Heterocyclic compounds as TYK2 pseudokinase domain inhibitors and synthesis methods and uses thereof

Abstract

The present invention provides heterocyclic compounds as TYK2 pseudokinase (JH2) domain inhibitors, synthesis methods and uses. Specifically, the present invention provides a heterocyclic compound as shown in Formula I or a pharmaceutically acceptable salt thereof, wherein each group is as defined in the text. The heterocyclic compound of the present invention has excellent TYK2 (JH2) selective inhibitory activity.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and specifically relates to a heterocyclic compound as a TYK2 pseudokinase (JH2) domain inhibitor, a synthesis method and use thereof Background Art

[0002] Janus kinase (JAK) is an intracellular non-receptor tyrosine kinase that mediates the signal transduction and activation of various cytokines. The JAK kinase family is divided into four subtypes: JAK1, 2, 3, and TYK2. Each subtype mediates different types of cytokine signaling pathways. JAK1, 2, and TYK2 are expressed in various tissue cells of the human body, and JAK3 is mainly expressed in various hematopoietic tissue cells.

[0003] TYK2 is the earliest subtype discovered in the JAK family, mediating the functions of cytokines such as IFN-α, IL-6, IL-10, IL-12 and IL-23. Studies have shown that TYK2 deletion mutations can effectively inhibit the occurrence of immune diseases such as allergies, autoimmunity and inflammation. IL-23 plays a vital role in the development of psoriasis. The latest studies have shown that the pathogenesis of psoriasis is that endogenous unknown antigens activate antigen-presenting cells (APCs) to secrete IL-23, and IL-23 activates Thi7 cells to secrete cytokines such as IL-17, inducing keratinocyte differentiation and division and secretion of IL-23, further stimulating inflammation and keratinocyte proliferation to produce psoriasis. TYK2 and JAK2 jointly mediate the downstream signaling pathway of IL-23. Inhibition of JAK2 can lead to anemia and other blood-related side effects. Therefore, inhibiting TYK2 while avoiding inhibiting JAK2 is a good strategy to inhibit the IL-23 signaling pathway. The first oral Tofacitinib is a non-selective JAK inhibitor with significant inhibitory activity against JAK1, 2, 3, and TYK2. It can inhibit the catalytic domain (JH1 or "JAK protein homology 1 domain") that binds to the adenosine triphosphate (ATP) site, preventing the phosphorylation of the catalytic activity of downstream kinases by blocking ATP, and the resulting pathway signal transduction. Tofacitinib's inhibition of the activity of other subtypes such as JAK1, 2, 3, and TYK2 increases the efficacy, but also brings more serious side effects, including infection, tuberculosis, tumors, anemia, liver damage, and increased cholesterol. Because JAK2 activity is related to erythroid cell differentiation and lipid metabolism, some of the above-mentioned adverse reactions such as anemia are believed to be related to Tofacitinib's insufficient selectivity for JAK2, which is caused by the drug's non-selective inhibition.

[0004] In view of the good efficacy of JAK non-selective inhibitors and multiple target-related serious side effects, there is an urgent need in the art for a TYK2 inhibitor that can be distinguished from the previous JAK inhibitors in terms of their binding to the catalytic domain (JH1), selectively bind to the TYK2 pseudokinase (JH2) domain, inhibit the function of the pseudokinase (JH2) domain, avoid inhibition of other subtypes such as JAK1, 2, 3, and TYK2 (JH1), and increase the safety of TYK2 pseudokinase (JH2) inhibitors, thereby It can be used for the treatment of various autoimmune and inflammatory diseases related to the function of the TYK2 pseudokinase (JH2) domain, including psoriasis, lupus erythematosus, inflammatory bowel disease, psoriatic arthritis, arthritis, dermatitis, lupus nephritis, neuroinflammation such as multiple sclerosis and Alzheimer's disease, ankylosing spondylitis, suppurative hidradenitis, respiratory diseases, diabetes, inflammatory eye diseases, hepatitis, cardiovascular diseases, systemic sclerosis, organ transplantation, alopecia areata, acne, eczema, vitiligo, Sjögren's syndrome, some cancers, etc., and has great clinical application potential.

[0005] Therefore, there is an urgent need in the art to develop novel TYK2 pseudokinase (JH2) domain inhibitors with high safety and excellent efficacy. Summary of the invention

[0006] The purpose of the present invention is to provide a TYK2 pseudokinase (JH2) domain inhibitor with novel structure, high safety and excellent efficacy.

[0007] In the first aspect of the present invention, a heterocyclic compound or a pharmaceutically acceptable salt thereof is provided, wherein the compound is as shown in Formula I

[0008]

[0009] in,

[0010] X and Y are each independently CR5 or N;

[0011] Z is CR6 or N;

[0012] W1 and W2 are each independently a divalent group selected from the group consisting of O, S, SO2, CO, NR7CO, CONR7;

[0013] W3 is a divalent group selected from the group consisting of CO, NR7, NR7CO, CONR7;

[0014] R3 is substituted or unsubstituted C 3-6 Cycloalkyl;

[0015] R4 is selected from the group consisting of: substituted or unsubstituted C 1-6 Alkyl, substituted or unsubstituted C 3-6Cycloalkyl; and R4 is further optionally substituted by R1 and R2;

[0016] R1 and R2 are each independently selected from the following group: H, substituted or unsubstituted C1-6 alkyl; or, R1 and R2 and the carbon atom connected thereto together form a substituted or unsubstituted C 3-6 Cycloalkyl;

[0017] R5, R6 and R7 are each independently selected from the group consisting of H, substituted or unsubstituted C 1-4 alkyl;

[0018] n = 1 or 2;

[0019] Unless otherwise specified, the substitution refers to the replacement of one or more hydrogen atoms in the group by a substituent selected from the group consisting of halogen, C1-4 alkyl, and C1-4 haloalkyl.

[0020] In another preferred embodiment, R3 is substituted or unsubstituted cyclopropyl.

[0021] In another preferred embodiment, R3 is cyclopropyl.

[0022] In another preferred embodiment, X, Y, Z, W1, W2, W3, R1, R2, R3, R4, R5, R6, R7 and n are corresponding groups in the specific compounds in the embodiments (such as the compounds shown in Table A, preferably compounds 2-5).

[0023] In another preferred embodiment, the heterocyclic compound is as shown in Formula IA;

[0024]

[0025] Wherein, each group is as defined above.

[0026] In another preferred embodiment, the heterocyclic compound is as shown in Formula IB;

[0027]

[0028] Wherein, each group is as defined above.

[0029] In another preferred embodiment, the heterocyclic compound is as shown in Formula IC;

[0030]

[0031] Wherein, each group is as defined above.

