Novel amide-substituted aromatic ring derivative and application thereof

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a novel amide-substituted aromatic ring derivative or pharmaceutically acceptable salt thereof, which comprises but is not limited to inflammatory or autoimmune diseases such as psoriasis, psoriatic arthritis, dermatitis, lupus erythematosus, inflammatory bowel disease, hidradenitis suppurativa, rheumatoid arthritis, uveitis and the like. The structure of the compound shown in the formula I is shown in the claims and the specification. As a novel TYK2 inhibitor, the amide-substituted aromatic ring derivative provided by the invention has a good TYK2 inhibition effect, has a good treatment effect in an animal in-vivo model of psoriasis and asthma, and has good druggability, high stability and good safety.

Description

Novel amide-substituted aromatic ring derivatives and uses thereof Technical Field

[0001] The present invention relates to the field of pharmaceutical chemistry technology, and in particular to a novel amide-substituted aromatic ring derivative and its use, including but not limited to the preparation of drugs for treating inflammatory or autoimmune diseases such as psoriasis, psoriatic arthritis, dermatitis, lupus erythematosus, inflammatory bowel disease, hidradenitis suppurativa, rheumatoid arthritis, and nephritis. Background Art

[0002] Janus kinases (JAKs) are a family of intracellular non-receptor tyrosine kinases that mediate the signaling of most cytokines, including interleukins (ILs), interferons (IFNs), erythropoietin (EPO), granulocyte- and macrophage-colony stimulating factor (GMCSF), growth hormone (GH), prolactin (PRL), thrombopoietin (TPO), platelet-derived growth factor (PDGF), and epidermal growth factor (EGF). Because JAK family members mediate multiple cytokine signaling pathways, currently approved JAK inhibitors (targeting JAK1-3) inevitably carry side effects. Most FDA-approved JAK inhibitors carry black box warnings in their labels, significantly limiting their clinical application. For example, early JAK inhibitors such as tofacitinib have promising efficacy, but they exhibit high inhibitory activity against JAK-1, JAK-2, and JAK-3, resulting in significant side effects. The FDA requires a black box warning in the tofacitinib package insert regarding the increased risk of thrombosis and death. Other JAK inhibitors, such as upadacitinib and baricitinib, carry the risk of "serious infection, malignancy, and thrombosis." TYK2 is a member of the JAK family and is crucial in the regulatory signal transduction cascade downstream of IL-12, IL-23, and type I interferon receptors. IL-12 and IL-23 are currently considered to be key cytokines that affect the progression of psoriasis. In addition, TYK2-mediated signal transduction is also associated with a variety of inflammatory and autoimmune diseases, such as arthritis, dermatitis, lupus erythematosus, and inflammatory bowel disease. Therefore, there is an urgent need to develop a new TYK2 inhibitor with both good efficacy and safety.

[0003] Summary of the Invention

[0004] This application provides the following technical solutions:

[0005] In the first aspect, embodiment 1 is provided: a novel aromatic ring derivative containing amide substitution or a pharmaceutically acceptable salt thereof, characterized in that the structural formula of the compound of formula I is as follows:

[0006] Wherein, X and P shown in the structural formula of the compound of formula I are selected independently of each other, and:

[0007] X is selected from CH, N;

[0008] P is selected from C1-C6 alkyl, substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted five-membered heteroaryl, substituted or unsubstituted six-membered heteroaryl, and the substituent is selected from one or more of the following: C1-C6 alkyl, C3-C6 cycloalkyl, halogen, C1-C6 alkoxy, C1-C6 haloalkyl, -CN;

[0009] When X is N, P is as defined above; or

[0010] When X is CH, P is as defined above.

[0011] Embodiment 2: The compound of formula I or a pharmaceutically acceptable salt thereof according to embodiment 1, characterized in that X and P shown in the structural formula of the compound of formula I are selected independently of each other, and:

[0012] X is selected from CH, N;

[0013] P is selected from C1-C4 alkyl, substituted or unsubstituted phenyl, substituted or unsubstituted five-membered heteroaryl containing 2-3 N, substituted or unsubstituted six-membered heteroaryl containing 1-2 N, and the substituent is selected from one or two of the following: C1-C4 alkyl, C3-C6 cycloalkyl, halogen, C1-C4 alkoxy, C1-C4 haloalkyl, -CN;

[0014] When X is N, P is as defined above; or

[0015] When X is CH, P is as defined above.

[0016] Embodiment 3: The compound of formula I or a pharmaceutically acceptable salt thereof according to embodiment 1, characterized in that X and P shown in the structural formula of the compound of formula I are selected independently of each other, and:

[0017] X is selected from CH, N;

[0018] P is selected from tert-butyl, substituted or unsubstituted phenyl, substituted or unsubstituted five-membered heteroaryl Substituted or unsubstituted six-membered heteroaryl The substituents are selected from one or two of the following: methyl, ethyl, isopropyl, tert-butyl, cyclopropyl, methoxy, cyano, fluorine, trifluoromethyl;

[0019] When X is N, P is as defined above; or

[0020] When X is CH, P is as defined above.

[0021] Embodiment 4: The compound of formula I or a pharmaceutically acceptable salt thereof according to embodiment 1, characterized in that X and P shown in the structural formula of the compound of formula I are selected independently of each other, and:

[0022] X is selected from CH, N;

[0023] P is selected from tert-butyl, substituted or unsubstituted phenyl, substituted or unsubstituted five-membered heteroaryl Substituted or unsubstituted six-membered heteroaryl

[0024] For the above-mentioned phenyl group, the substituent is selected from one or two of the following: methyl, ethyl, isopropyl, tert-butyl, cyclopropyl, methoxy, cyano, fluorine, trifluoromethyl;

[0025] For the above five-membered heteroaryl group, the substituent is selected from methyl:

[0026] For the above six-membered heteroaryl group, the substituent is selected from: methyl or fluorine;

[0027] When X is N, P is as defined above; or

[0028] When X is CH, P is as defined above.

[0029] Embodiment 5: The compound of formula I or a pharmaceutically acceptable salt thereof according to embodiment 1, characterized in that X and P shown in the structural formula of the compound of formula I are selected independently of each other, and:

[0030] X is selected from CH, N;

[0031] P is selected from tert-butyl,

[0032] When X is N, P is as defined above; or

[0033] When X is CH, P is as defined above.

[0034] Embodiment 6: The compound of formula I or a pharmaceutically acceptable salt thereof according to embodiment 1, characterized in that X and P shown in the structural formula of the compound of formula I are selected independently of each other, and:

[0035] X is selected from CH or N;

[0036] P is selected from substituted or unsubstituted phenyl, substituted or unsubstituted five-membered heteroaryl Substituted or unsubstituted six-membered heteroaryl

[0037] For the above-mentioned phenyl group, the substituent is selected from one or two of the following: methyl, fluorine;

[0038] For the above five-membered heteroaryl group, the substituent is selected from methyl:

[0039] For the above six-membered heteroaryl group, the substituent is selected from methyl;

[0040] Preferably, P is selected from:

[0041] When X is N, P is as defined above; or

[0042] When X is CH, P is as defined above.

[0043] Embodiment 7: The novel amide-substituted aromatic ring derivative or a pharmaceutically acceptable salt thereof according to any one of Embodiments 1-5, characterized in that the compound of formula I is selected from Compounds 1-106.

[0044] Embodiment 8: The novel amide-substituted aromatic ring derivative or a pharmaceutically acceptable salt thereof according to Embodiment 7, characterized in that the compound of formula I is selected from the following compounds: Compound 2, Compound 3, Compound 4, Compound 5, Compound 34, Compound 38, Compound 54, Compound 55, Compound 57, Compound 62, Compound 86, and Compound 91.

[0045] In a second aspect, embodiment 9 is provided: a pharmaceutical composition, characterized in that it comprises the novel amide-substituted aromatic ring derivative or a pharmaceutically acceptable salt thereof as described in any one of embodiments 1-8 and optionally one or more pharmaceutically acceptable carriers, diluents, excipients or adjuvants.

[0046] In a third aspect, embodiment 10 is provided: use of the novel amide-substituted aromatic ring derivative according to any one of embodiments 1 to 8 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to embodiment 9 in the preparation of a drug for treating TYK2-mediated related diseases;

[0047] Preferably, the disease is selected from inflammatory or autoimmune diseases;

[0048] Further preferably, the disease is selected from psoriasis, psoriatic arthritis, dermatitis, lupus erythematosus, inflammatory bowel disease, hidradenitis suppurativa, rheumatoid arthritis or uveitis.

[0049] Embodiment 11: A method for treating TYK2-mediated diseases, comprising providing a subject in need thereof with the novel amide-substituted aromatic ring derivative or a pharmaceutically acceptable salt thereof according to any one of Embodiments 1-8 or the pharmaceutical composition according to Embodiment 9;

[0050] Preferably, the disease is selected from inflammatory or autoimmune diseases;

[0051] Further preferably, the disease is selected from psoriasis, psoriatic arthritis, dermatitis, lupus erythematosus, inflammatory bowel disease, hidradenitis suppurativa, rheumatoid arthritis or uveitis.

