WO2019228442A1 - 一种jak3选择性抑制剂 - Google Patents

一种jak3选择性抑制剂 Download PDF

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WO2019228442A1
WO2019228442A1 PCT/CN2019/089213 CN2019089213W WO2019228442A1 WO 2019228442 A1 WO2019228442 A1 WO 2019228442A1 CN 2019089213 W CN2019089213 W CN 2019089213W WO 2019228442 A1 WO2019228442 A1 WO 2019228442A1
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compound
pharmaceutically acceptable
acceptable salt
alkyl
formula
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French (fr)
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潘峥婴
石莉扬
李锡涛
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北京大学深圳研究生院
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Priority to JP2020566808A priority Critical patent/JP7443252B2/ja
Priority to EP19809988.9A priority patent/EP3805214B1/en
Priority to CA3101525A priority patent/CA3101525A1/en
Priority to US17/059,211 priority patent/US11578060B2/en
Priority to AU2019278104A priority patent/AU2019278104B2/en
Priority to ES19809988T priority patent/ES2958835T3/es
Priority to KR1020207037750A priority patent/KR20210023902A/ko
Publication of WO2019228442A1 publication Critical patent/WO2019228442A1/zh

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    • 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
    • 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
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A61K31/454—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. pimozide, domperidone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4709—Non-condensed quinolines and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/472—Non-condensed isoquinolines, e.g. papaverine
    • A61K31/4725—Non-condensed isoquinolines, e.g. papaverine containing further heterocyclic rings
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D207/44—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having three double bonds between ring members or between ring members and non-ring members
    • C07D207/444—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having three double bonds between ring members or between ring members and non-ring members having two doubly-bound oxygen atoms directly attached in positions 2 and 5
    • C07D207/448—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having three double bonds between ring members or between ring members and non-ring members having two doubly-bound oxygen atoms directly attached in positions 2 and 5 with only hydrogen atoms or radicals containing only hydrogen and carbon atoms directly attached to other ring carbon atoms, e.g. maleimide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing three or more hetero rings
    • 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

Definitions

  • the invention relates to a JAK3 selective inhibitor.
  • JAK kinase and its downstream signaling and transcription factors play an important role in signal transduction of T cells.
  • JAK1 JAK2, JAK3 and TYK2
  • cytokine receptors bind to cytokine receptors in pairs and participate in regulating cytokine-mediated signaling pathways.
  • JAK3 only paired with JAK1 on ⁇ -common chain-containing cytokine receptors, and participated in the signals of interleukins IL-2, IL-4, IL-7, IL-9, IL-15, IL-21, etc. Conduction.
  • JAK3 is only expressed in the hematopoietic system, so it is generally believed that selective inhibition of JAK3 function achieves a safe and effective immune effect.
  • Tofacitinib a marketed drug developed by Pfizer, was initially developed as a selective JAK3 inhibitor. Later, it was found that it also has strong inhibitory activity on JAK1, which is actually a non-selective JAKs inhibitor.
  • JAK3 inhibitor NIBR3049 developed by Novartis has similar inhibitory activity as tofacitib at the level of enzyme activity, its inhibitory activity on the downstream substrate STAT5 phosphorylation is significantly weaker than tofacitib in cells.
  • An object of the present invention is to provide a JAK3 selective inhibitor having biological activity.
  • the invention provides a compound of formula I (including a stable isotope replacement thereof) or a pharmaceutically acceptable salt thereof,
  • Rh is H or methyl, preferably H
  • Rg is CH, -C-Rf or N, preferably CH;
  • Rf is a substituent, preferably, it is selected from methyl or halogen (such as F, Cl, Br or I);
  • n 0, 1, 2 or 3, preferably 0 or 1, and more preferably 0;
  • Rd is alkenyl or alkynyl, for example, said alkenyl or alkynyl has 2, 3, 4, 5, or 6 carbon atoms;
  • Ra, Rb and Rc are selected from a combination of:
  • Rb is C 1 -C 4 alkylene (such as C 1 -C 3 alkylene, such as methylene, ethylene, 1,3-propylene), and
  • Ra and Rc are hydrogen or C 1 -C 6 alkyl (such as C 1 -C 4 alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl);
  • Rb is C 1 -C 4 alkylene (such as C 1 -C 3 alkylene, such as methylene, ethylene, 1,3-propylene), and
  • Ra and Rc are joined together to form a C 2 -C 4 alkylene (eg, C 2 -C 3 alkylene, such as ethylene, 1,3-propylene);
  • a C 2 -C 4 alkylene eg, C 2 -C 3 alkylene, such as ethylene, 1,3-propylene
  • Ra is hydrogen or C 1 -C 6 alkyl (such as C 1 -C 4 alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl), and
  • Rb and Rc together with the N atom to which they are attached form a 5- or 6-membered saturated heterocyclic ring containing one N atom;
  • Rc is hydrogen or C 1 -C 6 alkyl (such as C 1 -C 4 alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl), and
  • Ra and Rb together with the N atom to which they are attached form a 5- or 6-membered saturated heterocyclic ring containing one N atom.
  • the invention provides a compound of formula II (including a stable isotope replacement thereof) or a pharmaceutically acceptable salt thereof:
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of formula I (including a stable isotope replacement thereof) or a pharmaceutically acceptable salt thereof, or a compound of formula II (including a stable isotope replacement thereof) or Its pharmaceutically acceptable salts, and pharmaceutically acceptable carriers.
  • Pharmaceutically acceptable carriers include inert solid fillers or excipients and sterile aqueous or organic solutions.
  • the compound should be present in the pharmaceutical composition in an amount sufficient to provide the desired dosage of the agent. Techniques for formulating and administering the compounds disclosed in the present invention are well known to those skilled in the art, and can be found, for example, in Remington: The Science and Practice of Pharmacy, Remington Pharmaceutical Science and Practice, 19th Edition, Mack Publishing Company, Easton, PA (1995).
  • the invention provides a compound of formula I (including a stable isotope substitute thereof) or a pharmaceutically acceptable salt thereof, a compound of formula II (including a stable isotope substitute thereof) or a pharmaceutically acceptable salt thereof, or Use of the pharmaceutical composition of the present invention in the preparation of a medicament for treating inflammation such as rheumatoid arthritis.
  • the invention provides compounds of Formula I or Formula II (including their stable isotope replacements) as selective inhibitors of JAK3.
  • Figure 1 Selective evaluation results of compounds in the kinase group
  • Figure 2-5 Cell viability evaluation results of the compound
  • Figures 10-12 Evaluation results of compounds inhibiting the release of stimulated inflammatory factors.
  • BTK Bruton's tyrosine kinase
  • CDI 1,1'-carbonyldiimidazole
  • EGFR epidermal growth factor receptor
  • GSK3 ⁇ Glycogen Synthase Kinase 3- ⁇
  • IL-2 interleukin-2
  • IL-6 interleukin-6
  • IL-15 interleukin-15
  • IFN- ⁇ interferon- ⁇
  • ITK Interleukin-2 Induced T Cell Kinase
  • JAK3 janus kinase 3
  • LPS lipopolysaccharide
  • MCP-1 monocyte chemotactic protein 1
  • PBMC peripheral blood mononuclear cells
  • PBS phosphate buffered saline
  • PE petroleum ether
  • PKC protein kinase C
  • HATU 1- [bis (dimethylamino) methylene] -1H-1,2,3-triazol [4,5-b] pyridinium 3-oxide hexafluorophosphate
  • RuPhos 2-dicyclohexylphosphino-2 ', 6'-diisopropoxybiphenyl.
  • the reagents used herein are all commercially available products without purification.
  • the solvents were re-distilled before use.
  • the reaction was monitored by a thin layer silica gel plate (TLC, GF254, 60-F250, 0.2mm, Yantai Jiangyou Silica Gel Thin Layer Chromatography).
  • Spectra Chemical Silica Gel ZCX-II, 200-300 mesh for flash column chromatography.
  • High-resolution mass spectrometry uses ABI Q-star Elite high-resolution mass spectrometer; final product purity is detected by high-performance liquid phase (HPLC) Agilent 1260 series chromatograph (Agilent PN959990-902 Eclipse Plus C18 (250mm ⁇ 4.6mm) column) The detection wavelength is 254 nm.
  • Step 2 3- (5-fluoro-2- (trifluoromethyl) phenyl) -4- (1H-indol-3-yl) -1H-pyrrole-2,5-dione (compound 40a).
  • Step 3 4- (3- (4- (1H-Indol-3-yl) -2,5-dioxo-2,5-dihydro-1H-pyrrole-3-yl) -4- (tri Fluoromethyl) phenyl) piperazine-1-carboxylic acid tert-butyl ester (Compound 41a).