[0032] In another preferred embodiment, the heterocyclic compound is as shown in Formula II,

[0033]

[0034] in,

[0035] X and Y are each independently CR5 or N;

[0036] Z is CR6 or N;

[0037] R1 and R2 are each independently selected from the following group: H, substituted or unsubstituted C1-6 alkyl; or, R1 and R2 and the carbon atom connected thereto together form a substituted or unsubstituted C 3-6 Cycloalkyl;

[0038] R5 and R6 are each independently selected from the group consisting of H, substituted or unsubstituted C 1-4 alkyl;

[0039] Unless otherwise specified, the substitution refers to the replacement of one or more hydrogen atoms in the group by a substituent selected from the group consisting of halogen, C1-4 alkyl, and C1-4 haloalkyl.

[0040] In another preferred embodiment, R5 and R6 are both H.

[0041] In another preferred embodiment, X, Y and Z are each independently CH or N.

[0042] In another preferred embodiment, X is N.

[0043] In another preferred embodiment, Y is CR5; preferably, Y is CH.

[0044] In another preferred embodiment, Z is N.

[0045] In another preferred embodiment, X and Z are N.

[0046] In another preferred embodiment, X is N and Y is CR5 (preferably, X is N and Y is CH).

[0047] In another preferred embodiment, X is N, Y is CR5 (preferably, Y is CH), and Z is N.

[0048] In another preferred embodiment, R1 and R2 are H.

[0049] In another preferred embodiment, R1 and R2 and the carbon atom connected thereto together form a substituted or unsubstituted C 3-6 Cycloalkyl.

[0050] In another preferred embodiment, R1 and R2 are H, or R1 and R2 and the carbon atom to which they are connected together form a cyclopropyl group.

[0051] In another preferred embodiment, X, Y, Z, R1 and R2 are corresponding groups in the specific compounds in the examples (such as the compounds shown in Table A, more preferably compounds 2-5).

[0052] In another preferred embodiment, the compound is a compound selected from Table A:

[0053] Table A

[0054]

[0055] In the second aspect of the present invention, an intermediate for preparing the heterocyclic compound as described in the first aspect is provided, wherein the intermediate is shown in Formula III, Formula IIIA, Formula IIIB, Formula IIIC or Formula IV:

[0056]

[0057]

[0058] Among them, R X is halogen; X, Y, Z, W1, W2, W3, R1, R2, R3, R4, R5, R6, R7 and n are as defined in the first aspect.

[0059] In another preferred embodiment, R X For Cl.

[0060] In the third aspect of the present invention, there is provided a method for preparing the heterocyclic compound as described in the first aspect, wherein the heterocyclic compound is as shown in Formula I, and wherein W3 is NR7CO; the method comprises the steps of:

[0061] Reacting the intermediate represented by formula III with the compound represented by formula V to obtain a compound represented by formula I;

[0062]

[0063] wherein R4 and R7 are as defined in the first aspect.

[0064] In another preferred embodiment, the intermediate shown in formula IV is reacted with the compound shown in formula VI to obtain a compound of formula II

[0065]

[0066] Wherein, R1 and R2 are as defined in the first aspect.

[0067] In a fourth aspect of the present invention, a pharmaceutical composition is provided, comprising:

[0068] (i) the heterocyclic compound according to the first aspect or a pharmaceutically acceptable salt thereof, and (ii) a pharmaceutically acceptable carrier or excipient.

[0069] In a fifth aspect of the present invention, there is provided a use of the heterocyclic compound as described in the first aspect in the preparation of (i) a medicament for treating or preventing a TYK2-mediated disease and / or (ii) a TYK2 inhibitor.

[0070] In another preferred embodiment, the TYK2-mediated disease is a disease associated with IL-23, IL-12 and / or IFNα.

[0071] In another preferred embodiment, the heterocyclic compound treats or prevents TYK2-JH2-mediated diseases by selectively inhibiting TYK2-JH2.

[0072] In another preferred embodiment, the TYK2-JH2-mediated disease includes: inflammation, autoimmune disease, or a combination thereof.

[0073] In another preferred embodiment, the TYK2-JH2 mediated diseases include: psoriasis, lupus erythematosus, inflammatory bowel disease, psoriatic arthritis, arthritis, dermatitis, lupus nephritis, neuroinflammation such as multiple sclerosis and Alzheimer's disease, ankylosing spondylitis, hidradenitis suppurativa, respiratory diseases, diabetes, inflammatory eye diseases, hepatitis, cardiovascular diseases, systemic sclerosis, organ transplantation, alopecia areata, acne, eczema, vitiligo, Sjögren's syndrome, cancer or a combination thereof.

[0074] In another preferred embodiment, the TYK2-JH2 inhibitor is a selective TYK2-JH2 inhibitor.

[0075] In a sixth aspect of the present invention, a method for selectively inhibiting TYK2 is provided, the method comprising:

[0076] The subject is contacted with the heterocyclic compound as described in the first aspect, thereby inhibiting the activity of TYK2-JH2 in the subject.

[0077] In another preferred embodiment, the object is a cell or TYK2 kinase.

[0078] In another preferred embodiment, the inhibition is selective inhibition of TYK2-JH2.

[0079] In another preferred embodiment, the method is non-therapeutic in vitro.

[0080] In a seventh aspect of the present invention, there is provided a method for treating or preventing a TYK2-mediated disease, comprising the steps of:

[0081] Administering a safe and effective amount of the heterocyclic compound of the first aspect or the pharmaceutical composition of the third aspect to a person in need, thereby inhibiting or

[0082] In another preferred embodiment, the TYK2-mediated disease is a disease associated with IL-23, IL-12 and / or IFNα.

[0083] In another preferred embodiment, the heterocyclic compound treats or prevents TYK2-JH2-mediated diseases by selectively inhibiting TYK2-JH2.

[0084] In another preferred embodiment, the TYK2-JH2-mediated disease includes: inflammation, autoimmune disease, or a combination thereof.

[0085] In another preferred embodiment, the TYK2-JH2 mediated diseases include: psoriasis, lupus erythematosus, inflammatory bowel disease, psoriatic arthritis, arthritis, dermatitis, lupus nephritis, neuroinflammation such as multiple sclerosis and Alzheimer's disease, ankylosing spondylitis, hidradenitis suppurativa, respiratory diseases, diabetes, inflammatory eye diseases, hepatitis, cardiovascular diseases, systemic sclerosis, organ transplantation, alopecia areata, acne, eczema, vitiligo, Sjögren's syndrome, cancer, or a combination thereof.

