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WO2021147790A1 - Dérivé de pyrazolo[1,5-a]pyrazine et procédé de préparation s'y rapportant et son utilisation - Google Patents

Dérivé de pyrazolo[1,5-a]pyrazine et procédé de préparation s'y rapportant et son utilisation Download PDF

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Publication number
WO2021147790A1
WO2021147790A1 PCT/CN2021/072362 CN2021072362W WO2021147790A1 WO 2021147790 A1 WO2021147790 A1 WO 2021147790A1 CN 2021072362 W CN2021072362 W CN 2021072362W WO 2021147790 A1 WO2021147790 A1 WO 2021147790A1
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cancer
kinase
compound
pharmaceutically acceptable
alkyl
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PCT/CN2021/072362
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Chinese (zh)
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张盼盼
颜孙力
李英
叶成
钱文建
胡泰山
陈磊
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浙江海正药业股份有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D519/00—Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00
    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/4995—Pyrazines or piperazines forming part of bridged 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
    • 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
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/02—Nasal agents, e.g. decongestants
    • 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
    • A61P11/06—Antiasthmatics
    • 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
    • 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
    • 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
    • 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
    • A61P37/08—Antiallergic agents

Definitions

  • the present invention relates to a pyrazolo[1,5-a]pyrazine derivative, a preparation method thereof, a pharmaceutical composition containing the derivative, and its use as a therapeutic agent, especially as a JAKS family kinase inhibitor.
  • Tyrosine kinase 2 is a non-receptor tyrosine kinase that belongs to the Janus kinase family (JAKS).
  • JAKS includes JAK1, JAK2, JAK3 and TYK2 subtypes.
  • JH1 is a highly conserved kinase domain with catalytic activity
  • JH2 is a kinase-like domain, which is the unique difference between JAK kinase and other tyrosine kinases At this point, this region does not have catalytic activity, but can regulate the activity of JH1
  • JH3-JH4 is the SH2 domain, which can specifically recognize and bind to activated tyrosine residues
  • JH5-JH7 is the FERM domain, which is relatively Conservative, regulates the binding of JAK to the receptor.
  • JAK1, JAK2 and TYK2 are widely present in the human body, while JAK3 only exists in hematopoietic tissues such as bone marrow and lymph.
  • JAK kinase/signal transducer and activator of transcription (The Janus kinase/signal transducer and activator of transcription, JAK/STAT) signaling pathway mediates a variety of cytokine signal transduction, including interleukin, interferon, erythropoietin , Granulocyte and macrophage colony stimulating factors.
  • the binding of cytokines to their receptors causes the dimerization of receptor molecules, so that JAK kinases coupled to the receptors approach each other and are activated by interactive tyrosine phosphorylation.
  • the activated JAK catalyzes the phosphorylation of the receptor itself.
  • the corresponding STAT docking site is formed to make STAT phosphorylate, and then STAT forms a homodimer or heterodimer and enters the nucleus, and combines with the corresponding target gene promoter to activate gene transcription and expression. Therefore, the signal pathway mediated by JAK kinase plays an important role in cell proliferation, differentiation, apoptosis, and inflammation.
  • cytokines In the pathological process, excessive secretion of cytokines leads to excessive activation of the JAK/STAT pathway, causing autoimmune diseases, tumors and other diseases, including psoriasis, systemic lupus erythematosus, rheumatoid arthritis, psoriasis, Vitiligo, Crohn's syndrome, colitis, etc.
  • JAK kinase participates in the signal transduction of cytokines and regulates the immune response. Therefore, JAK kinase can be used as a target for the treatment of autoimmune diseases such as psoriasis and rheumatoid arthritis.
  • tofacitinib developed by Pfizer has been successfully marketed, which can effectively inhibit JAK1, JAK2, and JAK3 for the treatment of rheumatoid arthritis; the JAK1 and JAK2 kinase inhibitor barectinib developed by Eli Lilly, As a drug for the treatment of rheumatoid arthritis, upadacitinib, a JAK1 kinase inhibitor developed by AbbVie, was launched in the United States in 2019. It is also a treatment for rheumatoid arthritis. The outstanding achievements made by other members of the JAKS kinase family have made TYK2 a research hotspot.
  • TYK2 is finally in the history of protein kinases. Began to show off.