  • Step 4 3- (5- (4-Acryloylpiperazin-1-yl) -2- (trifluoromethyl) phenyl) -4- (1H-indol-3-yl) -1H-pyrrole- 2,5-dione (Compound 5)
  • Step 1 2- (6-Fluoro-1H-indol-3-yl) -2-oxoethyl acetate (compound 43a).
  • Step 2 3- (6-Fluoro-1H-indol-3-yl) -4- (5-fluoro-2- (trifluoromethyl) phenyl) -1H-pyrrole-2,5-dione ( Compound 44a).
  • Step 3 4- (3- (4- (6-Fluoro-1H-indol-3-yl) -2,5-dioxo-2,5-dihydro-1H-pyrrole-3-yl)- 4- (Trifluoromethyl) phenyl) piperazine-1-carboxylic acid tert-butyl ester (Compound 45a).
  • Step 4 3- (5- (4-Acryloylpiperazin-1-yl) -2- (trifluoromethyl) phenyl) -4- (6-fluoro-1H-indol-3-yl)- 1H-pyrrole-2,5-dione (compound 20).
  • Step 1 2- (5- (4- (tert-butoxycarbonyl) piperazin-1-yl) -2-methoxyphenyl) acetic acid (compound 47d).
  • Step 2 4- (3- (2-Amino-2-oxoethyl) -4-methoxyphenyl) piperazine-1-carboxylic acid tert-butyl ester (compound 48d).
  • Step 3 4- (3- (4- (1H-Indol-3-yl) -2,5-dioxo-2,5-dihydro-1H-pyrrole-3-yl) -4-methoxy Phenyl) piperazine-1-carboxylic acid tert-butyl ester (compound 49d).
  • Step 4 3- (1H-Indol-3-yl) -4- (2-methoxy-5- (piperazin-1-yl) phenyl) -1H-pyrrole-2,5-dione ( Compound 50d).
  • Step 5 3- (5- (4-acryloylpiperazin-1-yl) -2-methoxyphenyl) -4- (1H-indol-3-yl) -1H-pyrrole-2,5 -Diketone (Compound 28).
  • Step 3 4- (3- (4- (1H-Indol-3-yl) -2,5-dioxo-2,5-dihydro-1H-pyrrole-3-yl) -4- (nitro Phenyl) piperazine-1-carboxylic acid tert-butyl ester (Compound 41b).
  • Step 4 3- (5- (4-acryloylpiperazin-1-yl) -2- (nitro) phenyl) -4- (1H-indol-3-yl) -1H-pyrrole-2, 5-dione (Compound 32)
  • Step 1 3-bromo-4- (1H-indol-3-yl) -1H-pyrrole-2,5-dione (Compound 52).
  • indole 0.3 g, 1.2 mmol was dissolved in anhydrous THF (8.0 mL), and a solution of ethyl magnesium bromide in ether (1.57 mL, 4.7 mmol) was added dropwise.
  • This reaction solution was heated under reflux for 2 hours.
  • a solution of 3,4-dibromo-1H-pyrrole-2,5-dione (compound 51, 0.55 g, 4.7 mmol) in THF was slowly added dropwise (about 1 hour) to the reaction system. The reaction was stirred at room temperature for 1 hour.
  • Step 2 4-((4- (1H-Indol-3-yl) -2,5-dioxo-2,5-dihydro-1H-pyrrole-3-yl) amino) piperidine-1- Tert-butyl formate (Compound 53).
  • Step 3 3-((1-acryloylpiperidin-4-yl) amino) -4- (1H-indol-3-yl) -1H-pyrrole-2,5-dione (Compound 34).
  • the in vitro enzyme activity was tested as follows: The kinase was purchased from Carna Biosciences. JAK3 enzyme activity use KinEase TM analysis was assessed with Km and 1 mM ATP concentrations, respectively. ATP kinase enzymatic analysis based KinEase (TM) analysis protocol (Cisbio Bioassays) specifies the protocol.
  • TM ATP kinase enzymatic analysis based KinEase
  • mice T cells For mouse T cells (CTLL-2 cells), after removing growth factors and starving overnight, incubate at 37 ° C with a specified concentration of compound (JAK3 inhibitor or DMSO) for 2 hours, and then perform the specified stimulation (500ng / ml IL) -2 or 500ng / ml IL-15, R & D Systems) for 30 minutes. Cells were collected and lysed in a cell lysis buffer containing a protease and a phosphatase inhibitor. After separation by SDS / PAGE electrophoresis, Western blot analysis was performed and transferred to a nitrocellulose membrane.
  • a specified concentration of compound JK3 inhibitor or DMSO
  • PBMC After thawing PBMC (purchased from Allcells) cells, PBMC was resuspended in RPMI-1640 containing 10% FBS overnight, and then cultured with a specified concentration of a compound (JAK3 inhibitor or DMSO) for 2 hours, and IL -2 (500ng / ml, R & D Systems), IL-15 (500ng / ml, R & D Systems), IL-6 (600ng / ml, R & D Systems), or IFN- ⁇ (400ng / ml, R & D Systems) was stimulated for 30 minutes. Cells were collected and lysed in a cell lysis buffer containing a protease and a phosphatase inhibitor.
  • a compound JAK3 inhibitor or DMSO
  • IL-15 signaling pathway depends on the activity of JAK3; the signal transduction of IL-6 depends on JAK1, JAK2, and TYK2; the IFN- ⁇ signal is only related to JAK1 and TYK2.
  • 300 sodium molybdenum compound 32 can effectively block STAT5 phosphorylation, and in the IL-6 and IFN- ⁇ signaling pathway, even the concentration of compound 32 as high as 10 micromolar can only partially inhibit the phosphorylation of STAT3 and STAT1 .
  • compound 32 Compared with compound NIBR3049, compound 32 not only improves cell viability, but also increases selectivity to other JAKs in a cellular environment with high concentrations of ATP. In contrast, the non-selective inhibitor tofacitib did not show significant selectivity for the inhibition of phosphorylation of downstream substrates stimulated by three cytokines.
  • the elution process was as follows: CTLL-2 cells were treated with the compound for 2 hours; then, for the elution group, the cells were thoroughly washed three times with PBS, and for the non-eluting group, they were kept stationary; then, the cells were stimulated with IL-15 for 30 minutes, Lyse and perform a standard Western blot.
  • the results are shown in Figure 9.
  • Cells were eluted multiple times with PBS after compound culture.
  • Compound 32 covalently bound to JAK3 in the cells could maintain continuous inhibition of STAT5 phosphorylation, while the inhibitory activity of the reversible inhibitor tofatib and compound NIBR3049 was eluted. After the basic loss.
  • JAK1 does not have cysteine residues at the same position as JAK3, 32 will not interfere with the activity of JAK1 after eluting with PBS. Therefore, this cell elution experiment demonstrated that the activity of the selective inhibitor JAK3 is sufficient to effectively inhibit the IL-15-mediated ⁇ c cytokine receptor signaling pathway without interfering with the function of JAK1.
  • erosive pain in joints is usually accompanied by increased release of inflammatory factors, including IL-6, IL-1 ⁇ , TNF- ⁇ , and MCP-1.
  • inflammatory factors including IL-6, IL-1 ⁇ , TNF- ⁇ , and MCP-1.
  • the release of IL-6, IL-1 ⁇ , TNF- ⁇ and MCP-1 inflammatory factors is regulated by negative feedback of the IL-10-JAKs-STAT3 signaling pathway.
  • LPS-induced IL-6 and TNF- ⁇ release analysis was performed as follows: Frozen PBMCs (from Allcells) were thawed in RPMI1640 (Thermo Fisher) containing 10% FBS and recovered overnight at 37 ° C. The next day, the cells were diluted to 1 x 106 cells / ml and seeded in 6-well plates (500 microliters). Compound or DMSO (5 ⁇ l, serially diluted in DMSO) was added to the plate and incubated with the cells for 2 hours at 37 ° C, then stimulated with LPS (5 ⁇ l, 1 ⁇ g / ml) and in 5% CO2 Incubate at 37 ° C for 24 hours. Supernatants were collected using human IL-6 or human THF- ⁇ Duoset ELISA kits (R & D Systems) according to the manufacturer's instructions to determine IL-6 and TNF- ⁇ levels.
  • compound 32 can significantly inhibit the release of LPS-stimulated inflammatory factors IL-6 and TNF- ⁇ ; the non-selective inhibitor tofatib promotes the release of inflammatory factors to varying degrees, because Fatib's inhibition of JAK1 blocked the negative feedback regulation mechanism of IL-10-JAKs-STAT3, while compound 32's selective inhibition of JAK3 maintained the regulatory function of the IL-10 signaling pathway.
  • Neither Compound 32 nor tofacitib affected the release of IL-6 stimulated inflammatory factor MCP-1 (the MCP-1 release signal is not mediated by JAKs), indicating that both compounds release inflammatory factors through the JAK-STAT signaling pathway Make adjustments.