[0086] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. DETAILED DESCRIPTION

[0087] After extensive and in-depth research, the inventors found that the novel structured compound of the present invention has excellent selective inhibition of TYK2 (JH2), and thus the present invention provides a compound with higher safety that can effectively inhibit the desired target without affecting other non-target targets. Based on this, the inventors completed the present invention.

[0088] the term

[0089] As used herein, the term "halogen" refers to F, Cl, Br or I. Accordingly, "halo" refers to a hydrogen atom in a group being replaced by F, Cl, Br or I.

[0090] Unless otherwise indicated, the term "alkyl" by itself or as part of another substituent refers to a straight or branched chain hydrocarbon radical (i.e., C 1-6 represents 1 to 6 carbons). Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, and the like.

[0091] The term "alkenyl" refers to an unsaturated alkyl group having one or more double bonds. Preferably, the alkenyl group includes 1 to 4 carbons, i.e., C1-4 alkenyl. Similarly, the term "alkynyl" refers to an unsaturated alkyl group having one or more triple bonds (preferably with one triple bond). Preferably, the alkenyl group includes 1 to 4 carbons. Examples of these unsaturated alkyl groups include vinyl, 2-propenyl, ethynyl, 1- and 3-propynyl, 3-butynyl, etc.

[0092] The term "cycloalkyl" refers to a cycloalkyl radical having the specified number of ring atoms (e.g., C 3-6 Cycloalkyl is a hydrocarbon ring having 3-6 ring atoms) and is fully saturated. "Cycloalkyl" also refers to bicyclic and polycyclic hydrocarbon rings, for example, bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane, and the like.

[0093] The term "alkylene" by itself or as part of another substituent refers to a divalent group derived from an alkane, such as -CH2- or -CH2CH2CH2-. Alkyl (or alkylene) groups typically have 1-4 carbon atoms. Similarly, "alkenylene" or "alkynylene" refer to unsaturated forms of "alkylene" groups having double or triple bonds, respectively.

[0094] Substituents for alkyl groups (including those groups commonly referred to as alkylene, alkenyl, alkynyl and cycloalkyl) may be various groups selected from the group consisting of -halogen, -OR', -NR'R", -SR', -OC(O)R', -C(O)R', -C02R', -CONR'R", -OC(O)NR'R", -NR"C(O)R', -NR'-C(O)NR"R"', -NR"C(O)2R', -NH-C(NH2)=NH, -NR'C(NH2)=NH, -NH-C(NH2)=NR', -S(O)R', -S(O)2R', -S(O)2NR'R", -NR'S(O)2R", -CN and -NO2, in an amount ranging from zero to (2m'+1), where m' is the total number of carbon atoms in such group. R', R" and R"' each independently represent hydrogen, unsubstituted C 1-8 Alkyl, unsubstituted heteroalkyl, unsubstituted aryl, aryl substituted with 1-3 halogens, unsubstituted C 1-8 Alkyl, C 1-8 Alkoxy or C 1-8 Thioalkoxy, or unsubstituted aryl-C 1-4Alkyl. When R' and R" are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 3-, 4-, 5-, 6- or 7-membered ring. For example, -NR'R" is meant to include 1-pyrrolidinyl and 4-morpholinyl. The term "acyl", used alone or as part of another group, refers to a group in which both substituents on the carbon closest to the point of attachment of the group are replaced by a substituent = O (e.g., -C(O)CH3, -C(O)CH2CH2OR', etc.).

[0095] As used herein, the term "heteroatom" is intended to include oxygen (O), nitrogen (N), sulfur (S) and silicon (Si); preferably, the heteroatom includes oxygen (O), nitrogen (N), sulfur (S).

[0096] For compounds provided herein, a bond from a substituent (typically an R group) to the center of an aromatic ring (e.g., benzene, pyridine, etc.) will be understood to mean a bond providing a connection at any available vertex of the aromatic ring. In some embodiments, this description also includes connections on a ring fused to an aromatic ring. For example, a bond drawn to the center of an indole benzene moiety will represent a bond connected to any available vertex of a six-membered or five-membered ring moiety of indole.

[0097] As used herein, the terms "comprising", "including" or "comprising" indicate that various components can be used together in the mixture or composition of the present invention. Therefore, the terms "consisting essentially of" and "consisting of" are included in the term "comprising".

[0098] As used herein, the term "pharmaceutically acceptable" ingredient refers to a substance that is suitable for use in humans and / or animals without excessive adverse side effects (such as toxicity, irritation, and allergic response), ie, at a reasonable benefit / risk ratio.

[0099] Unless otherwise specified, in the present invention, all compounds appearing are intended to include all possible optical isomers, such as single chiral compounds, or mixtures of various chiral compounds (i.e., racemates). Among all compounds of the present invention, each chiral carbon atom can be optionally in R configuration or S configuration, or a mixture of R configuration and S configuration.

[0100] Certain compounds of the present invention possess asymmetric carbon atoms (optical centers) or double bonds; racemates, diastereomers, geometric isomers, regioisomers and individual isomers (e.g., isolated enantiomers) are all intended to be included within the scope of the present invention. When compounds provided herein have defined stereochemistry (expressed as R or S, or indicated by dashed or wedge-shaped bonds), those compounds will be understood by those skilled in the art to be substantially free of other isomers (e.g., at least 80%, 90%, 95%, 98%, 99% and up to 100% free of other isomers).

[0101] The compounds of the invention may also contain unnatural proportions of atomic isotopes at one or more of the isotopic atoms that make up such compounds. An unnatural proportion of an isotope may be defined as the amount of the atom in question found in nature to 100% of that atom. For example, a compound may incorporate a radioactive isotope, such as tritium ( 3 H), iodine-125 ( 125 I) or carbon-14 ( 14 C), or non-radioactive isotopes such as deuterium ( 2 H) or carbon-13 ( 13 C). Such isotopic variants may provide additional uses in addition to those described herein. For example, isotopic variants of the compounds of the invention may have additional uses, including but not limited to as diagnostic and / or imaging agents, or as cytotoxic / radiotoxic therapeutic agents. In addition, isotopic variants of the compounds of the invention may have altered pharmacokinetic and pharmacodynamic characteristics, thereby helping to increase safety, tolerability or efficacy during treatment. All isotopic variants of the compounds of the invention, whether radioactive or not, are intended to be included within the scope of the invention.

[0102] Active ingredients

[0103] As used herein, the term "compound of the present invention" or "heterocyclic compound of the present invention" refers to a compound of formula (I) or formula (II). The term also includes various crystalline forms, pharmaceutically acceptable salts, hydrates or solvates of the compound of formula (I) or formula (II).

