CN110294696B - 巯乙基脲/硫脲类二聚体衍生物及其制备方法和药用用途 - Google Patents

巯乙基脲/硫脲类二聚体衍生物及其制备方法和药用用途 Download PDF

Info

Publication number
CN110294696B
CN110294696B CN201810248036.XA CN201810248036A CN110294696B CN 110294696 B CN110294696 B CN 110294696B CN 201810248036 A CN201810248036 A CN 201810248036A CN 110294696 B CN110294696 B CN 110294696B
Authority
CN
China
Prior art keywords
thiourea
compound
sepsis
substituted
thioethylurea
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN201810248036.XA
Other languages
English (en)
Other versions
CN110294696A (zh
Inventor
孙逊
彭鹏
唐美麟
阙兆麟
宋娇
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Fudan University
Original Assignee
Fudan University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Fudan University filed Critical Fudan University
Priority to CN201810248036.XA priority Critical patent/CN110294696B/zh
Publication of CN110294696A publication Critical patent/CN110294696A/zh
Application granted granted Critical
Publication of CN110294696B publication Critical patent/CN110294696B/zh
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • 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
    • 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
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C323/00—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups
    • C07C323/23—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton
    • C07C323/39—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton at least one of the nitrogen atoms being part of any of the groups, X being a hetero atom, Y being any atom
    • C07C323/43—Y being a hetero atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C335/00—Thioureas, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups
    • C07C335/04—Derivatives of thiourea
    • C07C335/06—Derivatives of thiourea having nitrogen atoms of thiourea groups bound to acyclic carbon atoms
    • C07C335/08—Derivatives of thiourea having nitrogen atoms of thiourea groups bound to acyclic carbon atoms of a saturated carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C335/00—Thioureas, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups
    • C07C335/04—Derivatives of thiourea
    • C07C335/16—Derivatives of thiourea having nitrogen atoms of thiourea groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C335/00—Thioureas, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups
    • C07C335/04—Derivatives of thiourea
    • C07C335/16—Derivatives of thiourea having nitrogen atoms of thiourea groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton
    • C07C335/18—Derivatives of thiourea having nitrogen atoms of thiourea groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton being further substituted by singly-bound oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
    • C07D295/04—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms
    • C07D295/12—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly or doubly bound nitrogen atoms
    • C07D295/125—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly or doubly bound nitrogen atoms with the ring nitrogen atoms and the substituent nitrogen atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings
    • C07D295/13—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly or doubly bound nitrogen atoms with the ring nitrogen atoms and the substituent nitrogen atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings to an acyclic saturated chain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
    • C07D307/02—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings
    • C07D307/34—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D307/38—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D307/52—Radicals substituted by nitrogen atoms not forming part of a nitro radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D317/00—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms
    • C07D317/08—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3
    • C07D317/44—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3 ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D317/46—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3 ortho- or peri-condensed with carbocyclic rings or ring systems condensed with one six-membered ring
    • C07D317/48—Methylenedioxybenzenes or hydrogenated methylenedioxybenzenes, unsubstituted on the hetero ring
    • C07D317/50—Methylenedioxybenzenes or hydrogenated methylenedioxybenzenes, unsubstituted on the hetero ring with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to atoms of the carbocyclic ring
    • C07D317/58—Radicals substituted by nitrogen atoms

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Engineering & Computer Science (AREA)
  • Rheumatology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Physical Education & Sports Medicine (AREA)
  • Immunology (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Pain & Pain Management (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

本发明属于药物化学和医药技术领域，具体涉及如下式(1)所示巯乙基脲/硫脲类二聚体衍生物及其药用盐及其制备方法和药用用途，特别是在制备治疗脓毒血症药物中的应用；式(1)中，X为氧、硫原子；R选自取代或未取代的C1‑6的烷基、取代或未取代的苯基、取代或未取代的杂环基、杂芳基等；经实验，结果显示，本发明的化合物及其药用盐具有良好的抗炎活性，对体外内毒素LPS诱导的RAW264.7细胞中肿瘤坏死因子(TNF‑α)等炎症因子的过表达具有良好的抑制作用，本发明所涉及的衍生物及其药用盐可用于制备治疗与炎症相关如关节炎、结肠炎或脓毒血症等疾病的小分子药物。

