CN116003417A

CN116003417A - Pyrrolo [2,3-d ] six-membered heteroaromatic ring derivatives, process for the preparation thereof and pharmaceutical use thereof

Info

Publication number: CN116003417A
Application number: CN202310064329.3A
Authority: CN
Inventors: 王永钢; 陈海杰; 廖辉; 胡双华
Original assignee: Hunan Nanxin Pharmaceutical Co ltd
Current assignee: Hunan Nanxin Pharmaceutical Co ltd
Priority date: 2022-02-14
Filing date: 2023-02-06
Publication date: 2023-04-25
Also published as: WO2023151069A1

Abstract

The invention relates to pyrrolo [2,3-d ] compounds of the general formula (I)]Six-membered heteroaromatic ring derivatives having Janus kinase (JAK) kinase inhibitory activity, in particular, selective for JAK3 kinase, high inhibitory activity, and excellent oral absorbability. The invention also relates to methods of preparation thereof comprising such compounds, pharmaceutical compositions comprising the same, and methods of treatment using the same. The compound of the present invention provides a medicine useful for preventing and/or treating diseases associated with abnormal JAK3 expression based on JAK3 inhibition.

Description

Pyrrolo [2,3-d ] six-membered heteroaromatic ring derivatives, process for the preparation thereof and pharmaceutical use thereof

Technical Field

The invention provides a pyrrolo [2,3-d ] six-membered heteroaromatic ring derivative which has pyrrolo [2,3-d ] pyrimidinyl or pyrrolo [2,3-d ] pyridinyl derivatives and Janus kinase (JAK) kinase inhibitory activity, and particularly has selectivity and higher inhibitory activity on JAK3 kinase. The invention is also directed to compositions comprising such compounds, methods for preparing such compounds, and methods for treating and preventing diseases mediated through JAK3 disorders.

Background

Janus kinases (JAKs) belong to the family of tyrosine kinases, altering the function of proteins containing them by their ability to phosphorylate tyrosine residues. Activated upon stimulation by specific growth factors, growth hormones, chemokines, cytokines and various cell surface receptors, have tyrosine kinase activity and bind in pairs, and dimer JAKs spontaneously phosphorylate, bind STAT proteins, phosphorylate STAT transcription factors and transfer into the nucleus, transfer extracellular signals from cell surface receptors to the nucleus, altering transcription of DNA and subsequent translation of proteins. The JAK-STAT pathway acts on more than 50 downstream cytokines and growth factors, and therefore JAK kinases are thought to be central communication nodes of the immune system. Janus kinases (JAKs) have four family members: JAK1, JAK2, JAK3 and TYK2. Among them, JAK1, JAK2 and TYK2 are widely present in various tissues and cells in the body, and JAK3 is mainly present in bone marrow cells, thymus cells, NK cells and activated B lymphocytes, T lymphocytes. Based on the functional characteristics and special tissue distribution of each subtype in the JAK kinase family, JAK1 has become a novel target in the fields of diseases such as immunity, inflammation, cancer and the like; JAK2 has become an exact target for treatment and prevention of diseases related to the blood system; JAK3 has become a popular target for the treatment of autoimmune diseases. Each cell surface receptor needs to signal through a pair of identical homodimers (e.g., JAK2/JAK 2) or heterodimers (e.g., JAK1/JAK 3), activate downstream STAT proteins (signal transducers and activators), regulate the corresponding target gene promoters and thus affect transcription of DNA and subsequent translation of the protein. Each pair of JAKs has a different activating ligand and acting downstream effector (Pharmacological Research,2019, 147, 104392).

JAK-STAT signaling pathway functions widely, and is involved in many important biological processes such as proliferation, differentiation, apoptosis, and immunomodulation of cells. The JAK-STAT pathway acts on more than 50 downstream cytokines and growth factors, including interleukins (IL-2-7, IL-9, IL-10, IL-15, IL-21), interferons (IFN- α, IFN- β, IFN- γ), erythropoietin (EPO), granulocyte and megacell colony stimulating factor (GM-CSF), somatotropin (GH), prolactin (PRL), thrombopoietin (TPO), etc., which play a key role in biological processes involved in proliferation, immunomodulation of immune cells and hematopoietic stem cells. Different receptors activate JAK kinases of different subtypes, thus achieving differentiated biological functions. JAK1 binds IL-10, IL-19, IL-20, IL-22, IL-26, IL-28, IFN- α, IFN- γ, IL-6 in the gp130 family, other receptors containing yc, etc. (Cell, 1998, 93:373-383). JAK1 Gene knockout experiments on mouse models indicate that this enzyme plays a key role in modulating the biological effects of the various cytokine receptors described above (Gene, 2002, 285:1-24). JAK1 is a novel target in the fields of diseases such as immunity, inflammation, cancer and the like. JAK1 inhibitors are useful in the treatment/prevention of autoimmune diseases, inflammation and tumors (Blood, 2010,115: 3287-3295), such as leukemia, lymphoma, melanoma, arthritis, psoriasis, crohn's disease, lupus erythematosus, acquired immunodeficiency syndrome, behcet's disease (hum. Genet.,2013,132: 1049-1058), and the like. JAK2 plays an important role in the regulation of a variety of receptor signaling including EPO, GH, PRL, IL-3, IFN- γ, etc. (Gene, 2002,285:1-24;

Nat.Rev.mol.CellBiol.,2002, 3:651-662). Knocking out JAK2 in a mouse model can lead to anaemia-induced animal death (j.biol. Chem.,2007, 282:20059-20063); one base mutation on the JAK2 gene in humans JAK2V617F is closely related to the occurrence of Polycythemia Vera (PV), essential Thrombocythemia (ET), idiopathic Myelofibrosis (IMF), chronic Myelogenous Leukemia (CML) and the like in myeloproliferative diseases (immunol.rev., 2009, 228:273-287). JAK2 has therefore become an exact target for the treatment/prevention of this type of disease. JAK3 regulates cell signaling by binding to the gamma co-chain (yc) in cytokine receptor complexes such as IL-2, IL-4, IL-7, IL-9, IL-15, IL-21, etc. Either JAK3 or yc mutations can lead to Severe Combined Immunodeficiency (SCID) (Blood, 1996, 88:817-823). Abnormal JAK3 activity is manifested by a massive decrease in T cells and NK cells, loss of B cell function, and serious effects on normal biological functions of the immune system and the like. Based on their functional characteristics and specific tissue distribution, JAK3 is an attractive drug target for immune system related diseases, and inhibitors thereof have important clinical application value in the treatment/prevention of Rheumatoid Arthritis (RA), crohn's disease and ulcerative colitis, systemic lupus erythematosus, multiple sclerosis, type i diabetes, psoriasis, allergic diseases, asthma, chronic obstructive pulmonary disease, leukemia, lymphoma, organ transplantation and other diseases (Trends pharm.sci.,2004, 25:558-562). TYK2 is the first member of the JAK family to be activated by a variety of receptors such as IFNs, IL-10, IL-6, IL-12, IL-23, IL-27, and the like. In mice, a deficiency in TYK2 function causes a defect in the signaling pathway of various cytokine receptors, which in turn leads to viral infection, a decrease in antibacterial immune function, and an increased likelihood of pulmonary infection, etc. (Gene, 2002, 285:1-24).

Early approved JAK inhibitors were all non-selective JAK inhibitors, and the first JAK inhibitor developed by Incyte corporation of the united states, ruxolitinib (Ruxolitinib), was approved for market in the united states in 2011 as the first drug specifically used to treat myelofibrosis. In 2012, as Tofacitinib (Tofacitinib) was FDA approved for the treatment of Rheumatoid Arthritis (RA), in 2017, baricitinib developed by the cooperation of Incyte and Eli Lilly first acquired a marketing license in europe, but was denied in FDA's marketing application; after perfecting the relevant clinical trial, baricitinib, month 1 of 2018, eventually obtained FDA approval. However, at present, these several pan-JAKs inhibitors are provided with a black box warning: serious infection, malignancy, risk of thrombosis. The pan-JAKs inhibitor Tofacitinib has side effects including causing decreased numbers of erythrocytes and leukocytes, increased cholesterol levels, etc., which may be associated with its high JAK2 inhibitory activity (j.med.chem., 2012, 55:6176-6193).

JAK3 is a member of the Janus family of protein kinases comprising JAKl, JAK2, JAK3 and TYK2 and is expressed at different levels in all tissues. Many cytokine receptors transmit signals through JAK kinases in the following combinations: JAKl/JAK2, JAKl/JAK3, JAK1/TYK2, JAK2/TYK2 or JAK2/JAK2. Animal studies have shown that the involvement of JAK3 in the development, function and homeostasis of the immune system to regulate immune activity by inhibiting JAK3 kinase activity may prove useful in the treatment of a variety of immune disorders (j.lmmunol., 178,2623-2629 (2007); gene,285,1-24 (2002); cell,109, (suppl.) Sl 21-S131 (2002)), while avoiding JAK 2-dependent Erythropoietin (EPO) and Thrombopoietin (TPO) signaling (Cell, 93 (3), 397-409 (1998); cell,93 (3), 385-95 (1998)). Currently, research and discovery of selective JAK inhibitors (particularly JAK 3) has become a major development direction of institutions such as pharmaceutical companies in the JAK inhibitor field.

Disclosure of Invention

The present invention relates to novel compounds which are selective JAK3 modulators useful in the treatment of diseases associated with JAK3 dysregulation. The invention also provides pharmaceutical compositions comprising such JAK3 modulators and methods of treating and/or preventing such diseases. Accordingly, the present invention provides a compound of formula (I), an optical isomer thereof or a mixture thereof, a pharmaceutically acceptable salt, solvate, N-oxide thereof or a prodrug thereof, having the structure:

wherein X is independently selected from N, CH or CCN;

r is independently selected from the group consisting of hydrogen, deuterium, fluorine, chlorine, cyano, C1-C8 alkynyl, C1-C8 haloalkynyl, C3-C5 cycloalkyl-substituted alkynyl, methyleneoxyalkyl-substituted alkynyl, methyleneoxyalkylated alkynyl, 5-to 6-membered aromatic or heteroaromatic ring-substituted alkynyl, C1-C6 linear or branched alkoxymethylene, halo C1-C6 linear or branched alkoxymethylene, C1-C6 linear or branched alkoxydifluoromethylene, halo C1-C6 linear or branched alkoxydifluoromethylene, C1-C6 linear or branched alkoxyfluoromethylene, halo C1-C6 linear or branched alkoxyfluoromethylene, C3-C6 cycloalkyl, C3-C6 alkyl-substituted cycloalkyl, C3-C6 halogen-substituted cycloalkyl, C6-C10 aryl, monocyclic or bicyclic heteroaryl containing 5-and/or 6-membered rings, (aryl) C1-C6 linear or branched alkyl, (heteroaryl) C1-C6 alkyl, (C6) heterocycle, (C1-C6 linear or branched alkoxycarbonyl, (C1-C6) linear or branched alkoxyfluorocarbonyl, (C1-C6 linear or branched alkoxyfluorocarbonyl, halo-C1-C6 linear or branched alkoxyfluorocarbonyl, (C1-C6 linear or branched alkoxyfluorocarbonyl), halo-C1-C6 linear or branched alkoxyfluorocarbonyl, C1-C6 alkyl-C3-C6 alkyl-substituted cycloalkyl, C3-C6 halo-substituted cycloalkyl, and C1-C6 heteroaryl, (C1-C6 linear or branched alkyl) aminocarbonyl, ethoxycarbonyl, isopropoxycarbonyl, isobutoxycarbonyl; wherein the alkyl, aryl, and heteroaryl groups are independently optionally substituted with one or more substituents selected from the group consisting of: alkyl, halogen, hydroxy, methoxy, amino, cyano, alkylamino, dialkylamino, CF3, aminocarbonyl, (C1-C6 linear or branched alkyl) aminocarbonyl and C3-C6 cycloalkyl.