[0052] Embodiment 12: The novel amide-substituted aromatic ring derivative or a pharmaceutically acceptable salt thereof according to any one of Embodiments 1 to 8, or the pharmaceutical composition according to Embodiment 9, for use in treating TYK2-mediated related diseases;

[0053] Preferably, the disease is selected from inflammatory or autoimmune diseases;

[0054] Further preferably, the disease is selected from psoriasis, psoriatic arthritis, dermatitis, lupus erythematosus, inflammatory bowel disease, hidradenitis suppurativa, rheumatoid arthritis or uveitis.

[0055] In addition, this application also relates to the following implementation schemes:

[0056] Embodiment 1A: The present invention provides a novel aromatic ring derivative containing amide substitution or a pharmaceutically acceptable salt thereof. The structural formula of the compound of formula I is as follows:

[0057] Wherein, X and P shown in the structural formula of the compound of formula I are selected independently of each other, and:

[0058] X is selected from CH, N;

[0059] P is selected from tert-butyl, substituted or unsubstituted phenyl, substituted or unsubstituted five-membered heteroaryl, substituted or unsubstituted six-membered heteroaryl, and the substituent is selected from C1-6 alkyl or C3-6 cycloalkyl, halogen, -OCH3, -CF3, and -CN.

[0060] Embodiment 2A: In the structural formula of the compound of formula I described in Embodiment 1A, X and P are selected independently of each other, and:

[0061] X is selected from CH, N;

[0062] P is selected from tert-butyl, substituted or unsubstituted phenyl, substituted or unsubstituted five-membered heteroaryl containing 2-3 N, substituted or unsubstituted six-membered heteroaryl containing 1-2 N, and the substituent is selected from C1-4 alkyl or C3-6 cycloalkyl, -F, -OCH3, -CF3, and -CN.

[0063] Embodiment 3A: A compound of formula I according to Embodiment 1A or 2A, which is selected from the following compounds 1-106:

[0064] The compounds and intermediates of the present invention may also exist in different tautomeric forms, and all such forms are included within the scope of the present invention.The term "tautomer" refers to structural isomers of different energies that are interconvertible via a low energy barrier.

[0065] The pharmaceutically acceptable salts of the compounds of the present invention refer to conventional non-toxic salts formed from pharmaceutically acceptable inorganic or organic acids.

[0066] The present invention also includes isotopically labeled compounds of the present invention that are identical to those described herein, but in which one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that may be incorporated into the compounds of the present invention include, but are not limited to, hydrogen ( 2 H. 3 H), carbon ( 11 C. 13 C. 14 C), nitrogen ( 13 N. 15 N), oxygen ( 15 O. 17 O. 18 O), phosphorus ( 31 P. 32 P), sulfur ( 35 S), fluorine ( 18 F), iodine ( 123 I. 125 I) and chlorine ( 36 Cl) isotopes.

[0067] In addition, the use of heavier isotopes such as deuterium (i.e. 2H)) substitution may provide certain therapeutic advantages resulting from greater metabolic stability (e.g., increased in vivo half-life or reduced dosage requirements) and may be preferred in certain circumstances, wherein deuterium substitution may be partial or complete, partial deuterium substitution means that at least one hydrogen is replaced by at least one deuterium, and all such forms of compounds are included within the scope of the present invention.

[0068] Embodiment 4A: The present invention also provides a pharmaceutical composition comprising the novel amide-substituted aromatic ring derivative or a pharmaceutically acceptable salt thereof according to any one of the above embodiments 1A-3A and optionally one or more pharmaceutically acceptable carriers, diluents, excipients or adjuvants.

[0069] Embodiment 5A: The present invention provides use of the novel amide-substituted aromatic ring derivative or a pharmaceutically acceptable salt thereof according to any one of Embodiments 1A to 3A above, or a pharmaceutical composition comprising the novel amide-substituted aromatic ring derivative or a pharmaceutically acceptable salt thereof according to Embodiment 4A, in the preparation of a drug for treating TYK2-mediated related diseases.

[0070] Embodiment 6A: The use according to embodiment 5A, wherein the disease is selected from an inflammatory or autoimmune disease.

[0071] Embodiment 7A: The use according to embodiment 5A or 6A, wherein the disease includes but is not limited to psoriasis, psoriatic arthritis, dermatitis, lupus erythematosus, inflammatory bowel disease, hidradenitis suppurativa, rheumatoid arthritis or uveitis; preferably, psoriasis, psoriatic arthritis, atopic dermatitis, dermatomyositis, systemic lupus erythematosus, cutaneous lupus erythematosus, lupus nephritis, hidradenitis suppurativa, Crohn's disease, ulcerative colitis, rheumatoid arthritis or uveitis.

[0072] Compared with the prior art, the present invention has the following beneficial effects:

[0073] The amide-substituted aromatic ring derivatives provided by the present invention are novel TYK2 inhibitors, which have good TYK2 inhibitory effects, good therapeutic effects in psoriasis and asthma animal models, good drugability, high stability and good safety. DETAILED DESCRIPTION

[0074] In the present invention, unless otherwise explicitly stated, the description method "...selected independently of each other" used throughout this document can mean that in different groups, the specific options expressed by the same or different symbols do not affect each other, and can also mean that in the same group, the specific options expressed by the same or different symbols do not affect each other.

[0075] The substituents of the compounds of the present invention are disclosed by group class or range. It is specifically noted that the present invention includes each independent subcombination of the individual members of these group classes and ranges. For example, the term "C1-C6 alkyl" specifically refers to the independently disclosed methyl, ethyl, C3 alkyl, C4 alkyl, C5 alkyl, and C6 alkyl.

[0076] Unless otherwise specified, the above groups and substituents have the common meanings in the field of medicinal chemistry.

[0077] The term "C1-C6 alkyl" refers to any straight-chain or branched group containing 1 to 6 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, tert-pentyl, n-hexyl, etc. Similarly, "C1-C4 alkyl" refers to any straight-chain or branched group containing 1 to 4 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, etc.

[0078] The terms "alkoxy" and "alkyloxy" refer to any of the above alkyl groups (e.g., C1-C6 alkyl, C1-C4 alkyl, etc.) connected to the rest of the molecule through an oxygen atom -O-, such as C1-C6 alkoxy, C1-C4 alkoxy, and specifically methoxy, ethoxy, etc.

[0079] The term "haloalkyl" refers to any of the above alkyl groups (e.g., C1-C6 alkyl, C1-C4 alkyl, etc.) substituted by one or more (e.g., 2-13 or 2-9) halogen atoms (e.g., fluorine, chlorine, bromine, iodine), examples of which are trifluoromethyl, difluoromethyl, dichloromethyl, trichloromethyl, iodomethyl, bromoethyl, 1,2-dichloroethyl, etc.

[0080] The term "C3-C6 cycloalkyl" refers to a saturated hydrocarbon monovalent ring containing 3-6 ring carbon atoms. The cycloalkyl group may be in the form of a single ring, a fused ring, a bridged ring, etc. Exemplary cycloalkyl groups include, but are not limited to, the following:

[0081] Examples of the term "C3-C6 cycloalkyl" include

[0082] Halogen refers to fluorine, chlorine, bromine or iodine.

[0083] The term "6-10 membered aryl" refers to an aromatic 6-10 membered monocyclic or bicyclic group, and specific examples include phenyl and naphthyl, with phenyl being preferred.

[0084] The term "five-membered / six-membered heteroaryl" refers to substituted and unsubstituted aromatic 5-membered or 6-membered monocyclic groups having at least one heteroatom (N, O, S or P) in at least one ring, the heteroatom-containing ring optionally further having 1, 2 or 3 heteroatoms selected from N, O, S or P.

[0085] Exemplary "five-membered / six-membered heteroaryl" include, but are not limited to: pyrrolyl / ring, pyrazolyl / ring, imidazolyl / ring, oxazolyl / ring, isoxazolyl / ring, thiazolyl / ring, thiadiazolyl / ring, isothiazolyl / ring, furanyl / ring, thienyl / ring, oxadiazolyl / ring, pyridinyl / ring, pyrazinyl / ring, pyrimidinyl / ring, pyridazinyl / ring, triazinyl / ring, triazolyl / ring, pyridazinyl / ring, 2-pyridone, etc.

[0086] The term "substituted" means optionally substituted by 1 or more (e.g., 2-9, such as 2, 3, 4, 5, 6, 7, 8, 9) halogen atoms, C1-C6 alkyl, C3-C6 cycloalkyl, C1-C6 alkoxy, C1-C6 haloalkyl, cyano and other groups.

[0087] As used herein, "treat" generally refers to obtaining a desired pharmacological and / or physiological effect. This effect can be prophylactic, in terms of completely or partially preventing a disease or its symptoms; and / or therapeutic, in terms of partially or completely stabilizing or curing a disease and / or causing side effects due to the disease. As used herein, "treat" encompasses any treatment of a disease in a patient, including: (a) preventing the onset of a disease or symptom in a patient who is susceptible to the disease or symptom but has not yet been diagnosed with the disease; (b) suppressing the symptoms of a disease, i.e., arresting its development; or (c) alleviating the symptoms of a disease, i.e., causing the disease or symptom to regress.