Description

巯乙基脲/硫脲类二聚体衍生物及其制备方法和药用用途
技术领域
本发明属于药物化学和医药技术领域，具体涉及巯乙基脲/硫脲类二聚体衍生物及其药用盐及其制备方法和药用用途，特别是在制备治疗如关节炎、结肠炎或脓毒血症药物中的应用。
背景技术
现有技术公开了脓毒血症是一种高致死率的全身炎症反应综合征，该疾患日益成为全球危重症的主要致死原因之一。调查显示，由于人口老龄化，耐药微生物增多，免疫抑制疾病以及高风险手术的增加，脓毒血症及脓毒性休克的发病率呈增加趋势。根据国际数据库的回顾性分析，在1995年至2015年期间，脓毒血症的全球发病率为每10万人中有437人患有脓毒血症。目前虽然对脓毒血症治疗有多种研究，但均显现没有确切的疗效，能用于治疗脓毒血症的药物严重缺乏。迄今为止，临床上治疗脓毒血症的药物有乌司他丁、血必净、小剂量的糖皮质激素表现出确切的疗效，因此寻求高效低副作用的治疗药物是当务之急。
有研究表明TLR4级联信号通路介导的炎症因子过度表达是脓毒血症发生发展中最为主要的因素。受全身炎症反应正反馈调节的肿瘤坏死因子(TNF-α)和白介素1β(IL-1β)是两种重要的促炎因子，当这类促炎因子大量产生并聚集在体内时，就导致脓毒性休克及继发性多器官功能障碍，因此，对LPS-TLR4通路及其下游信号通路中某些环节的干预有可能成为治疗脓毒血症有效的策略。
有研究报道，脲及硫脲是分子存在的两种重要的具有多种生物活性的药效团，能够提高母体分子的生物活性，优化理化性质，同时又能在简易的条件下通过化学反应构建。近年来，随着小分子药物研发的需要，该类药效团逐渐被引入到更多的化学实体结构中去发现新的先导化合物。
基于目前的研究现状，本申请的发明人拟提供一类巯乙基脲/硫脲类二聚体衍生物及其药用用途，所涉及的目标化合物及其相应的抗炎活性以及提高脓毒性休克小鼠存活率的活性等均未见报道。
发明内容
本发明的目的是克服现有技术的缺陷，提供一类具有抗炎活性的巯乙基脲/硫脲类二聚体小分子化合物，具体涉及巯乙基脲/硫脲类二聚体衍生物及其药用盐。
本发明提供了如下式(1)所示的巯乙基脲/硫脲类二聚体衍生物：
Figure BDA0001607036930000021
其中，X选自氧、硫原子；
R选自取代或未取代的C1-6的烷基、取代或未取代的苯基、取代或未取代的杂环基、取代或未取代的杂芳基。
在本发明的一些实施方式中，R选自取代或未取代的甲基、乙基、正丙基、异丙基、正丁基、异丁基、环戊基、环己基。
在本发明的一些实施方式中，R选自对位为氟、氯、溴、碘、甲基、甲氧基、三氟甲氧基、苄氧基取代的苯基；R选自邻位或间位为三氟甲基取代的苯基；R选自2,4-二甲氧基、2,4-二氯基、3,4,5-三甲氧基取代的苯基。
在本发明的一些实施方式中，R选自：
Figure BDA0001607036930000022
本发明所述的化合物选自所示化合物：
Figure BDA0001607036930000023
Figure BDA0001607036930000031
本发明提供了所述巯乙基脲/硫脲类二聚体衍生的制备方法，其包括：按下式，采用胱胺二盐酸盐与异氰酸酯(异硫氰酸酯)在三乙胺的催化作用下直接反应制得巯乙基脲/硫脲类二聚体衍生。
Figure BDA0001607036930000032
本发明进行了所述化合物其药用盐的体外对TNF-α的抑制活性，化合物的体外抗炎作用机制研究，细胞毒性，体外肝微粒体代谢，急性毒性，体内药效学研究等实验；结果表明，本发明的化合物具有药理学研究价值，可抑制LPS诱导的RAW264.7细胞中炎症因子TNF-α、IL-1β的过表达，进一步，可用于制备治疗炎症的药物，尤其可用于制备治疗与炎症因子TNF-α、IL-1β的过表达相关的炎症疾病的药物，如关节炎、结肠炎或脓毒血症等。
附图说明
图1：巯乙基硫脲类二聚体衍生物2a在不同浓度下对TNF-α、IL-1β和IL-6的抑制作用。
图2：化合物2a对体外巨噬细胞RAW264.7的细胞毒性。
图3：化合物2a对NF-κB和MAPK级联信号通路的抑制作用。
图4：化合物2a的体内药效学。
具体实施方式
为对本发明作进一步阐述，下面结合实施例对本发明进行阐述，但这些实施例绝不是对本发明的任何限制。
实施例1：巯乙基脲类二聚体衍生物1a的合成。
Figure BDA0001607036930000041
称取胱胺二盐酸盐(0.5mmol,112.5mg)于50ml单口瓶中，加入少量二氯甲烷溶解，搅拌下加入三乙胺(2mmol,0.28ml)和4-甲氧基苯异氰酸酯(1mmol,149mg)，室温下搅拌1-2h，TLC板监测。随着反应的进行，逐渐有白色粉末从反应液析出，抽滤，用二氯甲烷少量多次洗涤固体，得到白色固体(350mg)，收率为77％。
