Heterocyclic jak inhibitors

Abstract

The present application relates to heterocyclic compounds or pharmaceutically acceptable salts thereof as Janus kinase (JAK) inhibitors. In particular, the present application relates to a compound of general formula (I) or a pharmaceutically acceptable salt thereof. The present application also relates to a preparation method of the compound or the pharmaceutically acceptable salt thereof. The compound of the present application can be used for treating and / or preventing JAK-mediated related diseases, particularly inflammatory diseases, autoimmune diseases and cancers. In the general formula (I), each substituent is the same as defined in the specification.

Description

TECHNICAL FIELD

[0001] The present application relates to a kind of heterocyclic compounds or its pharmaceutically acceptable salt for regulating or inhibiting Janus kinase (JAK) activity.The present application also relates to the preparation method of the compound or its pharmaceutically acceptable salt.The present application further relates to the use and method of using of the compound or its pharmaceutically acceptable salt in the treatment and / or prevention of inflammatory diseases, autoimmune diseases and cancer. BACKGROUND

[0002] Janus kinase (JAK) is a kind of non-receptor tyrosine protein kinase, by four family members, namely JAK1, JAK2, JAK3 and TYK2.JAK has 7 homologous domains (JH) in structure, wherein JH1 domain is kinase region, JH2 domain is pseudo kinase region (regulates the kinase catalytic activity of JH1), JH6 and JH7 are receptor binding regions.When the cell surface region of Cytokine Receptor is combined with Cytokine, it causes the JAK combined with its intracellular region to be phosphorylated, thereby creating the parking site for signal transduction and transcription activator (STAT) protein, so that STAT protein is activated JAK phosphorylation, forms dimer, enters the nucleus, regulates the expression and transcription of related genes, realizes the signal transduction from cell membrane to nucleus (Lionard et al., Ann.Rev.Immunol.1998, 16, 293-322).Therefore, JAK transduces Cytokine-mediated signal through JAK-STAT pathway, plays an important role in many cell functions such as Cytokine-dependent regulation of cell proliferation, differentiation, apoptosis and immune response, and is one of the popular targets for treating inflammatory diseases, autoimmune diseases and cancer (Alicea-Velazquez et al., Curr.Drug Targets 2011, 12, 546-55).At present, several JAK inhibitor drugs have been approved for marketing, including JAK1 / JAK2 inhibitor ruxolitinib and JAK2 inhibitor fedratinib for treating myelofibrosis, pan-JAK inhibitor tofacitinib for treating rheumatoid arthritis, JAK1 / JAK2 inhibitor baricitinib, pan-JAK inhibitor peficitinib and JAK1 inhibitor upadacitinib, etc.

[0003] JAK and STAT play a highly specific role in the control of different immune responses. One JAK kinase can be involved in the signal transduction process of multiple cytokines, and one cytokine signal pathway can also activate multiple JAK kinases, but the activated STAT proteins have certain selectivity for cytokines, for example, interleukin (IL)-4 activates STAT1 / 3 / 5 / 6, while IL-12 specifically activates STAT4. JAK1, JAK2 and TYK2 are widely present in various tissues and cells, and JAK1 is closely related to the activation of inflammatory factors such as IL-6 and interferon (IFN). Therefore, JAK1 selective inhibitors are considered to have potential therapeutic effects on the treatment of autoimmune diseases such as rheumatoid arthritis (RA) and psoriasis. JAK2 can mediate the signal transduction of cytokines such as erythropoietin (EPO) and thrombopoietin (TPO) alone (Won et al., BMC Bioinformatics 2009, 10, S53), and is closely related to the proliferation and differentiation of blood cells. TYK2 is involved in the signal transduction of inflammatory cytokines such as interferon (IFN), IL-12 and IL-23, and plays a key role in innate immunity and adaptive immunity, so TYK2 as a target for autoimmune diseases has also attracted great attention, such as TYK2 inhibitors for the treatment of psoriasis, systemic lupus erythematosus (SLE) and inflammatory bowel disease (IBD) and the like. JAK3 only exists in the bone marrow and lymphatic system, and mediates the signal transduction of IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21, which play an important role in inducing the proliferation and differentiation of T cells, activating B cells to produce antibodies, activating macrophages, enhancing the activity of natural killer cells (NK cells) and inducing other cytokines such as IFN, so JAK3 selective inhibitors are expected to have important effects on the treatment of organ transplantation, autoimmune diseases, inflammatory pneumonia and the like.

[0004] Inflammatory bowel disease (IBD) is a common chronic intestinal inflammatory disease, including Crohn's disease (CD), ulcerative colitis (UC) and indeterminate IBD (IBDU). Inflammatory bowel disease affects 5 million people worldwide, and the prevalence is increasing year by year. The clinical symptoms are diarrhea, hematochezia, abdominal pain, fatigue, high fever, etc. The commonly used treatment drugs include 5-aminosalicylic acid (5-ASA), glucocorticoids, immunosuppressants (such as azathioprine) and biological agents (such as anti-TNF, IL-12 / IL-23 monoclonal antibodies) and the like, but many patients receiving treatment do not get relief, and as high as 80% of Crohn's disease patients and 30% of UC patients eventually need to undergo surgery. There is still a huge unmet medical need in this field, and more effective and safe drugs are needed.

[0005] During the pathogenesis of the disease, the expression of proinflammatory cytokines such as IL-13 and IL-17 is increased, thereby inducing inflammation through the JAK-STAT pathway. JAK inhibitors, as a new class of oral small molecule drugs, have potential use in the treatment of CD and UC, among which tofacitinib has been approved for the treatment of UC in several countries, but the non-selective inhibition of JAK1 / 2 / 3 by tofacitinib causes more serious side effects, such as severe infection and the initiation of malignancy. However, due to the high sequence similarity of the catalytic active sites of the JAK kinase family members, it is quite difficult to design an oral selective JAK inhibitor with a therapeutic systemic exposure. Therefore, the development of new JAK inhibitors, which only enrich the local exposure required for treatment at the action site (such as the colon, skin, etc.), but have very low blood solubility to avoid systemic adverse reactions, can not only exert the efficacy but also improve the safety. TD-1473 developed by Theravance company is a pan-JAK inhibitor with intestinal local absorption, which has entered clinical phase III trials and shown good tolerability. SUMMARY

[0006] DEFINITIONS

[0007] Unless otherwise indicated, the following terms have the meanings set forth below in the specification and claims.

[0008] “C x-y ” denotes a range of carbon atoms, wherein x and y are both integers, for example C 3-8 Cycloalkyl denotes a cycloalkyl group having 3 to 8 carbon atoms, i.e. a cycloalkyl group having 3, 4, 5, 6, 7 or 8 carbon atoms. It is also to be understood that “C 3-8 ” also encompasses any sub-range therein, for example C 3-7 , C 3-6 , C 4-7 , C 4-6 , C 5-6 , etc.

[0009] “Alkyl” refers to a saturated straight or branched chain hydrocarbon group containing one to twenty carbon atoms, for example, one to eight carbon atoms, one to six carbon atoms, or one to four carbon atoms. Non-limiting examples of alkyl groups include methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, and the like.

[0010] "Cycloalkyl" refers to saturated cyclic hydrocarbyl substituents containing from 3 to 14 carbon ring atoms. Cycloalkyl groups can be monocarbocyclic, typically containing from 3 to 8, 3 to 7, or 3 to 6 carbon ring atoms. Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and the like. Cycloalkyl groups can also be bicyclic or tricyclic fused, bridged, or spiro together, such as decalinyl, bicyclo[2.2.2]octane, spiro[3.3]heptane, and the like.

[0011] "Heterocyclyl or heterocycle" refers to saturated or partially unsaturated monocyclic or polycyclic ring systems, including from 3 to 20 ring atoms, for example, from 3 to 14, 3 to 12, 3 to 10, 3 to 8, 3 to 6, or 5 to 6 ring atoms, wherein one or more of the ring atoms are selected from nitrogen, oxygen, or S(O) m (wherein m is an integer from 0 to 2), but excluding -O-O-, -O-S-, or -S-S- ring members, with the remaining ring atoms being carbon. Preferred include from 3 to 12 ring atoms, more preferred from 3 to 10 ring atoms, more preferred from 4 to 7 ring atoms, more preferred from 4 to 6 ring atoms, most preferred 5 or 6 ring atoms, wherein from 1 to 4 are heteroatoms, more preferred from 1 to 3 are heteroatoms, most preferred from 1 to 2 are heteroatoms. Non-limiting examples of monocyclic heterocyclyl groups include pyrrolidinyl, oxetanyl, piperidinyl, piperazinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydrothiopyranyl, morpholinyl, thiomorpholinyl, homopiperazinyl, azetidinyl, and the like. Polycyclic heterocyclyl groups include fused, bridged, or spiro polycyclic heterocyclyl groups, such as octahydrocyclopenta[c]pyrrole, octahydropyrrolo[l,2-a]pyrazine, 3,8-diazabicyclo[3.2.1]octane, 5-azaspiro[2.4]heptane, 2-oxa-7-azaspiro[3.5]nonane, and the like.

[0012] "Aryl or arycyclic" refers to aromatic monocyclic or fused polycyclic groups containing from 6 to 14 carbon atoms, preferably from 6 to 10 members, for example, phenyl and naphthyl, most preferably phenyl. The arycyclic ring can be fused to a heteroaryl, heterocyclyl, or cycloalkyl ring, wherein the ring that is attached to the parent structure is the arycyclic ring, non-limiting examples include:

[0013]

[0014] and the like.

[0015] "Heteroaryl or heteroaromatic ring" means a heteroaromatic system containing 5 to 14 ring atoms, of which 1 to 4 ring atoms are selected from heteroatoms including oxygen, sulfur, and nitrogen. Heteroaryl is preferably 5 to 10 membered, more preferably the heteroaryl is 5 membered or 6 membered, for example furanyl, thienyl, pyridyl, pyrrolyl, pyrimidinyl, pyrazinyl, pyrazolyl, imidazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, quinolinyl, isoquinolinyl, indolyl, isoindolyl, and the like. The heteroaryl ring can be fused to an aryl, heterocyclyl, or cycloalkyl ring, where the ring that is attached to the parent structure is the heteroaryl ring, non-limiting examples include:

[0016]

[0017] and the like.

[0018] "Halo" means fluoro, chloro, bromo, or iodo.