When X is selected from N, the preferred structure is as in formula (II):

When X is selected from CH, the preferred structure is as in formula (III):

When X is selected from CCN, the preferred structure is as in formula (IV),

In particular, the present invention provides a compound selected from the group consisting of:

1- ((2S, 4S, 5S) -5- ((5- ((2-methyl-2H-tetrazol-5 yl) ethynyl) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) -4-fluoro-2-methylpiperidin-1-yl) prop-2-en-1-one;

1- ((2S, 4S, 5S) -5- ((5- ((1H-pyrrol-4 yl) ethynyl) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) -4-fluoro-2-methylpiperidin-1-yl) prop-2-en-1-one;

1- ((2S, 4S, 5S) -5- ((5- (3- (fluoromethoxy) propyn-1-yl) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) -4-fluoro-2-methylpiperidin-1-yl) prop-2-en-1-one;

1- ((2S, 4S, 5S) -5- ((5- (3-methoxy-propyn-1-yl) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) -4-fluoro-2-methylpiperidin-1-yl) prop-2-en-1-one;

1- ((2S, 4S, 5S) -5- ((5- (cyclopropylethynyl) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) -4-fluoro-2-methylpiperidin-1-yl) prop-2-en-1-one;

1- ((2S, 4S, 5S) -5- ((5- ((R) -2, 2-difluorocyclopropyl) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) -4-fluoro-2-methylpiperidin-1-yl) prop-2-en-1-one;

1- ((2S, 4S, 5S) -5- ((5- ((S) -2, 2-difluorocyclopropyl) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) -4-fluoro-2-methylpiperidin-1-yl) prop-2-en-1-one;

ethyl 4- (((3 s,4s,6 s) -1-acryloyl-4-fluoro-6-methylpiperidin-3-yl) amino) -7H-pyrrole [2,3-d ] pyrimidine-5-carboxylic acid;

Ethyl 4- (((3 r,4r,6 r) -1-acryloyl-4-fluoro-6-methylpiperidin-3-yl) amino) -7H-pyrrole [2,3-d ] pyrimidine-5-carboxylic acid;

1- ((2S, 4S, 5S) -5- ((5-cyano-7H-pyrrolo [2,3-d ] pyridin-4-yl) amino) -4-fluoro-2-methylpiperidin-1-yl) prop-2-en-1-one;

1- ((2 r,4r,5 r) -5- ((5-cyano-7H-pyrrolo [2,3-d ] pyridin-4-yl) amino) -4-fluoro-2-methylpiperidin-1-yl) prop-2-en-1-one;

1- ((2S, 4S, 5S) -5- ((7H-pyrrolo [2,3-d ] pyridin-4-yl) amino) -4-fluoro-2-methylpiperidin-1-yl) prop-2-en-1-one;

1- ((2 r,4r,5 r) -5- ((7H-pyrrolo [2,3-d ] pyridin-4-yl) amino) -4-fluoro-2-methylpiperidin-1-yl) prop-2-en-1-one;

1- ((2S, 4S, 5S) -5- ((7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) -4-fluoro-2-methylpiperidin-1-yl) prop-2-en-1-one;

1- ((2 r,4r,5 r) -5- ((7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) -4-fluoro-2-methylpiperidin-1-yl) prop-2-en-1-one;

or a pharmaceutically acceptable salt thereof.

In other aspects, the invention also provides:

a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a compound of the invention;

a method of treating or preventing a disorder or condition selected from the group consisting of inflammatory bowel disease, proctitis, eosinophilic gastroenteritis or mastocytosis, myositis, vasculitis, tendril sore, alzheimer's disease, lupus, nephritis, systemic lupus erythematosus, psoriasis, eczematous dermatitis, pruritis or other pruritic conditions, white-spotted wind, alopecia, autoimmune thyroiditis, multiple sclerosis, major depression, asthma, xerosis, systemic sclerosis, polyarteritis nodosa, dry eye syndrome, autoimmune hemolytic anemia, autoimmune atrophic gastritis of pernicious anemia, autoimmune encephalomyelitis, autoimmune orchitis, autoimmune thrombocytopenia, sympathogenic ophthalmia, myasthenia gravis, primary biliary cirrhosis, chronic active hepatitis, membranous glomerulopathy, organ transplant rejection, graft-versus-host disease, such as bone marrow, cartilage, cornea, heart, intervertebral disc, lung, kidney, muscle, pancreatic, adult cell, pancreatic cell, or pancreatic cell, or pancreatic transplant, and limb transplant rejection, chronic neuroinflammation, ocular diseases, disorders or conditions associated with neuropsychiatric disorders including ankylosing spondylitis, autoimmune alopecia, chronic obstructive pulmonary disease, acute respiratory disease, cachexia, and autoantibody mediated encephalopathy (including autoimmune diseases of the eye: keratoconjunctivitis, vernal conjunctivitis, uveitis including uveitis associated with Behcet's disease and lens-induced uveitis, keratitis, scar keratitis, keratoconus, corneal epithelial dystrophy, A method of treating keratoleukoplakia, iritis, keratoconjunctivitis sicca (dry eye), small scars, iridocyclitis, sarcoidosis, endocrinological eye disease, sympathogenic eye inflammation, allergic conjunctivitis and ocular neovascularization comprising the step of administering to a subject a therapeutically effective amount of a compound comprising a compound described herein, an optical isomer thereof or a mixture thereof, a pharmaceutically acceptable salt, solvate, N-oxide thereof or prodrug thereof, or a combination thereof. The method is by administering to a mammal in need thereof a therapeutically effective amount of a compound of the invention or a pharmaceutically acceptable salt thereof.

A method of treating a condition or disorder, including atopic dermatitis, eczema, psoriasis, scleroderma, lupus, pruritis, other pruritic conditions, summer eczema, inflammatory airway diseases, recurrent airway obstruction, airway hyperreactivity, and chronic obstructive pulmonary disease, by administering to a mammal in need thereof a therapeutically effective amount of a compound of the invention, an optical isomer thereof, or mixtures thereof, pharmaceutically acceptable salts, solvates thereof, N-oxides thereof, or prodrugs thereof, or a combination thereof; and a process for preparing the compounds of the present invention. The invention will be further understood by the following description (given by way of example only). The present invention relates to a class of pyrrolo [2,3-d ] pyrimidinyl and pyrrolo [2,3-d ] pyridine derivatives and analogs thereof. In particular, the present invention relates to compounds useful as inhibitors of JAK (particularly JAK 3) including pyrrolo [2,3-d ] pyrimidinyl and pyrrolo [2,3-d ] pyridine derivatives and analogs thereof. While the invention is not so limited, an appreciation of various aspects of the invention will be gained through a discussion and examples below.

The compounds of the invention may be administered alone or in combination with one or more additional agents that modulate the immune system of a mammal or with anti-inflammatory agents in pharmaceutically acceptable forms. These agents may include, but are not limited to, cyclosporin a (e.g., mountain mine) ^TM Or be as new as possible ^TM ) Rapamycin, FK-506 (tacrolimus), leflunomide, deoxyspergualin, mycophenolate (e.g., cellknow) ^TM ) Azathioprine (e.g. Epilobium) ^TM ) Daclizumab (e.g. cenipenem) ^TM ) OKT3 (e.g. orthoclone) ^TM )、AtGam ^TM Aspirin, acetaminophen, ibuprofen, naproxen, piroxicam and anti-inflammatory steroids (e.g. Deflazacor, prednisolone or dexamethasone), IFN-beta, teriflunomide, laquinimod, glatiramer acetate, dimethyl fumarate (dimethylf umerate), rituximab, fingolimod, natalizumab, alemtuzumab, mitoxantrone, sulfasalazinePyridine (Azulfidine), mesalazine (apris, an Sake, lialda, etc.), balsalazide (disodium balsalazide) and olsalazine (sodium olsalate (Dipentum)), mercaptopurine (Purinethol)), antibiotics (antimycobacterial agents such as metronidazole, ciprofloxacin), wu Sinu mab and vedolizumab. These agents may be administered in the same or separate dosage forms, by the same or different routes of administration, and according to the same or different administration schedules, according to standard pharmaceutical procedures known to those skilled in the art.

The present invention also provides a JAK3 selective inhibitor composition, optical isomers thereof or mixtures thereof, including the compounds of the present invention, pharmaceutically acceptable salts, solvates or prodrugs thereof. In the present invention, all isomers are intended to be included if not specifically stated. For example, double bonds, collective isomers in the ring (E-form, Z-form, cis-form, trans-form), alkyl groups include straight-chain alkyl groups and branched-chain alkyl groups, optical isomers resulting from the presence of asymmetric carbon atoms and the like (R, S-form), and mixtures thereof in any ratio, racemic mixtures, and all isomers resulting from tautomers are included in the present invention.

The compounds of formula I can be converted into the corresponding salts by known methods. Examples of salts which are preferably water-soluble pharmaceutically acceptable non-toxic acid addition salts are salts of amino groups with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid, or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid, or by using other methods known in the art, for example ion exchange. Other pharmaceutically acceptable salts include adipic acid salts, alginates, ascorbates, aspartic acid salts, benzenesulfonic acid salts, benzoic acid salts, bisulfate salts, boric acid salts, butyric acid salts, camphoric acid salts, citric acid salts, cyclopentanepropionic acid salts, digluconate, dodecylsulfate, ethanesulfonic acid salts, formate salts, fumaric acid salts, glucoheptonate, glycerophosphate, gluconate salts, hemisulfate salts, heptanoate salts, caproate salts, hydroiodides, 2-hydroxy-ethanesulfonic acid salts, lactobionate salts, Lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, and the like. Salts derived from suitable bases include alkali metal salts, alkaline earth metal salts, ammonium salts and N ⁺ (C _1-4 Alkyl group ₄ And (3) salt. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Additional pharmaceutically acceptable salts include nontoxic ammonium, quaternary ammonium, and amine cations formed using counter ions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates, and aryl sulfonates, as appropriate.