1HNMR(400MHz,DMSO):δ8.38(brs,2H),7.27(d,J＝9.0Hz,4H),6.80(d,J＝9.0Hz,4H),6.27(t,J＝5.8Hz,2H),3.68(s,6H),3.38(dd,J＝12.6,6.4Hz,4H),2.82(t,J＝6.6Hz,4H)ppm；13C NMR(150MHz,DMSO)δ155.2,153.8,133.3,119.4,113.7,54.9,38.1,37.9ppm.ESI-MS m/z:451.2[M+H]+.。
实施例2
采用与实施例1相同的方法分别制得衍生物1b-1d和2a-2n，
Figure BDA0001607036930000042
白色固体，收率为85％。1HNMR(400MHz,DMSO):δ8.74(brs,2H),7.41(d,J＝8.8Hz,4H),7.25(d,J＝8.8Hz,4H),6.39(t,J＝5.7Hz,2H),3.40(dd,J＝12.5,6.4Hz,4H),2.84(t,J＝6.6Hz,4H)ppm；13C NMR(150MHz,CDCl3)δ159.5,143.9,133.0,129.2,123.8,42.85,42.48ppm.
ESI-MS m/z:459.2[M+H]+.
Figure BDA0001607036930000051
白色固体，收率为89％。1HNMR(400MHz,DMSO):δ8.62(brs,2H),7.38(dd,J＝10.5,4.2Hz,4H),7.05(t,J＝8.0Hz,4H),6.34(t,J＝5.7Hz,2H),3.39(dd,J＝12.2Hz,6.2Hz,4H),2.83(t,J＝6.6Hz,4H)ppm；13C NMR(150MHz,CDCl3)δ162.3,160.7,159.8,141.3,123.9(d,J＝27.6Hz),119.7(t,J＝22.8Hz),42.9,42.6ppm.ESI-MS m/z:427.0[M+H]+.
Figure BDA0001607036930000052
白色固体，收率为81％。1HNMR(400MHz,DMSO):δ8.45(brs,2H),7.25(d,J＝8.4Hz,4H),7.01(d,J＝8.3Hz,4H),6.30(t,J＝5.7Hz,2H),3.39(dd,J＝12.2Hz,6.4Hz,4H),2.83(t,J＝6.6Hz,4H),2.20(s,6H)ppm；13C NMR(150MHz,DMSO)δ155.0,137.6,129.7,128.9,117.7,38.1,37.9,20.1ppm.ESI-MS m/z:419.0[M+H]+.
Figure BDA0001607036930000053
白色固体，收率为60％。1HNMR(400MHz,DMSO):δ7.92(brs,2H),7.62(brs,2H),6.85(d,J＝8.1Hz,4H),6.76(d,J＝7.9Hz,2H),5.98(s,4H),4.53(s,4H),3.70(s,4H),2.90(t,J＝6.7Hz,4H)；ESI-MS m/z:561.0[M+Na]+；HRMS m/z:calcd.for C22H26N4O4S4+Na561.0729,found 561.0728.
Figure BDA0001607036930000061
白色固体，收率为76％。1HNMR(400MHz,DMSO):δ9.64(brs,2H),7.86(brs,2H),7.37(s,4H),7.16(t,J＝8.2Hz,4H),3.77(s,4H),2.96(s,4H)ppm；ESI-MS m/z:481.0[M+Na]+；
Figure BDA0001607036930000062
白色固体，收率为72％。1HNMR(400MHz,CDCl3):δ7.65(brs,2H),7.17(d,J＝8.5Hz,4H),6.95(d,J＝8.4Hz,4H),6.29(brs,2H),3.92(q,J＝5.8Hz,4H),3.83(s,6H),2.92(t,J＝6.0Hz,4H)ppm；ESI-MS m/z:505.0[M+Na]+；HRMS m/z:calcd.for C20H26N4O2S4+Na505.0831,found 505.0833.
Figure BDA0001607036930000063
白色固体，收率为63％。1HNMR(400MHz,DMSO):δ9.80(s,2H),8.03(s,2H),7.52(d,J＝8.3Hz,4H),7.32(d,J＝7.9Hz,4H),3.79(s,4H),2.97(s,4H)ppm.ESI-MS m/z:613.0[M+Na]+.