  • compound 32 can selectively inhibit JAK3 and play an important role in regulating the release of inflammatory factors through the JAK3-STATs signaling pathway.

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Abstract

本发明涉及一种式(I)/(II)化合物或其药学上可接受的盐。式(I)中，Rh、Rg、Rf、m、Re、Rd、Ra、Rb和Rc如说明书中所定义。本发明还涉及一种包括上述式(I)/(II)化合物或其药学上可接受的盐的药物组合物，以及上述式(I)/(II)化合物或上述药物组合物在制备用于治疗炎症如类风湿关节炎的药物中的用途。

Description

一种JAK3选择性抑制剂 技术领域
本发明涉及一种JAK3选择性抑制剂。
背景技术
JAK激酶与其下游的信号传导和转录因子(STATs)对T细胞的信号转导具有重要作用。JAK激酶家族有四个亚型，分别是JAK1、JAK2、JAK3和TYK2，其成对地结合在细胞因子受体上，参与调节细胞因子介导的信号通路。其中，JAK3只与JAK1成对结合在含γ-公共链的细胞因子受体上，参与白介素IL-2、IL-4、IL-7、IL-9、IL-15、IL-21等的信号传导。不同于其他JAKs的广泛表达，JAK3只表达在造血系统中，因此人们通常认为选择性抑制JAK3的功能达到安全有效的免疫效果。
辉瑞公司研发的上市药物托法替布(Tofacitinib)一开始作为选择性JAK3抑制剂研发，之后发现其对JAK1也有较强的抑制活性，实际上为非选择性JAKs抑制剂。
Figure PCTCN2019089213-appb-000001
诺华公司发展的高选择性JAK3抑制剂NIBR3049虽然在酶活水平与托法替布有相似的抑制活性，但在细胞内对下游底物STAT5磷酸化的抑制活性却显著弱于托法替布。
Figure PCTCN2019089213-appb-000002
近年来，人们通过共价靶向JAK3特有的半胱氨酸残基Cys909获得了高选择性JAK3抑制剂，包括辉瑞公司发展的PF-06651600，目前已经在临床二期研究阶段。
Figure PCTCN2019089213-appb-000003
本领域中仍然亟需一种具有良好的酶活和细胞活性的JAK3选择性抑制剂。
发明内容
本发明的目的之一在于提供一种具有生物活性的JAK3选择性抑制剂。
在一个方面中，本发明提供了一种式I化合物(包括其稳定同位素替代物)或其药学上可接受的盐，
Figure PCTCN2019089213-appb-000004
其中，
Rh是H或甲基，优选H；
Rg是CH、-C-Rf或N，优选CH；
Rf是一种取代基，优选地，其选自甲基或卤素(如F，Cl，Br或I)；
m是0、1、2或3，优选0或1，更优选0；
Re是一种吸电子基团，选自：叔胺正离子(-N +R' 3，其中R'各自独立地选自H和C 1-C 6烷基)、硝基(-NO 2)、三卤甲基(-CX 3，X＝F、Cl、Br或I)、卤素(如F，Cl，Br和I)、甲酰基(-CHO)、酰基(-CO-C 1-4烷基)、羧基(-COOH)、氰基(-CN)、磺酸基(-SO 3H)；
Rd是烯基或炔基，例如所述烯基或炔基具有2、3、4、5或6个碳 原子；
Ra、Rb和Rc选自以下的组合：
(1)Rb为C 1-C 4亚烷基(如C 1-C 3亚烷基，例如亚甲基、亚乙基、1,3-亚丙基)，且
Ra和Rc为氢或C 1-C 6烷基(如C 1-C 4烷基，例如甲基、乙基、正丙基、异丙基、正丁基、叔丁基)；
(2)Rb为C 1-C 4亚烷基(如C 1-C 3亚烷基，例如亚甲基、亚乙基、1,3-亚丙基)，且
Ra和Rc连在一起形成C 2-C 4亚烷基(如C 2-C 3亚烷基，例如亚乙基、1,3-亚丙基)；
(3)Ra为氢或C 1-C 6烷基(如C 1-C 4烷基，例如甲基、乙基、正丙基、异丙基、正丁基、叔丁基)，且
Rb和Rc与它们所连接的N原子一起形成含有一个N原子的5或6元饱和杂环；
(4)Rc为氢或C 1-C 6烷基(如C 1-C 4烷基，例如甲基、乙基、正丙基、异丙基、正丁基、叔丁基)，且
Ra和Rb与它们所连接的N原子一起形成含有一个N原子的5或6元饱和杂环。
在一个方面中，本发明提供了式II化合物(包括其稳定同位素替代物)或其药学上可接受的盐：
Figure PCTCN2019089213-appb-000005
在一个方面中，本发明提供了一种药物组合物，其包括式I化合物(包括其稳定同位素替代物)或其药学上可接受的盐、或式II化合物(包括其稳定同位素替代物)或其药学上可接受的盐，和药学上可接受的载体。药学上可接受性载体包括惰性固体填充剂或者赋形剂以及无菌水溶 液或者有机溶液。所述化合物应当以足以提供期望的药剂剂量的量存在于所述的药物组合物中。配制并且施用本发明中公开的化合物的技术是本领域技术人员公知的，例如可以在Remington：the Science and Practice of Pharmacy《雷明顿药物科学与实践》，第19版，Mack出版公司，Easton，PA(1995年)中找到。
在一个方面中，本发明提供了式I化合物(包括其稳定同位素替代物)或其药学上可接受的盐、式II化合物(包括其稳定同位素替代物)或其药学上可接受的盐、或者本发明的药物组合物在制备用于治疗炎症如类风湿关节炎的药物中的用途。
在一个方面中，本发明提供了作为JAK3选择性抑制剂的式I或式II化合物(包括其稳定同位素替代物)。
附图说明
图1：化合物在激酶组内的选择性评价结果；
图2-5：化合物的细胞活性评价结果；
图6-8：化合物在细胞中的选择性评价结果；
图9：化合物在细胞洗脱实验中的评价结果；和
图10-12：化合物抑制受刺激的炎性因子释放的评价结果。
具体实施方式
本说明书中的缩写具有下述含义：
Ala：丙氨酸
ATP：三磷酸腺苷
AUC：曲线下面积
Boc：叔丁氧基羰基
BTK：布鲁顿氏酪氨酸激酶
CDI：1,1'-羰基二咪唑
Cys：半胱氨酸
DCM：二氯甲烷
DIEA：N,N-二异丙基乙胺
DMF：二甲基甲酰胺
DMSO：二甲基亚砜
EGFR：表皮生长因子受体
GSK3β：糖原合成酶激酶3-β
HTRF：均相时间分辨荧光
IL-2：白细胞介素-2
IL-6：白细胞介素-6
IL-15：白细胞介素-15
IFN-α：干扰素-α
ITK：白细胞介素-2诱导型T细胞激酶
JAK：janus激酶
JAK3：janus激酶3
Leu：亮氨酸
LPS：脂多糖
Lys：赖氨酸
MCP-1：单核细胞趋化蛋白1
Met：蛋氨酸
PBMC：外周血单核细胞
PBS：磷酸盐缓冲盐水
PE：石油醚
PK：药代动力学
PKC：蛋白激酶C
RA：类风湿性关节炎
RT：室温
STAT：信号转导子和转录激活子
TFA：三氟乙酸
THF：四氢呋喃
Val：缬氨酸
HATU：1-[双(二甲基氨基)亚甲基]-1H-1,2,3-三氮唑[4,5-b]吡啶鎓3-氧化物六氟磷酸盐
Pd 2(dba) 3：三(二亚苄基丙酮)二钯(0)
JohnPhos：2-(二叔丁基膦基)联苯
RuPhos：2-二环己基膦基-2',6'-二异丙氧基联苯。
按照下述方案I、II、III或IV制备化合物。
Figure PCTCN2019089213-appb-000006
Figure PCTCN2019089213-appb-000007
Figure PCTCN2019089213-appb-000008
方案IV
Figure PCTCN2019089213-appb-000009
反应试剂和主要条件：
(a)吲哚，EtMgBr/Et 2O，THF，回流，约1小时；
(b)Boc-保护的胺，DIEA，DMSO，100℃；
(c)(i)TFA/DCM，室温，约15分钟；
(ii)烯丙酰氯/DIEA，THF，H 2O，0℃至室温，约10分钟。
表：化合物的结构和IC 50值
Figure PCTCN2019089213-appb-000010
Figure PCTCN2019089213-appb-000011
Figure PCTCN2019089213-appb-000012
下面结合上述制备方案I-IV，以化合物5、20、28、32、34为例，具体说明化合物的合成过程。其他化合物可以参考上述制备方案I-IV，以类似的方法制备，这是本领域技术人员容易理解的。
如未特别说明，本文所用试剂均为市售商品，未经纯化。溶剂使用前均重蒸。监测反应用薄层硅胶板(TLC，GF254,60-F250,0.2mm，烟台江友硅胶薄层色谱)。快速柱层析用谱科化工硅胶(ZCX-II，200-300目)。NMR采用Bruker Advance 400(1H：400MHz；13C：100MHz)或Bruker Advance 500(1H：500MHz；13C：125MHz)核磁共振仪测定，并以TMS为内标，峰形以s(单峰)，d(双峰)，t(三重峰)，m(多重峰)描述。高分辨质谱(HRMS)采用ABI Q-star Elite高分辨质谱仪；终产物纯度检测采用高效液相(HPLC)Agilent 1260系列色谱仪(Agilent PN959990-902 Eclipse Plus C18(250mm×4.6mm)色谱柱)检测波长为254纳米。
化合物5的制备
3-(5-(4-丙烯酰哌嗪-1-基)-2-(三氟甲基)苯基)-4-(1H-吲哚-3-基)-1H- 吡咯-2,5-二酮(化合物5)的合成
步骤1：2-(5-氟-2-(三氟甲基)苯基)乙酰胺(化合物38a)
将5-氟-2-(三氟甲基)苯乙酸溶于DMF(4.0mL)，分批加入CDI(1.0g，4.5mmol)，室温下搅拌0.5小时后，滴加NH 3(3.6mL 7N甲醇溶液)，继续在室温下搅拌1小时。蒸除溶剂后加入水和乙酸乙酯萃取(2×120mL)，有机相用饱和食盐水(2×40mL)洗涤后收集有机相，无水Na 2SO 4干燥，浓缩后柱层析分离纯化，得到白色固体0.73g，产率73％。
1HNMR(400MHz,DMSO-d 6)δ7.75(dd,J＝8.7,5.6Hz,1H)，7.52(s,1H)，7.32(m，2H)，7.03(s,1H)，3.66(s，2H).；MS(ESI)m/z 222.0(M+H) +.