Unless otherwise indicated, the term "JAK3 inhibitor" is provided herein to include compounds of formula (i), formula (ii), formula (iii), formula (iv), each of which includes different stereoisomers of the same formula, wherein stereoisomers also include enantiomers, i.e., optical isomers, and diastereomers, i.e., stereoisomers that are not chiral enantiomers, and different isomers of the same formula as the compounds of the present invention are also within the scope of the present invention.

Unless otherwise indicated, the term "solvate" herein may also be referred to as "solvent compound", "solvate" refers to a solvent-containing compound in which solvent molecules may be associated with the compound molecules in other ways including coordinate bonds, covalent bonds, van der Waals forces, ionic bonds, hydrogen bonds, and the like.

The term "pharmaceutically acceptable salt" as used herein refers to a compound of the invention and/or the salt formed, which is chemically or physically compatible with the other ingredients comprising a pharmaceutical dosage form, and which is physiologically compatible with the recipient, unless otherwise indicated. The "pharmaceutically acceptable salts" may be acid and/or basic salts with inorganic and/or organic acids and bases, and also include zwitterionic salts (inner salts) and also include quaternary ammonium salts, such as alkylammonium salts. These salts may be obtained directly in the final isolation and purification of the compounds. Or by appropriately mixing the compound of the present invention or a stereoisomer or solvate thereof with a certain amount of an acid or a base. These salts may be obtained by precipitation in solution and collected by filtration, or recovered after evaporation of the solvent, or by reaction in an aqueous medium and then cooled and dried.

The term "alkyl" as used herein, unless otherwise indicated, refers to having from 1 to 4 carbon atoms ("C1-4 alkyl"). In some embodiments, the alkyl group has 1 to 3 carbon atoms ("C1-3 alkyl"). In some embodiments, the alkyl group has 1 to 2 carbon atoms ("C1-2 alkyl"). In some embodiments, the alkyl group has 1 carbon atom ("C1 alkyl"). Each instance of an alkyl group is independently optionally substituted, i.e., unsubstituted ("unsubstituted alkyl") or substituted with one or two substituents ("substituted alkyl").

Unless otherwise indicated, the term "5-membered heteroaryl" as used herein includes, unless otherwise indicated, exemplary 5-membered heteroaryl groups containing one heteroatom, including, but not limited to, pyrrolyl, furanyl, and thiophenyl. Exemplary 5-membered heteroaryl groups containing two heteroatoms include, but are not limited to, imidazolyl, pyrazolyl, oxazolinyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5-membered heteroaryl groups containing three heteroatoms include, but are not limited to, thiazolyl, oxadiazolyl, and thiadiazolyl. Exemplary 5-membered heteroaryl groups containing four heteroatoms include, but are not limited to, tetrazolyl.

Unless otherwise indicated, the term "heterocycloalkyl" as used herein refers to a group of a stable 3-10 membered saturated heterocyclic ring system consisting of carbon, in which one or more of the atoms constituting the ring of the non-aromatic ring is a heteroatom, including but not limited to nitrogen, oxygen, sulfur, etc. Unless specifically indicated otherwise in the specification, the heteroaryl group may be a monocyclic ("monocyclic heterocycloalkyl") or a bicyclic, tricyclic or higher ring system which may include a fused, bridged (bridged) or spiro ring system (e.g., bicyclic system ("bicyclic heterocycloalkyl"). The heterocycloalkyl bicyclic ring system may include one or more heteroatoms in one or both rings, and is saturated. Typical 5-6 membered monocyclic heterocyclyl groups contain 1 or more heteroatoms selected independently from N, O and S. In the scheme, the heterocyclic alkyl is 4-6 membered heterocyclic alkyl, wherein hetero atoms are selected from one or more of N, O and S, and the hetero atoms are 1, 2 or 3.

The term "heteroaryl" refers to an aromatic group containing heteroatoms, which may be a single ring or a fused ring, preferably containing 1 to 4 5-12 membered heteroaryl groups independently selected from N, O and S, including but not limited to pyrrolyl, furanyl, thienyl, imidazolyl, oxazolyl, isoxazolyl, pyrazolyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, quinolinyl, isoquinolinyl, triazolyl, tetrahydropyrrolyl. In one embodiment, a 5-6 membered monocyclic heteroaryl group typically containing 1 or more heteroatoms independently selected from N, O and S.

Where no substituent is explicitly indicated in a recited group, such a group is merely unsubstituted. For example, when the "C1-C4 alkyl" has no "substituted or unsubstituted" limitation before, "the" C1-C4 alkyl "itself or" unsubstituted C1-C4 alkyl "is only meant.

In the various parts of the invention, linking substituents are described. When the structure clearly requires a linking group, the markush variables recited for that group are understood to be linking groups. For example, if the structure requires a linking group and the markush group definition for that variable enumerates an "alkyl" or "aryl" group, it will be understood that the "alkyl" or "aryl" represents a linked alkylene group or arylene group, respectively.

In some specific structures, when an alkyl group is explicitly represented as a linking group, then the alkyl group represents a linked alkylene group, e.g., the C1-C6 alkyl in the group "halo-C1-C6 alkyl" is understood to be a C1-C6 alkylene.

The term "halogen" (halo and halogen) refers to fluorine (F), chlorine (Cl), bromine (Br) or iodine (I).

The term "mammal" refers to humans, domestic animals or cats and dogs.

Unless otherwise specified, all technical and scientific terms used herein have the standard meaning of the art to which the claimed subject matter belongs. In case there are multiple definitions for a term, the definitions herein control. As used herein, the singular forms "a", "an", and "the" are understood to include plural referents unless the context clearly dictates otherwise.

Furthermore, the term "comprising" is an open-ended limitation and does not exclude other aspects, i.e. it includes the content indicated by the invention.

Unless otherwise indicated, the present invention employs conventional methods of mass spectrometry, nuclear magnetism, and the like to identify compounds, and the procedures and conditions may be referred to procedures and conditions conventional in the art.

The present invention employs, unless otherwise indicated, standard nomenclature for analytical chemistry, organic synthetic chemistry and optics, and standard laboratory procedures and techniques. In some cases, standard techniques are used for chemical synthesis, chemical analysis, and light emitting device performance detection.

In addition, unless explicitly stated otherwise, the description that "…" is independently selected "as used in the present invention is to be construed broadly as meaning that each individual described is independently selected from the others. Thus, each substituent may be the same or different from the other substituents. In more detail, the description that "… is independently selected" may mean that specific options expressed between the same symbols in different groups do not affect each other; it may also be expressed that specific options expressed between the same symbols in the same group do not affect each other.

Those skilled in the art will appreciate that, in accordance with convention used in the art, the present application describes structural formulae of groups

The above preferred conditions can be arbitrarily combined on the basis of not deviating from the common knowledge in the art, and thus, each preferred embodiment of the present invention can be obtained.

The reagents and materials used in the present invention are commercially available.

In certain embodiments of the invention, a compound of the invention, or a stereoisomer or prodrug thereof, or a pharmaceutically acceptable salt of a stereoisomer or prodrug of the compound, may be in the form of a pharmaceutical composition comprising a pharmaceutically acceptable carrier, vehicle or diluent. They are also useful in the preparation of medicaments for the treatment of diseases associated with abnormal JAK3 kinase activity.

Detailed Description

The invention is further illustrated by way of examples below, which provide synthetic examples and biological examples described herein to illustrate the compounds, pharmaceutical compositions, and methods provided herein. The following examples are given for illustration of the present invention only and are not intended to limit the present invention, and modifications, changes, variations, etc. within the scope of the present invention are within the scope of the present invention.

The compounds provided herein can be prepared from readily available starting materials using the particular synthetic protocols set forth below, which will be well known to those skilled in the art. The experimental procedures, which do not address the specific conditions in the examples below, can be determined by one skilled in the art by routine optimization procedures, according to conventional methods and conditions.

In the examples which follow, the abbreviations explain:

Boc ₂ o: di-t-butyl dicarbonate;

DIEA: n, N-diisopropylethylamine

Xantphos:4, 5-bis (diphenylphosphine) -9, 9-dimethylxanthene

dppf Pd G3: (methanesulfonic acid (1, 1' -bis (diphenylphosphino) ferrocene) (2 ' -amino-1, 1' -biphenyl-2-yl) palladium (II)

Pd (PPh 3) 4: tetrakis (triphenylphosphine) palladium

Pd(Pd(PPh ₃ ) ₂ Cl ₂ : diphenyl phosphorus dichloropalladium

Pd(dppf)Cl ₂ : [1,1' -bis (diphenylphosphorus) ferrocene]Palladium dichloride

Pd ₂ (dba) ₃ : tris (dibenzylideneacetone) dipalladium

CuI: cuprous iodide

Pd/C: palladium carbon catalyst

KOtBu: potassium tert-butoxide

NaOtBu: sodium tert-butoxide

SEMCl:2- (trisilyl) ethoxymethyl chloride

NIS: n-iodosuccinamides

TEA: triethylamine

TMSOTF: trimethylsilane triflate

TFA: trifluoroacetic acid

TFAA: trifluoroacetic anhydride

TMS: trimethylsilyl group

PE: petroleum ether;

EA: ethyl acetate;

DMF: n, N-dimethylformamide;

DCM: dichloromethane;

THF: tetrahydrofuran (THF)

MeOH: methanol

Na ₂ CO ₃ : sodium carbonate

Prep-HPLC high pressure liquid chromatography

Rf: a ratio shift value;

g; gram (g)

mg: mg of (milligram)

h: hours of

And rt: room temperature

mol: molar (mol)

mmol: millimoles (milli)

mL: milliliters of (milliliters)

M: moles/liter

Example 1: preparation of 1- ((2S, 4S, 5S) -5- ((7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) -4-fluoro-2-methylpiperidin-1-yl) propen-2-en-1-one (Compound 1) and 1- ((2R, 4R, 5R) -5- ((7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) -4-fluoro-2-methylpiperidin-1-yl) propen-2-en-1-one (Compound 2)

Compound 1 and compound 2 were synthesized with reference to the following steps:

step 1: allyl amine 1-2 (13.07 mL,174 mmol) was added dropwise to acetaldehyde 1-1 (34.8 mL,174mmol,5 mol/L) at 0deg.C, the reaction stirred at 25deg.C for 1 hour, molecular sieves (15 g) and tetrahydrofuran (250 mL) were added and cooled to 0deg.C, then allyl magnesium bromide (1.0 mol/L in Et) ₂ O,191.4mL,191.4 mmol). After stirring for 30 minutes, a solution of benzyl chloroformate (35.70 g,208.8 mmol) in tetrahydrofuran (50 mL) was slowly added and the reaction mixture was warmed to 25℃and stirred for 1 hour. At the end of the reaction, the reaction was quenched with saturated ammonium chloride solution (900 mL), extracted with ethyl acetate (500 mL X3), the organic phase was washed with brine and dried over anhydrous sodium sulfate, and the filtrate was concentrated by filtration. Purification by silica gel column chromatography (petroleum ether/ethyl acetate=10:1) afforded compound 1-3 (23 g, purity 90%, yield 45%). LCMS (ESI) [ M+H ]] ⁺ m/z＝260.0。