Figure BDA0001607036930000071
白色固体，收率为67％。1H NMR(400MHz,CDCl3):δ3.89(s,4H),3.78(s,10H),3.50(s,2H),3.10(s,4H),2.61(s,12H)ppm；ESI-MS m/z:497.2[M+H]+；
Figure BDA0001607036930000072
白色固体，收率为78％。1HNMR(400MHz,DMSO):δ9.58(brs,2H),7.76(brs,2H),7.32-7.02(m,8H),3.76(s,4H),2.95(s,4H),2.27(s,6H)ppm；13C NMR(150MHz,DMSO):δ180.2,133.6,129.1,123.5,20.3ppm.ESI-MS m/z:473.2[M+Na]+；
Figure BDA0001607036930000073
白色固体，收率为74％。1HNMR(400MHz,CDCl3):δ3.93(s,4H),3.41(s,4H),2.98(t,J＝6.0Hz,4H),1.65-1.53(m,4H),1.48-1.34(m,4H),0.95(t,J＝7.3Hz,6H)ppm；13C NMR(150MHz,CDCl3)δ35.4,24.2,18.3ppm.ESI-MS m/z:405.2[M+Na]+；
Figure BDA0001607036930000074
白色固体，收率为70％。1HNMR(400MHz,DMSO):δ8.89(s,2H),7.56(s,2H),7.30(d,J＝8.6Hz,2H),6.61(d,J＝2.6Hz,2H),6.50(dd,J＝8.7,2.6Hz,2H),3.77(d,J＝6.1Hz,12H),3.73-3.71(m,4H),2.89(t,J＝6.8Hz,4H)ppm；13C NMR(150MHz,DMSO)δ180.8,158.3,154.2,128.0,104.2,98.9,55.4,55.2,42.9,36.4ppm.ESI-MS m/z:565.1[M+Na]+；
Figure BDA0001607036930000081
白色固体，收率为83％。1HNMR(400MHz,DMSO):δ9.48(s,2H),7.67(s,2H),7.50-7.28(m,10H),7.21(d,J＝8.1Hz,4H),6.98(d,J＝8.1Hz,4H),5.08(s,4H),3.74(s,4H),2.93(s,4H)ppm；13C NMR(150MHz,CDCl3)δ158.3,136.4,128.7,128.2,127.8,127.5,116.3,70.3,43.9,37.2ppm.ESI-MS m/z:635.2[M+H]+；
Figure BDA0001607036930000082
白色固体，收率为68％。1HNMR(400MHz,CDCl3):δ7.68(s,2H),6.54(t,J＝5.4Hz,2H),6.49(s,4H),3.95(q,J＝6.0Hz,4H),3.85(d,J＝3.9Hz,18H),2.99(t,J＝6.1Hz,4H)ppm；13C NMR(150MHz,CDCl3)δ154.2,137.3,131.3,103.1,60.9,56.4,43.8,37.1ppm.ESI-MS m/z:625.2[M+Na]+；
Figure BDA0001607036930000083
白色固体，收率为80％。1HNMR(400MHz,CDCl3):δ7.36-7.35(m,2H),6.34-6.33(m,2H),6.29(d,J＝3.2Hz,2H),4.64(s,4H),3.89(d,J＝5.3Hz,4H),2.95(t,J＝6.1Hz,4H)ppm；13C NMR(150MHz,CDCl3)δ182.3,150.3,142.5,110.7,108.3,43.71,41.25,37.91ppm.ESI-MS m/z:453.0[M+Na]+；
Figure BDA0001607036930000091
白色固体，收率为81％。1HNMR(400MHz,DMSO):δ9.94(s,2H),8.15(s,2H),7.96(s,2H),7.76-7.33(m,6H),3.81(s,4H),2.99(s,4H)ppm.ESI-MS m/z:557.0[M-H]+.
Figure BDA0001607036930000092