步骤2：3-(5-氟-2-(三氟甲基)苯基)-4-(1H-吲哚-3-基)-1H-吡咯-2,5-二酮(化合物40a).
将化合物38a(0.30g，1.3mmol)和化合物39(0.41g，2.0mmol)溶于无水THF(8.0mL)，0℃条件下，缓慢滴入叔丁醇钾(5.5mL，1M THF溶液)，恢复温度至10℃继续搅拌反应45分钟。TLC检测反应完全后加入HCl(5N)调节pH至6，蒸除溶剂后，加入乙酸乙酯萃取(2×120mL)，有机相用饱和食盐水(2×40mL)洗涤后收集有机相，无水Na 2SO 4干燥，浓缩后柱层析分离纯化，得到黄色固体0.35g，产率66％。
1HNMR(400MHz,DMSO-d 6)δ11.99(s,1H)，11.23(s,1H)，8.02(d,J＝3.0Hz,1H)，7.97(dd,J＝8.9,5.4Hz,1H)，7.60-7.52(m，1H)，7.49-7.38(m，2H)，7.07(ddd,J＝8.1,7.0,1.1Hz,1H)，6.75(ddd,J＝8.2,7.1,1.1Hz,1H)，6.46(d,J＝8.3Hz,1H).
13CNMR(101MHz,DMSO)δ172.48,172.01,165.30,162.80,137.08,136.25,132.67,126.18,125.48,125.25,122.91,120.95,120.56,120.19,119.96,117.31,117.09,112.94,105.14.
MS(ESI)m/z 375.1(M+H) +.
步骤3：4-(3-(4-(1H-吲哚-3-基)-2,5-二氧代-2,5-二氢-1H-吡咯-3-基)-4-(三氟甲基)苯基)哌嗪-1-甲酸叔丁酯(化合物41a).
将1-Boc-哌嗪(0.44g，2.4mmol)加入化合物40a(0.3g，0.55mmol)的DMSO(2.0mL)溶液中，加热至150℃回流搅拌过夜。TLC检测反应完全后，冷却至室温，加入乙酸乙酯萃取(2×120mL)，有机相用饱和食盐水(2×40mL)洗涤后收集有机相，无水Na 2SO 4干燥，浓缩后柱层析分离纯化，得到黄色固体0.35g，产率66％。
1HNMR(400MHz,DMSO-d 6)δ11.88(s,1H)，11.10(s,1H)，7.95(d,J＝2.9Hz,1H)，7.64(d,J＝9.0Hz,1H)，7.38(d,J＝8.1Hz,1H)，7.18-7.10(m，1H)，7.09-6.99(m，1H)，6.92(d,J＝2.6Hz,1H)，6.72(dd,J＝8.2,7.0Hz,1H)，6.61(d,J＝8.2Hz,1H)，3.34-3.22(m，5H)，3.24-3.09(m,4H)，1.38(s，9H).
13CNMR(101MHz,DMSO)δ172.85,172.40,154.34,152.82,136.97,135.64,132.09,131.86,131.85,128.70,128.42,126.50,126.50,125.39,125.38,122.69,121.37,120.70,117.97,114.87,112.64,105.55,79.57,49.00,47.32,30.63,29.52,28.55.
MS(ESI)m/z 541.2(M+H) +.
步骤4：3-(5-(4-丙烯酰哌嗪-1-基)-2-(三氟甲基)苯基)-4-(1H-吲哚-3-基)-1H-吡咯-2,5-二酮(化合物5)
将化合物41a(0.10g，0.18mmol)溶于2.0mL DCM，加入TFA(2.0mL)，室温搅拌15分钟。TLC检测反应完全后，蒸除TFA和DCM，得到的中间体不经进一步纯化，干燥后直接用于下一步反应。上一步反应中间体溶于THF(2.0mL)和水(1滴)的混合溶剂中，冰浴条件下加入N,N-二异丙基乙胺(0.10mL,0.36mmol)和丙烯酰氯(24μL,0.27mmol)。移除冰浴，反应液在室温下搅拌10分钟。TLC检测反应完全后，加入乙酸乙酯萃取(2×120mL)，有机相用饱和食盐水(2×40mL)洗涤后收集，无水Na 2SO 4干燥，浓缩后柱层析分离纯化，得到黄色固体73mg，两步产率82％。
1HNMR(400MHz,DMSO-d 6)δ11.88(d,J＝3.0Hz,1H)，11.11(s,1H)，7.96(d,J＝2.6Hz,1H)，7.65(d,J＝8.9Hz,1H)，7.38(d,J＝8.1Hz,1H)，7.18-7.10(m，1H)，7.08-7.00(m，1H)，6.96(d,J＝2.5Hz,1H)，6.76(m，2H)，6.65(d,J＝8.3Hz,1H)，6.11(dd,J＝16.6,2.4Hz,1H)，5.68(dd,J＝10.4,2.4Hz,1H)，3.66-3.46(m,4H)，3.23(m,4H).
13CNMR(101MHz,DMSO)δ172.64,172.20,164.62,152.49,136.73,135.43,131.85,131.65,128.49,128.34,127.86,126.28,125.18,123.57,122.50,121.15,120.51,117.67,114.54,112.42,105.35,47.58,47.01,44.37,40.90.
C 26H 21F 3N 4O 3[M+H] +的HRMS(ESI)m/z计算值：495.1566；实测值：495.1578.
化合物20的制备
3-(5-(4-丙烯酰哌嗪-1-基)-2-(三氟甲基)苯基)-4-(6-氟-1H-吲哚-3-基)-1H-吡咯-2,5-二酮(化合物20)的合成
步骤1：2-(6-氟-1H-吲哚-3-基)-2-氧代乙酸乙酯(化合物43a).
将化合物42a(0.50g，3.7mmol)溶于DCM(40mL)，在冰浴条件下，滴加Et 2AlCl(5.6mL 1M的己烷溶液)，保持0℃搅拌30分钟后，在此反应体系中滴加草酰氯单乙酯(0.61mL，5.5mmol)，继续保持0℃搅拌反应2小时。TLC检测反应完全后加入冰水淬灭。蒸除溶剂，加入水和乙酸乙酯萃取(3×50mL)，有机相用饱和食盐水(2×30mL)洗涤后收集，无水Na 2SO 4干燥，浓缩后柱层析分离纯化，得到灰白色固体0.38g，产率50％。
1HNMR(400MHz,DMSO-d 6)δ12.41(s,1H)，8.44(s,1H)，8.14(dd,J＝8.7,5.5Hz,1H)，7.35(dd,J＝9.5,2.4Hz,1H)，7.13(ddd,J＝9.8,8.7,2.4Hz,1H)，4.35(q，J＝7.1Hz，2H)，1.33(t，J＝7.1Hz，3H).