[0019] "Cyano" means -CN.

[0020] "Optional" means that the event or circumstance subsequently described can or can not occur, and this phrase contemplates the event or circumstance occurring or not occurring. For example, "heterocyclyl optionally substituted with alkyl" means that alkyl can or can not be present, and this phrase contemplates the heterocyclyl being substituted with alkyl and the heterocyclyl not being substituted with alkyl.

[0021] "Substituted" means that one or more hydrogen atoms, preferably 5, more preferably 1 to 3 hydrogen atoms, of a group are each, independently of one another, replaced with a corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, which can or cannot be possible (experimentally or theoretically) as determined by one of skill in the art without undue effort. For example, an amino or hydroxyl group with a free hydrogen can not be stable when bound to a carbon atom with an unsaturated (e.g., olefinic) bond. The substituents include, but are not limited to, halo, cyano, nitro, oxo, -SF5, C 1-4 alkyl, C 3-7 cycloalkyl, 4-7 membered heterocyclyl, phenyl, 5-6 membered heteroaryl, and the like.

[0022] "Isomers" means compounds having the same molecular formula but different physical structures or spatial arrangements of their atoms. Isomers that differ in their spatial arrangement of atoms are termed "stereoisomers." Stereoisomers include optical isomers, geometric isomers, and conformational isomers.

[0023] The compounds of this invention can exist as optical isomers. Optical isomers include enantiomers and diastereomers. Enantiomers are two stereoisomers that are mirror images of each other but cannot overlap. Racemic mixtures or racemates are mixtures in which the number of left- and right-handed enantiomers of a chiral molecule is equal. Diastereomers are two stereoisomers that are not mirror images of each other and cannot overlap. When an optical isomer is a single isomer and its absolute configuration is determined, it is an "R" or "S" absolute configuration based on the configuration of the substituents on the chiral carbon atom; when the absolute configuration of an optical isomer is not determined, it is (+) or (-) based on the measured optical rotation value. Methods for preparing and separating optical isomers are known in the art.

[0024] The compounds of this invention may also exist as geometric isomers. This invention considers various geometric isomers and mixtures thereof resulting from the distribution of substituents around carbon-carbon double bonds, carbon-nitrogen double bonds, cycloalkyl groups, or heterocyclic groups. Substituents around carbon-carbon double bonds or carbon-nitrogen bonds are designated as Z or E configurations, and substituents around cycloalkyl or heterocyclic groups are designated as cis or trans configurations.

[0025] The compounds of the present invention may also exhibit tautomerism, such as keto-enol tautomerism.

[0026] It should be understood that the present invention includes any tautomer or stereoisomer form and mixtures thereof, and is not limited to any one of the tautomer or stereoisomer forms used in the naming or chemical structural formula of the compound.

[0027] "Isotope" refers to all isotopes of atoms appearing in the compounds of this invention. Isotopes include atoms having the same atomic number but different mass numbers. Examples of isotopes suitable for inclusion in the compounds of this invention are hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, and chlorine, respectively, for example, but not limited to, [examples of isotopes]. 2 H(D), 3 H, 13 C 14 C 15 N、 17 O、 18 O、 31 P, 32 P, 35 S, 18 F and 36Cl. The isotope-labeled compounds of the present invention can generally be prepared by conventional techniques known to those skilled in the art or by methods similar to those described in the appended examples, using appropriate isotope-labeled reagents instead of non-isotope-labeled reagents. Such compounds have a variety of potential uses, for example, as standards and reagents in the determination of biological activity. In the case of stable isotopes, such compounds have the potential to advantageously alter biological, pharmacological, or pharmacokinetic properties. Deuterium (D) is a preferred isotope of the present invention, and hydrogen in, for example, methyl, methylene, or methine groups can be substituted with deuterium.

[0028] The compounds of the present invention can be administered in the form of prodrugs. A "prodrug" refers to a derivative which is converted into the bioactive compound of the present invention under physiological conditions in vivo, such as through oxidation, reduction, hydrolysis, etc. (each using an enzyme or without enzyme involvement). Examples of prodrugs include compounds in which the amino group is acylated, alkylated, or phosphorylated, for example, eicosanoylamino, alanylamino, neopentanoyloxymethylamino; or in which the hydroxyl group is acylated, alkylated, phosphorylated, or converted to a borate, for example, acetoxy, palmitoyloxy, neopentanoyloxy, succinoxy, fumaroyloxy, alanyloxy; or in which the carboxyl group is esterified or amidated; or in which the thiol group forms a disulfide bridge with a carrier molecule, such as a peptide, that selectively delivers drugs to a target and / or to the cytosol of cells. These compounds can be prepared from the compounds of the present invention according to known methods.

[0029] "Pharmaceutical-grade salt" or "pharmaceutically acceptable salt" refers to a salt made from a pharmaceutically acceptable base or acid, including inorganic bases or acids and organic bases or acids. In the case of compounds of the present invention containing one or more acidic or basic groups, the present invention also includes their corresponding pharmaceutically-grade salts. Thus, compounds of the present invention containing acidic groups can exist in salt form and can be used according to the present invention, for example, as alkali metal salts, alkaline earth metal salts, or as ammonium salts. More precise examples of such salts include sodium salts, potassium salts, calcium salts, magnesium salts, or salts formed with ammonia or organic amines, such as ethylamine, ethanolamine, triethanolamine, or amino acids. Compounds of the present invention containing basic groups can exist in salt form and can be used according to the present invention as addition salts of themselves with inorganic or organic acids. Examples of suitable acids include hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, nitric acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, oxalic acid, acetic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, formic acid, propionic acid, pentylamino acid, malonic acid, succinic acid, pimelic acid, fumaric acid, maleic acid, malic acid, aminosulfonic acid, phenylpropionic acid, gluconic acid, ascorbic acid, isonicotinic acid, citric acid, adipic acid, and other acids known to those skilled in the art. If the compounds of the present invention contain both acidic and basic groups in the molecule, the present invention also includes internal salts or internal ammonium salts in addition to the salt forms mentioned. Each salt can be obtained by those skilled in the art from known conventional methods, for example by contacting the compounds of the present invention with an organic or inorganic acid or base in a solvent or dispersant, or by anion or cation exchange with other salts.

[0030] "Pharmaceutical composition" refers to a composition containing one or more of the compounds described in this invention, or their pharmaceutically acceptable salts, stable isotope derivatives, isomers, prodrugs, mixtures thereof, and other components such as pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and enabling it to exert its biological activity.

[0031] Therefore, when referring to “compound,” “compound of the present invention,” or “compound of the present invention” in this application, it includes all said compound forms, such as pharmaceutically usable salts, stable isotope derivatives, isomers, prodrugs, and mixtures thereof.

[0032] In this article, the term "cancer / tumor" includes, but is not limited to, cancers of the digestive tract / gastrointestinal tract, colon cancer, liver cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, skin cancer, lymphoma, leukemia (including acute myeloid leukemia and chronic myeloid leukemia), kidney cancer, lung cancer, muscle cancer, bone cancer, bladder cancer, brain cancer, melanoma, multiple myeloma, and angiogenesis-related conditions / tumors.

[0033] In this article, the term "inflammatory disease or autoimmune disease" includes, but is not limited to, arthritis, Hashimoto's thyroiditis, autoimmune hemolytic anemia, autoimmune atrophic gastritis with pernicious anemia, autoimmune encephalomyelitis, autoimmune orchitis, Gudpassuia disease, autoimmune thrombocytopenic purpura, sympathetic ophthalmia, myasthenia gravis, Graves' disease, primary biliary cirrhosis, hepatitis, primary sclerosing cholangitis, chronic invasive hepatitis, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, ulcerative colitis, membranous glomerulonephritis, systemic lupus erythematosus, rheumatoid arthritis, psoriatic arthritis, Sjögren's syndrome, Reiter's syndrome, polymyositis, dermatomyositis, type I interferon diseases including Aicardi-Goutières syndrome and other systemic sclerosis that overexpress type I interferon, Mendelian diseases, polyarteritis nodosa, multiple sclerosis, and relapsing-remitting multiple sclerosis. Primary progressive multiple sclerosis, secondary progressive multiple sclerosis, bullous pemphigoid, intestinal inflammation such as Crohn's disease, ulcerative colitis, inflammatory bowel disease, celiac disease, proctitis, eosinophilic gastroenteritis, mastocytosis, skin diseases such as atopic dermatitis, eczema, psoriasis, scleroderma, pruritus or other pruritus symptoms, vitiligo, alopecia, and autoimmune diseases based on O-cell (humoral) or T-cells, including Kogan syndrome, ankylosing spondylitis. Inflammation, Wegener's granulomatosis, autoimmune alopecia, type I or juvenile diabetes, thyroiditis, etc.; allergic reactions such as allergic dermatitis, summer eczema, hoof pruritus, spasms, inflammatory airway diseases, recurrent airway obstruction, airway hyperresponsiveness, chronic obstructive pulmonary disease, etc.; asthma and other obstructive airway diseases including but not limited to chronic or excessive asthma, late-onset asthma, bronchitis, bronchial asthma, allergic asthma, endogenous asthma, extrinsic asthma, dust asthma.

[0034] In this document, the term "therapeuticly effective amount" refers to the amount of the compound of the present invention that can effectively treat or prevent the disease.

[0035] In this article, the term "patient" refers to mammals, especially humans.