  • BMS-986165 developed by BMS is used for the treatment of psoriasis. It is currently in Phase 3 clinical trials and is in a phase 2 trial for the treatment of patients with moderate to severe plaque psoriasis. It reaches the primary end point of curative effect in medium, with small side effects, and has high safety and effectiveness. It is believed that in the near future, TYK2 inhibitors will have good news and bring good news to patients with psoriasis.
  • the object of the present invention is to provide a pyrazolo[1,5-a]pyrazine derivative represented by the general formula (I), or its stereoisomers, tautomers, or pharmaceutically acceptable Salt:
  • Z is selected from bond or -(CH 2 ) h -, wherein one or more methylene units are optionally further selected from C 1 -C 3 alkyl, C 1 -C 6 alkoxy or halogen Wherein the alkyl or alkoxy group is optionally further substituted with one or more halogens;
  • R 1 is selected from a C 1 -C 4 alkyl group, a C 3 -C 6 cycloalkyl group or a 3-6 membered heterocyclic group, wherein the alkyl group, cycloalkyl group or heterocyclic group is optionally further substituted by one or more A substitution selected from C 1 -C 4 alkyl, C 1 -C 6 alkoxy, C 1 -C 4 haloalkyl, C 1 -C 6 haloalkoxy, halogen, hydroxyl, cyano, nitro or amino Substitution;
  • R 2 and R 3 are the same or different, and are each independently selected from hydrogen, C 1 -C 6 alkyl or halogen; wherein the alkyl group is optionally further substituted with one or more halogens;
  • R 4 is selected from hydrogen, C 1 -C 6 alkyl or C 3 -C 6 cycloalkyl, wherein the alkyl or cycloalkyl is optionally further substituted with one or more halogens;
  • R 5 is selected from hydrogen, amino or C 1 -C 6 alkyl, wherein the alkyl is optionally further substituted with one or more halogens;
  • n and n are each independently selected from 0, 1 or 2; preferably, m and n are 1;
  • h is selected from 1, 2 or 3.
  • the preferred technical solution of the present invention is a compound represented by general formula (I), a tautomer or a pharmaceutically acceptable salt thereof, which is a compound represented by general formula (II) or a stereoisomer thereof, Tautomers or pharmaceutically acceptable salts thereof,
  • R 6 is the same or different, each independently selected from C 1 -C 4 alkyl or halogen, wherein the alkyl is optionally further substituted with one or more halogens;
  • j is selected from 1, 2, 3 or 4;
  • k is selected from 0, 1 or 2;
  • R 2 to R 5 , Z, m or n are as shown in the general formula (I).
  • the preferred technical solution of the present invention is a compound represented by general formula (I), a tautomer or a pharmaceutically acceptable salt thereof, which is a compound represented by general formula (III) or a stereoisomer thereof, Tautomers or pharmaceutically acceptable salts thereof,
  • k 2;
  • R 2 to R 6 , Z, j, m or n are as shown in the general formula (II).
  • the preferred technical solution of the present invention is a compound represented by general formula (I), (II), (III) or its stereoisomers, tautomers, or pharmaceutically acceptable salts thereof, wherein R 4 is Methyl or ethyl.
  • the preferred technical solution of the present invention is a compound represented by the general formulas (I), (II), (III) or its stereoisomers, tautomers or pharmaceutically acceptable salts thereof, wherein:
  • R 2 and R 3 are the same or different, and are each independently selected from a hydrogen atom or a methyl group;
  • R 5 is selected from a hydrogen atom or a methyl group.
  • the preferred technical solution of the present invention is a compound represented by general formula (II) or (III) or its stereoisomers, tautomers or pharmaceutically acceptable salts thereof, wherein R 6 is selected from fluorine or tris Fluoromethyl.
  • the preferred technical solution of the present invention is a compound represented by general formula (II) or (III) or its stereoisomers, tautomers or pharmaceutically acceptable salts thereof, wherein j is selected from 1 or 2.
  • Typical compounds of the present invention include, but are not limited to:
  • the basic conditions are provided by an organic base selected from N,N-diisopropylethylamine, pyridine, triethylamine, piperidine, N-methylpiperazine, 4-dimethylaminopyridine or tert-butyl Potassium alkoxide, preferably N,N-diisopropylethylamine;
  • R 1 to R 5 , Z, m or n are as described in the general formula (I).