Step 2:benzyl allyl (pent-4-en-2-yl) carbamate 1-3 (23 g,88.7 mmol) was dissolved in dichloromethane (2L) and phenylmethylenebis (tricyclohexylphosphorus) ruthenium dichloride (3.65 g,4.4 mmol) was added. The reaction was stirred at 25℃for 5 hours under nitrogen. The mixture was concentrated and purified by silica gel column chromatography (petroleum ether/ethyl acetate=6:1) to give compound 1-4 (21 g, purity 85%, yield 87%). LCMS (ESI) [ M+H ]] ⁺ m/z＝232.1。

Step 3: benzyl 2-methyl-3, 6-dihydropyridine-1 (2H) -carboxylate 1-4 (21 g,90.8 mmol) was dissolved in acetonitrile (706 mL), disodium ethylenediamine tetraacetate aqueous solution (0.0004 mol/L, 457 mL,0.18 mmol) was added, the solution was cooled to 0deg.C, and 1, 1-trifluoroacetone (101.74 g, 328 mmol) was added. A mixture of potassium peroxomonosulphonate (279.1 g,454 mmol) and sodium hydrogen carbonate (61.02 g,726.4 mmol) was added in portions over 30 minutes and the reaction mixture was stirred at 0℃for a further 1 hour. At the end of the reaction, the reaction was quenched with saturated sodium sulfite solution (1.2L) and stirred for 0.5 h, extracted with ethyl acetate (800 mLX 3), the organic phase was washed with brine and dried over anhydrous sodium sulfate, filtered and the filtrate concentrated, and the crude product was purified by silica gel column chromatography (petroleum ether/ethyl acetate=3:1) to give compound 1-5 (14 g, purity 90%, yield 59%). LCMS (ESI) [ M+H ] ] ⁺ m/z＝248.1。

Step 4: (1R, 4S, 6S) -4-methyl-7-oxa-3-azabicyclo [4.1.0]Benzyl heptane-3-carboxylate 1-5 (12.4 g,50.1 mmol) was dissolved in methanol (90 mL) and water (30 mL) and sodium azide (9.77 g,150.3 mmol) and ammonium chloride (6.03 g,112.73 mmol) were added. The reaction mixture was stirred at 60 ℃ for 16 hours. The reaction was concentrated, diluted with water (150 mL) and extracted with ethyl acetate (150 mL x 2). The organic phase was washed with brine and dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated to give crude 1-6 (14 g) which was used in the next step without further purification. LCMS (ESI) [ M+H ]] ⁺ m/z＝291.1。

Step 5: (2S, 4S, 5S) -5-azido-4-hydroxy-2-methylpiperidine-1-carboxylic acid benzyl ester 1-6 (14 g,48.2 mmol) was dissolved in (100 mL) and triphenylphosphine (12.64 g,48.2 mmol) was added. The reaction was stirred at 85℃for 9 hours under nitrogen. The reaction solution was concentrated and purified by silica gel column chromatography (petroleum ether/ethyl acetate=1.5:1) to giveCompounds 1-7 (20 g, 55% purity). LCMS (ESI) [ M+H ]] ⁺ m/z＝247.2。

Step 6: compounds 1-7 (20 g,44.7mmol, purity 55%) were dissolved in anhydrous dichloromethane (120 mL) and triethylamine (9.05 g,89.4 mmol) and catalytic amounts of 4-dimethylaminopyridine (270 mg,2.235 mmol) were added at 0deg.C. The reaction mixture was stirred at 0deg.C for 15 min under nitrogen, then a solution of di-tert-butyl dicarbonate (19.51 g,89.4 mmol) in anhydrous dichloromethane (20 mL) was added dropwise. The reaction was stirred at 0℃for 1 hour. At the end of the reaction, quench the reaction by addition of ice water (300 mL), extract with dichloromethane (150 mL x 2), wash the organic phase with brine (300 mL x 2) and dry over anhydrous sodium sulfate, filter concentrate, purify by silica gel column chromatography (petroleum ether/ethyl acetate=3:1) to give compounds 1-8 (12 g, purity 90%, yield 69%). LCMS (ESI) [ M+Na ] ] ⁺ m/z＝369.2。

Step 7: compounds 1-8 (10 g,28.8 mmol) were dissolved in tetrahydrofuran (100 mL), tetrabutylammonium fluoride (1 mol/Lin THF,28.8mL,28.8 mmol) was added and the reaction stirred at 45℃for 48 hours under nitrogen. At the end of the reaction, dilute with saturated aqueous sodium bicarbonate (300 mL) and extract with ethyl acetate (200 mL x 2), wash the organic phase with saturated brine (250 mL), dry over anhydrous sodium sulfate, filter concentrate, purify by silica gel column chromatography (petroleum ether/ethyl acetate=3:1) to give compounds 1-9 (2.5 g, purity 90%, yield 16.6%). . LCMS (ESI) [ M+Na ]] ⁺ m/z＝389.2。

Step 8: 1-9 (2.5 g,6.8 mmol) was dissolved in dichloromethane (20 mL), a 1, 4-dioxane solution of hydrogen chloride (6 mL,4 mol/L) was added, and the mixture was stirred at 25℃for 4 hours. Concentration gave crude compound 1-10 (2 g) which was used in the next step without further purification. LCMS (ESI) [ M+H ]] ⁺ m/z＝267.14。

Step 9: 1-10 (4.32 g,16.55 mmol) was dissolved in N-butanol (50 mL), 1-11 (4.67 g,24.8 mmol) and N, N-diisopropylethylamine (5.97 g,46.2 mmol) were added, and the reaction was stirred at 140℃for 30 hours under nitrogen. After the reaction was completed, the PH was adjusted to ph=10 with a sodium carbonate solution, extracted with ethyl acetate (200 ml X3), the organic phase was washed with brine and dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated, and the extract was purified by Purification by silica gel column chromatography (dichloromethane/methanol=20:1) afforded compounds 1-12 (2.93 g, 42% yield). LCMS (ESI) [ M+H ]] ⁺ m/z＝418.1。

Step 10: compound 1-12 (700 mg,1.67 mmol) was dissolved in tetrahydrofuran (10 mL) and water (3 mL), 10% palladium on carbon (200 mg) was added, the reaction mixture was stirred under hydrogen protection (5 MPa) at 45℃for 24 hours, and after completion of the reaction, the crude product 1-13 (333 mg, yield 80%) was obtained by filtration and concentrated and used in the next step without further purification. LCMS (ESI) [ M+H ]] ⁺ m/z＝250.2.

Step 13: 1-13 (100 mg,0.4 mmol) was dissolved in tetrahydrofuran (2.5 mL), an aqueous solution (1 mL) of potassium phosphate (3411 mg,1.6 mmol) was added, the reaction mixture was stirred at 0deg.C, and a solution of 3-chloropropionyl chloride (62 mg,0.48 mmol) in tetrahydrofuran (0.5 mL) was added dropwise and stirring was continued at 0deg.C for 2 hours. An aqueous solution (1 mL) of sodium hydroxide (81 mg,2.01 mmol) was added dropwise to the above reaction mixture, and the reaction solution was stirred at room temperature for 18 hours. At the end of the reaction, it was diluted with saturated ammonium chloride solution (20 mL), extracted with ethyl acetate (10 mL X3), the organic phase was washed with brine and dried over anhydrous sodium sulfate, concentrated by filtration, and purified by Chiral-HPLC (40% EtOH/(NH) ₄ OH 0.2%) to give the crude product, which was further purified by preparative HPLC (0.1% fa/mecn=90% to 60%) to give the following white solid:

Compound 1 (21.9 mg, purity 99.442%), LCMS (ESI) [ M+H)] ⁺ m/z＝304.2. ¹ H NMR(400MHz,CD ₃ OD)δ7.20(s,1H),6.37(d,J＝2.0Hz,1H),6.13(dd,J＝12.9,8.9Hz,1H),5.97(s,1H),5.64(d,J＝13.9Hz,1H),5.28(d,J＝8.5Hz,1H),4.77-4.68(m,1H),4.67-4.61(m,0.5H),4.58(s,0.5H),4.50(s,0.5H),4.36(s,0.5H),4.19-4.09(m,1.5H),3.27 -2.98(m,1H),2.44(s,1H),2.20(d,J＝6.1Hz,1H),1.71(s,3H).

Compound 2 (15.6 mg, purity 99.724%), LCMS (ESI) [ M+H)] ⁺ m/z＝304.2. ¹ H NMR(400MHz,CD ₃ OD)δ7.20(s,1H),6.37(d,J＝2.6Hz,1H),6.13(dd,J＝13.3,8.6Hz,1H),5.96(d,J＝2.3Hz,1H),5.64(dd,J＝13.5,1.5Hz,1H),5.28(dd,J＝8.4,1.4Hz,1H),4.78-4.68(m,1H),4.67-4.58(m,1H),4.56-4.43(m,0.5H),4.36(s,0.5H),4.20-4.10(m,1.5H),3.26-3.01(m,1H),2.44(d,J＝4.5Hz,1H),2.25(s,1H),1.77-1.68(m,3H)..