[0104] The term "pharmaceutically acceptable salt" refers to a salt formed by a compound of the present invention and an acid or base that is suitable for use as a drug. Pharmaceutically acceptable salts include inorganic salts and organic salts. A preferred class of salts is a salt formed by a compound of the present invention and an acid. Acids suitable for forming salts include, but are not limited to, inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, and phosphoric acid; organic acids such as formic acid, acetic acid, trifluoroacetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, benzoic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, and naphthalenesulfonic acid; and amino acids such as proline, phenylalanine, aspartic acid, and glutamic acid. Another preferred salt is a salt of the compound of the present invention and a base, such as an alkali metal salt (e.g., sodium salt or potassium salt), an alkaline earth metal salt (e.g., magnesium salt or calcium salt), an ammonium salt (e.g., lower alkanolammonium salt and other pharmaceutically acceptable amine salts), for example, methylamine salt, ethylamine salt, propylamine salt, dimethylamine salt, trimethylamine salt, diethylamine salt, triethylamine salt, tert-butylamine salt, ethylenediamine salt, hydroxyethylamine salt, dihydroxyethylamine salt, trihydroxyethylamine salt, and amine salts formed from morpholine, piperazine, and lysine, respectively.

[0105] The term "solvate" refers to a complex formed by the coordination of the compound of the present invention with solvent molecules in a specific ratio. "Hydrate" refers to a complex formed by the coordination of the compound of the present invention with water.

[0106] In addition, the compounds of the present invention also include prodrugs of heterocyclic compounds represented by formula (I) or formula (II). The term "prodrug" includes a compound that can be biologically active or inactive, and when taken by an appropriate method, it undergoes metabolism or chemical reactions in the human body and is converted into a class of compounds of formula (I), or a salt or solution composed of a compound of formula (I). The prodrug includes (but is not limited to) carboxylate, carbonate, phosphate, nitrate, sulfate, sulfone, sulfoxide, amino compound, carbamate, azo compound, phosphoramide, glucoside, ether, acetal and the like forms of the compound.

[0107] Preparation method

[0108] The preparation method of the compound of formula (I) or formula (II) of the present invention is described in more detail below, but these specific methods do not constitute any limitation to the present invention. The compounds of the present invention can also be conveniently prepared by optionally combining various synthetic methods described in this specification or known in the art, and such combination can be easily carried out by a technician in the field to which the present invention belongs.

[0109] Pharmaceutical compositions and methods of administration

[0110] Since the compounds of the present invention have excellent selective inhibitory activity against the TYK2 pseudokinase domain (JH2), the compounds of the present invention and their various crystal forms, pharmaceutically acceptable inorganic or organic salts, hydrates or solvates, and pharmaceutical compositions containing the compounds of the present invention as the main active ingredient can be used to treat, prevent and alleviate diseases mediated by TYK2 kinase. According to the prior art, the compounds of the present invention can be used to treat the following diseases: psoriasis, lupus erythematosus, inflammatory bowel disease, psoriatic arthritis, arthritis, dermatitis, lupus nephritis, neuroinflammation such as multiple sclerosis and Alzheimer's disease, ankylosing spondylitis, suppurative hidradenitis, respiratory diseases, diabetes, inflammatory eye diseases, hepatitis, cardiovascular diseases, systemic sclerosis, organ transplantation, alopecia areata, acne, eczema, vitiligo, Sjögren's syndrome, some cancers, and other autoimmune and inflammatory related diseases.

[0111] As used herein, the term "selective" refers to an activity or potency (e.g., inhibitory activity) on a specified target (e.g., TYK2 (JH2)) that is higher than an activity or potency (e.g., inhibitory activity) on other targets (e.g., JAK1, 2, 3 (JH1 and JH2), and / or TYK2 (JH1)); for example, an activity or potency (e.g., inhibitory activity) on a specified target (e.g., TYK2 (JH2)) is at least 50 times higher than an activity or potency (e.g., inhibitory activity) on other targets (e.g., JAK1, 2, 3 (JH1 and JH2), and / or TYK2 (JH1)). For example, when the inhibitory activity is quantified by the IC50 value, the IC50 其他 / IC50 TYK2-JH2 >50, where IC50 TYK2-JH2 Refers to the inhibitory activity IC50 (nM) of the heterocyclic compound on TYK2-JH2 (pseudokinase domain of TYK2), IC50 其他 It refers to the inhibitory activity IC50 (nM) of the heterocyclic compound against one or more of the following kinases: TYK2-JH1 (kinase domain of TYK2), JAK1-JH1 (kinase domain of JAK1), JAK1-JH2 (pseudokinase domain of JAK1), JAK2-JH1 (kinase domain of JAK2), and JAK3-JH1 (kinase domain of JAK3).

[0112] The pharmaceutical composition of the present invention comprises a safe and effective amount of the compound of the present invention or a pharmacologically acceptable salt thereof and a pharmacologically acceptable excipient or carrier. Wherein "safe and effective amount" means: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects. Usually, the pharmaceutical composition contains 1-200 mg of the compound of the present invention per dose, and more preferably, contains 1-50 mg of the compound of the present invention per dose. Preferably, the "one dose" is a capsule or tablet.

[0113] "Pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with the compounds of the present invention and with each other without significantly reducing the efficacy of the compounds. Some examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as Tween ), wetting agents (such as sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0114] There is no particular limitation on the administration of the compound or pharmaceutical composition of the present invention. Representative administrations include, but are not limited to, oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and topical administration.

[0115] Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) fillers or extenders, for example, starches, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and acacia; (c) humectants, for example, glycerol; (d) disintegrants, for example, agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) solubilizers, for example, paraffin; (f) absorption accelerators, for example, quaternary ammonium compounds; (g) wetting agents, for example, cetyl alcohol and glyceryl monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents.

[0116] Solid dosage forms such as tablets, pills, capsules, pills and granules can be prepared using coatings and shell materials, such as enteric coatings and other materials known in the art. They may contain opacifiers, and the release of the active compound or compounds in such compositions can be delayed in a certain part of the digestive tract. Examples of embedding components that can be used are polymeric substances and waxes. If necessary, the active compound can also be formed into microencapsulated form with one or more of the above-mentioned excipients.

[0117] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compound, the liquid dosage form may contain an inert diluent conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide and oils, in particular cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances.

[0118] Besides such inert diluents, the composition may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0119] Suspensions, in addition to the active compounds, may contain suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methanol and agar, or mixtures of these substances, and the like.

[0120] Compositions for parenteral injection may include physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

[0121] Dosage forms for topical administration of the compounds of the invention include ointments, powders, patches, sprays and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be required.

[0122] The compounds of the present invention may be administered alone or in combination with other pharmaceutically acceptable compounds.