  • the present invention also relates to a compound represented by the general formula (IA) or its stereoisomers, tautomers or pharmaceutically acceptable salts thereof:
  • R 2 to R 5 , Z, m or n are defined as described in the general formula (I).
  • the compounds of general formula (IA) of the present invention include, but are not limited to:
  • the present invention provides a pharmaceutical composition containing an effective dose of the compound of general formula (I), (II) or (III) or its stereoisomers, tautomers Isomers or pharmaceutically acceptable salts thereof, and pharmaceutically acceptable carriers, excipients, or combinations thereof.
  • the present invention provides a pharmaceutical composition
  • the pharmaceutical composition also contains at least one of anti-inflammatory drugs, the anti-inflammatory drugs selected from non-steroidal anti-inflammatory drugs, non-specific epoxy Enzyme-2 inhibitors, specific cyclooxygenase-2 inhibitors, corticosteroids, tumor necrosis factor receptor antagonists, salicylates or salts, immunosuppressants, or methotrexate.
  • the anti-inflammatory drugs selected from non-steroidal anti-inflammatory drugs, non-specific epoxy Enzyme-2 inhibitors, specific cyclooxygenase-2 inhibitors, corticosteroids, tumor necrosis factor receptor antagonists, salicylates or salts, immunosuppressants, or methotrexate.
  • the present invention provides a method for inhibiting JAKS kinase, wherein the method includes administering a pharmaceutical composition to the patient, the pharmaceutical composition containing an effective dose of the general formula (I), (II) Or the compound described in (III) or its stereoisomers, tautomers, or pharmaceutically acceptable salts thereof, and pharmaceutically acceptable carriers, excipients, or combinations thereof.
  • the present invention provides a compound of the general formula (I), (II) or (III) or a stereoisomer, a tautomer or a pharmaceutically acceptable salt thereof, or a drug thereof
  • the use of the composition in the preparation of JAK1 kinase, JAK2 kinase, JAK3 kinase and/or TYK2 kinase inhibitors is preferably the use in the preparation of JAK1 kinase, JAK2 kinase and/or TYK2 kinase inhibitors.
  • the present invention provides a compound of the general formula (I), (II) or (III) or a stereoisomer, a tautomer or a pharmaceutically acceptable salt thereof, or a drug thereof
  • a medicine for the treatment of diseases mediated by JAK1 kinase, JAK2 kinase, JAK3 kinase and/or TYK2 kinase, wherein preferably in the preparation and treatment of diseases mediated by JAK1 kinase, JAK2 kinase and/or TYK2 kinase
  • the diseases mediated by JAK1 kinase, JAK2 kinase and/or TYK2 kinase include autoimmune diseases, inflammatory diseases and cancer; wherein the autoimmune diseases include asthma, psoriasis Disease, lupus, multiple sclerosis, allergic rhinitis, atopic dermatitis, contact dermatitis and delayed allergic reaction;
  • cancer colorectal cancer, melanoma, endometrial cancer, prostate cancer, bladder cancer, stomach cancer, liver cancer, gastrointestinal stromal tumor, thyroid cancer, leukemia, non-Hodgkin's lymphoma, nasopharyngeal cancer, esophageal cancer, brain Tumors, lymphomas, multiple myeloma, cholangiocarcinoma and solid tumors; the leukemia is further preferably selected from chronic myelogenous leukemia and acute myeloid leukemia.
  • the present invention provides a compound of the general formula (I), (II) or (III) or a stereoisomer, a tautomer or a pharmaceutically acceptable salt thereof, or a drug thereof
  • autoimmune diseases include asthma, psoriasis, lupus, multiple sclerosis, allergic rhinitis, atopic Dermatitis, contact dermatitis and delayed allergic reactions
  • the inflammatory diseases include inflammatory bowel disease, rheumatoid arthritis, and the inflammatory bowel disease includes Crohn’s disease and ulcerative colitis
  • the Cancers include non-small cell lung cancer, small cell lung cancer, breast cancer, pancreatic cancer, glioma, glioblastoma, ovarian cancer, cervical cancer, colorectal cancer, melanoma, endometrial cancer, prostate cancer, Bladder cancer, stomach cancer, liver cancer, gastrointestinal stromal
  • Alkyl when regarded as a group or a part of a group means to include a C 1 -C 20 linear or branched aliphatic hydrocarbon group. It is preferably a C 1 -C 10 alkyl group, more preferably a C 1 -C 6 alkyl group.
  • alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-di Methylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1 -Ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethyl Butyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl Wait. Alkyl groups can be substituted or unsubstituted.
  • Cycloalkyl refers to saturated or partially saturated monocyclic, fused, bridged, and spirocyclic carbocyclic rings. It is preferably a C 3 -C 12 cycloalkyl group, more preferably a C 3 -C 8 cycloalkyl group, and most preferably a C 3 -C 6 cycloalkyl group.
  • Examples of monocyclic cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptyl Alkenyl, cyclooctyl, etc., preferably cyclopropyl and cyclohexenyl.
  • Heterocyclyl “heterocyclic” or “heterocyclic” are used interchangeably in this application and all refer to non-aromatic heterocyclic groups, in which one or more ring-forming atoms are heteroatoms, such as oxygen, Nitrogen, sulfur atoms, etc., including monocyclic, polycyclic, fused, bridged, and spirocyclic rings. It preferably has a 5- to 7-membered monocyclic ring or a 7- to 10-membered bi- or tricyclic ring, which may contain 1, 2 or 3 atoms selected from nitrogen, oxygen and/or sulfur.
  • heterocyclyl examples include, but are not limited to, morpholinyl, oxetanyl, thiomorpholinyl, tetrahydropyranyl, 1,1-dioxo-thiomorpholinyl, piperidine Group, 2-oxo-piperidinyl, pyrrolidinyl, 2-oxo-pyrrolidinyl, piperazin-2-one, 8-oxa-3-aza-bicyclo[3.2.1]octyl or Piperazinyl,.
  • the heterocyclic group may be substituted or unsubstituted.
  • Alkoxy refers to a (alkyl-O-) group. Among them, the alkyl group is defined in this article. C 1 -C 6 alkoxy groups are preferred. Examples thereof include, but are not limited to: methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy and the like.
  • Halogen refers to fluorine, chlorine, bromine and iodine.
  • Amino refers to -NH 2 .
  • Cyano refers to -CN.
  • Niro refers to -NO 2 .
  • DMSO dimethyl sulfoxide
  • BOC means tert-butoxycarbonyl
  • Ts means p-toluenesulfonyl.
  • Substituted refers to one or more hydrogen atoms in the group, preferably up to 5, more preferably 1 to 3 hydrogen atoms, independently of each other, substituted with a corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and those skilled in the art can determine (by experiment or theory) possible or impossible substitutions without too much effort. For example, an amino group or a hydroxyl group having free hydrogen may be unstable when combined with a carbon atom having an unsaturated (e.g., olefinic) bond.
  • substituted or “substituted” mentioned in this specification, unless otherwise specified, mean that the group can be substituted by one or more groups selected from the following: alkyl, alkenyl, alkynyl, alkoxy , Alkylthio, alkylamino, amino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, Cycloalkylthio, heterocycloalkylthio, amino, haloalkyl, hydroxyalkyl, carboxyl, carboxylate.
  • “Pharmaceutically acceptable salts” refer to certain salts of the above compounds that can maintain the original biological activity and are suitable for medical use.
  • the pharmaceutically acceptable salt of the compound represented by formula (I) may be a metal salt or an amine salt formed with a suitable acid.
  • “Pharmaceutical composition” means a mixture containing one or more of the compounds described herein or their physiologically pharmaceutically acceptable salts or prodrugs and other chemical components, as well as other components such as physiologically pharmaceutically acceptable carriers and excipients. Shape agent. The purpose of the pharmaceutical composition is to promote the administration to the organism, which is beneficial to the absorption of the active ingredient and thus the biological activity.
  • the mass spectrum is measured by an LC/MS instrument, and the ionization method can be ESI or APCI.
  • the thin layer chromatography silica gel plate uses Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plate, the size of the silica gel plate used for thin layer chromatography (TLC) is 0.15mm ⁇ 0.2mm, and the size of the thin layer chromatography separation and purification product is 0.4mm. ⁇ 0.5mm.
  • the column chromatography uses Yantai Huanghai silica gel 200-300 mesh silica gel as the carrier.
  • CD 3 OD Deuterated methanol.
  • DMSO-d 6 Deuterated dimethyl sulfoxide.