Example 2:1- ((2S, 4S, 5S) -5- ((7H-pyrrolo [2,3-d ] pyridin-4-yl) amino) -4-fluoro-2-methylpiperidin-1-yl) propen-2-en-1-one (Compound 3) and 1- ((2R, 4R, 5R) -5- ((7H-pyrrolo [2,3-d ] pyridin-4-yl) amino) -4-fluoro-2-methylpiperidin-1-yl) propen-2-en-1-one (Compound 4)

Synthesis and compound 3 and compound 4 refer to the following steps:

step 1: compound 3-1 (4.6 g,30 mmoles) was dissolved in DMF (50 mL) and cooled to 0℃in a reaction flask, 60% NaH (1.4 g,36 mmoles) was added to the reaction system, and the mixture was stirred at the same temperature for 1h, followed by dropwise addition of SEMCl (6.8 mL,39 mmoles). After the addition was completed, the system was stirred at 0℃for 4 hours. LCMS monitored the end of the reaction. The reaction mixture was taken up in 100mL of saturated NH4Cl, extracted with ethyl acetate (50 mLX 3) and the extracted organic phase was taken up in Na ₂ SO ₄ The crude product obtained after concentrating under reduced pressure after drying and filtering was purified by thin layer chromatography plate preparation (ethyl acetate) to give compound 3-2 (7.67 g, yield 90%) as a white solid. LCMS (ESI) M/z [ M+H] ⁺ ＝283.1。

Step 2: a mixture of compound 3-2 (1.05 g,3.72 mmol) and compound 1-10 (5.62 g,18.6 mmol) was added to a round bottom flask with 2- (di-tert-butylphosphino) biphenyl (222 mg,0.75 mmol), cs ₂ CO ₃ (1.2 g,3.72 mmol) and Pd (OAc) ₂ (84 mg,0.36 mmol). Stirring was carried out at 110℃under nitrogen for 2h, cooling to room temperature and dilution with CHCl3 and MeOH was carried out. After stirring at room temperature for 10 minutes, the mixture was filtered through celite. The filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography (n-hexane/ethyl acetate=9/1 to 4/1) to give compound 3-3 (889 mg,50% yield). LCMS (ESI) M/z [ M+H] ⁺ ＝479.2。

Step 3: compound 3-3 (750 mg,1.84 mmol) was dissolved in 10mL THF, tetramethyl ethylenediamine (0.64 g,5.52 mmol) and 5.52mL TBAF THF (1M in THF,5.52mmol) were addedA solution. The reaction was warmed to 60 degrees overnight. 25 ml of ethyl acetate was added for extraction, and the mixture was washed three times with saturated brine, dried over anhydrous sodium sulfate, filtered and dried by spin-drying, and eluted with a silica gel column (PE: EA=10:1) to give a product of compound 3-4 (397 mg, yield: 62%). LCMS (ESI) M/z [ M+H] ⁺ ＝349.2。

Step 4: compound 3-4 (390 mg,1.12 mmol) was dissolved in tetrahydrofuran (15 mL) and water (5 mL), 10% palladium on carbon (300 mg) was added, the reaction mixture was stirred under hydrogen (5 MPa) at 45℃for 24 hours, the reaction was completed, and the concentration was filtered to give crude product 3-5 (237 mg, yield 85%) which was used in the next step without further purification. LCMS (ESI) [ M+H ]] ⁺ m/z＝249.14。

Step 5: 3-5 (99 mg,0.4 mmol) was dissolved in tetrahydrofuran (2.5 mL), an aqueous solution (1 mL) of potassium phosphate (3411 mg,1.6 mmol) was added, the reaction mixture was stirred at 0deg.C, and a solution of 3-chloropropionyl chloride (62 mg,0.48 mmol) in tetrahydrofuran (0.5 mL) was added dropwise and stirring was continued at 0deg.C for 2 hours. An aqueous solution (1 mL) of sodium hydroxide (81 mg,2.01 mmol) was added dropwise to the above reaction mixture, and the reaction solution was stirred at room temperature for 18 hours. At the end of the reaction, it was diluted with saturated ammonium chloride solution (20 mL), extracted with ethyl acetate (10 mL X3), the organic phase was washed with brine and dried over anhydrous sodium sulfate, concentrated by filtration, and purified by Chiral-HPLC (40% EtOH/(NH) ₄ OH 0.2%) to give the crude product, which was further purified by preparative HPLC (0.1% fa/mecn=90% to 60%) to give the following white solid:

compound 3 (35.1 mg, 99.2% purity), LCMS (ESI) [ M+H)] ⁺ m/z＝303.2. ¹ H NMR(400MHz,CD ₃ OD)δ8.26(d,J＝2.4Hz,1H),7.96(m,1H),7.01(d,J＝2.4Hz,1H),6.37(d,J＝2.0Hz,1H),6.13(dd,J＝12.9,8.9Hz,1H),5.97(s,1H),5.65(d,J＝13.9Hz,1H),5.27(d,J＝8.5Hz,1H),4.78-4.68(m,1H),4.67-4.61(m,0.5H),4.58(s,0.5H),4.50(s,0.5H),4.36(s,0.5H),4.20-4.10(m,1.5H),3.26 -3.15(m,0.5H),2.98(s,0.5H),2.45(s,1H),2.21(d,J＝6.1Hz,1H),1.73(s,3H)..

Compound 4 (25.3 mg, 99.5% purity), LCMS (ESI) [ M+H)] ⁺ m/z＝303.2. ¹ H NMR(400MHz,CD ₃ OD)δ8.27(d,J＝2.4Hz,1H),7.98(m,1H),7.02(d,J＝2.4Hz,1H),6.38(d,J＝2.0Hz,1H),6.15(dd,J＝12.9,8.9Hz,1H),5.97(s,1H),5.65(d,J＝13.9Hz,1H),5.27(d,J＝8.5Hz,1H),4.78-4.68(m,1H),4.67-4.62(m,0.5H),4.56(s,0.5H),4.52(s,0.5H),4.36(s,0.5H),4.20-4.10(m,1.5H),3.26 -2.98(m,1H),2.44(s,1H),2.20(d,J＝6.1Hz,1H),1.74(s,3H).

Example 3:1- ((2S, 4S, 5S) -5- ((5-cyano-7H-pyrrolo [2,3-d ] pyridin-4-yl) amino) -4-fluoro-2-methylpiperidin-1-yl) propen-2-en-1-one Compound 5 and 1- ((2R, 4R, 5R) -5- ((5-cyano-7H-pyrrolo [2,3-d ] pyridin-4-yl) amino) -4-fluoro-2-methylpiperidin-1-yl) propen-2-en-1-one (Compound 6)

The preparation steps of compound 5 and compound 6 are as follows:

step 1: 1-10 (700 mg,2.78 mmol) was dissolved in N-butanol (10 mL), 5-1 (602 mg,3.47 mmol) and N, N-diisopropylethylamine (896 mg,6.94 mmol) were added, and the reaction was stirred at 100℃for 10 hours under nitrogen. After the reaction was completed, the PH was adjusted to ph=10 with a sodium carbonate solution, extracted with ethyl acetate (30 ml X3), the organic phase was washed with brine and dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated, and purified by silica gel column chromatography (dichloromethane/methanol=20:1) to give compound 5-2 (678 mg, yield 72%). LCMS (ESI) [ M+H ]] ⁺ m/z＝408.18。

Step 2: compound 5-2 (678 mg,1.66 mmol) was dissolved in tetrahydrofuran (10 mL) and water (3 mL), 10% palladium on carbon (200 mg) was added, the reaction mixture was stirred under hydrogen protection (5 MPa) at 45℃for 24 hours, and after completion of the reaction, the crude product 5-3 (386 mg, yield 85%) was obtained by filtration and was used in the next step without further purification. LCMS (ESI) [ M+H ] ] ⁺ m/z＝274.14.

Step 3: 5-3 (200 mg,0.73 mmol) was dissolved in tetrahydrofuran (5 mL), an aqueous solution (2 mL) of potassium phosphate (680 mg,0.32 mmol) was added, the reaction mixture was stirred at 0deg.C, and a solution of 3-chloropropionyl chloride (124 mg,0.96 mmol) in tetrahydrofuran (0.5 mL) was added dropwise and stirring was continued at 0deg.C for 2 hours. An aqueous solution (1 mL) of sodium hydroxide (160 mg,4 mmol) was added dropwise to the above reaction mixture, and the reaction solution was stirred at room temperature for 18 hours. At the end of the reaction, dilute with saturated ammonium chloride solution (20 mL), extract with ethyl acetate (10 mL X3), wash the organic phase with brine and dry with anhydrous sodium sulfate, filter concentrate, purify by Chiral-HPLC (40% etoh/(NH 4OH 0.2%)) to give the crude product which is further purified by preparative HPLC (0.1% fa/mecn=90% to 60%) to give the following white solid:

compound 5 (45 mg, 99.2% purity), LCMS (ESI) [ M+H)] ⁺ m/z＝328.15. ¹ H NMR(400MHz,CD ₃ OD)δ11.12(s,1H),9.06(s,1H),7.93-7.85(m,1H),6.62(dd,J＝12.9,8.9Hz,1H),6.15-6.05(m,1H),5.58(d,J＝13.9Hz,1H),5.28(d,J＝8.5Hz,1H),4.77-4.68(m,1H),4.67-4.60(m,0.5H),4.58(s,0.5H),4.50(s,0.5H),4.36(s,0.5H),4.19-4.09(m,1.5H),3.26-2.98(m,1H),2.42(s,1H),2.21(d,J＝6.1Hz,1H),1.70(s,3H)..

Compound 6 (32 mg, 99.4% purity), LCMS (ESI) [ M+H)] ⁺ m/z＝328.15. ¹ H NMR(400MHz,CD ₃ OD)δ11.06(s,1H),9.05(s,1H),7.92-7.88(m,1H),6.64(dd,J＝12.9,8.9Hz,1H),6.16-6.08(m,1H),5.58(d,J＝13.9Hz,1H),5.28(d,J＝8.5Hz,1H),4.77-4.68(m,1H),4.67-4.60(m,0.5H),4.58(s,0.5H),4.50(s,0.5H),4.36(s,0.5H),4.20-4.06(m,1.5H),3.28-2.96(m,1H),2.44(s,1H),2.22(d,J＝6.1Hz,1H),1.73(s,3H).

Example 4: preparation of ethyl 4- (((3S, 4S, 6S) -1-propenoyl-4-fluoro-6-methylpiperidin-3-yl) amino) -7H-pyrrolo [2,3-d ] pyrimidine-5-carboxylic acid Compound 7 and ethyl 4- (((3R, 4R, 6R) -1-propenoyl-4-fluoro-6-methylpiperidin-3-yl) amino) -7H-pyrrolo [2,3-d ] pyrimidine-5-carboxylic acid (Compound 8)

Compound 7 and compound 8 were synthesized with reference to the following steps:

step 1: 1-10 (800 mg,2.64 mmol) was dissolved in N-butanol (10 mL), 7-1 (894 mg,3.86 mmol) and N, N-diisopropylethylamine (997 mg,7.72 mmol) were added, and the reaction was stirred at 140℃for 20 hours under nitrogen. Reverse-rotationAfter completion, the PH was adjusted to ph=10 with sodium carbonate solution, extracted with ethyl acetate (200 ml X3), the organic phase was washed with brine and dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated and purified by silica gel column chromatography (dichloromethane/methanol=20:1) to give compound 7-2 (782 g, yield 65%). LCMS (ESI) [ M+H ]] ⁺ m/z＝456.20。

Step 2: compound 7-2 (782 mg,1.71 mmol) was dissolved in tetrahydrofuran (10 mL) and water (3 mL), 10% palladium on carbon (200 mg) was added, the reaction mixture was stirred under hydrogen protection (5 MPa) at 45℃for 24 hours, the reaction was completed, and the concentration was filtered to give crude 7-3 (452 mg, yield 82%) which was used in the next step without further purification. LCMS (ESI) [ M+H ]] ⁺ m/z＝322.16.