[0036] This invention provides a compound of general formula (I) as a JAK inhibitor, or a pharmaceutically acceptable salt, stable isotope derivative, isomer, or prodrug thereof:

[0037]

[0038] in:

[0039] Bond 'a' can be a single bond or a double bond;

[0040] R 1 and R 2 Each is independently selected from H, D, CN, and C.1-6 Alkyl or C 3-6 Cycloalkyl, wherein one or more hydrogen atoms of the alkyl group are optionally replaced by D or fluorine;

[0041] A is C 3-10 Cycloalkyl, 4-10 membered heterocyclic groups, C 6-10 aryl or 5-10-membered heteroaryl, wherein the cycloalkyl, heterocyclic, aryl, and heteroaryl groups are optionally selected from one or more groups selected from D, halogen, cyano, -OR. a -NR a R b -C(O)R a -C(O)NR a R b -S(O)2R a -P(O)(CH3)2, C 1-6 Alkyl, C 3-6 The alkyl, 4-8-membered heterocyclic, or 5-6-membered heteroaryl groups are substituted; B is a phenyl or 5-6-membered heteroaryl group, wherein the phenyl and heteroaryl groups are optionally replaced by one or more substituents selected from D, halogen, cyano, -OR. a -NR a R b -COOR a -C(O)R a -NR a C(O)R b -C(O)NR a R b -S(O)2R a -S(O)2NR a R b -S(O)(NR) a )R b -P(O)(CH3)2, C 1-6 Alkyl, C 3-6 The alkyl, cycloalkyl, 4-8-membered heterocyclic, or 5-6-membered heteroaryl groups are substituted, wherein the alkyl, cycloalkyl, heterocyclic, or heteroaryl group is optionally further replaced by one or more substituents selected from D, halogen, CN, -OH, -NH2, C. 1-6 Alkyl, -OC 1-6 Alkyl, -COOR a -C(O)R a or -C(O)NR a R b The substituents are replaced;

[0042] R a and R b Each is independently selected from H and C. 1-6 Alkyl, C 3-6Cycloalkyl or 4-8 membered heterocyclic group, wherein the alkyl, cycloalkyl and heterocyclic group is optionally selected from one or more elements selected from D, halogen, C. 1-6 Alkyl groups are substituted.

[0043] In a preferred embodiment, R 1 and R 2 Both are H.

[0044] In one implementation, A is C 3-8 Cycloalkyl, 4-8 membered heterocyclic groups, C 6-10 aryl or 5-6-membered heteroaryl, wherein the cycloalkyl, heterocyclic, aryl, and heteroaryl groups are optionally selected from one or more groups selected from D, halogen, cyano, -OR a -NR a R b -C(O)R a -C(O)NR a R b -S(O)2R a C 1-6 Alkyl, C 3-6 Substituents include cycloalkyl, 4-8 membered heterocyclic or 5-6 membered heteroaryl groups.

[0045] In a preferred embodiment, A is C 3-8 Cycloalkyl, 4-8-membered heterocyclic, phenyl, or 5-6-membered heteroaryl, wherein the cycloalkyl, heterocyclic, phenyl, and heteroaryl groups are optionally selected from one or more halogens, -C(O)R a C 1-6 Alkyl and C 3-6 Substituents of cycloalkyl groups.

[0046] In a more preferred embodiment, A is a phenyl group, wherein one or more hydrogen atoms of the phenyl group are optionally replaced by halogens.

[0047] In one embodiment, B is a phenyl or a 5-6-membered heteroaryl group, wherein the phenyl and heteroaryl group is optionally selected from one or more halogens, -COOR a -C(O)R a -C(O)NR a R b -S(O)2R a C 1-6 Alkyl, C 3-6 The alkyl group is replaced by a substituent of a cycloalkyl group, a 4-8 membered heterocyclic group containing N, S, and / or O heteroatoms, wherein the alkyl group, cycloalkyl group, and heterocyclic group are optionally further replaced by one or more substituents selected from C10. 1-6 Alkyl, -C(O)R a and -C(O)NR a R bThe substituents are replaced by the substituents.

[0048] In one implementation, R a and R b Each is independently selected from H and C. 1-6 Alkyl, C 3-6 Cycloalkyl or 4-6 membered heterocyclic groups containing N, S and / or O heteroatoms, wherein one or more hydrogens of the alkyl, cycloalkyl and heterocyclic groups are optionally C-terminated. 1-6 Alkyl groups are substituted.

[0049] In some embodiments, compounds of general formula (I) are shown as those of general formula (II):

[0050]

[0051] in:

[0052] A is C 3-8 Cycloalkyl, 4-8-membered heterocyclic, phenyl, or 5-6-membered heteroaryl, wherein the cycloalkyl, heterocyclic, phenyl, and heteroaryl groups are optionally selected from one or more halogens, -C(O)R a C 1-6 Alkyl and C 3-6 Substituents of cycloalkyl groups.

[0053] In a preferred embodiment, A is a phenyl group, wherein one or more hydrogen atoms of the phenyl group are optionally replaced by halogens.

[0054] B is a phenyl or a 5-6-membered heteroaryl group, wherein the phenyl and heteroaryl group are optionally selected from one or more halogens, -COOR a -C(O)R a -C(O)NR a R b -S(O)2R a C 1-6 Alkyl, C 3-6 The alkyl group is replaced by a substituent of a cycloalkyl group, a 4-8 membered heterocyclic group containing N, S, and / or O heteroatoms, wherein the alkyl group, cycloalkyl group, and heterocyclic group are optionally further replaced by one or more substituents selected from C10. 1-6 Alkyl, -C(O)R a and -C(O)NR a R b The substituents are replaced by the substituents.

[0055] R a and R b Each is independently selected from H and C. 1-6 Alkyl, C 3-6 Cycloalkyl or 4-6 membered heterocyclic groups containing N, S and / or O heteroatoms, wherein one or more hydrogens of the alkyl, cycloalkyl and heterocyclic groups are optionally C-terminated.1-6 Alkyl groups are substituted.

[0056] In other embodiments, compounds of general formula (I) are shown as those of general formula (III):

[0057]

[0058] in:

[0059] A is a phenyl group, wherein one or more hydrogen atoms of the phenyl group are optionally replaced by halogens.

[0060] B is a phenyl or a 5-6-membered heteroaryl group, wherein the phenyl and heteroaryl group are optionally selected from one or more halogens, -COOR a -C(O)R a -C(O)NR a R b -S(O)2R a C 1-6 Alkyl, C 3-6 The alkyl group is replaced by a substituent of a cycloalkyl group, a 4-8 membered heterocyclic group containing N, S, and / or O heteroatoms, wherein the alkyl group, cycloalkyl group, and heterocyclic group are optionally further replaced by one or more substituents selected from C10. 1-6 Alkyl, -C(O)R a and -C(O)NR a R b The substituents are replaced by the substituents.

[0061] R a and R b Each is independently selected from H and C. 1-6 Alkyl, C 3-6 Cycloalkyl or 4-6 membered heterocyclic groups containing N, S and / or O heteroatoms, wherein one or more hydrogens of the alkyl, cycloalkyl and heterocyclic groups are optionally C-terminated. 1-6 Alkyl groups are substituted.

[0062] This invention also relates to compounds 1-40, or pharmaceutically acceptable salts, stable isotope derivatives, isomers, prodrugs, and mixtures thereof.

[0063]

[0064]

[0065]

[0066]

[0067]

[0068]

[0069]

[0070]

[0071] The compounds of the present invention can effectively inhibit the activity of JAK, preferably with an IC50 concentration. 50 Less than 100 nM, preferably its IC 50 Less than 10 nM.

[0072] The present invention also relates to a pharmaceutical composition comprising a compound of general formula (I) or a pharmaceutically acceptable salt thereof, a stable isotope derivative thereof, an isomer thereof, a prodrug thereof, and one or more pharmaceutically acceptable carriers or excipients.

[0073] Another aspect of the present invention provides a method for treating or preventing JAK-mediated diseases, the method comprising administering to a patient in need a therapeutically effective amount of a compound of general formula (I) or a pharmaceutically acceptable salt, stable isotope derivative, isomer, prodrug, or mixture thereof, or a pharmaceutical composition comprising said compound; said diseases include, but are not limited to, inflammatory diseases including enteritis, autoimmune diseases, cancer, etc., especially inflammatory bowel disease, dermatitis, eczema, rheumatoid arthritis, systemic lupus erythematosus, psoriasis, alopecia areata, etc.

[0074] According to the present invention, the drug can be any dosage form, including but not limited to tablets, capsules, solutions, lyophilized preparations, and injections.

[0075] The pharmaceutical formulations of the present invention can be administered in dose units containing a predetermined amount of the active ingredient per dose unit. Such units may contain, for example, 0.5 mg to 1 gram, preferably 1 mg to 700 mg, and particularly preferably 5 mg to 500 mg of the compound of the present invention, depending on the condition being treated, the method of administration, and the patient's age, weight, and condition. Furthermore, this type of pharmaceutical formulation can be prepared using methods known in the pharmaceutical industry, such as mixing the active ingredient with one or more excipients and / or adjuvants.

[0076] The pharmaceutical formulations of the present invention are suitable for administration by any desired and appropriate method, such as oral (including oral cavity or sublingual), rectal, nasal, local (including oral cavity, sublingual or percutaneous), vaginal or parenteral (including subcutaneous, intramuscular, intravenous or intradermal) administration.

[0077] This invention also provides a method for preparing the compound. The preparation of the compound of general formula (I) of this invention can be accomplished by the following exemplary methods and examples, but these methods and examples should not be considered in any way as limiting the scope of this invention. The compound of this invention can also be synthesized by synthetic techniques known to those skilled in the art, or by a combination of methods known in the art and the methods described in this invention. The product obtained from each reaction step is obtained using separation techniques known in the art, including but not limited to extraction, filtration, distillation, crystallization, chromatographic separation, etc. The starting materials and chemical reagents required for synthesis can be conventionally synthesized or purchased according to literature (available from SciFinder).

[0078] Synthesis method

[0079] The heterocyclic compounds of general formula (I) of this invention can be synthesized according to the following route: 1) Carboxylic acid A1 condenses with amine A2 to obtain A3; 2) A3 undergoes dehydration and cyclization under acidic conditions to obtain A4; 3) A4 is brominated with NBS to obtain A5; 4) A5 reacts with sodium methoxide to obtain A6; 5) A6 reacts with amine B-NH2 via a Bulkwald coupling reaction to obtain A7; 6) A7 is deprotected to obtain A8. Some functional groups FG1 and FG2 of A7 and A8 can be further derivatized to obtain various target compounds. For example, FG1 contains a protected amine, which is deprotected to obtain an amine, and the amine is further amidated or reductively amidated to obtain amides and substituted amines; another example is the ester in FG2 which is hydrolyzed by a base (e.g., LiOH) to generate an acid, which is further amidated to obtain an amide, etc.

[0080]

[0081] The heterocyclic compounds of general formula (I) of this invention can also be synthesized according to the route described below: 1) A8 is hydrogenated to obtain B1. Some functional groups FG1 and FG2 of B1 can be further derivatized to obtain various target compounds. For example, the ester in FG2 is hydrolyzed by a base (e.g., LiOH) to generate an acid, and the acid is further amidated to obtain an amide, etc.