  • the compound was purified using silica gel column chromatography eluent system and thin layer chromatography, wherein the eluent system was selected from: A: petroleum ether and ethyl acetate system; B: dichloromethane and methanol system; C: two Methyl chloride and ethyl acetate; the volume ratio of the solvent varies according to the polarity of the compound, and a small amount of acidic or alkaline reagents can also be added to adjust, such as acetic acid or triethylamine.
  • the eluent system was selected from: A: petroleum ether and ethyl acetate system; B: dichloromethane and methanol system; C: two Methyl chloride and ethyl acetate; the volume ratio of the solvent varies according to the polarity of the compound, and a small amount of acidic or alkaline reagents can also be added to adjust, such as acetic acid or trie
  • Test Example 1 The ADPGlo method determines the activity of the compounds of the present invention on TYK2 and JAK1 kinases
  • the following method is used to determine the degree of inhibition of recombinant human TYK2 and JAK1 kinase activity by the compounds of the present invention under in vitro conditions.
  • This method uses Promega's ADP-Glo TM Kinase Assay Kit (Cat. No. V9102).
  • the above kit is a luminescence kinase detection kit for detecting the content of ADP produced by the kinase reaction.
  • the content of ADP is positively correlated with kinase activity.
  • By measuring the content of ADP it reflects the inhibitory strength of the compound on the kinase activity of TYK2 and JAK1.
  • Recombinant human JAK1 was purchased from Carna (08-144), and TYK2 was purchased from Signalchem (T21-11G).
  • test compound is first dissolved in DMSO to prepare a storage solution, and then the buffer is configured according to the buffer formulation provided in the reagent manual (20mM MgCl 2 , 40mM Tris, 50uM DTT, 0.1mg/ml BSA, pH 7.4), using the buffer for gradient dilution, the final concentration of the test compound in the reaction system ranges from 16000 nM to 0.008 nM.
  • reagent manual 20mM MgCl 2 , 40mM Tris, 50uM DTT, 0.1mg/ml BSA, pH 7.4
  • the reaction is carried out in a 384-well microtiter plate. First, add the compound and a certain amount of TYK2 or JAK1 protein to the wells, and incubate at room temperature for 5 minutes, then add ATP solution and Axltide (TYK2 substrate, final concentration 0.1mg/mL) or IRS1(Y608) Peptide (JAK1 substrate, final concentration is 0.025mg/mL), and incubate with shaking at room temperature for 120 minutes or 60 minutes.
  • TYK2 substrate final concentration 0.1mg/mL
  • IRS1(Y608) Peptide JK1 substrate, final concentration is 0.025mg/mL
  • Table 1 The IC 50 data of the compound of the present invention for inhibiting the activity of JAK family enzymes
  • Test Example 2 HTRF method to determine the activity of the compounds of the present invention on JAK2 and JAK3 kinases
  • the following method is used to determine the degree of inhibition of recombinant human JAK2 and JAK3 kinase activity by the compounds of the present invention under in vitro conditions.
  • This method uses Cisbio's KinEASE-TK Tyrosine Kinase Kit (Cat. No. 62TK0PEB), the principle of this kit is based on time-resolved fluorescence energy resonance transfer (TF-FRET), which measures the phosphorylation of biotinylated peptide substrates mediated by JAK2 or JAK3 kinase
  • T-FRET time-resolved fluorescence energy resonance transfer
  • the degree reflects the inhibitory strength of the compound on the kinase activity.
  • Recombinant human JAK2 and JAK3 were purchased from Carna bioscience (product numbers are JAK2#08-045, JAK3#08-046, respectively).
  • the experimental procedure is briefly described as follows: the test compound is first dissolved in DMSO to prepare a storage solution, and then the test compound is gradually diluted with the buffer provided in the kit.
  • the final concentration of the test compound in the reaction system ranges from 16000M to 0.008nM.
  • the concentration of ATP Km of JAK2 and JAK3 proteins was determined using a gradiently diluted ATP solution (Sigma, A7699-1G). According to the obtained Km value, the ATP concentration in the reaction system was set to 2uM and 5uM, respectively.
  • the reaction is carried out in a 384-well microtiter plate.
  • the fluorescence intensity of each well at 620nM and 665nM emission wavelengths at the excitation wavelength of 304nM was measured on the microplate reader in the TF-FRET mode, and the fluorescence intensity ratio of each well 665/620 was calculated.