[0123] When using the pharmaceutical composition, a safe and effective amount of the compound of the present invention is applied to a mammal (such as a human) in need of treatment, wherein the dosage during administration is a pharmaceutically effective dosage, and for a person weighing 60 kg, the daily dosage is usually 1 to 200 mg, preferably 1 to 50 mg. Of course, the specific dosage should also take into account factors such as the route of administration and the health status of the patient, which are all within the skill of a skilled physician.

[0124] The main advantages of the present invention include:

[0125] 1. The compounds of the present invention have excellent selectivity. Compared with JH1 and JH2 of JAK1, 2, 3 and JH1 of TYK2, the compounds of the present invention have better inhibitory activity against JH2 of TYK2. Therefore, the compounds of the present invention are less likely to act on other similar kinases and have better safety.

[0126] 2. The compounds of the present invention have excellent selectivity and excellent inhibitory activity.

[0127] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

[0128] Preparation Example

[0129] Example 1: Synthesis of Compound 1

[0130]

[0131] Step A:

[0132]

[0133] Compound 1a (6.4 g, 0.045 mol, 1.0 eq), dimethyl sulfoxide (100 ml), and sodium cyclopropylsulfinate (6.4 g, 0.06 mol, 1.2 eq) were added to a 250 ml three-necked flask. The mixture was heated and stirred at 100 °C for 1 hr under nitrogen protection. TLC was used to detect the reaction until it was complete. The temperature was lowered, and the reaction solution was poured into ice water (600 ml). The mixture was extracted three times with methyl tert-butyl ether. The combined organic phases were washed twice with saturated brine, dried over Na2SO4, concentrated, and slurried with petroleum ether to obtain compound 1b (7.8 g, 0.034 mol, yield 76%) as a white powdery solid. 1H NMR (300MHz, CDCl3): 8.05 (m, 1H), 7.7-7.9 (m, 3H), 3.2-3.3 (m, 1H), 1.3-1.5 (m, 2H), 1.0-1.2 (m, 2H).

[0134] Step B:

[0135]

[0136] Compound 1b (3.2 g, 0.014 mol, 1.0 eq), ethanol (100 ml), Pd / C (10%, 1.0 g) were added to a 250 ml single-mouth bottle, and the reaction was carried out under hydrogen bag at room temperature for 3 hours. The raw material reacted completely by TLC, and the mixture was filtered, concentrated, and column chromatography was performed to obtain a white solid compound 1c (2.6 g, 0.013 mol, yield 94%). 1H NMR (300 MHz, CDCl3): 7.65 (m, 1H), 7.2-7.4 (m, 1H), 6.7-6.9 (m, 2H), 2.6-2.8 (m, 1H), 1.3-1.4 (m, 2H), 0.9-1.0 (m, 2H).

[0137] Step C:

[0138]

[0139] Compound 1c (0.8 g, 4 mmol, 1.0 eq), compound 1d (0.91 g, 4.4 mmol, 1.1 eq) and THF (40 ml) were added into a 100 ml three-necked flask under nitrogen protection. NaHMDS (2 M, 3 ml, 6 mmol) was added dropwise at 0-5 °C. After the addition was completed, the mixture was reacted at room temperature for 1 hr. TLC showed that the reaction of the raw materials was complete. The reaction solution was poured into cold saturated aqueous ammonium chloride solution (200 ml), extracted twice with ethyl acetate, and the combined organic phases were washed twice with saturated brine, dried over Na2SO4, concentrated, and subjected to column chromatography to obtain a light yellow solid compound 1f (0.4 g, 1 mmol, yield 94%). MS-ESI:[M+1]+=370.9.1H NMR(300MHz, CDCl3):11.4(s,1H),8.2(s,1H),8.0(m,1H),7.7(m,1H),7.4- 7.6(m,2H),7.1(s,1H),2.5-2.7(m,1H),1.2-1.4(m,2H),0.97-1.04(m,2H).

[0140] Step D:

[0141]

[0142] In a 100 ml single-necked bottle were added compound 1f (0.4 g, 1.0 mmol, 1.0 eq), 1,4-dioxane (30 ml), cyclopropylcarboxamide (0.14 g, 1.63 mol, 1.5 eq), Pd2(dba)3 (0.1 g), DPPF (0.01 g), and 2M K3PO4 aqueous solution (1.6 ml, 3.24 mol, 3.0 eq). The solution was protected by nitrogen and reacted at 110 °C overnight. The raw material did not react completely according to TLC. The solution was concentrated and subjected to column chromatography to obtain yellow powder compound 1 (90 mg, 0.22 mmol, yield 22%).

[0143] MS-ESI:[M+1]+=419.5.1H NMR(300MHz,d6-DMSO):11.35(s,1H),11.06(s,1H),9.09(s,1H),8.04(s,1H),7.89 (m,1H),7.7-7.73(m,2H),7.42(m,1H),2.75(m,1H),2.03(m,1H),0.75-1.02(m,8H).

[0144] Example 2: Synthesis of Compound 2

[0145]

[0146] Step A

[0147]

[0148] Compound 2a (10 g, 0.0575 mol, 1.0 eq), dimethyl sulfoxide (100 ml), sodium cyclopropylsulfinate (11 g, 0.086 mol, 1.5 eq), L-proline (1.32 g, 0.2 eq), cuprous iodide (1.1 g, 0.1 eq), and sodium hydroxide (0.46 g, 0.2 eq) were added to a 250 ml three-necked flask. The mixture was protected by nitrogen and reacted at 150 ° C for 48 hrs. The reaction was completed by TLC. The temperature was lowered and poured into ice water (2 L). The mixture was extracted twice with ethyl acetate. The combined organic phase was washed twice with saturated brine, dried, concentrated, and subjected to column chromatography to obtain compound 2b (6.6 g, 0.0331 mol, yield 57.6%) as a white powder. MS-ESI: [M+1]+=200.2. 1H NMR (300MHz, CDCl3): 8.24 (d, 1H), 8.01 (d, 1H), 2.8-2.9 (m, 1H), 1.4-1.5 (m, 2H), 1.05-1.15 (m, 2H).

[0149] Step B

[0150]

[0151] Compound 2b (0.4 g, 2 mmol, 1.0 eq) and THF (20 ml) were added to a 100 ml three-necked flask under nitrogen protection. NaHMDS (2M, 1.5 ml, 3 mmol) was added dropwise at 0-5°C, stirred at room temperature for 30 mins, and then compound 1d (0.42 g, 2 mmol, 1.0 eq) was added. TLC showed that the raw material did not react completely. Even after a delay, the reaction did not change. The product was poured into ice water, extracted with ethyl acetate, washed with saturated brine, dried and concentrated, and subjected to column chromatography to obtain compound 2c (0.53 g, 1.42 mmol, yield 71%) as a white powder. MS-ESI: [M+1]+=372.8.