[0088] In the present invention, an "effective amount" refers to an amount that is effective at the necessary dosage and time to achieve the desired therapeutic or preventive effect. The "therapeutically effective amount" of the substance / molecule of the present invention may vary according to factors such as the individual's disease state, age, sex and weight, and the ability of the substance / molecule to elicit the desired response in the individual. A therapeutically effective amount also encompasses an amount in which the therapeutically beneficial effects of the substance / molecule outweigh any toxic or deleterious consequences. A "prophylactically effective amount" refers to an amount that is effective at the necessary dosage and time to achieve the desired preventive effect. Usually, but not necessarily, since a prophylactic dose is used for a subject before the onset of the disease or in the early stages of the disease, the prophylactic effective amount will be lower than the therapeutically effective amount. In the case of cancer, a therapeutically effective amount of a drug can reduce the number of cancer cells; reduce the size of the tumor; inhibit (i.e., slow down to a certain extent, preferably stop) the infiltration of cancer cells into surrounding organs; inhibit (i.e., slow down to a certain extent, preferably stop) tumor metastasis; inhibit tumor growth to a certain extent; and / or alleviate one or more symptoms associated with cancer to a certain extent.

[0089] In the present invention, "subject" refers to a vertebrate. In certain embodiments, the vertebrate refers to a mammal. Mammals include, but are not limited to, livestock (such as cattle), pets (such as cats, dogs, and horses), primates, mice, and rats. In certain embodiments, the mammal refers to a human.

[0090] The pharmaceutical composition of the present invention can be prepared into various forms according to different routes of administration. For example, the pharmaceutical composition can be administered in any of the following ways: orally, by spray inhalation, rectally, nasally, vaginally, topically, parenterally such as by subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intrasternal, and intracranial injection or infusion, or via an explanted reservoir. Topical administration is preferred.

[0091] The compounds of the present invention may optionally be used in combination with one or more other active ingredients, and the dosage and ratio of each can be adjusted by those skilled in the art according to the specific disease and patient conditions and clinical needs.

[0092] As used herein, the term "pharmaceutically acceptable salt" refers to (i) salts formed by acidic functional groups present in the compounds provided by the present invention with appropriate inorganic or organic cations (bases), and includes, but is not limited to, alkali metal salts, such as sodium salts, potassium salts, lithium salts, etc.; alkaline earth metal salts, such as calcium salts, magnesium salts, etc.; other metal salts, such as aluminum salts, iron salts, zinc salts, copper salts, nickel salts, cobalt salts, etc.; inorganic base salts, such as ammonium salts; organic base salts, such as tert-octylamine salts, dibenzylamine salts, morpholine salts, glucosamine salts, phenylglycine alkyl ester salts, ethylenediamine salts, N-methylglucosamine salts, guanidine salts, diethylamine salts, triethylamine salts, dicyclohexylamine salts, N,N'-dibenzylethylenediamine salts, diethanolamine salts, N-benzyl-phenethylamine salts, piperazine salts, tetramethylamine salts, tris(hydroxymethyl)aminomethane salts. and (ii) salts formed by basic functional groups present in the compounds provided by the present invention and appropriate inorganic or organic anions (acids), including but not limited to hydrohalides, such as hydrofluorides, hydrochlorides, hydrobromides, hydroiodides, etc.; inorganic acid salts, such as nitrates, perchlorates, sulfates, phosphates, etc.; lower alkanesulfonates, such as methanesulfonates, trifluoromethanesulfonates, ethanesulfonates, etc.; arylsulfonates, such as benzenesulfonates, p-toluenesulfonates, etc.; organic acid salts, such as acetates, malates, fumarates, succinates, citrates, tartrates, oxalates, maleates, etc.; and amino acid salts, such as glycine, trimethylglycine, arginine, ornithine, glutamate, aspartate, etc.

[0093] The embodiments of the present invention will be described in detail below with reference to the examples, but those skilled in the art will appreciate that the following examples are intended only to illustrate the present invention and should not be construed as limiting the scope of the invention. Where specific conditions are not specified in the examples, conventional conditions were used. Where the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0094] The structures of the compounds of the present invention were determined by liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR). LC-MS measurements were performed using an AB Sciex TripleTOF 4600 mass spectrometer coupled to a Shimadzu LC-20AD XR high-performance liquid chromatograph and an Agela Venusil MP C18 (2.1x50, 3 μm) column. NMR measurements were performed using a BRUKER AVANCE NEO 400 MHz nuclear magnetic resonance spectrometer.

[0095] Unless otherwise specified, all reactions of the present invention were carried out under continuous magnetic stirring in dry nitrogen or argon, the solvent was a pre-dried solvent, and the reaction temperature was degrees Celsius.

[0096] Example 1

[0097] 6-(Cyclopropanecarboxamido)-4-(2-methoxy-3-(benzanilide)-N-(methyl-d3)pyridazine-3-carboxamide

[0098] Step 1

[0099] 2-Methoxy-3-nitrobenzoic acid (1.0 g, 5.1 mmol) was added to thionyl chloride (15.0 g), stirred, and heated to reflux until the reaction was complete as determined by TLC. The reaction solution was cooled to room temperature and concentrated under reduced pressure. The residue was dissolved in 5 ml of acetone and added dropwise to a 10 ml acetone solution containing aniline (0.4 g, 4.6 mmol) and triethylamine (1.0 g, 10.2 mmol). The temperature was controlled below 10°C during the addition. After completion of the addition, the reaction was allowed to react at room temperature until the reaction was complete as determined by TLC. 50 ml of water was added, the mixture was filtered, and the mixture was dried at room temperature for 24 h to obtain 2-methoxy-3-nitro-N-benzanilide (1.1 g, 71.9%).

[0100] MS m / z(ESI):273.08[M+H] + .

[0101] Step 2

[0102] 2-Methoxy-3-nitro-N-benzanilide (1.0 g, 3.7 mmol) and 10% palladium on carbon (0.15 g) were added to 30 ml of methanol, and hydrogen was passed through. The reaction was carried out at 25° C. under a hydrogen atmosphere until the reaction was complete as detected by TLC. The reaction solution was filtered and the filtrate was evaporated to dryness to give 3-amino-2-methoxy-N-benzanilide (0.81 g, 91.0%).

[0103] MS m / z(ESI):243.11[M+H] + .

[0104] Step 3

[0105] To a solution of 3-amino-2-methoxy-N-benzanilide (0.8 g, 3.3 mmol) and 4,6-dichloro-N-(methyl-d3)pyridazine-3-carboxamide (0.68 g, 3.3 mmol) in tetrahydrofuran (5 ml) was added dropwise a solution of lithium bistrimethylsilylamide (1 M, 9.9 ml, 9.9 mmol) in tetrahydrofuran at room temperature. The reaction was allowed to proceed at room temperature until the reaction was complete as detected by TLC. The reaction solution was diluted with dichloromethane and washed with saturated aqueous sodium chloride solution. The organic phase was dried over anhydrous magnesium sulfate and concentrated under reduced pressure. After column chromatography, 6-chloro-4-((2-methoxy-3-(benzanilide)-N-(methyl-d3)pyridazine-3-carboxamide (0.89 g, 55%) was obtained.

[0106] MS m / z(ESI):415.81[M+H] + .

[0107] Step 4

[0108] 6-Chloro-4-((2-methoxy-3-(benzanilide)-N-(methyl-d3)pyridazine-3-carboxamide (0.8 g, 1.9 mmol), cyclopropylcarboxamide (0.16 g, 1.9 mmol), tris(dibenzylideneacetone)dipalladium (0.1 g, 0.1 mmol), 4,5-bis(diphenylphosphine)-9,9-dimethylxanthene (0.12 g, 0.2 mmol), cesium carbonate (1.27 g, The mixture was stirred for 2 h at 4 ℃ for 30 minutes (1min). The mixture was stirred for 2 hours at 4 ℃ for 30 minutes. The mixture was stirred for 30 minutes (1min). The mixture was stirred for 2 hours. The mixture was stirred for 30 minutes (1min). The mixture was stirred for 30 minutes (1min). The mixture was stirred for 30 minutes (1min). The mixture was stirred for 2 hours (1min). The mixture was stirred for 30 minutes (1min). The mixture was stirred for 30 minutes (1min). The mixture was stirred for 30 minutes (1min). The mixture was stirred for 30 minutes (1min). The mixture was stirred for 30 minutes (1min). The mixture was stirred for 30 minutes (1min). The mixture was stirred for 30 minutes (1min).

[0109] MS m / z(ESI):464.20[M+H] + .

[0110] 1 H-NMR(DMSO-d6)б:11.34(1H,s),10.34(1H,s),9.14(1H,s),8.16(1H,s),7.73-7.71(2H,m),7.58-7 .56(1H,m),7.35-7.26(5H,m),7.11-7.07(1H,m),3.74(3H,s),2.08-2.06(1H,m),0.83-0.81(4H,m).

