WO2021159372A1 - Jak抑制剂在制备治疗jak激酶相关疾病药物中的应用 - Google Patents

Jak抑制剂在制备治疗jak激酶相关疾病药物中的应用 Download PDF

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WO2021159372A1
WO2021159372A1 PCT/CN2020/075024 CN2020075024W WO2021159372A1 WO 2021159372 A1 WO2021159372 A1 WO 2021159372A1 CN 2020075024 W CN2020075024 W CN 2020075024W WO 2021159372 A1 WO2021159372 A1 WO 2021159372A1
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compound
formula
application
alkyl
pharmaceutically acceptable
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PCT/CN2020/075024
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English (en)
French (fr)
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王帅
王德刚
吴守廷
于婷婷
房淼
穆利伟
方亮
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珠海联邦制药股份有限公司
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Priority to US17/760,233 priority Critical patent/US20230113620A1/en
Priority to EP20919255.8A priority patent/EP4105214A4/en
Priority to AU2020428591A priority patent/AU2020428591A1/en
Priority to JP2022549089A priority patent/JP7395762B2/ja
Priority to PCT/CN2020/075024 priority patent/WO2021159372A1/zh
Priority to KR1020227028080A priority patent/KR20220127900A/ko
Priority to CN202080094355.5A priority patent/CN115066425B/zh
Priority to CA3169832A priority patent/CA3169832A1/en
Publication of WO2021159372A1 publication Critical patent/WO2021159372A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/437—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a five-membered ring having nitrogen as a ring hetero atom, e.g. indolizine, beta-carboline
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/438—The ring being spiro-condensed with carbocyclic or heterocyclic ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents

Definitions

  • the present invention relates to the field of pharmacy, in particular to a [1,2,4]triazolo[1,5-a]pyridine compound used in the preparation and treatment of JAK kinase-related autoimmune, inflammatory, and allergic diseases, or Application in medicine for diseases such as graft-versus-host disease.
  • JAK is involved in inflammation, autoimmune diseases, proliferative diseases, transplant rejection (or graft-versus-host disease), diseases involving impaired cartilage turnover, congenital cartilage deformities, and/or diseases related to excessive IL6 secretion Tyrosine kinase family.
  • the present invention also provides a method for producing the compound, a pharmaceutical composition containing the compound, and prevention and/or treatment of inflammation, autoimmune disease, proliferative disease, transplant rejection, and disease involving impaired cartilage renewal by administering the compound of the present invention , Congenital cartilage deformities and/or diseases related to excessive secretion of IL6.
  • Janus kinase is a cytoplasmic tyrosine kinase that transduces cytokine signals from membrane receptors to STAT transcription factors.
  • JAK family members JAK1, JAK2, JAK3 and TYK2.
  • JAK family members autophosphorylate and/or transphosphorylate each other, and then STATs are phosphorylated and then migrate to the nucleus to regulate transcription.
  • JAK-STAT intracellular signal transduction is suitable for interferons, most interleukins, and a variety of cytokines and endocrine factors, such as EPO, TPO, GH, OSM, LIF, CNTF, GM-CSF and PRL (Vainchenker W. etc. People (2008)).
  • JAK3 is an immunosuppressive target (O’Shea J. et al. (2004)). JAK inhibitors were successfully used in clinical development, initially for organ transplant rejection, but later also used for other immuno-inflammatory indications, such as inflammatory bowel disease (IBD), allergic dermatitis (AD), rheumatoid arthritis (RA), psoriasis and Crohn’s disease (http://clinicaltrials.gov/).
  • IBD inflammatory bowel disease
  • AD allergic dermatitis
  • RA rheumatoid arthritis
  • psoriasis and Crohn’s disease http://clinicaltrials.gov/.
  • TYK2 is a potential target for immune inflammatory diseases, which has been confirmed by human genetics and mouse knockout studies (Levy D. and Loomis C. (2007)).
  • JAK1 is a new target in the field of immuno-inflammatory diseases. JAK1 is heterodimerized with other JAKs to transduce cytokine-driven pro-inflammatory signaling. Therefore, inhibition of JAK1 and/or other JAKs is expected to have therapeutic benefits for a series of inflammatory disorders and other diseases driven by JAK-mediated signal transduction.
  • Inflammatory bowel disease is an idiopathic intestinal inflammatory disease involving the ileum, rectum, and colon. The clinical manifestations are diarrhea, abdominal pain, and even bloody stools. Inflammatory bowel disease includes ulcerative colitis and Crohn's disease. Ulcerative colitis is a continuous inflammation of the mucosal layer and submucosa of the colon. The disease usually first involves the rectum and gradually spreads to the entire colon. Crohn's disease can involve the entire digestive tract. It is a non-continuous full-thickness inflammation. The most commonly involved parts are Terminal ileum, colon and perianal.
  • Allergic dermatitis is a skin disease caused by allergens. It mainly refers to skin diseases such as redness, swelling, itching, wind mass, peeling, etc. caused by human body contact with certain allergens. Specific allergens can include contact allergens, inhaled allergens, ingested allergens and injection allergens. Each type of allergen can cause a corresponding allergic reaction, the main manifestations are a variety of dermatitis, eczema, urticaria and so on.
  • Psoriasis commonly known as psoriasis, is a chronic inflammatory skin disease, which has a greater impact on the physical health and mental health of patients.
  • the clinical manifestations are mainly erythema and scaly, which can affect the whole body, and the scalp and extensible limbs are more common.
  • Systemic lupus erythematosus is an autoimmune disease whose etiology is not clear, and is characterized by multiple autoantibodies that cause damage to different target organs. Due to the large number of pathogenic autoantibodies and immune complexes in the body that cause tissue damage, clinical manifestations of damage to various systems and organs, such as skin, joints, serosal membrane, heart, kidney, central nervous system, blood system, etc. .
  • Graft-versus-host disease is caused by a series of "cytokine storm" stimuli initiated by the recipient of the T lymphocytes in the allogeneic donor transplant after transplantation, which greatly enhances its resistance to the recipient's antigen immune response.
  • US2009220688 discloses Filgotinib, which is a drug developed by Galapagos Company which is currently in clinical phase III for the treatment of rheumatoid arthritis.
  • JAK inhibitors [1,2,4] triazolo [1,5-a] pyridine compounds are developed and used in the preparation of inflammatory bowel disease, allergic dermatitis, psoriasis, The application of drugs for diseases such as systemic lupus erythematosus or graft-versus-host disease is necessary.
  • the present invention provides a JAK inhibitor [1,2,4]triazolo[1,5-a]pyridine compound of formula (I), its isomers or pharmaceutically acceptable
  • JAK inhibitor [1,2,4]triazolo[1,5-a]pyridine compound of formula (I) its isomers or pharmaceutically acceptable
  • E 1 and E 2 are each independently selected from a single bond, -CH 2 -or -(CH 2 ) 2 -;
  • n 1 or 2;
  • n 1 or 2;
  • g is 1, 2 or 3;
  • h is 1, 2 or 3;
  • R 1 is selected from H, CN, C 1-6 alkyl group or 3-6 membered cycloalkyl group, wherein said C 1-6 alkyl group and 3-6 membered cycloalkyl group are optionally grouped by 1, 2 or 3 R a replace
  • R 2 is selected from H, F, Cl, Br, I or C 1-3 alkyl, wherein the C 1-3 alkyl is optionally substituted with 1, 2 or 3 R b ;
  • R 3 , R 4 and R 5 are each independently selected from H, F, Cl, Br, I or C 1-3 alkyl, wherein the C 1-3 alkyl is optionally substituted by 1, 2 or 3 R c replace;
  • R 6 , R 7 and R 8 are each independently selected from H, F, Cl, Br, I or C 1-3 alkyl, wherein the C 1-3 alkyl is optionally substituted by 1, 2 or 3 R d replace;
  • Each R a is independently selected from H, F, Cl, Br, I, CN or C 1-3 alkyl, wherein said C 1-3 alkyl optionally substituted with 1, 2 or 3 R <
  • Each R b is independently selected from F, Cl, Br or I;
  • Each R c is independently selected from F, Cl, Br or I;
  • Each Rd is independently selected from F, Cl, Br or I;
  • Each R is independently selected from F, Cl, Br, or I.
  • the present invention provides JAK inhibitors of formula (I) [1,2,4]triazolo[1,5-a]pyridine compounds, their isomers or pharmacological properties
  • JAK inhibitors of formula (I) [1,2,4]triazolo[1,5-a]pyridine compounds, their isomers or pharmacological properties The application of the accepted salt in the preparation of a medicine for treating autoimmune diseases, inflammatory diseases, allergic diseases, or graft-versus-host disease.
  • the autoimmune disease includes systemic lupus erythematosus, psoriasis, psoriatic arthritis or lupus nephritis.
  • the inflammatory disease includes inflammatory bowel disease, ankylosing spondylitis or primary cholangitis and the like.
  • the allergic disease includes allergic dermatitis, contact dermatitis, allergic purpura, or bronchial asthma.
  • the graft-versus-host disease includes, but is not limited to, anti-acute rejection, anti-chronic rejection, or induction of immune tolerance.
  • the present invention provides a JAK inhibitor of formula (I) [1,2,4]triazolo[1,5-a]pyridine compound, its isomer or The use of pharmaceutically acceptable salts in the preparation of drugs for the treatment of inflammatory bowel disease.
  • the treatment of inflammatory bowel disease includes but is not limited to inhibiting colon shortening.
  • the present invention provides a JAK inhibitor of formula (I) [1,2,4]triazolo[1,5-a]pyridine compound, its isomer or The use of pharmaceutically acceptable salts in the preparation of medicines for the treatment of allergic dermatitis.
  • the present invention provides a JAK inhibitor of formula (I) [1,2,4]triazolo[1,5-a]pyridine compound, its isomer or Application of pharmaceutically acceptable salts in the preparation of medicines for the treatment of psoriasis.
  • the present invention provides a JAK inhibitor of formula (I) [1,2,4]triazolo[1,5-a]pyridine compound, its isomer or Application of pharmaceutically acceptable salts in the preparation of medicines for treating systemic lupus erythematosus.
  • the present invention provides a JAK inhibitor of formula (I) [1,2,4]triazolo[1,5-a]pyridine compound, its isomer or The use of pharmaceutically acceptable salts in the preparation of drugs for the treatment of graft-versus-host disease.
  • the graft-versus-host disease includes, but is not limited to, anti-acute rejection, anti-chronic rejection, or induction of immune tolerance.
  • the present invention prepares pharmaceutical applications for the treatment of inflammatory bowel disease, allergic dermatitis, psoriasis, systemic lupus erythematosus or graft-versus-host disease, such as the JAK inhibitor of formula (I) [1, 2,4]Triazolo[1,5-a]pyridine compounds, their isomers or their pharmaceutically acceptable salts:
  • E 1 and E 2 are each independently selected from a single bond, -CH 2 -or -(CH 2 ) 2 -;
  • n 1 or 2;
  • n 1 or 2;
  • g is 1, 2 or 3;
  • h is 1, 2 or 3;
  • R 1 is selected from H, CN, C 1-6 alkyl group or 3-6 membered cycloalkyl group, wherein said C 1-6 alkyl group and 3-6 membered cycloalkyl group are optionally grouped by 1, 2 or 3 R a replace
  • R 2 is selected from H, F, Cl, Br, I or C 1-3 alkyl, wherein the C 1-3 alkyl is optionally substituted with 1, 2 or 3 R b ;
  • R 3 , R 4 and R 5 are each independently selected from H, F, Cl, Br, I or C 1-3 alkyl, wherein the C 1-3 alkyl is optionally substituted by 1, 2 or 3 R c replace;
  • R 6 , R 7 and R 8 are each independently selected from H, F, Cl, Br, I or C 1-3 alkyl, wherein the C 1-3 alkyl is optionally substituted by 1, 2 or 3 R d replace;
  • Each R a is independently selected from H, F, Cl, Br, I, CN or C 1-3 alkyl, wherein said C 1-3 alkyl optionally substituted with 1, 2 or 3 R <
  • Each R b is independently selected from F, Cl, Br or I;
  • Each R c is independently selected from F, Cl, Br or I;
  • Each Rd is independently selected from F, Cl, Br or I;
  • Each R is independently selected from F, Cl, Br, or I.
  • each of the R a is independently selected from H, F, Cl, Br, I or CN, other variables are as defined in the present invention.
  • the R 1 is selected from H, CN, C 1-3 alkyl or 3 to 5 membered cycloalkyl, wherein the C 1-3 alkyl and 3 to 5 membered cycloalkyl optionally substituted with 1,2 or 3 substituents R a, the other variables are as defined in the present invention.
  • the R 1 is selected from H, CN, CH 3 , Where the CH 3 , Optionally substituted with 1,2 or 3 substituents R a, the other variables are as defined in the present invention.
  • the R 1 is selected from H, CN, CF 3 , CHF 2 , Other variables are as defined in the present invention.
  • the R 2 is selected from H, F, Cl, Br or I, and other variables are as defined in the present invention.
  • the R 3 , R 4 and R 5 are independently selected from H, F, Cl, Br or I, and other variables are as defined in the present invention.
  • the R 6 , R 7 and R 8 are independently selected from H, F, Cl, Br or I, and other variables are as defined in the present invention.
  • the compound of formula (I), its isomers or pharmaceutically acceptable salts thereof are selected from
  • L 1 , R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 are as defined in the present invention.
  • the compound of formula (I), its isomers or pharmaceutically acceptable salts thereof are selected from
  • R a, R 2 , R 3, R 4, R 5, R 6, R 7 and R 8 are as defined in the present invention.
  • the present invention also provides the following compounds, their isomers or their pharmaceutically acceptable salts:
  • the present invention further provides the following compounds, isomers or pharmaceutically acceptable salts thereof:
  • the present invention also provides a pharmaceutical composition, including a therapeutically effective amount of a compound of formula (I), its isomers, or pharmaceutically acceptable salts and pharmaceutically acceptable salts thereof as active ingredients. a.
  • JAK inhibitor [1,2,4]triazolo[1,5-a]pyridine compound of the present invention such as formula (I)
  • the series of compounds involved in the present invention have shown good selectivity to JAK1 and/or TYK2 in the in vitro activity experiment of the four subtypes of JAK kinases (JAK1, JAK2, JAk3 and TYK2) Inhibition, and these compounds showed higher exposure and good oral bioavailability in pharmacokinetic experiments in mice.
  • pharmaceutically acceptable refers to those compounds, materials, compositions and/or dosage forms that are within the scope of reliable medical judgment and are suitable for use in contact with human and animal tissues. , Without excessive toxicity, irritation, allergic reactions or other problems or complications, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable salt refers to a salt of the compound of the present invention, which is prepared from a compound with specific substituents discovered in the present invention and a relatively non-toxic acid or base.
  • a base addition salt can be obtained by contacting the neutral form of the compound with a sufficient amount of base in a pure solution or a suitable inert solvent.
  • Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine or magnesium salt or similar salts.
  • the acid addition salt can be obtained by contacting the neutral form of the compound with a sufficient amount of acid in a pure solution or a suitable inert solvent.
  • Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts including, for example, hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, hydrogen carbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, Hydrogen sulfate, hydroiodic acid, phosphorous acid, etc.; and organic acid salts, the organic acid includes, for example, acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, Similar acids such as fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid and methanesulfonic acid; also include salts of amino acids (such as arginine, etc.) , And salts of organic acids such as glucuronic acid. Certain specific compounds of the present invention contain basic and
  • the pharmaceutically acceptable salt of the present invention can be synthesized from the parent compound containing acid or base by conventional chemical methods.
  • such salts are prepared by reacting these compounds in free acid or base form with a stoichiometric amount of appropriate base or acid in water or an organic solvent or a mixture of both.
  • the compounds of the present invention may exist in specific geometric or stereoisomeric forms.
  • the present invention contemplates all such compounds, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers Isomers, (D)-isomers, (L)-isomers, and their racemic mixtures and other mixtures, such as enantiomers or diastereomer-enriched mixtures, all of these mixtures belong to this Within the scope of the invention.
  • Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All these isomers and their mixtures are included in the scope of the present invention.
  • enantiomer or “optical isomer” refers to stereoisomers that are mirror images of each other.
  • cis-trans isomer or “geometric isomer” is caused by the inability of free rotation of double bonds or single bonds of ring-forming carbon atoms.
  • diastereomer refers to a stereoisomer in which the molecule has two or more chiral centers and the relationship between the molecules is non-mirror-image relationship.
  • wedge-shaped solid line keys And wedge-shaped dashed key Represents the absolute configuration of a three-dimensional center, with a straight solid line key And straight dashed key Indicates the relative configuration of the three-dimensional center, using wavy lines Represents a wedge-shaped solid line key Or wedge-shaped dashed key Or use wavy lines Represents a straight solid line key And straight dashed key
  • formula (A) means that the compound exists as a single isomer of formula (A-1) or formula (A-2) or as two isomers of formula (A-1) and formula (A-2)
  • formula (B) means that the compound exists in the form of a single isomer of formula (B-1) or formula (B-2) or in the form of two of formula (B-1) and formula (B-2)
  • a mixture of isomers exists.
  • formula (C) represents that the compound exists in the form of a single isomer of formula (C-1) or formula (C-2) or in the form of two isomers of formula (C-1) and formula (C-2) Exist as a mixture.
  • tautomer or “tautomeric form” means that at room temperature, the isomers of different functional groups are in dynamic equilibrium and can be transformed into each other quickly. If tautomers are possible (such as in solution), the chemical equilibrium of tautomers can be reached.
  • proton tautomer also called prototropic tautomer
  • proton migration such as keto-enol isomerization and imine-ene Amine isomerization.
  • Valence isomers include some recombination of bonding electrons to carry out mutual transformations.
  • keto-enol tautomerization is the tautomerism between two tautomers of pentane-2,4-dione and 4-hydroxypent-3-en-2-one.
  • the term “enriched in one isomer”, “enriched in isomers”, “enriched in one enantiomer” or “enriched in enantiomers” refers to one of the isomers or pairs of
  • the content of the enantiomer is less than 100%, and the content of the isomer or enantiomer is greater than or equal to 60%, or greater than or equal to 70%, or greater than or equal to 80%, or greater than or equal to 90%, or greater than or equal to 95%, or 96% or greater, or 97% or greater, or 98% or greater, or 99% or greater, or 99.5% or greater, or 99.6% or greater, or 99.7% or greater, or 99.8% or greater, or greater than or equal 99.9%.
  • the term “isomer excess” or “enantiomeric excess” refers to the difference between the relative percentages of two isomers or two enantiomers. For example, if the content of one isomer or enantiomer is 90%, and the content of the other isomer or enantiomer is 10%, the isomer or enantiomer excess (ee value) is 80% .
  • optically active (R)- and (S)-isomers and D and L isomers can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If you want to obtain an enantiomer of a compound of the present invention, it can be prepared by asymmetric synthesis or derivatization with chiral auxiliary agents, in which the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide pure The desired enantiomer.
  • the molecule when the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), it forms a diastereomeric salt with an appropriate optically active acid or base, and then passes through a conventional method known in the art The diastereoisomers are resolved, and then the pure enantiomers are recovered.
  • the separation of enantiomers and diastereomers is usually accomplished through the use of chromatography, which uses a chiral stationary phase and is optionally combined with chemical derivatization (for example, the formation of amino groups from amines). Formate).
  • the compound of the present invention may contain unnatural proportions of atomic isotopes on one or more of the atoms constituting the compound.
  • compounds can be labeled with radioisotopes, such as tritium ( 3 H), iodine-125 ( 125 I), or C-14 ( 14 C).
  • deuterium can be substituted for hydrogen to form deuterated drugs.
  • the bond formed by deuterium and carbon is stronger than the bond formed by ordinary hydrogen and carbon.
  • deuterated drugs can reduce toxic side effects and increase drug stability. , Enhance the efficacy, extend the biological half-life of drugs and other advantages. All changes in the isotopic composition of the compounds of the present invention, whether radioactive or not, are included in the scope of the present invention.
  • “Optional” or “optionally” means that the event or condition described later may but not necessarily occur, and the description includes a situation in which the event or condition occurs and a situation in which the event or condition does not occur.
  • substituted means that any one or more hydrogen atoms on a specific atom are replaced by substituents, and may include deuterium and hydrogen variants, as long as the valence of the specific atom is normal and the substituted compound is stable of.
  • oxygen it means that two hydrogen atoms are replaced. Oxygen substitution does not occur on aromatic groups.
  • optionally substituted means that it can be substituted or unsubstituted. Unless otherwise specified, the type and number of substituents can be arbitrary on the basis that they can be chemically realized.
  • any variable such as R
  • its definition in each case is independent.
  • the group can optionally be substituted with up to two Rs, and R has independent options in each case.
  • combinations of substituents and/or variants thereof are only permitted if such combinations result in stable compounds.
  • linking group When the number of a linking group is 0, such as -(CRR) 0 -, it means that the linking group is a single bond.
  • substituents When a substituent is vacant, it means that the substituent is absent. For example, when X in A-X is vacant, it means that the structure is actually A.
  • substituents do not indicate which atom is connected to the substituted group, such substituents can be bonded via any atom.
  • a pyridyl group can pass through any one of the pyridine ring as a substituent. The carbon atom is attached to the substituted group.
  • the middle linking group L is -MW-, at this time -MW- can be formed by connecting ring A and ring B in the same direction as the reading order from left to right It can also be formed by connecting ring A and ring B in the opposite direction to the reading order from left to right
  • Combinations of the linking groups, substituents, and/or variants thereof are only permitted if such combinations result in stable compounds.
  • the number of atoms in a ring is generally defined as the number of ring members.
  • “5-7 membered ring” refers to a “ring” in which 5-7 atoms are arranged around.
  • 5-6 membered ring means cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, cycloalkynyl, heterocycloalkynyl, aromatic Group or heteroaryl.
  • the ring includes a single ring, as well as a double ring system such as a spiro ring, a fused ring and a bridged ring. Unless otherwise specified, the ring optionally contains 1, 2, or 3 heteroatoms independently selected from O, S, and N.
  • the 5-6 membered ring includes a 5-membered ring, a 6-membered ring, and the like.