[0152] Step C:

[0153]

[0154] Into a 100 ml single-necked bottle were added compound 2c (0.25 g, 0.67 mmol, 1.0 eq), dioxane (20 ml), cyclopropylcarboxamide (85 mg, 1 mmol, 1.5 eq), Pd2(dba)3 (50 mg), DPPF (60 mg), and the atmosphere was replaced with nitrogen three times. The mixture was stirred for 5 mins, and then K3PO4 aqueous solution (1 ml, 2 M, 3.0 eq) was added. The atmosphere was replaced with nitrogen three times and the mixture was reacted at 112 °C overnight. The raw material was not completely reacted according to TLC. The mixture was concentrated and slurried with methyl tert-butyl ether after column chromatography to give yellow powder compound 2 (80 mg, 0.19 mmol, yield 28.5%). MS-ESI:[M+1]+=421.5.1H NMR(300MHz,d6-DMSO):12.3(s,1H),11.5(s,1H),9.44(s,1H),9.18(s,1H),8.67(d ,1H),8.44(d,1H),3.16(m,1H),2.13(m,1H),1.10-1.22(m,4H),0.86-0.88(m,4H).

[0155] Example 3: Synthesis of Compound 3

[0156]

[0157] Step A

[0158]

[0159] Compound 3a (25 g, 0.144 mol, 1.0 eq), dimethyl sulfoxide (300 ml), sodium cyclopropylsulfinate (28 g, 0.218 mol, 1.5 eq), L-proline (3.3 g, 0.2 eq), cuprous iodide (2.74 g, 0.1 eq), and sodium hydroxide (1.2 g, 0.2 eq) were added to a 500 ml three-necked flask. The mixture was protected by nitrogen and reacted at 150 ° C for 48 hrs. The reaction was completed by TLC. The temperature was lowered and poured into ice water (2 L). The mixture was extracted with ethyl acetate twice. The combined organic phase was washed twice with saturated brine, dried over Na2SO4, concentrated, and slurried with petroleum ether-methyl tert-butyl ether to obtain beige powder compound 3b (16 g, 0.081 mol, yield 56.1%). MS-ESI:[M+1]+=199.2.1H NMR (300MHz, CDCl3): 8.25-8.27(d,1H),7.87-7.90(m,1H),6.73-6.78(m,1H),2.60-2.70(m,1H),1.29-1.35(m,2H),0.97-1.05(m,2H).

[0160] Step B

[0161]

[0162] Compound 3b (5.5 g, 0.028 mol, 1.0 eq) and THF (200 ml) were added into a 500 ml three-necked flask under nitrogen protection. NaHMDS (2M, 21 ml, 0.042 mol, 1.5 eq) was added dropwise at 0-5°C and stirred at room temperature for 30 mins. Compound 1d (6.3 g, 0.03 mol, 1.1 eq) was added under ice bath and stirred at room temperature for 1 hr. The reaction of the raw materials was complete by TLC. The mixture was poured into cold saturated aqueous ammonium chloride solution (100 ml), and methyl tert-butyl ether (100 ml) was added and stirred for 10 mins. Solid precipitated and was filtered, washed with water and ethyl acetate, and dried to obtain white powder compound 3c (3.1 g, 8.3 mmol, yield 30%). MS-ESI:[M+1]+=371.9.1H NMR(300MHz,d6-DMSO):12.3(s,1H),9.40(s,1H),8.88(s,1H),8.70(d,1H), 8.25-8.28(m,1H),8.37-8.41(m,1H),3.12(m,1H),1.18(m,2H),1.09(m,2H).

[0163] Step C

[0164]

[0165] Into a 250ml single-necked bottle were added compound 3c (3.1g, 8.3mmol, 1.0eq), dioxane (150ml), cyclopropylcarboxamide (1.1g, 13.0mmol, 1.6eq), Pd2(dba)3 (0.5g), DPPF (0.6g), and the mixture was replaced with nitrogen three times. The mixture was stirred for 5mins, and then K3PO4 aqueous solution (12.5ml, 2M, 3.0eq) was added. The mixture was replaced with nitrogen three times. The mixture was reacted at 112°C overnight. The raw material was not completely reacted by TLC. The mixture was concentrated and subjected to column chromatography to obtain compound 3 (1.25g, 3mmol, 36% yield) as a white powder. MS-ESI:[M+1]+=420.5.1H NMR(300MHz, CDCl3):12.4(s,1H),9.66(s,1H),9.23(s,1H),8.62(m,1H),8.16-8.23(m,2H),7.12 -7.16(m,1H),2.82-2.90(m,1H),1.64-1.82(m,1H),1.40(m,2H),1.20(m,2H),0.98-1.11(m,4H).

[0166] Example 4: Synthesis of Compound 4

[0167]

[0168] Here are the steps:

[0169]

[0170] Into a 100 ml single-necked bottle were added compound 3c (0.21 g, 0.56 mmol, 1.0 eq), dioxane (20 ml), compound 4a (94 mg, 0.85 mmol, 1.5 eq), Pd2(dba)3 (0.05 g), DPPF (0.06 g), and the mixture was replaced with nitrogen three times. The mixture was stirred for 5 mins, and then K3PO4 aqueous solution (1 ml, 2 M, 3.6 eq) was added. The mixture was replaced with nitrogen three times and the reaction was carried out at 112 °C overnight. The raw material was not completely reacted by TLC. The mixture was concentrated and subjected to column chromatography to obtain pink powder compound 4 (mixture of isomers) (0.07 g, 3 mmol, yield 28%). MS-ESI:[M+1]+=446.5.1H NMR(300MHz, CDCl3):12.4(s,1H),9.64(s,1H),8.86(s,1H),8.61-8.64(m,1H),8.14-8.23(m,2H),7.12-7 .17(m,1H),2.86-2.92(m,1H),2.18-2.22(m,1H),1.66(m,1H),1.55(m,1H),1.40(m,2H),1.0-1.13(m,6H).