[0111] Example 2

[0112] Referring to the synthesis method of Example 1, the aniline raw material in step 1 was replaced with 4-methylaniline to prepare compound 2.

[0113] MS m / z(ESI):478.22[M+H] + .

[0114] 1 H-NMR(DMSO-d6):11.34(1H,s),10.97(1H,s),10.25(1H,s),9.14(1H,s),8.16(1H,s),7.55-7.62(3H,m ),7.25-7.34(2H,m),7.13-7.15(2H,m),3.76(3H,s),2.26(3H,m),2.07-2.09(1H,m),0.81-0.83(4H,m).

[0115] Example 3

[0116] Referring to the synthesis method of Example 1, the aniline raw material in step 1 was replaced by 3-methylaniline to prepare compound 3.

[0117] MS m / z(ESI):478.22[M+H] + .

[0118] 1 H-NMR(DMSO-d6):11.34(1H,s),10.97(1H,s),10.25(1H,s),9.14(1H,s),8.16(1H,s),7.55-7.62(3H,m ),7.25-7.34(2H,m),7.13-7.15(2H,m),3.74(3H,s),2.26(3H,m),2.07-2.09(1H,m),0.81-0.83(4H,m).

[0119] Example 4

[0120] Referring to the synthesis method of Example 1, the aniline raw material in step 1 was replaced with 3,4-dimethylaniline to prepare compound 4.

[0121] MS m / z(ESI):492.21[M+H] + .

[0122] 1 H-NMR(DMSO-d6)б:11.34(1H,s),10.97(1H,s),10.17(1H,s),9.14(1H,s),8.16(1H,s),7.55-7.57(1H,m), 7.50-7.51(1H,m),7.41-7.44(1H,m),7.25-7.34(2H,m),7.07-7.09(1H,m),3.75(3H,s),2.17-2.20(6H,m), 2.06-2.09(1H,m),0.81-0.83(4H,m).

[0123] Example 5

[0124] Referring to the synthesis method of Example 1, the aniline raw material in step 1 was replaced by 4-fluoroaniline to prepare compound 5.

[0125] MS m / z(ESI):482.20[M+H] + .

[0126] 1 H-NMR(DMSO-d6):11.34(1H,s),10.98(1H,s),10.41(1H,s),9.14(1H,s),8.16(1H,s),7.77-7 .73(2H,m),7.65-7.56(1H,m),7.53-7.04(4H,m),3.74(3H,s),2.08-2.06(1H,m),0.83(4H,d).

[0127] Example 6-32

[0128] Referring to the synthesis method of Example 1, the aniline raw material in step 1 was replaced accordingly to obtain the following compound, the structure of which is shown in the following table:

[0129] Example 33

[0130] 6-(cyclopropanecarboxamido)-4-(2-methoxy-3-(1-methyl-1H-pyrazol-3-yl)carbamoyl)phenyl)amino)-N-(methyl-d3)pyridazine-3-carboxamide.

[0131] Step 1

[0132] 2-Methoxy-3-nitrobenzoic acid (1.0 g, 5.1 mmol) was added to thionyl chloride (15.0 g), stirred, and heated to reflux until the reaction was complete as determined by TLC. The reaction solution was cooled to room temperature and concentrated under reduced pressure. The residue was dissolved in 5 ml of acetone and added dropwise to a 10 ml acetone solution containing 1-methyl-1H-pyrazol-3-amine (0.4 g, 4.1 mmol) and triethylamine (1.0 g, 10.2 mmol). The temperature was controlled below 10°C during the addition. After completion of the addition, the mixture was allowed to react at room temperature until the reaction was complete as determined by TLC. 50 ml of water was added, the mixture was filtered, and the mixture was dried at room temperature for 24 h to obtain 2-methoxy-N-(1-methyl-1H-pyrazol-3-yl)-3-nitrobenzamide (1.0 g, 71.4%).

[0133] MS m / z(ESI):277.09[M+H] + .

[0134] Step 2

[0135] 2-Methoxy-N-(1-methyl-1H-pyrazol-3-yl)-3-nitrobenzamide (0.9 g, 3.2 mmol) and 10% palladium on carbon (0.15 g) were added to 30 ml of methanol and reacted at 25°C under a hydrogen atmosphere until the reaction was complete as detected by TLC. The reaction solution was filtered and the filtrate was evaporated to dryness to give 3-amino-2-methoxy-N-(1-methyl-1H-pyrazol-3-yl)benzamide (0.71 g, 88.8%).

[0136] MS m / z(ESI):247.10[M+H] + .

[0137] Step 3

[0138] To a solution of 3-amino-2-methoxy-N-(1-methyl-1H-pyrazol-3-yl)benzamide (0.7 g, 2.8 mmol) and 4,6-dichloro-N-(methyl-d3)pyridazine-3-carboxamide (0.58 g, 2.8 mmol) in tetrahydrofuran (5 ml) was added dropwise a solution of lithium bistrimethylsilylamide (1 M, 8.4 ml, 8.4 mmol) in tetrahydrofuran at room temperature. The reaction was allowed to proceed at room temperature until the reaction was complete as determined by TLC. The reaction solution was diluted with dichloromethane and washed with saturated aqueous sodium chloride solution. The organic phase was dried over anhydrous magnesium sulfate, concentrated under reduced pressure, and then purified by column chromatography to give 6-chloro-4-((2-methoxy-3-((1-methyl-1H-pyrazol-3-yl)carbamoyl)phenyl)amino)-N-(methyl-d3)pyridazine (0.59 g, 49.6%).

[0139] MS m / z(ESI):419.14[M+H] + .

[0140] Step 4

[0141] 6-Chloro-4-((2-methoxy-3-((1-methyl-1H-pyrazol-3-yl)carbamoyl)phenyl)amino)-N-(methyl-d3)pyridazine-3-carboxamide (0.5 g, 1.2 mmol), cyclopropylcarboxamide (0.10 g, 1.2 mmol), tris(dibenzylideneacetone)dipalladium (0.1 g, 0.1 mmol), 4,5-bis(diphenylphosphino-9,9-dimethylxanthene) (0.12 g, 0.2 mmol), cesium carbonate (1.27 g, 4% HCl) and 1.27 g sodium hydroxide were added. , 3.9mmol) was added to 1,4-dioxane (10ml), the temperature was raised to reflux, and the reaction was completed when detected by TLC. The reaction solution was diluted with dichloromethane and washed with a saturated aqueous sodium chloride solution. The organic phase was dried over anhydrous magnesium sulfate, concentrated under reduced pressure, and then column chromatography was performed to give 6-(cyclopropanecarboxamido)-4-(2-methoxy-3-(1-methyl-1H-pyrazol-3-yl)carbamoyl)phenyl)amino)-N-(methyl-d3)pyridazine-3-carboxamide (0.33g, 59.1%).

[0142] MS m / z(ESI):468.22[M+H] + .

[0143] 1H-NMR(DMSO-d6)б:11.3(1H,s),10.93(1H,s),10.57(1H,s),9.14(1H,s),8.13(1H,s),7.60-7.54(2H,m),7.3 8-7.35(1H,m),7.28-7.24(1H,m),6.57-6.56(1H,m),3,75-3.72(6H,m),2.07-2.05(1H,m),0.82-0.80(4H,m).

[0144] Example 34

[0145] Referring to the synthesis method of Example 33, 1-methyl-1H-pyrazol-3-amine in Step 1 was replaced with 1-methyl-1H-pyrazol-4-amine to prepare Compound 34.

[0146] MS m / z(ESI):468.22[M+H] + .

[0147] 1 H-NMR(DMSO-d6)δ:11.34(1H,s),10.98(1H,s),10.35(1H,s),9.14(1H,s),8.15(1H,s),8.00(1H,s),7.55-7.57(1H,m ),7.50(1H,s),7.31-7.33(1H,m),7.24-7.28(1H,m),3.80(3H,s),3.71(3H,s),2.06-2.08(1H,m),0.80-0.83(4H,m).

[0148] Examples 35-36

[0149] Referring to the synthetic method of Example 33, the 1-methyl-1H-pyrazol-3-amine in step 1 was replaced accordingly to obtain the following compound, the structure of which is shown in the following table:

[0150] Example 37

[0151] 6-(cyclopropanecarboxamido)-4-(2-methoxy-3-(4-methylpyridin-2-yl)carbamoyl)phenyl)amino)-N-(methyl-d3)pyridazine-3-carboxamide.

[0152] Step 1

[0153] 2-Methoxy-3-nitrobenzoic acid (1.0 g, 5.1 mmol) was added to thionyl chloride (15.0 g), stirred, and heated to reflux for reaction until the reaction was complete by TLC. The reaction solution was cooled to room temperature and concentrated under reduced pressure. The residue was dissolved in 5 ml of acetone and added dropwise to a 10 ml acetone solution containing 4-methylpyridin-2-amine (0.5 g, 4.6 mmol) and triethylamine (1.0 g, 10.2 mmol). The temperature was controlled below 10°C during the addition. After completion of the addition, the mixture was allowed to react at room temperature until the reaction was complete by TLC. 50 ml of water was added, the mixture was filtered, and the mixture was dried at room temperature for 24 h to obtain 2-methoxy-N-(4-methylpyridin-2-yl)-3-nitrobenzamide (1.2 g, 82.3%).