[0082] Example

[0083] The starting materials of this invention can be synthesized according to methods known in the art, or can be purchased from chemical companies such as ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, Accela ChemBio Inc., and Beijing Coupling Chemical Co.

[0084] The structures of the compounds of this invention were determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). NMR measurements were performed using a Bruker ASCEND-400 NMR spectrometer, with solvents such as deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), or deuterated methanol (CD3OD), and tetramethylsilane (TMS) as the internal standard. Chemical shifts were expressed as 10⁻⁶. -6 (ppm) are given as the unit. MS determinations were performed using an Agilent SQD (ESI) mass spectrometer (Agilent 6120).

[0085] HPLC was performed using an Agilent 1260DAD high-performance liquid chromatograph (Poroshell 120EC-C18, 50×3.0mm, 2.7μm column) or a Waters Arc high-performance liquid chromatograph (Sunfirc C18, 150×4.6mm, 5μm column).

[0086] Unless otherwise specified in the examples, the reaction temperature is room temperature (20-30°C).

[0087] Unless otherwise specified in the examples, the reactions were carried out under an argon or nitrogen atmosphere. An argon or nitrogen atmosphere refers to a reaction flask connected to an approximately 1L volume argon or nitrogen balloon.

[0088] A hydrogen atmosphere refers to a reaction flask that has been evacuated and then refilled with hydrogen (repeated 3 times) and then connected to a hydrogen balloon with a volume of about 1L.

[0089] The microwave reaction used a CEM Discover-SP type microwave reactor.

[0090] The reaction process in the examples was monitored using an Agilent liquid chromatography-mass spectrometry (LC-MS) system (1260 / 6120), or thin-layer chromatography (TLC) with a silica gel plate thickness of 0.15–0.2 mm (Qingdao Ocean GF254).

[0091] The compounds were purified by column chromatography or thin-layer chromatography. For column chromatography, 200-300 mesh silica gel from Qingdao Ocean was used, and for thin-layer chromatography, GF254 silica gel plates with a thickness of 0.4-0.5 mm from Qingdao Ocean were used.

[0092] The solvent systems used for column chromatography or thin-layer chromatography typically include a) dichloromethane and methanol, b) petroleum ether and ethyl acetate, or as shown in the examples. The volume ratio of the solvent is adjusted according to the polarity of the compound, and can also be further adjusted by adding a small amount of triethylamine or other acidic or basic reagents.

[0093] The compounds were purified using a Waters mass spectrometry-guided automated preparation system (mass spectrometer detector: SQD2), eluting a reversed-phase high-pressure column (XBridge-C18, 19×150mm, 5μm) with an appropriate acetonitrile / water gradient (containing 0.1% trifluoroacetic acid or formic acid, or 0.05% ammonia) at a flow rate of 20 mL / min, depending on the compound's polarity. In some examples, after purification using the automated preparation system, 1N dilute hydrochloric acid was added, followed by solvent removal under reduced pressure to obtain the hydrochloride salt.

[0094] The abbreviation DMF stands for N,N-dimethylformamide.

[0095] The abbreviation TFA stands for trifluoroacetic acid.

[0096] The abbreviation DIPEA refers to N,N-diisopropylethylamine.

[0097] The abbreviation NBS refers to N-bromosuccinimide.

[0098] The abbreviation HATU refers to 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate.

[0099] The abbreviation XantPhos refers to 4,5-bis(diphenylphosphine)-9,9-dimethyloxanthracene.

[0100] The abbreviation Pd2(dba)3 refers to tris(dibenzylacetone)dipalladium.

[0101] The abbreviation Brettphos refers to 2-(dicyclohexylphosphine)-3,6-dimethoxy-2',4',6'-triisopropyl-1,1'-biphenyl.

[0102] intermediate 1f

[0103] 1-Bromo-3-(2,6-difluorophenyl)-8-methoxyimidazo[1,5-a]pyrazine

[0104]

[0105] first step

[0106] N-((3-chloropyrazin-2-yl)methyl)-2,6-difluorobenzamide (1c)

[0107] 2,6-Difluorobenzoic acid 1a (5 g, 31.6 mmol) was dissolved in dichloromethane (100 mL), 3 drops of DMF were added, and the mixture was cooled to 0 °C. Then, oxalyl chloride (8 g, 63.3 mmol) was added dropwise. After stirring at room temperature for 1 hour, the mixture was concentrated to dryness. The residue was dissolved in dichloromethane (10 mL) to give solution A. In another 250 mL round-bottom flask, (3-chloropyrazine-2-yl)methylamine hydrochloride 1b (5.7 g, 31.6 mmol) was placed, followed by dichloromethane (100 mL) and triethylamine (9.6 g, 94.8 mmol) and cooled to 0 °C. Then, solution A was added dropwise. After stirring the mixture at room temperature for 1 hour, it was quenched with water (50 mL). The separated organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate / dichloromethane = 1 / 1) to obtain the target product 1c (6.8 g, 76%).

[0108] MS m / z(ESI): 284[M+1]

[0109] 1 H NMR (400MHz, CDCl3) δ8.47(d,J=2.5Hz,1H),8.39-8.30(m,1H),7.44-7.37(m,2H),7.03-6.90(m,2H),4.93(d,J=4.5Hz,2H).

[0110] Step 2

[0111] 8-Chloro-3-(2,6-difluorophenyl)imidazo[1,5-a]pyrazine (1d)

[0112] Add phosphine oxychloride (18.4 g, 120 mmol) to an acetonitrile (80 mL) solution of 1c (6.8 g, 24 mmol). Heat the mixture to 90 °C and stir for 16 hours. After cooling to room temperature, add phosphine oxychloride (18.4 g, 120 mmol) again, and heat the mixture to 90 °C and stir for 28 hours. After cooling to room temperature, concentrate to dryness, add saturated sodium bicarbonate solution (50 mL), and extract with dichloromethane (2 × 100 mL). Dry the combined organic phases with anhydrous sodium sulfate, filter, concentrate the filtrate to dryness under reduced pressure, and purify the residue by silica gel column chromatography (ethyl acetate / dichloromethane = 1 / 2) to give the target product 1d (5.9 g, 92%).

[0113] MS m / z(ESI): 266[M+1]

[0114] 1H NMR (400MHz, CDCl3) δ8.10 (s, 1H), 7.60-7.50 (m, 2H), 7.44 (d, J = 5.0Hz, 1H), 7.14 (t, J = 8.1Hz, 2H).

[0115] Step 3

[0116] 1-Bromo-8-chloro-3-(2,6-difluorophenyl)imidazo[1,5-a]pyrazine (1e)

[0117] NBS (590 mg, 3.31 mmol) was added to a solution of acetonitrile (40 mL) containing 1d (880 mg, 3.31 mmol). The mixture was stirred at room temperature for 18 hours and then concentrated to dryness. The residue was dissolved in ethyl acetate (100 mL), washed with water (2 × 50 mL), and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated to dryness under reduced pressure, and the residue was subjected to silica gel column chromatography (petroleum ether / ethyl acetate = 2 / 1) to give the target product 1e (940 mg, 82%).

[0118] MS m / z(ESI): 344[M+1]

[0119] 1 H NMR (400MHz, CDCl3) δ7.57(tt,J=8.5,6.3Hz,1H),7.49-7.46(m,1H),7.41(d,J=5.0Hz,1H),7.13(t,J=8.1Hz,2H).

[0120] Step 4

[0121] 1-Bromo-3-(2,6-difluorophenyl)-8-methoxyimidazo[1,5-a]pyrazine (1f)

[0122] 1e (590 mg, 1.71 mmol) was dissolved in methanol (40 mL), cooled to 0 °C, and sodium methoxide solution (463 mg, 2.57 mmol, 30% methanol solution) was added dropwise. The resulting mixture was stirred for 2 hours, then poured into a saturated ammonium chloride solution (100 mL), and extracted with ethyl acetate (3 × 50 mL). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 1 / 1) to give the target product 1f (570 mg, 98%).

[0123] MS m / z(ESI): 340[M+1]

[0124] 1H NMR (400MHz, CDCl3) δ7.56-7.49(m,1H),7.21(dt,J=5.0,1.9Hz,1H),7.17(d,J=5.1Hz,1H),7.13-7.08(m,2H),4.18(s,3H).

[0125] The intermediates in the table below are all operated according to the experimental procedure of intermediate 1f, but different carboxylic acids are used instead of 2,6-difluorobenzoic acid 1a in the first step.

[0126]

[0127] The NMR data for intermediates 3f and 19a are as follows:

[0128]

[0129] Intermediate 33c

[0130] 2-(4-aminophenyl)-2-methylpropionamide

[0131]

[0132] first step

[0133] 2-Methyl-2-(4-nitrophenyl)propionamide (33b)

[0134] Potassium carbonate (0.58 g, 4.21 mmol) and 30% hydrogen peroxide aqueous solution (35 mL) were added to a solution of 2-methyl-2-(4-nitrophenyl)propionitrile 33a (2.00 g, 10.52 mmol) in ethanol / water (20 mL, v / v) at 0 °C and stirred at 0 °C for 0.5 h. The mixture was then heated to room temperature and stirred for 16 h. After the reaction was complete, saturated sodium sulfite solution (50 mL) was added and stirred at room temperature for 0.5 h, followed by extraction with ethyl acetate (3 × 50 mL). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane / methanol = 20 / 1) to give the target product 33b (0.76 g, 35%).

[0135] MS m / z(ESI): 209[M+1]

[0136] Step 2

[0137] 2-(4-Aminophenyl)-2-methylpropionamide (33c)

[0138] Add 20% palladium on carbon (0.15 g) to a methanol (10 mL) solution of 33b (0.76 g, 3.65 mmol). Stir the mixture under a hydrogen atmosphere for 12 hours, then filter. Concentrate the filtrate to dryness under reduced pressure to give the target product 33c (0.65 g, 100%).

[0139] MS m / z(ESI): 179 [M+1]

[0140] Intermediate 36d

[0141] 4-(2-(4-aminophenyl)-2-methylpropionyl)piperazine-1-carboxylic acid tert-butyl ester

[0142]

[0143] first step

[0144] 2-Methyl-2-(4-nitrophenyl)propionic acid (36a)

[0145] Concentrated sulfuric acid (25 mL) was added to a mixture of 33a (5.00 g, 26.29 mmol) and water (25 mL) at 0 °C, and then the mixture was heated to 120 °C and stirred for 12 hours. After cooling to room temperature, water (50 mL) was added, and the pH was adjusted to 8 with saturated sodium bicarbonate solution, followed by extraction with ethyl acetate (3 × 50 mL). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure to give the target product 36a (5.00 g, 91%).