Step 3: 7-3 (100 mg,0.31 mmol) was dissolved in tetrahydrofuran (2.5 mL), an aqueous solution (1 mL) of potassium phosphate (3411 mg,1.6 mmol) was added, the reaction mixture was stirred at 0deg.C, and a solution of 3-chloropropionyl chloride (62 mg,0.48 mmol) in tetrahydrofuran (0.5 mL) was added dropwise and stirring was continued at 0deg.C for 2 hours. An aqueous solution (1 mL) of sodium hydroxide (81 mg,2.01 mmol) was added dropwise to the above reaction mixture, and the reaction solution was stirred at room temperature for 18 hours. At the end of the reaction, dilute with saturated ammonium chloride solution (20 mL), extract with ethyl acetate (10 mL X3), wash the organic phase with brine and dry with anhydrous sodium sulfate, filter concentrate, purify by Chiral-HPLC (40% etoh/(NH 4OH 0.2%)) to give the crude product, further purify the crude product by preparative HPLC (0.1% fa/mecn=90% to 60%) to give the following white solid:

Compound 7 (23 mg, 99.3% purity), LCMS (ESI) [ M+H)] ⁺ m/z＝376.17. ¹ H NMR(400MHz,CD ₃ OD)δ11.04(s,1H),8.96(s,1H),7.90-7.80(m,1H),6.66(dd,J＝12.9,8.9Hz,1H),6.16-6.08(m,1H),5.58(d,J＝12.9Hz,1H),5.26(d,J＝8.5Hz,1H),4.12(q,J＝8Hz,2H)，3.80-3.20(m,5H)1.78-1.62(m,2H)，1.30(t,J＝8Hz,3H)，1.26(d,J＝6.8Hz,3H).

Compound 8 (18 mg, 99.1% purity), LCMS (ESI) [ M+H)] ⁺ m/z＝376.17. ¹ H NMR(400MHz,CD ₃ OD)δ11.01(s,1H),8.98(s,1H),7.94-7.89(m,1H),6.64(dd,J＝12.9,8.9Hz,1H),6.16-6.08(m,1H),5.58(d,J＝12.9Hz,1H),5.28(d,J＝8.5hz, 1H), 4.14 (q, j=8 hz, 2H), 3.80-3.20 (m, 5H) 1.77-1.65 (m, 2H), 1.31 (t, j=8 hz, 3H), 1.26 (d, j=6.8 hz, 3H). Example 5:1- ((2S, 4S, 5S) -5- ((5- ((R) -2, 2-difluorocyclopropyl) -7H-pyrrole [2, 3-d)]Pyrimidin-4-yl) amino) -4-fluoro-2-methylpiperidin-1-yl) prop-2-en-1-one (compound 9); 1- ((2S, 4S, 5S) -5- ((5- ((S) -2, 2-difluorocyclopropyl) -7H-pyrrole [2, 3-d)]Preparation of pyrimidin-4-yl) amino) -4-fluoro-2-methylpiperidin-1-yl) prop-2-en-1-one (compound 10) was synthesized and compound 9 and compound 10 with reference to the following steps:

step 1: 4-chloro-7H-pyrrolo [2,3-d]Pyrimidine-5-carbaldehyde 9-1 (5 g,66.45 mmol) was dissolved in N, N-dimethylformamide (50 mL) and sodium hydride (0.80 g,34 mmol) was added under ice-bath conditions followed by the slow addition of 2- (trimethylsilyl) ethoxymethyl chloride (6.90 g,41.42 mol). The reaction was allowed to react at 25℃for 2 hours. LCMS detects completion of the reaction, water was added to the reaction, and extracted with ethyl acetate. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate and concentrated. The residue obtained was subjected to a silica gel column [ petroleum ether/ethyl acetate=3/1 ]After purification, 4-chloro-7- ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2,3-d]Pyrimidine-5-amino-carbaldehyde 9-2 (5.9 g, yellow solid) in 63% yield. LCMS (ESI) [ M+H ]] ⁺ m/z 312.10.

Step 2: methyl triphenylphosphine bromide (3, 42mg,9.63 mmol) was dissolved in toluene (45 mL) and potassium tert-butoxide (1.08 g,9.63 mmol) was added under ice-bath, followed by stirring at 0deg.C for 30 min under nitrogen. Compound 9-2 (1.5 g,4.8 mmol) was again added to the reaction system and stirred for 2 hours. LCMS detects completion of the reaction, spin-dries the solvent, then adds water to the reaction and extracts with dichloromethane. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate and concentrated. The residue obtained was subjected to a silica gel column [ petroleum ether/ethyl acetate=4/1]After purification, compound 9-3 (505 mg, white solid) was obtained in yield: 34%. LCMS (ESI) [ M+H ]] ⁺ m/z 310.1.

Step 3: compound 9-3 (500mg,1.6 mmol) was dissolved in acetonitrile (10 mL), followed by the addition of sodium iodide (323 mg,4.8 mmol) and trifluoromethyl trimethylsilane (686 mg,4.8 mmol). Stirring is carried out at 110 ℃ for 1 hour under the protection of nitrogen. LCMS detects completion of the reaction, spin-dries the solvent, then adds water to the reaction and extracts with dichloromethane. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate and concentrated. The residue obtained was subjected to a silica gel column [ petroleum ether/ethyl acetate=4/1 ]After purification, compound 9-4 (400 mg, yellow solid) was obtained in yield: 69%. LCMS (ESI) [ M+H ]] ⁺ m/z360.21.

Step 4: compound (2S, 4S, 5S) -1-10 (224 mg,0.72 mmol) was dissolved in N-butanol (5 mL), 9-4 (400 g,1.11 mmol) and N, N-diisopropylethylamine (0.266 g,2.06 mmol) were added, and the reaction solution was stirred at 140℃for 30 hours under nitrogen atmosphere. After the reaction was completed, the PH was adjusted to ph=10 with a sodium carbonate solution, extracted with ethyl acetate (10 ml X3), the organic phase was washed with brine and dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated, and purified by silica gel column chromatography (dichloromethane/methanol=20:1) to give compound 9-5 (294 mg, yield 45%). LCMS (ESI) [ M+H ]] ⁺ m/z＝590.27。

Step 5: compound 9-5 (290 mg,0.49 mmol) was dissolved in 5mL THF, and tetramethyl ethylenediamine (0.17 g,1.47 mmol) and 5.52mL TBAF THF (1M in THF,1.47mmol) solution were added. The reaction was warmed to 60 degrees overnight. 25 ml of ethyl acetate was added for extraction, and the mixture was washed three times with saturated brine, dried over anhydrous sodium sulfate, filtered and dried by spin-drying, and eluted with a silica gel column (PE: EA=10:1) to give a product of compound 9-6 (180 mg, yield: 80%). LCMS (ESI) M/z [ M+H] ⁺ ＝460.2。

Step 6: compound 9-6 (180 mg,0.39 mmol) was dissolved in tetrahydrofuran (5 mL) and water (1.5 mL), 10% palladium on carbon (100 mg) was added, the reaction mixture was stirred at 45℃for 24 hours under hydrogen protection (5 MPa), and the reaction was completed, filtered and concentrated to give crude product 9-7 (102 mg, yield 80%) which was used in the next step without further purification. LCMS (ESI) [ M+H ] ] ⁺ m/z＝326.15。

Step 7: 9-7 (102 mg,0.31 mmol) was dissolved in tetrahydrofuran (2.5 mL), an aqueous solution (1 mL) of potassium phosphate (264 mg,1.24 mmol) was added, the reaction mixture was stirred at 0deg.C, and a solution of 1-14-3-chloropropionyl chloride (48 mg,0.37 mmol) in tetrahydrofuran (0.5 mL) was added dropwise and stirring was continued at 0deg.C for 2 hours. An aqueous solution (1 mL) of sodium hydroxide (62 mg,1.55 mmol) was added dropwise to the above reaction mixture, and the reaction solution was stirred at room temperature for 18 hours. At the end of the reaction, dilute with saturated ammonium chloride solution (20 mL), extract with ethyl acetate (10 mL X3), wash the organic phase with brine and dry with anhydrous sodium sulfate, filter concentrate, purify by Chiral-HPLC (40% etoh/(NH 4OH 0.2%)) to give the crude product, further purify the crude product by preparative HPLC (0.1% fa/mecn=90% to 60%) to give the following white solid:

compound 9 (38 mg, 99.2% purity), LCMS (ESI) [ M+H)] ⁺ m/z＝380.16； ¹ H NMR(400MHz,CD ₃ OD)δ11.01(s,1H),8.98(s,1H),7.92-7.83(m,1H),6.68(dd,J＝12.9,8.9Hz,1H),6.16-6.08(m,1H),5.58(d,J＝12.9Hz,1H),5.26(d,J＝8.5Hz,1H),3.80-3.20(m,5H)1.88-1.62(m,2H)，1.26(d,J＝6.8Hz,3H).

Compound 10 (45.6 mg, 99.4% purity), LCMS (ESI) [ M+H)] ⁺ m/z＝380.16； ¹ H NMR(400MHz,CD ₃ OD)δ11.03(s,1H),8.96(s,1H),7.90-7.80(m,1H),6.68(dd,J＝12.9,8.9Hz,1H),6.18-6.08(m,1H),5.60(d,J＝12.9Hz,1H),5.28(d,J＝8.5Hz,1H),3.78-3.24(m,5H)1.86-1.60(m,2H)，1.27(d,J＝6.8Hz,3H)。

Example 6: preparation of 1- ((2S, 4S, 5S) -5- ((5- (cyclopropylethynyl) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) -4-fluoro-2-methylpiperidin-1-yl) prop-2-en-1-one (Compound 11)

Reference is made to the following procedure for the synthesis and compound 11.

Step 1: compound 1 (2 g,6.59mmol, dissolved in acetonitrile (60 mL), N-iodosuccinimide (1.63 g,7.25 mmol) was added at 0deg.C, the reaction mixture was stirred at 0deg.C for 8 hours, quenched with ice water (200 mL), extracted with ethyl acetate (100 mL. Times.2), the organic phase washed with brine (300 mL. Times.2) and dried over anhydrous sodium sulfate, filtered and concentrated Purification by silica gel column chromatography (petroleum ether/ethyl acetate=3:1) afforded compound 11-1 (2.12 g, yield 75%). LCMS (ESI) [ M+Na ]] ⁺ m/z＝430.05。

Step 2: compound 11-1 (106 mg,0.25 mmol) was dissolved in anhydrous dichloromethane (3 mL) and triethylamine (102 mg,1.0 mmol) and a catalytic amount of 4-dimethylaminopyridine (2 mg) were added at 0deg.C. The reaction mixture was stirred at 0deg.C for 15 min under nitrogen, then a solution of di-tert-butyl dicarbonate (109 mg,0.5 mmol) in anhydrous dichloromethane (2 mL) was added dropwise. The reaction was stirred at 0℃for 1 hour. At the end of the reaction, quench the reaction by adding ice water (10 mL), extract with dichloromethane (20 mL x 2), wash the organic phase with brine (20 mL x 2) and dry over anhydrous sodium sulfate, filter concentrate, purify by silica gel column chromatography (petroleum ether/ethyl acetate=3:1) to give compound 11-2 (111 mg, 85% yield). LCMS (ESI) [ M+Na ]] ⁺ m/z＝530.10。

Step 3: compound 11-2 (111 mg,0.21 mmol) was dissolved in tetrahydrofuran (4 mL) under nitrogen atmosphere, dppf Pd G3 (120 mg,0.21 mmol), cuprous iodide (13 mg,0.07 mmol), dppf (12 mg,0.021 mmol), 2-cyclopropylacetylene (67 mg,1.05 mmol) and triethylamine (0.6 mL) were added sequentially, and the reaction solution was stirred at 25℃for 3 hours. The solvent was dried by spin, and the residue obtained by concentration was purified by a silica gel column (petroleum ether/ethyl acetate=1/1) to give compound 11-3 (69 mg, yield: 71%) as a white solid. LCMS (ESI) [ M+H ] ] ⁺ m/z＝468.23.