MS(ESI)m/z 236.1(M+H) +.
步骤2：3-(6-氟-1H-吲哚-3-基)-4-(5-氟-2-(三氟甲基)苯基)-1H-吡咯-2,5-二酮(化合物44a).
将化合物43a(0.30g，1.3mmol)和化合物38a(0.19g，0.85mmol)溶于无水THF(4.0mL)，0℃条件下，缓慢滴入叔丁醇钾(4.0mL，1M的THF溶液)，恢复温度至10℃继续搅拌反应45分钟。TLC检测反应完全后加入HCl(5N)调节pH至6，蒸除溶剂后，加入乙酸乙酯萃取(3×50mL)，有机相用饱和食盐水(2×20mL)洗涤后收集有机相，无水Na 2SO 4干燥，浓缩后柱层析分离纯化，得到黄色固体0.25g，产率75％。
1HNMR(400MHz,DMSO-d6)δ12.06(s,1H)，11.26(s,1H)，8.03(d,J＝3.1Hz,1H)，8.00(dd,J＝8.9,5.4Hz,1H)，7.60(m，1H)，7.49(dd,J＝9.2,2.7Hz,1H)，7.42(dd,J＝8.9,4.8Hz,1H)，6.94(m，1H)，6.14(dd,J＝11.1,2.5Hz,1H).
MS(ESI)m/z 393.0(M+H) +.
步骤3：4-(3-(4-(6-氟-1H-吲哚-3-基)-2,5-二氧代-2,5-二氢-1H-吡咯-3-基)-4-(三氟甲基)苯基)哌嗪-1-甲酸叔丁酯(化合物45a).
将1-Boc-哌嗪(0.44g，2.4mmol)加入化合物44a(0.3g，0.55mmol) 的DMSO(2.0mL)溶液中，加热至150℃回流搅拌过夜。TLC检测反应完全后，冷却至室温，加入乙酸乙酯萃取(2×120mL)，有机相用饱和食盐水(2×40mL)洗涤后收集有机相，无水Na 2SO 4干燥，浓缩后柱层析分离纯化，得到黄色固体0.35g，产率30％。
1HNMR(400MHz,DMSO-d 6)δ11.87(s,1H)，11.13(s,1H)，7.89(s,1H)，7.64(d,J＝9.0Hz,1H)，7.17(dd,J＝9.5,2.3Hz,1H)，7.13(dd,J＝8.8,2.6Hz,1H)，6.94(d,J＝2.6Hz,1H)，6.70-6.57(m，2H)，3.33-3.25(m,4H)，3.22(m,4H)，1.39(s，9H).
MS(ESI)m/z 559.2(M+H) +.
步骤4：3-(5-(4-丙烯酰哌嗪-1-基)-2-(三氟甲基)苯基)-4-(6-氟-1H-吲哚-3-基)-1H-吡咯-2,5-二酮(化合物20).
将化合物45a(0.10g，0.18mmol)溶于2.0mL DCM，加入TFA(2.0mL)，室温搅拌15分钟。TLC检测反应完全后，蒸除TFA和DCM，得到的中间体不经进一步纯化，干燥后直接用于下一步反应。上一步反应中间体溶于THF(2.0mL)和水(1滴)的混合溶剂中，冰浴条件下加入N,N-二异丙基乙胺(0.10mL,0.36mmol)和丙烯酰氯(24μL,0.27mmol)。移除冰浴，反应液在室温下搅拌10分钟。TLC检测反应完全后，加入乙酸乙酯萃取(2×120mL)，有机相用饱和食盐水(2×40mL)洗涤后收集，无水Na 2SO 4干燥，浓缩后柱层析分离纯化，得到黄色固体73mg，两步产率80％。
1HNMR(400MHz,DMSO-d 6)δ11.86(s,1H)，11.12(s,1H)，7.87(s,1H)，7.64(d,J＝9.0Hz,1H)，7.21-7.08(m，2H)，6.97(d,J＝2.3Hz,1H)，6.78(m，1H)，6.69(m，1H)，6.62(m，1H)，6.11(dd,J＝16.7,2.3Hz,1H)，5.68(dd,J＝10.4,2.3Hz,1H)，3.55(m,4H)，3.24(m,4H).
13CNMR(101MHz,DMSO-d 6)δ172.70,172.30,164.88,158.20,152.82,137.14,137.02,135.41,132.57,129.48,128.63,128.05,122.60,122.51,122.17,117.72,114.79,109.16,108.92,105.65,98.84,98.59,47.75,47.16,44.65,41.14.
C 26H 20F 4N 4O 3[M+H] +的HRMS(ESI)m/z计算值：513.1472；实测值：513.1479.纯度99.2％。
化合物28的制备
3-(5-(4-丙烯酰哌嗪-1-基)-2-甲氧基苯基)-4-(1H-吲哚-3-基)-1H-吡咯 -2,5-二酮(化合物28)的合成
步骤1：2-(5-(4-(叔丁氧基羰基)哌嗪-1-基)-2-甲氧基苯基)乙酸(化合物47d).
将化合物46d(0.52g，2.0mmol)，1-Boc-哌嗪(0.49g，2.6mmol)，叔丁醇钠(0.59g，2.6mmol)，2-(二叔丁基膦基)联苯(JohnPhos，0.16g，0.41mmol)和Pd 2(dba) 3(0.19g，0.20mmol)溶于无水甲苯(15mL)中，通入氩气除氧后，微波加热至110℃反应1小时。冷却至室温，TLC监测反应完全后，硅藻土过滤，将滤液pH调至5并用乙酸乙酯萃取(3×100mL)，有机相用饱和食盐水(2×40mL)洗涤后收集，无水Na 2SO 4干燥，浓缩后柱层析分离纯化，得到白色固体0.35g，产率49％。
1HNMR(500MHz,DMSO-d 6)δ6.87-6.84(m，1H)，6.84(s,1H)，6.82(d,J＝2.8Hz,1H)，3.68(s,3H)，3.45(s，2H)，3.45-3.41(m,4H)，2.95-2.93(m,4H)，1.42(s，9H).
13CNMR(126MHz,DMSO)δ173.04,154.32,152.01,145.32,124.49,121.12,116.45,111.74,79.40,56.13,50.34,36.23,28.53.
MS/ESI 351.2(M+1) +
步骤2：4-(3-(2-氨基-2-氧代乙基)-4-甲氧基苯基)哌嗪-1-甲酸叔丁酯(化合物48d).
将化合物47d(0.30g，0.86mmol)溶于DMF(4.0mL)中，分批加入CDI(0.29g，1.29mmol)，室温下搅拌0.5小时后，滴加NH 3(3.6mL，7N的甲醇溶液)，继续在室温下搅拌1小时。蒸除溶剂后加入水和乙酸乙酯萃取(2×120mL)，有机相用饱和食盐水(2×40mL)洗涤后收集有机相，无水Na 2SO 4干燥，浓缩后柱层析分离纯化，得到白色固体0.73g，产率73％。
1HNMR(400MHz,DMSO-d 6)δ7.75(dd,J＝8.7,5.6Hz,1H)，7.52(s,1H)，7.32(m，2H)，7.03(s,1H)，3.66(s，2H)；
MS(ESI)m/z 222.0(M+H) +.
步骤3：4-(3-(4-(1H-吲哚-3-基)-2,5-二氧代-2,5-二氢-1H-吡咯-3-基)-4-甲氧基苯基)哌嗪-1-甲酸叔丁酯(化合物49d).
将化合物48d(0.2g，0.57mmol)和化合物38a(0.17g，0.85mmol)溶于无水THF(4.0mL)，0℃条件下，缓慢滴入叔丁醇钾(2.2mL，1M的THF溶液)，恢复温度至10℃继续搅拌反应45分钟。TLC检测反应 完全后加入HCl(5N)调节pH至5，蒸除溶剂后，加入乙酸乙酯萃取(3×40mL)萃取，有机相用饱和食盐水(2×20mL)洗涤后收集有机相，无水Na 2SO 4干燥，浓缩得到中间体49d。
MS(ESI)m/z 503.2(M+H) +.
步骤4：3-(1H-吲哚-3-基)-4-(2-甲氧基-5-(哌嗪-1-基)苯基)-1H-吡咯-2,5-二酮(化合物50d).