[0171] Example 5: Synthesis of Compound 5

[0172]

[0173] Into a 100 ml single-necked bottle were added compound 2c (0.25 g, 0.67 mmol, 1.0 eq), dioxane (20 ml), compound 4a (111 mg, 1 mmol, 1.5 eq), Pd2(dba)3 (0.05 g), DPPF (0.06 g), and the mixture was replaced with nitrogen three times. The mixture was stirred for 5 mins, and then K3PO4 aqueous solution (1 ml, 2 M, 3.0 eq) was added. The mixture was replaced with nitrogen three times and the mixture was reacted at 112 °C overnight. According to TLC, the raw material was not completely reacted. The mixture was concentrated, column chromatographed, and slurried with methyl tert-butyl ether to obtain yellow powder compound 5 (mixture of isomers) (0.05 g, 0.112 mml, yield 16.7%). MS-ESI:[M+1]+=447.4.1H NMR(300MHz, CDCl3):12.6(s,1H),9.68(s,1H),8.84(s,1H),8.60(d,1H),8.29(d,1H) ,8.16(s,1H),3.03-3.09(m,1H),2.17-2.21(m,1H),1.5-1.7(m,4H),1.0-1.3(m,6H).

[0174] Example 6: Synthesis of Compound 6

[0175]

[0176] Step A

[0177]

[0178] Compound 3b (532 mg, 2.68 mmol, 1.0 eq) and DMF (20 ml) were added to a 200 ml three-necked flask under nitrogen protection. NaH (539 mg, 13.4 mmol, 60%) was added at 0-5°C and stirred at room temperature for 30 mins. Compound 6a (700 mg, 3.38 mol, 1.2 eq) was added under ice bath and stirred at room temperature for 1 hr. The mixture was reacted at 50°C for 3 h, poured into cold saturated aqueous ammonium chloride solution (100 ml), extracted twice with ethyl acetate, and the combined organic phases were washed twice with saturated brine, dried over Na2SO4, concentrated, and subjected to column chromatography to obtain white powder compound 6b (220 mg, 0.6 mmol, yield 22%). MS-ESI:[M+1]+=370.1.1H NMR(400MHz, CDCl3):11.7(s,1H),8.64(s,1H),8.60(s,1H),8.56(m,1H),8.20(m,1 H),7.16(m,1H),6.88(m,1H),2.83-2.9(m,1H),1.41-1.43(m,2H),1.05-1.1(m,2H).

[0179] Step B

[0180]

[0181] Into a 100 ml single-necked bottle were added compound 6b (200 mg, 0.51 mmol, 1.0 eq), dioxane (150 ml), cyclopropylcarboxamide (230 mg, 2.7 mmol), Pd2(dba)3 (43 mg), DPPF (94 mg), and the atmosphere was replaced with nitrogen three times. The mixture was stirred for 5 mins, and Cs2CO3 (355 mg, 1.08 mmol) was added. The atmosphere was replaced with nitrogen three times and the mixture was reacted at 130 °C overnight. The raw material was not completely reacted by TLC. The mixture was concentrated and subjected to column chromatography to obtain compound 6 (55 mg, 0.13 mmol, yield 25.5%) as a white powder. MS-ESI:[M+1]+=419.2.1H NMR(400MHz,d6-DMSO):11.42(s,1H),10.86(s,1H),8.93(s,1H),8.5-8.54(m,2H),8.17- 8.2(m,1H),7.22-7.26(m,1H),3.0(m,1H),2.0(m,1H),1.2(m,2H),1.0(m,2H),0.8(m,4H).

[0182] Test Example 1

[0183] FACS detection of the inhibitory effect of compounds on pSTAT5 expression in CD3+ cells

[0184] 1 Experimental Materials and Instruments

[0185] 1.1 Experimental Reagents

[0186] DMSO, Sigma, Cat# D2650-100ml, stored at room temperature.

[0187] Perm buffer III, BD Biosciences, Cat#558050, stored at 4°C.

[0188] Lysis / Fixation Buffer, BD Biosciences, Cat# 558049, stored at room temperature.

[0189] EDTA, Invitrogen, Cat#15575-038, stored at room temperature.

[0190] PBS, Hydrone, Cat#SH30256.01, stored at 4°C.

[0191] PE mouse anti-human CD3, BD Biosciences, Cat#555333, stored at 4℃.

[0192] Mouse anti-human phosphorylated STAT5 (pY694) (Alexa 647 conjugate), BD Biosciences, Cat#562076, stored at 4°C.

[0193] IFN-α, Biolegend, Cat#592702

[0194] 1.2 Experimental consumables

[0195] Microplate, 96 wells, PP, v-bottom, Greiner, Cat# GN651201-100EA.

[0196] 5 ml polystyrene round bottom tube, FALCON, Cat# 04318011.

[0197] 96 square well storage plate, Thermo, Cat# AB-0661.

[0198] · 96-well plate, Corning, Cat#3599.

[0199] 1.3 Instruments

[0200] CO2 cell incubator: MCO-15AC (Thermo)

[0201] Pipette: 0.2-10μl, 20-200μl, 200-1000μl (Thermo)

[0202] Multichannel pipette: 0.2-10μl, 5-50μl, 20-300μl (Raining)

[0203] Centrifuge: Thermo Centrifuge ST 40R; Thermo LEGEND Micro 21R

[0204] Water system: Millipore Milli-Q Reference system

[0205] Freezer: Haier ultra-low temperature freezer

[0206] Haier 4 degree refrigerator

[0207] Haier -20 degrees freezer

[0208] Eddy current: EARTH REQUIRED

[0209] Plate making machine: Qilin Beier; MH-2

[0210] Flow cytometer: BD FACSVerseTM flow cytometer

[0211] 2 Experimental methods

[0212] 2.1 Compound dilution

[0213] 1) On the day of the experiment, the compound was prepared into a 10 mM solution in DMSO, diluted to 1.5 mM in DMSO, and then diluted 3-fold to 8 gradient concentrations.

[0214] 2) Transfer 5 μl of the diluted compound to 120 μl of DPBS solution containing 0.1% BSA.

[0215] 3) Positive and negative control groups were set up, and 0.5% DMSO was added to the positive and negative control groups.

[0216] 2.2 Experimental process

[0217] 1) Add 67.5ul of fresh human whole blood into a 96-well cell culture plate.

[0218] 2) Add 3.5ul of diluted compound and mix well.

[0219] 3) Incubate in a 37°C incubator for 60 minutes.

[0220] 4) Dilute IFN-α to 400 ng / ml in DPBS containing 0.1% BSA, and dilute PE-anti-hCD3 antibody to 5 times in DPBS containing 0.1% BSA. After the above 60-minute incubation, add 5ul of diluted PE-anti-hCD3 antibody to each well, and add 4ul of diluted IFN-α to each well, that is, the final concentration of IFN-α in each well is 20 ng / ml.

[0221] 5) Incubate in a 37°C incubator for 15 minutes.

[0222] 6) Transfer all cells to a 96-well deep-well plate and add 1 ml of lysis / fixation buffer preheated at 37°C.

[0223] 7) Incubate at 37°C in the dark for 10 minutes.