Step 4: compound 11-3 (69 mg,0.14 mmol) was dissolved in dichloromethane (1 mL) and trifluoroacetic acid (0.5 mL) was added under an ice-water bath. The reaction solution was stirred at 0℃for 2 hours. The solvent was dried to give the title product compound 11 (52 mg, yield: 68%) as a white solid. LCMS (ESI) [ M+H ]] ⁺ m/z＝368.18； ¹ H NMR(400MHz,CD ₃ OD)δ11.01(s,1H),8.98(s,1H),7.95-7.82(m,1H),6.68(dd,J＝12.9,8.9Hz,1H),6.16-6.05(m,1H),5.62(d,J＝12.9Hz,1H),5.26(d,J＝8.5Hz,1H),3.80-3.22(m,5H)1.27(d,J＝6.8Hz,3H)，0.95-0.60(m,2H)。

Example 7: preparation of 1- ((2S, 4S, 5S) -5- ((5- (3-methoxy-propyn-1-yl) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) -4-fluoro-2-methylpiperidin-1-yl) prop-2-en-1-one (Compound 12)

Synthesis and Compound 12 with reference to the following procedure

Step 1: compound 11-2 (100 mg,0.19 mmol) was dissolved in tetrahydrofuran (4 mL) under nitrogen, dppf Pd G3 (110 mg,0.19 mmol), cuprous iodide (13 mg,0.07 mmol), dppf (11 mg,0.019 mmol), 3-methoxypropyne (68 mg,0.95 mmol) and triethylamine (0.6 mL) were added sequentially, and the reaction was stirred at 25℃for 3 hours. The solvent was dried by spin, and the residue obtained by concentration was purified by a silica gel column (petroleum ether/ethyl acetate=1/1) to give compound 12-1 (58 mg, yield: 65%) as a white solid. LCMS (ESI) [ M+H ]] ⁺ m/z＝472.23.

Step 2: compound 12-1 (58 mg,0.12 mmol) was dissolved in dichloromethane (1 mL) and trifluoroacetic acid (0.5 mL) was added under an ice-water bath. The reaction solution was stirred at 0℃for 2 hours. Spin-drying the solvent to give the title product 1- ((2S, 4S, 5S) -5- ((5- (3-methoxy-propyn-1-yl) -7H-pyrrole [2, 3-d) ]Pyrimidin-4-yl) amino) -4-fluoro-2-methylpiperidin-1-yl) prop-2-en-1-one compound 12 (37 mg, yield: 81%) of a white solid. LCMS (ESI) [ M+H ]] ⁺ m/z＝372.18； ¹ H NMR(400MHz,CD ₃ OD)δ11.01(s,1H),8.98(s,1H),7.95-7.82(m,1H),6.68(dd,J＝12.9,8.9Hz,1H),6.16-6.05(m,1H),5.62(d,J＝12.9Hz,1H),5.26(d,J＝8.5Hz,1H),4.15(s,3H),3.80-3.22(m,5H)，3.30(s,3H),1.27(d,J＝6.8Hz,3H)。

Example 8: preparation of 1- ((2S, 4S, 5S) -5- ((5- (3- (fluoromethoxy) propyn-1-yl) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) -4-fluoro-2-methylpiperidin-1-yl) prop-2-en-1-one (Compound 13)

Reference is made to the following procedure for the synthesis and compound 13

Step 1: compound 11-2 (100 mg,0.19 mmol) was dissolved in tetrahydrofuran (4 mL) under nitrogen, and added sequentiallydppf Pd G3 (110 mg,0.19 mmol), cuprous iodide (13 mg,0.07 mmol), dppf (11 mg,0.019 mmol), 3- (fluoromethoxy) propyne (71 mg,0.95 mmol) and triethylamine (0.6 mL), and the reaction mixture was stirred at 25℃for 3 hours. The solvent was dried by spin, and the residue obtained by concentration was purified by a silica gel column (petroleum ether/ethyl acetate=1/1) to give compound 13-1 (53 mg, yield: 58%) as a white solid. LCMS (ESI) [ M+H ]] ⁺ m/z＝490.22.

Step 2: compound 13-1 (58 mg,0.11 mmol) was dissolved in dichloromethane (1 mL), and trifluoroacetic acid (0.5 mL) was added under an ice-water bath. The reaction solution was stirred at 0℃for 2 hours. The solvent was dried to give the title product compound 13 (26 mg, yield: 71%) as a white solid. LCMS (ESI) [ M+H ] ] ⁺ m/z＝390.18； ¹ H NMR(400MHz,CD ₃ OD)δ11.02(br s,1H),8.96(s,1H),7.96-7.82(m,1H),6.67(dd,J＝12.9,8.9Hz,1H),6.16(s,3H),6.15-6.05(m,1H),5.62(d,J＝12.9Hz,1H),5.26(d,J＝8.5Hz,1H),4.16(s,3H),3.80-3.22(m,5H)，1.26(d,J＝6.8Hz,3H)。

Example 9: preparation of 1- ((2S, 4S, 5S) -5- ((5- ((1H-pyrrol-4 yl) ethynyl) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) -4-fluoro-2-methylpiperidin-1-yl) prop-2-en-1-one (Compound 14)

The synthesis and compound 14 were referred to the following steps:

step 1: compound 11-2 (100 mg,0.19 mmol) was dissolved in tetrahydrofuran (4 mL) under nitrogen, dppf Pd G3 (110 mg,0.19 mmol), cuprous iodide (13 mg,0.07 mmol), dppf (11 mg,0.019 mmol), (1H-pyrrol-4 yl) acetylene (85 mg,0.95 mmol) and triethylamine (0.6 mL) were added sequentially, and the reaction was stirred at 25℃for 3 hours. The solvent was dried by spin, and the residue obtained by concentration was purified by a silica gel column (petroleum ether/ethyl acetate=1/1) to give compound 14-1 (44 mg, yield: 48%) as a white solid. LCMS (ESI) [ M+H ]] ⁺ m/z＝494.22.

Step 2: compound 14-1 (44 mg,0.09 mmol) was dissolved in dichloromethane (1 mL) and trifluoroacetic acid (0.5 mL) was added under an ice-water bath. The reaction solution was 0Stirred at c for 2 hours. The solvent was dried to give the title product compound 14 (24 mg, yield: 69%) as a white solid. LCMS (ESI) [ M+H ]] ⁺ m/z＝394.18； ¹ H NMR(400MHz,CD ₃ OD)δ13.85(brs,1H),11.02(br s,1H),8.96(s,1H),8.94-8.85(m,2H),7.96-7.82(m,1H),6.67(dd,J＝12.9,8.9Hz,1H),6.15-6.05(m,1H),5.62(d,J＝12.9Hz,1H),5.26(d,J＝8.5Hz,1H),3.80-3.25(m,5H)，1.26(d,J＝6.8Hz,3H)。

Example 10: preparation of 1- ((2S, 4S, 5S) -5- ((5- ((2-methyl-2H-tetrazol-5 yl) ethynyl) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) amino) -4-fluoro-2-methylpiperidin-1-yl) prop-2-en-1-one (Compound 15)

The synthesis and compound 15 are referred to the following steps:

step 1: compound 11-2 (100 mg,0.19 mmol) was dissolved in tetrahydrofuran (4 mL) under nitrogen atmosphere, dppf Pd G3 (110 mg,0.19 mmol), cuprous iodide (13 mg,0.07 mmol), dppf (11 mg,0.019 mmol), (1H-pyrrol-4 yl) acetylene (101 mg,0.95 mmol) and triethylamine (0.6 mL) were added sequentially, and the reaction solution was stirred at 25℃for 3 hours. The solvent was dried by spin, and the residue obtained by concentration was purified by a silica gel column (petroleum ether/ethyl acetate=1/1) to give compound 15-1 (43 mg, yield: 45%) as a white solid. LCMS (ESI) [ M+H ]] ⁺ m/z＝510.23.

Step 2: compound 15-1 (43 mg,0.085 mmol) was dissolved in dichloromethane (1 mL) and trifluoroacetic acid (0.5 mL) was added under an ice-water bath. The reaction solution was stirred at 0℃for 2 hours. The solvent was dried to give the title product compound 15 (23 mg, yield: 65%) as a white solid. LCMS (ESI) [ M+H ]] ⁺ m/z＝410.18； ¹ H NMR(400MHz,CD ₃ OD)δ11.03(br s,1H),8.98(s,1H),7.98-7.82(m,1H),6.66(dd,J＝12.9,8.9Hz,1H),6.15-6.05(m,1H),5.65(d,J＝12.9Hz,1H),5.28(d,J＝8.5Hz,1H),3.80-3.25(m,5H)，3.64(s,3H),1.27(d,J＝6.8Hz,3H)。

Example 11: determination of kinase inhibitory Activity

Four LanthaScreen JAThe groups of K biochemical assays (JAK 1, 2, 3 and Tyk 2) were carried in common kinase reaction buffers (50mM HEPES,pH 7.5,0.01%Brij-35, 10mM MgCl ₂ And 1mM EGTA). Recombinant GST-tagged JAK enzyme and GFP-tagged STAT1 peptide substrates were obtained from Life Technologies.

Serial dilutions of the compound were pre-incubated with each of the four JAK enzymes and substrate in white 384 well microplates (Corning) for 1 hour at ambient temperature. ATP with 1% DMSO was then added to initiate the kinase reaction in a total volume of 10. Mu.L. The final enzyme concentrations for JAK1, 2, 3 and Tyk2 were 1nM, 0.1nM and 0.25nM, respectively; the corresponding Km ATP concentrations used were 25. Mu.M, 3. Mu.M, 1.6. Mu.M and 10. Mu.M; while the substrate concentration for all four assays was 200nM. Kinase reactions were allowed to proceed for 1 hour at ambient temperature, after which 10 μl of formulation of EDTA (10 mM final concentration) and Tb anti-pSTAT 1 (pTyr 701) antibody (Life Technologies,2nM final concentration) in TRFRET dilution buffer (Life Technologies) was added. The plates were incubated for 1 hour at ambient temperature, after which they were read on an EnVision reader (Perkin Elmer). Emission ratio signals (520 nm/495 nm) were recorded and used to calculate percent inhibition values based on DMSO and background controls.

For dose response analysis, percent inhibition data was plotted versus compound concentration and IC was determined using a 4-parameter robust fit model with Prism software (GraphPad Software) ₅₀ Values. Fitting these data resulted in best fit IC50 values in the case of the test compounds titrating and resulting in inhibition of peptide product formation.

Compound inhibition (% inh) = (average negative control-compound)/(average negative control-average positive control) ×100%

Negative control: blank DMSO

Positive control: PF06651600

b) IC of the compound was obtained using the following nonlinear fitting equation ₅₀ (half inhibition concentration):

Y＝Bottom+(Top-Bottom)/(1+10^((LogIC ₅₀ -X)*HillSlope))

log of compound concentration

Inhibition of the compounds (% inh)

The results of these assays are shown in table 1 below, where "a" represents a calculated IC50 of less than 1nM; "B" means that the calculated IC50 is 1nM to less than 100nM; "C" means that the calculated IC50 is 100nM to less than 1. Mu.M; "D" means the calculated IC ₅₀ Greater than or equal to 1 μm, "NT" means that the indicated compound was not tested in the indicated assay.

TABLE 1 inhibitory Activity of selected Compounds of the invention against JAK1, JAK2, JAK3 and TYK2

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The results show that the compounds of the invention have good selective inhibition effect on JAK 3.

Example 12: determination of human Whole blood cell (HWB) IL-15-induced STAT5 phosphorylation inhibition Activity

Test compounds were serially diluted 1:2 to the desired concentration (final 500X) with DMSO, then the compounds were further diluted in PBS (by adding 4 μl of compound to 96 μl of PBS, [ DMSO]=4%, final 20X). Phase 96 well Polypropylene plates were added with 90. Mu.l HWB (heparin treated human whole blood)/well followed by 5. Mu.l/well of 4% DMSO in D-PBS or different concentrations of D-PBS (w/o Ca) ⁺² Or Mg (Mg) ⁺² ) 20X inhibitor in 4% dmso to give a 1X concentration in 0.2% dmso. Mix and incubate for 45 min at 37℃and then add 5. Mu. l D-PBS (unstimulated control) or 5. Mu.l of 20 Xstock of human IL-15 (final concentration 50 ng/ml) mix three times. After incubation for 15 min at 37 ℃, 1 Xlysis/immobilization buffer (BDPhosflow 5 Xlysis/immobilization buffer) was added to all wells at 1000 μl/well, followed by incubation for 20 min at 37 ℃ and rotation at 1200rpm for 5 min. After washing with 1000. Mu.l FACS buffer and spinning at 1200rpm for 5 minutes, 400. Mu.l ice-cold Perm buffer III was added to each well. Mix gently (1-2X) and incubate on iceAfter 30 minutes, the mixture was spun at 1200rpm for 5 minutes without interruption and washed 1X with cold 1000ml of FACS buffer (D-PBS containing 0.1% BSA and 0.1% sodium azide) and the desired AlexaFluor647 conjugated anti-phosphorylated STAT5 antibody diluted 1:125 with FACS buffer was added at 250 μl/well. After overnight incubation at 4 ℃, all samples were transferred to a 96-well polypropylene U-bottom plate and examined by flow cytometry (gating total lymphocytes). The resulting IC ₅₀ The values are listed in the table.

TABLE 2 inhibition of HWB IL-15 induced STAT5 phosphorylation by selected Compounds of the invention

Compounds of formula (I)	HWB IL15-pSTAT5 IC ₅₀ (nM)
		PF06651600	136
1	60
		3	55
5	198
		7	87
9	351
		11	1850
12	685
		13	96
14	560
		15	990

The results show that the compound has obvious inhibition effect on IL-15 induced STAT5 phosphorylation of HWB.

Claims

1. A compound having a structure represented by general formula (I), an enantiomer or diastereomer thereof, or a mixture thereof, a pharmaceutically acceptable salt, solvate, atropisomer, isotopically labeled derivative, crystalline form, or prodrug thereof:

wherein X is independently selected from N, CH or CCN;

2. The compound, enantiomer or diastereomer thereof, or mixture thereof, pharmaceutically acceptable salt, solvate, atropisomer, isotopically labeled derivative, crystalline form or prodrug thereof according to claim 1, having the structure of formula (II) when X is N:

wherein R is independently selected from the group consisting of hydrogen, deuterium, fluorine, chlorine, cyano, C1-C8 alkynyl, C1-C8 haloalkynyl, C3-C5 cycloalkyl-substituted alkynyl, methyleneoxyalkyl-substituted alkynyl, 5-to 6-membered aromatic or heteroaromatic ring-substituted alkynyl, C1-C6 linear or branched alkoxymethylene, halo-C1-C6 linear or branched alkoxymethylene, C1-C6 linear or branched alkoxydifluoromethylene, halo-C1-C6 linear or branched alkoxydifluoromethylene, C1-C6 linear or branched alkoxyfluoromethylene, halo-C1-C6 linear or branched alkoxyfluoromethylene, C3-C6 cycloalkyl, C3-C6 alkyl-substituted cycloalkyl, C3-C6 halogen-substituted cycloalkyl, C6-C10 aryl, mono-or bicyclic heteroaryl containing 5-and/or 6-membered rings, (aryl) C1-C6 or branched alkyl, (heteroaryl) C1-C6 linear or branched alkyl, (C6) C1-or branched alkyl, (C1-C6) linear or branched alkoxycarbonyl, (C1-C6 linear or branched alkoxyfluorocarbonyl), halo-C1-C6 linear or branched alkoxyfluorocyclo, C1-C6 linear or branched alkoxyfluorocycloalkyl, C3-C6 cycloalkyl, C3-C6 alkyl-substituted cycloalkyl, C6-C1-C6 cycloalkyl, and/C6 heteroaryl, mono-or bicyclic heteroaryl, (aryl) containing 5-and/or C1-C6 membered cyclic, (C1-C6 linear or branched alkyl) aminocarbonyl, ethoxycarbonyl, isopropoxycarbonyl, isobutoxycarbonyl; wherein the alkyl, aryl, and heteroaryl groups are independently optionally substituted with one or more substituents selected from the group consisting of: alkyl, halogen, hydroxy, methoxy, amino, cyano, alkylamino, dialkylamino, CF3, aminocarbonyl, (C1-C6 linear or branched alkyl) aminocarbonyl and C3-C6 cycloalkyl.

3. The compound, enantiomer or diastereomer thereof, or mixture thereof, pharmaceutically acceptable salt, solvate, atropisomer, isotopically labeled derivative, crystalline form or prodrug thereof according to claim 1, having a structure represented by the following formula (III) when X is CH:

4. The compound, enantiomer or diastereomer thereof, or mixture thereof, pharmaceutically acceptable salt, solvate, atropisomer, isotopically labeled derivative, crystalline form or prodrug thereof according to claim 1, having a structure represented by formula (IV) below when X is CCN:

5. A compound according to any one of claims 1-4, an enantiomer or diastereomer thereof, or mixture thereof, a pharmaceutically acceptable salt, solvate, atropisomer, isotopically-labeled derivative, crystalline form or prodrug thereof, comprising the following compound:

6. a pharmaceutical composition comprising a compound according to any one of claims 1 to 5, a pharmaceutically acceptable salt, solvate, atropisomer, isotopically labeled derivative, crystalline form or prodrug thereof, and a pharmaceutically acceptable adjuvant, diluent or carrier.

7. Use of a compound according to any one of claims 1-5, a pharmaceutically acceptable salt, solvate, atropisomer, isotopically labeled derivative, crystalline form or prodrug thereof, or a pharmaceutical composition according to claim 6, in the manufacture of a medicament for the treatment or prevention of a disorder or condition selected from vitiligo, alopecia, rheumatoid arthritis, inflammatory bowel disease including crohn's disease and ulcerative colitis, proctitis, eosinophilic gastroenteritis or mastocytosis, myositis, vasculitis, pemphigus, bullous pemphigoid, lupus, nephritis, systemic lupus erythematosus, psoriasis, eczematoid dermatitis, pruritis or other pruritic conditions, autoimmune thyroiditis, multiple sclerosis, asthma, xerosis, systemic sclerosis, polyarteritis nodosa, dry eye syndrome, sympathogenic ophthalmitis, organ transplant rejection, graft versus host disease, autoimmune alopecia chronic obstructive pulmonary disease, acute respiratory disease, ocular disease, disorder or condition including: autoimmune diseases of the eye, keratoconjunctivitis, vernal conjunctivitis, uveitis including uveitis associated with behcet's disease and lens-induced uveitis, keratitis, herpetic keratitis, keratoconus, leukoplakia, pemphigus oculi, keratoconjunctivitis sicca (dry eye), iridocyclitis, sarcoidosis, endocrinopathy, sympathogenic ophthalmitis, allergic conjunctivitis.

8. A method of treating a disorder or condition comprising administering a compound of any one of claims 1-5, a pharmaceutically acceptable salt, solvate, atropisomer, isotopically labeled derivative, crystalline form or prodrug thereof, or a pharmaceutical composition of claim 6 to a subject in need thereof, wherein the disorder or condition is selected from vitiligo, alopecia, rheumatoid arthritis, inflammatory bowel disease including crohn's disease and ulcerative colitis, proctitis, eosinophilic gastroenteritis or mastocytosis, myositis, vasculitis, pemphigus, bullous pemphigoid, lupus, nephritis, systemic lupus erythematosus, psoriasis, eczematoid dermatitis, pruritus or other pruritic conditions, autoimmune thyroiditis, multiple sclerosis, asthma, xerosis, systemic sclerosis, sarcoidosis, polyarteritis, dry eye syndrome, sympathogenic ophthalmitis, organ transplant rejection, graft versus host disease, autoimmune alopecia chronic obstructive pulmonary disease, acute respiratory disease, an ocular disease, disorder or condition including: autoimmune diseases of the eye, keratoconjunctivitis, vernal conjunctivitis, uveitis including uveitis associated with behcet's disease and lens-induced uveitis, keratitis, herpetic keratitis, keratoconus, leukoplakia, pemphigus oculi, keratoconjunctivitis sicca (dry eye), iridocyclitis, sarcoidosis, endocrinopathy, sympathogenic ophthalmitis, allergic conjunctivitis.

9. The use of claim 7 or the method of claim 8, wherein the disorder or condition is inflammatory bowel disease.

10. The use of claim 7 or the method of claim 8, wherein the disorder or condition is rheumatoid arthritis.

11. The use of claim 7 or the method of claim 8, wherein the disorder or condition is alopecia.

12. The use of claim 7 or the method of claim 8, wherein the disorder or condition is vitiligo.