  • the percentage inhibition rate of the compound at each concentration was calculated, and the non-linear regression analysis of the compound concentration vs. the inhibition rate was performed by GraphPad Prism 5 software to obtain the compound's IC 50 value, see Table 2.
  • Test Example 3 HTRF method to determine the inhibitory activity of the compound of the present invention on p-STAT3 in Jurkat cells induced by IFN- ⁇ 2b
  • the following method is used to determine the effect of the compound of the present invention on p-STAT3 in Jurkat cells induced by IFN- ⁇ 2b.
  • Jurkat cells were purchased from the cell bank of the Type Culture Collection Committee of the Chinese Academy of Sciences. The cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum, 100U penicillin and 100 ⁇ g/mL streptomycin; cultured in a 37°C, 5% CO 2 incubator.
  • p-STAT3 was measured by LANCE Ultra Phosphorylated STAT3 (Y705) Cellular Detection Kit (PerkinElmer, #TRF4004M).
  • test compound was first dissolved in DMSO to prepare a stock solution, and then gradually diluted with the medium of the corresponding cell to prepare a test sample.
  • the final concentration of the compound was in the range of 10000nM -0.61nM. Inoculate a certain number of Jurkat cells into T25 or T75 culture flasks, place them in a 37°C, 5% CO 2 incubator and starve them overnight, and then plate the starved cells at 200,000 cells (8 ⁇ l cell suspension)/well to 384 Orifice plate.
  • test method of the kit is briefly described as follows: Use LANCE Ultra Lysis Buffer 1 to lyse the cells for 40 minutes, and then add 5ul 4X MIX Eu-labeled anti-STAT3 (Y705) Antibody (0.5nM final) and ULight labeled anti-STAT3 Antibody to a 384-well plate. (5nM final). After 4 hours of incubation at room temperature, the fluorescence intensity of each well at 620nM and 665nM emission wavelengths at the excitation wavelength of 304nM was measured in the TF-FRET mode in a microplate reader, and the fluorescence intensity ratio of each well 665/620 was calculated.
  • Test Example 3 Study on oral pharmacokinetics of the compound of the present invention in SD rats
  • the LC/MS/MS method was used to determine the intravenous injection or intragastric administration of the compound of the present invention in rats, and to determine the drug concentration in plasma at different times to study the pharmacokinetics of the compound of the present invention in rats. Kinetic characteristics.

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Abstract

La présente invention concerne un dérivé de pyrazolo[1,5-a]pyrazine et un procédé de préparation s'y rapportant et une utilisation de celui-ci dans des médicaments. En particulier, la présente invention concerne un dérivé de pyrazolo[1,5-a]pyrazine représenté par la formule générale (I), un procédé de préparation s'y rapportant, un sel pharmaceutiquement acceptable de celui-ci et une utilisation de celui-ci en tant qu'agent thérapeutique, en particulier en tant qu'inhibiteur de kinases de la famille JAK. La définition de chaque groupe substituant dans la formule générale (I) est la même que celle dans la description.
PCT/CN2021/072362 2020-01-22 2021-01-18 Dérivé de pyrazolo[1,5-a]pyrazine et procédé de préparation s'y rapportant et son utilisation WO2021147790A1 (fr)

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Citations (3)

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WO2013033862A1 (fr) * 2011-09-05 2013-03-14 浙江海正药业股份有限公司 Dérivés 4-substitué-(3-substitué-1h-pyrazole-5-amino)-pyrimidines ayant une activité d'inhibition de protéine kinase et son utilisation
CN107278203A (zh) * 2014-12-05 2017-10-20 阵列生物制药公司 作为JANUS激酶抑制剂的4,6‑取代的吡唑并[1,5‑a]吡嗪
CN110267960A (zh) * 2017-01-18 2019-09-20 阿雷生物药品公司 作为RET激酶抑制剂的取代的吡唑并[1,5-a]吡嗪化合物

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CN109400610A (zh) * 2017-08-18 2019-03-01 浙江海正药业股份有限公司 吡咯并三嗪类衍生物、其制备方法及其在医药上的用途

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WO2013033862A1 (fr) * 2011-09-05 2013-03-14 浙江海正药业股份有限公司 Dérivés 4-substitué-(3-substitué-1h-pyrazole-5-amino)-pyrimidines ayant une activité d'inhibition de protéine kinase et son utilisation
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