[0154] MS m / z(ESI):288.12[M+H] + .

[0155] Step 2

[0156] 2-Methoxy-N-(4-methylpyridin-2-yl)-3-nitrobenzamide (1.1 g, 3.8 mmol) and 10% palladium on carbon (0.15 g) were added to 30 ml of methanol and reacted at 25°C under a hydrogen atmosphere until the reaction was complete as detected by TLC. The reaction solution was filtered and the filtrate was evaporated to dryness to give 3-amino-2-methoxy-N-(4-methylpyridin-2-yl)benzamide (0.92 g, 93.4%).

[0157] MS m / z(ESI):258.13[M+H] + .

[0158] Step 3

[0159] To a solution of 3-amino-2-methoxy-N-(4-methylpyridin-2-yl)benzamide (0.9 g, 3.5 mmol) and 4,6-dichloro-N-(methyl-d3)pyridazine-3-carboxamide (0.72 g, 3.5 mmol) in tetrahydrofuran (5 ml) was added dropwise a solution of lithium bistrimethylsilylamide (1 M, 10.5 ml, 10.5 mmol) in tetrahydrofuran at room temperature. The reaction was allowed to proceed at room temperature until the reaction was complete as determined by TLC. The reaction solution was diluted with dichloromethane and washed with a saturated aqueous sodium chloride solution. The organic phase was dried over anhydrous magnesium sulfate, concentrated under reduced pressure, and then purified by column chromatography to give 6-chloro-4-((2-methoxy-3-((4-methylpyridin-2-yl)carbamoyl)phenyl)amino)-N-(methyl-d3)pyridazine-3-carboxamide (0.88 g, 58%).

[0160] MS m / z(ESI):430.03[M+H] + .

[0161] Step 4

[0162] 6-Chloro-4-((2-methoxy-3-((4-methylpyridin-2-yl)carbamoyl)phenyl)amino)-N-(methyl-d3)pyridazine-3-carboxamide (0.8 g, 1.8 mmol), cyclopropylcarboxamide (0.15 g, 1.8 mmol), tris(dibenzylideneacetone)dipalladium (0.1 g, 0.1 mmol), 4,5-bis(diphenylphosphine)-9,9-dimethylxanthene (0.12 g, 0.2 mmol), cesium carbonate (1.27 g, 3.9 mmol) were added to 1,4-dioxane (10 ml) The mixture was heated to reflux and reacted until the reaction was complete as detected by TLC. The reaction solution was diluted with dichloromethane and washed with a saturated aqueous sodium chloride solution. The organic phase was dried over anhydrous magnesium sulfate and concentrated under reduced pressure. Column chromatography gave 6-(cyclopropanecarboxamido)-4-(2-methoxy-3-(4-methylpyridin-2-yl)carbamoyl)phenyl)amino)-N-(methyl-d3)pyridazine-3-carboxamide (0.42 g, 47%).

[0163] MS m / z(ESI):479.24[M+H] + .

[0164] 1 H-NMR(DMSO-d6):11.34(1H,s),10.96(1H,s),10.58(1H,s),9.15(1H,s),8.20-8.08(3H,m),7.62-7.59(1 H,m),7.50-7.48(2H,m),7.32-7.29(1H,m),3,75(3H,s),2.30(3H,s),2.07-2.05(1H,m),0.81-0.79(4H,m).

[0165] Example 38

[0166] Referring to the synthesis method of Example 37, the 4-methylpyridin-2-amine in step 1 was replaced with 5-methylpyridin-3-amine to prepare compound 38.

[0167] MS m / z(ESI):479.24[M+H] + .

[0168] 1H-NMR(DMSO-d6)δ:11.35(1H,s),10.99(1H,s),10.51(1H,s),9.15(1H,s),8.65(1H,s),8.15-8.16(2H,m),8.0 3(1H,s),7.58-7.61(1H,m),7.28-7.37(2H,m),3.75(3H,s),2.30(3H,s),2.07-2.08(1H,m),0.81-0.83(4H,m).

[0169] Examples 39-52, 105

[0170] Referring to the synthetic method of Example 37, 4-methylpyridin-2-amine in step 1 was replaced accordingly to obtain the following compound, the structure of which is shown in the following table:

[0171] Example 57

[0172] 6-(Cyclopropanecarboxamido)-4-((3-((4-fluorophenyl)carbamoyl)-2-methoxyphenyl)amino)-N-(methyl-d3)nicotinamide

[0173] Step 1

[0174] 2-Methoxy-3-nitrobenzoic acid (1.0 g, 5.1 mmol) was added to thionyl chloride (15.0 g), stirred, and heated to reflux for reaction until the reaction was complete as determined by TLC. The reaction solution was cooled to room temperature and concentrated under reduced pressure. The residue was dissolved in 5 ml of acetone and added dropwise to a 10 ml acetone solution containing 4-fluoroaniline (0.5 g, 4.5 mmol) and triethylamine (1.0 g, 10.2 mmol). The temperature was controlled below 10°C during the addition. After completion of the addition, the mixture was reacted at room temperature until the reaction was complete as determined by TLC. 50 ml of water was added, filtered, and dried at room temperature for 24 h to obtain N-(4-fluorophenyl)-2-methoxy-3-nitrobenzamide (1.1 g, 74.8%).

[0175] MS m / z(ESI):291.38[M+H] + .

[0176] Step 2

[0177] N-(4-Fluorophenyl)-2-methoxy-3-nitrobenzamide (1.0 g, 3.4 mmol) and 10% palladium on carbon (0.15 g) were added to 30 ml of methanol and reacted at 25°C under a hydrogen atmosphere until the reaction was complete as detected by TLC. The reaction solution was filtered and the filtrate was evaporated to dryness to give 3-amino-N-(4-fluorophenyl)-2-methoxybenzamide (0.82 g, 91.4%).

[0178] MS m / z(ESI):261.02[M+H] + .

[0179] Step 3

[0180] To a solution of 3-amino-N-(4-fluorophenyl)-2-methoxybenzamide (0.8 g, 3.1 mmol) and 4,6-dichloro-N-(methyl-d3)pyridine-3-carboxamide (0.64 g, 3.1 mmol) in tetrahydrofuran (5 ml) was added dropwise a solution of lithium bistrimethylsilylamide (1 M, 9.3 ml, 9.3 mmol) in tetrahydrofuran at room temperature. The reaction was allowed to proceed at room temperature until the reaction was complete as determined by TLC. The reaction solution was diluted with dichloromethane and washed with a saturated aqueous sodium chloride solution. The organic phase was dried over anhydrous magnesium sulfate and concentrated under reduced pressure. After column chromatography, 6-chloro-4-((3-((4-fluorophenyl)carbamoyl)-2-methoxyphenyl)amino)-N-(methyl-d3)nicotinamide (0.92 g, 69.3%) was obtained.

[0181] MS m / z(ESI):432.06[M+H] + .

[0182] Step 4

[0183] 6-Chloro-4-((3-((4-fluorophenyl)carbamoyl)-2-methoxyphenyl)amino)-N-(methyl-d3)nicotinamide (0.9 g, 2.0 mmol), cyclopropylcarboxamide (0.17 g, 2.0 mmol), tris(dibenzylideneacetone)dipalladium (0.1 g, 0.1 mmol), 4,5-bis(diphenylphosphino-9,9-dimethylxanthene) (0.12 g, 0.2 mmol), cesium carbonate (1.27 g, 3 .9mmol) was added to 1,4-dioxane (10ml), the temperature was raised to reflux for reaction, and the reaction was completed as detected by TLC. The reaction solution was diluted with dichloromethane and washed with a saturated aqueous sodium chloride solution. The organic phase was dried over anhydrous magnesium sulfate and concentrated under reduced pressure. After column chromatography, 6-(cyclopropanecarboxamido)-4-((3-((4-fluorophenyl)carbamoyl)-2-methoxyphenyl)amino)-N-(methyl-d3)nicotinamide (0.42g, 42%) was obtained.

[0184] MS m / z(ESI):481.16[M+H] + .

[0185] 1 H-NMR(DMSO-d6)б:10.78(1H,s),10.69(1H,s),10.39(1H,s),8.59(1H,s),8.52(1H,s),8.05(1H,s), 7.77-7.73(2H,m),7.56-7.53(1H,m),7.27-7.16(4H,m),3.73(3H,s),2.00-1.94(1H,m),0.77(4H,m).

[0186] Referring to the synthesis method of Example 57, the 4-fluoroaniline in step 1 was replaced accordingly to obtain the following compound, the structure of which is shown in the following table:

[0187] BMS-986165 was prepared according to CN201380069692.9, and its specific structure is:

[0188] Biological test evaluation

[0189] The present invention is further described and explained below in conjunction with test examples, but these examples are not intended to limit the scope of the present invention.

[0190] Test Example 1: In vitro cell inhibition model

[0191] The inhibitory effect of the compound of formula (I) of the present invention on the cell TYK2 signaling pathway can be determined by the following method:

[0192] Experimental instrument: SpectraMax Paradigm plate reader.

[0193] Experimental method: This experiment uses the TYK2-expressing Ba / F3 cell line to evaluate the effect of compounds on the inhibition of cell proliferation by measuring the effects of compounds on cell activity in vitro.

[0194] Experimental Procedure: Thaw CellTiter-Glo Buffer and bring to room temperature. Bring CellTiter-Glo Substrate to room temperature and add buffer to dissolve the substrate to prepare CellTiter-Glo working solution. Add 98 μL of cell culture medium to a flat-bottomed 96-well clear plate, then add 2 μL of the serially diluted compound solution. Incubate for 10 minutes before adding 50 μL of CellTiter-Glo working solution. Shake on an orbital shaker for 2 minutes and let stand at room temperature for 10 minutes. Finally, measure the luminescence signal on a SpectraMax Paradigm plate reader.

[0195] Experimental data processing method: Cell proliferation inhibition rate (Inhibition Rate) data is processed using the following formula:

[0196] Inhibition Rate (Inh%) = 100-(RLUDrug-RLUMin) / (RLUMax-RLUMin)*100%. The inhibition rates corresponding to different concentrations of the compound were calculated in EXCEL, and then the inhibition rate curve was plotted using GraphPad Prism software and related parameters were calculated, including the maximum and minimum inhibition rates of the cells, IC 50 value.

[0197] The inhibitory activity of the compounds obtained by the above scheme is as follows: Note: A represents 1nM <IC 50 <5nM, B indicates 5nM <IC 50 <10nM.

[0198] Experimental conclusion: The compound of the present invention has a good inhibitory effect on the cell TYK2 signaling pathway.

[0199] Test Example 2: hERG channel inhibition activity experiment

[0200] Experimental equipment: HEKA EPC 10 patch clamp amplifier.

[0201] Experimental Methods: This experiment uses manual patch clamp technology to detect the blocking effect of compounds on the current of HEK-293 cell lines stably expressing hERG channels, and evaluates the risk of compound inhibition of cardiac hERG potassium channels by fitting the concentration-effect relationship.

[0202] Experimental instrument: SpectraMax Paradigm plate reader.

[0203] Experimental Methods: This study uses expression cell lines to evaluate the cardiotoxic effects of compounds by measuring their inhibition of cell activity in vitro.

[0204] Experimental Procedure: Patch clamping involves first pulling a recording electrode from a glass capillary using a microelectrode puller. The electrode, filled with intracellular solution, is then inserted into a microelectrode holder. Under an inverted microscope, the microelectrode manipulator is used to immerse the electrode in the extracellular solution and record the electrode resistance (Rpip). The electrode is then gently brought into contact with the cell surface and negative pressure is applied to form a GΩ seal. Fast capacitance compensation is then performed, and negative pressure is continued to rupture the cell membrane, establishing whole-cell recording mode. Finally, slow capacitance compensation is performed and experimental parameters such as series resistance (Rs) are recorded. No leakage compensation is applied. Drug administration is initiated after the whole-cell hERG current has stabilized. Each drug concentration is applied for approximately 5 minutes (or until the current stabilizes) before the next concentration is measured. The coverslip containing cells is placed in the recording bath under an inverted microscope. A blank external solution and the test compound working solution are passed through the recording bath sequentially by gravity, from low to high concentrations. A peristaltic pump is used for fluid exchange during recording. The current measured in the compound-free external solution for each cell serves as its control. Each concentration was tested in duplicate. All electrophysiological experiments were performed at room temperature.

[0205] Experimental data processing method: First, the peak tail current after each drug concentration is compound ) and blank control tail current (Peak tail current control ) normalized, and then calculated the inhibition rate corresponding to each drug concentration The mean (Mean), standard deviation (SD) and standard error (SE) of the inhibition rate at each concentration were calculated, and the data were expressed as Mean ± SE. 50 Calculation of values ​​and dose-effect curve fitting were performed using GraphPad Prism software.

[0206] Experimental conclusion: The hERG test results of the compounds of Examples 2, 3, 4, 5, 34, 38, 54, 55, 57, 62, 86, and 91 of the present invention were all negative, that is, they had no cardiotoxicity and had good safety.

[0207] Test Example 3: Kinase Selectivity Assay

[0208] 3.1 JAK1-3 / TYK2 JH1 in vitro enzyme binding assay

[0209] Experimental instruments: Envision microplate reader (PerkinElmer), ECHO550 (LABCYTE)

[0210] Experimental method: This experiment uses the fluorescence resonance energy transfer (TR-FRET) method to test the inhibitory effect of compounds on JAK1-3 / TYK2 JH1 kinase.

[0211] Experimental Procedure: Compounds were prepared in DMSO to a 10 mM stock solution. Compounds were further diluted in DMSO to create a gradient of compound dilutions. The 100x compound dilutions were transferred to a 384-well plate using an Echo instrument. Assay buffer, SEB, TK-Substrate-Biotin, and detection reagents were all from the TK kit (Cisbio, Cat# 62TK0PEC). Prepare the following three 1× working solutions: 1. Working solution for JAK1 JH1 assay: 1× final concentration of assay buffer, 5 mM final concentration of MgCl2, 0.625 mM final concentration of EGTA, 60 nM final concentration of SEB, 0.01% final concentration of Brij-35, and 1 mM final concentration of DTT. 2. Working solution for JAK2-3 JH1 assay: 1× final concentration of assay buffer, 5 mM final concentration of MgCl2, and 1 mM final concentration of DTT. 3. Working solution for TYK2 JH1 assay: 1× final concentration of assay buffer, 5 mM final concentration of MgCl2, 1 mM final concentration of MnCl2, 12.5 nM final concentration of SEB, and 1 mM final concentration of DTT. Use each 1× working solution to prepare 2× final concentrations of JAK1-3 / TYK2 JH1 kinase and 2× final concentrations of TK-Substrate-biotin substrate. Add 5 μL of JAK1-3 / TYK2 JH1 kinase to a 384-well plate, centrifuge at 1000 rpm for 30 seconds, and incubate at room temperature for 15 minutes. Add 5 μL of TK-Substrate-biotin substrate to the 384-well plate, centrifuge at 1000 rpm for 30 seconds, incubate at room temperature for 45 minutes for JAK1-2 JH1 and 60 minutes for JAK3 / TYK2 JH1. Prepare 2× detection reagent and add 10 μL to the 384-well plate. Centrifuge at 1000 rpm for 30 seconds, incubate at room temperature for 60 minutes for JAK1-2 JH1 and 120 minutes for JAK3 / TYK2 JH1, and then incubate all at 4°C overnight. Read the 665 nm / 615 nm fluorescence signal ratio on an Envision microplate reader (PerkinElmer).

[0212] Experimental data processing method: XLfit, a software developed by IDBS and integrated into the Microsoft Excel environment, was used for test data processing and analysis. First, the average reaction signals of the high-signal control wells and the low-signal control wells were calculated respectively, and then the reaction inhibition rate of each compound well was calculated according to the formula "single-well inhibition rate % = 100% - (average value of high-signal control group - single-well signal value) / (average value of high-signal control group - average value of low-signal control group) * 100%". Then, the concentration and corresponding inhibition rate data were imported into the XLfit software, and the Dose Response One Site 205 model in the software was used to fit the inhibition rate-concentration curve using the four-parameter method, and the IC of the compound was calculated. 50 value.

[0213] 3.2 JAK1 JH2 in vitro enzyme binding assay

[0214] Experimental instruments: Envision microplate reader (PerkinElmer), Echo (LABCYTE)

[0215] Experimental method: This experiment uses the fluorescence resonance energy transfer (TR-FRET) method to test the inhibitory effect of compounds on JAK1 JH2 pseudokinase.

[0216] Experimental Procedure: Prepare the compound to a 10mM stock solution in DMSO. Further dilute the compound dilutions in DMSO to create a gradient of compound dilutions. Transfer the compound dilutions to a 384-well plate using an Echo instrument at 200× the final concentration. Prepare the following 1× working solution: HEPES pH 7.5 (final concentration 20mM), MgCl2 (final concentration 10mM), Brij-35 (final concentration 0.015%), DTT (final concentration 2mM), and BSA (final concentration 50µg / mL). Prepare 3× the final concentrations of JAK1 JH2 pseudokinase, Tb antibody, and Tracer using the 1× working solution. Add 5µL of JAK1 JH2 pseudokinase to a 384-well plate and centrifuge at 1000rpm for 30 seconds. Add 5µL of Tb antibody to a 384-well plate and centrifuge at 1000rpm for 30 seconds. Add 5µL of Tracer to a 384-well plate and centrifuge at 1000rpm for 30 seconds. The cells were incubated at room temperature for 60 minutes and then overnight at 4°C. The fluorescence signal ratio at 495 nm / 520 nm was read using an Envision microplate reader (PerkinElmer).