将化合物49d(0.12g，0.24mmol)溶于2.0mL DCM，加入TFA(2.0mL)，室温搅拌15分钟。TLC检测反应完全后，蒸除TFA和DCM，加入水和乙酸乙酯(2×60mL)萃取，有机相用饱和食盐水(2×30mL)洗涤后收集，无水Na 2SO 4干燥，浓缩后柱层析分离纯化，得到黄色固体0.15g，两步产率52％。
1HNMR(400MHz,DMSO-d 6)δ11.80(s,1H)，7.91(s,1H)，7.37(d,J＝8.1Hz,1H)，7.02(ddd,J＝8.2,7.0,1.1Hz,1H)，6.95(dd,J＝9.0,3.0Hz,1H)，6.88(d,J＝9.1Hz,1H)，6.78(d,J＝2.9Hz,1H)，6.65(ddd,J＝8.2,7.0,1.1Hz,1H)，6.51-6.41(m，1H)，3.26(s,3H)，2.89-2.81(m,4H)，2.80-2.78(m,4H).
13CNMR(101MHz,DMSO)δ173.08,172.67,152.06,145.93,136.87,134.69,130.87,128.29,125.38,122.39,121.29,121.12,120.23,119.96,118.58,113.08,112.38,106.29,55.97,50.91,45.78.
MS/ESI 503.2(M+1) +.
步骤5：3-(5-(4-丙烯酰哌嗪-1-基)-2-甲氧基苯基)-4-(1H-吲哚-3-基)-1H-吡咯-2,5-二酮(化合物28).
将化合物50d(0.12g，0.24mmol)溶于THF(2.0mL)和水(1滴)的混合溶剂中，冰浴条件下加入N,N-二异丙基乙胺(0.16mL,0.96mmol)和丙烯酰氯(30μL,0.36mmol)。移除冰浴，反应液在室温下搅拌10分钟。TLC检测反应完全后，加入乙酸乙酯萃取(2×60mL)，有机相用饱和食盐水(2×30mL)洗涤后收集，无水Na 2SO 4干燥，浓缩后柱层析分离纯化，得到黄色固体90mg，两步产率82％。
1HNMR(400MHz,DMSO-d 6)δ11.81(s,1H)，10.93(s,1H)，7.93(d,J＝2.8Hz,1H)，7.38(d,J＝8.1Hz,1H)，7.07-6.98(m，2H)，6.92(d,J＝9.0Hz,1H)，6.84(d,J＝2.9Hz,1H)，6.80(dd,J＝16.7,10.5Hz,1H)，6.66(t，J＝7.3Hz,1H)，6.45(d,J＝8.1Hz,1H)，6.10(dd,J＝16.7,2.4Hz,1H)， 5.68(dd,J＝10.4,2.4Hz,1H)，3.59(m,4H)，3.29(s,3H)，2.91(m,4H).
13CNMR(101MHz,DMSO-d 6)δ173.05,172.62,164.74,152.47,144.97,136.88,134.75,130.96,128.69,128.10,127.93,125.33,122.42,121.25,121.20,120.60,120.26,119.13,113.12,112.42,106.23,55.98,50.83,50.22,45.27,41.73.
C 26H 24N 4O 4[M+H] +的HRMS(ESI)m/z计算值：457.1798；实测值：457.1794.
化合物32的制备
步骤1：2-(5-氟-2-(硝基)苯基)乙酰胺(化合物38b)
将5-氟-2-(硝基)苯乙酸(1.0g，5.0mmol)溶于DMF(4.0mL)，分批加入CDI(1.2g，7.5mmol)，室温下搅拌0.5小时后，滴加NH 3(3.5mL 7N甲醇溶液)，继续在室温下搅拌1小时。蒸除溶剂后加入水和乙酸乙酯萃取(2×120mL)，有机相用饱和食盐水(2×40mL)洗涤后收集有机相，无水Na 2SO 4干燥，浓缩后柱层析分离纯化，得到白色固体0.70g，产率70％。
1H NMR(400MHz,DMSO-d 6)δ8.11(dd,J＝9.0,5.2Hz,1H),7.54(s,1H),7.46–7.29(m,2H),7.02(s,1H),3.88(s,2H).
MS(ESI)m/z 199.1(M+H) +.
步骤2：3-(5-氟-2-(硝基)苯基)-4-(1H-吲哚-3-基)-1H-吡咯-2,5-二酮(化合物40b)
Figure PCTCN2019089213-appb-000013
将化合物38b(0.30g，1.5mmol)和化合物39(0.45g，2.2mmol)溶于无水THF(15mL)，0℃条件下，缓慢滴入叔丁醇钾(7.5mL，1M THF溶液)，恢复温度至10℃继续搅拌反应45分钟。TLC检测反应完全后加入HCl(5N)调节pH至6，蒸除溶剂后，加入乙酸乙酯萃取(2×120mL)，有机相用饱和食盐水(2×40mL)洗涤后收集有机相，无水Na 2SO 4干燥，浓缩后柱层析分离纯化，得到黄色固体0.30g，产率72％。
MS(ESI)m/z 352.2(M+H) +.
步骤3：4-(3-(4-(1H-吲哚-3-基)-2,5-二氧代-2,5-二氢-1H-吡咯-3- 基)-4-(硝基)苯基)哌嗪-1-甲酸叔丁酯(化合物41b).
Figure PCTCN2019089213-appb-000014
将1-Boc-哌嗪(0.64g，3.4mmol)加入化合物40b(0.3g，0.85mmol)的DMSO(2.0mL)溶液中，加热至150℃回流搅拌过夜。TLC检测反应完全后，冷却至室温，加入乙酸乙酯萃取(2×120mL)，有机相用饱和食盐水(2×40mL)洗涤后收集有机相，无水Na 2SO 4干燥，浓缩后柱层析分离纯化，得到黄色固体0.27g，产率62％。
1H NMR(400MHz,DMSO-d 6)δ11.94(s,1H),11.12(s,1H),8.17(d,J＝9.4Hz,1H),8.03(s,1H),7.42(d,J＝8.1Hz,1H),7.16–6.94(m,2H),6.74(t,J＝7.6Hz,1H),6.65–6.49(m,2H),3.14(d,J＝48.7Hz,4H),2.97(s,4H),1.37(s,9H).
13C NMR(101MHz,DMSO)δ172.88,171.47,154.25,153.43,137.86,137.08,132.84,131.86,129.73,129.47,127.83,124.65,122.61,121.04,120.56,116.08,114.18,112.80,104.45,79.61,60.29,57.90,46.77,28.52.
MS(ESI)m/z 518.4(M+H) +.
步骤4：3-(5-(4-丙烯酰哌嗪-1-基)-2-(硝基)苯基)-4-(1H-吲哚-3-基)-1H-吡咯-2,5-二酮(化合物32)
Figure PCTCN2019089213-appb-000015
将化合物41b(0.10g，0.19mmol)溶于2.0mL DCM，加入TFA(2.0 mL)，室温搅拌15分钟。TLC检测反应完全后，蒸除TFA和DCM，得到的中间体不经进一步纯化，干燥后直接用于下一步反应。上一步反应中间体溶于THF(2.0mL)和水(1滴)的混合溶剂中，冰浴条件下加入N,N-二异丙基乙胺(70μL,0.38mmol)和丙烯酰氯(26μL,0.28mmol)。移除冰浴，反应液在室温下搅拌10分钟。TLC检测反应完全后，加入乙酸乙酯萃取(2×120mL)，有机相用饱和食盐水(2×40mL)洗涤后收集，无水Na 2SO 4干燥，浓缩后柱层析分离纯化，得到黄色固体70mg，两步产率78％。
1H NMR(400MHz,DMSO-d 6)δ11.96(s,1H),11.14(s,1H),8.18(d,J＝9.3Hz,1H),8.03(s,1H),7.41(d,J＝8.1Hz,1H),7.07(d,J＝9.6Hz,1H),7.02(d,J＝7.9Hz,1H),6.82–6.71(m,1H),6.70–6.62(m,1H),6.60–6.50(m,2H),6.08(d,J＝16.6Hz,1H),5.66(d,J＝10.5Hz,1H),3.26(m,4H),3.10(m,4H).
13C NMR(101MHz,DMSO-d 6)δ173.17,171.51,164.85,153.40,138.29,137.06,133.13,131.87,129.76,128.52,128.09,127.85,124.69,122.67,121.05,120.60,115.95,114.10,112.83,104.46,80.45,47.53,46.29,44.15,43.52.
C 25H 21N 5O 5[M+H] +的HRMS(ESI)m/z的计算值：472.1543；实测值：472.1539.
化合物34的制备
3-((1-丙烯酰哌啶-4-基)氨基)-4-(1H-吲哚-3-基)-1H-吡咯-2,5-二酮(化合物34)的合成
步骤1：3-溴-4-(1H-吲哚-3-基)-1H-吡咯-2,5-二酮(化合物52).
