WO2021023207A1 - Jak激酶抑制剂及其用途 - Google Patents

Jak激酶抑制剂及其用途 Download PDF

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WO2021023207A1
WO2021023207A1 PCT/CN2020/107028 CN2020107028W WO2021023207A1 WO 2021023207 A1 WO2021023207 A1 WO 2021023207A1 CN 2020107028 W CN2020107028 W CN 2020107028W WO 2021023207 A1 WO2021023207 A1 WO 2021023207A1
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substituted
unsubstituted
independently selected
ethyl
triazole
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PCT/CN2020/107028
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English (en)
French (fr)
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秦引林
苏梅
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江苏柯菲平医药股份有限公司
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Priority to EP20849907.9A priority Critical patent/EP4011880A4/en
Priority to US17/631,596 priority patent/US20220274983A1/en
Publication of WO2021023207A1 publication Critical patent/WO2021023207A1/zh

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    • 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
    • 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
    • A61P11/00—Drugs for disorders of the respiratory system
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal 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
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • 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 belongs to the technical field of medicine, and specifically relates to a novel nitrogen-containing five-membered heterocyclic pyridine compound, its preparation method and pharmaceutical composition containing it, and its use for regulating the activity of Janus kinase (JAK) and for treatment And/or the use of preventing diseases related to JAK activity.
  • JAK Janus kinase
  • Chondrocytes are the main cell component of avascular tissue. In normal articular cartilage, chondrocytes account for about 5% of the tissue volume, while the extracellular matrix accounts for 95% of the remaining tissue. Chondrocytes secrete matrix components, mainly proteoglycans and collagen, which in turn provide chondrocytes with an environment suitable for their survival under mechanical pressure. In cartilage, type II collagen and type IX protein collagen form a fibrous structure, which provides the cartilage with great mechanical strength. Proteoglycans can absorb water and are responsible for the elasticity and shock absorption properties of cartilage. One of the functional roles of cartilage is to provide a smooth joint connection between the bones.
  • cartilage degradation is caused by proteases (collagenase) secreted by inflamed tissues, such as inflamed synovium.
  • Cartilage cells in damaged cartilage often show a decrease in cartilage synthesis activity and/or an increase in cartilage degeneration activity.
  • Cartilage degeneration is a major disease marker for rheumatoid arthritis and osteoarthritis.
  • Rheumatoid arthritis is a chronic joint degeneration disease, which is characterized by inflammation and destruction of joint structures, which may lead to functional disorders of the joints, causing substantial disability and pain, and even premature death. Therefore, the purpose of RA treatment is not only to delay the disease, relieve pain, and protect joint destruction.
  • JAK kinases belong to the Janus kinase (JAK) family of cytoplasmic tyrosine kinases, which are involved in cytokine receptor-mediated intracellular signal transduction.
  • the JAK kinase family includes 4 members: JAK1, JAK2, JAK3 and TYK2. JAK recruits cytokine receptors, binds to cytokines, and then dimerizes cytokine receptors and shared receptor subunits. JAK is then activated by autophosphorylation and/or transphosphorylation of another JAK, leading to receptor phosphorylation and the recruitment and phosphorylation of signal transducers and activator of transcription (STAT) members.
  • JAK Janus kinase
  • STAT signal transducers and activator of transcription
  • JAK Phosphorylated SATA dimerizes and transfers to the nucleus, where they bind to the enhancer region of cytokine response genes.
  • JAK plays an important role in regulating the biological response of multiple cytokine receptor families. JAK1 knockout mice have an early postnatal lethal factor phenotype, and the nervous system is also damaged, resulting in birth defects in young mice. Studies have shown that JAK1 gene knockout mice will have thymocyte and B cell secretion defects, and JAK1 gene knockout tissues have significantly weakened response to IL-6 and IL-10.
  • JAK is associated with autoimmune diseases.
  • mutations in JAK and the upstream signaling component r-c receptor chain and IL7 receptor have been detected. Therefore, targeting the JAK family can provide new therapeutic drugs in the immune-inflammatory field.
  • the JAK family member JAK2 involves diseases, including myeloproliferative disorders, cancers, especially leukemias such as acute myeloid leukemia, acute lymphoblastic leukemia, or solid tumors such as uterine leiomyosarcoma, prostate cancer and other related diseases. Therefore, drugs targeting the JAK family may also provide new options for the treatment of the aforementioned diseases.
  • the present invention provides novel JAK kinase inhibitors and preparation methods, pharmaceutical compositions containing these kinase inhibitors and applications thereof.
  • the present invention relates to a compound represented by formula (I) or its stereoisomers, tautomers, or pharmaceutically acceptable salts or solvates or prodrugs thereof:
  • B is independently selected from substituted or unsubstituted C 3 -C 6 cycloalkyl, aryl, 5- to 6-membered heteroaryl ring having 1 to 2 ring heteroatoms independently selected from N, O and S, wherein The 5- to 6-membered heteroaromatic ring is optionally independently selected from halogen, fluorinated or non-fluorinated C 1 -C 6 alkyl, fluorinated or non-fluorinated C 1 -C 6 alkoxy, fluorine One or more substituents of substituted or unfluorinated C 1 -C 6 alkylamino groups;
  • D is independently selected from C, N;
  • F, G, H, K are independently selected from C, N, S, O;
  • L 1 does not exist or is independently selected from a single bond, -CH 2 -, -(CH 2 ) x O(CH 2 ) y -, -(CH 2 ) x NH(CH 2 ) y -, -CH 2 O-, -C(O)-,-CON(R 4 )-,-CH 2 N(R 4 )-,-CONH(CH 2 ) y -,-N(R 4 )-,-SO 2 N(R 4 ) -,-S(O) 2 -,-N(Me)-;
  • R 4 is independently selected from H, C 1 -C 6 alkyl, substituted C 1 -C 6 alkyl, C 1 -C 3 ether C 1 -C 3 alkyl, methylsulfonyl;
  • R 1 is independently selected from H, -NH 2 , -COOH, substituted or unsubstituted C 1 -C 6 alkyl, acyl, substituted or unsubstituted acylamino, substituted or unsubstituted C 1 -C 6 Alkoxy, halogen, hydroxyl, substituted or unsubstituted C 1 -C 3 ester group, substituted or unsubstituted heteroaryl group;
  • R 2 does not exist or is independently selected from H, F, Cl, Me;
  • R 3 is independently selected from H, substituted or unsubstituted groups: sulfonyl, sulfonamide, 5- to 6-membered heterocyclic ring, which has at least one heteroatom and optionally has a third group independently selected from N and S Bicyclic heteroatoms, substituted or unsubstituted C 1 -C 6 alkanes, the substituents are independently selected from 5 to 6-membered heteroaryl groups and methoxy substituted, substituted or unsubstituted C 3 -C 7 cycloalkyls , Substituted or unsubstituted 4-7 membered heterocycloalkyl, substituted or unsubstituted 5-7 membered heteroaryl;
  • n 0, 1, 2, 3
  • p 0, 1, 2
  • t 0, 1, 2, 3.
  • F, G, H, K are not C at the same time.
  • B is selected from the following substituted or unsubstituted groups: cyclopropane, cyclobutane, pyrazolyl, pyridyl, imidazolyl; the substituent is selected from F, Cl, Br, methyl, ethyl Group, propyl, isopropyl, trifluoromethyl.
  • B is substituted or unsubstituted cyclopropane; the substituent is selected from F, Cl, Br, methyl, ethyl, propyl, isopropyl, and trifluoromethyl.
  • D is C or N.
  • D is N.
  • n 1
  • K is S, F, G, and H are C.
  • R 1 is independently selected from H, -NH 2 , -COOH, hydroxyl, halogen, substituted or unsubstituted C 1 -C 3 alkyl, substituted or unsubstituted C 1 -C 3 acyl, A substituted or unsubstituted C 1 -C 3 acylamino group, a substituted or unsubstituted C 1 -C 3 alkoxy group, a substituted or unsubstituted C 1 -C 3 ester group; in a preferred implementation
  • R 1 is independently selected from H, -NH 2 , -COOH, hydroxyl, halogen, methyl, ethyl, propyl, isopropyl, formylamino, and carbomethoxy.
  • n is 0 or 1, more preferably, m is 0.
  • R 2 is absent or is H.
  • R 2 is not present.
  • p is zero.
  • L 1 is absent or independently selected from a single bond, -CH 2 -, -(CH 2 ) x O(CH 2 ) y -, -(CH 2 ) x NH(CH 2 ) y- ,-CONH(CH 2 ) y -,-N(R 4 )-;
  • R 4 is independently selected from H, methyl, ethyl, propyl, ethyl methyl ether, methyl methyl ether, ethyl ethyl ether;
  • x or y is independently selected from 0, 1, or 2.
  • L 1 is absent or independently selected from a single bond, -CH 2 -, -(CH 2 ) x O(CH 2 ) y -, -(CH 2 ) x NH(CH 2 ) y -,-CONH(CH 2 ) y -,-N(R 4 )-;
  • L 1 is selected from -CH 2 -, -(CH 2 ) x O(CH 2 ) y -, -CONH(CH 2 ) y -, R 4 is independently selected from H, methyl, and ethyl methyl ether;
  • x or y is independently selected from 0, 1 or 2; in some specific embodiments, x Or y is independently selected from 1 or 2.
  • t is 1.
  • R 3 is independently selected from H, substituted or unsubstituted groups: sulfonyl, sulfonamido, thiomorpholine 1,1-dioxide, piperidine, pyrazole, thiazole , Imidazole, 1,3,4-thiadiazole, piperazine, morpholine, thiophene, oxazole, 1,3,4-oxadiazole, furan, pyrrole, 3-pyrroline, 2-pyrazoline, 1 ,2,3-azole, 1,2,3-triazole, 1,2,4-triazole, pyran; the substituent is selected from H, halogen, C 1 -C 3 alkyl, C 1 -C 3 haloalkane Group, hydroxy C 1 -C 3 alkyl, C 3 -C 6 cycloalkyl, ethyl methyl ether, methyl methyl ether, ethyl ethyl ether; in some embodiment
  • the present invention also provides a compound represented by the following structure or a stereoisomer, a tautomer or a pharmaceutically acceptable salt or a solvate or prodrug thereof:
  • the present invention also provides methods for synthesizing the compounds of the present invention, as well as disclosed representative synthetic schemes and routes.
  • the present invention also provides a pharmaceutical composition, which comprises the compound of formula I of the present invention or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable diluent or carrier.
  • the present invention also provides the use of the compound of formula I of the present invention in the preparation of drugs for the prevention or treatment of JAK kinase-related diseases, especially in the preparation of drugs for the prevention and/or treatment of cartilage degradation, bone and/or joint degradation, Applications in medicines involving inflammation or immune response disorders, endotoxin-driven disease states, cancer and organ transplant rejection.
  • the present invention also provides a method for preventing and/or treating diseases involving cartilage degradation, bone and/or joint degradation, disorders involving inflammation or immune response, endotoxin-driven disease states, cancer and organ transplantation by administering the compounds of the present invention Methods of rejection.
  • examples of diseases involving cartilage degradation, bone and/or joint degradation, disorders involving inflammation or immune response, endotoxin-driven disease states, cancer and organ transplant rejection include but not Limited to: osteoarthritis, Crohn's disease, rheumatoid arthritis, psoriasis, allergic airway diseases (such as asthma, rhinitis), juvenile idiopathic arthritis, colitis, inflammatory bowel disease, endomycin-driven The disease state, cartilage renewal damage disease (such as cartilage cell anabolic excitement disease), congenital cartilage deformity, organ transplant rejection, cartilage degeneration, joint degeneration, myeloproliferative disorder, leukemia (acute myelogenous leukemia, acute lymphoid Cell leukemia), solid tumors (uterine leiomyosarcoma, prostate cancer), etc.
  • osteoarthritis Crohn's disease, rheumatoid arthritis, psoriasis, allergic airway diseases (such as asthma, rhinitis), juvenile
  • amino used in the present application refers to a functional group having 1 nitrogen atom and 1 to 2 hydrogen atoms. "Amino” is generally used herein to describe primary, secondary or tertiary amines and those skilled in the art can easily determine the amino group based on the context in which the term is used in this disclosure.
  • amine or “amine group” or “amino group” refers to a functional group containing a nitrogen atom derived from ammonia (NH 3 ).
  • the amine group is preferably a primary amine, which means that the nitrogen is bonded to two hydrogen atoms and one substituent containing a substituted or unsubstituted alkyl or aryl group or an aliphatic or aromatic group.
  • the amine group may be a secondary amine, which means that the nitrogen is bonded to one hydrogen atom and two substituents containing a substituted or unsubstituted alkyl or aryl group or aliphatic or aromatic group as defined below.
  • the amine group may be a tertiary amine, which means but is bonded to three substituents containing substituted or unsubstituted alkyl or aryl groups or aliphatic or aromatic groups.
  • the amine group can also be a quaternary amine, which means that the specified amine group is bound to the fourth group, which produces a positively charged ammonium group;
  • any or all of the amines in the present invention may be in free amine form (i.e. -NH 2 for primary amines) or protonated form with pharmaceutically acceptable anions (i.e. for primary amines It is -NH 3 + Y -, wherein Y - is a pharmaceutically acceptable anion);
  • amide group refers to a functional group containing a carbonyl group attached to nitrogen.
  • alkane refers to a saturated hydrocarbon bonded by a single bond. Alkanes can be linear or branched. "Cycloalkane” is a saturated hydrocarbon ring bonded by a single bond;
  • C 1 -C 6 alkyl refers to a saturated linear or branched or cyclic hydrocarbon, which is basically composed of 1 to 6 carbon atoms and a corresponding number of hydrogen atoms. Typically, straight or branched chain groups have 1 to 10 carbons, or more typically 1 to 5 carbons. Exemplary C 1 -C 6 alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, and the like. According to the teachings of the present disclosure, other C 1 -C 6 alkyl groups will be apparent to those skilled in the art;
  • C 2 -C 9 heteroalkyl refers to a saturated linear or branched or cyclic hydrocarbon consisting essentially of 2 to 10 atoms, of which 2 to 9 atoms are carbon and the remaining atoms Selected from nitrogen, sulfur and oxygen. According to the teachings of the present disclosure, exemplary C 2 -C 9 heteroalkyl groups will be obvious to those skilled in the art;
  • C 3 -C 10 cycloalkyl refers to a non-aromatic saturated hydrocarbon group that forms at least one ring consisting essentially of 3 to 10 carbon atoms and a corresponding number of hydrogen atoms.
  • the C 3 -C 10 cycloalkyl group may be monocyclic or polycyclic.
  • each ring of a polycyclic cycloalkyl group may have different connectivity, such as fused, bridged, spiro ring, etc.;
  • C 2 -C 9 heterocycloalkyl refers to a non-aromatic group having 3 to 10 atoms to form at least one ring, wherein 2 to 9 ring atoms are carbon and the remaining atoms are selected from Nitrogen, sulfur and oxygen.
  • the C 2 -C 9 heterocycloalkyl group may be monocyclic or polycyclic. In addition to covalent bond substitution, each ring of such a polycyclic heterocycloalkyl group may have different connectivity, such as fused, bridged, spiro ring, and the like. According to the teachings of the present disclosure, exemplary C 2 -C 9 heterocycloalkyl groups will be obvious to those skilled in the art;
  • aliphatic group refers to a non-aromatic group composed of carbon and hydrogen and may optionally include one or more double bonds and/or triple bonds.
  • an aliphatic group is any group that is composed of carbon and hydrogen and does not contain aromatic functionality.
  • Aliphatic groups can be linear, branched or cyclic and typically contain 1 to 24 carbon atoms;
  • aryl group can be used interchangeably with “aryl”, “aryl ring”, “aromatic group”, “aromatic group” and “aromatic ring”.
  • Aryl groups include carbocyclic aromatic groups, which typically have 6 to 14 ring carbon atoms.
  • Aryl groups also include heteroaryl groups, which typically have 5 to 14 ring atoms, with one or more heteroatoms selected from nitrogen, oxygen, and sulfur;
  • C 6 -C 14 aryl refers to an aromatic functional group having 6 to 14 carbon atoms to form at least one ring
  • C 2 -C 9 heteroaryl refers to an aromatic functional group having 5 to 10 atoms to form at least one ring, wherein 2 to 9 ring atoms are carbon and the remaining ring atoms are selected from nitrogen, Sulfur and oxygen.
  • the C 2 -C 9 heteroaryl group may be monocyclic or polycyclic. In addition to covalent bond substitution, each ring of such polycyclic heteroaryl groups may have different connectivity, such as fused, bridged, spiro ring, and the like.
  • the C 2 -C 9 heteroaryl group is typically connected to the main structure via a carbon atom, however, those skilled in the art will understand when certain other atoms such as heterocyclic atoms are connected to the main structure. According to the teachings of the present disclosure, other C 2 -C 9 heteroaryl groups will be obvious to those skilled in the art;
  • alkylamine refers to a C 1 -C 6 alkyl group containing a primary, secondary or tertiary amine group in place of a hydrogen atom, which is composed of C 1 -C 6 alkylamine and (C 1- C 6 alkyl) 2 amine means;
  • alkyl ester refers to a C 1 -C 6 alkyl group containing an ester group in place of one hydrogen atom, which is represented by -O(O)C (C 1 -C 6 alkyl);
  • alkyl acid refers to a C 1 -C 6 alkyl group containing a carboxylic acid group instead of a hydrogen atom, which is represented by C 1 -C 6 alkyl-COOH;
  • fatty acid refers to non-aromatic hydrocarbon acids, which are represented by C 1 -C 6 alkyl-COOH and C 3 -C 6 alkyl-COOH;
  • halogen refers to fluorine (F), chlorine (Cl), bromine (Br), iodine (I) or astatine (At) ion;
  • methoxy refers to a (C 1 )alkyl group containing oxygen instead of one hydrogen atom, which is represented by -(O)CH 3 ;
  • polyol refers to an alcohol containing multiple hydroxyl groups
  • Substituted means that the carbon of an alkyl, heterocyclyl or aryl group is substituted with one or more non-carbon substituents.