[0224] 8) Centrifuge at 600 g for 5 minutes, discard the supernatant, add 1 ml PBS, wash twice and centrifuge again.

[0225] 9) Add 1 ml of Perm buffer III to the cell pellet.

[0226] 10) Incubate at 4°C in the dark for 30 minutes.

[0227] 11) Centrifuge at 600 g for 5 minutes, discard the supernatant, add 1 ml PBS, wash twice and centrifuge again.

[0228] 12) Dilute APC anti-human pSTAT5 antibody in staining buffer, add 100ul per well to the cell wells, and mix well.

[0229] 13) Incubate at room temperature for 40 minutes.

[0230] 14) Wash twice with staining buffer (1 ml per well) and centrifuge at 600 g for 5 minutes.

[0231] 15) Discard the supernatant and resuspend the cell pellet in 300ul staining buffer.

[0232] 16) Load the sample into the flow cytometer for analysis. The IC50 of the sample to be tested is obtained by calculation. The results are shown in Table 1.

[0233] Table 1

[0234] Compound No. IFN-α / pSTAT5 IC50(nM) 1 2800 2 867 3 64 4 168 5 2111 6 128

[0235] Test Example 2

[0236] Comparison of JAK family selectivity

[0237] 1. Experimental Methods

[0238] 1.1. Pseudokinase (JH2) experimental operation is as follows:

[0239] 1.1.1. Dissolve compounds in DMSO to a stock concentration of 10 mM.

[0240] 1.1.2. Prepare a compound dilution plate with a compound concentration 200 times the final concentration, dilute from the highest concentration point using the 27-fold dilution method, for a total of 4 concentration points, and transfer to the Echo plate.

[0241] 1.1.3. Pulse the compounds from the Echo plate to a 384-well assay plate using an Echo instrument, so that the compounds become a 3-fold dilution matrix with 11 concentration points.

[0242] 1.1.4. Add 5ul of TYK2-JH2 or JAK1-JH2 pseudokinase at 3 times the final concentration to a 384-well assay plate.

[0243] 1.1.5. Add 5ul of Tb at 3 times the final concentration to a 384-well assay plate.

[0244] 1.1.6. Add 5ul of Tracer at 3 times the final concentration to a 384-well assay plate.

[0245] 1.1.7. Centrifuge for 30 seconds and incubate at room temperature for 60 minutes.

[0246] 1.1.8. The fluorescence ratio at 520 nm and 495 nm was read using Envision microplate reader (PerkinElmer).

[0247] 1.2. Kinase (JH1) experiment operation is as follows:

[0248] 1.2.1. Dissolve compounds in DMSO to a stock concentration of 10 mM.

[0249] 1.2.2. Prepare a compound dilution plate with a compound concentration 100 times the final concentration, dilute from the highest concentration point using the 27-fold dilution method, for a total of 4 concentration points, and transfer to the Echo plate.

[0250] 1.2.3. Pulse the compounds from the Echo plate to the 384 assay plate using the Echo instrument, so that the compounds become a 3-fold dilution matrix with 11 concentration points.

[0251] 1.2.4. Prepare kinase working solution at 2 times the final concentration and add 5ul to each well of a 384-well assay plate. Incubate the compound and kinase at room temperature for 15 minutes.

[0252] 1.2.5. Add 5ul of substrate (containing ATP) at twice the final concentration into a 384-well plate.

[0253] 1.2.6. Incubate at room temperature for 45 minutes.

[0254] 1.2.7. Add the detection reagent mixture to the 384-well plate, centrifuge for 30 seconds, and incubate at room temperature for 60 minutes.

[0255] 1.2.8. The fluorescence ratio at 665 nm and 615 nm was read using Envision microplate reader (PerkinElmer).

[0256] Data Analysis

[0257] 1.3.1. XL-Fit software was used for data analysis and IC50 of compounds was calculated. See Table 2.

[0258] Table 2

[0259] Compound No. TYK2(JH1) TYK2(JH2) JAK1(JH1) JAK1(JH2) JAK2(JH1) JAK3(JH1) 3 4313.5nM 0.32nM 3185.5nM 53.1nM 2812.9nM 2378.3nM <![CDATA[IC50 其他 / IC50 TYK2-JH2 ]]> 13480 1 9955 166 8790 7432

[0260] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A heterocyclic compound or a pharmaceutically acceptable salt thereof, characterized in that: The compound is shown in formula II in, X is N; Y is CH; Z is CR6 or N; R1 and R2 are each independently selected from the following group: H, substituted or unsubstituted C1-6 alkyl; or, R1 and R2 and the carbon atom connected thereto together form a substituted or unsubstituted C 3-6 Cycloalkyl; R6 is selected from the group consisting of H, substituted or unsubstituted C 1-4 alkyl; Furthermore, the substitution refers to the substitution of one or more hydrogen atoms in the group by a substituent selected from the group consisting of halogen, C 1-4 Alkyl, C 1-4 Halogenated alkyl.

2. The heterocyclic compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: R6 is selected from the following group: H, C 1-4 alkyl; R1 and R2 are each independently selected from the following group: H, C1-6 alkyl; or, R1 and R2 and the carbon atom connected thereto together form a substituted or unsubstituted C 3-6 Cycloalkyl; wherein the substitution refers to one or more hydrogen atoms in the group being replaced by a substituent selected from the group consisting of: C 1-4 alkyl.

3. The heterocyclic compound or pharmaceutically acceptable salt thereof according to claim 1 or 2, characterized in that: Z is N.

4. The heterocyclic compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The heterocyclic compound is a compound selected from the following table: Table A 5. The heterocyclic compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The heterocyclic compound is:

6. An intermediate for preparing the heterocyclic compound according to claim 1, characterized in that: The intermediate is shown in Formula IV: Among them, R X is halogen; X, Y and Z are as defined in claim 1.

7. A pharmaceutical composition, characterized in that include: (i) the heterocyclic compound according to claim 1 or a pharmaceutically acceptable salt thereof, and (ii) a pharmaceutically acceptable carrier or excipient.

8. Use of the heterocyclic compound according to claim 1 or a pharmaceutically acceptable salt thereof in the preparation of (i) a medicament for treating or preventing a TYK2-JH2-mediated disease and / or (ii) a selective TYK2-JH2 inhibitor.

9. The use according to claim 8, characterized in that The TYK2-JH2 mediated disease is selected from the group consisting of inflammation, autoimmune disease, or a combination thereof.

10. An in vitro non-therapeutic method for selectively inhibiting TYK2-JH2, characterized in that: The method comprises: The subject is contacted with the heterocyclic compound or a pharmaceutically acceptable salt thereof as claimed in claim 1, thereby inhibiting TYK2-JH2 activity in the subject.

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