在带滴液漏斗的两口圆底烧瓶中，将吲哚(0.3g，1.2mmol)溶于无水THF(8.0mL)，滴加乙基溴化镁的乙醚溶液(1.57mL，4.7mmol)，此反应液加热回流2小时。冷却至室温后，向反应体系中缓慢滴加(约1小时)3,4-二溴-1H-吡咯-2,5-二酮(化合物51,0.55g，4.7mmol)的THF溶液，继续在室温下搅拌反应1小时。TLC检测反应完全后，用稀盐酸至pH至9，加入氯化铵饱和溶液和乙酸乙酯萃取(2×60mL)，有机相用饱和食盐水(2×30mL)洗涤后收集，无水Na 2SO 4干燥，浓缩后柱层析分离纯化，得到黄色固体0.28g，产率82％。
1HNMR(400MHz,DMSO-d 6)δ12.10(s,1H)，11.35(s,1H)，8.03(d, J＝2.9Hz,1H)，7.89(dt，J＝8.1,1.0Hz,1H)，7.51(dt，J＝8.1,1.0Hz,1H)，7.22(ddd,J＝8.1,7.0,1.2Hz,1H)，7.14(ddd,J＝8.1,7.1,1.2Hz,1H).
13CNMR(101MHz,DMSO)δ170.75,167.99,138.54,137.01,131.54,125.05,122.95,122.77,120.92,115.13,112.84,104.25.
MS/ESI 291.0(M+1) +
步骤2：4-((4-(1H-吲哚-3-基)-2,5-二氧代-2,5-二氢-1H-吡咯-3-基)氨基)哌啶-1-甲酸叔丁基酯(化合物53).
将化合物52(0.13g，0.45mmol)和Boc保护的4-氨基哌啶(0.18g，0.89mmol)溶于DMSO(1.5mL)，加入DIEA(0.15mL,0.89mmol)，加热至126℃搅拌反应过夜。冷却至室温，TCL监测反应完全后加入水和乙酸乙酯萃取(2×40mL)，有机相用饱和食盐水(2×20mL)洗涤后收集，无水Na 2SO 4干燥，浓缩后柱层析分离纯化，得到黄色固体0.11g，产率60％。
1HNMR(400MHz,DMSO-d 6)δ11.21(d,J＝2.5Hz,1H)，10.34(s,1H)，7.40(d,J＝8.1Hz,1H)，7.37-7.27(m，2H)，7.14-7.06(m，1H)，7.04-6.95(m，1H)，6.86(d,J＝9.0Hz,1H)，3.67(m，2H)，3.43(m，1H)，1.97(m，2H)，1.47(m，2H)，1.31(s，9H)，1.26(m，2H). 13CNMR(101MHz,DMSO)
13CNMR(101MHz,DMSO)δ173.82,169.57,154.19,143.23,136.10,128.71,126.48,121.70,119.91,119.37,112.05,104.59,100.05,93.48,79.22,50.31,32.01,28.52.
MS/ESI 410.1(M+1) +.
步骤3：3-((1-丙烯酰哌啶-4-基)氨基)-4-(1H-吲哚-3-基)-1H-吡咯-2,5-二酮(化合物34).
将化合物53(0.10g，0.18mmol)溶于2.0mL DCM，加入TFA(2.0mL)，室温搅拌15分钟。TLC检测反应完全后，蒸除TFA和DCM，得到的中间体不经进一步纯化，干燥后直接用于下一步反应。上一步反应中间体溶于THF(2.0mL)和水(1滴)的混合溶剂中，冰浴条件下加入N,N-二异丙基乙胺(0.10mL,0.36mmol)和丙烯酰氯(24μL,0.27mmol)。移除冰浴，反应液在室温下搅拌10分钟。TLC检测反应完全后，加入乙酸乙酯萃取(2×120mL)，有机相用饱和食盐水(2×40mL)洗涤后收集，无水Na 2SO 4干燥，浓缩后柱层析分离纯化，得到黄色固体80mg， 两步产率90％。
1HNMR(400MHz,DMSO-d 6)δ11.23(s,1H)，10.43(s,1H)，7.54-7.20(m，3H)，7.09(d,J＝7.1Hz,1H)，7.01(t，J＝7.0Hz,1H)，6.64(d,J＝7.5Hz,1H)，5.75(t，J＝14.6Hz,1H)，5.27(d,J＝7.8Hz,1H)，3.87(d,J＝12.2Hz,1H)，2.93(m，1H)，2.82(m，1H)，2.56(m，1H)，1.75(m，1H)，1.57(m，2H)，0.84(m，1H).
13CNMR(101MHz,DMSO)δ173.79,169.60,164.78,142.34,136.13,128.48,128.46,127.84,127.35,126.68,121.84,119.96,119.59,112.24,104.14,50.10,49.39,41.81,30.50,28.52.
C 20H 20N 4O 3[M+H] +的HRMS(ESI)m/z的计算值：365.1535；实测值：365.1541.纯度99.3％.
生物活性测试
(1)体外酶活实验
在K m ATP条件(0.6微摩)下和在高浓度ATP(1毫摩)条件下，进行体外酶活实验。体外酶活实验的结果列于上述表格中。
体外酶活的测试方法如下：激酶购自Carna Biosciences。JAK3的酶活性使用
Figure PCTCN2019089213-appb-000016
KinEase TM分析分别用Km和1mM的ATP浓度评估。ATP激酶酶学分析根据
Figure PCTCN2019089213-appb-000017
KinEase TM分析说明书(Cisbio Bioassays)指定的方案进行。
(2)选择性评价
为评价化合物32在激酶组内的选择性，我们选择了50种代表性激酶进行初步选择性实验，结果如图1所示。
结果显示化合物32具有良好的选择性，在1微摩的测试浓度下，对大多数激酶的抑制率不超过50％，只对PKCα、PKCγ和GSK3β三种激酶有超过50％的抑制率，这与NIBR3049的选择性结果类似。化合物32在JAKs家族成员及Cys909同等位置上有半胱氨酸的其他10种激酶间都表现出良好的选择性，意味着化合物32可作为小分子探针用于JAK3的功能及JAKs-STATs信号通路的研究。
(3)细胞活性评价
为评价化合物32在细胞中的活性情况，我们在细胞中检测化合物32抑制下游底物STAT5磷酸化的能力。
对于小鼠T细胞(CTLL-2细胞)，在除去生长因子且饥饿过夜后，在37℃用规定浓度的化合物(JAK3抑制剂或DMSO)培养2小时，然后进行指定的刺激(500ng/毫升IL-2或500ng/毫升IL-15，R&D Systems)30分钟。收集细胞并在含有蛋白酶和磷酸酶抑制剂的细胞裂解缓冲液中裂解。然后通过SDS/PAGE电泳分离后进行Western印迹分析并转移至硝酸纤维素膜。磷酸化STAT5、STAT5和β-actin(全部抗体来自Cell Signaling Technologies)分别用特异性抗体印迹。结果见图2和3。量化条带强度，并使用GraphPad Prism软件计算EC50值。
在CTLL-2中，600纳摩的化合物32几乎能完全抑制IL-2诱导的STAT5磷酸化(EC 50＝305纳摩)，而化合物NIBR3049需要6000纳摩才能完全阻断STAT5的激活(EC 50＝1999纳摩)。化合物32对IL-15刺激的STAT5磷酸化抑制更敏感(EC 50＝141纳摩)。
类似地，对于人外周血单核细胞(PBMC)，与NIBR3049比较，化合物32对IL-2及IL-15诱导的STAT5磷酸化亦显示出更强的抑制活性。结果见图4和5。
分析方法：在将PBMC(购自Allcells)细胞解冻后，将PBMC重悬于含有10％FBS的RPMI-1640中过夜，然后用规定浓度的化合物(JAK3抑制剂或DMSO)培养2小时，并用IL-2(500ng/毫升,R&D Systems)、IL-15(500ng/毫升,R&D Systems)、IL-6(600ng/毫升,R&D Systems)或IFN-α(400ng/毫升,R&D Systems)刺激30分钟。收集细胞并在含有蛋白酶和磷酸酶抑制剂的细胞裂解缓冲液中裂解。然后通过SDS/PAGE电泳分离后进行Western印迹分析并转移至硝酸纤维素膜。磷酸化STAT5、磷酸化STAT3和磷酸化β-actin(全部抗体来自Cell Signaling Technologies)分别用特异性抗体印迹。β-actin被等量加样印迹。量化条带强度，并使用GraphPad Prism软件计算EC 50值。
(4)细胞选择性评价
为进一步探究化合物32在细胞中的选择性，我们用不同细胞因子(IL-15、IL-6或IFN-α)刺激PBMC后，分别检测抑制剂化合物对JAKs下游底物磷酸化的抑制程度。分析方法如(4)中所述，结果见图6、7和8。