[0146] MS m / z(ESI): 210[M+1]

[0147] Step 2

[0148] 2-Methyl-2-(4-nitrophenyl)propionyl chloride (36b)

[0149] 36a (5.00 g, 23.90 mmol) was added to thionyl chloride at 0 °C, and then heated to 85 °C and stirred for 12 hours. After cooling to room temperature, the solution was concentrated to dryness to obtain the target product 36b (5.20 g, 96%).

[0150] Step 3

[0151] 4-(2-methyl-2-(4-nitrophenyl)propionyl)piperazine-1-carboxylic acid tert-butyl ester

[0152] Triethylamine (2.00 g, 19.77 mmol) was added to a 20 mL solution of piperazine-1-carboxylate (1.84 g, 9.89 mmol) in dichloromethane and stirred for 10 minutes at room temperature. The mixture was then cooled to 0 °C and a 10 mL solution of 36c (1.50 g, 6.59 mmol) in dichloromethane was added. After stirring at room temperature for 2 hours, water (20 mL) was added, and the mixture was extracted with dichloromethane (3 × 30 mL). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness. The residue was purified by silica gel column chromatography (dichloromethane / methanol = 100 / 1) to give the target product 36c (1.50 g, 60%).

[0153] MS m / z(ESI): 378[M+1]

[0154] Step 4

[0155] 4-(2-(4-aminophenyl)-2-methylpropionyl)piperazine-1-carboxylic acid tert-butyl ester (36d)

[0156] Add 20% palladium on carbon (0.3 g) to a methanol (250 mL) solution of 36c (1.50 g, 3.97 mmol), and then stir for 12 hours under a hydrogen atmosphere. Filter and concentrate the filtrate to dryness to give the target product 36d (1.38 g, 100%).

[0157] MS m / z(ESI): 348[M+1]

[0158] Intermediate 35d was synthesized following the same experimental procedure as intermediate 36d, but in the third step, N-methylpiperidine was used instead of tert-butyl piperazine-1-carboxylate.

[0159]

[0160] Example 1

[0161] 3-(2,6-difluorophenyl)-1-((4-(morpholino-4-carbonyl)phenyl)amino)imidazo[1,5-a]pyrazin-8(7H)-one

[0162]

[0163] first step

[0164] (4-((3-(2,6-difluorophenyl)-8-methoxyimidazo[1,5-a]pyrazin-1-yl)amino)phenyl)(morpholino)methyl ketone (1h)

[0165] Sodium tert-butoxide (144 mg, 1.5 mmol) was added to a mixture of 1f (200 mg, 0.59 mmol), (4-aminophenyl)(morpholino) methyl ketone 1 g (121 mg, 0.59 mmol), and 1,4-dioxane (2 mL), followed by XantPhos (69 mg, 0.12 mmol) and Pd2(dba)3 (55 mg, 0.06 mmol). The mixture was heated to 100 °C and stirred for 1 hour in a microwave reactor. After cooling to room temperature, the reaction mixture was diluted with water (20 mL) and extracted with ethyl acetate (3 × 20 mL). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane / methanol = 20 / 1) to give the target product 1h (260 mg, 95%).

[0166] MS m / z(ESI): 466[M+1]

[0167] Step 2

[0168] 3-(2,6-difluorophenyl)-1-((4-(morpholino-4-carbonyl)phenyl)amino)imidazo[1,5-a]pyrazin-8(7H)-one(1)

[0169] To a 1-hour (260 mg, 0.56 mmol) solution of acetonitrile (10 mL), 10 mL of 6 N hydrochloric acid was added, and the mixture was heated to 70 °C and stirred for 1 hour. After cooling to room temperature, the mixture was concentrated to dryness, and the residue was diluted with water (10 mL), then adjusted to pH 8 with saturated sodium bicarbonate solution and extracted with ethyl acetate (2 × 50 mL). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane / methanol = 20 / 1) to give target product 1 (solid, 150 mg, 59%).

[0170] MS m / z(ESI): 452[M+1]

[0171] 1 H NMR(400MHz,DMSO-d6)δ10.56(d,J=5.3Hz,1H),8.42(s,1H),7.78-7.63(m,3H),7.43-7.2 7(m,4H),6.84(d,J=5.3Hz,1H),6.59(t,J=5.7Hz,1H),3.59(d,J=4.5Hz,4H),3.50(s,4H).

[0172] The examples or intermediates in the table below are all operated according to the experimental steps of Example 1, but in the first step, different compounds are used instead of 1f and 1g.

[0173]

[0174]

[0175]

[0176]

[0177] The NMR data for Examples 3, 4, 6, 7, 9, 11, 14, 22, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, and 38 are as follows:

[0178]

[0179]

[0180]

[0181]

[0182] Example 2

[0183] 3-(2,6-difluorophenyl)-1-((4-(morpholino-4-carbonyl)phenyl)amino)-6,7-dihydroimidazo[1,5-a]pyrazin-8(5H)-one(2)

[0184]

[0185] Mix 1 (97 mg, 0.215 mmol), methanol (30 mL), and 10% palladium on carbon (97 mg), then heat to 30 °C and stir for 16 hours under a hydrogen atmosphere. After filtration, the filtrate was concentrated to dryness, and the residue was purified by reversed-phase preparative high-performance liquid chromatography to obtain target product 2 (solid, 25 mg, 26%).

[0186] MS m / z(ESI): 490[M+1]

[0187] 1H NMR (400MHz, CD3OD) δ7.73(d,J=6.3Hz,1H),7.66(td,J=6.2,2.8Hz,3H),7.58(d,J=8.3Hz,2H),7.28(ddd,J=9.1,7.6,3.3Hz,1H),7.19(dd,J=9. 1,4.2Hz,1H),6.94(d,J=6.4Hz,1H),4.73(s,2H),4.52(t,J=11.5Hz,1H ),3.99(s,3H),3.76-3.70(m,1H),2.21-2.03(m,6H),1.63-1.53(m,2H).

[0188] The examples in the table below are all performed following the experimental steps of Example 2, but different compounds are used instead of 1 in the operation.

[0189]

[0190]

[0191] The NMR data for Examples 5, 8, 10, 12, 16, 18, and 23 are as follows:

[0192]

[0193]

[0194] Example 13

[0195] 1-((4-(1-(azacyclobutane-1-yl)-2-methyl-1-oxopropane-2-yl)phenyl)amino)-3-(2,6-difluorophenyl)-6,7-dihydroimidazol[1,5-a]pyrazine-8(5H)-one

[0196]

[0197] first step

[0198] 2-(4-((3-(2,6-difluorophenyl)-8-methoxyimidazo[1,5-a]pyrazin-1-yl)amino)phenyl)-2-methylpropionate tert-butyl ester (13b)

[0199] Under a nitrogen atmosphere, tert-butyl 2-(4-aminophenyl)-2-methylpropionate 13a (280 mg, 1.17 mmol), Brettphos (62 mg, 0.118 mmol), Pd2(dba)3 (53 mg, 0.058 mmol), and cesium carbonate (600 mg, 1.76 mmol) were added to a 5 mL solution of 1f (200 mg, 0.588 mmol). The mixture was then heated to 90 °C and stirred for 1 hour in a microwave reactor. After cooling to room temperature, the mixture was concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 100 / 0 to 7 / 3) to give the target product 13b (200 mg, 68%).

[0200] MS m / z(ESI): 495[M+1]

[0201] Step 2

[0202] Ethyl 2-(4-((3-(2,6-difluorophenyl)-8-oxo-7,8-dihydroimidazo[1,5-a]pyrazin-1-yl)amino)phenyl)-2-methylpropionate (13c)

[0203] A solution of HCl in ethanol (12N, 2 mL) was added to a 4 mL ethanol solution of 13b (100 mg, 0.1 mmol). The solution was stirred at 70 °C for 2 hours, then cooled to room temperature and concentrated to dryness to give the target product 13c (90 mg). This product was used directly in the next reaction without further purification.

[0204] MS m / z(ESI): 453[M+1]

[0205] Step 3

[0206] Ethyl 2-(4-((3-(2,6-difluorophenyl)-8-oxo-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazin-1-yl)amino)phenyl)-2-methylpropionate (13d)

[0207] 13c (90 mg, 0.2 mmol), ethanol (5 mL) and 10% palladium on carbon (100 mg) were mixed and stirred for 3 hours under a hydrogen atmosphere. The mixture was then filtered, and the filtrate was concentrated to dryness to obtain the target product 13d (80 mg, 88%).

[0208] MS m / z(ESI): 455[M+1]

[0209] Step 4

[0210] 2-(4-((3-(2,6-difluorophenyl)-8-oxo-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazin-1-yl)amino)phenyl)-2-methylpropionic acid (13e)

[0211] Lithium hydroxide monohydrate (17 mg, 0.4 mmol) was added to a 5 mL solution of 13d (90 mg, 0.2 mmol) in tetrahydrofuran. The mixture was heated to 60 °C and stirred for 18 hours. After cooling to room temperature, the reaction solution was concentrated to dryness. The residue was purified by reversed-phase preparative high-performance liquid chromatography to obtain the target product 13e (60 mg, containing TFA).

[0212] MS m / z(ESI): 427[M+1]

[0213] Step 5

[0214] 1-((4-(1-(azacyclobutane-1-yl)-2-methyl-1-oxopropane-2-yl)phenyl)amino)-3-(2,6-difluorophenyl)-6,7-dihydroimidazole[1,5-a]pyrazine-8(5H)-one(13)

[0215] Diisopropylethylamine (26 mg, 0.2 mmol) was added to a mixture of 13e (17 mg, 0.04 mmol), DMF (2 mL), HATU (20 mg, 0.052 mmol), and aziridine (10 mg, 0.12 mmol). The resulting solution was stirred at room temperature for 1 hour and then purified directly by reversed-phase preparative high-performance liquid chromatography to give the target product 13 (solid, 2.69 mg, 14%).