[0217] Experimental data processing method: XLfit, a software developed by IDBS and integrated into the Microsoft Excel environment, was used for test data processing and analysis. First, the average reaction signals of the high-signal control wells and the low-signal control wells were calculated respectively, and then the reaction inhibition rate of each compound well was calculated according to the formula "single-well inhibition rate % = 100% - (high-signal control group average value - single-well signal value) / (high-signal control group average value - low-signal control group average value) * 100%". The concentration and corresponding inhibition rate data were then imported into the XLfit software, and the Dose Response One Site 205 model in the software was used to fit the inhibition rate-concentration curve using the four-parameter method, and the IC of the compound was calculated. 50 value.

[0218] 3.3 TYK2 JH2 in vitro enzyme binding assay

[0219] Experimental instruments: Envision microplate reader (PerkinElmer), ECHO550 (LABCYTE).

[0220] Experimental method: Fluorescence resonance energy transfer (TR-FRET) method was used to test the inhibitory effect of compounds on TYK2 JH2 pseudokinase.

[0221] Experimental Procedure: Dissolve the compound in DMSO to a 10 mM stock solution. Further dilute in DMSO to a 200× compound dilution series. Transfer the 200× compound dilution series to a 384-well plate using an Echo instrument. Prepare dilution buffer (20 mM HEPES pH 7.5, 10 mM MgCl2, 0.015% Brij-35, 2 mM DTT, and 50 μg / mL BSA). Prepare the TYK2 JH2 pseudokinase, Tb antibody, and Tracer working solutions in dilution buffer. Add 5 μL of the TYK2 JH2 pseudokinase working solution (final concentration 0.5 nM) to a 384-well plate and centrifuge at 1000 rpm for 30 seconds. Add 5 μL of the Tb antibody (final concentration 1×) to a 384-well plate and centrifuge at 1000 rpm for 30 seconds. Add 5 μL of Tracer (final concentration 0.5 nM) to a 384-well plate and centrifuge at 1000 rpm for 30 seconds. Incubate at room temperature for 60 minutes and then at 4°C overnight. Finally, read the 495 nm / 520 nm fluorescence signal ratio using an Envision microplate reader (PerkinElmer).

[0222] Experimental data processing method: XLfit, a software developed by IDBS and integrated into the Microsoft Excel environment, was used for test data processing and analysis. First, the signal averages of the high-signal control group and the low-signal control group were calculated respectively, and then the reaction inhibition rate of each compound well was calculated according to the formula "single-well inhibition rate % = 100% - (high-signal control group average value - single-well signal value) / (high-signal control group average value - low-signal control group average value) * 100%". The concentration and corresponding inhibition rate data were then imported into the XLfit software, and the Dose Response One Site 205 model in the software was used to fit the inhibition rate-concentration curve using the four-parameter method, and the IC of the compound was calculated. 50 value.

[0223] Experimental conclusion: The JH1 target selectivity of the compound of the present invention for JAK 1-3 / TYK2 is comparable to that of BMS-986165, and both have no inhibitory activity. In addition, the inhibitory activity against JAK1 JH2 is significantly reduced compared with BMS-986165. The compound of the present invention has good TYK2 JH2 selectivity and low off-target risk.

[0224] Test Example 4: In vivo-mouse psoriasis model

[0225] The efficacy of the compounds of the present invention in the imiquimod-induced psoriasis-like mouse model can be determined by the following method:

[0226] Sample preparation: Prepared with reference to commercially available Benvimod cream. The compound (compound of Example 3), an oil phase (cetyl alcohol, petrolatum, liquid paraffin, and glyceryl mono- and distearate), an aqueous phase (propylene glycol, Tween 80, and water), and an appropriate additive (ethyl hydroxybenzoate) were mixed and vacuum emulsified to prepare a 1% cream sample.

[0227] The test area on the back of the animals was shaved, covering an area of ​​2×3 square centimeters. Imiquimod ointment was applied to the back skin of the mice continuously for 6 days to establish a psoriasis mouse model. On Days 1 to 6, each group of animals was given the drug according to the experimental protocol. The experimental design of the imiquimod-induced psoriasis-like mouse model is shown in the table below:

[0228] On the seventh day, the severity of skin inflammation was assessed using a 5-point scale (0-4) (PASI score):

[0229] Skin thickness: 0: smooth and wrinkle-free skin; 1: slight wrinkles appear at the edge of the applied area; 2: slight wrinkles appear on the entire applied area; 3: wrinkles in the applied area become more severe; 4: based on a score of 3, the mouse experiences weight loss or poor condition.

[0230] Scabbing: 0: smooth skin without scaling; 1: slight scaling in the applied area; 2: the applied area is completely covered with scaling; 3: the scaling in the applied area is further deepened; 4: based on a score of 3, the mouse loses weight or is in poor condition.

[0231] Erythema: 0: smooth skin; 1: slight redness of the skin in the applied area; 2: the skin in the applied area turns completely red; 3: the redness of the applied area deepens further; 4: based on a score of 3, the mouse loses weight or is in poor condition.

[0232] The comparison results of PASI scores of different compounds in the imiquimod-induced psoriasis model in mice are shown in the table below: Note: Compared with the model group, *** P<0.001, ** P<0.01, data represent the mean PASI score within the group.

[0233] Experimental Conclusion: The compound of Example 3 effectively improved psoriasis symptoms in an imiquimod-induced psoriasis-like mouse model, with a significant difference compared to the model group (P < 0.001), and was superior to benvimod cream and BMS-986165. Compounds of Examples 2, 4, 5, 34, 38, 54, 55, 57, 62, 86, and 91 exhibited similar efficacy to that of the compound of Example 3 and are not further detailed here.

[0234] In the imiquimod-induced psoriasis-like model in mice, compounds such as Examples 2, 3, 4, 5, 34, 38, 54, 55, 57, 62, 86, and 91 were effectively improved by oral administration (oral administration, 10 mg / kg, 25 mg / kg, 50 mg / kg, bid), with significant differences compared with the model group.

[0235] The efficacy test found that the same compound had certain differences in anti-psoriasis efficacy when administered orally or topically. Test Example 5: In vivo - Mouse Asthma Model

[0236] The efficacy of the compounds of the present invention in the OVA-induced asthma model in mice can be determined by the following method:

[0237] 5.1 Test sample preparation method

[0238] Test sample: Accurately weigh the compound of Example 3 and prepare a solution with a concentration of 2 mg / ml using DMSO.

[0239] 5.2 Model preparation

[0240] Thirty female BALB / c mice were adaptively fed. After one week, 10 mice were randomly selected as the blank control group. Twenty mice were selected to prepare an asthma model. The model mice were sensitized by intraperitoneal injection of 0.2 mL of sensitization solution containing 50 μg OVA and 2 mg Al(OH)3 on days 0, 7, and 14, respectively. The normal group was injected with an equal amount of normal saline.

[0241] On days 21-23 after sensitization, mice in the model group and the drug-treated group were challenged with 5% OVA by aerosol for 30 min, and mice in the normal group were challenged with physiological saline by aerosol for the same period of time.

[0242] 5.3 Grouping and Dosing

[0243] 30 minutes before daily challenge, mice in the blank and model groups were intratracheally administered with 50 μl of normal saline, and mice in the Example 3 compound group were intratracheally administered with 50 μl of the corresponding drug, once daily for 3 consecutive days. The animal grouping and dosing schedule are as follows:

[0244] 5.4 Airway Hyperresponsiveness Measurement

[0245] Airway hyperresponsiveness was measured in each group of mice after the final challenge. Mice were placed in a body scan chamber, and after measuring the baseline expiratory pause (Penh) value, they were challenged with nebulized methacholine. The concentrations of methacholine ranged from 0, 6.25, and 12.5 mg / mL, with a nebulized dose of 100 μL per session for 60 seconds. After each concentration, the mice were observed for signs of hypoxia, such as shortness of breath, head scratching, and irritability. The Penh values ​​were then recorded for 3 minutes, and the average values ​​were taken to compare airway hyperresponsiveness among the groups.

[0246] 5.5 Detection of inflammatory cells in alveolar lavage fluid

[0247] After the mouse airway hyperresponsiveness test is completed, the mouse is killed by dislocating the cervical vertebrae and fixed on a dissecting tray. The mouse abdomen and chest cavity are opened, the neck skin and excess tissue are cut off, the mouse trachea is exposed, and a small incision is made on the transverse axis of the trachea with small scissors. A 1ml syringe needle that has been processed in advance is inserted and the needle is fixed with surgical thread. 0.5ml of pre-cooled phosphate buffered saline (PBS) is drawn with a 1ml syringe and slowly injected into the mouse lungs, and then slowly aspirated. Each mouse is lavaged once, and a total of about 0.4ml of lavage fluid is collected. The number of inflammatory cells in the alveolar lavage fluid is detected by cell counter.

