 65 °C for 1 h and 0 °C for 3 h, and then filtered. The solid was washed successively with aqueous acetonitrile (33%, 4.5 mL) and acetonitrile (4.5 mL), dried under vacuum at 65 °C for 8 h to afford the desired product 3 (811 mg, solid) in 56% yield.

MS mass/charge (IER): 426 [M+1]

¹ H NMR (400 MHz, DMSO-d ₆ ) 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).

Compound 3 can be converted to the HCl salt by the following method:

Compound 3 (5.00 g, 11.752 mmol) and DMSO (27 mL) were added to the reaction flask. The resulting mixture was heated to 50~55 °C with stirring until the solid was completely dissolved to form a homogeneous solution. Then, concentrated hydrochloric acid (36%~38%, 1.18 g) was added to the mixture, followed by water (3 mL) and crystal seed (25 mg). The resulting mixture was stirred at 50~55 °C for half an hour, cooled to 35~40 °C, isopropanol (60 mL) was added dropwise over 0.5~1.0 hour, and stirred at 35~40 °C for half an hour. The mixture was slowly cooled to 20~25 °C over an hour, stirred overnight, and then filtered. The filter cake was washed with isopropanol (2×15 mL) and dried under reduced pressure at 65°C overnight to give the HCl monosalt of compound 3 (4.5 g, solid) in 83% yield.

MS mass/charge (IER): 426 [M+1]

¹ H NMR (400 MHz, DMSO-d ₆ ) 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).

Example 4. 6-(cyclopropanecarboxamido)-4-((2-(methoxy-d ₃ )-3-(1-methyl-1H-1,2,4-triazol-3-yl)phenyl)amino)-N- (methyl-d ₃ )pyridazine-3-carboxamide (4)

Compound 4 was synthesized according to the methods of Example 3, except that _{deuterated methylamine hydrochloride (CD3NH2⋅HCl )} was used in step _{8 instead of methylamine hydrochloride (CH3NH2⋅HCl )} .

MS mass/charge (IEC): 429 [M+1]

^1H NMR (400 MHz, DMSO- _d6 ) δ 11.32 (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).

Example 5 (S)-6-(2,2-difluorocyclopropane-1-carboxamido)-4-((2-methoxy-3-(1-methyl-1H-1,2,4-triazol-3-yl) phenyl)amino)-N-(methyl-d ₃ )pyridazine-3-carboxamide (5)

Stage 1

(S)-N-(2,4-dimethoxybenzyl)-2,2-difluorocyclopropane-1-carboxamide (5b)

To a mixture of (5)-2,2-difluorocyclopropane-1-carboxylic acid 5a (1.5 g, 12.3 mmol), HATU (5.7 g, 15 mmol), diisopropylamine (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 purified by reversed-phase preparative HPLC to give the title compound 5b (4.4 g, solid).

MS mass/charge (IEC): 272 [M+1]

Stage 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 purified by silica gel column chromatography (dichloromethane/methanol 100/0 to 9/1) to give the title compound 5d (1.4 g, solid) in 93% yield in two steps.

MS mass/charge (IEC): 122 [M+1]

Stage 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) at 0 °C were added sequentially 5-chloro-2-methoxybenzonitrile (20 g, 120 mmol) and methylformylhydrazide 3b (22 g, crude). After stirring at room temperature for 72 h, water (500 mL) was added and the mixture was extracted with ethyl acetate (3 x 300 mL). The organic phases were combined, washed with brine (2 x 300 mL), dried over anhydrous sodium sulfate, filtered and concentrated to dryness under reduced pressure to give the title compound 5d (17.1 g, solid) in 88% yield.

MS mass/charge (IEC): 224 [M+1]

Stage 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, the resulting solution was added to a mixture of water (250 g) and methanol (150 g) at 0 °C. Then, the mixture was 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 (petroleum ether/ethyl acetate 100/0 to 3/7) to give the desired product 5e (17.1 g, solid) in 88% yield.

MS mass/charge (IEC): 269 [M+1]

Stage 5

2-methoxy-3-(1-methyl-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 were added 10% palladium on carbon (3 g) and sodium bicarbonate (10.5 g, 126 mmol). The mixture was stirred under hydrogen atmosphere for 5 h and then filtered. The filtrate was concentrated to dryness under reduced pressure and the residue was purified by reversed-phase preparative HPLC to afford the title compound 5f (8.8 g, solid) in 68% yield.