[0216] MS m / z(ESI): 466[M+1]

[0217] 1 H NMR (400MHz, DMSO-d6) δ8.04(s,1H),7.81(s,1H),7.75-7.65(m,1H),7.59(d,J=8.7Hz,2H),7.42-7.29(m,2H),7.10(d,J =8.7Hz,2H),4.06-3.93(m,2H),3.86-3.71(m,2H),3.53-3.47(m,2H),2.59-2.51(m,2H),2.03-1.84(m,2H),1.37(s,6H).

[0218] Example 15

[0219] 3-(2-chloro-6-fluorophenyl)-1-((4-(morpholino-4-carbonyl)phenyl)amino)-6,7-dihydroimidazo[1,5-a]pyrazin-8(5H)-one (15)

[0220]

[0221] To a solution of 3 (140 mg, 0.3 mmol) in ethanol (10 mL), add chlorobenzene (1 mL) and platinum dioxide (70 mg). Stir the mixture under a hydrogen atmosphere for 70 minutes, then filter. Concentrate the filtrate to dryness, and purify the residue by reversed-phase preparative high-performance liquid chromatography to give the target product 15 (solid, 62.7 mg, 44%).

[0222] MS m / z(ESI): 312[M+1]

[0223] 1 H NMR (400MHz, DMSO-d6) δ8.29(s,1H),7.87(s,1H),7.72-7.63(m,3H),7.57(d,J=8.1Hz,1H),7.47 (t,J=8.7Hz,1H),7.31(d,J=8.6Hz,2H),3.95-3.90(m,2H),3.61-3.55(m,4H),3.54-3.45(m,6H).

[0224] Example 17

[0225] 3-(2,6-difluorophenyl)-1-((4-(2-methyl-1-morpholino-1-oxopropane-2-yl)phenyl)amino)imidazo[1,5-a]pyrazin-8(7H)-one

[0226]

[0227] first step

[0228] 2-(4-((3-(2,6-difluorophenyl)-8-oxo-7,8-dihydroimidazo[1,5-a]pyrazin-1-yl)amino)phenyl)-2-methylpropionic acid (17a)

[0229] A solution of 13b (420 mg, 0.848 mmol) in 10 mL of trifluoroacetic acid was heated to 70 °C and stirred for 1 hour. After cooling to room temperature, the reaction solution was concentrated to dryness under reduced pressure to obtain the target product 17a (360 mg). This product was used directly in the next step without further purification.

[0230] MS m / z(ESI): 425[M+1]

[0231] Step 2

[0232] 3-(2,6-difluorophenyl)-1-((4-(2-methyl-1-morpholino-1-oxopropane-2-yl)phenyl)amino)imidazo[1,5-a]pyrazine-8(7H)-one hydrochloride (17)

[0233] 17a (360 mg, 0.85 mmol), DMF (5 mL), HATU (420 mg, 1.1 mmol), morpholine (220 mg, 2.55 mmol), and DIPEA (442 mg, 3.2 mmol) were mixed. After stirring at room temperature for 1 hour, the reaction mixture was directly purified by reversed-phase preparative high-performance liquid chromatography to give the target product 17 (solid, 320 mg, 76%, hydrochloride).

[0234] MS m / z(ESI): 494[M+1]

[0235] 1 H NMR (400MHz, DMSO-d6) δ10.47(d,J=5.3Hz,1H),8.12(s,1H),7.78-7.67(m,1H),7.64(d,J=8.7Hz,2H),7.42- 7.31(m,2H),7.08(d,J=8.7Hz,2H),6.81(d,J=5.5Hz,1H),6.62-6.49(m,1H),3.69-3.41(m,8H),1.40(s,6H).

[0236] Example 19

[0237] 3-(1-acetylpiperidin-4-yl)-1-((4-(morpholin-4-carbonyl)phenyl)amino)imidazo[1,5-a]pyrazin-8(7H)-one

[0238]

[0239] first step

[0240] 4-(8-methoxy-1-((4-(morpholino-4-carbonyl)phenyl)amino)imidazo[1,5-a]pyrazin-3-yl)piperidine-1-carboxylic acid benzyl ester (19b)

[0241] XantPhos (116 mg, 0.2 mmol) and Pd2(dba)3 (92 mg, 0.1 mmol) were added to a mixture of 19a (206 mg, 1 mmol), 1,4-dioxane (8 mL), and cesium carbonate (813 mg, 2.5 mmol). The mixture was heated to 90 °C and stirred for 1 hour in a microwave reactor. After cooling to room temperature, the mixture was filtered, and the filtrate was concentrated to dryness. The residue was purified by silica gel column chromatography (dichloromethane / methanol = 100 / 0 to 19 / 1) to give the target product 19b (490 mg, 86%).

[0242] MS m / z(ESI): 571 [M+1]

[0243] Step 2

[0244] (4-((8-methoxy-3-(piperidin-4-yl)imidazo[1,5-a]pyrazin-1-yl)amino)phenyl)(morpholino)methyl ketone (19c)

[0245] Add 10% palladium on carbon (115 mg) to a methanol (10 mL) solution of 19b (230 mg, 0.403 mmol). Stir the mixture under a hydrogen atmosphere for 30 minutes, then filter. Concentrate the filtrate to dryness to give the target product 19c (190 mg). This product was used directly in the next reaction without further purification.

[0246] MS m / z(ESI): 437[M+1]

[0247] Step 3

[0248] 1-(4-(8-methoxy-1-((4-(morpholin-4-carbonyl)phenyl)amino)imidazo[1,5-a]pyrazin-3-yl)piperidin-1-yl)ethane-1-one (19d)

[0249] Triethylamine (85 mg, 0.837 mmol) was added to a solution of 19c (122 mg, 0.279 mmol) in dichloromethane (5 mL). After cooling to 0 °C, acetyl chloride (22 mg, 0.279 mmol) was added dropwise. After stirring at room temperature for 30 minutes, the mixture was concentrated to dryness, and the residue was purified by silica gel column chromatography (dichloromethane / methanol = 100 / 0 to 19 / 1) to give the target product 19d (81 mg, 61%).

[0250] MS m / z(ESI): 479[M+1]

[0251] Step 4

[0252] 3-(1-acetylpiperidin-4-yl)-1-((4-(morpholin-4-carbonyl)phenyl)amino)imidazo[1,5-a]pyrazin-8(7H)-one (19)

[0253] To a solution of acetonitrile (2 mL) containing 19d (81 mg, 0.17 mmol), HCl ethanol solution (33%, 1 mL) was added, and the mixture was heated to 50 °C and stirred for 30 minutes. After cooling to room temperature, the reaction mixture was concentrated to dryness to give a yellow solid (85 mg). 45 mg of this solid was removed and purified by reversed-phase preparative high-performance liquid chromatography to give the target product 19 (solid, 31.1 mg, 69%).

[0254] MS m / z(ESI): 465[M+1]

[0255] 1 H NMR (400MHz, CD3OD) δ7.50-7.42(m,3H),7.34-7.28(m,2H),6.88(d,J=6.1Hz,1H),4.72(d,J=13.5Hz,1H),4.14(d,J=13.8Hz,1 H),3.85-3.57(m,9H),3.38(dd,J=19.4,7.5Hz,1H),2.88(t,J=11.9Hz,1H),2.19(s,3H),2.17-2.06(m,2H),1.97-1.75(m,2H).

[0256] Example 20

[0257] 1-((4-(morpholino-4-carbonyl)phenyl)amino)-3-(piperidin-4-yl)imidazo[1,5-a]pyrazin-8(7H)-one(20)

[0258]

[0259] To a solution of acetonitrile (2 mL) containing 19c (40 mg, 0.092 mmol), HCl solution (1 mL of 30% ethanol) was added, and the mixture was heated to 50 °C and stirred for 30 minutes. After cooling to room temperature, the mixture was concentrated to dryness, and the residue was purified by reversed-phase preparative high-performance liquid chromatography to obtain the target product 20 (solid, 10.3 mg, 25%).

[0260] MS m / z(ESI): 423[M+1]

[0261] 1H NMR (400MHz, CD3OD) δ7.66-7.61(m,2H),7.44-7.39(m,2H),7.28(d,J=6.1Hz,1H),6.63(d,J=6.0Hz,1H),3.72 (s,8H),3.62(t,J=3.5Hz,1H),3.58(t,J=3.5Hz,1H),3.56-3.49(m,1H),3.30-3.20(m,2H),2.29-2.17(m,4H).

[0262] Example 21

[0263] 3-(1-Cyclopropylpiperidin-4-yl)-1-((4-(morpholin-4-carbonyl)phenyl)amino)imidazo[1,5-a]pyrazin-8(7H)-one

[0264]

[0265] first step

[0266] (4-((3-(1-Cyclopropylpiperidin-4-yl)-8-methoxyimidazo[1,5-a]pyrazin-1-yl)amino)phenyl)(morpholino)methyl ketone (21a)

[0267] To a mixture of 19c (150 mg, 0.344 mmol), methanol (2 mL), and tetrahydrofuran (2 mL), (1-ethoxycyclopropoxy)trimethylsilane (120 mg, 0.687 mmol) was added, followed by acetic acid (103 mg, 1.72 mmol) and sodium cyanoborohydride (32 mg, 0.86 mmol). The mixture was heated to 60 °C and stirred for 20 hours. After cooling to room temperature, a saturated sodium bicarbonate solution (20 mL) was added, followed by extraction with ethyl acetate (3 × 20 mL). The combined organic phases were washed with saturated brine (20 mL) and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated to dryness, and the residue was purified by silica gel column chromatography (dichloromethane / methanol = 100 / 0 to 19 / 1) to give the target product 21a (46 mg, 28%).

[0268] MS m / z(ESI): 477[M+1]

[0269] Step 2

[0270] 3-(1-Cyclopropylpiperidin-4-yl)-1-((4-(morpholin-4-carbonyl)phenyl)amino)imidazo[1,5-a]pyrazin-8(7H)-one (21)

[0271] A solution of hydrogen chloride (30% ethanol, 1 mL) was added to a 2 mL acetonitrile solution of 21a (46 mg, 0.0965 mmol), and the mixture was heated to 50 °C and stirred for 30 minutes. After cooling to room temperature, the mixture was concentrated to dryness, and the residue was purified by reversed-phase preparative high-performance liquid chromatography to give the target product 21 (solid, 16.9 mg, 38%).