  • 5-6 membered ring includes, for example, phenyl, pyridyl, piperidinyl and the like; on the other hand, the term “5-6 membered heterocycloalkyl” includes piperidinyl and the like, but does not include phenyl.
  • ring also includes a ring system containing at least one ring, where each "ring" independently meets the above definition.
  • C 1-6 alkyl is used to indicate a linear or branched saturated hydrocarbon group composed of 1 to 6 carbon atoms.
  • the C 1-6 alkyl group includes C 1-5 , C 1-4 , C 1-3 , C 1-2 , C 2-6 , C 2-4 , C 6 and C 5 alkyl groups, etc.; it may Is monovalent (such as methyl), divalent (such as methylene) or multivalent (such as methine).
  • C 1-6 alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), butyl (including n-butyl, isobutyl) , S-butyl and t-butyl), pentyl (including n-pentyl, isopentyl and neopentyl), hexyl, etc.
  • C 1-3 alkyl is used to indicate a linear or branched saturated hydrocarbon group composed of 1 to 3 carbon atoms.
  • the C 1-3 alkyl group includes C 1-2 and C 2-3 alkyl groups, etc.; it can be monovalent (such as methyl), divalent (such as methylene) or multivalent (such as methine) .
  • Examples of C 1-3 alkyl include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), and the like.
  • C 3-6 cycloalkyl means a saturated cyclic hydrocarbon group composed of 3 to 6 carbon atoms, which is a monocyclic and bicyclic ring system, and the C 3-6 cycloalkyl includes C 3-5 , C 4-5 and C 5-6 cycloalkyl, etc.; it can be monovalent, divalent or multivalent.
  • Examples of C 3-6 cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like.
  • C n-n+m or C n -C n+m includes any specific case of n to n+m carbons, for example, C 1-12 includes C 1 , C 2 , C 3 , C 4 , C 5 , C 6 , C 7 , C 8 , C 9 , C 10 , C 11 , and C 12 , including any range from n to n+m, for example, C 1-12 includes C 1-3 , C 1-6 , C 1-9 , C 3-6 , C 3-9 , C 3-12 , C 6-9 , C 6-12 , and C 9-12, etc.; similarly, from n to n +m member means that the number of atoms in the ring is from n to n+m, for example, 3-12 membered ring includes 3-membered ring, 4-membered ring, 5-membered ring, 6-membered ring, 7-membered ring, 8-membered ring, 9-membered
  • the compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, the embodiments formed by combining them with other chemical synthesis methods, and those well known to those skilled in the art Equivalent alternatives, preferred implementations include but are not limited to the embodiments of the present invention.
  • the solvent used in the present invention is commercially available.
  • the present invention uses the following abbreviations: aq stands for water; HATU stands for O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate ; EDC stands for N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride; m-CPBA stands for 3-chloroperoxybenzoic acid; eq stands for equivalent and equivalent; CDI stands for Carbonyl diimidazole; DCM stands for dichloromethane; PE stands for petroleum ether; DIAD stands for diisopropyl azodicarboxylate; DMF stands for N,N-dimethylformamide; DMSO stands for dimethyl sulfoxide; EtOAc stands for ethyl acetate Esters; EtOH stands for ethanol; MeOH stands for methanol; CBz
  • Figure 2 Curve chart of the results of mouse body weight changes in Experimental Example 3.
  • Figure 3 Histogram of colon length in Experimental Example 3.
  • Figure 4 Histogram of the spleen index in Experimental Example 3.
  • Figure 5 A graph of the skin scoring results in Experimental Example 4.
  • Figure 6 The histogram of the thickness of the right ear in Experimental Example 4.
  • Figure 7 Histogram of the spleen index in Experimental Example 4.
  • Figure 8 Bar graph of serum lgE concentration in Experimental Example 4.
  • Figure 9 The body weight change curve of the mice in Experimental Example 4.
  • reaction was quenched with 250 mL saturated ammonium chloride, diluted with 200 mL water, and then extracted with ethyl acetate (200 mL*3). The organic phases were combined, washed with saturated brine, dried over sodium sulfate, filtered and concentrated to obtain compound 1-2. The crude product was directly used in the next reaction without purification.
  • Step 2 Add potassium acetate (12.7g, 129.3mmol) and Pd( dppf) Cl 2 .CH 2 Cl 2 (3.5 g, 4.3 mmol), replaced with nitrogen 3 times and kept stirring at 70° C. for 3 hours in a nitrogen atmosphere.
  • the TLC board shows that the raw materials are completely consumed and new points are generated.
  • the reaction solution was dispersed in a mixture of 300 mL of water and 400 mL of ethyl acetate. The organic phase was separated, washed with saturated brine, dried over sodium sulfate, filtered and concentrated to obtain a crude product. The crude product was purified by silica gel chromatography to obtain compound 1-3.
  • Step 3 In a nitrogen atmosphere, compound 1-3 (3.5g, 10.0mmol) and N-(5-bromo-[1,2,4]triazolo[1,5-a]pyridine-2 -Base) cyclopropane carboxamide (2.6g, 9.1mmol) in dioxane (60mL) and water (15mL) was added potassium carbonate (3.8g, 27.3mmol) and Pd(dppf)Cl 2 .CH 2 Cl 2 (744 mg, 911.0 ⁇ mol). The reaction solution was stirred at 90°C for 3 hours. LCMS showed that the raw material was consumed completely, and the target molecular ion peak was monitored. The reaction solution was concentrated, and the obtained crude product was separated and purified by column chromatography to obtain compound 1-4. LCMS (ESI) m/z: 424.3 [M+H] + .
  • Step 4 To a dichloromethane (10 mL) solution of compound 1-4 (3.5 g, 8.2 mmol) was added hydrochloric acid/ethyl acetate (4M, 30 mL), and the reaction solution was stirred at 25° C. for 0.5 hours. LCMS showed that the raw materials were consumed, and the target molecular ion peak was monitored. The solid precipitated, filtered and dried to obtain compound 1-5 (3.3g hydrochloride, crude product), which was directly used in the next reaction without purification. LCMS (ESI) m/z: 324.1.
  • Step 5 In a nitrogen atmosphere, Pd/C (1 g, 10%) was added to a methanol (100 mL) solution in which compound 1-5 (3.0 g, 8.34 mmol, hydrochloride) was dissolved. This suspension was replaced with hydrogen three times, and then stirred under a hydrogen atmosphere (30 psi) at 30°C for 12 hours. LCMS showed that the raw materials were consumed, and the target molecular ion peak was monitored. The reaction solution was filtered and concentrated to obtain compound 1-6 (3g hydrochloride, crude product), which was directly used in the next reaction without purification. LCMS (ESI) m/z: 326.2 [M+H] + .
  • Step 6 Dissolve compound 1-6 (0.87g, 2.40mmol, hydrochloride) in N,N-dimethylformamide (10mL), add HOBt (487mg, 3.6mmol,) and EDCI (691mg, 3.6 mmol), and then (1S)-2,2-difluorocyclopropylcarboxylic acid (323 mg, 2.6 mmol) and diisopropylethylamine (621 mg, 4.8 mmol) were added, and the reaction solution was reacted at 15°C for 12 hours. LC-MS showed that the reaction was complete.
  • Step 1 Under the protection of nitrogen at -78°C, dissolve tert-butyl 9-oxo-3-azaspiro[5.5]undecane-3-carboxylic acid (3-1) (5g, 18.7mmol) in anhydrous In tetrahydrofuran (150 mL), lithium bis(trimethylsilyl)amide (1M, 22.4 mL) was slowly added dropwise, and the reaction solution was stirred at -78°C for 1 hour.
  • Step 2 Dissolve compound 3-2 (8g, 20.0mmol) and pinacol diborate (5.59g, 22.0mmol) in N,N-dimethylformamide (100mL), add potassium acetate (5.90g) ,60.1mmol) and [1,1-bis(diphenylphosphine)ferrocene]palladium dichloride dichloromethane (1.64g, 2.0mmol), the reaction solution was stirred and reacted at 70°C for 3 hours. TLC showed that the reaction was complete. The reaction solution was diluted with water (300 mL) and extracted with ethyl acetate (200 mL*2).
  • Step 3 Under the protection of nitrogen, N-(5-bromo-[1,2,4]triazole[1,5-a]pyridin-2-yl)cyclopropylcarboxamide (2g, 7.1 mmol), compound 3-3 (3.49g, 9.3mmol), potassium carbonate (2.95g, 21.3mmol), and [1,1-bis(diphenylphosphine)ferrocene]palladium dichloride dichloromethane ( A mixed solution of 581 mg, 711.5 ⁇ mol) of dioxane (40 mL) and water (10 mL) was replaced with nitrogen three times, and the reaction solution was heated to 90° C. for 3 hours. LC-MS showed that the reaction was complete.
  • Step 4 Dissolve compound 3-4 (3.5 g, 7.8 mmol) in dichloromethane (15 mL), add hydrochloric acid/ethyl acetate (4M, 30 mL), and react the reaction solution at 20° C. for 30 minutes. LC-MS showed that the reaction was complete. The solid precipitated, filtered and dried to obtain compound 3-5. LCMS (ESI) m/z: 352.2 [M+H] + .
  • Step 5 Under N 2 protection, dissolve compound 3-5 (2.9g, 7.4mmol, hydrochloride) in methanol (100mL) solution, add catalyst dry palladium/carbon (1g, 10%), the reaction solution Hydrogen replacement 3 times. Under the hydrogen pressure (30 Psi) and the reaction temperature of 25°C, the reaction solution was stirred for 12 hours. LC-MS showed that the reaction was complete. The solid was filtered through celite, and the filtrate was concentrated under reduced pressure to obtain compound 3-6 ((2.6 g hydrochloride). LCMS (ESI) m/z: 354.7 [M+H] + .
  • Step 6 Dissolve compound 3-6 (1g, 2.6mmol, hydrochloride) in N,N-dimethylformamide (20mL), add HOBt (573mg, 4.2mmol) and EDCI (813mg, 4.2mmol) Then, (1S)-2,2-difluorocyclopropylcarboxylic acid (380mg, 3.1mmol) and diisopropylethylamine (731mg, 5.7mmol) were added, and the reaction solution was reacted at 15°C for 12 hours. LC-MS showed that the reaction was complete.
  • reaction solution was diluted with water (100mL), extracted with dichloromethane/methanol (10/1, 150mL*2), the combined organic phase was washed with saturated brine (100mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure , The residue was subjected to preparative HPLC (neutral system) to obtain compound 3-7.
  • Step 1 Dissolve 5-bromo-[1,2,4]triazole[1,5-a]pyridine-2-amino(4-1) (5g, 23.5mmol) in acetonitrile ( 50mL), triethylamine (11.87g, 117.4mmol) and cyclopropylformyl chloride (6.13g, 58.7mmol) were added, and the reaction solution was reacted at 25°C for 12 hours. TLC showed that the reaction was complete. The acetonitrile solvent was removed by concentration under reduced pressure, and the residue was separated through a rapid silica gel column (0-5% methanol/dichloromethane) to obtain compound 4-2. LCMS (ESI) m/z: 350.8 [M+H] + .
  • Step 2 Under N 2 protection, dissolve compound 4-2 (1.99 g, 5.7 mmol) and compound 1-1 (1.5 g, 6.3 mmol) in anhydrous tetrahydrofuran (30 mL), slowly at -70°C A solution of n-butyl lithium (2.5M, 5.7 mL) was added, and the reaction solution was stirred at 10°C for 30 minutes. LC-MS showed that the reaction was complete. The reaction solution was quenched with saturated ammonium chloride (50 mL) at 0°C, and extracted with ethyl acetate (150 mL*2).
  • Step 3 Dissolve compound 4-3 (0.8g, 1.81mmol) in anhydrous dichloromethane (10mL) at 0°C, add diethylaminosulfur trifluoride (DAST) (351mg, 2.17mmol) The reaction solution was reacted at 0°C for 15 minutes, and then heated to 25°C for 1 hour. LC-MS showed that the reaction was complete. The reaction solution was quenched with saturated aqueous sodium bicarbonate solution (5 mL) at 0°C, diluted with water (10 mL), extracted with dichloromethane (50 mL*3), and the combined organic phase was washed with saturated brine (20 mL), anhydrous Dry over sodium sulfate, filter and concentrate under reduced pressure. The residue was separated via a flash silica gel column (0-100% ethyl acetate/petroleum ether) to obtain compound 4-4. LCMS (ESI) m/z: 444.3 [M+H] + .
  • Step 4 Dissolve compound 4-4 (410 mg, 924.4 ⁇ mol) in dichloromethane (5 mL), add hydrochloric acid/ethyl acetate (4M, 10 mL), and react the reaction solution at 20° C. for 30 minutes. LC-MS showed that the reaction was complete. A solid precipitated, filtered and dried to obtain compound 4-5 (390 mg hydrochloride). LCMS (ESI) m/z: 344.2 [M+H] + .
  • Step 5 Dissolve compound 4-5 (130mg, 342.2 ⁇ mol, hydrochloride) in N,N-dimethylformamide (10mL), add HOBt (77mg, 567.9 ⁇ mol) and EDCI (109mg, 567.9 ⁇ mol) Then, 2-cyanoacetic acid (35mg, 416.4 ⁇ mol) and diisopropylethylamine (98mg, 757.1 ⁇ mol) were added, and the reaction solution was reacted at 15°C for 12 hours. LC-MS showed that the reaction was complete. The reaction solution was concentrated under reduced pressure, and the residue was subjected to preparative HPLC (neutral conditions) to obtain compound 4-6.
  • HOBt 77mg, 567.9 ⁇ mol
  • EDCI 109mg, 567.9 ⁇ mol
  • 2-cyanoacetic acid 35mg, 416.4 ⁇ mol
  • diisopropylethylamine 98mg, 757.1 ⁇ mol
  • reaction was quenched with 10 mL of saturated ammonium chloride solution, then 20 mL of water was added, and extraction was performed with ethyl acetate (30 mL*3). The organic phases were combined, washed with saturated brine (40 mL), dried over anhydrous sodium sulfate, filtered and concentrated to obtain compound 5-2, which was directly used in the next reaction without purification.
  • Step 2 Add KOAc (191mg, 2.0mmol) and Pd(dppf) to the DMF (10mL) solution containing compound 5-2 (0.25g, 648.7 ⁇ mol) and pinacol diborate (165mg, 648.7 ⁇ mol) Cl 2 (48 mg, 64.9 ⁇ mol).
  • the reaction solution was stirred at 70°C for 12 hours.
  • Step 3 Dissolve compound 5-3 (0.13g, 357.8 ⁇ mol), N-(5-bromo-[1,2,4]triazolo[1,5-a]pyridin-2-yl)cyclopropane
  • a solution of formamide (101mg, 357.8 ⁇ mol), K 2 CO 3 (149mg, 1.1mmol), Pd(dppf)Cl 2 (26mg, 35.8 ⁇ mol) in dioxane (4mL) and water (1mL) was replaced with nitrogen 3 Second-rate. The mixture was stirred at 90°C for 12 hours in a nitrogen atmosphere. LCMS showed that the raw material was consumed, and the target molecular ion peak was monitored.
  • Step 4 Under an argon atmosphere, Pd/C (10%, 50 mg) was added to a methanol (10 mL) solution in which compound 5-4 (0.2 g, 457.1 ⁇ mol) was dissolved. The mixture was replaced with hydrogen three times, and then stirred under a hydrogen atmosphere (15 psi) at 25°C for 2 hours. LCMS showed that the raw materials were consumed, and the target molecular ion peak was monitored. The reaction solution was filtered and concentrated to obtain compound 5-5, which was used directly in the next step without purification. LCMS (ESI) m/z: 440.4 [M+H] + .
  • Step 5 The methylene chloride (10 mL) solution in which compound 5-5 (150 mg, 341.3 ⁇ mol) and trifluoroacetic acid (4 mL) were dissolved was replaced with nitrogen three times, and then the reaction solution was stirred at 25° C. for 30 minutes. LCMS showed that the raw materials were consumed, and the target molecular ion peak was monitored. The reaction solution was concentrated to remove the solvent, and compound 5-6 (0.15 g, trifluoroacetate) was obtained without purification, which was directly used in the next reaction. LCMS (ESI) m/z: 340.2 [M+H] + .
  • Step 6 Add EDCI (104mg, 541.1 ⁇ mol), HOBt (73mg, 541.1 ⁇ mol), DIEA ( 140 mg, 1.1 mmol, 189 ⁇ L) was stirred and reacted at 25°C for 5 minutes, then compound 5-6 (122 mg, 270 ⁇ mol, trifluoroacetate) was added, and the mixture was stirred at 25°C for 16 hours. LCMS showed that the raw material was consumed, and the target molecular ion peak was monitored.
  • the crude product is prepared and separated (neutral separation conditions, chromatographic column: Waters Xbridge 150mm*25mm 5 ⁇ m; mobile phase: [H 2 O(10mM NH 4 HCO 3 )-ACN]; B(CH 3 CN)%: 25%-55 %, 7min) and SFC chiral separation (column: DAICEL CHIRALCEL OD-H (250mm*30mm, 5 ⁇ m); mobile phase: [0.1% NH 3 H 2 O EtOH]; B(CO 2 )%: 40%) Compound 5-7 was obtained, SFC retention time: 3.685min.
  • Step 3 Dissolve compound 6-3 (186mg, 512 ⁇ mol), N-(5-bromo-[1,2,4]triazolo[1,5-a]pyridin-2-yl)cyclopropanecarboxamide (144mg, 512 ⁇ mol), K 2 CO 3 (212mg, 1.5 mmol), Pd(dppf)Cl 2 .CH 2 Cl 2 (42mg, 51.2 ⁇ mol) in dioxane (4mL) and water (1mL) solution with nitrogen Replace 3 times. The mixture was stirred at 90°C for 12 hours in a nitrogen atmosphere. LCMS showed that the raw material was consumed, and the target molecular ion peak was monitored.
  • Step 4 Under an argon atmosphere, Pd/C (10%, 50 mg) was added to a methanol (10 mL) solution in which compound 6-4 (196 mg, 448 ⁇ mol) was dissolved. The mixture was replaced with hydrogen three times, and then stirred under a hydrogen atmosphere (15 psi) at 25°C for 16 hours. LCMS showed that the raw materials were consumed, and the target molecular ion peak was monitored. The reaction solution was filtered and concentrated to obtain compound 6-5, which was used directly in the next step without purification. LCMS (ESI) m/z: 440.3 [M+H] + .
  • Step 5 The dichloromethane (10 mL) solution in which compound 6-5 (130 mg, 296 ⁇ mol) and trifluoroacetic acid (4 mL) were dissolved was replaced with nitrogen three times, and then the reaction solution was stirred at 25° C. for 30 minutes. LCMS showed that the raw materials were consumed, and the target molecular ion peak was monitored. The reaction solution was concentrated to remove the solvent, and compound 6-6 (134 mg, trifluoroacetate) was obtained without purification, which was directly used in the next reaction. LCMS (ESI) m/z: 340.2 [M+H] + .
  • Step 6 Add EDCI (85mg, 443.3 ⁇ mol), HOBt (60mg, 443.3 ⁇ mol), DIEA( 115 mg, 886.5 ⁇ mol, 154.4 ⁇ L) was stirred and reacted at 25°C for 5 minutes, then compound 6-6 (134 mg, 295.5 ⁇ mol, trifluoroacetate) was added, and the mixture was stirred at 25°C for 16 hours. LCMS showed that the raw material was consumed, and the target molecular ion peak was monitored. The crude product was prepared and separated (neutral conditions.
  • Step 3 Dissolve compound 7-3 (0.15g, 447.43 ⁇ mol), N-(5-bromo-[1,2,4]triazolo[1,5-a]pyridin-2-yl)cyclopropane Formamide (126mg, 447.43 ⁇ mol), K 2 CO 3 (186mg, 1.34mmol), Pd(dppf)Cl 2 .CH 2 Cl 2 (37mg, 44.7 ⁇ mol) of dioxane (4mL) and water (1mL) The solution was replaced with nitrogen 3 times. The mixture was stirred at 90°C for 12 hours in a nitrogen atmosphere. LCMS showed that the raw material was completely consumed, and the target molecular ion peak was monitored.
  • Step 4 Under an argon atmosphere, Pd/C (10%, 0.05 g) was added to a methanol (10 mL) solution in which compound 7-4 (0.15 g, 366.3 ⁇ mol) was dissolved. The mixture was replaced with hydrogen three times, and then stirred at 25°C for 16 hours in a hydrogen atmosphere (15 psi). LCMS showed that the raw material was completely consumed, and the target molecular ion peak was monitored. The reaction solution was filtered and concentrated to obtain compound 7-5, which was used directly in the next step without purification. LCMS (ESI) m/z: 412.2 [M+H] + .
  • Step 5 The methylene chloride (10 mL) solution in which compound 7-5 (0.13 g, 315.9 ⁇ mol) and trifluoroacetic acid (4 mL) were dissolved was replaced with nitrogen three times, and then the reaction solution was stirred at 25° C. for 30 minutes. LCMS showed that the raw material was completely consumed, and the target molecular ion peak was monitored. The reaction solution was concentrated to remove the solvent, and compound 7-6 (130 mg, trifluoroacetate) was obtained without purification, which was directly used in the next reaction. LCMS (ESI) m/z: 312.1 [M+H] + .
  • Step 6 Add EDCI (88mg, 458.4 ⁇ mol), HOBt (62mg, 458.4 ⁇ mol), DIEA( 119 mg, 916.8 ⁇ mol, 160 ⁇ L) was stirred and reacted at 25°C for 5 minutes, then compound 7-6 (0.13 g, 305.6 ⁇ mol, trifluoroacetate) was added, and the mixture was stirred at 25°C for 16 hours. LCMS showed that the raw material was completely consumed, and the target molecular ion peak was monitored.
  • the crude product was prepared and separated (chromatographic column: Waters Xbridge 150*25 5 ⁇ m; mobile phase: [H 2 O(10mM NH 4 HCO 3 )-ACN]; B(CH 3 CN)%: 20%-50%, 7min) and Chiral separation (column: DAICEL CHIRALPAK AD (250mm*30mm, 10 ⁇ m); mobile phase, A%: (0.1% NH 3 H 2 O EtOH); B (CO 2 )%: (40%-40%) Compound 7-7, SFC retention time: 3.714 min.