[0248] 5.6 Statistics

[0249] SPSS software was used for statistical analysis. The measurement data were expressed as mean ± standard deviation. One-way ANOVA was used for comparison between groups. LSD test was used for homogeneous variances, and Dunnett-t test was used for unequal variances. P < 0.05 was considered statistically significant.

[0250] 5.7 Results

[0251] 5.7.1 Effects on the Penh Value of Mice

[0252] Here are the results: Note: Compared with the blank control group, # P<0.05, ### P<0.001; compared with the model group, * P<0.05.

[0253] After sensitization with OVA+Al(OH)3 for 3 weeks, mice were stimulated with 5% OVA for 3 consecutive days and then stimulated with 6.25 mg / ml and 12.5 mg / ml methacholine. The Penh value was significantly increased compared with the blank control group (P<0.001 or P<0.05), proving that the mouse asthma model was successfully established.

[0254] Compared with the model group, the administration of the compound of Example 3 to mice before challenge significantly reduced the expiratory pause (Penh) value of the mice (P<0.05).

[0255] 5.7.2 Effects on Inflammatory Cells in Mouse Bronchoalveolar Lavage Fluid

[0256] Here are the results: Note: Compared with the blank control group, ## P<0.01, ### P<0.001; compared with the model group, * P<0.05.

[0257] In mice sensitized with OVA+Al(OH)3 for 3 weeks and then challenged with 5% OVA for 3 consecutive days, the number of inflammatory cells in the bronchoalveolar lavage fluid increased significantly (P<0.001 or P<0.01). Administration of the compound of Example 3 prior to challenge significantly reduced the number of inflammatory cells (leukocytes and lymphocytes) compared to the model group.

[0258] Test Example 6: Repeated 4-week toxicity study in rats

[0259] 6.1 Test materials:

[0260] Animals: SD rats, SPF grade, half male and half female, 200±20g;

[0261] Test sample: Example 3 compound cream (2%), prepared with reference to commercially available benvimod cream;

[0262] Positive control: BMS-986165;

[0263] Reagents: urethane, special reagents for blood cell analyzer, special reagents for fully automatic biochemical analyzer, special reagents for fully automatic coagulation analyzer, special reagents for electrolyte analyzer;

[0264] Instruments: electronic balance, blood cell analyzer, fully automatic biochemistry analyzer, fully automatic coagulation analyzer, electrolyte analyzer.

[0265] 6.2 Grouping and Dosing:

[0266] Grouping: The animals were randomly divided into three groups according to their body weight, namely, a blank control group, a group treated with the compound of Example 3, and a BMS-986165 group, with 10 animals in each group, half of them male and half female.

[0267] Dosage regimen: The Example 3 compound group was administered with skin application twice daily, and the BMS-986165 group was administered with oral gavage twice daily for 4 consecutive weeks (28 days). The specific dosing regimen is shown in the table below:

[0268] 6.3 Index detection:

[0269] Daily clinical observation and weight testing;

[0270] After the last administration, the rats were fasted for more than 16 hours and anesthetized the next day. Blood was collected from the abdominal aorta for hematology (EDTA-2K anticoagulation), blood biochemistry (serum), and coagulation function (sodium citrate anticoagulation and plasma separation).

[0271] After the animals were sacrificed, tissues and organs were removed for gross observation and organ weights and coefficients (heart, liver, spleen, kidney, and thymus) were measured;

[0272] Skin and lung tissues at the administration site were collected and stained with HE to observe the changes in epidermal thickness and inflammatory cells in the dermis as well as the lung lesions.

[0273] 6.4 Experimental Results Note: Compared with the blank control group: * P<0.05, ** P<0.01.

[0274] WBC: white blood cell; Lymph: lymphocyte; Mono: monocyte; Gran: granulocyte; PLT: platelet; APTT: partial thromboplastin time; TB: total bilirubin.

[0275] Hematological results showed that BMS-986165 could significantly reduce the number of white blood cells (P<0.01) and platelets (P<0.05) in rats, while the compound of Example 3 had no significant effect on the number of white blood cells (P>0.05);

[0276] The coagulation results showed that BMS-986165 could significantly prolong APTT (P<0.01), while the compound of Example 3 had no significant effect on APTT (P>0.05);

[0277] The blood biochemistry results showed that BMS-986165 could significantly increase the total bilirubin (TB) level in serum (P<0.05), while the compound of Example 3 had no significant effect on total bilirubin (P>0.05).

[0278] In addition, the compound of Example 3 had no significant effect on the general condition, body weight, organ coefficient, skin and lung pathology, and other hematological, coagulation, and blood biochemical indices of rats, demonstrating good safety.

[0279] In addition, the compound of the present invention showed good safety in skin irritation test, skin allergy test and oral acute toxicity test.

[0280] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A novel amide-substituted aromatic ring derivative or a pharmaceutically acceptable salt thereof, characterized in that: The structural formula of the compound of formula I is as follows: Wherein, X and P shown in the structural formula of the compound of formula I are selected independently of each other, and: X is selected from CH, N; P is selected from C1-C6 alkyl, substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted five-membered heteroaryl, substituted or unsubstituted six-membered heteroaryl, and the substituent is selected from one or more of the following: C1-C6 alkyl, C3-C6 cycloalkyl, halogen, C1-C6 alkoxy, C1-C6 haloalkyl, -CN; When X is N, P is as defined above; or When X is CH, P is as defined above.

2. The compound of formula I or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: X and P shown in the structural formula of the compound of formula I are selected independently of each other, and: X is selected from CH, N; P is selected from C1-C4 alkyl, substituted or unsubstituted phenyl, substituted or unsubstituted five-membered heteroaryl containing 2-3 N, substituted or unsubstituted six-membered heteroaryl containing 1-2 N, and the substituent is selected from one or two of the following: C1-C4 alkyl, C3-C6 cycloalkyl, halogen, C1-C4 alkoxy, C1-C4 haloalkyl, -CN; When X is N, P is as defined above; or When X is CH, P is as defined above.

3. The compound of formula I or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: X and P shown in the structural formula of the compound of formula I are selected independently of each other, and: X is selected from CH, N; P is selected from tert-butyl, substituted or unsubstituted phenyl, substituted or unsubstituted five-membered heteroaryl Substituted or unsubstituted six-membered heteroaryl The substituent is selected from One or two of the following: methyl, ethyl, isopropyl, tert-butyl, cyclopropyl, methoxy, cyano, fluorine, trifluoromethyl; When X is N, P is as defined above; or When X is CH, P is as defined above.

4. The compound of formula I or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: X and P shown in the structural formula of the compound of formula I are selected independently of each other, and: X is selected from CH, N; P is selected from tert-butyl, substituted or unsubstituted phenyl, substituted or unsubstituted five-membered heteroaryl Substituted or unsubstituted six-membered heteroaryl For the above-mentioned phenyl group, the substituent is selected from one or two of the following: methyl, ethyl, isopropyl, tert-butyl, cyclopropyl, methoxy, cyano, fluorine, trifluoromethyl; For the above five-membered heteroaryl group, the substituent is selected from methyl: For the above six-membered heteroaryl group, the substituent is selected from: methyl or fluorine; When X is N, P is as defined above; or When X is CH, P is as defined above.

5. The compound of formula I or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: X and P shown in the structural formula of the compound of formula I are selected independently of each other, and: X is selected from CH, N; P is selected from tert-butyl, When X is N, P is as defined above; or When X is CH, P is as defined above.

6. The compound of formula I or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: X and P shown in the structural formula of the compound of formula I are selected independently of each other, and: X is selected from CH or N; P is selected from substituted or unsubstituted phenyl, substituted or unsubstituted five-membered heteroaryl Substituted or unsubstituted six-membered heteroaryl For the above-mentioned phenyl group, the substituent is selected from one or two of the following: methyl, fluorine; For the above five-membered heteroaryl group, the substituent is selected from methyl: For the above six-membered heteroaryl group, the substituent is selected from methyl; Preferably, P is selected from: When X is N, P is as defined above; or When X is CH, P is as defined above.

7. The novel amide-substituted aromatic ring derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, characterized in that: The compound of formula I is selected from the following compounds:

8. The novel amide-substituted aromatic ring derivative or a pharmaceutically acceptable salt thereof according to claim 7, characterized in that: The compound of formula I is selected from the following compounds: Compound 2, Compound 3, Compound 4, Compound 5, Compound 34, Compound 38, Compound 54, Compound 55, Compound 57, Compound 62, Compound 86, and Compound 91.

9. A pharmaceutical composition, characterized in that The invention comprises the novel amide-substituted aromatic ring derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 8 and optionally one or more pharmaceutically acceptable carriers, diluents, excipients or adjuvants.

10. Use of the novel amide-substituted aromatic ring derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 8 or the pharmaceutical composition according to claim 9 in the preparation of a drug for treating TYK2-mediated related diseases.

11. The use according to claim 10, characterized in that The disease is selected from inflammatory or autoimmune diseases.

12. The use according to claim 10 or 11, characterized in that The disease is selected from psoriasis, psoriatic arthritis, dermatitis, lupus erythematosus, inflammatory bowel disease, hidradenitis suppurativa, rheumatoid arthritis or uveitis.