[0272] MS m / z(ESI): 463[M+1]

[0273] 1 H NMR (400MHz, CD3OD) δ7.77(d,J=8.6Hz,2H),7.38(d,J=8.6Hz,2H),7.15(d,J=6.0Hz,1H),6.46(d,J=6.0Hz,1H),3.71(s,4H),3. 68(s,4H),3.28(d,J=11.9Hz,2H),3.15-3.06(m,1H),2.57(t,J=10.5Hz,2H),2.13-1.94(m,4H),1.90(s,1H),0.66-0.49(m,4H).

[0274] Example 24

[0275] 3-(2,6-difluorophenyl)-1-((1-(2-morpholino-2-oxoethyl)-1H-pyrazol-4-yl)amino)-6,7-dihydroimidazo[1,5-a]pyrazin-8(5H)-one

[0276]

[0277] first step

[0278] 2-(4-nitro-1H-pyrazole-1-yl)tert-butyl acetate (24b)

[0279] 4 g (17.7 mmol) of tert-butyl 2-bromoacetate was added to a mixture of 4-nitro-1H-pyrazole 24a (2 g, 17.7 mmol), potassium carbonate (4.8 g, 35 mmol), and DMF (10 mmol) at room temperature. The resulting mixture was stirred at room temperature for 12 hours, diluted with water (200 mL), and extracted with ethyl acetate (2 × 150 mL). The combined organic phases were washed with saturated brine (2 × 150 mL) and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane / methanol = 100 / 0 to 99 / 1) to give the target product 24b (5 g, 110%).

[0280] MS m / z(ESI): 228[M+1]

[0281] Step 2

[0282] 2-(4-amino-1H-pyrazole-1-yl)tert-butyl acetate (24c)

[0283] 10% palladium on carbon (700 mg) was added to a 20 mL ethanol solution of 24b (5 g, 20 mmol), and the mixture was stirred for 12 hours under a hydrogen atmosphere, then filtered. The filtrate was concentrated to dryness under reduced pressure to give the target product 24c (3.45 g, 77%).

[0284] MS m / z(ESI): 198[M+1]

[0285] Step 3

[0286] 2-(4-((3-(2,6-difluorophenyl)-8-methoxyimidazo[1,5-a]pyrazin-1-yl)amino)-1H-pyrazin-1-yl)tert-butyl acetate (24d)

[0287] A mixture of 1f (600 mg, 1.77 mmol), 1,4-dioxane (15 mL), 24c (720 mg, 3.53 mmol), Brettphos (192 mg, 0.353 mmol), Pd2(dba)3 (180 mg, 0.177 mmol), and cesium carbonate (1.7 g, 5.32 mmol) was heated to 90 °C and stirred for 1 hour under a nitrogen atmosphere in a microwave reactor. After cooling to room temperature, the solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 100 / 0 to 1 / 4) to give the target product 24d (450 mg, 47%).

[0288] MS m / z(ESI): 457[M+1]

[0289] Step 4

[0290] 2-(4-((3-(2,6-difluorophenyl)-8-oxo-7,8-dihydroimidazo[1,5-a]pyrazin-1-yl)amino)-1H-pyrazin-1-yl)acetic acid(24e)

[0291] A solution of 24d (450 mg, 0.98 mmol) in 10 mL of trifluoroacetic acid was heated to 80 °C and stirred for 12 hours. After cooling to room temperature, the reaction solution was concentrated to dryness under reduced pressure to obtain the target product 24e (815 mg). This product was used directly in the next reaction without further purification.

[0292] MS m / z(ESI): 387[M+1]

[0293] Step 5

[0294] 2-(4-((3-(2,6-difluorophenyl)-8-oxo-5,6,7,8-tetrahydroimidazo[1,5-a]pyrazin-1-yl)amino)-1H-pyrazin-1-yl)acetic acid(24f)

[0295] 10% palladium on carbon (300 mg) was added to a 24e (800 mg, 2.07 mmol) ethanol (30 mL) solution, and the mixture was stirred for 12 hours under a hydrogen atmosphere. After filtration, the filtrate was concentrated to dryness under reduced pressure to obtain the target product 24f (280 mg, 35%).

[0296] MS m / z(ESI): 389[M+1]

[0297] Step 6

[0298] 3-(2,6-Difluorophenyl)-1-((1-(2-morpholino-2-oxoethyl)-1H-pyrazol-4-yl)amino)-6,7-dihydroimidazo[1,5-a]pyrazin-8(5H)-one(24)

[0299] DIPEA (10 mg, 0.0771 mmol) was added to 24f (10 mg, 0.0257 mmol), HATU (13 mg, 0.0335 mmol), morpholine (0.025 mL), and DMF (0.5 mL). After stirring at room temperature for 1 hour, the reaction solution was directly purified by reversed-phase preparative high-performance liquid chromatography to obtain the target product 24 (solid, 2.12 mg, 18%).

[0300] MS m / z(ESI): 458[M+1]

[0301] 1 H NMR(400MHz,DMSO-d6)δ7.84-7.81(m,1H),7.80(s,1H),7.72-7.62(m,2H),7.55(s,1H), 7.36-7.29(m,2H),5.03(s,2H),3.97-3.90(m,2H),3.60-3.52(m,2H),3.50-3.39(m,8H).

[0302] Example 25 follows the experimental procedure of Example 24, but in step 6, N-methylpiperazine is used instead of morpholine.

[0303]

[0304] The NMR data for Example 25 are as follows:

[0305]

[0306]

[0307] Example 39

[0308] 4-((3-(2,6-difluorophenyl)-8-oxo-7,8-dihydroimidazo[1,5-a]pyrazin-1-yl)amino)benzoic acid (39)

[0309]

[0310] Lithium hydroxide monohydrate (155 mg, 3.7 mmol) was added to a mixed solution of 39a (150 mg, 0.37 mmol) in methanol, tetrahydrofuran, and water (3 / 3 / 1 v / v / v, 7 mL), and the mixture was heated to 50 °C and stirred for 12 hours. After cooling to room temperature, the organic solvent was removed by concentration under reduced pressure. The residue was adjusted to pH 1 with 1 N hydrochloric acid and stirred at room temperature for 5 hours, then concentrated to dryness. The residue was purified by reversed-phase preparative high-performance liquid chromatography to give the target product 39 (solid, 25 mg, 18%).

[0311] MS m / z(ESI): 383[M+1]

[0312] 1 H NMR (400MHz, DMSO-d6) δ12.36(s,1H),10.61(d,J=5.5Hz,1H),8.62(s,1H),7.81(d,J=8.8 Hz,2H),7.70(d,J=8.9Hz,3H),7.39(d,J=8.2Hz,2H),6.87(d,J=5.6Hz,1H),6.60(s,1H).

[0313] Example 40 follows the experimental procedure of Example 39, but 40a is used instead of 39a in the operation.

[0314]

[0315] The NMR data for Example 40 are as follows:

[0316]

[0317]

[0318] Biological experiments

[0319] JAK2 activity inhibition test

[0320] The effect of the compounds of this invention on JAK2 activity was evaluated using in vitro kinase assays.

[0321] The experimental methods are summarized as follows:

[0322] The enzyme activity of JAK2 was determined by detecting the phosphorylation level of the substrate in the kinase reaction using a homogeneous time-resolved fluorescence (HTRF) kinase assay kit (Cisbio, 62TK0PEC). The reaction buffer contained the kit's own enzyme reaction buffer (1×), 5 mM MgCl2, 1 mM DTT, and 0.01% Brij 35; human recombinant JAK2 protein (Carna Biosciences, 08-045) was diluted with the reaction buffer to a kinase reaction solution of 0.15 ng / μL; the substrate reaction solution consisted of biotin-labeled tyrosine kinase substrate diluted with the reaction buffer to 0.25 μM and 2.5 μM ATP; the assay buffer consisted of Eu phosphate diluted with the reaction buffer to 0.1 ng / μL. 3+ Labeled cage-like antibody (Cisbio, 61T66KLB) and 12.5 nM streptavidin-labeled XL665 (Cisbio, 610SAXLB); the compounds were dissolved and diluted to 10 μM with DMSO, and then serially diluted 4-fold with DMSO to a minimum concentration of 0.061 nM. Each concentration point was then diluted 40-fold with reaction buffer.

[0323] Add 4 μL of compound solutions of varying concentrations and 2 μL of kinase reaction solution to a 384-well detection plate (Corning, 3674), mix thoroughly, and incubate at room temperature for 15 minutes. Then add 4 μL of substrate reaction solution and incubate the reaction mixture at room temperature for 30 minutes. Next, add 10 μL of detection buffer, mix thoroughly, and let stand at room temperature for 30 minutes. Detect the reaction progress using an Envision plate reader (Perkin Elmer) at wavelengths of 620 nm and 665 nm. Signal values ​​(absorbance) are then recorded. 665nm Absorbance 620nm The activity of JAK2 kinase was positively correlated with the degree of phosphorylation of the substrate, thus allowing the detection of JAK2 kinase activity. In this experiment, the group without JAK2 kinase protein was designated as the 100% inhibition group, and the group with JAK2 kinase protein but without the compound was designated as the 0% inhibition group. XLfit software was used to plot the compound inhibition curves and calculate their IC50 values. 50 The experimental results are shown in Table 1.

[0324] TYK2 activity inhibition test

[0325] The effect of the compounds of this invention on TYK2 activity was evaluated using in vitro kinase assays.

[0326] The experimental methods are summarized as follows:

[0327] The enzyme activity of TYK2 was determined by detecting the phosphorylation level of the substrate in the kinase reaction using a homogeneous time-resolved fluorescence (HTRF) kinase assay kit (Cisbio, 62TK0PEC). The reaction buffer contained the kit's own enzyme reaction buffer (1×), 5 mM MgCl2, 1 mM DTT, and 0.01% Brij 35; human recombinant TYK2 protein (Carna Biosciences, 08-147) was diluted with the reaction buffer to a kinase reaction solution of 0.25 ng / μL; the substrate reaction solution consisted of biotin-labeled tyrosine kinase substrate diluted with the reaction buffer to 0.5 μM and 11.25 μM ATP; the assay buffer consisted of Eu phosphate diluted with the reaction buffer to 0.1 ng / μL. 3+ Labeled cage-like antibody (Cisbio, 61T66KLB) and 25 nM streptavidin-labeled XL665 (Cisbio, 610SAXLB); the compounds were dissolved and diluted to 10 μM with DMSO, and then serially diluted 4-fold with DMSO to the lowest concentration of 0.061 nM. Each concentration point was then diluted 40-fold with reaction buffer.