  • Step 1 Under the protection of nitrogen, compound 8-1 (1.11g, 5.21mmol), compound 1-3, potassium carbonate (2.16g, 15.6mmol), and [1,1-bis(diphenylphosphine) )Ferrocene]palladium dichloride dichloromethane (425mg, 520.6 ⁇ mol) in a mixed solution of dioxane (40mL) and water (10mL). After nitrogen replacement 3 times, the reaction solution is heated to 90°C for 3 hours . LC-MS showed that the reaction was complete. The reaction solution was concentrated under reduced pressure, and the residue was separated through a rapid silica gel column (0-4% methanol/dichloromethane) to obtain compound 8-2. LCMS (ESI) m/z: 356.3 [M+H] + .
  • Step 2 Under N 2 protection, dissolve compound 8-2 (2g, 5.6mmol) in methanol (100mL) solution, add catalyst dry palladium/carbon (0.5g, 10%), and replace the reaction solution with hydrogen for 3 times . Under the hydrogen pressure (30 Psi) and the reaction temperature of 30°C, the reaction solution was stirred for 12 hours. LC-MS showed that 50% starting material remained. The catalyst was filtered out, new catalyst dry palladium/carbon (1 g) was added, and the reaction was continued for 3 hours. LCMS showed that the reaction was complete. The solid was filtered through celite, and the filtrate was concentrated under reduced pressure to obtain compound 8-3. LCMS (ESI) m/z: 358.2 [M+H] + .
  • Step 3 Dissolve (1R)-2,2-difluorocyclopropylcarboxylic acid (282mg, 2.3mmol) in pyridine (10mL), add EDCI (4.0g, 21.0mmol) and compound 8-3 (0.75g , 2.1mmol), the reaction solution was stirred at 10°C for 12 hours. LC-MS showed that the reaction was complete. The reaction solution was diluted with water (30mL), extracted with dichloromethane/methanol (10/1, 50mL*3), the combined organic phase was washed with saturated brine (30mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure .
  • Step 4 Dissolve compound 8-4 (300 mg, 650.1 ⁇ mol) in dichloromethane (5 mL), add hydrochloric acid/ethyl acetate (4M, 10 mL), and react the reaction solution at 15° C. for half an hour. LC-MS showed that the reaction was complete. The reaction solution was concentrated to obtain compound 8-5 (hydrochloride). LCMS (ESI) m/z: 362.2 [M+H] + .
  • Step 5 Dissolve compound 8-5 (100mg, 251.4 ⁇ mol, HCl) in N,N-dimethylformamide (5mL), add HOBt (51mg, 377.0 ⁇ mol) and EDCI (72.28mg, 377.0 ⁇ mol), Then (1S)-2,2-difluorocyclopropylcarboxylic acid (34mg, 276.5 ⁇ mol) and diisopropylethylamine (65mg, 502.7 ⁇ mol) were added, and the reaction solution was reacted at 15°C for 12 hours. LC-MS showed that the reaction was complete. The reaction solution was concentrated under reduced pressure, and the residue was subjected to preparative HPLC (neutral conditions) to obtain compound 8-6.
  • compound 8-5 was prepared by the same acid amine condensation synthesis and separation method as compound 8-6 (addition of a different substituted carboxylic acid from compound 8-6) compound 8-7, 8-8 characterization data as follows:
  • Step 1 Dissolve (S)-2,2-difluorocyclopropylcarboxylic acid (1.13g, 9.2mmol) in pyridine (150mL), add EDCI (16.1g, 84mmol) and compound 8-3 (3g, 8.4mmol), the reaction solution was stirred at 10°C for 12 hours. LC-MS showed that the reaction was complete. The reaction solution was diluted with water (100 mL), extracted with dichloromethane/methanol (10/1, 100 mL*3), the combined organic phase was washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure.
  • Step 2 Dissolve compound 9-1 (2.3 g, 4.9 mmol) in dichloromethane (5 mL), add hydrochloric acid/ethyl acetate (4M, 20 mL), and react the reaction solution at 15° C. for half an hour. LC-MS showed that the reaction was complete and the target molecular ion peak was detected. The precipitated solid was filtered and dried to obtain compound 9-2 (hydrochloride). LCMS (ESI) m/z: 362.2 [M+H] + .
  • Step 3 Dissolve compound 9-2 (1.23g, 3.1mmol, HCl) in N,N-dimethylformamide (20mL), add HOBt (626mg, 4.6mmol) and EDCI (889mg, 4.6mmol), Then (1S)-2,2-difluorocyclopropylcarboxylic acid (414.92mg, 3.40mmol) and diisopropylethylamine (798.70mg, 6.18mmol) were added, and the reaction solution was reacted at 15°C for 12 hours. LC-MS showed that the reaction was complete.
  • reaction solution was diluted with water (10 mL), extracted with dichloromethane/methanol (10/1, 50 mL), the organic phase was washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was subjected to preparative HPLC (neutral conditions) to obtain compound 9-3.
  • Step 1 Dissolve compound 10-1 (100mg, 334.3 ⁇ mol), compound 3-3 (126mg, 334.3 ⁇ mol), Pd(dppf)Cl 2 (25mg, 33.4 ⁇ mol), potassium carbonate (139mg, 1.00mmol)
  • the mixed solution of dioxane (12 mL) and H 2 O (3 mL) was replaced with nitrogen 3 times.
  • the reaction solution was stirred at 90°C for 2 hours in a nitrogen atmosphere.
  • LCMS showed that the raw materials were consumed, and the main peak was monitored as the target molecular ion peak.
  • the reaction solution was filtered and concentrated to remove the solvent, and then separated and purified by a preparation plate to obtain compound 10-2.
  • Step 2 A solution of compound 10-2 (130 mg, 276.9 ⁇ mol) and HCl/EtOAc (4M, 2 mL) in dichloromethane (1 mL) was stirred at 25° C. for 5 minutes. LCMS showed that the raw materials were consumed, and the main peak was monitored as the target molecular ion peak. The reaction solution was concentrated under reduced pressure to obtain yellow solid compound 10-3 (120 mg, hydrochloride), which was directly used in the next reaction without purification. LCMS (ESI) m/z: 370.6 [M+H] + .
  • Step 3 In a nitrogen atmosphere, Pd/C (20 mg, 10%) was added to a MeOH (25 mL) solution containing compound 10-3 (120 mg, 295.6 ⁇ mol, hydrochloride). The suspension was replaced with hydrogen three times, and then stirred at 25°C for 12 hours in a hydrogen atmosphere (15 Psi). LCMS showed that the raw materials were consumed, and the main peak was monitored as the target molecular ion peak. The reaction solution was filtered and concentrated under reduced pressure to remove the solvent to obtain compound 10-4 (130 mg, hydrochloride), which was directly used in the next reaction without further purification. LCMS (ESI) m/z: 372.3 [M+H] + .
  • Step 4 Dissolve compound 10-4 (130mg, 318.7 ⁇ mol, hydrochloride), 2-cyanoacetic acid (33mg, 382.4 ⁇ mol), EDCI (92mg, 478 ⁇ mol), HOBt (65mg, 478 ⁇ mol) and DIEA (206mg , 1.6mmol, 277.6 ⁇ L) DMF (5mL) solution was stirred at 25°C for 12 hours. LCMS showed that the raw material was consumed, and the target molecular ion peak was monitored. The reaction solution was concentrated under reduced pressure to remove the solvent, and then prepared and separated to obtain (neutral system) compound 10-5.
  • JAK2, JAK3 and TYK2 dilution 20mM 3-(N-morpholine) propanesulfonic acid (MOPS), 1mM EDTA, 0.01% Brij-35.5% glycerol, 0.1% ⁇ -mercaptoethanol, 1mg/mL BSA; JAK1 dilution: 20mM TRIS, 0.2mM EDTA, 0.1% ⁇ -mercaptoethanol, 0.01% Brij-35.5% glycerol. All compounds were prepared into 100% DMSO solution and reached 50 times the final measured concentration. The test compound was diluted by a 3-fold concentration gradient, and the final concentration was 9 concentrations from 10 ⁇ M to 0.001 ⁇ M. The content of DMSO in the detection reaction was 2%. The working stock solution of this compound is added as the first component of the reaction to the measurement well, and then the remaining components are added according to the protocol detailed in the measurement below.
  • MOPS 3-(N-morpholine) propanesulfonic acid
  • JAK1(h) was incubated with 20mM Tris/HCl pH7.5, 0.2mM EDTA, 500 ⁇ M MGEEPLYWSFPAKKK, 10mM magnesium acetate and [ ⁇ - 33 P]-ATP (activity and concentration as required). The reaction was started by adding Mg/ATP mixture. After incubating at room temperature for 40 minutes, the reaction was terminated by adding 0.5% phosphoric acid. Then 10 ⁇ L of the reactant was spotted on the P30 filter pad and washed three times with 0.425% phosphoric acid and once with methanol within 4 minutes, dried, and scintillated counted.
  • JAK2(h) was incubated with 8mM MOPS pH 7.0, 0.2mM EDTA, 100 ⁇ M KTFCGTPEYLAPEVRREPRILSEEEQEMFRDFDYIADWC, 10mM magnesium acetate and [ ⁇ - 33 P]-ATP (activity and concentration as required).
  • the reaction was started by adding Mg/ATP mixture. After incubating at room temperature for 40 minutes, the reaction was terminated by adding 0.5% phosphoric acid. Then 10 ⁇ L of the reactant was spotted on the P30 filter pad and washed three times with 0.425% phosphoric acid and once with methanol within 4 minutes, dried, and scintillated counted.
  • JAK3(h) was incubated with 8mM MOPS pH 7.0, 0.2mM EDTA, 500 ⁇ M GGEEEEYFELVKKKK, 10mM magnesium acetate and [ ⁇ - 33 P]-ATP (activity and concentration as required). The reaction was started by adding Mg/ATP mixture. After incubating at room temperature for 40 minutes, the reaction was terminated by adding 0.5% phosphoric acid. Then 10 ⁇ L of the reactant was spotted on the P30 filter pad and washed three times with 0.425% phosphoric acid and once with methanol within 4 minutes, dried, and scintillated counted.
  • TYK2(h) was incubated with 8mM MOPS pH 7.0, 0.2mM EDTA, 250 ⁇ M GGMEDIYFEFMGGKKK, 10mM magnesium acetate and [ ⁇ - 33 P]-ATP (activity and concentration as required). The reaction was started by adding Mg/ATP mixture. After incubating at room temperature for 40 minutes, the reaction was terminated by adding 0.5% phosphoric acid. Then 10 ⁇ L of the reactant was spotted on the P30 filter pad and washed three times with 0.425% phosphoric acid and once with methanol within 4 minutes, dried, and scintillated counted.
  • the IC 50 result is analyzed by IDBS's XLFIT5 (205 formula). See Table 1 for details.
  • the compound of the present invention shows good selective inhibition of JAK1 and/or TYK2 in the in vitro activity test of the 4 kinase subtypes JAK1, JAK2, JAk3 and TYK2.
  • the clear solution obtained by dissolving the test compound was injected via tail vein and intragastrically administered to male mice (C57BL/6) or rats (SD) (overnight fasting, 7-8 weeks old).
  • the intravenous group (2mg/kg) at 0.117, 0.333, 1, 2, 4, 7 and 24 hours
  • the intragastric group (15 mg/kg) at 0.25, 0.5, 1, 2, 4, 8 At and 24 hours
  • blood was collected from the mandibular vein and centrifuged to obtain plasma.
  • the LC-MS/MS method was used to determine the plasma drug concentration
  • the WinNonlin TM Version 6.3 pharmacokinetic software was used to calculate the relevant pharmacokinetic parameters by the non-compartmental model linear logarithmic trapezoidal method.
  • the test results are as follows:
  • AUC 0-inf the area under the plasma concentration-time curve from time 0 to extrapolated to infinity
  • Bioavailability Bioavailability.
  • the compound of the present invention has good oral bioavailability in mice, and higher exposure is beneficial to produce good in vivo efficacy.
  • DSS configuration method weigh 20g DSS, add 1,000ml of drinking water, make a 2% DSS solution, store it in a refrigerator at 4°C, and it will be effective within one month.
  • Vehicle solution is an aqueous solution containing 0.5% methyl cellulose and 0.5% Tween 80. First dissolve 5g of methyl cellulose in 990mL of pure water, then add 5g of Tween 80 in small amounts several times, mix well and store at room temperature.
  • Compound preparation method weigh the compound, add an aqueous solution with a mass ratio of 0.5% methyl cellulose + 0.5% Tween 80, and ultrasound until it dissolves. After mixing the solution thoroughly, dispense into glass bottles and store in a refrigerator at 4°C.
  • mice 70 male C57BL/6 mice were weighed and randomly divided into 7 groups according to their body weight, with 10 mice in each group.
  • the grouping and dosage design are shown in Table 3-1.
  • the animals were adapted for 3 days and weighed every day after adaptation.
  • the blank group was given drinking water, and the other groups were given drinking water containing 2% DSS.
  • the amount of water filled was 5ml/head/day; the animals in the test drug group were given intragastric administration, 1 day a day Second, the blank group and the DNCB group were given the same volume of Vehicle with a volume of 10ml/kg. Every day, evaluate the consistency of stool and collect stool for fecal occult blood testing, and calculate the Disease Activity Index (DAI) score; test DSS consumption every day, and replace fresh DSS solution every 2 days.
  • DAI Disease Activity Index
  • the dosing cycle is The intragastric administration was continued for 7 days, and the drug was stopped for 4 days, and then the intragastric administration was continued for 7 days. When the intragastric administration was stopped, the normal water was changed.
  • DAI is the sum of the three index scores of body weight, stool viscosity, and fecal occult blood.
  • the scoring standards are shown in Table 3-2.
  • the method of operation is to add pirami hole to the specimen first, and then add hydrogen peroxide and ethanol solution. The color is read and scored within 2 minutes, and purple is immediately produced. Blue is recorded as 4+, purple-blue produced within 10s is recorded as 3+, purple-red produced within 1 minute is recorded as 2+, and purple-red only gradually produced within 1-2 minutes is recorded as 1+, and there is no purple blue within the interpretation time. Or purple-red color reaction is recorded as negative (-). After the experiment, the colon and spleen were taken, and the length of the colon and the weight of the spleen were measured. The spleen index was the ratio of the weight of the spleen to the body weight.
  • the experimental data are expressed by Mean ⁇ SEM, and all data are calculated by One-way ANOVA, and P ⁇ 0.05 is considered statistically different.
  • the DAI scores of compounds 1-8, 1-13 and the positive drug Filgotinib began to decrease, and there were significant differences on the 16th and 18th days compared with the DSS group; at the same time, on the 16th day and On the 18th day, the DAI score of compound 1-13 was significantly lower than that of the positive drug Filgotinib, indicating that compound 1-13 has a better effect on reducing the DAI score than the positive drug Filgotinib, suggesting that compound 1-13 may be used in the treatment of IBD Has a better therapeutic effect.
  • Compound 1-13 showed significant differences on days 11, 15, 16 and 18, while compound 1-8 had no significant difference; Compared with the positive drug Filgotinib, compound 1-13 can reduce the weight loss of mice caused by DSS, and there is a significant increase on the 9, 10, 14 and 18 days, indicating that compound 1-13 has a better reduction in mice The effect of weight loss in mice caused by DSS suggests that compound 1-13 may have a better therapeutic effect on IBD.
  • DSS can significantly increase the spleen index of mice (DSS group); after drug intervention, compounds 1-8, 1-13, 9- 3. Both 1-11 and Filgotinib can reduce the increase in mouse spleen index caused by DSS. Compounds 1-8, 1-13 and the positive drug Filgotinib have significant inhibitory effects.
  • the compound of the present invention exhibits a good disease treatment effect, and has the same or even better efficacy in mice at half the dose of Filgotinib.
  • Allergic dermatitis is a chronic inflammatory skin disease that is prone to recurring attacks.
  • the clinical manifestations are severe itching, pleomorphic skin lesions and xeroderma-like symptoms. Its pathogenesis is related to many factors such as heredity, immunity and infection.
  • the development of drugs for the treatment of AD can help relieve the skin symptoms of patients and improve the quality of life of patients.
  • the animal model of allergic dermatitis induced by 1-chloro-2,4-dinitrobenzene (DNCB) is basically the same as the clinical manifestations of AD patients. It is a classic and commonly used model for preclinical evaluation of drug efficacy.
  • the purpose of this experiment is to investigate the therapeutic effects of compound 1-8, compound 1-11, compound 9-3, and compound 1-13 on DNCB-induced allergic dermatitis in mice, and provide relevant information about preclinical pharmacodynamics for clinical research.
  • Solvent preparation method Vehicle solution is 30% PEG-400+70% (5% HP- ⁇ -CD), pH 4-5. First dissolve 35g of HP- ⁇ -CD in 700ml of MilliQ pure water, then add 300ml of PEG-400, adjust the pH to 4-5 after mixing, and store at room temperature.
  • Compound preparation method weigh the compound, add it to a solution with a volume ratio of 30% PEG-400+70% (5% HP- ⁇ -CD), and sonicate it. After mixing the solution thoroughly, dispense into glass bottles and store in a refrigerator at 4°C.
  • mice Seventy male Balb/c mice were weighed and randomly divided into 7 groups according to their body weight, each with 10 mice. The detailed grouping and dosage design information are shown in Table 4-1.
  • the adaptation period of the animal is 3 days. After adaptation, the back is shaved. After 2 days, it is sensitized with the modeling solution. Apply 20 ⁇ l 0.5% DNCB solution to the right ear of the mouse and 200 ⁇ l 0.5% DNCB solution on the back of the mouse, once a day for 3 days ; Start the challenge from the 4th day, apply 10 ⁇ l 1% DNCB solution to the right ear of the mouse, and 100 ⁇ l 1% DNCB solution on the back of the mouse, once every 3 days; at the same time, from the 4th day, give each day Gavage the mice with compounds 1-8, 1-11, 9-3 and 1-13, and smear the skin of ears and back with the positive drug Iloxone.
  • DNCB atopic dermatitis modeling success indicators: DNCB successfully induced BALB/c mice to produce erythema, erosion, hemorrhage, edema, epidermal shedding, epidermal thickening and other typical atopic dermatitis-like skin lesions. In 3 days, 9 days, 15 days, 21 days, 27 days, the four clinical atopic dermatitis inflammation indicators of erythema/hemorrhage, scar/dryness, edema, and infiltration/erosion of the back skin of mice were evaluated to treat atopic dermatitis. Severity evaluation. Each evaluation index has four levels: 0, none; 1, slight; 2, moderate; 3, obvious; 4, very obvious. Add the scores to score the skin.
  • spleen index spleen weight (mg)/body weight (g) .
  • the experimental data is expressed by Mean ⁇ SEM, and all data are calculated by One-way ANOVA, and P ⁇ 0.05 is considered statistically different.
  • the skin score results show that, as shown in Table 4-2 and Figure 5, after the DNCB group was given DNCB induction, the skin score continued to increase. From the 9th day, the skin score was significantly higher than that of the blank control group and continued until the end of the experiment. Compared with the DNCB group, the skin score gradually decreased after drug intervention, and the compound 1-13 group showed a significant decrease after 21 days of administration, showing a better effect than the positive drug Iloxone. At the 27th day of administration, all administration groups including Compounds 1-8, 1-11, 9-3 and 1-13 and Iloxone group showed a significant reduction in skin score.
  • the experimental results showed (as shown in Table 4-3 and Figure 6) that the thickness of the right ear of the mice was significantly increased after DNCB induction (DNCB group), and after drug intervention (including compounds 1-8, 1-11, 9-3 and 1- 13 and Iloxone group) the thickness of the right ear of the mice decreased significantly, indicating that compounds 1-8, 1-11, 9-3 and 1-13 have significant therapeutic effects on DNCB-induced allergic dermatitis in mice. Its effect is equivalent to that of Iloxone.
  • the four compounds (compounds 1-8, 1-11, 9-3 and 1-13) had the strongest effect on reducing the thickness of the right ear with compound 1-13, but there was no statistical difference between the groups.
  • mice The experimental results show (as shown in Table 4-4 and Figure 7) that the spleen of mice (DNCB group) increased after DNCB induction, which was manifested as a significant increase in spleen index.
  • DNCB group mice spleen index decreased significantly, indicating that compounds 1-8, 1-11, 9-3 and 1-13 have a significant therapeutic effect on DNCB-induced allergic dermatitis in mice Its effect is equivalent to that of Iloxone.
  • the four compounds (compounds 1-8, 1-11, 9-3 and 1-13) had the strongest effect on reducing the spleen index with compound 1-13, but there was no statistical difference between the groups.
  • mice serum lgE concentration decreased significantly, indicating that compounds 1-8, 1-11, 9-3 and 1-13 have a significant therapeutic effect on DNCB-induced allergic dermatitis in mice .
  • the experimental results show (as shown in Table 4-6 and Figure 9) that the weight of the mice in the blank group and the DNCB group increased slowly, and the weight of the mice in the DNCB group was slightly lower than that of the blank group, and there was no statistical difference between the groups.
  • the body weight of the four compounds (compounds 1-8, 1-11, 9-3, and 1-13) is similar to that of the DNCB group, which is higher than that of the Iloxone group, but there is no statistical difference between the groups, indicating that the compounds 1-8, 1 -11, 9-3 and 1-13 had no significant effect on the body weight of mice.
  • the compounds 1-8, 1-11, 9-3 and 1-13 of the present invention can significantly reduce the skin score, right ear thickness, and spleen of mice Index and serum lgE concentration, and have no significant effect on body weight, showing a good disease treatment effect, and its effect is equivalent to the effect of the glucocorticoid Iloxone.

Abstract

一种JAK抑制剂[1,2,4]三唑并[1,5-a]吡啶类化合物在制备治疗自身免疫性、炎症性或变应性疾病，或者移植排斥等疾病的应用，所述JAK抑制剂[1,2,4]三唑并[1,5-a]吡啶类化合物包括式(Ⅰ)所示化合物、其异构体或其药学上可接受的盐。所述JAK抑制剂在自身免疫性、炎症性或变应性等疾病的动物模型试验中具有良好药效。

Description

JAK抑制剂在制备治疗JAK激酶相关疾病药物中的应用 技术领域
本发明涉及制药领域，具体涉及一种[1,2,4]三唑并[1,5-a]吡啶类化合物在制备治疗JAK激酶相关的自身免疫性、炎症性、变应性疾病、或者移植物抗宿主病等疾病的药物中的应用。
背景技术
JAK属于参与炎症、自身免疫疾病、增殖性疾病、移植排斥(或移植物抗宿主病)、涉及软骨更新(turnover)受损的疾病、先天软骨畸形和/或与IL6分泌过多相关的疾病的酪氨酸激酶家族。有研究证实，抑制JAK信号通路被认为可以调控多条与炎症、自身免疫性疾病、增殖性疾病、移植排斥、涉及软骨更新(turnover)受损的疾病、先天软骨畸形和/或与IL6分泌过多相关疾病的信号通路。本发明还提供所述化合物、含有所述化合物的药物组合物的生产方法和通过施用本发明化合物预防和/或治疗炎症、自身免疫疾病、增殖性疾病、移植排斥、涉及软骨更新受损的疾病、先天软骨畸形和/或与IL6分泌过多相关的疾病的方法。
Janus激酶(JAK)是转导细胞因子信号从膜受体到STAT转录因子的细胞质酪氨酸激酶。现有技术已经描述了四种JAK家族成员：JAK1、JAK2、JAK3和TYK2。当细胞因子与其受体结合时，JAK家族成员自磷酸化和/或彼此转磷酸化，随后STATs磷酸化，然后迁移至细胞核内以调节转录。JAK-STAT细胞内信号转导适用于干扰素、大多数白细胞介素以及多种细胞因子和内分泌因子，例如EPO、TPO、GH、OSM、LIF、CNTF、GM-CSF和PRL(Vainchenker W.等人(2008))。
遗传学模型和小分子JAK抑制剂的组合研究揭示了几种JAKs的治疗潜能。通过小鼠和人遗传学确证JAK3是免疫抑制靶点(O’Shea J.等人 (2004))。JAK抑制剂成功用于临床开发，最初用于器官移植排斥，但后来也用于其它免疫炎性适应证，例如炎症性肠病(IBD)、过敏性皮炎(AD)、类风湿性关节炎(RA)、银屑病和克罗恩病(http：//clinicaltrials.gov/)。TYK2是免疫炎性疾病的潜在靶点，已经通过人遗传学和小鼠剔除研究确证(Levy D.和Loomis C.(2007))。JAK1是免疫炎性疾病领域的新靶点。将JAK1与其它JAKs杂二聚化以转导细胞因子驱动的促炎信号传导。因此，预期抑制JAK1和/或其它JAK对于一系列炎性病症和其它由JAK介导的信号转导驱动的疾病是具有治疗益处的。
而炎症性肠病为累及回肠、直肠、结肠的一种特发性肠道炎症性疾病。临床表现腹泻、腹痛，甚至可有血便。炎症性肠病包括溃疡性结肠炎和克罗恩病。溃疡性结肠炎是结肠黏膜层和黏膜下层连续性炎症，疾病通常先累及直肠，逐渐向全结肠蔓延，克罗恩病可累及全消化道，为非连续性全层炎症，最常累及部位为末端回肠、结肠和肛周。
过敏性皮炎是由过敏原引起的皮肤病，主要指人体接触到某些过敏源而引起皮肤红肿、发痒、风团、脱皮等皮肤病症。具体的过敏原可有接触性过敏原、吸入性过敏原、食入性过敏原和注射性过敏等。每类过敏原都可以引起相应的过敏反应，主要的表现为多种皮炎、湿疹、荨麻疹等。
银屑病俗称牛皮癣，是一种慢性炎症性皮肤病，其对患者的身体健康和精神状况影响较大。临床表现以红斑，鳞屑为主，全身均可发病，以头皮，四肢伸侧较为常见。
系统性红斑狼疮是一种病因尚不明确的、以多种自身抗体导致不同靶器官损害为特点的自身免疫病。由于体内有大量致病性自身抗体和免疫复合物而造成组织损伤，临床上可出现各个系统和脏器损伤的表现，如皮肤、关节，浆膜、心脏、肾脏，中枢神经系统、血液系统等。
移植物抗宿主病移植物抗宿主病(GVHD)是由于移植后异体供者移植物中的T淋巴细胞，经受者发动的一系列“细胞因子风暴”刺激，大大增强了其对受者抗原的免疫反应。
US2009220688公开了Filgotinib，该化合物为Galapagos公司开发的目前处于临床三期用于类风湿性关节炎治疗的药物。
Figure PCTCN2020075024-appb-000001
而在此基础上开发新的JAK抑制剂[1,2,4]三唑并[1,5-a]吡啶类化合物及其在用于制备炎症性肠病、过敏性皮炎、银屑病、系统性红斑狼疮或移植物抗宿主病的药物应用等疾病的药物的应用是有必要的。
发明内容
针对以上技术现状，本发明提供一种如式(Ⅰ)的JAK抑制剂[1,2,4]三唑并[1,5-a]吡啶类化合物、其异构体或其药学上可接受的盐在制备治疗JAK激酶相关疾病的药物的应用：
Figure PCTCN2020075024-appb-000002
其中，
E 1和E 2分别独立地选自单键、-CH 2-或-(CH 2) 2-；
L 1选自单键、-(CH 2) g-、-C(＝O)-或-C(＝O)-(CH 2) h-；
m为1或2；
n为1或2；
g为1、2或3；
h为1、2或3；
R 1选自H、CN、C 1-6烷基或3～6元环烷基，其中所述C 1-6烷基和3～6元环烷基任选被1、2或3个R a取代；
R 2选自H、F、Cl、Br、I或C 1-3烷基，其中所述C 1-3烷基任选被1、2或3个R b取代；
R 3、R 4和R 5分别独立地选自H、F、Cl、Br、I或C 1-3烷基，其中所述C 1-3烷基任选被1、2或3个R c取代；
R 6、R 7和R 8分别独立地选自H、F、Cl、Br、I或C 1-3烷基，其中所述C 1-3烷基任选被1、2或3个R d取代；
每一个R a分别独立地选自H、F、Cl、Br、I、CN或C 1-3烷基，其中所述C 1-3烷基任选被1、2或3个R取代；
每一个R b分别独立地选自F、Cl、Br或I；
每一个R c分别独立地选自F、Cl、Br或I；
每一个R d分别独立地选自F、Cl、Br或I；
每一个R分别独立地选自F、Cl、Br或I。
本发明应用中，作为实施方案之一，本发明提供如式(Ⅰ)JAK抑制剂[1,2,4]三唑并[1,5-a]吡啶类化合物、其异构体或药学可接受的盐在制备治疗自身免疫性疾病、炎症性疾病、变应性疾病、或移植物抗宿主病的药物中的应用。
本发明应用中，作为实施方案之一，所述自身免疫性疾病包括系统性红斑狼疮、银屑病、银屑病性关节炎或狼疮肾炎。
本发明应用中，作为实施方案之一，所述炎症性疾病包括炎症性肠病、强直性脊柱炎或原发性胆道炎等。
本发明应用中，作为实施方案之一，所述变应性疾病包括过敏性皮炎、接触性皮炎、过敏性紫癜或支气管哮喘等。
本发明应用中，作为实施方案之一，所述移植物抗宿主病包括但不限于抗急性排斥反应、抗慢性排斥反应或诱导免疫耐受等。
本发明应用中，作为实施方案之一，本发明提供一种如式(Ⅰ)JAK 抑制剂[1,2,4]三唑并[1,5-a]吡啶类化合物、其异构体或药学可接受的盐在制备治疗炎症性肠病的药物中的应用。
本发明应用中，作为实施方案之一，所述治疗炎症性肠病包括但不限于抑制结肠缩短。
本发明应用中，作为实施方案之一，本发明提供一种如式(Ⅰ)JAK抑制剂[1,2,4]三唑并[1,5-a]吡啶类化合物、其异构体或药学可接受的盐在制备治疗过敏性皮炎的药物中的应用。
本发明应用中，作为实施方案之一，本发明提供一种如式(Ⅰ)JAK抑制剂[1,2,4]三唑并[1,5-a]吡啶类化合物、其异构体或药学可接受的盐在制备治疗银屑病的药物中的应用。
本发明应用中，作为实施方案之一，本发明提供一种如式(Ⅰ)JAK抑制剂[1,2,4]三唑并[1,5-a]吡啶类化合物、其异构体或药学可接受的盐在制备治疗系统性红斑狼疮的药物中的应用。
本发明应用中，作为实施方案之一，本发明提供一种如式(Ⅰ)JAK抑制剂[1,2,4]三唑并[1,5-a]吡啶类化合物、其异构体或药学可接受的盐在制备治疗移植物抗宿主病的药物中的应用。
作为实施方案之一，所述移植物抗宿主病包括但不限于抗急性排斥反应、抗慢性排斥反应或诱导免疫耐受。
作为实施方案之一，本发明制备治疗炎症性肠病、过敏性皮炎、银屑病、系统性红斑狼疮或移植物抗宿主病的药物应用中，如式(Ⅰ)的JAK抑制剂[1,2,4]三唑并[1,5-a]吡啶类化合物、其异构体或其药学上可接受的盐：
Figure PCTCN2020075024-appb-000003
其中，
E 1和E 2分别独立地选自单键、-CH 2-或-(CH 2) 2-；
L 1选自单键、-(CH 2) g-、-C(＝O)-或-C(＝O)-(CH 2) h-；
m为1或2；
n为1或2；
g为1、2或3；
h为1、2或3；
R 1选自H、CN、C 1-6烷基或3～6元环烷基，其中所述C 1-6烷基和3～6元环烷基任选被1、2或3个R a取代；
R 2选自H、F、Cl、Br、I或C 1-3烷基，其中所述C 1-3烷基任选被1、2或3个R b取代；
R 3、R 4和R 5分别独立地选自H、F、Cl、Br、I或C 1-3烷基，其中所述C 1-3烷基任选被1、2或3个R c取代；
R 6、R 7和R 8分别独立地选自H、F、Cl、Br、I或C 1-3烷基，其中所述C 1-3烷基任选被1、2或3个R d取代；
每一个R a分别独立地选自H、F、Cl、Br、I、CN或C 1-3烷基，其中所述C 1-3烷基任选被1、2或3个R取代；
每一个R b分别独立地选自F、Cl、Br或I；
每一个R c分别独立地选自F、Cl、Br或I；
每一个R d分别独立地选自F、Cl、Br或I；
每一个R分别独立地选自F、Cl、Br或I。
本发明应用中，作为实施方案之一，所述每一个R a分别独立地选自H、F、Cl、Br、I或CN，其他变量如本发明所定义。
本发明应用中，作为实施方案之一，所述R 1选自H、CN、C 1-3烷基或3～5元环烷基，其中所述C 1-3烷基和3～5元环烷基任选被1、2或3个R a取代，其他变量如本发明所定义。
本发明应用中，作为实施方案之一，所述R 1选自H、CN、CH 3、
Figure PCTCN2020075024-appb-000004
Figure PCTCN2020075024-appb-000005
其中所述CH 3、
Figure PCTCN2020075024-appb-000006
任选被1、2或3个R a取 代，其他变量如本发明所定义。
本发明应用中，作为实施方案之一，所述R 1选自H、CN、CF 3、CHF 2、
Figure PCTCN2020075024-appb-000007
其他变量如本发明所定义。
本发明应用中，作为实施方案之一，所述R 2选自H、F、Cl、Br或I，其他变量如本发明所定义。
本发明应用中，作为实施方案之一，所述R 3、R 4和R 5分别独立地选自H、F、Cl、Br或I，其他变量如本发明所定义。
本发明应用中，作为实施方案之一，所述R 6、R 7和R 8分别独立地选自H、F、Cl、Br或I，其他变量如本发明所定义。
本发明应用中，作为实施方案之一，所述L 1选自单键、-CH 2-、-(CH 2) 2-、-C(＝O)-或-C(＝O)-(CH 2)-，其他变量如本发明所定义。
本发明应用中，作为实施方案之一，所述结构单元
Figure PCTCN2020075024-appb-000008
选自
Figure PCTCN2020075024-appb-000009
Figure PCTCN2020075024-appb-000010
其他变量如本发明所定义。
本发明应用中，作为实施方案之一，所述结构单元
Figure PCTCN2020075024-appb-000011
选自
Figure PCTCN2020075024-appb-000012
Figure PCTCN2020075024-appb-000013
Figure PCTCN2020075024-appb-000014
其他变量如本发明所定义。
本发明应用中，作为实施方案之一，所述结构单元
Figure PCTCN2020075024-appb-000015
选自
Figure PCTCN2020075024-appb-000016
Figure PCTCN2020075024-appb-000017
其他变量如本发明所定义。
本发明应用中，作为实施方案之一，所述结构单元
Figure PCTCN2020075024-appb-000018
选自
Figure PCTCN2020075024-appb-000019
Figure PCTCN2020075024-appb-000020
其他变量如本发明所定义。
本发明还有一些方案是有上述各变量任意组合而来。
本发明应用中，作为实施方案之一，所述式(Ⅰ)化合物、其异构体或其药学上可接受的盐，其选自
Figure PCTCN2020075024-appb-000021
其中，
L 1、R 1、R 2、R 3、R 4、R 5、R 6、R 7和R 8如本发明所定义。
本发明应用中，作为实施方案之一，所述式(Ⅰ)化合物、其异构体或其药学上可接受的盐，其选自
Figure PCTCN2020075024-appb-000022
其中，
L 1、R a、R 2、R 3、R 4、R 5、R 6、R 7和R 8如本发明所定义。
本发明应用中，作为实施方案之一，本发明还提供了下列化合物、 其异构体或其药学上可接受的盐：
Figure PCTCN2020075024-appb-000023
Figure PCTCN2020075024-appb-000024
本发明应用中，作为实施方案之一，本发明还进一步提供了下列化合物、其异构体或其药学上可接受的盐：
Figure PCTCN2020075024-appb-000025
Figure PCTCN2020075024-appb-000026
作为实施方案之一，本发明还提供了一种药物组合物，包括治疗有效量的作为活性成分的如式(I)化合物、其异构体或其药学上可接受的盐和药学上可接受的载体。
实验证明，本发明的如式(I)的JAK抑制剂[1,2,4]三唑并[1,5-a]吡啶类化合物在制备治疗自身免疫性、炎症性或变应性疾病，或者移植排斥等疾病的药物中的应用，本发明涉及的系列化合物在JAK激酶4个亚型(JAK1、JAK2、JAk3和TYK2)的体外活性实验中展现了对JAK1和/或TYK2的良好选择性抑制作用，并且这些化合物在小鼠的药代动力学实验中表现出较高的暴露量,良好的口服生物利用度。在自身免疫性、炎症性或变应性等疾病的动物模型试验中具有良好药效，尤其在炎症性肠病和过敏性皮炎的动物体内药效评价实验中展现更好的体内药效结果。实验中显示了本发明中化合物1-13在Filgotinib一半剂量下具有同等甚至更优的治疗炎症性肠病的作用，化合物1-8、1-11、1-13和9-3均具有显著治疗过敏性皮炎的作用，其作用与艾洛松作用相当。
定义和说明
除非另有说明，本文所用的下列术语和短语旨在具有下列含义。一个特定的术语或短语在没有特别定义的情况下不应该被认为是不确定的或不清楚的，而应该按照普通的含义去理解。当本文中出现商品名时，意在指代其对应的商品或其活性成分。
这里所采用的术语“药学上可接受的”，是针对那些化合物、材料、组合物和/或剂型而言，它们在可靠的医学判断的范围之内，适用于与人类和动物的组织接触使用，而没有过多的毒性、刺激性、过敏性反应或其它问题或并发症，与合理的利益/风险比相称。
术语“药学上可接受的盐”是指本发明化合物的盐，由本发明发现的具有特定取代基的化合物与相对无毒的酸或碱制备。当本发明的化合物中含有相对酸性的功能团时，可以通过在纯的溶液或合适的惰性溶剂中用足够量的碱与这类化合物的中性形式接触的方式获得碱加成盐。药学上可接受的碱加成盐包括钠、钾、钙、铵、有机胺或镁盐或类似的盐。当本发明的化合物中含有相对碱性的官能团时，可以通过在纯的溶液或合适的惰性溶剂中用足够量的酸与这类化合物的中性形式接触的方式获得酸加成盐。药学上可接受的酸加成盐的实例包括无机酸盐，所述无机酸包括例如盐酸、氢溴酸、硝酸、碳酸，碳酸氢根，磷酸、磷酸一氢根、磷酸二氢根、硫酸、硫酸氢根、氢碘酸、亚磷酸等；以及有机酸盐，所述有机酸包括如乙酸、丙酸、异丁酸、马来酸、丙二酸、苯甲酸、琥珀酸、辛二酸、反丁烯二酸、乳酸、扁桃酸、邻苯二甲酸、苯磺酸、对甲苯磺酸、柠檬酸、酒石酸和甲磺酸等类似的酸；还包括氨基酸(如精氨酸等)的盐，以及如葡糖醛酸等有机酸的盐。本发明的某些特定的化合物含有碱性和酸性的官能团，从而可以被转换成任一碱或酸加成盐。
本发明的药学上可接受的盐可由含有酸根或碱基的母体化合物通过常规化学方法合成。一般情况下，这样的盐的制备方法是：在水或有机溶剂或两者的混合物中，经由游离酸或碱形式的这些化合物与化学计量的适当的碱或酸反应来制备。
本发明的化合物可以存在特定的几何或立体异构体形式。本发明设想所有的这类化合物，包括顺式和反式异构体、(-)-和(+)-对映体、(R)-和(S)-对映体、非对映异构体、(D)-异构体、(L)-异构体，及其外消旋混合物和其他混合物，例如对映异构体或非对映体富集的混合物，所有这些混合物都属于本发明的范围之内。烷基等取代基中可存在另外的不对称碳原子。所有这些异构体以及它们的混合物，均包括在本发明的范围之内。
除非另有说明，术语“对映异构体”或者“旋光异构体”是指互为镜像关系的立体异构体。
除非另有说明，术语“顺反异构体”或者“几何异构体”系由因双键或者 成环碳原子单键不能自由旋转而引起。
除非另有说明，术语“非对映异构体”是指分子具有两个或多个手性中心，并且分子间为非镜像的关系的立体异构体。
除非另有说明，“(D)”或者“(+)”表示右旋，“(L)”或者“(-)”表示左旋，“(DL)”或者“(±)”表示外消旋。
除非另有说明，用楔形实线键
Figure PCTCN2020075024-appb-000027
和楔形虚线键
Figure PCTCN2020075024-appb-000028
表示一个立体中心的绝对构型，用直形实线键
Figure PCTCN2020075024-appb-000029
和直形虚线键
Figure PCTCN2020075024-appb-000030
表示立体中心的相对构型，用波浪线
Figure PCTCN2020075024-appb-000031
表示楔形实线键
Figure PCTCN2020075024-appb-000032
或楔形虚线键
Figure PCTCN2020075024-appb-000033
或用波浪线
Figure PCTCN2020075024-appb-000034
表示直形实线键
Figure PCTCN2020075024-appb-000035
和直形虚线键
Figure PCTCN2020075024-appb-000036
除非另有说明，当化合物中存在双键结构，如碳碳双键、碳氮双键和氮氮双键，且双键上的各个原子均连接有两个不同的取代基时(包含氮原子的双键中，氮原子上的一对孤对电子视为其连接的一个取代基)，如果该化合物中双键上的原子与其取代基之间用波浪线
Figure PCTCN2020075024-appb-000037
连接，则表示该化合物的(Z)型异构体、(E)型异构体或两种异构体的混合物。例如下式(A)表示该化合物以式(A-1)或式(A-2)的单一异构体形式存在或以式(A-1)和式(A-2)两种异构体的混合物形式存在；下式(B)表示该化合物以式(B-1)或式(B-2)的单一异构体形式存在或以式(B-1)和式(B-2)两种异构体的混合物形式存在。下式(C)表示该化合物以式(C-1)或式(C-2)的单一异构体形式存在或以式(C-1)和式(C-2)两种异构体的混合物形式存在。
Figure PCTCN2020075024-appb-000038
Figure PCTCN2020075024-appb-000039
除非另有说明，术语“互变异构体”或“互变异构体形式”是指在室温下，不同官能团异构体处于动态平衡，并能很快的相互转化。若互变异构体是可能的(如在溶液中)，则可以达到互变异构体的化学平衡。例如，质子互变异构体(proton tautomer)(也称质子转移互变异构体(prototropic tautomer))包括通过质子迁移来进行的互相转化，如酮-烯醇异构化和亚胺-烯胺异构化。价键异构体(valence tautomer)包括一些成键电子的重组来进行的相互转化。其中酮-烯醇互变异构化的具体实例是戊烷-2,4-二酮与4-羟基戊-3-烯-2-酮两个互变异构体之间的互变。
除非另有说明，术语“富含一种异构体”、“异构体富集”、“富含一种对映体”或者“对映体富集”指其中一种异构体或对映体的含量小于100％，并且，该异构体或对映体的含量大于等于60％，或者大于等于70％，或者大于等于80％，或者大于等于90％，或者大于等于95％，或者大于等于96％，或者大于等于97％，或者大于等于98％，或者大于等于99％，或者大于等于99.5％，或者大于等于99.6％，或者大于等于99.7％，或者大于等于99.8％，或者大于等于99.9％。
除非另有说明，术语“异构体过量”或“对映体过量”指两种异构体或两种对映体相对百分数之间的差值。例如，其中一种异构体或对映体的含量为90％，另一种异构体或对映体的含量为10％，则异构体或对映体过量(ee值)为80％。