  • the non-carbon substituents are selected from nitrogen, oxygen and sulfur;
  • Ester group means a -C(O)O-R' group, wherein R'is as defined above, but R'cannot be hydrogen.
  • Alkylsulfonyl represents a group represented by the formula -SO 2 R, and R represents an alkyl group.
  • Thiomorpholine 1,1-dioxide means When it is used as a substituent group, it is connected to the parent structure of the compound through N, which is
  • the synthetic route is as follows:
  • SM1 5-Bromo-[1,2,4]triazolo[1,5-a]pyridin-2-amine
  • SM2 cyclopropylformyl chloride
  • N-(5-Bromo-[1,2,4]triazolo[1,5-a]pyridin-2-yl)-N-(cyclopropanecarbonyl)cyclopropanecarboxamide (1) (3.3g crude product, 9.4 mmol) was dissolved in methanol (30 mL) solution, potassium carbonate (3.9 g, 28.2 mmol) was added, and the mixture was stirred at room temperature for 1 hour. The reaction solution was concentrated in vacuo at room temperature. The obtained crude product was dissolved and dispersed in ethyl acetate (250 mL) and then filtered. The filter cake was washed with dichloromethane (250 mL) and tetrahydrofuran (250 mL) successively.
  • N-(5-Bromo-[1,2,4]triazolo[1,5-a]pyridin-2-yl)cyclopropanecarboxamide (Int-1) (300mg, 1.1mmol), (5-methyl Acyl-2-thienyl)boronic acid (SM3) (332.9mg, 2.1mmol) and potassium carbonate (442.5mg, 3.2mmol) are dissolved in dioxane (5mL) and water (1mL), replaced with nitrogen three times and then quickly added 1,1-bis(diphenylphosphorus)ferrocene palladium chloride (39.0mg, 53.4umol), replaced with nitrogen again three times, and stirred at 90°C for 30 minutes under nitrogen protection.
  • SM3 5-methyl Acyl-2-thienyl)boronic acid
  • potassium carbonate 442.5mg, 3.2mmol
  • reaction solution was diluted with water (20mL) and dichloromethane for extraction (20mL ⁇ 3), the organic phase was washed with saturated brine (20mL), dried over anhydrous sodium sulfate and filtered, the filtrate was concentrated in vacuo to obtain the crude product, which was purified by silica gel column chromatography (washing Removal agent: 1% ⁇ 2% methanol/dichloromethane) to obtain the product N-[5-(5-formyl-2-thienyl)-[1,2,4]triazolo[1,5-a] Pyridin-2-yl]cyclopropanecarboxamide (2) (350mg semi-pure product, containing part of raw material 1, conversion yield 49.4%), yellow solid.
  • N-[5-(5-formyl-2-thienyl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl]cyclopropanecarboxamide (2) (220mg half Pure product, 331.0umol) and 1,1-dioxothiomorpholine (SM4) (53.7mg, 397.3umol) dissolved in methanol (6mL), add acetic acid dropwise to adjust the pH to about 6, and stir at 50°C for 1 hour After cooling to room temperature, sodium cyanoborohydride (41.6mg, 662.1umol) was added, and stirring was continued for 15 hours at 30°C.
  • SM4 1,1-dioxothiomorpholine
  • the reaction solution was treated with saturated sodium bicarbonate aqueous solution (3mL), diluted with water (10mL) and extracted with dichloromethane (20mL ⁇ 3). The organic phases were combined and washed with saturated brine (20mL), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated in vacuo to obtain a crude product, and the target compound EXP-1 (28.9 mg, yield 20%) was obtained as a white solid by alkaline preparation, separation and purification.
  • the synthetic route is as follows:
  • 6-Bromopyridin-2-amine (SM16) (5g, 28.90mmol) and ethyl bromopyruvate (SM17) (6.20g, 31.79mmol) were dissolved in ethanol (30mL) and stirred at 85°C for 16 hours.
  • the reaction solution was filtered, the filter cake was collected, and the mixture was slurried with petroleum ether/ethyl acetate (5/1, v/v, 50 mL), and the solid was filtered and dried to obtain 5-bromoimidazole [1,2-a]pyridine-2-carboxylic acid Ethyl ester (22) (8.41 g, 83% yield, hydrogen bromide salt, yellow solid).
  • reaction solution was filtered, the filtrate was concentrated in vacuo, and the crude product was purified by silica gel chromatography (methanol/dichloromethane, from 0% to 2%) to obtain tert-butyl 5-bromoimidazole [1,2-a]pyridine-2-carbamate ( 24) (1.01 g, 49% yield, 78% purity, yellow solid).
  • reaction solution (combined with batch EB4-106, 100 mg of substrate 1 was charged) was quenched by adding saturated sodium bicarbonate solution (40 mL), and extracted with ethyl acetate (60 mL ⁇ 3). The organic phases were combined, washed with brine (40 mL), dried with anhydrous sodium sulfate, filtered, concentrated in vacuo, and purified by silica gel column (methanol/dichloromethane, from 0% to 2%) to obtain trans-N-(5- Bromoimidazole [1,2-a]pyridin-2-yl)-2-fluoro-cyclopropylcarboxamide (26) (587 mg, 69% yield, yellow solid).
  • reaction solution was concentrated and purified by silica gel column (methanol/dichloromethane, from 0% to 2%) to obtain trans-2-fluoro-N-[5-(5-formyl-2-thiophene)imidazole [1, 2-a]pyridin-2-yl]cyclopropylcarboxamide (27) (90 mg, 41% yield, 100% purity, yellow solid).
  • reaction solution was quenched by adding saturated sodium bicarbonate (30 mL), stirred at room temperature for half an hour, and extracted with dichloromethane (50 mL ⁇ 3). The organic phases were combined, washed with brine (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo.
  • the crude product was passed through preparative HPLC (Column: Waters Xbridge 150*25mm*5um; Condition: water (0.04% NH 3.
  • the synthetic route is as follows:
  • reaction solution was filtered, and the filtrate was concentrated in vacuo to obtain a crude product, which was then dissolved in tetrahydrofuran (2mL), and diisopropylethylamine (0.1mL) and diethyl phosphite (74uL) were sequentially added, and stirred at 10°C for 25 minutes to obtain 2- Bromomethylthiazole-5-boronic acid lanalol ester (3) (135mg, crude product, dissolved in 2mL tetrahydrofuran brown solution), used directly in the next step.
  • reaction solution was concentrated in vacuo and quickly purified by a column machine (eluent: 0% ⁇ 0.5% methanol/dichloromethane) to obtain the crude product, and the target molecule N-[5-[2-[(1,1- Dioxy-1,4-thiazine-4-)methyl]thiazole-5-]-[1,2,4]triazole[1,5-a]pyridine-2-]cyclopropylcarboxamide (EXP -4) (5.6 mg, 6% yield, 93% purity, yellow solid).
  • the synthetic route is as follows:
  • cis-2-fluorocyclopropanecarboxylic acid (SM6) (50 mg, 0.48 mmol) was dissolved in SOCl 2 (1 mL) and stirred at 75° C. for 1 hour. Concentrate under reduced pressure below 30°C to obtain compound cis-2-fluorocyclopropane acid chloride (5) (58 mg, 98% yield), and the yellow oily substance was used directly in the next step.
  • N-[5-(2-Formyl)-thiophene-[1,2,4]triazole[1,5-a]pyridin-2-yl]-cis-2-fluoro-cyclopropanecarboxamide (6 ) (100mg, 0.302mmol) and 1,1-Dioxythiomorpholine (SM4) (82mg, 0.605mmol) were dissolved in methanol (5mL), added with acetic acid (9mg, 0.151mmol), and then heated to 80°C for reaction After 0.2 hours, reduce to room temperature and add sodium cyanoborohydride (152mg, 2.42mmol), then heat to 80°C to react for 0.5 hours, cool down, add saturated sodium bicarbonate (10mL), stir at room temperature for 0.5 hours, concentrate to remove most of the methanol and then Extract with ethyl acetate (20mL ⁇ 2), concentrate, and dissolve in methanol (5mL) for preparation, separation and purification to obtain the title product N-[5-[4-[
  • the synthetic route is as follows:
  • the synthetic route is as follows:
  • Methyl 6-aminopyridine-2-carboxylate (SM8) (3 g, 19.72 mmol) was dissolved in dioxane (30 mL) and ethoxycarbonyl isothiocyanate (SM9) (2.84 g, 21.69 mmol) was added. Stir at 10 degrees for 16 hours. The reaction solution was concentrated and beaten with ethyl acetate/petroleum ether (1/15, 30mL) to obtain methyl 6-(3-(ethoxy)thiourea)picolinate (8) (5.25g, 94% yield, 100%) Purity, pale yellow solid).
  • SM9 ethoxycarbonyl isothiocyanate
  • reaction solution was added to water (100mL), filtered, the solid was washed with water (20mL), ethanol (40mL), and dried to obtain N-[5-[[[2-(1,1-diox-1,4-thiazine- 4-)Acetyl]amino]carbamoyl]-[1,2,4]triazole[1,5-a]pyridine-2-]cyclopropylcarboxamide (12) (3.26g, 89% yield Rate, 91% purity, white solid).
  • the synthetic route is as follows:
  • the synthetic route is as follows:
  • N-[5-(5-formyl-2-thienyl)-[1,2,4]triazole[1,5-a]pyridin-2-yl]cyclopropanecarboxamide (2) (30mg, 0.1mmol) in methanol (2ml), then sodium cyanoborohydride (12mg, 0.19mmol) was added. Stir at 50°C for 0.2 hours under nitrogen protection. Then sodium bicarbonate (1 mL) was added and extracted with ethyl acetate (10 mL ⁇ 3).
  • the synthetic route is as follows:
  • N-(5-Bromo-[1,2,4]triazolo[1,5-a]pyridin-2-yl)-cyclopropanecarboxamide (Int-1) (100mg, 0.36mmol), 5-carboxy -2-thiophene boronic acid (SM13) (122mg, 0.71mmol) and sodium carbonate (113mg, 1.07mmol) were dissolved in tetrahydrofuran (10mL) and water (5mL), replaced with nitrogen three times and then added 1,1-bis(diphenyl) Phosphorus) Ferrocene palladium chloride (50mg, 0.07mmol), replaced with nitrogen three times and then raised to 100°C and stirred for 2 hours.
  • SM13 5-carboxy -2-thiophene boronic acid
  • sodium carbonate 113mg, 1.07mmol
  • reaction solution was concentrated and then prepared by HPLC [Column: Phenomenex Gemini-NX 150*30mm*5um; Condition: 25-55% B (A: water (0.04% NH 3. H 2 O + 10mM NH 4 HCO 3 ); B: CH 3 CN); Flow rate: 25mL/min] Purify to obtain 5-[2-[2-thiazole-2-ethyl]carboxamido]-thiophene-[1,2,4]triazole[1,5-a ] Pyridine-2-cyclopropanecarboxamide (EXP-17) (6.3 mg, 8% yield, white solid).
  • the synthetic route is as follows:
  • reaction solution was poured into water (20mL), the aqueous phase was extracted with ethyl acetate (20mL ⁇ 3), the organic phase was dried with anhydrous sodium sulfate, filtered, concentrated and spin-dried, and then subjected to silica gel column chromatography (eluent: 0% ⁇ 10% ethyl acetate/petroleum ether) to obtain the light yellow target product N-tert-butoxycarbonylpiperidine-4-methylxanthate (18) (1.6 g, yield 55%).
  • 1,3-Dibromo-5,5-dimethylhydantoin (2.21g, 7.72mmol) was dissolved in dichloromethane (20mL), and hydrogen fluoride pyridine solution (7.14g) was added in portions in a nitrogen atmosphere at -75°C , 72.06mmol) and N-tert-Butoxycarbonylpiperidine-4 methylxanthate (18) (500mg, 1.72mmol) in dichloromethane (2mL).
  • the reaction solution was reacted at 15-20°C for 16 hours and then quenched with saturated sodium bicarbonate solution (20 mL), and the aqueous phase was extracted with ethyl acetate (20 mL ⁇ 3).
  • the organic phase was dried with anhydrous sodium sulfate, filtered, concentrated and spin-dried to obtain the yellow target product 4-(trifluoromethoxy)piperidine (19) (400 mg, crude product).
  • the mixed reaction solution was reacted at 15-20°C for 16 hours and then concentrated and spin-dried to obtain the brown target product trans-2-fluoro-N-[5-(5-chloromethyl-2-thiophene)-[1,2,4 ]Triazole[1,5-a]pyridin-2-yl]cyclopropanecarboxamide (21) (60 mg, yield 71%).
  • reaction mixture was diluted with dichloromethane (20mL) and water (20mL), the aqueous phase was extracted with dichloromethane (20mL ⁇ 3), the organic phase was washed with saturated brine (20mL ⁇ 3), dried over anhydrous sodium sulfate and filtered and concentrated After spin-drying, it was purified by HPLC (separation column: Waters Xbridge 150*25mm*5um; mobile phase: A: water (0.04% NH 3.
  • Example 11 JAK kinase inhibitory activity detection
  • the compounds of the above examples are applied to the detection and screening of JAK kinase inhibitory activity.
  • the inhibition percentage is calculated based on the following formula:
  • Inhibition % (Ratio max -Ratio compound )/(Ratio max -Ratio min )] ⁇ 100%
  • Ratio compound is the HTRF ratio at a given compound concentration
  • Ratio min is the HTRF ratio added to the blank hole
  • Ratio max is the HTRF ratio without adding the compound.
  • Filgotinib represents a compound with the following structure:
  • IC 50 (nM) values of the tested compounds are shown in Table 5 below.
  • IC 50 values 0-10nM labeled A; 10-30nM labeled B; 30-50nM labeled C; 50-500nM labeled D; greater than 500nM labeled E; NT not tested representatives.
  • Example 12-1 Based on detection of proliferation inhibition on Hela cells
  • Cell plating Collect cultured cells and count viable cells with a Vi-Cell XR cell counter. Adjust the cell suspension to an appropriate concentration with culture medium and add it to a 96-well cell culture plate. Add 50 ⁇ L-100 ⁇ L of cell suspension to each well on a 96-well cell culture plate. The cell plating density depends on the growth rate of the cells. Place in the corresponding incubator for cultivation.
  • T is the luminescence reading of the drug treatment group
  • C is the average luminescence reading of the solvent control group.
  • IC 50 value between 0-5uM is marked as A; 5-30uM is marked as B; undetected marked as NT.
  • Example 12-2 Based on detection of proliferation inhibition on BaF3 cells
  • Cell plating Collect cultured cells with RPMI1640+10% FBS (containing IL-3) medium and count viable cells with a Vi-CellXR cell counter. Adjust the cell suspension to an appropriate concentration with RPMI1640+10% FBS (containing IL-3) medium and add it to a 96-well cell culture plate. Add a certain amount of cell suspension to each well on the 96-well cell culture plate. Cultivate in the corresponding incubator.
  • Drug treatment firstly prepare a series of test compound concentration (work solution-1) with 3X times gradient dilution with DMSO, and then use RPMI1640+10% FBS (containing IL-3) to dilute it multiple times to obtain work solution-2; RPMI1640+10% FBS (containing IL-3) is used to prepare Cisplatin's work solution-2, and then 1-10 ⁇ L of each from the work solution-2 is added to the prepared cell wells containing medium. The cells are cultured in the corresponding incubator. Add appropriate CTG solution to each well, shake and mix for 5-15 minutes, then measure the luminescence signal with Envision2104 plate reader.
  • T is the luminescence reading of the drug treatment group
  • C is the average luminescence reading of the solvent control group.
  • IC 50 value between 0-5uM is marked as A; 5-30uM is marked as B; undetected marked as NT.
  • Example 12-3 Based on the detection of proliferation inhibition on THP1 cells
  • Cell plating Collect cultured cells and count viable cells with a Vi-Cell XR cell counter. Adjust the cell suspension to an appropriate concentration with culture medium and add it to a 96-well cell culture plate. Add 80 ⁇ L of cell suspension per well to the 96-well cell culture plate. The cell plating density depends on the growth rate of the cells. Cultivate in the corresponding incubator.
  • Drug treatment and IL-4 stimulation firstly prepare a 3X series of test compound concentration (work solution-1) with DMSO, and then use RPMI1640+10% FBS to dilute it multiple times to obtain work solution-2; use RPMI1640 +10% FBS is used to prepare Cisplatin's work solution-2, and then 1-10 ⁇ L from the work solution-2 is added to the prepared cell wells containing medium. The cells are cultured in the corresponding incubator. After incubation, add 1-10 ⁇ L of IL-4 medium to each well to continue the culture. Add appropriate CTG solution to each well, shake and mix for 5-15 minutes, then measure the luminescence signal with Envision2104 plate reader.
  • T is the luminescence reading of the drug treatment group
  • C is the average luminescence reading of the solvent control group.