白色固体，收率为72％。1HNMR(400MHz,DMSO):δ9.25(s,2H),8.04(s,2H),7.88-7.31(m,8H),3.77(s,4H),2.94(s,4H)ppm.ESI-MS m/z:557.0[M-H]+
Figure BDA0001607036930000093
白色固体，收率为85％。1HNMR(400MHz,DMSO):δ9.25(s,2H),8.04(s,2H),7.78-7.59(m,4H),7.57-7.38(m,3H),3.77(s,4H),2.94(s,4H)ppm.ESI-MS m/z:557.0[M-H]+。
实施例3：巯乙基脲/硫脲类二聚体衍生物对体外LPS诱导的RAW264.7细胞中TNF-α的抑制作用实验
采用Real-Time PCR方法来检测巯乙基脲/硫脲类二聚体衍生物对体外LPS诱导的RAW264.7细胞中TNF-α的抑制作用。
本方法具体操作如下：收集处于对数生长期的RAW264.7细胞，用含有10％FBS的RPMI-1640培养基稀释，调整细胞悬液浓度，每孔加入1ml接种于6孔无菌细胞培养板中，置于5％CO2、37℃培养箱中培养；待细胞密度达到80-90％后，吸去培养液，每孔加入2ml无血清RPMI-1640培养基继续培养4-6h，弃掉培养基，加入2ml含有不同化合物(50μM)和LPS(1μg/ml)的培养基继续培养6h，同时设置对照孔；洗掉培养基，每孔加入1ml Trizol，用移液枪吸打几次转移到EP管中，逐个加入200μl氯仿，用力振摇15s，室温放置3min，4℃、12000rpm离心15min，离心后小心吸取500μl上层无色液体移入新的EP管中，加入500μl异丙醇轻轻振摇，室温放置10min沉淀水相中的RNA，4℃、12000rpm离心10min，离心后底部有胶状沉淀，弃上清加入1ml 75％乙醇洗涤RNA沉淀，4℃、7500rpm离心5min，弃上清，室温放置干燥3-5min，根据沉淀的量，加入适量的DEPC水，吸打数次使RNA充分溶解。
待测定RNA的浓度后，使用Takara公司生产的逆转录试剂盒并按照操作说明书，在microtube管中配置下列20μl体系的反转录反应液：
Figure BDA0001607036930000101
充分混匀后在PCR扩增仪中进行逆转录得到cDNA，反应条件为37℃,15min；85℃,5s；4℃,∞。
按照Thermo Scientific SYBR Green qPCR的说明书在96孔板中配置下列20μl反应体系：
Figure BDA0001607036930000102
反应条件为：步骤1：95℃,10min；步骤2：95℃,15s；60℃,60s；40个循环反复。计算每个化合物对TNF-α的抑制率。
实验结果表明巯乙基硫脲类二聚体衍生物在浓度为50μM时，对体外LPS诱导的RAW264.7细胞中TNF-α具有良好的抑制作用，其中巯乙基硫脲类二聚体衍生物2a对TNF-α、IL-1β和IL-6呈现出浓度依赖性抑制作用，结果如表1，图1所示。
表1
Figure BDA0001607036930000111
实施例,4：化合物2a对RAW264.7细胞的细胞毒性实验
本实施例采用MTT法检测化合物2a对RAW264.7细胞的细胞毒性：收集处于对数生长期的RAW264.7细胞，用含有10％FBS的RPMI-1640培养基稀释，调整细胞悬液浓度，每孔加入100μl接种于96孔无菌细胞培养板中，边缘孔用无菌PBS进行填充，置于5％CO2、37℃培养箱中培养；待细胞密度达到80-90％后，吸去培养液，每孔加入200μl无血清RPMI-1640培养基继续培养4-6h，弃掉培养基，加入含有优选化合物2a的培养基，使其终浓度梯度分别为100μM、50μM、25μM、12.5μM，同时设置调零孔和对照孔，置于5％CO2、37℃培养箱中培养24h；每孔加入20μl MTT(5mg/ml)，继续培养4h；然后终止培养，小心吸去孔内培养液，每孔加入150μl二甲基亚砜(DMSO)，置酶标仪中低速振荡10min，使结晶物臜充分溶解，并于OD560nm下检测各孔的吸光值。
图2显示了化合物2a在不同浓度下对巨噬细胞RAW264.7均无明显毒性，说明化合物2a对LPS刺激后TNF-α、IL-1β以及IL-6表达量上调的抑制作用与巨噬细胞RAW264.7的活力无关。
实施例5：化合物2a的体外抗炎作用机制研究