MS mass/charge (IER): 205 [M+1]

Stage 6

Zinc 6-chloro-4-((2-methoxy-3-(1-methyl-1H-1,2,4-triazol-3-yl)phenyl)amino)pyridazine-3-carboxylate (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), kept for 30 min and then filtered. The solid was washed with water (2×30 mL) and tetrahydrofuran (2×30 mL) and dried under vacuum to give the title compound 5g (7.6 g, solid) in 93% yield.

MS mass/charge (IEC): 361 [M+1]

Stage 7

(S)-6-(2,2-difluorocyclopropan-1-carboxamido)-4-((2-methoxy-3-(1-methyl-1H-1,2,4-triazol-3-yl)phenyl)amino )zinc pyridazine-3-carboxylate (5h)

A mixture of zinc 6-chloro-4-((2-methoxy-3-(1-methyl-1H-1,2,4-triazol-3-yl)phenyl)amino)pyridazine-3-carboxylate 5g (1.6 g, 3.93 mmol), (5)-2,2-difluorocyclopropane-1-carboxamide 5c (1.2 g, 9.8 mmol), (2R)-1-[(1R)-1-[bis(1,1-dimethylethyl)phosphino]ethyl]-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 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 x 30 mL). Then water (50 mL) was added and left for 3 h. The mixture was filtered and the solid was dried in vacuo to give the title compound 5h (1.1 g, solid) in 62% yield.

MS mass/charge (IEC): 446 [M+1]

Stage 8

(S)-6-(2,2-difluorocyclopropan-1-carboxamido)-4-((2-methoxy-3-(1-methyl-1H-1,2,4-triazol-3-yl)phenyl)amino )-N-(methyl-d ₃ )pyridazine-3-carboxamide

To a mixture of (5)-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 (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 h. After cooling to room temperature, the mixture was concentrated to dryness under reduced pressure and the residue was purified by reversed-phase preparative HPLC to afford the title compound 5 (420 mg, solid) in 37% yield.

MS mass/charge (IEC): 462 [M+1]

^1H 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).

Example 6 (S)-6-(2,2-difluorocyclopropane-1-carboxamido)-4-((2-methoxy-3-(1-methyl-1H-1,2,4-triazol-3-yl) phenyl)amino)-N-methylpyridazine-3-carboxamide (6)

Compound 6 was synthesized according to the methods of Example 5, except that the deuterated _{methylamine hydrochloride (CD3NH2⋅HCl )} in step _{8 was replaced by methylamine hydrochloride (CH3NH2⋅HCl )} .

MS mass/charge (IER): 459 [M+1]

¹ H NMR (400 MHz, DMSO-d ₆ ) 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).

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-d ₃ )pyridazine-3-carboxamide (7)

Compound 7 was synthesized according to the methods of Example 5, except that (S)-2,2-difluorocyclopropane-1-carboxylic acid (5a) in step 1 was replaced by (R)-2,2-difluorocyclopropane-1-carboxylic acid.

MS mass/charge (IEC): 462 [M+1]

¹ H NMR (400 MHz, DMSO-d ₆ ) 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).

Example 8. (R)-6-(2,2-difluorocyclopropane-1-carboxamido)-4-((2-methoxy-3-(1-methyl-1H-l,2,4-triazol-3-yl) phenyl)amino)-N-methylpyridazine-3-carboxamide (8)

Compound 8 was synthesized according to the methods of Example 5, except that (i) (S)-2,2-difluorocyclopropane-1-carboxylic acid (5a) in step 1 was replaced with (R)-2,2-difluorocyclopropane-1-carboxylic acid, and (ii) deuterated methylamine hydrochloride ( _CD3NH2⋅HCl ) in step 8 was replaced with _{methylamine hydrochloride (CH3NH2⋅HCl )} .

MS mass/charge (IER): 459 [M+1]

¹ H NMR (400 MHz, DMSO-d ₆ ) 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).

Example 9. Analysis of the enzymatic activity of the JAK2 kinase domain

The effect of the compounds of the present invention on the enzymatic activity of the kinase domain of recombinant JAK2 (JH1) was assessed by determining the level of phosphorylation of the substrate in the kinase reaction using the HTRF kinase assay detection kit (Cisbio, cat. no. 62TK0PEC) (Table 1).