  • IC 50 value between 0-5uM is marked as A; 5-30uM is marked as B; undetected marked as NT.
  • liver microsomes of different species are used to react with the compound of the present invention for a certain period of time, and the residual rate is calculated by comparing the reaction sample with the unreacted sample, and the degree of liver metabolism of the compound of the present invention is evaluated.
  • Compound EXP-3 human liver microsomes: 59.32%; rat liver microsomes: 55.3%.
  • CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4 Use commercially available mixed human liver microsomes as the typical substrate metabolism reaction of the main human CYP molecular species (CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4), and the reaction conditions are as follows: Substrate, CYP1A2 : 30 ⁇ M phenacetin; CYP2C9: diclofenac 10 ⁇ M; CYP2C19: 35 ⁇ M S-methphenytoin; CYP3A4: midazolam 5 ⁇ M, testosterone 80 ⁇ M; CYP2D6: Bufurolol, 10 ⁇ M; CYP2C8: 10 ⁇ M paclitaxel ; CYP2B6: bupropion 70 ⁇ M; reaction time, CYP1A2, 2C9, 2D6, 2C8, 2B6: 10 minutes; CYP2C19: 45 minutes; CY
  • Reaction temperature 37°C.
  • Inhibitor positive control
  • CYP1A2 ⁇ -naphthoflavone
  • CYP2C9 Sulfafloxazole
  • CYP2C19 Omeprazole
  • CYP3A4 Ketoconazole
  • CYP2D6 Quinidine
  • CYP2C8 Nicardipine
  • CYP2B6 Clopidogrel .
  • the s-type (non-linear) dose-response model (GraphPad Prism 5.0 or Xlfit model 205) is used to calculate the IC50 by curve fitting.
  • the data calculation formula is as follows:
  • X is the logarithm of the concentration.
  • Y is the s-shaped response from bottom to top when the concentration goes from high to low.
  • the compound is formulated in 5% DMSO + 95% hydroxypropyl- ⁇ -cyclodextrin aqueous solution.
  • the test animals were administered orally at 1-10 mg/kg and intravenously at 1-5 mg/kg. Each group included 3 rats.
  • About 0.2 mL of blood was collected by jugular vein puncture. Heparin sodium anticoagulant was collected at the following time points: 0.083h, 0.25h, 0.5h, 1h, 2h, 4h, 6h, 8h, 10h and 24h. After the blood sample was collected, the plasma was separated by centrifugation at 5000-13000 rpm for 10 min within 1 hour, and the plasma sample was analyzed on LC-MS/MS.
  • Use Phoenix WinNonlin 7.0 to calculate pharmacokinetic parameters through blood drug concentration data at different time points, and provide parameters such as AUC 0-t , AUC 0- ⁇ , MRT 0- ⁇ , C max , T max , and T 1/2 .
  • Use Phoenix WinNonlin 7.0 to calculate pharmacokinetic parameters through blood drug concentration data at different time points, and provide parameters such as AUC 0-t , AUC 0- ⁇ , MRT 0- ⁇ , C max , T max , and T 1/2 .
  • AUC is 12043h*ng/mL
  • Cmax is 2626ng/mL
  • T 1/2 is 2.68h.
  • AUC is 2849h*ng/mL
  • Cmax is 426ng/mL
  • T 1/2 is 3.9h.
  • HEK293 cells expressing humanether-a-go-related gene (hERG) channels were used to study the important role of the compounds of the present invention in the process of ventricular repolarization
  • the role of delayed rectification K+ current (Ikr) was used to study the important role of the compounds of the present invention in the process of ventricular repolarization.
  • Extracellular fluid NaCl, 137; KCl, 4; CaCl 2 , 1.8; MgCl 2 , 1; HEPES, 10; glucose 10; pH 7.4 (NaOH titration).
  • the intracellular fluid (mM) is: K Aspartate, 130; MgCl2, 5; EGTA 5; HEPES, 10; Tris-ATP 4; pH 7.2 (KOH titration).
  • Example 17 Back mutation test of Salmonella typhimurium
  • Saturnella TA98, TA100, TA1535, TA1537 and Escherichia coli as test strains (strain source Molecular Toxicology, Inc.), Ames test was performed under non-metabolic activation conditions and activation conditions to investigate whether the compounds of the present invention have gene mutations Induced.
  • the cryopreserved strains were resuscitated and inoculated into nutrient broth medium at 100-120 rpm, 37°C constant temperature shaker for 11 hours. Pour the bottom agar medium (containing an appropriate amount of agar, V-B buffer, 20% glucose solution, 20% magnesium sulfate solution) into a dish to cool for later use. In each test tube, add 0.1mL test substance/negative control substance (DMSO)/positive control substance solution, 0.1mL bacterial solution, 0.5mL 0.2mol/L phosphate buffer saline (PBS)(-S9)/0.5mL S9 mixture (+S9) and 2.5mL top medium, vortex and mix, and quickly and evenly spread on the bottom agar medium plate.
  • DMSO test substance/negative control substance
  • PBS phosphate buffer saline
  • S9 mixture (+S9) 0.5mL S9 mixture (+S9)
  • the plate was placed upside down and placed in a mold incubator at 37°C for 48 hours. Take out the plate count after culture and compare it with the DMSO group for evaluation.
  • the average number of reverted colonies is twice or more than the number of reverted colonies in the negative control group (can be regarded as spontaneous reverted number) (TA1535: 3 times or Above), if there is a dose-effect relationship, the result is positive, and the test product can be determined to be a mutagenic agent. Otherwise, it is negative.
  • Example 18 In vivo drug effect model
  • CFA Complete Freund's adjuvant
  • type II bovine collagen were purchased from Chondrex.
  • Lewis rats male, 10-12 weeks old were obtained from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. The rats were alternated with light and dark for 12 hours, and the environment was kept at a temperature of 23 ⁇ 2°C and a humidity of 40-70%.
  • type II collagen solution (2mg/mL) with 0.05M acetic acid solution, make it fully dissolved, and place it overnight at 4°C in the dark.
  • 2 mg/mL type II collagen (C II) solution and 4 mg/mL complete Freund's adjuvant (CFA) solution were mixed in equal volume under ice bath conditions to fully emulsify.
  • C II type II collagen
  • CFA complete Freund's adjuvant
  • an emulsion prepared by mixing equal volumes of C II (2 mg/mL) and CFA (4 mg/mL) was injected intracutaneously into the base of the tail of experimental rats for the first immunization, and the second booster immunization was performed on the seventh day.
  • the therapeutic effect of the test compound was tested in a rat CIA model.
  • the rats were randomly divided into groups with 8 animals in each group. All animals were immunized on day 0 and boosted on day 7.
  • Blank control Group
  • the vehicle control group Vehicle group
  • the positive control group was treated with Filgotinib (10 mg/kg, 2 weeks of administration), Tofacitinib citrate (Tofacitinib Group, 2mg/kg, 2 weeks of administration) and Upadacitinib (Upadacitinib group, 2mg/kg, 2 weeks of administration)
  • the experimental group is 2mg/kg, 3mg/kg, 5mg/kg, 10mg/kg
  • the EXP-3 was administered for two weeks.
  • Foot volume was scored for the swelling of four toes at the same time, each foot was 0-4 points, and the highest score for each rat was 16 points.
  • Pathology score data was analyzed using SPSS non-parametric test Mann-Whitney U test, and p ⁇ 0.05 was considered as a significant difference.
  • weight loss and foot volume loss are associated with arthritis. Therefore, the difference between body weight and foot volume after the onset of arthritis and the negative group can be used to evaluate the therapeutic effect in the rat model.
  • the experimental data are expressed as Mean ⁇ SEM; the data between the two groups was unpaired t-test, and p ⁇ 0.05 was considered as a significant difference.
  • the foot volume increased significantly on the 14th to 28th day after the first vaccination, and the foot volume began to decrease on the 24th day.
  • the upadacitinib 2mg/kg group had significantly lower foot volume on day 17-28 (p ⁇ 0.05-0.001).
  • the foot volume of the animals in the rest of the administration groups was significantly reduced from day 21 to 28 (p ⁇ 0.05-0.001).
  • EXP-3 had the strongest effect at 10mg/kg, and was significantly stronger than filgotinib at 10mg/kg, followed by Upadacitinib at 2mg/kg.
  • EXP-3 5mg/kg ⁇ 2mg/kg has similar effects to Tofacitinib 2mg/kg, but slightly weaker than Upadacitinib 2mg/kg. (Table 10). It shows that the test substance has a certain effect on improving the foot volume of CIA model rats.
  • EXP-3 10mg/kg has the strongest effect on reducing swelling of limbs, followed by tofacitinib 2mg/kg.
  • EXP-3 5mg/kg, EXP-3 2mg/kg and Upadacitinib 2mg/kg have similar effects, slightly weaker than tofacitinib 2mg/kg. Filgotinib is the least effective. (Table 11). It shows that the test substance has a certain effect on improving the condition of arthritis of the extremities of rats in the CIA model.

Abstract

本发明公开JAK激酶抑制剂及其用途，所述JAK激酶抑制剂为式(I)所示的化合物或其立体异构体、互变异构体或其药学上可接受的盐或其溶剂合物或者前药，本发明还提供本发明式I的化合物在用于制备预防或治疗JAK激酶相关疾病药物中的用途，特别是在制备用于预防和/或治疗涉及软骨退化、骨和/或关节退化的疾病、涉及炎症或免疫响应的病症、内毒素驱动的疾病状态、癌症和器官移植排斥的药物中的应用。

Description

JAK激酶抑制剂及其用途 技术领域
本发明属于医药技术领域，具体涉及一种新颖的含氮五元杂环并吡啶类化合物、其制备方法及含有其的药物组合物，以及其用于调节Janus激酶(JAK)活性并且用于治疗和/或预防与JAK活性相关的疾病的用途。
背景技术
软骨细胞是无血管组织的主要细胞组分，正常关节软骨中软骨细胞占组织体积的约5％，而细胞外基质占组织剩余的95％。软骨细胞分泌基质组分，主要是蛋白聚糖和胶原，其反过来为软骨细胞在机械压力下提供适合于它们生存的环境。在软骨中，II型胶原与IX型蛋白胶原形成纤维样结构，为软骨提供很大的机械强度。蛋白聚糖可以吸收水并且负责软骨的弹性和减震性质。软骨的功能性作用之一是为骨头部分提供彼此光滑的关节连接。因此，关节软骨的损耗引起骨彼此摩擦，导致疼痛和运动丧失。在炎性关节炎例如类风湿性关节炎中，软骨退化是由发炎组织(例如发炎的滑膜)分泌的蛋白酶(胶原酶)引起的。受损软骨中的软骨细胞常常显示出软骨合成活性的减弱和/或软骨退化活性的增强，软骨变性是发生类风湿性关节炎和骨关节炎的主要疾病标志。类风湿性关节炎(RA)是慢性关节变性疾病，其特征在于关节结构的炎症和破坏，可能导致关节功能性的失常引起实质性的失能和疼痛，甚至过早死亡。因此，RA治疗的目的不仅是延缓疾病，减轻病痛，保护关节破坏。
JAK激酶属于细胞质酪氨酸激酶的Janus激酶(JAK)家族，其参与细胞因子受体介导的细胞内信号转导。JAK激酶家族包括4个成员：JAK1、JAK2、JAK3和TYK2。JAK募集细胞因子受体，结合细胞因子，随后将细胞因子受体和共享的受体亚基进行二聚化。然后JAK通过自磷酸化和/或另一个JAK的转磷酸化而活化，导致受体磷酸化以及信号转导物和转录激活物(STAT)成员的募集和磷酸化。磷酸化的SATA二聚化并且转移至细胞核内，在细胞核内它们结合至细胞因子应答基因的增强子区。JAK在调控多种细胞因子受体家族的生物学应答功能中起着重要的作用。JAK1基因敲除的小鼠具有早期的出生后致死因子显型，神经***也受到损害，导致幼鼠出现先天缺陷。研究表明JAK1基因敲除小鼠会出现胸腺细胞和B细胞的分泌缺陷，JAK1基因敲除的组织对IL-6、IL-10的反应明显减弱。
临床已经确定JAK与自身免疫疾病的关联，约70％的重症联合免疫缺陷病例中检测到JAK以及上游信号传导组分r-c受体链和IL7受体的突变。因此，靶向JAK家族可以在免疫-炎症领域提供新的治疗药物。
临床上JAK家族成员JAK2涉及的病症，包括骨髓增殖性障碍、癌症特别是白血病例如急性髓性白血病、急性淋巴细胞白血病或实体瘤例如子宫平滑肌肉瘤、***癌等相关疾病。因此，靶向JAK家族的药物也可能为上述的疾病的治疗提供新的选择。
发明内容
本发明提供新颖的JAK激酶抑制剂及制备方法、包含有这些激酶抑制剂的药物组合物及其应用。
更具体而言，本发明涉及结构如式(I)所示的化合物或其立体异构体、互变异构体或其药学上可接受的盐或其溶剂合物或者前药：
Figure PCTCN2020107028-appb-000001
其中：
B独立选自取代或未取代的C 3-C 6环烷基、芳基、具有1至2个独立选自N、O和S的环杂原子的5至6元杂芳基环，其中所述的5至6元杂芳环任选地被独立选自卤素、氟代或未氟代的C 1-C 6烷基、氟代或未氟代的C 1-C 6烷氧基、氟代或未氟代的C 1-C 6烷基胺基的一个或多个取代基取代；
D独立选自C,N；
F、G、H、K独立选自C、N、S、O；
L 1不存在或独立选自单键,-CH 2-，-(CH 2) xO(CH 2) y-,-(CH 2) xNH(CH 2) y-,-CH 2O-,-C(O)-,-CON(R 4)-,-CH 2N(R 4)-,-CONH(CH 2) y-,-N(R 4)-,-SO 2N(R 4)-,-S(O) 2-,-N(Me)-；
R 4独立选自H、C 1-C 6烷基、取代的C 1-C 6烷基、C 1-C 3醚C 1-C 3烷基、甲磺酰基；
R 1独立选自H、-NH 2、-COOH、取代或未取代的C 1-C 6烷基、酰基、取代的或未取代的酰基胺基、取代的或未取代的C 1-C 6烷氧基、卤素、羟基、取代的或未取代的C 1-C 3酯基、取代或未取代的杂芳基；
R 2不存在或独立选自H、F、Cl、Me；
R 3独立地选自H、取代或未取代的以下基团：磺酰基、磺酰胺基、5至6元杂环，其具有至少一个杂原子且任选地具有独立选自N和S的第二环杂原子、取代的或未取代的C 1-C 6烷烃,取代基独立选自5至6元杂芳基和甲氧基取代、取代的或未取代的C 3-C 7环烷基、取代的或未取代的4-7元杂环烷基、取代的或未取代的5-7元杂芳基；
m是0、1、2、3；n是0，1、2、3；p是0、1、2；t是0、1、2、3。
在一些实施例中，F、G、H、K不同时为C。
在一种实施方式中，B选自取代或未取代的以下基团：环丙烷、环丁烷、吡唑基、吡啶基、咪唑基；取代基选自F、Cl、Br、甲基、乙基、丙基、异丙基、三氟甲基。
在一种具体的实施例中，B为取代或未取代的环丙烷；取代基选自F、Cl、Br、甲基、乙基、丙基、异丙基、三氟甲基。
在一种实施方式中，D为C或N。
在一种具体的实施例中，D为N。
在一种实施方式中，n为1。
在一种实施方式中，K为S,F、G、H为C。
在一种实施方式中，R 1独立选自H、-NH 2、-COOH、羟基、卤素、取代或未取代的C 1-C 3烷基、取代或未取代的C 1-C 3酰基、取代的或未取代的C 1-C 3酰基胺基、取代的或未取代的C 1-C 3烷氧基、取代的或未取代的C 1-C 3酯基；在一种优选的实施方式中，R 1独立选自H、-NH 2、-COOH、羟基、卤素、甲基、乙基、丙基、异丙基、甲酰基胺基、甲酯基。
在一种实施方式中，m为0或者1，更优选的，m为0。
在一种实施方式中，R 2不存在或为H。
在一种具体的实施例中，R 2不存在。
在一种实施方式中，p为0。
在一种实施方式中，L 1不存在或独立选自单键，-CH 2-，-(CH 2) xO(CH 2) y-,-(CH 2) xNH(CH 2) y-,-CONH(CH 2) y-,-N(R 4)-；R 4独立选自H、甲基、乙基、丙基、乙基甲醚、甲基甲醚、乙基***；x或y独立的选自0、1或2。在一种更优选的实施方式中，L 1不存在或独立选自单键，-CH 2-，-(CH 2) xO(CH 2) y-,-(CH 2) xNH(CH 2) y-,-CONH(CH 2) y-,-N(R 4)-；在一些更为具体的实施例中，L 1选自-CH 2-，-(CH 2) xO(CH 2) y-,-CONH(CH 2) y-,R 4独立选自H、甲基、乙基甲醚；x或y独立的选自0、1或2；在一些具体的实施例中，x或y独立的选自1或2。
在一种实施方式中，t为1。
在一种实施方式中，R 3独立地选自H、取代或未取代的以下基团：磺酰基、磺酰胺基、硫代吗啉1,1-二氧化物、哌啶、吡唑、噻唑、咪唑、1,3,4-噻二唑、哌嗪、吗啉、噻吩、噁唑、1,3,4-噁二唑、呋喃、吡咯、3-吡咯啉、2-吡唑啉、1,2,3-唑、1,2,3-***、1,2,4-***、吡喃；取代基选自H、卤素、C 1-C 3烷基、C 1-C 3卤代烷基、羟基C 1-C 3烷基、C 3-C 6环烷基、乙基甲醚、甲基甲醚、乙基***；在一些实施例中，取代基还可以选自C 1-C 3卤代烷氧基；优选的，R 3独立地选自H、取代或未取代的以下基团：磺酰基、磺酰胺基、硫代吗啉1,1-二氧化物、哌啶、吡唑、噻唑、咪唑、1,3,4-噻二唑、哌嗪、吗啉、吡嗪、噻吩、噁唑、1,3,4-噁二唑、呋喃、吡咯、3-吡咯啉、2-吡唑啉、1,2,3-唑、1,2,3-***、1,2,4-***、吡喃；取代基选自H、卤素、甲基、乙基、丙基、异丙基、三氟甲基、羟乙基、羟甲基、环丙烷、环丁烷、乙基甲醚、甲基甲醚、乙基***；在一些实施例中，取代基还可以选自三氟甲氧基。
本发明还提供以下结构所示的化合物或其立体异构体、互变异构体或其药学上可接受的盐或其溶剂合物或者前药：
Figure PCTCN2020107028-appb-000002
Figure PCTCN2020107028-appb-000003
本发明还提供了合成本发明化合物的方法以及公开的代表性的合成方案和路线。
本发明还提供一种药物组合物，其包含本发明所述的式I的化合物或其药学上可接受的盐，和药学上可接受的稀释剂或载体。
本发明还提供本发明式I的化合物在用于制备预防或治疗JAK激酶相关疾病药物中的用途，特别是在制备用于预防和/或治疗涉及软骨退化、骨和/或关节退化的疾病、涉及炎症或免疫响应的病症、内毒素驱动的疾病状态、癌症和器官移植排斥的药物中的应用。
本发明还提供一种通过施用本发明所述化合物预防和/或治疗涉及软骨退化、骨和/或关节退化的疾病、涉及炎症或免疫响应的病症、内毒素驱动的疾病状态、癌症和器官移植排斥的方法。
在一种实施例中，本发明所述的涉及软骨退化、骨和/或关节退化的疾病、涉及炎症或免疫响应的病症、内毒素驱动的疾病状态、癌症和器官移植排斥的实例包括但不限于：骨关节炎、克隆病、类风湿性关节炎、银屑病、过敏性气道疾病(例如哮喘、鼻炎)、幼年特发性关节炎、结肠炎、炎性肠病、内霉素驱动的疾病状态、软骨更新损伤的疾病(例如软骨细胞的合成代谢兴奋的疾病)、先天软骨畸形、器官移植排斥、涉及软骨退化、关节退化、骨髓增殖性障碍、白血病(急性髓性白血病、急性淋巴细胞白血病)、实体瘤(子宫平滑肌肉瘤、***癌)等。
定义
本申请使用的术语“氨基”是指具有1个氮原子及1至2个氢原子的官能团。“氨基”在本文通常用于描述伯胺、仲胺或叔胺且本领域技术人员能够根据本公开中使用该术语的上下文而容易地确定所述氨基。术语“胺”或“胺基团”或“氨基团”是指含有衍生自氨(NH 3)的氮原子的官能团。胺基团优选为伯胺，其意指氮结合至两个氢原子和一个包含取代或未取代的烷基或芳基或脂族或芳族基团的取代基。胺基团可以是仲胺，其意指氮结合至一个氢原子和两个包含如下文所定义的取代或未取代的烷基或芳基或脂族或芳族基团的取代基。胺基团可以是叔胺，其意指但结合至三个包含取代或未取代的烷基或芳基或脂族或芳族基团的取代基。胺基团也可以是季胺，其意指所指定的胺基团结合至第四个基团，这产生带正电荷的铵基团；
应理解的是，本发明中的任何或所有胺可以为游离胺形式(即对于伯胺而言为-NH 2)或与药学上可接受的阴离子形成的质子化形式(即对于伯胺而言为-NH 3 +Y -，其中Y -为药学上可接受的阴离子)；
如本文所用，术语“酰胺基团”是指包含连接至氮的羰基的官能团。“羰基”是指包含以双键键合至氧原子的碳原子的官能团，其以(C＝O)表示；
术语“烷烃”是指通过单键键合的饱和烃。烷烃可以是直链或支链的。“环烷烃”是通过单键键合的饱和烃环；
如本文所用，术语“C 1-C 6烷基”是指饱和直链或支链或环状烃，其基本上由1至6个碳原子和相应数量的氢原子构成。典型地，直链或支链基团具有1至10个碳，或更典型地1至5个碳。示例性的C 1-C 6烷基包括甲基、乙基、正丙基、异丙基、正丁基、异丁基等。根据本公开的教导，其他C 1-C 6烷基对于本领域技术人员将显而易见的；
如本文所用，术语“C 2-C 9杂烷基”是指饱和直链或支链或环状烃，其基本上由2至10个原子构成，其中2至9个原子为碳且其余原子选自氮、硫和氧。根据本公开的教导，示例性的C 2-C 9杂烷基对于本领域的技术人员而言将是显而易见的；
如本文所用，术语“C 3-C 10环烷基”是指非芳族饱和烃基团，其形成至少一个基本上由3至10个碳原子和相应数量的氢原子构成的环。C 3-C 10环烷基可以是单环的或多环的。除了共价键以外，多环环烷基的各个环可具有不同的连接性，例如稠合、桥连、螺环等；
如本文所用，术语“C 2-C 9杂环烷基”是指具有3至10个原子以形成至少一个环的非芳族基团，其中2至9个环原子为碳且其余原子选自氮、硫和氧。C 2-C 9杂环烷基可以是单环的或多环的。除了共价键取代以外，此类多环杂环烷基的各个环可能具有不同的连接性，例如稠合、桥连、螺环等。根据本公开的教导，示例性的C 2-C 9杂环烷基对于本领域的技术人员而言将是显而易见的；
术语“脂族基团”或“脂族基”是指由碳和氢构成的非芳族基团且可任选包括一个或多个双键和/或叁键。换言之，脂族基团是由碳和氢构成且不含芳族官能性的任何基团。脂族基团可以是直链的、支链的或环状的且典型地含有1至24个碳原子；
术语“芳基基团”可与“芳基”、“芳基环”、“芳族基”、“芳族基团”和“芳族环”互换使用。芳基包括碳环芳族基团，其典型地具有6至14个环碳原子。芳基也包括杂芳基，其典型地具有5至14个环原子，其中一个或多个杂原子选自氮、氧和硫；
如本文所用，术语“C 6-C 14芳基”是指具有6至14个碳原子以形成至少一个环的芳族官能团；
如本文所用，术语“C 2-C 9杂芳基”是指具有5至10个原子以形成至少一个环的芳族官能团，其中2至9个环原子为碳且其余环原子选自氮、硫和氧。C 2-C 9杂芳基可以是单环的或多环的。除了共价键取代以外，此类多环杂芳基的各个环可具有不同的连接性，例如稠合、桥连、螺环等。C 2-C 9杂芳基典型地经碳原子而连接至主结构，然而本领域技术人员将了解某些其他原子例如杂环原子何时连接至主结构。根据本公开的教导，其他C 2-C 9杂芳基对于本领域技术人员而言将是显而易见的；
如本文所用，术语“烷基胺”是指含伯、仲或叔胺基团以代替一个氢原子的C 1-C 6烷基，其以C 1-C 6烷基胺和(C 1-C 6烷基) 2胺表示；
术语“烷基酯”是指含有酯基团以代替一个氢原子的C 1-C 6烷基，其以-O(O)C(C 1-C 6烷基)表示；
术语“烷基酸”是指含有羧酸基团以代替一个氢原子的C 1-C 6烷基，其以C 1-C 6烷基-COOH表示；
术语“脂肪酸”是指非芳族烃的酸，其以C 1-C 6烷基-COOH和C 3-C 6烷基-COOH表 示；
术语“卤素”是指氟(F)、氯(Cl)、溴(Br)、碘(I)或砹(At)离子；
术语“甲氧基”是指含有氧以代替一个氢原子的(C 1)烷基，其以-(O)CH 3表示；
术语“多元醇”是指含有多个羟基的醇；
“取代”是指烷基、杂环基或芳基的碳经一个或多个非碳取代基取代。非碳取代基选自氮、氧和硫；
“未取代”是指基团仅包含氢和碳。
“酯基”表示-C(O)O-R’基团，其中R’定义同上，但是R’不能是氢。
“烷基磺酰基”代表式-SO 2R所示的基团，R表示烷基。
“磺酰基”表示-S(O 2)-；
“磺酰胺基”表示-S(O 2)NH-；
“1,3,4-噻二唑”表示
Figure PCTCN2020107028-appb-000004
“1,3,4-噁二唑”表示
Figure PCTCN2020107028-appb-000005
“3-吡咯啉”表示
Figure PCTCN2020107028-appb-000006
“2-吡唑啉”表示
Figure PCTCN2020107028-appb-000007
“1,2,3-唑”表示
Figure PCTCN2020107028-appb-000008
“1,2,3-***”表示
Figure PCTCN2020107028-appb-000009
“硫代吗啉1,1-二氧化物”表示
Figure PCTCN2020107028-appb-000010
当其作为取代基团时，通过N与化合物母体结构连接，即为
Figure PCTCN2020107028-appb-000011
具体实施方式
以下结合实施例进一步描述本发明，但这些实施例并非限制着本发明的范围，凡在本发明的构思前提下对本发明制备方法的简单改进都属于本发明的保护范围之内。下面实施例未注明具体条件的实验方法，通常按照本领域的公知手段。下述实施例中所用的试验材料，如无特殊说明，均为自常规生化试剂商店购买得到的。
实施例1：化合物EXP-1的制备
合成路线如下：
Figure PCTCN2020107028-appb-000012
步骤1.1化合物1的制备
Figure PCTCN2020107028-appb-000013
5-溴-[1,2,4]***并[1,5-a]吡啶-2-胺(SM1)(2g，9.4mmol)和三乙胺(2.4g，23.5mmol) 溶于乙腈(10mL)中，在0℃下滴加环丙基甲酰氯(SM2)(2.45g，23.5mmol)，自然升到室温搅拌16小时。反应液真空浓缩得到双酰基化中间体N-(5-溴-[1,2,4]***并[1,5-a]吡啶-2-基)-N-(环丙烷羰基)环丙烷甲酰胺(1)(3.3g粗品，黄色固体)，无需进步纯化可直接用于下一步。
LCMS:t R＝0.785min in 5-95AB_1.5min_220&254_Shimadzu.lcm chromatography(Agilent Pursult 5 C18 20*2.0mm),MS(ESI)m/z＝350.9[M+2+H] +.
步骤1.2化合物Int-1的制备
Figure PCTCN2020107028-appb-000014
N-(5-溴-[1,2,4]***并[1,5-a]吡啶-2-基)-N-(环丙烷羰基)环丙烷甲酰胺(1)(3.3g粗品，9.4mmol)溶于甲醇(30mL)溶液中，加入碳酸钾(3.9g，28.2mmol)，室温搅拌1小时。反应液室温下真空浓缩，得到的粗品用乙酸乙酯(250mL)溶解分散后过滤，滤饼用二氯甲烷(250mL)和四氢呋喃(250mL)依次洗涤，收集滤液真空浓缩，用石油醚/乙酸乙酯(22mL,10:1v/v)进步打浆后得到产物N-(5-溴-[1,2,4]***并[1,5-a]吡啶-2-基)环丙烷甲酰胺(Int-1)(2.14g，收率80％，纯度99％)，黄色固体。
LCMS:t R＝0.699min in 5-95AB_1.5min_220&254_Shimadzu.lcm chromatography(Merk RP18e 25-3mm),MS(ESI)m/z＝282.9[M+H+2] +.
1H NMR(400MHz,DMSO-d 6):δ＝11.21(br s,1H),7.71(dd,J＝8.4,0.8Hz,1H),7.56(t,J＝7.2Hz,1H),7.50(dd,J＝7.6,0.6Hz,1H),2.04(br s,1H),0.85-0.82(m,4H).
步骤1.3化合物2的制备
Figure PCTCN2020107028-appb-000015
N-(5-溴-[1,2,4]***并[1,5-a]吡啶-2-基)环丙烷甲酰胺(Int-1)(300mg,1.1mmol)，(5-甲酰基-2-噻吩基)硼酸(SM3)(332.9mg,2.1mmol)和碳酸钾(442.5mg,3.2mmol)溶于二氧六环(5mL)和水(1mL)中，置换氮气三次后快速加入1,1-双(二苯基磷)二茂铁氯化钯(39.0mg,53.4umol)，再次置换氮气三次，在氮气保护下90℃搅拌30分钟。反应液用水(20mL)和二氯甲烷稀释萃取(20mL×3)，有机相用饱和食盐水(20mL)洗涤，无水硫酸钠干燥后过滤，滤液真空浓缩得到粗品，硅胶柱层析纯化(洗脱剂：1％～2％甲醇/二氯甲烷)得到产品N-[5-(5-甲酰基-2-噻吩基)-[1,2,4]***并[1,5-a]吡啶-2-基]环丙烷甲酰胺(2)(350mg半纯品，含有部分原料1，折算收率49.4％)，黄色固体。
LCMS:t R＝0.783min in 5-95AB_1.5min_220&254_Shimadzu.lcm chromatography(Agilent Pursult 5 C18 20*2.0mm),MS(ESI)m/z＝312.8[M+H] +.
步骤1.4化合物EXP-1的制备
Figure PCTCN2020107028-appb-000016
N-[5-(5-甲酰基-2-噻吩基)-[1,2,4]***并[1,5-a]吡啶-2-基]环丙烷甲酰胺(2)(220mg半纯品,331.0umol)和1,1-二氧硫代吗啉(SM4)(53.7mg,397.3umol)溶于甲醇(6mL)中，滴加醋酸调节pH值到6左右，50℃搅拌1小时，冷却到室温后加入氰基硼氢化钠(41.6mg,662.1umol)，30℃继续搅拌15小时。反应液用饱和碳酸氢钠水溶液(3mL)处理，加水(10mL)稀释并用二氯甲烷萃取(20mL×3)，有机相合并后用饱和食盐水(20mL)洗涤，无水硫酸钠干燥后过滤，滤液真空浓缩得到粗品，碱性制备分离纯化得到目标化合物EXP-1(28.9mg，收率20％),白色固体。
LCMS:t R＝1.893min in 10-80AB_7min_220&254_Shimadzu.lcm chromatography(Xtimate C18 2.1*30mm),MS(ESI)m/z＝432.0[M+H] +.
HPLC:t R＝2.50min in 10-80AB_8min_Shimadzu.lcm chromatography(Ultimate 3.0 *50mm 3um).
1H NMR(400MHz,CDCl 3):δ＝8.77(s,1H),8.12(d,J＝3.6Hz,1H),7.61-7.51(m,2H),7.37(dd,J＝6.4,2.8Hz,1H),7.07(d,J＝3.6Hz,1H),3.96(s,2H),3.18-3.06(m,8H),1.66-1.58(m,1H),1.30-1.20(m,2H),1.03-0.93(m,2H).
依据与上述实施例相同的方法，使用市售化合物或参考所示的中间化合物的制备方法而制备以下表1的实施例化合物。
【表1】
Figure PCTCN2020107028-appb-000017
实施例2：化合物EXP-3的制备
合成路线如下：
Figure PCTCN2020107028-appb-000018
步骤2.1化合物22的制备
Figure PCTCN2020107028-appb-000019
将6-溴吡啶-2-胺(SM16)(5g,28.90mmol)和溴代丙酮酸乙酯(SM17)(6.20g,31.79mmol)溶于乙醇(30mL)，于85度下搅拌16小时。反应液过滤，滤饼收集，用石油醚/乙酸乙酯(5/1,v/v,50mL)打浆，固体过滤，干燥得到5-溴咪唑[1,2-a]吡啶-2-羧酸乙酯(22)(8.41g,83％yield,溴化氢盐，黄色固体)。
1H NMR(400MHz,DMSO-d 6):δ＝8.56(s,1H),7.75(d,J＝8.8Hz,1H),7.52-7.44(m,2H),4.36(q,J＝7.2Hz,2H),1.34(t,J＝7.2Hz,3H).
步骤2.2化合物23的制备
Figure PCTCN2020107028-appb-000020
将5-溴咪唑[1,2-a]吡啶-2-羧酸乙酯(22)(2g,5.71mmol,溴化氢盐)溶于水，四氢呋喃和甲醇各5毫升溶剂中，加入一水合氢氧化锂(839mg,20.00mmol)。反应液于15度搅拌2小时。反应液加水(10mL)，用二氯甲烷(20mL)。水相用稀盐酸(1N)调至pH＝5，过滤，滤饼干燥得到5-溴咪唑[1,2-a]吡啶-2-甲酸(23)(1.24g,90％收率，白色固体)用于下一步。
LCMS:t R＝0.292min in 5-95AB_1.5min_220&254_Shimadzu.lcm,chromatography(Chromolith Flash RP-18,5um,3.0*25mm),MS(ESI)m/z＝241.1[M+H] +.
1H NMR(400MHz,DMSO-d 6):δ＝8.37(s,1H),7.72(d,J＝8.8Hz,1H),7.43(dd,J＝8.8,1.2Hz,1H),7.39-7.28(m,1H).
步骤2.3化合物24的制备
Figure PCTCN2020107028-appb-000021
将5-溴咪唑[1,2-a]吡啶-2-甲酸(23)(1.24g,5.14mmol)，4A分子筛(2.48g,10.29mmol)和三乙胺(1.56g,15.43mmol)悬浮于叔丁醇(12mL)中，加入叠氮磷酸二苯酯(2.12g,7.72mmol)。反应液于氮气氛围下在90度搅拌16小时。反应液过滤，滤液真空浓缩，粗品通过硅胶色谱纯化(甲醇/二氯甲烷，从0％到2％)得到5-溴咪唑[1,2-a]吡啶-2-胺基甲酸叔丁酯(24)(1.01g,49％收率,78％纯度，黄色固体)。
LCMS:t R＝1.443min in 10-80AB_2min_220&254_Shimadzu.lcm,chromatography(Xtimate C18 2.1*3mm,3um),MS(ESI)m/z＝312.1[M+H] +.
步骤2.4化合物25的制备
Figure PCTCN2020107028-appb-000022
将5-溴咪唑[1,2-a]吡啶-2-胺基甲酸叔丁酯(24)(1.01g,3.24mmol)溶于二氯甲烷(20mL)中加入三氟乙酸(12.32g,108.05mmol)，反应液于15度搅拌2小时。反应液浓缩，用饱和碳酸氢钠调节pH＝9，然后用二氯甲烷(30mL×3)萃取。有机相合并，用食盐水(20mL)洗涤，无水硫酸钠干燥，过滤，浓缩得到粗品，通过硅胶色谱柱纯化(甲醇/二氯甲烷，从0％到5％)得到5-溴咪唑[1,2-a]吡啶-2-胺(25)(400mg,58％收率，黄色固体)。
1H NMR(400MHz,DMSO-d 6):δ＝7.24(d,J＝8.0Hz,1H),7.06(d,J＝7.2Hz,1H),7.02-6.97(m,2H),5.30(brs,2H).
步骤2.5化合物26的制备
Figure PCTCN2020107028-appb-000023
将反式-2-氟环丙基甲酸(SM18)(491mg,4.72mmol)溶于二氯甲烷(1mL)中，零度下加入草酰氯(539mg,4.24mmol)和一滴二甲基甲酰胺，反应液于零度下搅拌1小时，然后零度下加入5-溴咪唑[1,2-a]吡啶-2-胺(25)(500mg,2.36mmol)的二甲基乙酰胺(4mL)溶液，室温下搅拌2小时。反应液(和批次EB4-106合并，投料100mg的底物1)加入饱和碳酸氢钠溶液(40mL)淬灭，用乙酸乙酯(60mL×3)萃取。有机相合并，食盐水(40mL)洗涤，无水硫酸钠干燥，过滤，真空浓缩，通过硅胶色谱柱(甲醇/二氯甲烷，从0％到2％)纯化得到反式-N-(5-溴咪唑[1,2-a]吡啶-2-基)-2-氟-环丙基甲酰胺(26)(587mg,69％收率，黄色固体)。
1H NMR(400MHz,DMSO-d 6):δ＝11.31(s,1H),8.02(s,1H),7.53(d,J＝8.8Hz,1H),7.32-7.17(m,2H),5.07-4.75(m,1H),2.47-2.41(m,1H),1.61-1.47(m,1H),1.30-1.20(m,1H).
步骤2.6化合物27的制备
Figure PCTCN2020107028-appb-000024
将(5-甲酰基-2-噻吩)硼酸(SM3)(419mg,2.68mmol)，反式-N-(5-溴咪唑[1,2-a]吡啶-2-基)-2-氟-环丙基甲酰胺(26)(200mg,0.671mmol)和碳酸钾(278mg,2.01mmol)溶于二氧六环(4mL)和水(0.5mL)中，氮气置换3次，然后加入1,1-双(二苯基磷)二茂铁氯化钯(49mg,0.0671mmol)，再次氮气置换3次，反应液100度下搅拌1小时。反应液浓缩，通过硅胶色谱柱(甲醇/二氯甲烷，从0％到2％)纯化得到反式-2-氟-N-[5-(5-甲酰基-2-噻吩)咪唑[1,2-a]吡啶-2-基]环丙基甲酰胺(27)(90mg,41％收率,100％纯度，黄色 固体)。
LCMS:t R＝0.832min in 5-95AB_1.5min_220&254_Shimadzu.lcm,chromatography(Xtimate C18 2.1*30mm,3um),MS(ESI)m/z＝330.1[M+H] +.
步骤2.7化合物EXP-3的制备
Figure PCTCN2020107028-appb-000025
将反式-2-氟-N-[5-(5-甲酰基-2-噻吩)咪唑[1,2-a]吡啶-2-基]环丙基甲酰胺(27)(90mg,0.273mmol)和硫代吗啉-1,1-二氧化物(SM4)(55mg,0.410mmol)溶于甲醇(4mL)，二氯甲烷(2mL)和四氢呋喃(2mL)中，加入醋酸(21mg,0.355mmol)，室温下搅拌2小时，加入氰基硼氢化钠(69mg,1.09mmol)，反应液室温下搅拌16小时。反应液加入饱和碳酸氢钠(30mL)淬灭，室温下搅拌半小时，加入二氯甲烷(50mL×3)萃取。有机相合并，食盐水(30mL)洗涤，无水硫酸钠干燥，过滤，真空浓缩，粗品通过制备HPLC(Column:Waters Xbridge 150*25mm*5um；Condition:water(0.04％NH 3.H 2O+10mM NH 4HCO 3)-ACN；Begin B,25％；End B,55％；Gradient Time:(8min)；Flow Rate:(25mL/min))纯化得到反式-N-[5-[5-[(1,1-二氧代-1,4-噻嗪-4-基)甲基]-2-噻吩]咪唑[1,2-a]吡啶-2-基]-2-氟-环丙基甲酰胺(EXP-3)(22.4mg,17％收率,96.17％纯度，黄色固体)。
LCMS:t R＝1.765min in 10-80AB_4min_220&254_Shimadzu.lcm,chromatography(Xtimate C18 2.1*3mm,3um),MS(ESI)m/z＝449.2[M+H] +.
HPLC:t R＝2.36min in10-80AB_8min_215&220&254.met.
1H NMR(400MHz,DMSO-d 6):δ＝11.25(s,1H),8.30(s,1H),7.56(d,J＝3.6Hz,1H),7.49(d,J＝9.2Hz,1H),7.32(dd,J＝9.2,7.2Hz,1H),7.20(d,J＝3.6Hz,1H),7.07(d,J＝7.2Hz,1H), 5.03-4.70(m,1H),4.01(s,2H),3.17-3.10(m,4H),3.03-2.93(m,4H),2.48-2.40(m,1H),1.62-1.45(m,1H),1.28-1.16(m,1H).
实施例3：化合物EXP-4的制备
合成路线如下：
Figure PCTCN2020107028-appb-000026
步骤3.1化合物3的制备
Figure PCTCN2020107028-appb-000027
将2-甲基噻唑-5-硼酸嚬哪醇酯(SM5)(100mg,0.444mmol)溶于四氯化碳中(2mL)，加入N-溴代丁二酰亚胺(103mg,0.577mmol)和偶氮二异丁腈(7.3mg,0.0444mmol)。反应液在氮气保护下于80℃搅拌0.5小时。反应液过滤，滤液真空浓缩得粗品，然后溶于四氢呋喃(2mL)中，依次加入二异丙基乙胺(0.1mL)和亚磷酸二乙酯(74uL),在10℃搅拌25分钟得到2-溴甲基噻唑-5-硼酸嚬哪醇酯(3)(135mg,粗品，溶于2mL四氢呋喃的棕色溶液)，直接用于下一步。
LCMS:t R＝0.590min in 5-95AB_1.5min_220&254_Shimadzu.lcm chromatography(Merk RP18e 25-3mm),MS(ESI)m/z＝221.7[M-81] +.
步骤3.2化合物4的制备
Figure PCTCN2020107028-appb-000028
将2-溴甲基噻唑-5-硼酸嚬哪醇酯(3)(135mg,0.444mmol)和碳酸钾(246mg,1. 78mmol)悬浮在四氢呋喃(2mL)中，加入硫代吗啉-1，1-二氧化物(SM4)(72mg,0.533mmol)。反应液氮气保护下在10℃反应24小时。反应液真空浓缩得到4-[[5-(4,4,5,5-四甲基-1,3,2-二氧硼烷-2-)噻唑-2-]甲基]-1,4-噻嗪1,1-二氧化物(4)(159mg，粗品，红色固体)，直接用于下一步。
LCMS:t R＝0.287min in 5-95AB_1.5min_220&254_Shimadzu.lcm chromatography(Merk RP18e 25-3mm),MS(ESI)m/z＝276.8[M-81] +.
步骤3.3化合物EXP-4的制备
Figure PCTCN2020107028-appb-000029
将N-(5-溴-[1,2,4]***[1,5-a]吡啶-2-基)环丙烷甲酰胺(Int-1)(20mg,0.071mmol)，4-[[5-(4,4,5,5-四甲基-1,3,2-二氧硼烷-2-)噻唑-2-]甲基]-1,4-噻嗪1,1-二氧化物(4)(76.5mg,0.213mmol)，碳酸钾(29.5mg,0.213mmol)悬浮在二氧六环(1mL)和水(0.2mL)中，反应液氮气置换三次，然后快速加入1,1-二(叔丁基磷)二茂铁氯化钯(9.3mg,0.014mmol)，反应液再次氮气置换三次，在85℃下搅拌半个小时。反应液真空浓缩，快速过柱子机纯化(洗脱剂：0％～0.5％甲醇/二氯甲烷)得到粗品，碱性机分得到目标分子N-[5-[2-[(1,1-二氧-1,4-噻嗪-4-)甲基]噻唑-5-]-[1,2,4]***[1,5-a]吡啶-2-]环丙基甲酰胺(EXP-4)(5.6mg,6％收率,93％纯度，黄色固体)。
LCMS:t R＝1.793min in 10-80AB_7min_220&254_Shimadzu.lcm,chromatography(Xtimate C18,2.1*30mm),MS(ESI)m/z＝433.0[M+H] +.
HPLC:t R＝2.54min in 10-80AB_8min.met(Ultimate C18 3*50mm 3um).
1H NMR(400MHz,DMSO-d 6):δ＝11.22(br s,1H),8.93(s,1H),7.81-7.63(m,3H),4.16(s,2H),3.23-3.04(m,8H),2.13-2.06(m,1H),0.95-0.81(m,4H).
实施例4：化合物EXP-5的制备
合成路线如下：
Figure PCTCN2020107028-appb-000030
步骤4.1化合物5的制备
Figure PCTCN2020107028-appb-000031
氮气保护下，顺式2-氟环丙羧酸(SM6)(50mg,0.48mmol)溶于SOCl 2(1mL)中，在75℃下搅拌1小时。低于30℃减压浓缩，得到化合物顺式-2-氟环丙烷酰氯(5)(58mg,98％yield)，黄色油状物直接用于下一步。
步骤4.2化合物Int-2的制备
Figure PCTCN2020107028-appb-000032
氮气保护下，5-溴-[1,2,4]***[1,5-a]吡啶-2-胺(SM1)(100mg,0.469mmol)和顺式-2-氟环丙烷酰氯(5)(58mg,0.469mmol)溶于吡啶(1mL)，加热至60℃，搅拌1小时。浓缩得粗品，溶于二氯甲烷(3mL)和(1mL)甲醇中。过滤除去不溶性固体,二氯甲烷(1mL×2)洗涤，滤液浓缩得粗品N-[5-溴-[1,2,4]***[1,5-a]吡啶-2-基]-顺式-2-氟环丙烷甲酰胺(I nt-2)(130mg,粗品)黄色固体，直接用于下一步。
LCMS:t R＝0.609min in 5-95AB_1.5min_220&254_Shimadzu.lcm,chromatography(Agilent Pursult 5 C18 20*2.0mm),MS(ESI)m/z＝300.6[M+H] +.
1H NMR(400MHz,CDCl 3):δ＝8.01(s,1H),7.65(d,J＝8.8Hz,1H),7.40(d,J＝8.4Hz,1H),7.25-7.24(m,1H),4.95-4.76(m,1H),2.08-1.94(m,1H),1.89-1.77(m,1H),1.26-1.21(m,1H).
步骤4.3化合物6的制备
Figure PCTCN2020107028-appb-000033
N-[5-溴-[1,2,4]***[1,5-a]吡啶-2-基]-顺式-2-氟环丙烷甲酰胺(Int-2)(100mg,0.334mmol)和(5-甲酰-2-噻吩)硼酸(SM3)(261mg,1.67mmol)，和碳酸钠(106mg,1.00mmol)溶于四氢呋喃(2mL)和水(1mL)中，氮气置换三次后加入1,1-双(二苯基磷)二茂铁氯化钯(47mg,0.067mmol)，氮气置换三次后升到100℃搅拌0.5小时。反应液加水(20mL)搅拌，乙酸乙酯(40mL)萃取，有机相用饱和食盐水(10mL)洗涤，无水硫酸钠干燥后过滤，滤液真空浓缩得到粗品黄色固体N-[5-(2-甲酰基)-噻吩-[1,2,4]***[1,5-a]吡啶-2-基]-顺式-2-氟-环丙烷甲酰胺(6)(100mg,粗品)直接用于下一步。
步骤4.4化合物EXP-5的制备
Figure PCTCN2020107028-appb-000034
N-[5-(2-甲酰基)-噻吩-[1,2,4]***[1,5-a]吡啶-2-基]-顺式-2-氟-环丙烷甲酰胺(6)(100mg,0.302mmol)和1,1-二氧硫代吗啉(SM4)(82mg,0.605mmol)溶于甲醇(5mL)中， 加入醋酸(9mg,0.151mmol)，然后加热到80℃反应0.2小时，降至室温加入氰基硼氢化钠(152mg,2.42mmol)，再加热至80℃反应0.5小时，降温，加入饱和碳酸氢钠(10mL)，室温搅拌0.5小时，浓缩掉大部分甲醇然后乙酸乙酯(20mL×2)萃取，浓缩，溶于甲醇(5mL)中，制备分离纯化得到标题产品N-[5-[4-[(1,1-二氧硫代吗啉)甲基]噻吩基]-[1,2,4]***[1,5-a]吡啶-2-基]-顺式-2-氟-环丙烷甲酰胺(EXP-5)(10.3mg,8％yield)，白色固体。
LCMS:t R＝1.545min in 10-80AB_4min_220&254_Shimadzu.lcm,chromatography(Xtimate C18 2.1*30mm),MS(ESI)m/z＝450.0[M+H] +.
HPLC:t R＝2.43min in 10-80AB_8min_met,chromatography(Ultimate 3.0*50mm*3um).
1H NMR(400MHz,DMSO-d 6):δ＝11.23(brs,1H),8.26(brs,1H),7.77-7.56(m,3H),7.20(brs,1H),5.07-4.91(m,1H),4.00(brs,2H),3.14-2.98(m,8H),2.34-2.31(m,1H),1.73-1.68(m,1H),1.22-1.20(m,1H).
依据与上述实施例相同的方法，使用市售化合物或参考所示的中间化合物的制备方法而制备以下表2的实施例化合物。
【表2】
Figure PCTCN2020107028-appb-000035
Figure PCTCN2020107028-appb-000036
Figure PCTCN2020107028-appb-000037
实施例5：化合物EXP-12的制备
合成路线如下：
Figure PCTCN2020107028-appb-000038
步骤5.1化合物7的制备
Figure PCTCN2020107028-appb-000039
将反式-N-(5-溴-[1,2,4]***[1,5-a]吡啶-2-基)-2-氟-环丙烷甲酰胺(Int-3)(100mg，0.33mmol)，(5-甲酰-2-噻吩基)硼酸(SM3)(111mg，0.71mmol)，碳酸钾(98mg，0.71mmol)，双(三苯基磷)二氯化钯(50mg，0.07mmol)溶于水(0.5mL)和四氢呋喃(1mL)中，氮气置换3次，100℃搅拌1小时。过滤，滤饼干燥，得到粗产物，柱分离(二氯甲烷:甲醇＝100∶1～20∶1)纯化，得到反式-N-(5-(5-甲酰基-2-噻吩基)-[1,2，4]***并[1,5 -a]吡啶-2-基]-2-氟-环丙烷酰胺(7)(50毫克，粗品，黄色固体)。
LCMS:t R＝2.054min in 10-80AB_4.0min_220&254_Shimadzu.lcm,chromatography(Xtimate C18,3um,2.1*30mm),MS(ESI)m/z＝301.0[M+H] +.
步骤5.2化合物EXP-12的制备
Figure PCTCN2020107028-appb-000040
反式-N-(5-(5-甲酰基-2-噻吩基)-[1,2，4]***并[1,5-a]吡啶-2-基]-2-氟-环丙烷酰胺(7)(25mg，0.08mmol)和4,4-二氟哌啶(SM7)(18mg，0.15mmol)溶于甲醇(3ml)中，然后加入醋酸(2mg，0.04mmol)，50℃搅拌0.5小时，然后加入氰基硼氢化钠(38mg，0.61mmol)，50℃再搅拌0.5小时。加入饱和碳酸氢钠(10mL)，过滤，滤液浓缩，得到粗品，制备薄层色谱分离(二氯甲烷:甲醇＝10:1)纯化，得到反式-N-[5-[5-[(4,4-二氟-1-哌啶基)甲基]-2-噻吩基]-[1,2,4]***[1,5-a]吡啶-2-基]-2-氟环丙烷甲酰胺(EXP-12)(5.0mg，13％收率，白色固体)。
LCMS:t R＝0.985min in 5-95AB_1.5min_220&254_Shimadzu.lcm,chromatography(Xtimate C18 2.1*30mm),MS(ESI)m/z＝436.1[M+H] +.
HPLC:t R＝2.15min in 10-80AB_8min_met,chromatography(XBridge Shield RP18 5um).
1H NMR(400MHz,CD 3OD)δ＝8.27(d,J＝4.0Hz,1H),7.80-7.74(m,2H),7.67-7.64(m,1H),7.47(d,J＝3.6Hz,1H),5.01-4.88(m,1H),4.69(s,2H),3.49(brs,4H),2.62-2.42(m,1H),2.38(brs,4H),1.67-1.56(m,1H),1.48-1.44(m,1H).
依据与上述实施例相同的方法，使用市售化合物或参考所示的中间化合物的制备方法而制备以下表3的实施例化合物。
【表3】
Figure PCTCN2020107028-appb-000041
实施例6：化合物EXP-14的制备
合成路线如下：
Figure PCTCN2020107028-appb-000042
步骤6.1化合物8的制备
Figure PCTCN2020107028-appb-000043
将6-氨基吡啶-2-甲酸甲酯(SM8)(3g,19.72mmol)溶在二氧六环(30mL)中加入乙氧羰基异硫氰酸酯(SM9)(2.84g,21.69mmol)。10度下搅拌16小时。反应液浓缩，用乙酸乙酯/石油醚(1/15,30mL)打浆得到6-(3-(乙氧)硫脲)吡啶甲酸甲酯(8)(5.25g,94％收率,100％纯度，淡黄色固体)。
LCMS:t R＝0.840min in 5-95AB_220&254_Agilent,chromatography(MERCK RP1 8e 25*3.0mm),MS(ESI)m/z＝284.1[M+H] +.
步骤6.2化合物9的制备
Figure PCTCN2020107028-appb-000044
将6-(3-(乙氧)硫脲)吡啶甲酸甲酯(8)(5.25g,18.53mmol)溶于甲醇(75mL)加入盐酸羟按(1.42g,20.38mmol)和二异丁基乙胺(3.11g,24.09mmol)，反应液60度搅拌16小时。反应液冷却，过滤，固体收集干燥得到2.5g白色固体产物，滤液浓缩得到粗品然后过柱子纯化(甲醇/二氯甲烷，0％到2％)得到1.5g的白色固体。两批产物合并得到2-氨基-[1,2,4]***[1,5-a]吡啶-5-甲酸甲酯(9)(4g,crude，白色固体)。
1H NMR(400MHz,DMSO-d 6):δ＝7.62-7.58(m,1H),7.53-7.47(m,1H),7.41(dd,J＝7.6,1.2Hz,1H),6.26(s,2H),3.92(s,3H).
步骤6.3化合物10的制备
Figure PCTCN2020107028-appb-000045
将2-氨基-[1,2,4]***[1,5-a]吡啶-5-甲酸甲酯(9)(3.56g,18.52mmol)溶在二甲基乙酰胺(20mL)然后0度下加入环丙基酰氯(SM2)(3.87g,37.05mmol)。20度下搅拌3.5小时。反应液用饱和碳酸氢钠调节至pH＝8。过滤，固体水洗(10mL×3)干燥得到2-(环丙甲酰氨基)-[1,2,4]***[1,5-a]吡啶-5-甲酸甲酯(10)(2.11g,44％收率,100％纯度，白色固体)。
LCMS:t R＝0.573min in 5-95AB_1.5min_220&254_Agilent,chromatography(Merck RP-18e 25-3mm),MS(ESI)m/z＝261.1[M+H] +.
步骤6.4化合物11的制备
Figure PCTCN2020107028-appb-000046
将2-(环丙甲酰氨基)-[1,2,4]***[1,5-a]吡啶-5-甲酸甲酯(10)(2.11g,8.11mmol)溶于乙醇(40mL)加入水合肼(2.48g,48.65mmol,98％purity)。13度下搅拌2小时。反应液过滤，固体用乙醇(10mL)洗涤，干燥得到N-[5-(肼基酰基)-[1,2,4]***[1,5-a]吡啶-2-]环丙甲酰胺(11)(2g,95％收率,100％纯度，白色固体)。
1H NMR(400MHz,DMSO-d 6):δ＝11.45(br s,1H),11.02(br s,1H),7.93-7.86(m,1H),7.85-7.76(m,2H),4.96(J＝4.4Hz,2H),2.02(br s,1H),0.94-0.81(m,4H).
步骤6.5化合物12的制备
Figure PCTCN2020107028-appb-000047
将2-(1,1-二氧-1,4-噻嗪-4-)乙酸(SM10)(1.63g,8.45mmol)溶于二甲基甲酰胺(20mL)中，依次加入O-(7-氮杂苯并三氮唑-1-)-N,N,N,N-四甲基脲六氟膦盐(3.51g,9.22mmol)，N-[5-(肼基酰基)-[1,2,4]***[1,5-a]吡啶-2-]环丙甲酰胺(11)(2g,7.68mmol)，最后加入二异丙基乙胺(1.99g,15.37mmol)。20度下搅拌3.5小时。反应液加入水(100mL)中，过滤，固体水洗(20mL)，乙醇(40mL)洗，干燥得到N-[5-[[[2-(1,1-二氧-1,4-噻嗪-4-)乙酰基]胺基]氨甲酰基]-[1,2,4]***[1,5-a]吡啶-2-]环丙基甲酰胺(12)(3.26g,89％收率,91％纯度，白色固体)。
LCMS:t R＝0.546min in 5-95AB_1.5min_220&254_Shimadzu.lcm,chromatography(Merck RP-18e 25-3mm),MS(ESI)m/z＝436.1[M+H] +.
步骤6.6化合物EXP-14的制备
Figure PCTCN2020107028-appb-000048
将N-[5-[[[2-(1,1-二氧-1,4-噻嗪-4-)乙酰基]胺基]氨甲酰基]-[1,2,4]***[1,5-a]吡啶-2-]环丙基甲酰胺(12)(300mg,0.689mmol)悬浮溶于吡啶(12mL)加入五硫化二磷二吡啶盐(524mg,1.38mmol)。反应液100度搅拌16小时。反应液过滤，滤液浓缩，制备HPLC分离纯化(basic condition:column:YMC-Triart Prep C18 250*50mm*10um；mobilephase:[water(0.04％NH 3H 2O+10mM NH 4HCO 3)-ACN]；B％:15％-45％,8min)，得到N-[5-[5-[(1,1-二氧-1,4-噻嗪-4-)甲基]-1,3,4-噻二唑-2-]-[1,2,4]***[1,5-a]吡啶-2-]环丙基甲酰胺(EXP-14)(30.4mg,10％收率,97.03％纯度，白色固体)。
LCMS:t R＝1.598min in 10-80AB_7min_220&254_Shimadzu.lcm,chromatography(Xtimate C18 2.1*30mm,3um),MS(ESI)m/z＝434.1[M+H] +.
HPLC:t R＝2.55min in 10-80AB_8min.met,chromatography(Ultimate C18 3um,3.0*50mm).
1H NMR(400MHz,DMSO-d 6):δ＝11.35(br s,1H),8.22(dd,J＝7.2,0.8Hz,1H),7.96-7.91(m,1H),7.89-7.83(m,1H),4.35(s,2H),3.21-3.13(m,4H),3.13-3.05(m,4H),2.08(br s,1H),0.95-0.84(m,4H).
实施例7：化合物EXP-15的制备
合成路线如下：
Figure PCTCN2020107028-appb-000049
步骤7.1化合物EXP-15的制备
Figure PCTCN2020107028-appb-000050
将N-[5-[5-[(1,1-二氧-1,4-噻嗪-4-)甲基]-1,3,4-噻二唑-2-]-[1,2,4]***[1,5-a]吡啶-2-]环丙基甲酰胺(12)(180mg,0.413mmol)溶于二氯亚砜(3mL)和二甲基甲酰胺(0.3mL)于20度搅拌4小时。反应液浓缩，然后加入饱和碳酸氢钠调节pH＝8，再用二氯甲烷(40mL×3)。有机相合并，食盐水(10mL),硫酸钠干燥，过滤，浓缩，制备HPLC分离纯化(HCl condition:column:ACE 5 C18-AR 150*30mm*5μm；mobile phase:[water(0.05％HCl)-ACN]；B％:10％-40％,8min)，得到N-[5-[5-[(1,1-二氧代-1,4-噻嗪-4-)甲基]-1,3,4-恶二唑-2-]-[1,2,4]***[1,5-a]吡啶-2-]环丙基甲酰胺(EXP-15)(27.1mg,14.40％收率,99.73％纯度,盐酸盐，黄色固体)。
LCMS:t R＝2.341min in 0-60AB_7min_220&254_Shimadzu.lcm,chromatography(Xtimate C18 2.1*30mm,3um),MS(ESI)m/z＝418.2[M+H] +.
HPLC:t R＝3.12min in 0-60AB_8min.met,chromatography(Ultimate C18 3um,3.0*50mm).
1H NMR(400MHz,DMSO-d 6):δ＝11.28(br s,1H),8.00-7.99(m,1H),7.87-7.82(m,2H),4.31(s,2H),3.22(s,8H),2.07(br s,1H),0.93-0.77(m,4H).
实施例8：化合物EXP-16的制备
合成路线如下：
Figure PCTCN2020107028-appb-000051
步骤8.1化合物13的制备
Figure PCTCN2020107028-appb-000052
将2-(1H-吡唑-4-基)乙醇(SM12)(200mg,1.78mmol),三乙胺(361mg,3.57mmol),二甲氨基吡啶(44mg,0.36mmol)和Boc酸酐(467mg,2.14mmol)溶于二氯甲烷(3mL)中，0-20℃氮气保护下搅拌12小时。将反应液浓缩，得到4-(2-羟乙基)吡唑-1-羧酸叔丁酯(13)(110mg，粗品)黄色油状物直接用于下一步。
步骤8.2化合物14的制备
Figure PCTCN2020107028-appb-000053
4-(2-羟乙基)吡唑-1-甲酸叔丁基酯(13)(100mg,0.47mmol)，对甲苯磺酰氯(99mg,0.52mmol)和三乙胺(95mg,0.94mmol)溶于二氯甲烷(3mL)中，在0-20℃氮气保护下搅拌2小时，反应液浓缩，得到黄色油状物4-[2-(对甲苯磺酰氧基)乙基]吡唑(1 4)(120mg,粗品)，直接用于下一步。
LCMS:t R＝2.850min in 10-80AB_4min_220&254_Shimadzu,chromatography(Xtimate C18,3um,2.1*30mm),MS(ESI)m/z＝367.2[M+H] +.
1H NMR(400MHz,DMSO-d 6):δ＝8.05-7.98(m,1H),7.74(d,J＝8.0Hz,2H),7.62(s,1H),7.45(d,J＝8.0Hz,2H),4.20(t,J＝6.4Hz,2H),2.77(t,J＝6.4Hz,2H),2.42(s,3H),1.57(s,9H).
步骤8.3化合物15的制备
Figure PCTCN2020107028-appb-000054
将N-[5-(5-甲酰-2-噻吩基)-[1,2,4]***[1,5-a]吡啶-2-基]环丙烷甲酰胺(2)(30mg，0.1mmol)于甲醇(2ml)中，然后加入氰基硼氢化钠(12mg，0.19mmol)。氮气保护下50℃搅拌0.2小时。然后加入碳酸氢钠(1mL)，乙酸乙酯(10mL×3)萃取。有机相合并，饱和食盐水(5ml)洗涤，无水硫酸钠干燥，过滤，浓缩，得到N-[5-[5-(羟甲基)-2-噻吩基]-[1,2,4]***[1,5-a]吡啶-2-基]环丙烷甲酰胺(15)(30mg，粗)黄色固体。
LCMS:t R＝2.099min in 10-80AB_4.0min_220&254_Shimadzu.lcm,chromatography(Xtimate C18,3um,2.1*30mm),MS(ESI)m/z＝315.1[M+H] +.
1H NMR(400MHz,DMSO-d 6):δ＝11.14(s,1H),8.15(d,J＝3.6Hz,1H),7.60-7.52(m,3H),7.03(d,J＝3.6Hz,1H),5.58(t,J＝5.6Hz,1H),4.64(d,J＝5.6Hz,2H),2.01-1.98(m,1H),0.81-0.76(m,4H).
步骤8.4化合物16的制备
Figure PCTCN2020107028-appb-000055
将4-[2-(对甲苯磺酰氧基)乙基]吡唑-1-甲酸叔丁酯(14)(23mg，0.06mmol)，N-[5-[5-(羟甲基)-2-噻吩基]-[1,2,4]***[1,5-a]吡啶-2-基]环丙烷甲酰胺(15)(20mg，0.06mmol)，碳酸铯(21mg，0.06mmol)溶于二甲亚砜(2mL)中的，氮气置换3次，60℃下搅拌0.5小时。加水(6mL),乙酸乙酯(10mL×3)萃取，有机相合并，饱和食盐水(5mL)洗涤，无水硫酸钠干燥，过滤，浓缩，得到N-[5-[5-(4-(2-羟乙基)吡唑-1-羧酸叔丁酯-羟甲基)-2-噻吩基]-[1,2,4]***[1,5-a]吡啶-2-基]环丙烷甲酰胺(16)(27mg，粗)作为黄色固体。
LCMS:t R＝1.156min in 5-95AB_1.5min_220&254_Shimadzu.lcm,chromatography(Xtimate C18,3um,2.1*30mm),MS(ESI)m/z＝509.2[M+H] +.
步骤8.5化合物EXP-16的制备
Figure PCTCN2020107028-appb-000056
N-[5-[5-(4-(2-羟乙基)吡唑-1-羧酸叔丁酯-羟甲基)-2-噻吩基]-[1,2,4]***[1,5-a]吡啶-2-基]环丙烷甲酰胺(16)(27mg,0.05mmol)溶于二氯甲烷(2mL)，室温加入三氟乙酸(29mg,0.256mmol)，室温氮气保护下搅拌0.5小时，反应液浓缩的粗产品，制备薄层色谱(二氯甲烷:甲醇＝10:1)纯化得到标题产品N-[5-[5-[2-(1H-吡唑-4-基)-乙氧基甲基]-2-噻吩基]-[1,2,4]***[1,5-a]吡啶-2-基]环丙烷甲酰胺(EXP-16)(3.0mg,14％收率，白色固体)。
LCMS:t R＝1.872min in 10-80AB_4min_220&254_Shimadzu.lcm,chromatography(Xtimate C18 2.1*30mm),MS(ESI)m/z＝409.3[M+H] +.
HPLC:t R＝2.39min in 10-80CD_8min_met,chromatography(XBridge Shield RP18 5um).
1H NMR(400MHz,CD 3OD):δ＝8.13(d,J＝4.0Hz,1H),7.80-7.70(m,2H),7.63(dd,J＝8.4and 1.2Hz,1H),7.49(s,2H),7.16(d,J＝3.6Hz,1H),4.86-4.84(m,2H),4.24-4.20(m,2H),2.93-2.89(m,2H),2.53-2.47(m,1H),1.06-1.03(m,2H),0.90-0.89(m,2H).
实施例9：化合物EXP-17的制备
合成路线如下：
Figure PCTCN2020107028-appb-000057
步骤9.1化合物17的制备
Figure PCTCN2020107028-appb-000058
N-(5-溴-[1,2,4]***并[1,5-a]吡啶-2-基)-环丙甲酰胺(Int-1)(100mg,0.36mmol)，5-羧基-2-噻吩硼酸(SM13)(122mg,0.71mmol)和碳酸钠(113mg,1.07mmol)溶于四氢呋喃(10mL)和水(5mL)中，氮气置换三次后加入1,1-双(二苯基磷)二茂铁氯化钯(50mg,0.07mmol)，氮气置换三次后升到100℃搅拌2小时。反应液加水(10mL)用乙酸乙酯(20mL)萃取，有机相用饱和食盐水(10mL)洗涤，无水硫酸钠干燥后过滤，滤液真空浓缩得到5-(2-甲酰基)-噻吩-[1,2,4]***[1,5-a]吡啶-2-环丙烷甲酰胺(17)(50mg，粗品)黄色固 体，直接用于下一步。
LCMS:t R＝0.783min in 5-95AB_1.5min_220&254_Shimadzu.lcm,chromatography(Xtimate C18,3um,2.1*30mm),MS(ESI)m/z＝329.0[M+H] +.
步骤9.2化合物EXP-17的制备
Figure PCTCN2020107028-appb-000059
将5-(2-羧基)-噻吩-[1,2,4]***[1,5-a]吡啶-2-环丙烷甲酰胺(17)(50mg,0.15mmol)和2-噻唑-2-基乙胺盐酸盐(SM14)(46mg,0.23mmol)溶于二甲基甲酰胺(2mL)中加入O-(7-氮杂苯并三氮唑-1-基)-N,N,N,N-四甲基脲六氟膦盐(75mg,0.20mmol)和三乙胺(46mg,0.46mol)。反应液于25度下搅拌0.2小时。
反应液浓缩然后制备HPLC[Column:Phenomenex Gemini-NX 150*30mm*5um；Condition:25-55％B(A:water(0.04％NH 3.H 2O+10mM NH 4HCO 3)；B:CH 3CN)；Flow rate:25mL/min]纯化得到5-[2-[2-噻唑-2-乙基]甲酰胺基]-噻吩-[1,2,4]***[1,5-a]吡啶-2-环丙甲酰胺(EXP-17)(6.3mg，8％收率，白色固体)。
LCMS:t R＝2.468min in 10-80AB_4min_220&254_Shimadzu.lcm,chromatography(Xtimate C18 2.1*30mm),MS(ESI)m/z＝439.2[M+H] +.
HPLC:t R＝3.51min in 50-100AB_8min_met,chromatography(XBridge Shield RP18 5um).
1H NMR(400MHz,DMSO-d 6):δ＝11.21(brs,1H),8.90(t,J＝4.8Hz,1H),8.32(d,J＝4.0Hz,1H),7.87-7.81(m,2H),7.78-7.68(m,3H),7.61(d,J＝3.2Hz,1H),3.65(dd,J＝12.8and 7.2Hz,2H),2.58-2.54(m,2H),2.14-2.12(m,1H),0.92-0.86(m,4H).
依据与上述实施例相同的方法，使用市售化合物或参考所示的中间化合物的制备方法而制备以下表4的实施例化合物。
【表4】
Figure PCTCN2020107028-appb-000060
实施例10：化合物EXP-19的制备
合成路线如下:
Figure PCTCN2020107028-appb-000061
步骤10.1化合物18的制备
Figure PCTCN2020107028-appb-000062
4-羟基-N-Boc-哌啶(SM15)(2g,9.94mmol)溶解在四氢呋喃(20mL)中，0℃下分批加入钠氢(1.19g,29.81mmol,60％purity)。搅拌30分钟后，加入二硫化碳(3.03g,39.75mmol,2.40mL)继续搅拌30分钟。碘甲烷(2.82g,19.87mmol)加入后室温反应1 小时。反应液倒入水里(20mL)，水相用乙酸乙酯(20mL×3)萃取，有机相用无水硫酸钠干燥后过滤浓缩旋干后用硅胶柱层析(洗脱剂：0％～10％乙酸乙酯/石油醚)得到淡黄色目标产物N-叔丁氧羰基哌啶-4-甲基黄原酸酯(18)(1.6g,收率55％)。
1H NMR(400MHz,DMSO-d 6):δ＝5.74-5.60(m,1H),3.62-3.48(m,2H),3.32-3.23(m,2H),2.55(s,3H),2.02-1.86(m,2H),1.71-1.63(m,2H),1.40(s,9H).
步骤10.2化合物19的制备
Figure PCTCN2020107028-appb-000063
1,3-二溴-5,5-二甲基海因(2.21g,7.72mmol)溶解在二氯甲烷(20mL)中，在-75℃下氮气氛围中分批加入氟化氢吡啶溶液(7.14g,72.06mmol)和N-叔丁氧羰基哌啶-4甲基黄原酸酯(18)(500mg,1.72mmol)的二氯甲烷(2mL)溶液。反应液在15-20℃下反应16小时后用饱和碳酸氢钠溶液(20mL)淬灭，水相用乙酸乙酯萃取(20mL×3)。有机相用无水硫酸钠干燥后过滤浓缩旋干后得到黄色目标产物4-(三氟甲氧基)哌啶(19)(400mg,粗品)。
1H NMR(400MHz,DMSO-d 6):δ＝8.79(s,1H),4.81-4.52(m,1H),3.30-3.17(m,2H),3.16-3.03(m,2H),2.16-1.99(m,2H),1.94-1.72(m,2H).
步骤10.3化合物20的制备
Figure PCTCN2020107028-appb-000064
反式-2-氟-N-[5-(5-醛基-2-噻吩)-[1,2,4]***[1,5-a]吡啶-2-基]环丙甲酰胺(7)(180mg,0.54mmol)溶解在四氢呋喃(3mL)中，0℃下加入四氢铝锂(41mg,1.09mmol)。混合反应液在15-20℃下反应2小时后用1M HCl溶液(20mL)淬灭，水相用二氯甲烷萃取(20mL×3)。有机相用无水硫酸钠干燥后过滤浓缩旋干后得到深黄色目标产物反式- 2-氟-N-[5-(5-羟甲基-2-噻吩)-[1,2,4]***[1,5-a]吡啶-2-基]环丙甲酰胺(20)(80mg,收率44％)。
1H NMR(400MHz,DMSO-d 6):δ＝11.33(s,1H),8.26(d,J＝4.0Hz,1H),7.74-7.60(m,3H),7.13(d,J＝4.0Hz,1H),5.10-4.83(m,1H),4.74(s,2H),2.42-2.36(m,1H),1.65-1.50(m,1H),1.37-1.27(m,1H).
步骤10.4化合物21的制备
Figure PCTCN2020107028-appb-000065
反式-2-氟-N-[5-(5-羟甲基-2-噻吩)-[1,2,4]***[1,5-a]吡啶-2-基]环丙甲酰胺(20)(80mg,0.24mmol)溶解在二氯甲烷(2mL)中，0℃下向其中加入二氯亚砜(286mg,2.41mmol)。混合反应液在15-20℃下反应16小时后浓缩旋干得到棕色目标产物反式-2-氟-N-[5-(5-氯甲基-2-噻吩)-[1,2,4]***[1,5-a]吡啶-2-基]环丙甲酰胺(21)(60mg,收率71％)。
步骤10.5化合物EXP-19的制备
Figure PCTCN2020107028-appb-000066
反式-2-氟-N-[5-(5-氯甲基-2-噻吩)-[1,2,4]***[1,5-a]吡啶-2-基]环丙甲酰胺(21)(60mg,0.17mmol)溶解在N,N-二甲基甲酰胺(2mL)中，向其中加入碳酸钾(70mg,0.51mmol)和4-(三氟甲氧基)哌啶(19)(58mg,0.34mmol),反应液在15-25℃下反应2小时。 反应混合物用二氯甲烷(20mL)和水(20mL)稀释，水相用二氯甲烷(20mL×3)萃取，有机相用饱和食盐水(20mL×3)洗涤，无水硫酸钠干燥后过滤浓缩旋干后采用HPLC纯化(分离柱：Waters Xbridge 150*25mm*5um；流动相:A:水(0.04％NH 3.H 2O+10mM NH 4HCO 3)和B:乙腈；梯度：B 50-80％)纯化得到白色固体反式2-氟-N-[5-[5-[5-[[4-(三氟甲基)-1-哌啶]甲氧基]-2-噻吩]-[1,2,4]***[1,5-a]吡啶-2-基]环丙甲酰胺(EXP-19)(1.8mg,收率2％,纯度98.56％)。
LCMS:t R＝1.773min in 10-80AB_4min_220&254_Shimadzu.lcm(Xtimate C18,3um,2.1*3mm),MS(ESI)m/z＝484.30[M+H] +.
HPLC:t R＝2.58min in 10-80AB_8min.met,chromatography.
1H NMR(400MHz,DMSO-d 6):δ＝11.31(s,1H),8.25(d,J＝4.0Hz,1H),7.80-7.55(m,3H),7.15(d,J＝4.0Hz,1H),5.06-4.83(m,1H),4.53-4.41(m,1H),3.79(s,2H),2.79-2.71(m,2H),2.43-2.41(m,1H),2.39-2.33(m,2H),1.97-1.87(m,2H),1.80-1.67(m,2H),1.65-1.51(m,1H),1.40-1.27(m,1H).
实施例11：JAK激酶抑制活性检测
将上述实施例的化合物应用于对JAK激酶抑制活性的检测和筛选。
1.方法
在第一块96孔板上每孔加入相应体积DMSO。每孔分别加入20～30μL的100～200μM不同供试化合物储存液。震荡混合3min。对所有化合物进行3X系列稀释。在第二块96孔板上每孔加入60～80μL Kinase buffer，从第一块96孔板每孔取1～2μL溶液加入在第二块96孔板相应孔中。震荡混合3min。从第二块化合物稀释板每孔取5μL化合物稀释液转移到384孔测试板相应孔。在测试板每孔加1～2μL TK Substrate–biotin。每孔加1～2μL酶混合液。同时设置不加酶的blank孔。每孔加1～2μL ATP溶液，封好板子后室温进行反应。反应时间分别为：JAK1:2小时,JAK2:30min,JAK3:30min,TYK2:50min。每孔加3～5μL of Streptavidin-XL665，3～5μL of TK Antibody-cryptate，封好板子后室温放置30分钟以结束反应。PerkinElmer EnVision机器上读取665nm和620nm的fluorescence。
IC50计算
计算每个孔的HTRF ratio：(665signal/620signal)x 10^4.
抑制百分率基于以下公式计算：
抑制％＝(Ratio max-Ratio 化合物)/(Ratio max-Ratio min)]×100％
其中Ratio 化合物为给定化合物浓度下的HTRF ratio，Ratio min为加入blank孔的HTRF ratio，Ratio max为不加入化合物的情况下的HTRF ratio。通过使用GraphPad Prism 5.0软件，使用非线性回归模型绘制S型剂量-抑制率曲线并计算IC 50值。
Filgotinib表示具有如下结构的化合物：
Figure PCTCN2020107028-appb-000067
2.实验结果
各送检化合物测得的IC 50(nM)检测值如下表5所示。
【表5】化合物对JAK的抑制活性
Figure PCTCN2020107028-appb-000068
Figure PCTCN2020107028-appb-000069
备注：IC 50值在0-10nM标记为A；10-30nM标记为B；30-50nM标记为C；50-500nM标记为D；大于500nM标记为E；NT代表为未测试。
实施例12：细胞增殖抑制检测
实施例12-1：基于Hela细胞上的增殖抑制检测
方法：
1、细胞铺板：收集培养的细胞并用Vi-Cell XR细胞计数仪进行活细胞计数。用培养基将细胞悬液调整到适当的浓度后加入96-孔细胞培养板中，每孔加50μL-100μL细胞悬液于96-孔细胞培养板，细胞铺板密度取决于细胞的生长速度，细胞置于相应的培养箱 中培养。
2、药物处理与OSM刺激
首先用DMSO配制3X倍梯度稀释的系列测试化合物浓度(work solution-1)，再用RPMI1640+10％FBS对其进行倍数稀释得到work solution-2；用RPMI1640+10％FBS配制Cisplatin的work solution-2，然后从work solution-2中各取1-10μL加入到已准备好的含培养基的细胞孔中。孵育后，每孔加入1-10μL的OSM的培养基，继续培养。每孔加入适当CTG溶液，震荡混匀1-5分钟后用Envision2104读板仪测定luminescence信号
细胞存活率计算
公式：T/C×100％
其中T为药物处理组的luminescence读数，C为溶剂对照组的luminescence读数平均值。应用GraphPad Prism 5.0软件,使用非线性回归模型绘制S型剂量-抑制率曲线并计算IC50(T/C×100％＝50％)值。
实验结果
各送检化合物测得的IC 50(nM)检测值如下表6所示。
【表6】化合物对Hela细胞的增殖抑制活性检测
Code ID IC 50(uM)
EXP-1 A
EXP-2 A
EXP-3 A
EXP-4 B
EXP-5 B
EXP-6 NT
EXP-7 NT
EXP-8 NT
EXP-9 NT
EXP-10 NT
EXP-11 NT
EXP-12 NT
EXP-18 NT
EXP-19 NT
Filgotinib B
备注：IC 50值在0-5uM标记为A；5-30uM标记为B；未检测标记为NT.
实施例12-2：基于BaF3细胞上的增殖抑制检测
方法：
1、细胞铺板：用RPMI1640+10％FBS(含有IL-3)培养基收集培养的细胞并用Vi-CellXR细胞计数仪进行活细胞计数。用RPMI1640+10％FBS(含有IL-3)培养基将细胞悬液调整到适当的浓度后加入96-孔细胞培养板中，每孔加一定细胞悬液于96-孔细胞培养板，细胞置于相应的培养箱中培养。
2、药物处理：首先用DMSO配制3X倍梯度稀释的系列测试化合物浓度(work solution-1)，再用RPMI1640+10％FBS(含有IL-3)对其进行倍数稀释得到work solution-2；用RPMI1640+10％FBS(含有IL-3)配制Cisplatin的work solution-2，然后从work solution-2中各取1-10μL加入到已准备好的含培养基的细胞孔中。细胞置相应的培养箱中培养。每孔加入适当CTG溶液，震荡混匀5-15分钟后用Envision2104读板仪测定luminescence信号。
细胞存活率计算
公式：T/C×100％
其中T为药物处理组的luminescence读数，C为溶剂对照组的luminescence读数平均值。应用GraphPad Prism 5.0软件,使用非线性回归模型绘制S型剂量-抑制率曲线并计算IC50(T/C×100％＝50％)值。
【表7】化合物对BaF3细胞的增殖抑制活性检测
Code ID IC 50(uM)
EXP-1 A
EXP-2 A
EXP-3 A
EXP-4 B
EXP-5 A
EXP-6 NT
EXP-7 NT
EXP-8 NT
EXP-9 NT
EXP-10 NT
EXP-11 NT
EXP-12 NT
EXP-18 NT
EXP-19 NT
Filgotinib B
备注：IC 50值在0-5uM标记为A；5-30uM标记为B；未检测标记为NT.
实施例12-3：基于THP1细胞上的增殖抑制检测
方法：
1、细胞铺板：收集培养的细胞并用Vi-Cell XR细胞计数仪进行活细胞计数。用培养基将细胞悬液调整到适当的浓度后加入96-孔细胞培养板中，每孔加80μL细胞悬液于96-孔细胞培养板，细胞铺板密度取决于细胞的生长速度，细胞置于相应的培养箱中培养。
2、药物处理与IL-4刺激：首先用DMSO配制3X倍梯度稀释的系列测试化合物浓 度(work solution-1)，再用RPMI1640+10％FBS对其进行倍数稀释得到work solution-2；用RPMI1640+10％FBS配制Cisplatin的work solution-2，然后从work solution-2中各取1-10μL加入到已准备好的含培养基的细胞孔中。细胞置相应的培养箱中培养。孵育后，每孔加入1-10μL的IL-4的培养基，继续培养。每孔加入适当CTG溶液，震荡混匀5-15分钟后用Envision2104读板仪测定luminescence信号。
细胞存活率计算
公式：T/C×100％
其中T为药物处理组的luminescence读数，C为溶剂对照组的luminescence读数平均值。应用GraphPad Prism 5.0软件,使用非线性回归模型绘制S型剂量-抑制率曲线并计算IC50(T/C×100％＝50％)值。
【表8】化合物对THP1细胞的增殖抑制活性检测
Code ID IC 50(uM)
EXP-1 A
EXP-2 A
EXP-3 A
EXP-4 B
EXP-5 A
EXP-6 NT
EXP-7 NT
EXP-8 NT
EXP-9 NT
EXP-10 NT
EXP-11 NT
EXP-12 NT
EXP-18 NT
EXP-19 NT
Filgotinib B
备注：IC 50值在0-5uM标记为A；5-30uM标记为B；未检测标记为NT.
实施例13：肝微粒体的稳定性
使用不同种属(人、大鼠)肝微粒体分别与本发明化合物反应一定时间，通过反应样品与未反应样品的比较而算出残存率，对本发明化合物被肝代谢的程度进行评价。
(结果)表示化合物在10umol/L下的氧化代谢中的残存率。
化合物EXP-3:人的肝微粒体：59.32％；大鼠肝微粒：55.3％。
实施例14：CYP抑制试验
使用市售的混合人肝微粒体，以作为人类主要CYP分子种类(CYP1A2、2B6、2C8、2C9、2C19、2D6和3A4)的典型底物代谢反应的，反应条件如下所述：底物，CYP1A2：30μM的非那西丁；CYP2C9：双氯芬酸10μM；CYP2C19：35μM的S-甲吩妥英；CYP3A4：咪达***为5μM，***为80μM；CYP2D6：丁呋洛尔，10μM；CYP2C8：10μM紫杉醇；CYP2B6：安非他酮70μM；反应时间，CYP1A2、2C9、2D6、2C8、2B6:10分钟；CYP2C19:45分钟；CYP3A4:5分钟。反应温度，37℃。抑制剂(阳性对照)，CYP1A2：α-萘黄酮；CYP2C9：磺胺苯唑；CYP2C19：奥美拉唑；CYP3A4：酮康唑；CYP2D6：奎尼丁；CYP2C8：尼卡地平；CYP2B6：氯吡格雷。
在96孔板中准备用于测试化合物和阳性对照的一系列稀释液，将8μL的10mM测试化合物转移到12μL的ACN中。在DMSO：ACN混合物(v/v：40:60)中进行1：3系列稀释。在测定孔中加入HLM，然后在冰上向指定的孔中加入测试化合物或血清稀释的参比抑制剂溶液。在37℃下预孵育96孔板和NADPH溶液5分钟后，将15μL预热的8mM NADPH溶液添加到测定板中以启动反应。孵育时间：3A4 5分钟，1A2、2B6、2C8、2C9和2D6 10分钟，2C19 45分钟。加入含IS的ACN终止反应。
采用s型(非线性)剂量-响应模型(GraphPad Prism 5.0或Xlfit模型205)进行曲线拟合计算IC50，数据计算公式如下:
Y＝Bottom+(Top-Bottom)/(1+10^((LogEC50-X)*HillSlope))
X是浓度的对数。Y为浓度由高到低时从下到上呈s形的响应。
结果：化合物EXP-3没有抑制作用。
实施例15：药代动力学研究
对于静脉内途径和口服途径，将化合物配制在5％DMSO+95％羟丙基-β-环糊***溶液中。将试验动物以1-10mg/kg口服给药和以1-5mg/kg静脉内给药。每组包括3只大鼠，经颈静脉穿刺采血约0.2mL，肝素钠抗凝剂，在下面范围内的时间点下采集：0.083h、0.25h、0.5h、1h、2h、4h、6h、8h、10h和24h。将血样采集后于1小时之内以5000-13000rpm离心10min分离血浆，并将血浆样品在LC-MS/MS上分析。
药代动力学参数
通过不同时间点的血药浓度数据，运用Phoenix WinNonlin 7.0计算药代动力学参数，提供AUC 0-t、AUC 0-∞、MRT 0-∞、C max、T max、和T 1/2等参数。通过不同时间点的血药浓度数据，运用Phoenix WinNonlin 7.0计算药代动力学参数，提供AUC 0-t、AUC 0-∞、MRT 0-∞、C max、T max、和T 1/2等参数。
化合物EXP-1药代动力学参数：
口服：AUC是12043h*ng/mL,Cmax是2626ng/mL,T 1/2是2.68h.
filgotinib药代动力学参数：
口服：AUC是2849h*ng/mL,Cmax是426ng/mL,T 1/2是3.9h.
实施例16：hERG试验
以本发明化合物的心电图QT间隔延长危险性评价为目的，使用表达出humanether-a-go-go related gene(hERG)通道的HEK293细胞，而研究本发明化合物对于心室再极化过程中发挥重要作用的延迟整流K+电流(Ikr)的作用。
使用全自动膜片钳***(patch clamp PC-505B(WARNER instruments))，并通过全自动膜片钳法，将细胞保持在–80mV，然后用持续4秒方波去极化到40mV，再用持续2秒方波超极化到-40mV，以得到hERG尾电流。检测第二个方波引发的最大电流，待其稳定后，灌流测试化合物，当反应稳定后，计算阻断的强度。
细胞外液(mM)为:NaCl,137；KCl,4；CaCl 2,1.8；MgCl 2,1；HEPES,10；glucose 10；pH 7.4(NaOH滴定)。细胞内液(mM)为：K Aspartate,130；MgCl2,5；EGTA 5；HEPES,10；Tris-ATP 4；pH 7.2(KOH滴定)。
试验结果：EXP-3:IC 50大于10μM。
实施例17：鼠伤寒沙门氏菌回复突变试验
使用沙氏杆菌TA98、TA100、TA1535、TA1537及大肠杆菌作为试验菌株(菌株来源Molecular Toxicology,Inc.)，于非代谢活化条件下和活化条件下实施Ames试验，调查本发明的化合物有无基因突变诱发性。
将冷冻保存的菌株进行复苏，接种于营养肉汤培养基中，100～120转/分，37℃恒温振荡器培养11小时。将底层琼脂培养基(含适量琼脂、V-B缓冲液、20％葡萄糖溶液、20％硫酸镁溶液)倒入皿中冷却备用。每个试管中，分别加入0.1mL供试品/阴性对照品(DMSO)/阳性对照品溶液、0.1mL菌液、0.5mL 0.2mol/L磷酸盐缓冲液(PBS)(-S9)/0.5mL S9混合液(+S9)及2.5mL顶层培养基，涡旋混匀后迅速均匀平铺在已铺好的底层琼脂培养基平皿上。自然冷却凝固后，将平皿倒置放入37℃霉菌培养箱内培养48小时。取出培养后的平皿计数，并与DMSO组进行比较而评价，平均回变菌落数为阴性对照组回变菌落数(可视为自发回变数)的2倍或2倍以上(TA1535：3倍或以上)，呈剂量-效应关系，则结果为阳性，可判定供试品为致突变剂。反之为阴性。
试验结果：化合物EXP-3为阴性。
实施例18：体内药效模型
18.1 CIA模型
18.1.1材料
完全Freund佐剂(CFA)和II型牛胶原购自Chondrex。
18.1.2动物
Lewis大鼠(雌性，10-12周龄)从北京维通利华实验动物技术有限公司获得。将大鼠进行12小时的明暗交替，环境保持温度23±2℃,湿度40～70％。
18.1.3胶原诱导的关节炎(CIA)
用0.05M醋酸溶液配制Ⅱ型胶原溶液(2mg/mL)，使之充分溶解，4℃避光放置过夜。试验时在冰浴条件下将2mg/mL的Ⅱ型胶原(C Ⅱ)溶液与4mg/mL的完全弗氏佐剂(CFA)溶液等体积混合充分乳化。第0天在实验大鼠尾根部皮内注射等体积混合C Ⅱ (2mg/mL)和CFA(4mg/mL)制备的乳剂进行第一次免疫，在第7天进行第二次增强免疫。
18.1.4研究设计
试验化合物的治疗效果是在大鼠CIA模型中试验的。将大鼠随机分组，每组8只动物。将所有动物在第0天免疫，第7天加强免疫。空白对照(
Figure PCTCN2020107028-appb-000070
组)，溶媒对照组(Vehicle组)是含DMSO的20％HP-β-CD溶液，并且阳性对照组分别用Filgotinib(10mg/kg，给药2周)、枸橼酸托法替布(Tofacitinib组，2mg/kg，给药2周)和乌帕替尼(Upadacitinib组，2mg/kg，给药2周)，实验组为分别用2mg/kg、3mg/kg、5mg/kg、10mg/kg的EXP-3给药两周。
18.1.5关节炎的临床评价
足容积同时对四个足趾肿胀进行评分，每只足0～4分，每只大鼠最高评分为16分。0-无肿胀，外观正常；1-踝关节、腕关节或指关节的轻度肿胀或红肿；2-踝关节、腕关节或指关节的中度肿胀或红肿；3-踝关节、腕关节或指关节的重度肿胀或红肿；4-踝关节、腕关节或指关节非常严重的肿胀或红肿。病理评分数据使用SPSS非参数检验Mann-Whitney U test分析，p<0.05认为是有显著性差异。
18.1.6关节炎发作后的体重、足体积
在临床上，体重减轻和足体积减轻与关节炎相关。因此，关节炎发作后体重和足体积分别与阴性组的差异可以用于评价大鼠模型中的治疗效果。实验数据以Mean±SEM表示；两组间数据采用非配对t检验，p<0.05认为是有显著性差异。
18.1.7试验结果
18.1.7.1受试物对实验大鼠临床观察和体重的影响
所有动物实验期间无肉眼可见明显异常。
Figure PCTCN2020107028-appb-000071
组动物体重在实验期间稳定增长。Vehicle组动物体重在第1次免疫后8～28天均显著低于
Figure PCTCN2020107028-appb-000072
组(p<0.01～0.001)。枸橼酸托法替布(Tofacitinib)2mg/kg和EXP-3 10mg/kg组动物体重在第11～17天略有增长，但后续体重又出现回落，其余各给药组动物体重与Vehicle组相近，除了
Figure PCTCN2020107028-appb-000073
组，其余组均无统计学的显著性差异，说明受试物各组对大鼠体重影响不大。
表9.每天1次连续2w经口灌胃给予受试物对CIA大鼠体重的影响(n＝8，
Figure PCTCN2020107028-appb-000074
)
Figure PCTCN2020107028-appb-000075
Figure PCTCN2020107028-appb-000076
18.1.7.2受试物对实验大鼠后足足容积的影响
Vehicle组动物在一免后第14～28天足容积显著升高，第24天足容积开始降低。Upadacitinib 2mg/kg组与Vehicle相比在第17～28天足容积显著降低(p<0.05～0.001)。其余各给药组与Vehicle组相比在第21～28天动物足容积均显著降低(p<0.05～0.001)。其中EXP-3 10mg/kg作用最强，且明显强于filgotinib 10mg/kg，其次是Upadacitinib 2mg/kg。EXP-3 5mg/kg～2mg/kg与枸橼酸托法替布(Tofacitinib)2mg/kg作用相近，略弱于乌西替尼(Upadacitinib)2mg/kg。(表10)。表明受试物对CIA模型的大鼠足容积具有一定改善效果。
表10.每天1次连续2w经口灌胃给予受试物对CIA大鼠后足足容积的影响(n＝8，
Figure PCTCN2020107028-appb-000077
)
Figure PCTCN2020107028-appb-000078
*p<0.05,**p<0.01,***p<0.001vs.Vehicle
18.1.7.3受试物对实验大鼠四肢关节炎指数(AI)的影响
实验动物在一免后第9～10天开始出现四肢肿胀，在第14天大部分发病足达到2～3分，开始分组给予药物治疗。结果显示，Vehicle组动物AI评分在第11～24天持续升高，第28天略有下降。EXP-3 10mg/kg对降低四肢肿胀的作用最强，其次是tofacitinib 2mg/kg。EXP-3 5mg/kg、EXP-3 2mg/kg与Upadacitinib 2mg/kg作用相近，略弱于tofacitinib 2mg/kg。filgotinib药效最弱。(表11)。表明受试物对CIA模型的大鼠四肢关节炎病情具有一定改善效果。
表11.每天1次连续2w经口灌胃给予受试物对CIA大鼠关节炎指数的影响(n＝8，
Figure PCTCN2020107028-appb-000079
)
Figure PCTCN2020107028-appb-000080
*p<0.05,**p<0.01,***p<0.001vs.Vehicle

Claims (10)

  1. 式(I)所示的化合物或其立体异构体、互变异构体或其药学上可接受的盐或其溶剂合物或者前药：
    Figure PCTCN2020107028-appb-100001
    其中：
    B独立选自取代或未取代的C 3-C 6环烷基、芳基、具有1至2个独立选自N、O和S的环杂原子的5至6元杂芳基环，其中所述的C 3-C 6环烷基或5至6元杂芳环任选地被独立选自卤素、氟代或未氟代的C 1-C 6烷基、氟代或未氟代的C 1-C 6烷氧基、氟代或未氟代的C 1-C 6烷基胺基的一个或多个取代基取代；
    D独立选自C,N；
    F、G、H、K独立选自C、N、S、O；
    L 1不存在或独立选自单键,-CH 2-，-(CH 2) xO(CH 2) y-,-(CH 2) xNH(CH 2) y-,-CH 2O-,-C(O)-,-CON(R 4)-,-CH 2N(R 4)-,-CONH(CH 2) y-,-N(R 4)-,-SO 2N(R 4)-,-S(O) 2-,-N(Me)-；
    R 4独立选自H、C 1-C 6烷基、取代的C 1-C 6烷基、C 1-C 3醚C 1-C 3烷基、甲磺酰基；
    R 1独立选自H、-NH 2、-COOH、取代或未取代的C 1-C 6烷基、酰基、取代的或未取代的酰基胺基、取代的或未取代的C 1-C 6烷氧基、卤素、羟基、取代的或未取代的C 1-C 3酯基、取代或未取代的杂芳基；
    R 2不存在或独立选自H、F、Cl、Me；
    R 3独立地选自H、取代或未取代的以下基团：磺酰基、磺酰胺基、5至6元杂环，其具有至少一个杂原子且任选地具有独立选自N和S的第二环杂原子、取代的或未取代的C 1-C 6烷烃,取代基独立选自5至6元杂芳基和甲氧基取代、取代的或未取代的C 3-C 7环烷基、取代的或未取代的4-7元杂环烷基、取代的或未取代的5-7元杂芳基；
    m是0、1、2、3；n是0，1、2、3；p是0、1、2；t是0、1、2、3。
  2. 权利要求1所述的式(I)所示的化合物或其立体异构体、互变异构体或其药学上可接受的盐或其溶剂合物或者前药，其特征在于，B选自取代或未取代的以下基团：环丙烷、环丁烷、吡唑基、吡啶基、咪唑基；取代基选自F、Cl、Br、甲基、乙基、丙基、异丙基、三氟甲基。
  3. 权利要求1所述的式(I)所示的化合物或其立体异构体、互变异构体或其药学上可接受的盐或其溶剂合物或者前药，其特征在于，F、G、H、K不同时为C；优选的，K为S,F、G、H为C。
  4. 权利要求1所述的式(I)所示的化合物或其立体异构体、互变异构体或其药学上可接受的盐或其溶剂合物或者前药，其特征在于，D为C或N；n为1；m为0或者1，优选的，m为0；R 2不存在或为H；p为0；t为1。
  5. 权利要求1所述的式(I)所示的化合物或其立体异构体、互变异构体或其药学上可接受的盐或其溶剂合物或者前药，其特征在于，R 1独立选自H、-NH 2、-COOH、羟基、卤素、取代或未取代的C 1-C 3烷基、取代或未取代的C 1-C 3酰基、取代的或未取代的C 1-C 3酰基胺基、取代的或未取代的C 1-C 3烷氧基、取代的或未取代的C 1-C 3酯基；优选的，R 1独立选自H、-NH 2、-COOH、羟基、卤素、甲基、乙基、丙基、异丙基、甲酰基胺基、甲酯基；L 1不存在或独立选自单键，-CH 2-，-(CH 2) xO(CH 2) y-,-(CH 2) xNH(CH 2) y-,-CONH(CH 2) y-,-N(R 4)-；R 4独立选自H、甲基、乙基、丙基、乙基甲醚、甲基甲醚、乙基***；x或y独立的选自0、1或2；优选的，L 1不存在或独立选自单键，-CH 2-，-(CH 2) xO(CH 2) y-,-(CH 2) xNH(CH 2) y-,-CONH(CH 2) y-,-N(R 4)-；R 4独立选自H、甲基、乙基甲醚；x或y独立的选自0、1或2。
  6. 权利要求1所述的式(I)所示的化合物或其立体异构体、互变异构体或其药学上可接受的盐或其溶剂合物或者前药，其特征在于，R 3独立地选自H、取代或未取代的以下基团：磺酰基、磺酰胺基、硫代吗啉1,1-二氧化物、哌啶、吡唑、噻唑、咪唑、1,3,4-噻二唑、哌嗪、吗啉、噻吩、噁唑、1,3,4-噁二唑、呋喃、吡咯、3-吡咯啉、2-吡唑啉、1,2,3-唑、1,2,3-***、1,2,4-***、吡喃；取代基选自H、卤素、C 1-C 3烷基、C 1-C 3卤代烷基、羟基C 1-C 3烷基、C 3-C 6环烷基、乙基甲醚、甲基甲醚、乙基***； 优选的，R 3独立地选自H、取代或未取代的以下基团：磺酰基、磺酰胺基、硫代吗啉1,1-二氧化物、哌啶、吡唑、噻唑、咪唑、1,3,4-噻二唑、哌嗪、吗啉、吡嗪、噻吩、噁唑、1,3,4-噁二唑、呋喃、吡咯、3-吡咯啉、2-吡唑啉、1,2,3-唑、1,2,3-***、1,2,4-***、吡喃；取代基选自H、卤素、甲基、乙基、丙基、异丙基、三氟甲基、羟乙基、羟甲基、环丙烷、环丁烷、乙基甲醚、甲基甲醚、乙基***。
  7. 权利要求1所述的式(I)所示的化合物或其立体异构体、互变异构体或其药学上可接受的盐或其溶剂合物或者前药，其特征在于，R 3独立地选自H、取代或未取代的以下基团：磺酰基、磺酰胺基、硫代吗啉1,1-二氧化物、哌啶、吡唑、噻唑、咪唑、1,3,4-噻二唑、哌嗪、吗啉、噻吩、噁唑、1,3,4-噁二唑、呋喃、吡咯、3-吡咯啉、2-吡唑啉、1,2,3-唑、1,2,3-***、1,2,4-***、吡喃；取代基选自C 1-C 3卤代烷氧基；优选的，R 3独立地选自H、取代或未取代的以下基团：磺酰基、磺酰胺基、硫代吗啉1,1-二氧化物、哌啶、吡唑、噻唑、咪唑、1,3,4-噻二唑、哌嗪、吗啉、吡嗪、噻吩、噁唑、1,3,4-噁二唑、呋喃、吡咯、3-吡咯啉、2-吡唑啉、1,2,3-唑、1,2,3-***、1,2,4-***、吡喃；取代基选自三氟甲氧基。
  8. 以下结构所示的化合物或其立体异构体、互变异构体或其药学上可接受的盐或其溶剂合物或者前药：
    Figure PCTCN2020107028-appb-100002
    Figure PCTCN2020107028-appb-100003
    Figure PCTCN2020107028-appb-100004
  9. 一种药物组合物，其包含权利要求1～7任一项所述的化合物或其立体异构体、互变异构体或其药学上可接受的盐，和药学上可接受的载体。
  10. 权利要求1～6任一项所述的化合物在用于制备预防或治疗JAK激酶相关疾病药物中的用途，优选是在制备用于预防和/或治疗涉及软骨退化、骨和/或关节退化的疾病、涉及炎症或免疫响应的病症、内毒素驱动的疾病状态、癌症和器官移植排斥的药物中的应用；更优选的，是在制备用于预防和/或治疗骨关节炎、克隆病、类风湿性关节炎、银屑病、过敏性气道疾病、幼年特发性关节炎、结肠炎、炎性肠病、内霉素驱动的疾病状态、软骨更新损伤的疾病、先天软骨畸形、器官移植排斥、涉及软骨退化、关节退化、骨髓增殖性障碍、白血病、实体瘤的药物中的应用。
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