这些细胞因子中只有IL-15信号通路依赖JAK3的活性；IL-6的信 号传导依赖JAK1、JAK2和TYK2；IFN-α信号只与JAK1和TYK2相关。300钠摩的化合物32能有效阻断STAT5的磷酸化，而在IL-6和IFN-α信号通路中，即使化合物32浓度高达10微摩也只能对STAT3和STAT1的磷酸化产生部分抑制作用。与化合物NIBR3049比较，化合物32不仅提高了细胞活性，还在高浓度ATP的细胞环境中提高了对其他JAKs的选择性。相反，非选择性抑制剂托法替布对三种细胞因子刺激的下游底物磷酸化抑制并未表现出明显的选择性。
(5)细胞洗脱实验
为进一步证明化合物32在细胞中与JAK3共价结合，我们进行细胞洗脱(wash-out)实验。
洗脱过程如下：用化合物处理CTLL-2细胞2小时；然后，对于洗脱组，用PBS三次充分洗涤细胞，对于非洗脱组，保持不动；然后，细胞用IL-15刺激30分钟，裂解并进行标准Western印迹。结果如图9所示。细胞用化合物培养后经PBS多次洗脱，与细胞中的JAK3共价结合的化合物32能保持对STAT5磷酸化的持续抑制，而可逆抑制剂托法替布和化合物NIBR3049的抑制活性在洗脱后却基本丧失。由于JAK1在JAK3相同位置上不存在半胱氨酸残基，经PBS洗脱后32不会对JAK1的活性产生干扰。因此，这种细胞洗脱实验在不干扰JAK1功能的条件下证明了选择性抑制剂JAK3的活性足以有效抑制IL-15介导的γ c细胞因子受体信号通路。
(6)抑制LPS刺激的炎性因子释放
在类风湿性关节炎(rheumatoid arthritis，RA)病人中，关节的侵蚀疼痛通常伴随炎性因子释放的增加，包括IL-6、IL-1β、TNF-α和MCP-1等。其中IL-6、IL-1β、TNF-α和MCP-1炎性因子的释放受IL-10-JAKs-STAT3信号通路的负反馈调节。
如下进行LPS诱导的IL-6和TNF-α释放分析：将冷冻的PBMC(来自Allcells)在含有10％FBS的RPMI1640(Thermo Fisher)中解冻，并在37℃回收过夜。第二天，将细胞稀释至1×106个细胞/毫升并在6孔板中接种(500微升)。将化合物或DMSO(5微升，在DMSO中连续稀释)加入平板中，并在37℃下与细胞一起培养2小时，然后用LPS(5微升，1微克/毫升)刺激并在5％CO2、37℃培养24小时。根据制造商的说明， 使用人IL-6或人THF-αDuoset ELISA试剂盒(R&D Systems)收集上清液以测定IL-6和TNF-α水平。
以类似方式，进行IL-6刺激的炎性因子MCP-1释放分析。实验结果如图10-12所示。
在PBMC细胞中，化合物32能显著抑制LPS刺激的炎性因子IL-6和TNF-α释放；而非选择性抑制剂托法替布却不同程度地促进炎性因子的释放，这是因为托法替布对JAK1的抑制阻断了IL-10-JAKs-STAT3的负反馈调节机制，而化合物32对JAK3的选择性抑制保持了IL-10信号通路的调节功能。化合物32与托法替布都不影响IL-6刺激的炎性因子MCP-1释放(MCP-1释放信号不由JAKs介导)，说明两个化合物均通过JAK-STAT信号通路炎性因子的释放进行调节。综上，化合物32能选择性地抑制JAK3，并通过JAK3-STATs信号通路对炎性因子的释放起到重要的调节作用。
(7)药代动力学评价
通过静脉与口服两种给药途径，我们在小鼠体内对化合物32的药代动力学性质进行评价。
对于体内药代动力学研究，雄性ICR小鼠(n＝3)禁食过夜并以静脉内剂量(2mg/kg)或通过口服强饲法(5mg/kg)的方式接受化合物32。在0.08、0.25、0.5、1、2、4、8和24小时(iv)以及0.25、0.5、1、2、4、8和24小时(po)收集血液样品。血浆样品用含有内标的乙腈脱蛋白。在4℃离心后，收集上清液用于LC/MS/MS分析。
通过在指定的时间点分析血浆浓度来测量药代动力学，结果见下表，其中数据代表单剂量2.0mg/kg静脉内剂量和5mg/kg口服剂量后血浆中的平均浓度(n＝3)。
表：在ICR小鼠中化合物32的药代动力学评价结果
化合物32 iv(2mg/kg) po(5mg/kg)
AUC 0-t(ng·hr/mL) 995±181 578±47
AUC 0-∞(ng·hr/mL) 997±181 608
T 1/2(hr) 0.44±0.02 1.66±1.06
V z(L/kg) 1.3±0.21 18.8±10.06
Cl(mL min -1kg -1) 34.1±5.62 138.47±16.3
MRT(hr) 0.48±0.05 1.51±0.69
生物利用度(％)   24.4
5mg/kg的口服给药剂量下，化合物32在小鼠体内的半衰期(T 1/2)为1.66小时，药时曲线下面积(AUC)为608ng·hr/mL，并且有良好的口服生物利用度(F＝24.4％)。这些良好的药代动力学性质表明化合物32可作为一个口服抑制剂或探针在动物体内进行进一步的药效学评价与生物功能的探究。
应当理解的是，上述实例是对本发明的举例说明，而不构成对本发明的保护范围的任何限制。本发明的保护范围由所附的权利要求确定，其不仅包括权利要求中的技术方案的字面解释的含义，也包括权利要求中的技术方案的等同方案，例如，化合物的稳定同位素替代物也包括在本发明的保护范围中。

Claims (8)

  1. 一种式I化合物或其药学上可接受的盐，
    Figure PCTCN2019089213-appb-100001
    其中，
    Rh是H或甲基，优选H；
    Rg是CH、-C-Rf或N，优选CH；
    Rf是一种取代基，优选地，其选自甲基或卤素(如F，Cl，Br或I)；
    m是0、1、2或3，优选0或1，更优选0；
    Re是一种吸电子基团，选自：叔胺正离子(-N +R' 3，其中R'各自独立地选自H和C 1-C 6烷基)、硝基(-NO 2)、三卤甲基(-CX3，X＝F、Cl、Br或I)、卤素(如F，Cl，Br和I)、甲酰基(-CHO)、酰基(-CO-C 1-4烷基)、羧基(-COOH)、氰基(-CN)、磺酸基(-SO 3H)；
    Rd是烯基或炔基，例如所述烯基或炔基具有2、3、4、5或6个碳原子；
    Ra、Rb和Rc选自以下的组合：
    (1)Rb为C 1-C 4亚烷基(如C 1-C 3亚烷基，例如亚甲基、亚乙基、1,3-亚丙基)，且
    Ra和Rc为氢或C 1-C 6烷基(如C 1-C 4烷基，例如甲基、乙基、正丙基、异丙基、正丁基、叔丁基)；
    (2)Rb为C 1-C 4亚烷基(如C 1-C 3亚烷基，例如亚甲基、亚乙基、1,3-亚丙基)，且
    Ra和Rc连在一起形成C 2-C 4亚烷基(如C 2-C 3亚烷基，例如亚乙基、1,3-亚丙基)；
    (3)Ra为氢或C 1-C 6烷基(如C 1-C 4烷基，例如甲基、乙基、正丙基、异丙基、正丁基、叔丁基)，且
    Rb和Rc与它们所连接的N原子一起形成含有一个N原子的5或6元饱和杂环；
    (4)Rc为氢或C 1-C 6烷基(如C 1-C 4烷基，例如甲基、乙基、正丙基、异丙基、正丁基、叔丁基)，且
    Ra和Rb与它们所连接的N原子一起形成含有一个N原子的5或6元饱和杂环。
  2. 权利要求1的化合物或其药学上可接受的盐，
    其中-N(Ra)-Rb-N(Rc)-形成
    Figure PCTCN2019089213-appb-100002
    Figure PCTCN2019089213-appb-100003
  3. 权利要求1-2中任一项的化合物或其药学上可接受的盐，
    其中Rh为H，Rg为CH，和m为0。
  4. 权利要求1-3中任一项的化合物或其药学上可接受的盐，
    其中Re为-NO 2。
  5. 权利要求1-4中任一项的化合物或其药学上可接受的盐，
    其中Rd为乙烯基。
  6. 式II化合物或其药学上可接受的盐：
    Figure PCTCN2019089213-appb-100004
  7. 一种药物组合物，其包括权利要求1-5中任一项的式I化合物或其药学上可接受的盐、或权利要求6的式II化合物或其药学上可接受的盐，和药学上可接受的载体。
  8. 权利要求1-5中任一项的式I化合物或其药学上可接受的盐、权利要求6的式II化合物或其药学上可接受的盐、或者权利要求3的药物组合物在制备用于治疗炎症如类风湿关节炎的药物中的用途。
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