本实施例采用Western-blot实验研究化合物2a的体外抗炎作用机制，具体操作方法如下：收集处于对数生长期的RAW264.7细胞，调整细胞悬液浓度，每孔加入1ml接种于6孔无菌细胞培养板中，置于5％CO2、37℃培养箱中培养；待细胞密度达到80-90％后，吸去培养液，加入2ml分别含有浓度梯度的优选化合物2a(50μM、25μM、12.5μM)的培养基继续培养30min，同时设置对照孔；30min后再加入LPS(1μg/ml)，继续培养30min，弃去培养液，用无菌PBS清洗3次，加入含有蛋白酶抑制剂(cocktail)的RIPA裂解液，充分吹打细胞并转移到EP管中，置于冰上反复涡旋吹打至细胞彻底裂解。裂解混合物于4℃，12000rpm离心10min，吸取上清于新的EP管中用于下游实验。采用BCA试剂盒，按照说明书操作步骤对蛋白浓度进行定量。蛋白样品用5×LB(Loading Buffer)和PBS调节到一致的浓度和体积，涡旋混匀并于沸水中煮5min，-40℃保存用于下步实验。
采用碧云天生产的SDS-PAGE凝胶快速配制试剂盒，按照使用说明配制10％SDS-PAGE胶。待其凝固后装入电泳槽中，加入预先配制好的电泳液，拔去梳子，每孔上样体积为20μl，上样量为30μg；浓缩胶用80V，分离胶用120V，电泳至溴酚兰刚跑出即可终止电泳，进行转膜；根据凝胶大小剪切NC膜，按照海绵－滤纸－凝胶－NC膜－滤纸—海绵顺序进行转膜；将转移槽置于冰浴中，放入装配好的膜和胶，加转膜缓冲液，恒压100V转60min；待转膜结束后，取出NC膜，用5％脱脂牛奶室温封闭1-2h；封闭完后取出NC膜，用TBST清洗，根据目的蛋白的分子量对NC膜进行剪切，并加入预先配好的一抗，4℃孵育过夜；次日取出NC膜用TBST清洗5min×3次，选择相应的二抗室温孵育1-2h，用TBST清洗5min×3次；采用ECL法进行显影，并采用ImageJ对条带图片进行分析。
实验结果如图3所示，与LPS刺激组相比，不同浓度下的化合物2a对IκB、NF-κB p65以及p38磷酸化均表现出明显的抑制作用，但对ERK1/2和JNK通路没有抑制作用，说明化合物2a可以通过抑制NF-κB和p38的活化，从而抑制炎症因子的产生，发挥抗炎作用。
实施例6：化合物2a的体外肝微粒体代谢实验
本实施例采用人肝微粒体(Human liver microsomes,HLM)和大鼠肝微粒体(Ratliver microsomes,RLM)及辅酶NADPH与优选化合物2a共同孵育，利用LC-MS-MS检测不同时间点下化合物的浓度，计算内在清除率。具体操作方法如下：在96孔板中加入肝微粒体工作液(100μl/孔)，然后再分别加入2μl化合物(100μM)于37℃孵育10min，其中NCF孔中添加98μl PH为7.4的磷酸缓冲液，预热后每孔加入98μl辅酶NADPH，启动反应；分别在0min、5min、10min、20min、30min、60min加入预冷的乙腈溶液(100ng/ml甲苯磺丁脲和100ng/ml拉贝洛尔)终止反应，震荡10min后4℃下4000rpm，离心20min，取上清液用LC-MS-MS检测化合物浓度。实验数据用一阶动力学方程处理。
Figure BDA0001607036930000121
Figure BDA0001607036930000131
实验结果显示化合物2a在HLM和RLM中的消除半衰期无明显差异，且在两者中的内在清除率数据表明，化合物2a具有良好的代谢稳定性。
实施例7：化合物2a的体内药效学实验
本实施例选取腹腔注射LPS诱导的脓毒性休克小鼠模型，尾静脉注射不同浓度的化合物2a(5mg/kg、10mg/kg、15mg/kg)，同时选取地塞米松(5mg/kg)作为阳性对照药，考察化合物是否能够提高脓毒性休克小鼠的存活率。
研究结果如图4所示，表明不同浓度的化合物2a均能提高脓毒性休克小鼠的存活率。当尾静脉注射5mg/kg化合物2a时，小鼠存活率提高40％；当尾静脉注射10mg/kg化合物2a时，小鼠存活率提高60％。
实施例8：化合物2a的急性毒性实验
本实施例对化合物2a进行小鼠体内急性毒性实验研究。将体重为20±2g的50只ICR小鼠随机分成5组，每组10只，雌雄各半，依次为空白溶剂组、100mg/kg浓度组、200mg/kg浓度组、300mg/kg浓度组和400mg/kg浓度组，对每组小鼠进行单次腹腔注射给药，给药体积为0.4ml/20g。给药后，观察小鼠的体征行为、运动状态以及对刺激的反应性，同时记录小鼠的体重变化及死亡状况，为期14天。实验结果采用SPSS软件(IBM SPSS Statistics 19)进行机率单位加权回归法(Bliss法)计算半数致死量LD50和95％置信限度。
Figure BDA0001607036930000132
实验结果表明按Bliss法计算小鼠经腹腔注射化合物2a的LD50值为336.11mg/kg，95％的可信限为284.91～395.06mg/kg。