The experimental method is outlined below:

Reaction buffer containing the following components: enzyme buffer (1x), 5 mM _MgCl2 , 1 mM DTT, and 0.01% Brij35 from the kit; recombinant human JAK2 kinase domain protein (Carna Biosciences, cat. no. 08-045) diluted with reaction buffer to a concentration of 0.15 ng/µL; substrate reaction solution containing 2.5 µM ATP and biotinylated tyrosine kinase substrate diluted to 0.25 µM with reaction buffer; detection solution containing 0.1 ng/µL Eu3 ⁺ -labeled antibody (Cisbio, cat. no. 61T66KLB) and 12.5 nM streptavidin-labeled XL665 (Cisbio, cat. no. 610SAXLB) in reaction buffer.

The test compound was dissolved to 1 mM in DMSO, followed by serial 4-fold dilutions with DMSO to a minimum concentration of 61 nM. Each concentration was further diluted 40-fold with reaction buffer.

In a 384-well assay plate (Corning, cat. no. 3674), 4 μl of compound solution and 2 μl of JAK2 kinase solution were added. The mixture was incubated at room temperature for 15 min, and then 4 μl of substrate reaction solution was added. After a further incubation at room temperature for 30 min, an equal volume (10 μl) of detection solution was added to the reaction mixture and left at room temperature for 30 min. An Envision plate spectrophotometer (Perkin Elmer) was then used to measure the progress of the reaction at 620 nm and 665 nm. The ratio of absorbance at 665 nm and 620 nm positively correlated with the degree of substrate phosphorylation, thus revealing the activity of JAK2 kinase. In this experiment, the group without JAK2 kinase protein was the 100% inhibition group, and the group with JAK2 kinase protein but no test compound was the 0% inhibition group. The percentage of inhibition of JAK2 kinase activity by the test compound was calculated by the following formula:

Percent inhibition = 100 - 100×( _compound ratio - _{100% inhibition} ratio)/( _{0% inhibition} ratio - _{100% inhibition} ratio)

The IC ₅₀ value of the test compound is calculated from 8 concentration points using the XLfit software (ID Business Solutions Ltd., UK) according to the following formula:

Y=Bottom + (Top - Bottom)/(1+10 ^{(logIC50-X)×slope factor} )

where Y is the percentage of inhibition, 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 Slope is the slope of the curve.

Example 10. Analysis of the enzymatic activity of the TYK2 kinase domain

The effect of the compounds of the present invention on the enzymatic activity of the kinase domain of recombinant TYK2 (JH1) was assessed by determining the level of phosphorylation of the substrate in the kinase reaction using the HTRF kinase assay detection kit (Cisbio, cat. no. 62TK0PEC) (Table 1).

The experimental method is outlined below:

Reaction buffer containing the following components: enzyme buffer (1×), 5 mM _MgCl2 , 1 mM DTT, 10 nM SEB (Cisbio, cat. no. 61SEBALB), 0.625 mM EGTA, and 0.01% Brij35 from the kit; recombinant human TYK2 kinase domain (JH1) protein (Carna Biosciences, cat. no. 08-147) diluted with reaction buffer to a concentration of 0.25 ng/µl; substrate reaction solution containing 11.25 µM ATP and biotinylated tyrosine kinase substrate diluted to 0.5 µM with reaction buffer; detection solution containing 0.1 ng/µl Eu ³⁺ -labeled antibody (Cisbio, cat. no. 61T66KLB) and 25 nM streptavidin-labeled XL665 (Cisbio, cat. no. 610SAXLB) in reaction buffer.

The test compound was dissolved to 1 mM in DMSO, followed by serial 4-fold dilutions with DMSO to a minimum concentration of 61 nM. Each concentration was further diluted 40-fold with reaction buffer.

In a 384-well assay plate (Corning, cat. no. 3674), 4 μl of compound solution and 2 μl of TYK2 kinase solution were added. The mixture was incubated at room temperature for 15 min, and then 4 μl of substrate reaction solution was added. After a further incubation at room temperature for 40 min, an equal volume (10 μl) of detection solution was added to the reaction mixture and left at room temperature for 30 min. An Envision plate spectrophotometer (Perkin Elmer) was then used to measure the progress of the reaction at 620 nm and 665 nm. The ratio of absorbance at 665 nm and 620 nm positively correlated with the degree of substrate phosphorylation, thus revealing TYK2 kinase activity. In this experiment, the group without TYK2 kinase protein was the 100% inhibition group, and the group with TYK2 kinase protein but without the test compound was the 0% inhibition group. The percentage of inhibition of TYK2 kinase activity by the test compound was calculated by the following formula:

Percent inhibition = 100-100×( _compound ratio - _{100% inhibition} ratio)/( _{0% inhibition} ratio - _{100% inhibition} ratio)

The IC ₅₀ value of the test compound is calculated from 8 concentration points using the XLfit software (ID Business Solutions Ltd., UK) according to the following formula:

Y=Bottom + (Top - Bottom)/(1+10 ^{(logIC50-X)×slope factor} )

where Y is the percentage of inhibition, 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 Slope is the slope of the curve.

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

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 in the full-length protein) was expressed and purified using the Protein Purification and Identification Platform at Tsinghua University. The test compound was dissolved at 0.1 mM in DMSO, followed by serial 4-fold dilution with DMSO to a minimum concentration of 61 nM. Each concentration was further diluted 40-fold with reaction buffer.

To a 384-well assay plate (Corning, Cat. #4512), 5 μl of compound solution and 5 μl of TYK2 JH2 domain solution (160 nM) were added. The mixture was incubated at room temperature for 30 min, and then 10 μl of a mixture of fluorescein-labeled probe (ThermoFisher, Cat. #PV5593) (20 nM) and GST-Europium (Eu)-labeled antibody (Cisbio, Cat. #61GSTKLA) (40 ng/ml) were added. After further incubation at room temperature for 30 min, the HTRF signal (the ratio of fluorescence intensity at 615 nm and 665 nm for the fluorescein acceptor and europium donor, respectively) was measured using an Envision plate reader (Perkin Elmer). The percentage of inhibition was calculated by comparison with a positive control without test compound and a negative control without protein using the following formula:

% inhibition = 100-100×(signal _compound - signal _{negative control} )/(signal _{positive control} - signal _{negative control} )

The IC ₅₀ value of the test compound is calculated from 8 concentration points using the XLfit software (ID Business Solutions Ltd., UK) according to the following formula:

Y=Bottom + (Top - Bottom)/(1+10 ^{(1ogIC50-X)×slope factor} )

where Y is the percentage of inhibition, 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 Slope is the slope of the curve.

The compounds of the present invention have weak or low inhibitory activity against the kinase domains of JAK2 or TYK2. Table 1 shows that compounds 2, 3, 4, 7 and 8 had IC ₅₀ >10 μM for direct inhibition of kinase activity, while the reference compound had a lower IC ₅₀ of 2.9 μM. The test compounds and reference compounds showed strong binding to JH2 TYK2 (IC ₅₀ in the nM range).

Example 12. Inhibition of IL-12-induced IFN-γ secretion in NK92 cells

The effect of the compounds of the present invention on TYK2-induced IFN-γ secretion in NK92 cells was assessed by enzyme-linked immunosorbent assay (ELISA) (Table 2).

The IL-12 receptor is mainly expressed in activated T cells, NK cells (NK92 - NK cell line), dendritic cells and B cells. By binding to IL-12, it activates the JAK2/TYK2 signaling pathways within NK cells and T lymphocytes, thereby inducing the secretion of IFN-γ.

The experimental method is outlined below:

The test compound was dissolved to 2.5 μM in DMSO, followed by serial 4-fold dilutions with DMSO to a minimum concentration of 0.31 μM. Each concentration was further diluted 50-fold with FBS-free MEM a (Gibco, Cat. No. 12561-056).

NK92 cells (Nan jing 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. .number 202-1L), and 100 U/ml penicillin (ThermoFisher, cat. no. 15140122). When 80-90% of the surface of the culture container is covered, the cells are dispersed and seeded onto a 96-well plate (ThermoFisher, cat. no. 167425) at 100,000 cells per well (80 µl complete MEMα medium without IL-2). The 96-well The plate is incubated overnight in an incubator at 37°C/5% _CO2 .

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, mixed gently, and the 96-well plate was incubated in a 37°C/5% CO ₂ incubator for another 24 h. The plates were centrifuged at 800 rpm for 10 min at room temperature and 50 μl of the supernatant from each well was transferred to another 96-well plate (Sigma, cat. no. CLS3695) coated with anti-IFN-γ antibody. IFN-γ secretion was quantified according to the instructions in the Human IFN-gamma DuoSet ELISA kit (R&D Systems, cat. no. DY285B). In the experiment, the IL-12 group in which the test compound was replaced with MEMα medium was the unstimulated control group (100% inhibition), and the IL-12 and 0.2% DMSO group was the stimulated group (0% inhibition). The inhibition percentage of IL-12-induced IFN-γ secretion in NK-92 cells by the test compound was calculated by the following formula:

Percent inhibition = 100-100×(signal _compound - signal _{unstimulated control} )/(signal _{stimulated control} - signal _{unstimulated control} )

The IC ₅₀ value of the test compound is calculated from 8 concentration points using the XLfit software (ID Business Solutions Ltd., UK) according to the following formula:

Y=Bottom + (Top - Bottom)/(1+10 ^{(1ogIC50-X)×slope factor} )

where Y is the percentage of inhibition, 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 Slope is the slope of the curve.

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

Example 13. Determination of pharmacokinetics in rats in vivo

The pharmacokinetics of compound 3 of the present invention and the reference compound BMS-986165 were evaluated. Compound 3 has an OCD ₃ on the benzene ring, while the reference compound has a CD ₃ on the amide group. The methyl group is generally labile in vivo, undergoing hydrolysis by amidase in the case of methylamide and oxidative demethylation by CYP in the case of methoxy and methyltriazole. Replacing methyl with trideuterated methyl improves the bioavailability and exposure of the compound in vivo and provides better potency of the compound at the same dose.

Compound 3 and the reference compound in a 0.5 mg/mL solution containing 5% N,N-dimethylacetamide + 20% solutol + 75% saline were orally administered to three male Sprague Dawley rats at a dose of 5 mg/kg. Blood samples were collected at 0.25, 0.5, 1, 2, 4, 8 and 24 hours after administration. The plasma concentrations of the compound were quantified by LC-MS/MS using an API-4500 mass spectrometer. The limit of quantification (LOQ) of the assay was 1 ng/mL. The pharmacokinetic (PK) parameters calculated by a non-compartmental method using WinNonlin are shown in Table 3. The results indicate that compound 3 of the present invention has better in vivo distribution parameters than the reference compound.

Example 14. Evaluation of in vivo efficacy in an animal model of CD40 antibody-induced colitis

Female CB17-Scid mice (8-10 weeks old, 18-20 g) from Beijing Vital River Laboratory were randomly divided into 5 groups (n=8 per group). On day 0, mice were induced to develop colitis by a single intraperitoneal injection of 100 μg of the anti-CD40 monoclonal antibody FGK4.5 (BioXCell, cat. no. EB0016-2) in PBS. From day 0 to day 7, mice in the treatment groups were orally administered 0, 1.5, 5, 15 mg/kg compound 3 or 5 mg/kg BMS-986165 in DMSO/Solutol/PEG-400 vehicle (10:5:30) twice daily, while mice in the vehicle group were orally administered the above vehicle. Mice were weighed daily and monitored for signs of colitis, including weight loss and associated loose stools and diarrhea. On day 8, all animals were euthanized. Spleen tissues were collected and weighed. The results show that compound 3 at doses of 1.5 mg/kg, 5 mg/kg, and 15 mg/kg and the reference compound at a dose of 5 mg/kg significantly protected mice from colitis by preventing weight loss (Fig. 2, Table 4) and spleen enlargement (Table 4) compared to mice in the vehicle group.

It should be understood that the foregoing describes preferred embodiments of the present invention and that modifications can be made therein without departing from the scope of the present invention as set forth in the claims.

Claims (8)

1. A compound selected from: or a pharmaceutically acceptable salt thereof. 2. The compound of claim 1, which is Compound 3 or a pharmaceutically acceptable salt thereof. 3. The compound of claim 1, which is Compound 2 or a pharmaceutically acceptable salt thereof. 4. The compound of claim 1, which is Compound 4 or a pharmaceutically acceptable salt thereof.