可以通过的手性合成或手性试剂或者其他常规技术制备光学活性的(R)-和(S)-异构体以及D和L异构体。如果想得到本发明某化合物的一种对映体，可以通过不对称合成或者具有手性助剂的衍生作用来制备，其中将所得非对映体混合物分离，并且辅助基团裂开以提供纯的所需对映异构体。或者，当分子中含有碱性官能团(如氨基)或酸性官能团(如羧基)时，与适当的光学活性的酸或碱形成非对映异构体的盐，然后通过本领域所公知的常规方法进行非对映异构体拆分，然后回收得到纯的 对映体。此外，对映异构体和非对映异构体的分离通常是通过使用色谱法完成的，所述色谱法采用手性固定相，并任选地与化学衍生法相结合(例如由胺生成氨基甲酸盐)。本发明的化合物可以在一个或多个构成该化合物的原子上包含非天然比例的原子同位素。例如，可用放射性同位素标记化合物，比如氚( 3H)，碘-125( 125I)或C-14( 14C)。又例如，可用重氢取代氢形成氘代药物，氘与碳构成的键比普通氢与碳构成的键更坚固，相比于未氘化药物，氘代药物有降低毒副作用、增加药物稳定性、增强疗效、延长药物生物半衰期等优势。本发明的化合物的所有同位素组成的变换，无论放射性与否，都包括在本发明的范围之内。“任选”或“任选地”指的是随后描述的事件或状况可能但不是必需出现的，并且该描述包括其中所述事件或状况发生的情况以及所述事件或状况不发生的情况。
术语“被取代的”是指特定原子上的任意一个或多个氢原子被取代基取代，可以包括重氢和氢的变体，只要特定原子的价态是正常的并且取代后的化合物是稳定的。当取代基为氧(即＝O)时，意味着两个氢原子被取代。氧取代不会发生在芳香基上。术语“任选被取代的”是指可以被取代，也可以不被取代，除非另有规定，取代基的种类和数目在化学上可以实现的基础上可以是任意的。
当任何变量(例如R)在化合物的组成或结构中出现一次以上时，其在每一种情况下的定义都是独立的。因此，例如，如果一个基团被0-2个R所取代，则所述基团可以任选地至多被两个R所取代，并且每种情况下的R都有独立的选项。此外，取代基和/或其变体的组合只有在这样的组合会产生稳定的化合物的情况下才是被允许的。
当一个连接基团的数量为0时，比如-(CRR) 0-，表示该连接基团为单键。
当其中一个变量选自单键时，表示其连接的两个基团直接相连，比如A-L-Z中L代表单键时表示该结构实际上是A-Z。
当一个取代基为空缺时，表示该取代基是不存在的，比如A-X中X为空缺时表示该结构实际上是A。当所列举的取代基中没有指明其通过 哪一个原子连接到被取代的基团上时，这种取代基可以通过其任何原子相键合，例如，吡啶基作为取代基可以通过吡啶环上任意一个碳原子连接到被取代的基团上。
当所列举的连接基团没有指明其连接方向，其连接方向是任意的，例如，
Figure PCTCN2020075024-appb-000040
中连接基团L为-M-W-，此时-M-W-既可以按与从左往右的读取顺序相同的方向连接环A和环B构成
Figure PCTCN2020075024-appb-000041
也可以按照与从左往右的读取顺序相反的方向连接环A和环B构成
Figure PCTCN2020075024-appb-000042
所述连接基团、取代基和/或其变体的组合只有在这样的组合会产生稳定的化合物的情况下才是被允许的。
除非另有规定，环上原子的数目通常被定义为环的元数，例如，“5-7元环”是指环绕排列5-7个原子的“环”。
除非另有规定，“5-6元环”表示由5至6个环原子组成的环烷基、杂环烷基、环烯基、杂环烯基、环炔基、杂环炔基、芳基或杂芳基。所述的环包括单环，也包括螺环、并环和桥环等双环体系。除非另有规定，该环任选地包含1、2或3个独立选自O、S和N的杂原子。所述5-6元环包括5元、6元环等。“5-6元环”包括例如苯基、吡啶基和哌啶基等；另一方面，术语“5-6元杂环烷基”包括哌啶基等，但不包括苯基。术语“环”还包括含有至少一个环的环系，其中的每一个“环”均独立地符合上述定义。
除非另有规定，术语“C 1-6烷基”用于表示直链或支链的由1至6个碳原子组成的饱和碳氢基团。所述C 1-6烷基包括C 1-5、C 1-4、C 1-3、C 1-2、C 2-6、C 2-4、C 6和C 5烷基等；其可以是一价(如甲基)、二价(如亚甲基)或者多价(如次甲基)。C 1-6烷基的实例包括但不限于甲基(Me)、乙基(Et)、丙基(包括n-丙基和异丙基)、丁基(包括n-丁基，异丁基，s-丁基和t- 丁基)、戊基(包括n-戊基，异戊基和新戊基)、己基等。
除非另有规定，术语“C 1-3烷基”用于表示直链或支链的由1至3个碳原子组成的饱和碳氢基团。所述C 1-3烷基包括C 1-2和C 2-3烷基等；其可以是一价(如甲基)、二价(如亚甲基)或者多价(如次甲基)。C 1-3烷基的实例包括但不限于甲基(Me)、乙基(Et)、丙基(包括n-丙基和异丙基)等。
除非另有规定，“C 3-6环烷基”表示由3至6个碳原子组成的饱和环状碳氢基团，其为单环和双环体系，所述C 3-6环烷基包括C 3-5、C 4-5和C 5-6环烷基等；其可以是一价、二价或者多价。C 3-6环烷基的实例包括，但不限于，环丙基、环丁基、环戊基、环己基等。
除非另有规定，C n-n+m或C n-C n+m包括n至n+m个碳的任何一种具体情况，例如C 1-12包括C 1、C 2、C 3、C 4、C 5、C 6、C 7、C 8、C 9、C 10、C 11、和C 12，也包括n至n+m中的任何一个范围，例如C 1-12包括C 1-3、C 1-6、C 1-9、C 3-6、C 3-9、C 3-12、C 6-9、C 6-12、和C 9-12等；同理，n元至n+m元表示环上原子数为n至n+m个，例如3-12元环包括3元环、4元环、5元环、6元环、7元环、8元环、9元环、10元环、11元环、和12元环，也包括n至n+m中的任何一个范围，例如3-12元环包括3-6元环、3-9元环、5-6元环、5-7元环、6-7元环、6-8元环、和6-10元环等。
本发明的化合物可以通过本领域技术人员所熟知的多种合成方法来制备，包括下面列举的具体实施方式、其与其他化学合成方法的结合所形成的实施方式以及本领域技术上人员所熟知的等同替换方式，优选的实施方式包括但不限于本发明的实施例。
本发明所使用的溶剂可经市售获得。本发明采用下述缩略词：aq代表水；HATU代表O-(7-氮杂苯并三唑-1-基)-N，N，N'，N'-四甲基脲六氟磷酸盐；EDC代表N-(3-二甲基氨基丙基)-N'-乙基碳二亚胺盐酸盐；m-CPBA代表3-氯过氧苯甲酸；eq代表当量、等量；CDI代表羰基二咪唑；DCM代表二氯甲烷；PE代表石油醚；DIAD代表偶氮二羧酸二异丙酯；DMF代表N，N-二甲基甲酰胺；DMSO代表二甲亚砜；EtOAc代表乙酸乙酯；EtOH代表乙醇；MeOH代表甲醇；CBz代表苄氧羰基， 是一种胺保护基团；BOC代表叔丁氧羰基是一种胺保护基团；HOAc代表乙酸；NaCNBH 3代表氰基硼氢化钠；r.t.代表室温；O/N代表过夜；THF代表四氢呋喃；Boc 2O代表二-叔丁基二碳酸酯；TFA代表三氟乙酸；DIPEA代表二异丙基乙基胺；SOCl 2代表氯化亚砜；CS 2代表二硫化碳；TsOH代表对甲苯磺酸；NFSI代表N-氟-N-(苯磺酰基)苯磺酰胺；NCS代表1-氯吡咯烷-2,5-二酮；n-Bu 4NF代表氟化四丁基铵；iPrOH代表2-丙醇；mp代表熔点；LDA代表二异丙基胺基锂；Pd(dppf)Cl 2·CH 2Cl 2代表[1,1'-双(二苯基膦基)二茂铁]二氯化钯的二氯甲烷络合物；EDCI代表碳化二亚胺；DIEA代表N,N-二异丙基乙胺；IPA代表异丙醇；HOBt代表1-羟基苯并三唑；LiHMDS代表六甲基二硅基胺基锂；TEA代表三乙基胺；HEPES代表4-羟乙基哌嗪乙磺酸；LiHMDS代表六甲基二硅基胺基锂；EDCI代表碳化二亚胺；Pd/C代表钯碳；METHANOL代表甲醇；KOAc代表醋酸钾；K 2CO 3代表碳酸钾。
化合物经手工或者
Figure PCTCN2020075024-appb-000043
软件命名，市售化合物采用供应商目录名称。
附图说明
图1：实验例3中DAI评分结果曲线图。
图2：实验例3中小鼠体重变化结果曲线图。
图3：实验例3中结肠长度柱状图。
图4：实验例3中脾脏指数柱状图。
图5：实验例4中皮肤评分结果曲线图。
图6：实验例4中的右耳厚度柱状图。
图7：实验例4中脾脏指数柱状图。
图8：实验例4中血清lgE浓度柱状图。
图9：实验例4中小鼠体重变化曲线图。
具体实施方式
下面通过实施例对本发明进行详细描述，但并不意味着对本发明任何不利限制。本文已经详细地描述了本发明，其中也公开了其具体实施例方式，对本领域的技术人员而言，在不脱离本发明精神和范围的情况下针对本发明具体实施方式进行各种变化和改进将是显而易见的。
实施例1
Figure PCTCN2020075024-appb-000044
步骤1:在-78℃下，向溶有化合物1-1(10.2g,42.6mmol)的THF(150mL)溶液中滴加LiHMDS(1M,51.2mL)。该反应液在-78℃下搅拌1小时后将1,1,1-三氟-N-苯基-N-(三氟甲基磺酰基)甲磺酰胺(16.7g,46.9mmol)的THF(150mL)溶液加入到该反应液中,然后在15℃下搅拌12小时。TLC(PE:EA＝10:1)显示原料被消耗完全，并有新点产生。用250mL饱和氯化铵淬灭反应，用200mL水稀释，然后用乙酸乙酯(200mL*3)萃取。合并有机相，经饱和食盐水洗涤，硫酸钠干燥，过滤浓缩得到化合物1-2。粗品未经纯化直接用于下一步反应。
1H NMR(400MHz,CDCl 3)δ5.63(br s,1H),3.50-3.65(m,4H),2.34(br s,4H),1.88(br t,J＝5.90Hz,2H),1.37(s,9H)。
步骤2：向溶有化合物1-2(16g,43.1mmol)和联硼酸频那醇酯(12.0g,47.4mmol)的DMF(100mL)溶液中加入醋酸钾(12.7g,129.3mmol)和Pd(dppf)Cl 2.CH 2Cl 2(3.5g,4.3mmol)，用氮气置换3次并保持在氮气氛围 中70℃下搅拌3小时。TLC板显示原料消耗完全，并有新点产生。将反应液分散在300mL水和400mL乙酸乙酯混合液中。将有机相分离并经饱和食盐水洗涤，硫酸钠干燥，过滤浓缩得到粗品。粗品经硅胶色谱柱法纯化得到化合物1-3。 1H NMR(400MHz,CDCl 3)δ6.46(br s,1H),3.71-3.53(m,4H),2.31(br d,J＝3.0Hz,2H),2.24-2.16(m,2H),1.74(t,J＝6.3Hz,2H),1.44(s,9H),1.26(s,12H)。
步骤3：在氮气氛围中，向溶有化合物1-3(3.5g,10.0mmol)和N-(5-溴-[1,2,4]三唑并[1,5-a]吡啶-2-基)环丙烷甲酰胺(2.6g,9.1mmol)的二氧六环(60mL)和水(15mL)的溶液中加入碳酸钾(3.8g,27.3mmol)和Pd(dppf)Cl 2.CH 2Cl 2(744mg,911.0μmol)。该反应液在90℃下搅拌3小时。LCMS显示原料消耗完全，并监测到目标分子离子峰。将反应液浓缩，所得粗品经柱色谱分离纯化得到化合物1-4。LCMS(ESI)m/z:424.3[M+H] +。
步骤4：向溶有化合物1-4(3.5g,8.2mmol)的二氯甲烷(10mL)溶液中加入盐酸/乙酸乙酯(4M,30mL)，该反应液在25℃下搅拌0.5小时。LCMS显示原料被消耗完，并监测到目标分子离子峰。固体析出，过滤并干燥，得到化合物1-5(3.3g盐酸盐，粗品)，未经纯化，直接用于下一步反应。LCMS(ESI)m/z:324.1。
步骤5：在氮气氛围中，向溶有化合物1-5(3.0g,8.34mmol,盐酸盐)的甲醇(100mL)溶液中加入Pd/C(1g,10％)。该悬浊液用氢气置换3次，然后在氢气氛围(30psi)30℃下搅拌12小时。LCMS显示原料被消耗完，并监测到目标分子离子峰。将反应液过滤，浓缩得到化合物1-6(3g盐酸盐，粗品)，未经纯化，直接用于下一步反应。LCMS(ESI)m/z:326.2[M+H] +。
步骤6：将化合物1-6(0.87g,2.40mmol,盐酸盐)溶解在N,N-二甲基甲酰胺(10mL)中，加入HOBt(487mg,3.6mmol,)和EDCI(691mg,3.6mmol)，之后加入(1S)-2,2-二氟环丙基甲酸(323mg,2.6mmol)和二异丙基乙胺(621mg,4.8mmol)，反应液在15℃反应12小时。LC-MS显示反应完全。反应液减压浓缩，残余物经由制备型HPLC(中性体系)得到化 合物1-13： 1H NMR(400MHz,METHANOL-d 4)δ7.32-7.73(m,2H),6.95(br s,1H),3.62-4.22(m,4H),3.45(br s,1H),3.18-3.37(m,1H),2.61(br s,1H),1.45-2.27(m,10H),0.78-1.17(m,4H)。LCMS(ESI)m/z:430.0[M+H] +。
以化合物1-6为共同中间体，运用同化合物1-13相同的合成及分离方法(即在酸胺缩合反应步骤中将合成化合物1-13的羧酸替换为下列目标分子中相应的羧酸)得到下列化合物，其表征数据如下：
Figure PCTCN2020075024-appb-000045
化合物1-7： 1H NMR(400MHz,DMSO-d 6)δ11.08(br s,1H),7.48-7.78(m,2H),7.03(d,J＝7.0Hz,1H),3.86-4.25(m,2H),3.61-3.80(m,2H),3.29-3.38(m,1H),2.69-2.88(m,1H),1.85-2.19(m,7H),1.51-1.79(m,4H),0.83-0.96(m,4H).LCMS(ESI)m/z:430.0[M+H] +。
化合物1-8： 1H NMR(400MHz,DMSO-d 6)δ11.14(br s,1H),7.52-7.66(m,2H),7.00(d,J＝7.03Hz,1H),3.54-3.83(m,6H),3.29(br t,J＝11.54Hz,1H),1.94-2.09(m,5H),1.41-1.70(m,4H),0.77-0.90(m,4H).LCMS(ESI)m/z:393.1[M+H] +。
化合物1-9： 1H NMR(400MHz,DMSO-d 6)δ10.99(br s,1H),7.49-7.65(m,2H),6.99(br d,J＝7.03Hz,1H),4.02-4.20(m,2H),3.61-3.78(m,2H), 1.94-2.13(m,5H),1.48-1.72(m,4H),1.14-1.32(m,4H),0.77-0.87(m,4H).LCMS(ESI)m/z:412.1[M+H] +。
化合物1-10： 1H NMR(400MHz,METHANOL-d 4)δ7.77-7.87(m,1H),7.62(d,J＝8.78Hz,1H),7.20(dd,J＝7.28,11.80Hz,1H),4.08(s,1H),3.96(s,1H),3.83(s,1H),3.72(s,1H),3.43-3.56(m,1H),3.22(dq,J＝6.90,10.75Hz,2H),2.06-2.23(m,4H),1.94(br s,1H),1.56-1.84(m,3H),1.56-2.00(m,1H),0.94-1.14(m,4H).LCMS(ESI)m/z:436.1[M+H] +。
化合物1-11： 1H NMR(400MHz,METHANOL-d 4)δ7.56-7.67(m,1H),7.50(d,J＝8.78Hz,1H),7.00(t,J＝7.15Hz,1H),4.26-4.48(m,2H),3.70-3.90(m,2H),3.42-3.59(m,1H),2.08-2.24(m,4H),1.48-1.99(m,9H),0.87-1.10(m,4H).LCMS(ESI)m/z:419.1[M+H] +。
化合物1-12： 1H NMR(400MHz,METHANOL-d 4)δ7.56-7.64(m,1H),7.48(d,J＝9.03Hz,1H),6.97(d,J＝7.28Hz,1H),3.91-4.17(m,2H),3.78-3.86(m,1H),3.67-3.75(m,1H),3.40-3.54(m,1H),2.53-2.69(m,1H),1.92-2.21(m,6H),1.72-1.85(m,3H),1.50-1.69(m,2H),0.86-1.08(m,4H).LCMS(ESI)m/z:430.1[M+H] +。
化合物1-14： 1H NMR(400MHz,METHANOL-d 4)δ7.57-7.66(m,1H),7.50(d,J＝8.78Hz,1H),6.95-7.03(m,1H),6.01-6.38(m,1H),4.62(s,1H),3.65-4.10(m,4H),3.43-3.58(m,1H),2.76-2.93(m,2H),2.04-2.21(m,4H),1.51-1.87(m,4H),1.01-1.07(m,2H),0.93(qd,J＝3.74,7.34Hz,2H).LCMS(ESI)m/z:418.1[M+H] +。
化合物1-15： 1H NMR(400MHz,DMSO-d 6)δ11.01(br s,1H),7.48-7.66(m,2H),7.00(dd,J＝7.53,9.79Hz,1H),4.11-4.33(m,2H),3.60-3.81(m,2H),3.25-3.32(m,1H),2.03(br t,J＝9.03Hz,5H),1.53-1.73(m,4H),1.49(d,J＝4.77Hz,6H),0.76-0.88(m,4H).LCMS(ESI)m/z:421.1[M+H] +。
Figure PCTCN2020075024-appb-000046
化合物1-16的合成:将化合物1-6(100mg,227.6μmol,TFA)溶解在N,N-二甲基甲酰胺(5mL)中，加入碳酸钾(94mg,682.7μmol)和2-溴乙腈(30mg,250.3μmol)，反应液在10℃搅拌12小时。LC-MS显示反应完全。反应液用水(5mL)稀释，二氯甲烷/甲醇(10/1,10mL)萃取，将有机相用饱和食盐水(10mL)洗涤，无水硫酸钠干燥，过滤并减压浓缩。残余物经由制备型HPLC纯化(中性体系)，得到化合物1-16。 1H NMR(400MHz,METHANOL-d 4)δ7.83(t,J＝8.03Hz,1H),7.64(br d,J＝8.78Hz,1H),7.21(d,J＝7.53Hz,1H),4.51(s,2H),4.23(s,2H),4.08(s,2H),3.49(br t,J＝11.92Hz,1H),2.14-2.30(m,4H),1.79-1.97(m,3H),1.59-1.74(m,2H),0.95-1.12(m,4H).LCMS(ESI)m/z:365.0[M+H] +。
以化合物1-6为共用中间体运用同化合物1-16相同的合成及分离方法(将溴乙腈换为目标分子中相应的溴丙腈)得到下列化合物，其表征数据如下：
化合物1-17： 1H NMR(400MHz,DMSO-d 6)δ11.00(br s,1H),7.50-7.63(m,2H),6.96(d,J＝6.27Hz,1H),3.33-3.34(m,2H),3.23-3.30(m,1H),2.95(s,2H),3.05(s,2H),2.58-2.69(m,2H),1.99(br d,J＝10.29Hz,5H),1.40-1.65(m,4H),0.74-0.88(m,4H)。LCMS(ESI)m/z:379.0[M+H] +。
实施例2
Figure PCTCN2020075024-appb-000047
步骤1：在-78℃氮气保护下，将叔丁基9-氧-3-氮杂螺[5.5]十一烷-3-羧酸(3-1)(5g,18.7mmol)溶解在无水四氢呋喃(150mL)中，缓慢滴加双(三甲基硅基)氨基锂(1M,22.4mL)，反应液在-78℃搅拌1小时。然后，加入溶有1,1,1-三氟-N-苯基-N-((三氟甲基)磺酰)甲磺酰胺(7.35g,20.6mmol)的无水四氢呋喃(50mL)溶液，反应液在15℃搅拌12小时。TLC显示反应完全。反应液用饱和氯化铵(50mL)淬灭，用乙酸乙酯(200mL*2)萃取。将合并的有机相用饱和食盐水(50mL)洗涤，无水硫酸钠干燥，过滤并减压浓缩，得到化合物3-2，该产物无需纯化直接用于下一步反应。
步骤2：将化合物3-2(8g,20.0mmol)和联硼酸频那醇酯(5.59g,22.0mmol)溶解在N,N-二甲基甲酰胺(100mL)中，加入醋酸钾(5.90g,60.1mmol)和[1,1-双(二苯基膦)二茂铁]二氯化钯二氯甲烷(1.64g,2.0mmol)，反应液在70℃搅拌反应3小时。TLC显示反应完全。反应液加水(300mL)稀释，用乙酸乙酯(200mL*2)萃取。将合并的有机相用饱和食盐水(150mL)洗涤，用无水硫酸钠干燥，过滤并减压浓缩。残余物经由快速硅胶柱分离(0～10％乙酸乙酯/石油醚)得到化合物3-3。 1H NMR(400MHz,CDCl 3)δ6.41(br s,1H),6.34-6.47(m,1H),3.32-3.44(m,2H),3.14-3.29(m,2H),2.00-2.10(m,2H),1.90(br d,J＝3.01Hz,2H),1.38(s,9H),1.28(br t,J＝5.52Hz,4H),1.19(s,12H)。
步骤3：在氮气保护下，将溶有N-(5-溴-[1,2,4]三氮唑[1,5-a]吡啶-2-基)环丙基甲酰胺(2g,7.1mmol),化合物3-3(3.49g,9.3mmol),碳酸钾(2.95g,21.3mmol),和[1,1-双(二苯基膦)二茂铁]二氯化钯二氯甲烷(581mg,711.5μmol)的二氧六环(40mL)和水(10mL)的混合溶液，氮气置换3次后，反应液加热至90℃反应3小时。LC-MS显示反应完全。反应液减压浓缩，残余物经由快速硅胶柱分离(0～4％甲醇/二氯甲烷)得到化合物3-4。LCMS(ESI)m/z:452.4[M+H] +。