Claims (4)

1.一种巯乙基脲/硫脲类二聚体衍生化合物或其药学上可接受的盐，所述的巯乙基脲/硫脲类二聚体衍生化合物的结构如式(1)所示：
Figure FDA0003000051880000011
在结构式(1)中，X为氧或硫原子；R为4-甲氧基苯基、4-氯苯基、4-氟苯基、3,4-二甲氧基苄基、4-三氟甲氧基苯基、4-甲基苯基、正丁基、2,4-二甲氧基苯基、4-苄氧基苯基、3,4,5-三甲氧基苯基、3-三氟甲基苯基、2，4-二氯苯基、2-三氟甲基苯基、
Figure FDA0003000051880000012
或呋喃-2-甲基
Figure FDA0003000051880000013
2.巯乙基脲/硫脲类二聚体衍生化合物(1)及其药学上可接受的盐，其中所述化合物(1)的结构选自：
Figure FDA0003000051880000014
Figure FDA0003000051880000021
3.权利要求1、2任一项所述的巯乙基脲/硫脲类二聚体衍生化合物(1)及其药学上可接受的盐，在制备用于治疗与体内炎症因子TNF-α、IL-1β过表达相关的自身免疫性疾病的药物中的应用，所述免疫炎症性疾病为类风湿关节炎、溃疡性结肠炎、脓毒血症。
4.巯乙基脲类二聚体衍生化合物(1d)及其药学上可接受的盐，在制备用于治疗与体内炎症因子TNF-α、IL-1β过表达相关的自身免疫性疾病的药物中的应用，所述免疫炎症性疾病为类风湿关节炎、溃疡性结肠炎、脓毒血症，
Figure FDA0003000051880000022
CN201810248036.XA 2018-03-24 2018-03-24 巯乙基脲/硫脲类二聚体衍生物及其制备方法和药用用途 Active CN110294696B (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201810248036.XA CN110294696B (zh) 2018-03-24 2018-03-24 巯乙基脲/硫脲类二聚体衍生物及其制备方法和药用用途