[0328] Add 4 μL of compound solution of varying concentrations and 2 μL of kinase reaction solution to a 384-well detection plate (Corning, 3674), mix thoroughly, and incubate at room temperature for 15 minutes. Then add 4 μL of substrate reaction solution and incubate the reaction mixture at room temperature for 40 minutes. Next, add 10 μL of detection buffer, mix thoroughly, and let stand at room temperature for 30 minutes. Detect the reaction progress using an Envision plate reader (Perkin Elmer) at wavelengths of 620 nm and 665 nm. Signal values ​​(absorbance) are then recorded. 665nm Absorbance 620nm The activity of TYK2 kinase was positively correlated with the degree of phosphorylation of the substrate, thus allowing the detection of TYK2 kinase activity. In this experiment, the group without TYK2 kinase protein was designated as the 100% inhibition group, and the group with TYK2 kinase protein but without the compound was designated as the 0% inhibition group. XLfit software was used to plot the compound inhibition curves and calculate their IC50 values. 50 The experimental results are shown in Table 1.

[0329] Table 1

[0330]

[0331]

[0332] The compounds in the embodiments of the present invention have an inhibitory effect on the activity of both JAK2 and TYK2, preferably with an IC50 concentration of 1. 50 Less than 100 nM, preferably its IC 50 Less than 10 nM.

[0333] Determination of the inhibition of IL-12-induced IFN-γ secretion in NK92 cells

[0334] The effect of the compounds of the present invention on IFN-γ secretion in IL-12-induced NK92 cells was evaluated by enzyme-linked immunosorbent assay (ELISA).

[0335] The experimental principle is summarized as follows: IL-12R is mainly expressed by activated T cells, NK cells (NK92 is an NK cell line), DC cells and B cells. It binds to IL-12 and activates the JAK2 / TYK2 signaling pathway in NK cells and activated T lymphocytes, thereby inducing the production of IFN-γ.

[0336] The experimental methods are summarized as follows:

[0337] The compound was dissolved and diluted to 2.5 mM with DMSO, and then serially diluted 4-fold with DMSO to the lowest concentration of 0.31 μM. Each concentration point was then diluted 50-fold with FBS-free MEMα medium (Thermofisher, 12561-056).

[0338] NK92 cells (Nanjing Kebai, CBP60980) were subjected to treatment with 12.5% ​​FBS (Ausbian, VS500T), 12.5% ​​horse serum (Thermofisher, 16050-122), 0.02 mM folic acid (Sigma, F8758), 0.2 mM inositol (Sigma, 17850), 0.55 mM β-mercaptoethanol (Thermofish, 21985-023), and 200 U / mL IL-2 (R&D). Cells were cultured in MEMα complete medium containing a mixture of 100 U / mL penicillin and streptomycin (Systems, 202-1L) and 100 U / mL penicillin and streptomycin (Thermofisher, 15140122). When the cell coverage in the culture vessel reached 80-90%, the cells were dispersed and seeded into 96-well plates (Thermofish, 167425) with 100,000 cells per well (80 μL of MEMα complete medium without IL-2). The 96-well plates were then incubated overnight at 37°C with 5% CO2.

[0339] After overnight incubation, 10 μL of the diluted compound and 10 μL of 50 ng / mL IL-12 (R&D Systems, 219-1L) were added to each well, gently mixed, and then the 96-well plate was incubated at 37°C with 5% CO2. After 24 hours, the plate was centrifuged at 800 rpm for 10 minutes at room temperature. 50 μL of the supernatant was transferred to a 96-well plate (Sigma, CLS3695) coated with anti-IFN-γ antibody. IFN-γ secretion was detected according to the Human IFN-γ DuoSet ELISA kit (R&D Systems, DY285B). The unstimulated control group received neither IL-12 nor the tested compound, and MEMα medium was used instead (100% inhibition). The stimulated control group received IL-12 and 0.2% DMSO (0% inhibition). Inhibition curves were plotted using XLfit software, and the IC50 of each compound was calculated. 50 The experimental results are shown in Table 2.

[0340] Table 2

[0341] Compound No. IC 50 (NK92 IL12 / IFN-γ) (nM) 1 57 2 95 3 66 4 175 5 1308 6 198 10 122 11 242 12 110 13 1061 14 113 15 193 16 1443 17 520 18 1406 26 149 27 60 28 38 29 75 30 318 31 56 32 425 33 66 34 65 35 191 36 3477 37 268 38 2854 39 4095 40 406

[0342] Mouse pharmacokinetics experiment

[0343] Prepare a 5 mg / mL sample (suspension or solution) of the test compound in 20% HP-β-CD solvent.

[0344] Three female C57 mice were administered the drug sample at a dose of 25 mg / kg via gavage (PO). Blood samples were collected 4 hours after administration. The mice were then euthanized with CO2. The colon, about 4-6 cm in length, near the rectum, was cut open, rinsed with cold saline, dried with absorbent paper, and weighed.

[0345] The concentrations of the analytes in plasma and intestinal homogenate samples were quantitatively analyzed by LC-MS / MS using an API-4500 mass spectrometer. The limit of quantitation (LOQ) for plasma was 1 ng / mL. Pharmacokinetic (PK) parameters were calculated using WinNonlin, and the results are summarized in Table 3.

[0346] Table 3

[0347] Compound Exposure in plasma (ng / mL) Exposure in colon (ng / g) 1 249 5210 28 15 2853 29 75 3925 30 BLOQ 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 12200 34 50 2705 36 18 5093

Claims

1. A compound of general formula (I), or a pharmaceutically acceptable salt or stable isotopic derivative thereof: in: Bond 'a' can be a single bond or a double bond; R 1 and R 2 Each is independently selected from H, D, CN, and C. 1-6 Alkyl or C 3-6 Cycloalkyl, wherein one or more hydrogen atoms of the alkyl group are optionally replaced by D or fluorine; A is a 4-10 membered heterocyclic group or C is a heterocyclic group. 6-10 Aryl, wherein the heterocyclic group and aryl group are optionally selected from one or more groups selected from D, halogen, cyano, -OR a -NR a R b -C(O)R a -C(O)NR a R b -S(O)2R a -P(O)(CH3)2, C 1-6 Alkyl or C 3-6 Substituents of cycloalkyl groups; B is a phenyl or a 5-6-membered heteroaryl group, wherein the phenyl and heteroaryl group is optionally selected from one or more groups chosen from D, halogen, cyano, -OR. a -NR a R b -COOR a -C(O)R a -NR a C(O)R b -C(O)NR a R b -S(O)2R a -S(O)2NR a R b -S(O)(NR) a )R b -P(O)(CH3)2, C 1-6 Alkyl, C 3-6 The alkyl, cycloalkyl, 4-8-membered heterocyclic, or 5-6-membered heteroaryl groups are substituted, wherein the alkyl, cycloalkyl, heterocyclic, or heteroaryl group is optionally further replaced by one or more substituents selected from D, halogen, CN, -OH, -NH2, C. 1-6 Alkyl, -OC 1-6 Alkyl, -COOR a -C(O)R a or -C(O)NR a R b The substituents are replaced; R a and R b Each is independently selected from H and C. 1-6 alkyl.

2. The compound according to claim 1, or its pharmaceutically acceptable salt or stable isotope derivative, R 1 and R 2 Both are H.

3. The compound according to claim 1, or its pharmaceutically acceptable salt or stable isotope derivative, wherein it is a compound represented by general formula (II): in: A is a 4-8 membered heterocyclic group or phenyl group, wherein the heterocyclic group and phenyl group are optionally selected from one or more halogens, -C(O)R a C 1-6 Alkyl and C 3-6 Substituents of cycloalkyl groups; B is a phenyl or a 5-6-membered heteroaryl group, wherein the phenyl and heteroaryl group are optionally selected from one or more halogens, -COOR a -C(O)R a -C(O)NR a R b -S(O)2R a C 1-6 Alkyl, C 3-6 The alkyl group is replaced by a substituent of a cycloalkyl group, a 4-8 membered heterocyclic group containing N, S, and / or O heteroatoms, wherein the alkyl group, cycloalkyl group, and heterocyclic group are optionally further replaced by one or more substituents selected from C10. 1-6 Alkyl, -C(O)R a and -C(O)NR a R b The substituents are replaced; R a and R b Each is independently selected from H or C 1-6 alkyl.

4. The compound according to claim 1 or 3, or a pharmaceutically acceptable salt or stable isotope derivative thereof, wherein A is a phenyl group, and one or more hydrogen atoms of the phenyl group are optionally substituted with halogens.

5. The compound according to claim 1, or a pharmaceutically acceptable salt or stable isotope derivative thereof, wherein it is a compound represented by general formula (III): in: A is a phenyl group, wherein one or more hydrogen atoms of the phenyl group may optionally be replaced by halogens; B is a phenyl or a 5-6-membered heteroaryl group, wherein the phenyl and heteroaryl group are optionally selected from one or more halogens, -COOR a -C(O)R a -C(O)NR a R b -S(O)2R a C 1-6 Alkyl, C 3-6 The alkyl group is replaced by a substituent of a cycloalkyl group, a 4-8 membered heterocyclic group containing N, S, and / or O heteroatoms, wherein the alkyl group, cycloalkyl group, and heterocyclic group are optionally further replaced by one or more substituents selected from C10. 1-6 Alkyl, -C(O)R a and -C(O)NR a R b The substituents are replaced; R a and R b Each is independently selected from H and C. 1-6 alkyl.

6. The compound according to claim 1, or its pharmaceutically acceptable salt or stable isotope derivative, has the following structure:

7. The compound of claim 6, wherein:

8. A pharmaceutical composition comprising the compound of any one of claims 1-7 or a pharmaceutically acceptable salt or stable isotope derivative thereof, and one or more pharmaceutically acceptable carriers or excipients.

9. Use of the compound of any one of claims 1-7 or its pharmaceutically acceptable salt or stable isotope derivative, or the pharmaceutical composition of claim 8, in the preparation of a medicament for the prevention or treatment of JAK-mediated diseases, wherein the JAK-mediated diseases are inflammatory diseases, autoimmune diseases, or cancer.

10. The use according to claim 9, wherein the JAK-mediated disease is inflammatory bowel disease, dermatitis, eczema, rheumatoid arthritis, systemic lupus erythematosus, psoriasis, or alopecia areata.

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