步骤4：将化合物3-4(3.5g,7.8mmol)溶解在二氯甲烷(15mL)中，加入盐酸/乙酸乙酯(4M,30mL)，反应液在20℃下反应30分钟。LC-MS显示反应完全。固体析出，过滤并干燥，得到化合物3-5。LCMS(ESI)m/z:352.2[M+H] +。
步骤5：在N 2保护下，将化合物3-5(2.9g,7.4mmol,盐酸盐)溶解在甲醇(100mL)溶液中，加入催化剂干钯/碳(1g，10％)，反应液经氢气置换3次。在氢气压力(30Psi)，反应温度25℃下，反应液搅拌12小时。LC-MS显示反应完全。固体经硅藻土过滤，滤液减压浓缩，得到化合物3-6((2.6g盐酸盐)。LCMS(ESI)m/z:354.7[M+H] +。
步骤6：将化合物3-6(1g,2.6mmol,盐酸盐)溶解在N,N-二甲基甲酰胺(20mL)中，加入HOBt(573mg,4.2mmol)和EDCI(813mg,4.2mmol)，之后加入(1S)-2,2-二氟环丙基羧酸(380mg,3.1mmol)和二异丙基乙胺(731mg,5.7mmol)，反应液在15℃反应12小时。LC-MS显示反应完全。反应液加水(100mL)稀释，用二氯甲烷/甲醇(10/1,150mL*2)萃取，将合并的有机相用饱和食盐水(100mL)洗涤，并用无水硫酸钠干燥，过滤并减压浓缩，残余物经由制备型HPLC(中性体系)得到化合物3-7。
1H NMR(400MHz,METHANOL-d 4)δ7.57-7.64(m,1H),7.48(d,J＝8.78Hz,1H),7.02(d,J＝7.28Hz,1H),3.63-3.77(m,3H),3.41-3.61(m,2H),2.93(dt,J＝8.28,11.80Hz,1H),1.36-2.06(m,15H),1.04(quin,J＝3.76Hz,2H),0.93(qd,J＝3.66,7.34Hz,2H)。LCMS(ESI)m/z:458.1[M+H] +。
以化合物3-6为共同中间体，运用同化合物3-7相同的合成及分离方法(即在酸胺缩合反应步骤中将合成化合物3-7的羧酸替换为下列目标分 子中相应的羧酸)得到下列化合物，
Figure PCTCN2020075024-appb-000048
其表征数据如下：
化合物3-8： 1H NMR(400MHz,METHANOL-d 4)δ7.57-7.67(m,1H),7.49(d,J＝8.53Hz,1H),7.03(dd,J＝3.76,6.78Hz,1H),4.61(s,1H),3.83-3.91(m,1H),3.62(td,J＝3.76,7.53Hz,2H),3.42-3.54(m,3H),1.68-2.08(m,9H),1.40-1.56(m,4H),1.05(quin,J＝3.76Hz,2H),0.88-0.97(m,2H)。LCMS(ESI)m/z:421.1[M+H] +。
化合物3-9： 1H NMR(400MHz,METHANOL-d 4)δ7.61(dd,J＝7.28,8.78Hz,1H),7.49(d,J＝8.78Hz,1H),7.02(dd,J＝4.52,6.78Hz,1H),3.63(td,J＝3.83,7.40Hz,2H),3.43-3.58(m,5H),1.67-2.07(m,9H),1.39-1.54(m,4H),1.01-1.08(m,2H),0.93(qd,J＝3.68,7.28Hz,2H)。LCMS(ESI)m/z:464.1[M+H] +。
实施例3
Figure PCTCN2020075024-appb-000049
步骤1：在0℃下，将5-溴-[1,2,4]三氮唑[1,5-a]吡啶-2-氨基(4-1) (5g,23.5mmol)溶解在乙腈(50mL)中，加入三乙胺(11.87g,117.4mmol)和环丙基甲酰氯(6.13g,58.7mmol)，反应液在25℃下反应12小时。TLC显示反应完全。减压浓缩除去乙腈溶剂，残余物经由快速硅胶柱分离(0～5％甲醇/二氯甲烷)得到化合物4-2。LCMS(ESI)m/z:350.8[M+H] +。
步骤2：在N 2保护下，将化合物4-2(1.99g,5.7mmol)和化合物1-1(1.5g,6.3mmol)溶解在无水四氢呋喃(30mL)中，在-70℃下，缓慢加入正丁基锂(2.5M,5.7mL)溶液，反应液在10℃下搅拌30分钟。LC-MS显示反应完全。反应液在0℃下用饱和氯化铵(50mL)淬灭，用乙酸乙酯(150mL*2)萃取，将合并的有机相用饱和食盐水(10mL)洗涤，无水硫酸钠干燥，过滤并减压浓缩。残余物经由经由快速硅胶柱分离(0～3％甲醇/二氯甲烷)得到化合物4-3。LCMS(ESI)m/z:442.3[M+H] +。
步骤3：在0℃下，将化合物4-3(0.8g,1.81mmol)溶解在无水二氯甲烷(10mL)中，加入二乙胺基三氟化硫(DAST)(351mg,2.17mmol)，反应液在0℃下反应15分钟，然后升温至25℃反应1小时。LC-MS显示反应完全。反应液在0℃用饱和碳酸氢钠水溶液(5mL)淬灭，加水(10mL)稀释，用二氯甲烷(50mL*3)萃取，将合并的有机相用饱和食盐水(20mL)洗涤，无水硫酸钠干燥，过滤并减压浓缩。将残余物经由快速硅胶柱分离(0～100％乙酸乙酯/石油醚)得到化合物4-4。LCMS(ESI)m/z:444.3[M+H] +。
步骤4：将化合物4-4(410mg,924.4μmol)溶解在二氯甲烷(5mL)中，加入盐酸/乙酸乙酯(4M,10mL)，反应液在20℃下反应30分钟。LC-MS显示反应完全。固体析出，过滤并干燥，得到化合物4-5(390mg盐酸盐)。LCMS(ESI)m/z:344.2[M+H] +。
步骤5：将化合物4-5(130mg,342.2μmol,盐酸盐)溶解在N,N-二甲基甲酰胺(10mL)中，加入HOBt(77mg,567.9μmol)和EDCI(109mg,567.9μmol)，之后加入2-氰基乙酸(35mg,416.4μmol)和二异丙基乙胺(98mg,757.1μmol)，反应液在15℃反应12小时。LC-MS显示反应完全。反应液减压浓缩，残余物经由制备型HPLC(中性条件)得到化合 物4-6。 1H NMR(400MHz,METHANOL-d 4)δ7.66-7.72(m,1H),7.56-7.62(m,1H),7.25(t,J＝7.28Hz,1H),4.29(s,1H),4.03(s,1H),3.97(s,1H),3.76(s,1H),3.26-3.30(m,2H),2.97-3.28(m,2H),1.74-2.08(m,7H),0.89-1.13(m,4H)。LCMS(ESI)m/z:411.1[M+H] +。
以化合物4-5为共用中间体，运用与化合物4-6相同的酸胺缩合的合成及分离方法制备(加入与化合物4-6不同取代的羧酸化合物)化合物4-7、4-8表征数据如下：
Figure PCTCN2020075024-appb-000050
化合物4-7： 1H NR(400MHz,METHANOL-d 4)δ＝7.65-7.73(m,1H),7.59(dt,J＝1.13,9.72Hz,1H),7.20-7.29(m,1H),4.33(s,1H),4.01(d,J＝11.29Hz,2H),3.76(s,1H),2.99-3.30(m,4H),1.74-2.10(m,7H),0.88-1.12(m,4H)。LCMS(ESI)m/z:454.1[M+H] +。
化合物4-8： 1H NMR(400MHz,METHANOL-d 4)δ＝7.64-7.73(m,1H),7.59(dt,J＝1.25,9.16Hz,1H),7.20-7.28(m,1H),6.01-6.44(m,1H),4.30(s,1H),3.99(d,J＝11.80Hz,2H),3.73(s,1H),2.99-3.27(m,2H),2.76-2.99(m,2H),1.75-2.10(m,7H),0.89-1.10(m,4H)。LCMS(ESI)m/z:436.1[M+H] +。
实施例4
Figure PCTCN2020075024-appb-000051
步骤1：在-78℃下，向溶有化合物5-1(0.15g,592.1μmol)的THF(8mL)中滴加LiHMDS(1M,770μL)。混合物在-78℃下搅拌1小时。在-78℃下，向反应液中滴加1,1,1-三氟-N-苯基-N-(三氟甲基磺酰基)甲磺酰胺(233mg,651μmol)的四氢呋喃(4mL)溶液，然后在15℃下搅拌12小时。TLC(PE:EA＝5:1)显示原料反应完全，并有新点生成。用10mL饱和氯化铵溶液淬灭反应，然后加入20mL水，用乙酸乙酯(30mL*3)萃取。合并有机相，经饱和食盐水(40mL)洗涤，无水硫酸钠干燥，过滤浓缩得到化合物5-2，未经纯化直接用于下一步反应。
步骤2：向溶有化合物5-2(0.25g,648.7μmol)和联硼酸频那醇酯(165mg,648.7μmol)的DMF(10mL)溶液中加入KOAc(191mg,2.0mmol)和Pd(dppf)Cl 2(48mg,64.9μmol)。该反应液在70℃下搅拌12小时。TLC(PE:EA＝5:1)显示原料反应完全，并监测到有新点生成。加入20mL水，用乙酸乙酯(30mL*3)萃取。合并有机相，经饱和食盐水(40mL)洗涤，无水硫酸钠干燥，过滤浓缩得到粗品，然后经柱色谱(SiO 2,PE:EA＝50:0～20:1)分离纯化得到无色油状化合物5-3。 1H NMR(400MHz,METHANOL-d4)δ6.50(br s,1H),3.35-3.49(m,2H),3.07-3.15(m,2H),2.02-2.22(m,4H),1.54-1.81(m,4H),1.47(s,9H),1.27(s,12H)。
步骤3：将溶有化合物5-3(0.13g,357.8μmol),N-(5-溴-[1,2,4]三唑并[1,5-a]吡啶-2-基)环丙烷甲酰胺(101mg,357.8μmol),K 2CO 3(149mg,1.1mmol),Pd(dppf)Cl 2(26mg,35.8μmol)的二氧六环(4mL)和水(1mL) 溶液用氮气置换3次。该混合物在氮气氛围内，90℃下搅拌12小时。LCMS显示原料被消耗，并监测到目标分子离子峰。将反应液浓缩除掉溶剂，然后分散在10mL水中，DCM/MeOH(10:1,30mL*3)萃取。合并有机相，用饱和食盐水(40mL)洗涤并用无水硫酸钠干燥，过滤，滤液减压蒸馏得到的粗产品。用硅胶色谱柱法(SiO 2,DCM:MeOH＝1:0至20:1)纯化得到化合物5-4。LCMS(ESI)m/z:438.3[M+H] +。
步骤4：在氩气氛围下，向溶有化合物5-4(0.2g,457.1μmol)的甲醇(10mL)溶液中加入Pd/C(10％,50mg)。该混合物用氢气置换3次，然后在氢气氛围(15psi)25℃下搅拌2小时。LCMS显示原料被消耗完，并监测到目标分子离子峰。将反应液过滤，浓缩得到化合物5-5，未经纯化直接用于下一步。LCMS(ESI)m/z:440.4[M+H] +。
步骤5：将溶有化合物5-5(150mg,341.3μmol)和三氟乙酸(4mL)的二氯甲烷(10mL)溶液用氮气置换3次，然后该反应液在25℃下搅拌30分钟。LCMS显示原料被消耗完，并监测到目标分子离子峰。将反应液浓缩除掉溶剂，得到化合物5-6(0.15g,三氟乙酸盐)未经纯化，直接用于下一步反应。LCMS(ESI)m/z:340.2[M+H] +。
步骤6：向溶有(1S)-2,2-二氟环丙基甲酸(44mg,360.7μmol)的DMF(4mL)加入EDCI(104mg,541.1μmol),HOBt(73mg,541.1μmol)，DIEA(140mg,1.1mmol,189μL)在25℃搅拌反应5分钟，然后加入化合物5-6(122mg,270μmol,三氟乙酸盐)，该混合物在25℃下搅拌16小时。LCMS显示原料被消耗，并监测到目标分子离子峰。粗品经制备分离(中性分离条件,色谱柱:Waters Xbridge 150mm*25mm 5μm；流动相:[H 2O(10mM NH 4HCO 3)-ACN]；B(CH 3CN)％:25％-55％,7min)和SFC手性分离(色谱柱:DAICEL CHIRALCEL OD-H(250mm*30mm,5μm)；流动相:[0.1％NH 3H 2O EtOH]；B(CO 2)％:40％)得到化合物5-7，SFC保留时间:3.685min. 1H NMR(400MHz,METHANOL-d 4)δ7.48-7.55(m,1H),7.39(d,J＝8.78Hz,1H),6.92(dd,J＝6.90,3.39Hz,1H),3.35-3.76(m,5H),2.66-2.94(m,1H),1.51-2.10(m,13H),0.94(br s,2H),0.78-0.88(m,2H).LCMS(ESI)m/z:444.1[M+H] +。化合物5-8,SFC保留时间:4.283min. 1H NMR(400MHz,METHANOL-d 4)δ7.48-7.59(m,1H),7.40(br d,J＝8.53Hz,1H),6.94(br d,J＝6.78Hz,1H),3.23-3.78(m,5H),2.65-2.81(m,1H),1.54-2.06(m,13H),0.95(br s,2H),0.77-0.87(m,2H).LCMS(ESI)m/z:444.2[M+H] +。
以化合物5-6为共用中间体，运用与化合物5-7相同的酸胺缩合的合成及分离方法制备(加入与化合物5-7不同取代的羧酸)下列化合物，表征数据如下:
Figure PCTCN2020075024-appb-000052
化合物5-9：HPLC(中性分离条件,色谱柱:Waters Xbridge150mm*25mm 5μm；流动相:[H 2O(10mM NH 4HCO 3)-ACN]；B(CH 3CN)％:18％-32％,9min)制备分离，保留时间2.117min。 1H NMR(400MHz,METHANOL-d 4)δ7.60-7.68(m,1H),7.52(d,J＝8.6Hz,1H),7.05(dd,J＝3.4,6.8Hz,1H),3.44-3.73(m,4H),3.31(br s,2H),2.11(td,J＝7.6,14.9Hz,3H),2.01(br t,J＝7.3Hz,1H),1.96(br s,1H),1.66-1.88(m,6H),1.31(br s,1H),1.02-1.10(m,2H),0.91-0.99(m,2H)。LCMS(ESI)m/z:407.2[M+H] +。
化合物5-10：SFC手性拆分条件，色谱柱:DAICEL CHIRALCEL OD-H(250mm*30mm,5μm)；流动相:[0.1％NH 3H 2O EtOH]；B(CO 2)％:40％-40％,保留时间4.114min.。 1H NMR(400MHz,METHANOL-d 4)δ＝7.52(br d,J＝8.0Hz,1H),7.40(br d,J＝8.8Hz,1H),6.89-6.98(m,1H),3.57(t,J＝7.0Hz,1H),3.25-3.51(m,6H),1.78-2.09(m,5H),1.51-1.76(m,6H),0.94(br d,J＝3.8Hz,2H),0.79-0.88(m,2H).LCMS(ESI)m/z:450.2[M+H] +。
实施例5
Figure PCTCN2020075024-appb-000053
步骤1：在-78℃下，向溶有化合物6-1(250mg,986.8μmol)的THF(8mL)中滴加LiHMDS(1M,1.3mL)。混合物在-78℃下搅拌1小时。在-78℃下，向反应液中滴加1,1,1-三氟-N-苯基-N-(三氟甲基磺酰基)甲磺酰胺(388mg,1.1mmol)的四氢呋喃(4mL)溶液，然后在25℃下搅拌12小时。TLC(PE:EA＝5:1)显示原料反应完全，并有新点生成。用10mL饱和氯化铵溶液淬灭反应，然后加入20mL水，用乙酸乙酯(30mL*3)萃取。合并有机相，经饱和食盐水(40mL)洗涤，无水硫酸钠干燥，过滤浓缩得到粗品，然后经柱色谱(SiO 2,PE:EA＝20:1～10:1)分离纯化得到化合物6-2。
步骤2：向溶有化合物6-2(386mg,1.0mmol)和联硼酸频那醇酯(254mg,1.0mmol)的DMF(5mL)溶液中加入KOAc(295mg,3.0mmol)和Pd(dppf)Cl 2.CH 2Cl 2(82mg,100μmol)。该反应液在70℃下搅拌12小时。TLC(PE:EA＝5:1)显示原料反应完全，并监测到有新点生成。加入20mL水，用乙酸乙酯(30mL*3)萃取。合并有机相，经饱和食盐水(40mL)洗涤，无水硫酸钠干燥，过滤浓缩得到粗品，然后经柱色谱(SiO 2,PE:EA＝50:0～20:1)分离纯化得到化合物6-3。
步骤3：将溶有化合物6-3(186mg,512μmol),N-(5-溴-[1,2,4]三唑并[1,5-a]吡啶-2-基)环丙烷甲酰胺(144mg,512μmol),K 2CO 3(212mg,1.5 mmol),Pd(dppf)Cl 2.CH 2Cl 2(42mg,51.2μmol)的二氧六环(4mL)和水(1mL)溶液用氮气置换3次。该混合物在氮气氛围内，90℃下搅拌12小时。LCMS显示原料被消耗，并监测到目标分子离子峰。将反应液浓缩除掉溶剂，然后分散在10mL水中，DCM:MeOH(10:1,30mL*3)萃取。合并有机相，用饱和食盐水(40mL)洗涤并用无水硫酸钠干燥，过滤，滤液减压蒸馏得到的粗产品。用硅胶色谱柱法(SiO 2,DCM:MeOH＝1:0～20:1)纯化得到化合物6-4。LCMS(ESI)m/z:438.7[M+H] +。
步骤4：在氩气氛围下，向溶有化合物6-4(196mg,448μmol)的甲醇(10mL)溶液中加入Pd/C(10％,50mg)。该混合物用氢气置换3次，然后在氢气氛围(15psi)25℃下搅拌16小时。LCMS显示原料被消耗完，并监测到目标分子离子峰。将反应液过滤，浓缩得到化合物6-5，未经纯化直接用于下一步。LCMS(ESI)m/z:440.3[M+H] +。
步骤5：将溶有化合物6-5(130mg,296μmol)和三氟乙酸(4mL)的二氯甲烷(10mL)溶液用氮气置换3次，然后该反应液在25℃下搅拌30分钟。LCMS显示原料被消耗完，并监测到目标分子离子峰。将反应液浓缩除掉溶剂，得到化合物6-6(134mg,三氟乙酸盐)未经纯化，直接用于下一步反应。LCMS(ESI)m/z:340.2[M+H] +。
步骤6：向溶有(1S)-2,2-二氟环丙基甲酸(36mg,295.5μmol)的DMF(4mL)加入EDCI(85mg,443.3μmol),HOBt(60mg,443.3μmol)，DIEA(115mg,886.5μmol,154.4μL)在25℃搅拌反应5分钟，然后加入化合物6-6(134mg,295.5μmol，三氟乙酸盐)，该混合物在25℃下搅拌16小时。LCMS显示原料被消耗，并监测到目标分子离子峰。粗品经制备分离(中性条件.色谱柱:Waters Xbridge 150*25 5μm；流动相:[water(10mM NH 4HCO 3)-ACN]；B％:30％-50％,7min)和SFC手性分离(色谱柱:YMC CHIRAL Amylose-C(250mm*30mm,10μm；流动相:[0.1％NH 3H 2O EtOH]；B％:50％)。得到化合物6-7,SFC保留时间:2.339min. 1H NMR(400MHz,DMSO-d 6)δ11.01(br s,1H),7.51-7.63(m,2H),7.08(br d,J＝6.78Hz,1H),3.83(br s,1H),3.41-3.66(m,4H),3.15(br d,J＝5.02Hz,1H),2.14-2.35(m,2H),2.06(br s,1H),1.75-1.95(m,4H),1.40-1.73 (m,6H),0.84(br s,4H)。LCMS(ESI)m/z:444.1[M+H] +。化合物6-8:SFC保留时间,4.142min. 1H NMR(400MHz,DMSO-d 6)δ11.01(br s,1H),7.54-7.65(m,2H),7.08(br s,1H),3.80-3.90(m,1H),3.45-3.66(m,4H),3.09-3.22(m,1H),2.18-2.36(m,2H),2.06(br s,1H),1.75-1.95(m,4H),1.68(br d,J＝7.28Hz,3H),1.50(br d,J＝4.77Hz,3H),0.77-0.88(m,4H).LCMS(ESI)m/z:444.1[M+H] +。
实施例6
Figure PCTCN2020075024-appb-000054
步骤1：在-78℃下，向溶有化合物7-1(0.3g,1.33mmol)的THF(8mL)中滴加LiHMDS(1M,1.7mL)。混合物在-78℃下搅拌1小时后，向反应液中滴加1,1,1-三氟-N-苯基-N-(三氟甲基磺酰基)甲磺酰胺(523mg,1.46mmol)的四氢呋喃(4mL)溶液，然后在25℃下搅拌12小时。TLC(PE:EA＝5:1)显示原料反应完全，并有新点生成。用饱和氯化铵(10mL)溶液淬灭反应，然后加入20mL水，用乙酸乙酯(30mL*3)萃取。合并有机相，经饱和食盐水(40mL)洗涤，无水硫酸钠干燥，过滤浓缩得到粗品，然后经柱色谱(SiO 2,PE:EA＝20:1～10:1)分离纯化得到化合物7-2。 1H NMR(400MHz,CDCl 3)δ5.72(s,1H),3.86-3.92(m,2H),3.76-3.82(m,2H),2.53-2.61(m,2H),2.18-2.25(m,2H),1.37(s,9H).
步骤2：向溶有化合物7-2(0.46g,1.3mmol)和联硼酸频那醇酯(327mg,1.3mmol)的DMF(5mL)溶液中加入KOAc(379mg,3.9mmol)和Pd(dppf)Cl 2.CH 2Cl 2(105mg,128.7μmol)。该反应液在70℃下搅拌12小时。TLC(PE:EA＝5:1)显示原料反应完全，并监测到有新点生成。加入20mL水淬灭反应，用乙酸乙酯(30mL*3)萃取。合并有机相，经饱和食盐水(40mL)洗涤，无水硫酸钠干燥，过滤浓缩得到粗品，然后经柱色谱(SiO 2,PE:EA＝50:0～20:1)分离纯化得到化合物7-3。 1H NMR(400MHz,CDCl 3)δ6.45(t,J＝1.88Hz,1H),3.83-3.88(m,2H),3.73-3.78(m,2H),2.36-2.42(m,2H),2.04(t,J＝7.03Hz,2H),1.37(s,9H),1.21(s,12H).
步骤3：将溶有化合物7-3(0.15g,447.43μmol),N-(5-溴-[1,2,4]三唑并[1,5-a]吡啶-2-基)环丙烷甲酰胺(126mg,447.43μmol),K 2CO 3(186mg,1.34mmol),Pd(dppf)Cl 2.CH 2Cl 2(37mg,44.7μmol)的二氧六环(4mL)和水(1mL)溶液用氮气置换3次。该混合物在氮气氛围内，90℃下搅拌12小时。LCMS显示原料被消耗完全，并监测到目标分子离子峰。将反应液浓缩除掉溶剂，然后分散在10mL水中，DCM:MeOH(10:1,30mL*3)萃取。合并有机相，用饱和食盐水(40mL)洗涤并用无水硫酸钠干燥，过滤，滤液减压蒸馏得到的粗产品。用硅胶色谱柱法(SiO 2,DCM:MeOH＝1:0～20:1)纯化得到化合物7-4。LCMS(ESI)m/z:410.2[M+H] +。
步骤4：在氩气氛围下，向溶有化合物7-4(0.15g,366.3μmol)的甲醇(10mL)溶液中加入Pd/C(10％,0.05g)。该混合物用氢气置换3次，然后在氢气氛围(15psi)中25℃下搅拌16小时。LCMS显示原料被消耗完全，并监测到目标分子离子峰。将反应液过滤，浓缩得到化合物7-5，未经纯化直接用于下一步。LCMS(ESI)m/z:412.2[M+H] +。
步骤5：将溶有化合物7-5(0.13g,315.9μmol)和三氟乙酸(4mL)的二氯甲烷(10mL)溶液用氮气置换3次，然后该反应液在25℃下搅拌30分钟。LCMS显示原料被消耗完全，并监测到目标分子离子峰。将反应液浓缩除掉溶剂，得到化合物7-6(130mg，三氟乙酸盐)未经纯化，直接用于下一步反应。LCMS(ESI)m/z:312.1[M+H] +。
步骤6：向溶有(1S)-2,2-二氟环丙基甲酸(37mg,305.6μmol)的DMF (4mL)加入EDCI(88mg,458.4μmol),HOBt(62mg,458.4μmol)，DIEA(119mg,916.8μmol,160μL)在25℃下搅拌反应5分钟，然后加入化合物7-6(0.13g,305.6μmol,三氟乙酸盐)，该混合物在25℃下搅拌16小时。LCMS显示原料被消耗完全，并监测到目标分子离子峰。粗品经制备分离(色谱柱:Waters Xbridge 150*25 5μm；流动相:[H 2O(10mM NH 4HCO 3)-ACN]；B(CH 3CN)％:20％-50％,7min)和手性分离(色谱柱:DAICEL CHIRALPAK AD(250mm*30mm,10μm)；流动相，A％:(0.1％NH 3H 2O EtOH)；B(CO 2)％:(40％-40％)得到化合物7-7,SFC保留时间：3.714min. 1H NMR(400MHz,CDCl 3)δ＝9.59(br d,J＝12.05Hz,1H),7.34-7.57(m,2H),6.70-6.84(m,1H),4.06-4.21(m,2H),3.92-3.99(m,1H),3.74-3.92(m,2H),1.81-2.38(m,8H),1.59(dtd,J＝11.36,7.62,7.62,3.76Hz,1H),1.08-1.24(m,2H),0.79-0.96(m,2H).LCMS(ESI)m/z:416.0[M+H] +。
分离得到化合物7-8，SFC保留时间:4.468min. 1H NMR(400MHz,CDCl 3)δ＝9.48(br s,1H),7.34-7.54(m,2H),6.76(br d,J＝7.03Hz,1H),4.03-4.25(m,2H),3.73-3.99(m,3H),2.50(ddd,J＝16.81,13.18,8.16Hz,1H),1.80-2.40(m,8H),1.53-1.66(m,1H),1.05-1.23(m,2H),0.80-0.95(m,2H).LCMS(ESI)m/z:416.0[M+H] +。
实施例7
Figure PCTCN2020075024-appb-000055
步骤1：在氮气保护下，将溶有化合物8-1(1.11g,5.21mmol)，化合物1-3,碳酸钾(2.16g,15.6mmol),和[1,1-双(二苯基膦)二茂铁]二氯化钯二氯甲烷(425mg,520.6μmol)的二氧六环(40mL)和水(10mL)的混合溶液，氮气置换3次后，反应液加热至90℃反应3小时。LC-MS显示反应完全。反应液减压浓缩，残余物经由快速硅胶柱分离(0～4％甲醇/二氯甲烷)得到化合物8-2。LCMS(ESI)m/z:356.3[M+H] +。
步骤2：在N 2保护下，将化合物8-2(2g,5.6mmol)溶解在甲醇(100mL)溶液中，加入催化剂干钯/碳(0.5g，10％)，反应液经氢气置换3次。在氢气压力(30Psi)，反应温度30℃下，反应液搅拌12小时。LC-MS显示50％原料剩余。滤出催化剂，加入新的催化剂干钯/碳(1g)，继续反应3小时，LCMS显示反应完全。固体经硅藻土过滤，滤液减压浓缩，得到化合物8-3。LCMS(ESI)m/z:358.2[M+H] +。
步骤3：将(1R)-2,2-二氟环丙基羧酸(282mg,2.3mmol)溶解在吡啶(10mL)中，加入EDCI(4.0g,21.0mmol)和化合物8-3(0.75g,2.1mmol)，反应液在10℃下搅拌12小时。LC-MS显示反应完全。反应液加水(30mL)稀释，用二氯甲烷/甲醇(10/1,50mL*3)萃取，将合并的有机相用饱和食盐水(30mL)洗涤，无水硫酸钠干燥，过滤并减压浓缩。残余物经由快速硅胶柱分离(0～3％甲醇/二氯甲烷)，再经乙酸乙酯打浆纯化，得到化合物8-4。LCMS(ESI)m/z:462.3[M+H] +。
步骤4：将化合物8-4(300mg,650.1μmol)溶解在二氯甲烷(5mL)中，加入盐酸/乙酸乙酯(4M,10mL)，反应液在15℃下反应半小时。LC-MS显示反应完全。反应液浓缩，得到化合物8-5(盐酸盐)。LCMS(ESI)m/z:362.2[M+H] +。
步骤5：将化合物8-5(100mg,251.4μmol,HCl)溶解在N,N-二甲基甲酰胺(5mL)中，加入HOBt(51mg,377.0μmol)和EDCI(72.28mg,377.0μmol)，之后加入(1S)-2,2-二氟环丙基羧酸(34mg,276.5μmol)和二异丙基乙胺(65mg,502.7μmol)，反应液在15℃反应12小时。LC-MS显示反应完全。反应液减压浓缩，残余物经由制备型HPLC(中性条件)得到化合物8-6。 1H NMR(400MHz,METHANOL-d 4)δ7.59-7.67(m,1H),7.51 (d,J＝8.78Hz,1H),7.01(br d,J＝7.53Hz,1H),3.92-4.20(m,2H),3.79-3.88(m,1H),3.67-3.77(m,1H),3.43-3.57(m,1H),2.81(br s,1H),2.62(dq,J＝7.78,11.96Hz,1H),2.07-2.24(m,5H),1.52-2.05(m,7H)。LCMS(ESI)m/z:466.2[M+H] +。
以化合物8-5为共用中间体，运用与化合物8-6相同的酸胺缩合的合成及分离方法制备(加入与化合物8-6不同取代的羧酸)化合物8-7、8-8表征数据如下:
Figure PCTCN2020075024-appb-000056
化合物8-7，粗产品通过制备型HPLC纯化(中性条件)。 1H NMR(400MHz,METHANOL-d 4)δ7.59-7.66(m,1H),7.51(d,J＝8.78Hz,1H),6.97-7.04(m,1H),4.30(d,J＝4.27Hz,1H),4.18(d,J＝4.27Hz,1H),3.87(s,1H),3.76(s,1H),3.49(br t,J＝11.80Hz,1H),2.81(br s,1H),2.05-2.28(m,5H),1.74-1.96(m,3H),1.53-1.70(m,2H),1.23-1.33(m,4H)。LCMS(ESI)m/z:448.2[M+H] +。
化合物8-8,粗产品通过制备型HPLC纯化(中性条件)。 1H NMR(400MHz,METHANOL-d 4)δ7.59-7.67(m,1H),7.51(d,J＝8.78Hz,1H),7.00(t,J＝7.40Hz,1H),4.61(s,2H),3.69-4.11(m,4H),3.41-3.54(m,1H),2.82(br s,1H),2.04-2.26(m,5H),1.72-1.93(m,3H),1.61(q,J＝11.80Hz,2H)。LCMS(ESI)m/z:429.0[M+H] +。
Figure PCTCN2020075024-appb-000057
化合物8-9的合成：将中间体8-5(100mg,227.6μmol,TFA)溶解在N,N-二甲基甲酰胺(5mL)中，加入碳酸钾(94mg,682.7μmol)和2-溴乙腈(30mg,250.3μmol)，反应液在10℃搅拌12小时。LC-MS显示反应完全。反应液用水(5mL)稀释，二氯甲烷/甲醇(10/1,10mL)萃取，将有机相用饱和食盐水(10mL)洗涤，无水硫酸钠干燥，过滤并减压浓缩。残余物经由制备型HPLC(中性条件)纯化，得到化合物8-9。 1H NMR(400MHz,METHANOL-d 4)δ7.59-7.66(m,1H),7.50(d,J＝8.78Hz,1H),6.99(d,J＝7.53Hz,1H),3.62(s,2H),3.46(br t,J＝12.05Hz,1H),3.33(s,2H),3.21(s,2H),2.80(br s,1H),2.14(br d,J＝9.79Hz,5H),1.82-1.95(m,1H),1.52-1.77(m,4H)。LCMS(ESI)m/z:401.0[M+H] +。
实施例8
Figure PCTCN2020075024-appb-000058
步骤1：将(S)-2,2-二氟环丙基羧酸(1.13g,9.2mmol)溶解在吡啶(150mL)中，加入EDCI(16.1g,84mmol)和化合物8-3(3g,8.4mmol)，反应液在10℃下搅拌12小时。LC-MS显示反应完全。反应液加水(100mL)稀释，用二氯甲烷/甲醇(10/1,100mL*3)萃取，将合并的有机相用饱和食盐水(30mL)洗涤，无水硫酸钠干燥，过滤并减压浓缩。残余物经由快速硅胶柱分离(0～3％甲醇/二氯甲烷)，再经乙酸乙酯打浆纯化，得到化合物9-1。LCMS(ESI)m/z:462.3[M+H] +。
步骤2：将化合物9-1(2.3g,4.9mmol)溶解在二氯甲烷(5mL)中，加入盐酸/乙酸乙酯(4M,20mL)，反应液在15℃下反应半小时。LC-MS显示反应完全，检测到目标分子离子峰。析出的固体过滤，干燥，得到化合物9-2(盐酸盐)。LCMS(ESI)m/z:362.2[M+H] +。
步骤3：将化合物9-2(1.23g,3.1mmol,HCl)溶解在N,N-二甲基甲酰 胺(20mL)中，加入HOBt(626mg,4.6mmol)和EDCI(889mg,4.6mmol)，之后加入(1S)-2,2-二氟环丙基羧酸(414.92mg,3.40mmol)和二异丙基乙胺(798.70mg,6.18mmol)，反应液在15℃反应12小时。LC-MS显示反应完全。反应液加水(10mL)稀释，用二氯甲烷/甲醇(10/1,50mL)萃取，将有机相用饱和食盐水(10mL)洗涤，无水硫酸钠干燥，过滤并减压浓缩。残余物经由制备型HPLC(中性条件)得到化合物9-3。 1H NMR(400MHz,METHANOL-d 4)δ7.63(dd,J＝7.53,8.78Hz,1H),7.51(d,J＝8.78Hz,1H),7.01(br d,J＝7.28Hz,1H),3.92-4.19(m,2H),3.79-3.87(m,1H),3.67-3.76(m,1H),3.44-3.55(m,1H),2.52-2.92(m,2H),1.53-2.25(m,12H)。LCMS(ESI)m/z:466.1[M+H] +。
以化合物9-2为共用中间体，运用与化合物9-3相同的酸胺缩合的合成及分离方法制备(加入与化合物9-3不同取代的羧酸)化合物9-4、9-5表征数据如下：
Figure PCTCN2020075024-appb-000059
化合物9-4： 1H NMR(400MHz,METHANOL-d 4)δ7.58-7.68(m,1H),7.51(d,J＝8.78Hz,1H),6.97-7.05(m,1H),4.30(d,J＝4.02Hz,1H),4.18(d,J＝4.27Hz,1H),3.87(s,1H),3.76(s,1H),3.49(br t,J＝11.80Hz,1H),2.82(br s,1H),2.07-2.25(m,5H),1.73-1.95(m,3H),1.51-1.70(m,2H),1.24-1.35(m,4H)。LCMS(ESI)m/z:448.2[M+H] +。
化合物9-5： 1H NMR(400MHz,METHANOL-d 4)δ7.58-7.68(m,1H),7.51(d,J＝9.03Hz,1H),7.00(t,J＝7.53Hz,1H),4.61(s,2H),3.69-4.10(m,4H),3.43-3.55(m,1H),2.82(br s,1H),2.05-2.25(m,5H),1.72-1.97(m,3H),1.51-1.69(m,2H)。LCMS(ESI)m/z:429.0[M+H] +。
Figure PCTCN2020075024-appb-000060
化合物9-6的合成：中间体化合物9-2(190mg,525.8μmol)溶解在N,N-二甲基甲酰胺(5mL)中，加入碳酸钾(218mg,1.6mmol)和2-溴乙腈(70mg,578.3μmol)，反应液在10℃搅拌12小时。LC-MS显示反应完全。反应液用水(5mL)稀释，二氯甲烷/甲醇(10/1,10mL)萃取，将有机相用饱和食盐水(10mL)洗涤，无水硫酸钠干燥，过滤并减压浓缩。残余物经由制备型HPLC(中性条件)纯化，得到化合物9-6。 1H NMR(400MHz,METHANOL-d 4)δ7.58-7.66(m,1H),7.50(d,J＝8.53Hz,1H),6.99(d,J＝7.28Hz,1H),3.62(s,2H),3.46(br t,J＝11.42Hz,1H),3.33(s,2H),3.21(s,2H),2.81(br s,1H),2.14(br d,J＝10.29Hz,5H),1.81-1.95(m,1H),1.51-1.78(m,4H)。LCMS(ESI)m/z:401.2[M+H] +。
实施例9
Figure PCTCN2020075024-appb-000061
步骤1：将溶有化合物10-1(100mg,334.3μmol),化合物3-3(126mg,334.3μmol),Pd(dppf)Cl 2(25mg,33.4μmol),碳酸钾(139mg,1.00mmol)的二氧六环(12mL)和H 2O(3mL)混合溶液中用氮气置换3次。 该反应液在氮气氛围内、90℃下搅拌2小时。LCMS显示原料被消耗完，并监测到主峰为目标分子离子峰。将反应液过滤浓缩除掉溶剂，再经制备板分离纯化得到化合物10-2。LCMS(ESI)m/z:470.4[M+H] +。
步骤2：将溶有化合物10-2(130mg,276.9μmol)和HCl/EtOAc(4M,2mL)的二氯甲烷(1mL)溶液在25℃下搅拌5分钟。LCMS显示原料被消耗完，并监测到主峰为目标分子离子峰。反应液经减压浓缩得到黄色固体化合物10-3(120mg，盐酸盐)，未经纯化直接用于下一步反应。LCMS(ESI)m/z：370.6[M+H] +。
步骤3：氮气氛围内，向溶有化合物10-3(120mg,295.6μmol,盐酸盐)的MeOH(25mL)溶液中加入Pd/C(20mg,10％)。将悬浊液用氢气置换3次，然后在氢气氛围内(15Psi)、25℃下搅拌12小时。LCMS显示原料被消耗完，并监测到主峰为目标分子离子峰。反应液经过滤减压浓缩除掉溶剂，得到化合物10-4(130mg,盐酸盐)，未经进一步纯化直接用于下一步反应。LCMS(ESI)m/z:372.3[M+H] +。
步骤4：将溶有化合物10-4(130mg,318.7μmol,盐酸盐),2-氰基乙酸(33mg,382.4μmol),EDCI(92mg,478μmol),HOBt(65mg,478μmol)和DIEA(206mg,1.6mmol,277.6μL)的DMF(5mL)溶液在25℃下搅拌12小时。LCMS显示原料被消耗，并监测到目标分子离子峰。反应液经减压浓缩除掉溶剂，再经制备分离得到(中性体系)化合物10-5。 1H NMR(400MHz,METHANOL-d 4)δ＝7.57-7.65(m,1H),7.49-7.55(m,1H),3.59-3.81(m,3H),3.44-3.54(m,2H),3.34-3.38(m,2H),2.48(br s,2H),1.81-2.04(m,5H),1.64-1.77(m,2H),1.36-1.58(m,4H),0.86-1.12(m,4H)。LCMS(ESI)m/z:439.1[M+H] +。
生物活性测试
实验例1：Jak1,Jak2,Jak3,Tyk2激酶体外活性测试
实验材料
重组人源JAK1、JAK2、JAK3、Tyk2蛋白酶、主要仪器及试剂均由英国的Eurofins公司提供
实验方法
JAK2,JAK3和TYK2稀释：20mM 3-(N-吗啉)丙磺酸(MOPS),1mM EDTA,0.01％Brij-35.5％甘油,0.1％β-巯基乙醇,1mg/mL BSA；JAK1稀释：20mM TRIS,0.2mM EDTA,0.1％β-巯基乙醇,0.01％Brij-35.5％甘油。将所有化合物制备成100％的DMSO溶液并达到最终测定浓度50倍。测试化合物进行3倍浓度梯度稀释，终浓度为10μM到0.001μM共9个浓度，DMSO在检测反应中的含量为2％。将该化合物的工作储备液作为反应的第一组分添加到测定孔中，然后按照下面测定详述的方案加入其余组分。
JAK1(h)酶反应
JAK1(h)与20mM Tris/HCl pH7.5,0.2mM EDTA，500μM MGEEPLYWSFPAKKK，10mM乙酸镁和[γ- 33P]-ATP(根据需要制定活性和浓度)一起孵育。添加Mg/ATP混合物开始反应，在室温下孵育40分钟后，加入0.5％浓度的磷酸终止反应。然后将10μL反应物点在P30滤垫上并于4分钟内用0.425％磷酸洗涤三次和甲醇洗涤一次，干燥、闪烁计数。
JAK2(h)酶反应
JAK2(h)与8mM MOPS pH 7.0,0.2mM EDTA,100μM KTFCGTPEYLAPEVRREPRILSEEEQEMFRDFDYIADWC,10mM乙酸镁和[γ- 33P]-ATP(根据需要制定活性和浓度)一起孵育。添加Mg/ATP混合物开始反应，在室温下孵育40分钟后，加入0.5％浓度的磷酸终止反应。然后将10μL反应物点在P30滤垫上并于4分钟内用0.425％磷酸洗涤三次和甲醇洗涤一次，干燥、闪烁计数。
JAK3(h)酶反应
JAK3(h)与8mM MOPS pH 7.0,0.2mM EDTA,500μM GGEEEEYFELVKKKK,10mM乙酸镁和[γ- 33P]-ATP(根据需要制定活性和浓度)一起孵育。添加Mg/ATP混合物开始反应，在室温下孵育40分钟后，加入0.5％浓度的磷酸终止反应。然后将10μL反应物点在P30滤垫上并于4分钟内用0.425％磷酸洗涤三次和甲醇洗涤一次，干燥、闪 烁计数。
TYK2(h)酶反应
TYK2(h)与8mM MOPS pH 7.0,0.2mM EDTA,250μM GGMEDIYFEFMGGKKK,10mM乙酸镁和[γ- 33P]-ATP(根据需要制定活性和浓度)一起孵育。添加Mg/ATP混合物开始反应，在室温下孵育40分钟后，加入0.5％浓度的磷酸终止反应。然后将10μL反应物点在P30滤垫上并于4分钟内用0.425％磷酸洗涤三次和甲醇洗涤一次，干燥、闪烁计数。
数据分析
IC 50结果由IDBS公司的XLFIT5(205公式)进行分析得到，具体见表1。
表1.本发明化合物体外筛选试验结果
Figure PCTCN2020075024-appb-000062
Figure PCTCN2020075024-appb-000063
结论：本发明的化合物在激酶4个亚型JAK1、JAK2、JAk3和TYK2的体外活性测试中展现了对JAK1和/或TYK2的良好的选择性抑制。
实验例2：药代动力学(PK)试验
将试验化合物溶解后得到的澄清溶液分别经尾静脉注射和灌胃给予雄性小鼠(C57BL/6)或大鼠(SD)体内(过夜禁食，7～8周龄)。给予受试化合物后，静脉注射组(2mg/kg)在0.117,0.333,1,2,4,7和24小时，灌胃组(15mg/kg)在0.25,0.5,1,2,4,8和24小时，分别从下颌静脉采血并离心后获得血浆。采用LC-MS/MS法测定血药浓度，使用 WinNonlin TM Version 6.3药动学软件，以非房室模型线性对数梯形法计算相关药代动力学参数。测试结果如下：
表2-1 化合物1-11在小鼠中的PK测试结果
PK参数 结果
T 1/2(hr) 2.99
C max(nM) 5745
AUC 0-inf(nM.hr) 9918
Bioavailability(％) a 42.1％
表2-2 化合物1-13在小鼠中的PK测试结果
PK参数 结果
T 1/2(hr) 1.61
C max(nM) 5105
AUC 0-inf(nM.hr) 9917
Bioavailability(％) a 38.1％
表2-3 化合物3-7在小鼠中的PK测试结果
PK参数 结果
T 1/2(h) 4.74
C max(nM) 7380
AUC 0-inf(nM.h) 17969
Bioavailability(％) a 50.1％
注：T 1/2：半衰期；C max：达峰浓度；
AUC 0-inf：从0时间到外推至无穷大时的血浆浓度-时间曲线下面积；
Bioavailability：生物利用度。
结论：本发明的化合物在小鼠中都有良好的口服生物利用度，较高的暴露量，有利于产生良好的体内药效。
实验例3：化合物对炎症性肠病(IBD)的体内药效研究
实验目的：
炎症性肠病(inflammatory bowl disease,IBD)是一类反复发病的疾病，治愈难度大、复发率高、愈后较差，与结肠癌发病存在一定相关性。研发治疗IBD的药物，有助于缓解溃疡性结肠炎和克罗恩病的症状，改善患者的生存质量。葡聚糖硫酸钠(dextran sulfate sodium,DSS)诱导的小鼠炎症模型与人类疾病溃疡性结肠炎和克罗恩病情况较为相似，是临床前评价药物药效作用常用的经典模型。本实验目的是考察化合物1-8、化合物1-11、化合物9-3和化合物1-13对DSS诱导的小鼠溃疡性结肠炎和克罗恩病的治疗效果，为临床研究提供临床前药效学相关信息。
实验方法：
1.造模液及灌胃用药物的配制
DSS配置方法：称量20g DSS，加入1,000ml饮用水，配成2％DSS溶液，保存于4℃冰箱中，一个月内有效。
溶媒配制方法：Vehicle溶液为含有0.5％甲基纤维素和0.5％Tween 80的水溶液。先将5g甲基纤维素溶解在990mL纯水中，然后少量多次滴加5g Tween 80，混合均匀并常温保存。
化合物配制方法：称取化合物，加入质量比为0.5％甲基纤维素+0.5％Tween 80的水溶液，超声直至溶解。将此溶液充分混匀后，分装至玻璃瓶中，储存在4℃冰箱中。
2.IBD的诱导及给药
C57BL/6小鼠雄性70只，称重，按体重随机分为7组，每组10只，分组及剂量设计如表3-1。
动物适应期3天，适应后每天称重，空白组给予饮用水，其他组给予含2％DSS的饮用水，装水量按照5ml/只/天；受试药物组动物灌胃给药，每天1次，空白组和DNCB组给予相同体积的Vehicle，给药体积为10ml/kg。每隔1天，评价粪便粘稠度并收集粪便进行粪便隐血检测，计算疾病活动指数(Disease Activity Index,DAI)得分；每天检测DSS消耗量，每隔2天更换新鲜DSS溶液，给药周期为连续灌胃给药7天，后停 药4天，再连续灌胃7天，停止灌胃给药时改换正常引用水。
表3-1.分组及剂量设计
Figure PCTCN2020075024-appb-000064
注：p.o.：口服；qd：每日一次。
3.IBD疾病严重程度检测
DAI为体重、粪便粘稠度、粪便隐血三个指标评分之和，评分标准如表3-2。其中，粪便隐血检测采用匹拉米洞半定量检测法，操作方法为向标本中先滴加匹拉米洞，后滴加过氧化氢和乙醇溶液，在2min内判读颜色并评分，立即产生紫蓝色记为4+，10s内产生紫蓝色记为3+，1min内产生紫红色记为2+，1-2min内才逐渐产生紫红色记为1+，于判读时间内无任何紫兰或紫红的颜色反应记为阴性(-)。实验结束后取结肠和脾脏，测量结肠长度和脾脏重量，脾脏指数为脾脏重量与体重的比值。
表3-2.DAI评分标准
Figure PCTCN2020075024-appb-000065
4.统计学处理
实验数据采用均数±标准误(Mean±SEM)表示，所有数据均采用 单因素方差分析(One-way ANOVA)进行统计，以P<0.05为具有统计学差异。
实验结果：
1.DAI评分
从DAI评分结果看，如表3-3和附图1所示，在给药4天后DSS组DAI评分迅速增大，与空白对照组相比，DSS组在第6天DAI评分出现显著性增加，并持续到实验结束。各给药组在给药1周内与DSS组相比未发现有显著性差异，停药前后(即第8天与第12天)相比，所有DSS引用水组DAI均有明显下降。在第12天给药后，化合物1-8、1-13和阳性药Filgotinib DAI评分开始下降，与DSS组相比在第16天和第18天出现显著性差异；同时，在第16天和第18天，化合物1-13与阳性药Filgotinib相比DAI评分出现显著性降低，表明化合物1-13比阳性药Filgotinib具有更好的降低DAI评分的作用，提示化合物1-13用于治疗IBD可能具有更优的治疗效果。
表3-3 本发明的DAI评分
Figure PCTCN2020075024-appb-000066
注：与空白组相比#P<0.05,##P<0.01；与DSS组相比*P<0.05,**P<0.01；与DSS+005组相比∝P<0.05∝∝P<0.01
2.体重
实验结果如表3-4和附图2所示，从第5天开始，给予DSS各组小鼠体重开始下降，与空白对照组小鼠相比，DSS组在第5天体重开始降低，在第7天出现显著性差异并持续到实验结束。与DSS组相比，化合物1-8和1-13能够减少DSS引起的体重减少，化合物1-13在第11、15、16和18天出现显著性差异，化合物1-8没有显著性差异；与阳性药 Filgotinib相比，化合物1-13能够减少因DSS导致的小鼠体重下降，并在第9、10、14和18天出现显著性增加，表明化合物1-13具有更好的减少小鼠因DSS导致的小鼠体重下降的作用，提示化合物1-13对IBD的治疗作用可能更优。
表3-4本发明小鼠体重
Figure PCTCN2020075024-appb-000067
注：与空白组相比#P<0.05,##P<0.01；与DSS组相比*P<0.05,**P<0.01；与DSS+005组相比∝P<0.05，∝∝P<0.01
3.结肠长度
实验结果如表3-5和附图3，与空白对照组小鼠相比，DSS组小鼠结肠长度显著减少；给予药物干预后，与DSS组相比，化合物1-8、1-13、9-3、1-11和Filgotinib均具有减少DSS引起的小鼠结肠缩短的作用，其中化合物1-8和化合物1-13作用具有显著性，且化合物1-13作用显著优于阳性药Filgotinib。
表3-5本发明结肠长度
Figure PCTCN2020075024-appb-000068
注：与空白组相比##P<0.01；与DSS组相比*P<0.05；与DSS+005组相比∝P<0.05
4.脾脏指数
实验结果如表3-6和附图4，与空白对照组小鼠相比，DSS能够显著增加小鼠脾脏指数(DSS组)；给予药物干预后，化合物1-8、1-13、9-3、1-11和Filgotinib均能够降低DSS引起的小鼠脾脏指数的增加，其中化合物1-8、1-13和阳性药Filgotinib具有显著抑制作用。
表3-6本发明脾指数
Figure PCTCN2020075024-appb-000069
注：与空白组相比##P<0.01；与DSS组相比*P<0.05
结论：在DSS诱导的小鼠炎症性肠病(IBD)模型中，本发明的化合物展现出良好的疾病治疗效果，并且在Filgotinib一半剂量下具有同等甚至更优的小鼠体内药效。
实验例4：化合物对过敏性皮炎(AD)的体内药效研究
实验目的：
过敏性皮炎(Atopicdermatitis,AD)是一种慢性炎症性皮肤病，易反复发作，临床表现为剧烈瘙痒、多形性皮损以及干皮病样症状。其发病机制与遗传、免疫、感染等众多因素相关。近年来，AD发病率呈逐年升高的趋势,研发治疗AD的药物，有助于缓解患者皮肤症状，改善患者生存质量。1-氯-2,4-二硝基苯(DNCB)诱导的动物过敏性皮炎模型与AD患者临床表现基本一致，是临床前评价药物药效的经典常用模型。本实验目的是考察化合物1-8、化合物1-11、化合物9-3和化合物1-13对DNCB诱导的小鼠过敏性皮炎的治疗效果，为临床研究提供临床前药效学相关信息。
1.造模液及灌胃用药物的配制
溶媒配制方法：Vehicle溶液为30％PEG-400+70％(5％HP-β-CD)，pH 4-5。先将35g HP-β-CD溶解在700毫升的MilliQ纯水中，然后加入300毫升PEG-400，混匀后调解pH至4-5，并常温保存。
化合物配制方法：称取化合物，加入体积比为30％PEG-400+70％(5％HP-β-CD)的溶液中，超声。将此溶液充分混匀后，分装至玻璃瓶中，储存在4℃冰箱中。
2.过敏性皮炎的诱导及给药
Balb/c小鼠雄性70只，称重，按体重随机分为7组，每组10只，详细分组及剂量设计信息见表4-1。
动物适应期3天，适应后背部剃毛，2天后用造模液敏化，在小鼠右耳涂20μl 0.5％DNCB溶液，在小鼠背部涂200μl 0.5％DNCB溶液，每天1次共3天；从第4天开始进行激发，分别在小鼠右耳涂10μl 1％DNCB溶液，在小鼠背部涂100μl 1％DNCB溶液，每3天进行1次；同时从第4天开始，每天分别给小鼠灌胃化合物1-8、1-11、9-3和1-13，阳性药艾洛松涂抹耳部和背部皮肤。
表4-1.分组及剂量设计
Figure PCTCN2020075024-appb-000070
注：p.o.：口服；qd：每日一次。
3.疾病严重程度检测
DNCB特应性皮炎造模成功指标：DNCB成功诱导BALB/c小鼠产生红斑、侵蚀、出血，水肿，表皮脱落，表皮增厚等典型的特应性皮炎样皮损。在3天，9天，15天，21天，27天通过评价小鼠背部皮肤的红斑/出血，瘢痕/干燥，水肿，浸润/侵蚀这四种临床特应性皮炎炎症指标来 对特异性皮炎严重度评价。每一个评价指标都有四种等级：0，无；1，轻微；2，中度；3，明显；4，十分明显。对评分进行加和为皮肤评分。实验结束后测量小鼠右耳厚度，并眼部取血，离心(3000rpm，15min)分离血清，检测lgE的浓度；取脾脏并计算脾脏指数：脾脏指数＝脾脏重量(mg)/体重(g)。
4.统计学处理
实验数据采用均数±标准误(Mean±SEM)表示，所有数据均采用单因素方差分析(One-way ANOVA)进行统计，以P<0.05为具有统计学差异。
实验结果：
1.皮肤评分
皮肤评分结果显示，如表4-2和附图5，DNCB组给予DNCB诱导后，皮肤评分不断升高，从第9天开始，皮肤评分显著高于空白对照组，并持续到实验结束。与DNCB组相比，给予药物干预后皮肤评分逐渐下降，化合物1-13组在给药21天时出现显著性降低，展现出比阳性药艾洛松更好的作用效果。在给药27天时，所有给药组包括化合物1-8、1-11、9-3和1-13以及艾洛松组均表现出显著降低皮肤评分的作用。
表4-2本发明皮肤评分
Figure PCTCN2020075024-appb-000071
注：与空白组相比##P<0.01；与DNCB组相比*P<0.05,**P<0.01
2.右耳厚度
实验结果显示(如表4-3和附图6)，DNCB诱导后(DNCB组)小鼠右耳厚度显著增加，药物干预后(包括化合物1-8、1-11、9-3和1-13以及艾洛松组)小鼠右耳厚度均出现显著性下降，表明化合物1-8、1-11、9-3和1-13对DNCB诱导的小鼠过敏性皮炎具有显著的治疗作用，其作 用与艾洛松作用相当。四个化合物(化合物1-8、1-11、9-3和1-13)对右耳厚度的降低作用以化合物1-13作用最强，但组间无统计学差异。
表4-3本发明右耳厚度
Figure PCTCN2020075024-appb-000072
注：与空白组相比##P<0.01；与DNCB组相比**P<0.01
3.脾脏指数
实验结果显示(如表4-4和附图7)，DNCB诱导后(DNCB组)小鼠脾脏增大，表现为脾脏指数显著升高，通过药物干预(包括化合物1-8、1-11、9-3和1-13以及艾洛松组)小鼠脾脏指数显著下降，表明化合物1-8、1-11、9-3和1-13对DNCB诱导的小鼠过敏性皮炎具有显著的治疗作用，其作用与艾洛松作用相当。四个化合物(化合物1-8、1-11、9-3和1-13)对脾脏指数的降低作用以化合物1-13作用最强，但组间无统计学差异。
表4-4本发明脾脏指数
Figure PCTCN2020075024-appb-000073
注：与空白组相比##P<0.01；与DNCB组相比**P<0.01
4.血清lgE浓度
实验结果显示(如表4-5和附图8)，DNCB诱导后(DNCB组)小鼠血清中lgE浓度显著性升高，通过药物干预(包括化合物1-8、1-11、9-3和1-13以及艾洛松组)小鼠血清中lgE浓度显著下降，表明化合物1-8、1-11、9-3和1-13对DNCB诱导的小鼠过敏性皮炎具有显著的治疗作用。
表4-5本发明血清lgE浓度
Figure PCTCN2020075024-appb-000074
注：与空白组相比##P<0.01；与DNCB组相比**P<0.01
5.体重
实验结果显示(如表4-6和附图9)，空白组和DNCB组小鼠体重缓慢增加，DNCB组小鼠体重略低于空白组，组间无统计学差异。四个化合物(化合物1-8、1-11、9-3和1-13)组与DNCB组体重相当，高于艾洛松组，但组间无统计学差异，表明化合物1-8、1-11、9-3和1-13对小鼠体重无明显影响。
表4-6本发明小鼠体重
Figure PCTCN2020075024-appb-000075
结论：在DNCB诱导的小鼠过敏性皮炎(AD)模型中，本发明的化合物1-8、1-11、9-3和1-13能够显著降低小鼠的皮肤评分、右耳厚度、脾脏指数和血清中lgE浓度，并且对体重无显著性影响，展现出良好的疾病治疗效果，其作用与糖皮质激素艾洛松作用相当。

Claims (23)

  1. JAK抑制剂[1,2,4]三唑并[1,5-a]吡啶类化合物在制备治疗自身免疫性疾病、炎症性疾病、变应性疾病、或移植物抗宿主病的药物中的应用，其特征在于，所述JAK抑制剂[1,2,4]三唑并[1,5-a]吡啶类化合物包括式(Ⅰ)化合物、其异构体或其药学上可接受的盐：
    Figure PCTCN2020075024-appb-100001
    其中，
    E 1和E 2分别独立地选自单键、-CH 2-或-(CH 2) 2-；
    L 1选自单键、-(CH 2) g-、-C(＝O)-或-C(＝O)-(CH 2) h-；
    m为1或2；
    n为1或2；
    g为1、2或3；
    h为1、2或3；
    R 1选自H、CN、C 1-6烷基或3～6元环烷基，其中所述C 1-6烷基和3～6元环烷基任选被1、2或3个R a取代；
    R 2选自H、F、Cl、Br、I或C 1-3烷基，其中所述C 1-3烷基任选被1、2或3个R b取代；
    R 3、R 4和R 5分别独立地选自H、F、Cl、Br、I或C 1-3烷基，其中所述C 1-3烷基任选被1、2或3个R c取代；
    R 6、R 7和R 8分别独立地选自H、F、Cl、Br、I或C 1-3烷基，其中所述C 1-3烷基任选被1、2或3个R d取代；
    每一个R a分别独立地选自H、F、Cl、Br、I、CN或C 1-3烷基，其中所述C 1-3烷基任选被1、2或3个R取代；
    每一个R b分别独立地选自F、Cl、Br或I；
    每一个R c分别独立地选自F、Cl、Br或I；
    每一个R d分别独立地选自F、Cl、Br或I；
    每一个R分别独立地选自F、Cl、Br或I。
  2. 根据权利要求1所述的应用，其特征在于，所述自身免疫性疾病包括系统性红斑狼疮、银屑病、银屑病性关节炎或狼疮肾炎；所述炎症性疾病包括炎症性肠病、强直性脊柱炎或原发性胆道炎；所述变应性疾病包括过敏性皮炎、接触性皮炎、过敏性紫癜或支气管哮喘。
  3. 根据权利要求2所述的应用，其特征在于，所述应用为式(Ⅰ)化合物、其异构体或其药学上可接受的盐在制备治疗系统性红斑狼疮的药物的应用。
  4. 根据权利要求2所述的应用，其特征在于，所述应用为式(Ⅰ)化合物、其异构体或其药学上可接受的盐在制备治疗银屑病的药物的应用。
  5. 根据权利要求2所述的应用，其特征在于，所述应用为式(Ⅰ)化合物、其异构体或其药学上可接受的盐在制备治疗过敏性皮炎的药物的应用。
  6. 根据权利要求2所述的应用，其特征在于，所述式(Ⅰ)化合物、其异构体或其药学上可接受的盐在制备治疗移植物抗宿主病的药物的应用包括抗急性排斥反应、抗慢性排斥反应或诱导免疫耐受。
  7. 根据权利要求2所述的应用，其特征在于，所述应用为式(Ⅰ)化合物、其异构体或其药学上可接受的盐在制备治疗炎症性肠病的药物的应用。
  8. 根据权利要求1所述的应用，其特征在于，所述式(Ⅰ)化合物中每一个R a分别独立地选自H、F、Cl、Br、I或CN。
  9. 根据权利要求1所述的应用，其特征在于，所述式(Ⅰ)化合物中R 1选自H、CN、C 1-3烷基或3～5元环烷基，其中所述C 1-3烷基和3～5 元环烷基任选被1、2或3个R a取代。
  10. 根据权利要求9所述的应用，其特征在于，所述式(Ⅰ)化合物中R 1选自H、CN、CH 3、
    Figure PCTCN2020075024-appb-100002
    其中所述CH 3、
    Figure PCTCN2020075024-appb-100003
    Figure PCTCN2020075024-appb-100004
    任选被1、2或3个R a取代。
  11. 根据权利要求10所述的应用，其特征在于，所述式(Ⅰ)化合物中R 1选自H、CN、CF 3、CHF 2、
    Figure PCTCN2020075024-appb-100005
  12. 根据权利要求1所述的应用，其特征在于，所述式(Ⅰ)化合物中R 2选自H、F、Cl、Br或I。
  13. 根据权利要求1所述的应用，其特征在于，所述式(Ⅰ)化合物中R 3、R 4和R 5分别独立地选自H、F、Cl、Br或I。
  14. 根据权利要求1所述的应用，其特征在于，所述式(Ⅰ)化合物中R 6、R 7和R 8分别独立地选自H、F、Cl、Br或I。
  15. 根据权利要求1所述的应用，其特征在于，所述式(Ⅰ)化合物中L 1选自单键、-CH 2-、-(CH 2) 2-、-C(＝O)-或-C(＝O)-(CH 2)-。
  16. 根据权利要求1所述的应用，其特征在于，所述式(Ⅰ)化合物中结构单元
    Figure PCTCN2020075024-appb-100006
    选自
    Figure PCTCN2020075024-appb-100007
  17. 根据权利要求1所述的应用，其特征在于，所述式(Ⅰ)化合物中结构单元
    Figure PCTCN2020075024-appb-100008
    选自
    Figure PCTCN2020075024-appb-100009
    Figure PCTCN2020075024-appb-100010
  18. 根据权利要求1所述的应用，其特征在于，所述式(Ⅰ)中结构单元
    Figure PCTCN2020075024-appb-100011
    选自
    Figure PCTCN2020075024-appb-100012
    Figure PCTCN2020075024-appb-100013
  19. 根据权利要求1～18任一所述的应用，其特征在于，所述式(Ⅰ)化合物、其异构体或其药学上可接受的盐，其选自：
    Figure PCTCN2020075024-appb-100014
    Figure PCTCN2020075024-appb-100015
    其中，
    L 1如权利要求1或15所定义；
    R a如权利要求1或8所定义；
    R 2如权利要求1或12所定义；
    R 3、R 4和R 5如权利要求1或13所定义；
    R 6、R 7和R 8如权利要求1或14所定义。
  20. 根据权利要求19所述的应用，其特征在于，所述式(Ⅰ)化合物、其异构体或其药学上可接受的盐，其选自：
    Figure PCTCN2020075024-appb-100016
    其中，
    L 1如权利要求1或15所定义；
    R a如权利要求1或8所定义；
    R 2如权利要求1或12所定义；
    R 3、R 4和R 5如权利要求1或13所定义；
    R 6、R 7和R 8如权利要求1或14所定义。
  21. 根据权利要求1所述的应用，其特征在于，所述式(Ⅰ)化合物、 其异构体或其药学上可接受的盐，其选自：
    Figure PCTCN2020075024-appb-100017
  22. 根据权利要求21所述的应用，其特征在于，所述式(Ⅰ)化合 物、其异构体或其药学上可接受的盐，其选自：
    Figure PCTCN2020075024-appb-100018
  23. 一种药物组合物，其特征在于，包括治疗有效量的作为活性成分的根据权利要求1～22任一所述应用中的式(Ⅰ)化合物、其异构体或其药学上可接受的盐和药学上可接受的载体。
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JP2022549089A JP7395762B2 (ja) 2020-02-13 2020-02-13 Ｊａｋキナーゼ関連疾患の治療のための薬物の調製におけるｊａｋ阻害剤の使用
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