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201810248036.XA CN110294696B (zh) 2018-03-24 2018-03-24 巯乙基脲/硫脲类二聚体衍生物及其制备方法和药用用途

Publications (2)

Publication Number Publication Date
CN110294696A CN110294696A (zh) 2019-10-01
CN110294696B true CN110294696B (zh) 2021-07-02

Family

ID=68026168

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201810248036.XA Active CN110294696B (zh) 2018-03-24 2018-03-24 巯乙基脲/硫脲类二聚体衍生物及其制备方法和药用用途

Country Status (1)

Country Link
CN (1) CN110294696B (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116283990B (zh) * 2022-09-08 2023-12-15 沈阳药科大学 取代吡咯烷酮类化合物在作为肿瘤坏死因子抑制剂方面的应用

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3694141A (en) * 1967-08-02 1972-09-26 Oreal Method of hair conditioning
US3957774A (en) * 1972-04-17 1976-05-18 L'oreal N-morpholinomethyl-n-'-substituted ethyl and propylureas
FR2459231A1 (fr) * 1979-06-19 1981-01-09 Anvar Nouveaux derives de la cystamine utiles comme medicaments
IT1175808B (it) * 1984-03-07 1987-07-15 Ausonia Farma Srl Procedimento per la preparazione di composto con attivita' antiulcera
CN1305846C (zh) * 1998-03-26 2007-03-21 参天制药株式会社 脲衍生物
JP2000086660A (ja) * 1998-09-10 2000-03-28 Wyeth Lederle Japan Ltd カルバペネム化合物
EP1368309B2 (en) * 2001-02-22 2014-10-01 Anika Therapeutics Inc. Thiol-modified hyaluronan
SE0400213D0 (sv) * 2004-02-04 2004-02-04 Active Biotech Ab Diurea Derivatives

Also Published As

Publication number Publication date
CN110294696A (zh) 2019-10-01

Similar Documents

Publication Publication Date Title
CN109232477B (zh) 查尔酮-氨基二硫代甲酸酯类过氧化氢酶抑制剂及其合成方法和应用
EP4248997A1 (en) Therapeutic agent or prophylactic agent for covid-19
CN104010501A (zh) 含src同源区2蛋白酪胺酸磷酸酶-1增效剂及其治疗方法
CN103664785A (zh) 一类新颖的二氢吡唑磺胺衍生物的合成及在抗癌药物中的应用
CN113666824B (zh) 一种大麻二酚-2-丙酸酯及其应用
JPS60136592A (ja) Ｎ↑６‐（１‐および２‐ベンゾシクロアルキル）アデノシンおよびその製法
CN110294696B (zh) 巯乙基脲/硫脲类二聚体衍生物及其制备方法和药用用途
CN106243183B (zh) 熊果酸-硫化氢供体试剂衍生物及其合成方法
CN104523664A (zh) 姜黄素类抗肿瘤药物及其应用
CN106243182A (zh) 甘草次酸‑硫化氢供体试剂衍生物及其合成方法和应用
CN111440105B (zh) 阿法骨化醇氨基甲酸酯衍生物及其制备方法和用途
CN109721579A (zh) 7,8-脱氢葡萄藤戊素衍生物、其制法和药物组合物与用途
CN108299255A (zh) 组蛋白去乙酰化酶8选择性抑制剂及其制备方法和应用
CN109516926B (zh) 一种荜茇明碱衍生物制备及用途
CN104003966B (zh) 5,7,2，,4，-四羟基-3-烃基黄酮类似物及其制备方法和应用
CN112679409B (zh) 一种4-吲哚-取代硫半脲衍生物及其制备方法和应用
JPS6344577A (ja) ピリミジン誘導体
CN110698403A (zh) 三取代吡唑衍生物及应用
CN112961096B (zh) 具有改善顺铂肾毒性和抗肿瘤活性的查尔酮色氨酸衍生物
CN107827828B (zh) 含苯酰肼骨架的喹喔啉衍生物及其制备方法和在制备抗肿瘤药物中的应用
CN110078717B (zh) 一种具有抗肿瘤活性的双苯并呋喃—苯并咪唑盐类化合物及其制备方法
CN111825608A (zh) 四氢喹啉类与四氢异喹啉类化合物及其用途
CN106674180B (zh) 一种槲皮素衍生物及其制备方法和应用
CN103919770A (zh) 一种含噻喃酮结构的姜黄素类似物s5在制备抗炎药物方面的应用
CN111635446B (zh) 灯盏乙素酰胺衍生物及其制备方法和用途

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant