CN115650973A - 一种nsc69187及其衍生物的合成方法与应用 - Google Patents
一种nsc69187及其衍生物的合成方法与应用 Download PDFInfo
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- CN115650973A CN115650973A CN202210634329.8A CN202210634329A CN115650973A CN 115650973 A CN115650973 A CN 115650973A CN 202210634329 A CN202210634329 A CN 202210634329A CN 115650973 A CN115650973 A CN 115650973A
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Abstract
本发明公开了一种NSC69187及其衍生物的合成方法与应用,具体的说,发明所制备的NSC69187衍生物CX24能够特异性抑制TLR3,且具有良好的体内外抗炎活性;在体内动脉粥样硬化模型中,可以抑制斑块形成、提高斑块稳定性以及减少病变部位炎症浸润,表现出良好的抗动脉粥样硬化作用,为临床动脉粥样硬化治疗提供新的分子选择和理论依据。
Description
技术领域
本发明涉及化合物有机合成的技术领域,具体涉及一种 NSC69187及其衍生物的合成方法与应用。
背景技术
长链dsRNA是TLR3的天然配体,包括外源性dsRNA病毒,如脊髓灰质炎病毒、脑心肌炎病毒、柯萨奇病毒等;DNA病毒,如疱疹病毒等;感染复制过程中释放的dsRNA;人工合成dsRNA类似物,如Poly I:C,Poly A:U等;以及由损伤和坏死细胞等释放的内源性dsRNA。其能够选择性地与细胞内体的TLR3形成dsRNA-TLR3 复合物,导致TLR3二聚化,启动下游信号级联反应。TLR3是TLR 家族中,唯一不依赖MyD88衔接蛋白,而是招募TRIF衔接蛋白触发下游信号级联反应的TLR。TLR3识别dsRNA后,招募TRIF衔接蛋白,活化IFN通路,可以通过下游TRIF/TRAF3/TBK1/IRF3信号级联传递信号,使干扰素转录因子3(IRF3)进入细胞核,促进IFN α和IFNβ等I型干扰素分泌;也可以通过下游TRIF/TRAF6/TAK1信号级联传递信号,活化MAPK和NF-κB通路,使AP-1和核转录因子NF-κB进入细胞核,促进IL-6、TNF-α和IL-1β等炎症因子、MCP-1 和CCL5等趋化因子,以及内皮粘附因子的释放,激活先天性免疫反应,和调节获得性免疫反应。TLR3信号通路的激活在机体应对病原体感染和肿瘤免疫等方面起重要作用,通过人工合成dsRNA类似物激活TLR3,以模拟体内TLR3介导的免疫反应,在肿瘤治疗、疫苗佐剂研发和抗病毒感染研究中取得很好的成果。然而,相关研究也显示TLR3的异常活化和表达参与到肿瘤恶化、炎症和中枢神经系统等相关疾病的调节中。
TLR3的异常表达对于动脉粥样硬化疾病发生、发展过程有重要影响,抑制TLR3是潜在的动脉粥样硬化治疗方法,而目前尚未有药理学研究报道TLR3抑制剂的动脉粥样硬化治疗作用。
发明内容
针对现有技术中存在的上述技术问题,本发明提出了一种 NSC69187及其衍生物的合成方法与应用,本发明所制备的 NSC69187衍生物CX24能够特异性抑制TLR3,且具有良好的体内外抗炎活性;在体内动脉粥样硬化模型中,可以抑制斑块形成、提高斑块稳定性以及减少病变部位炎症浸润,表现出良好的抗动脉粥样硬化作用,为临床动脉粥样硬化治疗提供新的分子选择和理论依据。
为了实现上述目的,本发明采用如下技术方案:
一种NSC69187的合成方法,所述合成方法包括:
A 5-甲基吲哚、2,5-己二酮和对甲苯磺酸一水合物溶解于无水乙醇中,80℃回流3h;乙醇旋干,反应物加硅胶粉拌匀、干燥后,转移至索氏提取器中,正己烷69℃回流、提取;收集提取瓶中的液体,旋干得到白色粉末状固体化合物CX1-a;
B N-甲基甲酰苯胺、三氯氧磷和甲苯混合于洁净干燥的反应瓶中,室温反应半小时;将化合物CX1-a溶解于甲苯中,并逐滴加入反应瓶混合物中,升温至105℃,反应4h;甲苯旋干,超声条件下,加入乙醇溶解不溶固体;硅胶柱纯化,得到淡黄色固体粉末化合物 CX1-b;
C将化合物CX1-b与氨基乙醛二乙缩醛混合于洁净干燥的反应瓶中,升温至115℃,反应3h;反应液冷却至室温,加入无水乙醇和硼氢化钠,室温搅拌1h;旋干乙醇,使用饱和氯化钠溶液和乙酸乙酯萃取,收集有机相,旋干乙酸乙酯;硅胶柱纯化(洗脱液为乙酸乙酯/石油醚/三乙胺,得到淡黄色油状液体化合物CX1-c;
D将化合物CX1-c、对甲苯磺酰氯、碳酸钠、水和四氢呋喃混合,室温反应2h;乙酸乙酯和水萃取,收集有机相,旋干;反应物溶于1,4-二氧六环中,边搅拌边滴入6M盐酸溶液,室温反应过夜;加入二氯甲烷和水萃取,收集水相;往水相中加入边搅拌边滴加1M 氢氧化钠溶液,调pH至8.0,有大量固体析出,过滤,收集滤渣,得到黄色NSC69187粗产物;硅胶柱纯化,得到黄色固体化合物 NSC69187;其中,化合物NSC69187的结构式为:
本发明还提供一种基于化合物NSC69187制备的衍生物,将所述步骤A中的5-甲基吲哚替换为吲哚参加反应,即可得到黄色固体CX5,其中,化合物CX5的结构式为:
将化合物CX5和乌洛托品混合于三氟乙酸中,73℃加热回流20 min;旋干三氟乙酸,水和乙酸乙酯萃取,收集有机相;硅胶柱纯化,得到黄色固体CX9,其中,化合物CX9的结构式为:
将化合物CX9和异丙胺混合于无水乙醇中,37℃反应3h;反应液冷却至室温,加入硼氢化钠,室温反应1h;旋干乙醇,水和乙酸乙酯萃取,收集有机相;硅胶柱纯化,得到黄色固体CX24,其中,化合物CX24的结构式为:
本发明再提供一种NSC69187衍生化合物用于TLR3的抑制,化合物CX24用于制备TLR3的抑制剂,其中,所述化合物CX24结构式为:
本发明再提供一种含有NSC69187衍生化合物的抑制剂,所述抑制剂含有所述化合物CX24。
本发明有益技术的效果在于:
通过对照实验,本发明的NSC69187衍生物CX24在治疗组中,可使小鼠主动脉斑块面积减少,占整条主动脉面积的3.87±0.15%,抑制率为49.2%,在另一治疗组中,可显著降低主动脉根部瓣膜脂质成分堆积,占比2.23±0.26%,抑制率为57.7%。所以,通过实验可知,本发明的NSC69187衍生物CX24在处理动脉粥样硬化模型小鼠,能够减少小鼠主动脉和主动脉根部的脂质堆积和斑块形成,表现出良好的体内抗动脉粥样硬化效果。
附图说明
图1为化合物CX24对TLR3抑制作用曲线图;
图2为化合物CX24抑制细胞I型干扰素的分泌,能够呈剂量依赖显著抑制IFNα和IFNβ的表达的示意图;
图3为收集处理后小鼠的整条主动脉和心脏,通过油红O染色分析药物处理对主动脉斑块形成的影响的示意图。
具体实施方式
以下将结合实施例对本发明作进一步的描述,需要说明的是,本实施例以本技术方案为前提,给出了详细的实施方式和具体的操作过程,但本发明的保护范围并不限于本实施例。
本发明为一种NSC69187的合成方法,所述合成方法包括:
A 5-甲基吲哚、2,5-己二酮和对甲苯磺酸一水合物溶解于无水乙醇中,80℃回流3h;乙醇旋干,反应物加硅胶粉拌匀、干燥后,转移至索氏提取器中,正己烷69℃回流、提取;收集提取瓶中的液体,旋干得到白色粉末状固体化合物CX1-a;
B N-甲基甲酰苯胺、三氯氧磷和甲苯混合于洁净干燥的反应瓶中,室温反应半小时;将化合物CX1-a溶解于甲苯中,并逐滴加入反应瓶混合物中,升温至105℃,反应4h;甲苯旋干,超声条件下,加入乙醇溶解不溶固体;硅胶柱纯化,得到淡黄色固体粉末化合物 CX1-b;
C将化合物CX1-b与氨基乙醛二乙缩醛混合于洁净干燥的反应瓶中,升温至115℃,反应3h;反应液冷却至室温,加入无水乙醇和硼氢化钠,室温搅拌1h;旋干乙醇,使用饱和氯化钠溶液和乙酸乙酯萃取,收集有机相,旋干乙酸乙酯;硅胶柱纯化(洗脱液为乙酸乙酯/石油醚/三乙胺,得到淡黄色油状液体化合物CX1-c;
D将化合物CX1-c、对甲苯磺酰氯、碳酸钠、水和四氢呋喃混合,室温反应2h;乙酸乙酯和水萃取,收集有机相,旋干;反应物溶于1,4-二氧六环中,边搅拌边滴入6M盐酸溶液,室温反应过夜;加入二氯甲烷和水萃取,收集水相;往水相中加入边搅拌边滴加1M 氢氧化钠溶液,调pH至8.0,有大量固体析出,过滤,收集滤渣,得到黄色NSC69187粗产物;硅胶柱纯化,得到黄色固体化合物 NSC69187;其中,化合物NSC69187的结构式为:
需要指出的是,本发明化合物NSC69187的合成路线如下:
本发明还提供一种基于化合物NSC69187制备的衍生物,将所述步骤A中的5-甲基吲哚替换为吲哚参加反应,即可得到黄色固体 CX5,其中,化合物CX5的结构式为:
将化合物CX5和乌洛托品混合于三氟乙酸中,73℃加热回流20 min;旋干三氟乙酸,水和乙酸乙酯萃取,收集有机相;硅胶柱纯化,得到黄色固体CX9,其中,化合物CX9的结构式为:
将化合物CX9和异丙胺混合于无水乙醇中,37℃反应3h;反应液冷却至室温,加入硼氢化钠,室温反应1h;旋干乙醇,水和乙酸乙酯萃取,收集有机相;硅胶柱纯化,得到黄色固体CX24,其中,化合物CX24的结构式为:
本发明再提供一种NSC69187衍生化合物用于TLR3的抑制,化合物CX24用于制备TLR3的抑制剂,其中,所述化合物CX24结构式为:
本发明再提供一种含有NSC69187衍生化合物的抑制剂,所述抑制剂含有所述化合物CX24。
实施例
化合物NSC69187和中间体的合成和结构表征:
6-methoxy-1,4-dimethyl-9H-carbazole(CX1-a)
5-甲基吲哚(5.15g,34.99mmol)、2,5-己二酮(4.1mL,34.95 mmol)和对甲苯磺酸一水合物(3.325g,17.47mmol)溶解于无水乙醇(50mL)中,80℃回流3h;乙醇旋干,反应物加硅胶粉拌匀、干燥后,转移至索氏提取器中,正己烷69℃回流、提取;收集提取瓶中的液体,旋干得到白色粉末状固体化合物CX1-a,产率62%;1H NMR(400MHz,CDCl3)δ7.87(s,1H),7.72(d,J=2.4Hz,1H), 7.41(d,J=8.7Hz,1H),7.14(d,J=7.3Hz,1H),7.10(dd,J=8.7,2.5Hz,1H),6.93(d,J=7.3Hz,1H),3.96(s,3H),2.87(s,3H),2.55 (s,3H).13C NMR(101MHz,CDCl3)δ153.66,139.72,134.48, 130.76,126.14,125.01,121.39,120.43117.18,113.50 110.97, 106.40,56.22,20.46,16.52.
6-methoxy-1,4-dimethyl-9H-carbazole-3-carbaldehyde (CX1-b)
N-甲基甲酰苯胺(1.2mL,8.87mmol)、三氯氧磷(1.4mL,9.13 mmol)和甲苯(5mL)混合于洁净干燥的反应瓶中,室温反应半小时;CX1-a(1.27g,5.63mmol)溶解于甲苯(10mL)中,并逐滴加入反应瓶混合物中,升温至105℃,反应4h;甲苯旋干,超声条件下,加入乙醇溶解不溶固体;硅胶柱纯化(洗脱液为乙酸乙酯/石油醚(4/1)),得到淡黄色固体粉末化合物CX1-b,产率48%;1H NMR(400MHz,CDCl3)δ10.47(s,1H),8.23(s,1H),7.88-7.68(m, 2H),7.45(d,J=8.7Hz,1H),7.15(dd,J=8.7,2.0Hz,1H),3.97(s, 3H),3.20(s,3H),2.57(s,3H).13C NMR(101MHz,DMSO)δ191.80,154.00,143.32,136.44,135.59,128.17,125.75,124.41, 121.57,118.51,114.72,112.54,106.59,56.07,16.94,14.99.
2,2-diethoxy-N-(6-methoxy-1,4-dimethyl-9H-carbazol-3-yl)met hyl)ethanamine(CX1-c)
CX1-b(59mg,0.53mmol)与氨基乙醛二乙缩醛(118μL,0.88 mmol)混合于洁净干燥的反应瓶中,升温至115℃,反应3h;反应液冷却至室温,加入无水乙醇(2mL)和硼氢化钠(34mg,0.89mmol),室温搅拌1h;旋干乙醇,使用饱和氯化钠溶液和乙酸乙酯萃取,收集有机相,旋干乙酸乙酯;硅胶柱纯化(洗脱液为乙酸乙酯/石油醚/ 三乙胺(4/1/0.01)),得到淡黄色油状液体化合物CX1-c,产率78%;1H NMR(400MHz,CDCl3)δ8.33(s,1H),7.75(d,J=2.4Hz,1H), 7.34(d,J=8.7Hz,1H),7.13(s,1H),7.07(dd,J=8.7,2.4Hz,1H), 4.70(s,1H),3.98(s,2H),3.95(s,3H),3.72(dd,J=9.4,7.1Hz, 2H),3.56(dd,J=9.4,7.1Hz,2H),2.90(d,J=5.6Hz,2H),2.85(s, 3H),2.43(s,3H),2.19(s,1H),1.24(t,J=7.0Hz,6H).13CNMR (101MHz,CDCl3)δ153.52,139.11,134.87,129.07,128.57, 128.51,125.08,122.00,116.72,113.21,110.85,106.85,102.17, 62.26,56.20,51.81,51.64,16.42,15.72,15.37.
9-methoxy-5,11-dimethyl-6H-pyrido[4,3-b]carbazole (NSC69187)
化合物CX1-c(126mg,0.34mmol)、对甲苯磺酰氯(90mg,0.47 mmol)、碳酸钠(58mg,0.54mmol)、水(5mL)和四氢呋喃(5 mL)混合,室温反应2h;乙酸乙酯和水萃取,收集有机相,旋干;反应物溶于1,4-二氧六环(2mL)中,边搅拌边滴入6M盐酸溶液 (2mL),室温反应过夜;加入二氯甲烷和水萃取,收集水相;往水相中加入边搅拌边滴加1M氢氧化钠溶液,调pH至8.0,有大量固体析出,过滤,收集滤渣,得到黄色NSC69187粗产物;硅胶柱纯化(洗脱液为乙酸乙酯/石油醚/三乙胺(2/1/0.01)),得到黄色固体化合物NSC69187;产率65%;1H NMR(400MHz,DMSO)δ 11.18(s,1H),9.69(s,1H),8.41(d,J=6.1Hz,1H),7.96–7.87(m, 1H),7.49(d,J=8.7Hz,1H),7.20(dd,J=8.7,2.5Hz,1H),3.91(s, 3H),3.27(s,3H),2.78(s,3H).13C NMR(101MHz,DMSO)δ 153.49,150.11,141.59,140.76,137.72,132.71,128.58,123.96, 123.78,122.07,116.16,115.57,111.54,108.26,108.17,56.17, 14.62,12.26.ESI-HRMS m/z:calculated for C18H16N2O[M+H]+ 277.1341,found 277.1345.
本发明还提供一种基于化合物NSC69187制备的衍生物,将所述步骤A中的5-甲基吲哚替换为吲哚参加反应,即可得到黄色固体 CX5,产率为41%;1H NMR(400MHz,CDCl3)δ8.21(d,J=7.9Hz, 1H),7.52(d,J=8.1Hz,1H),7.44(t,J=7.6Hz,1H),7.28(d,J=14.9Hz,2H),7.16(d,J=7.3Hz,1H),6.97(d,J=7.3Hz,1H),2.89 (s,3H),2.57(s,3H).13CNMR(101MHz,DMSO)δ150.05, 143.08,140.97,140.83,132.87,128.44,127.50,124.20,123.80, 123.54,122.36,119.57,116.27,111.09,108.43,14.74,12.35. ESI-HRMS m/z:calculated for C17H14N2[M+H]+247.1235,found 247.1238.其中,化合物CX5的结构式为:
将化合物CX5和乌洛托品混合于三氟乙酸中,73℃加热回流20 min;旋干三氟乙酸,水和乙酸乙酯萃取,收集有机相;硅胶柱纯化,得到黄色固体CX9,产率为36%;1H NMR(400MHz,DMSO)δ 12.14(s,1H),10.10(s,1H),9.80(s,1H),8.84(s,1H),8.47(d,J=6.1Hz,1H),8.08(dd,J=11.6,7.3Hz,2H),7.68(d,J=8.3Hz,1H), 3.28(s,3H),2.79(s,3H).13C NMR(101MHz,DMSO)δ192.31, 150.17,147.14,141.35,141.06,133.29,129.15,128.84,128.53, 127.47,123.58,123.14,122.74,116.40,111.36,109.76,14.68,12.28.ESI-HRMS m/z:calculated for C18H14N2O[M+H]+275.1184, found 275.1192.其中,化合物CX9的结构式为:
将化合物CX9和异丙胺混合于无水乙醇中,37℃反应3h;反应液冷却至室温,加入硼氢化钠,室温反应1h;旋干乙醇,水和乙酸乙酯萃取,收集有机相;硅胶柱纯化,得到黄色固体CX24,产率为54%;1H NMR(400MHz,DMSO)δ11.29(s,1H),9.69(s,1H), 8.42(d,J=6.0Hz,1H),8.34(s,1H),7.92(d,J=6.0Hz,1H),7.50 (s,2H),3.89(s,2H),3.29(s,3H),2.84–2.77(m,4H),1.99(s,1H), 1.06(d,J=6.2Hz,6H).13C NMR(101MHz,DMSO)δ150.04,142.00,141.26,140.82,132.76,132.51,128.27,127.94,123.85, 123.53,123.42,122.32,116.22,110.60,108.28,51.45,47.71, 23.30,14.80,12.32.ESI-HRMS m/z:calculated for C21H23N3[M+ H]+318.1970,found 318.1963.其中,化合物CX24的结构式为:
实施例2
化合物CX24对TLR3的抑制活性实验
1.实验步骤
(1)细胞培养:HEK Blue hTLR3为贴壁细胞,用含有10%FBS、 1%青霉素/链霉素及Zeocin(100μg/mL)的DMEM完全培养基进行培养;所有细胞均置于37℃、5%CO2和饱和湿度的细胞孵育箱中培养;
(2)接种HEK Blue TLR3细胞:用含10%胎牛血清(60℃加热30分钟)的DMEM培养基配成单个细胞悬液,以每孔20000个细胞接种到384孔细胞培养板,每孔接种体积20μL;
(3)加入待测化合物,培养:向各个孔加入20μL用含10%胎牛血清(60℃加热30分钟)的DMEM培养基稀释成相应浓度的待测化合物和Poly I:C(10μg/mL),在37℃,5%CO2培养条件下培养24小时;
(4)QUANTI-blue工作液配制:按照试剂盒说明书配制工作液(美国Invivogen,Rep-qb2),将QUANTI-blue粉末与无菌水50mL 混合,37℃温热30min使充分溶解,0.22μm滤膜过滤,滤液分装, -20℃避光保存;
(5)将细胞培养液和QUANTI-blue工作按照1:1比例加到96孔板中,室温避光孵育10-60min,至溶液由紫色变成蓝色,于620nm 波长处读取各孔的吸光度值。IC50通过Growth中的Hill1进行非线性拟合。
2.实验结果
化合物CX24对TLR3抑制作用如图1所示,化合物CX24对 TLR3抑制活性IC50为18.87±2.21nM。该化合物有较好的抑制TLR3 的活性,具有良好的开发潜力。
化合物CX24抑制TLR3活化的NF-κB/MAPK/IRF3信号通路
1.实验步骤
(1)4%巯乙醇酸盐溶液的配制:4g硫乙醇酸盐,定容于100mL 超纯水中,100℃加热煮沸至完全溶解;溶液经过高压灭菌后,分装并保存于4℃冰箱备用;
(2)腹腔巨噬细胞的诱导和分离:8-12周龄的C57BL/6J小鼠,给予腹腔注射4%巯乙醇酸盐溶液4mL刺激4天;小鼠摘眼球放血,浸泡至75%酒精中消毒10min;小鼠转移至在生物安全柜中,腹部朝上,四肢躺平固定,用75%酒精消毒腹腔表面;用注射器吸取无菌PBS灌洗腹腔3次,每次10mL,收集灌洗液于50mL离心管中; 1000rmp室温离心5min,弃去上清,细胞重悬于PBS中,并通过 200目无菌尼龙网过滤,收集滤液;1000rmp离心5min,弃去上清,细胞重悬于RPMI培养基中;
(3)腹腔巨噬细胞以每孔(8×105个)细胞接种到6孔细胞培养板,每孔接种体积2mL。37℃孵育2h使细胞贴壁;弃去不贴壁的细胞,RPMI培养基洗2遍,加入新鲜RPMI培养基(含10%FBS 和1%P/S)
(4)腹腔巨噬细胞经不同浓度的化合物CX24(0.01μM、0.1μ M和1μM)提前处理半小时,再用Poly I:C(10μg/mL)刺激1h 后收集细胞总蛋白,进行Western blot分析。
2.实验结果
化合物CX24能够呈剂量依赖的地抑制NF-κB通路的磷酸化p65 蛋白表达,缓解IkBα蛋白的降解;也可以降低MAPK通路的磷酸化 p38和磷酸化ERK蛋白表达。化合物CX24能够呈剂量依赖抑制TRIF-IRF3通路的磷酸TBK-1蛋白表达。这些结果表明,SMU-24 对TLR3活化的下游NF-κB、MAPK和TRIF-IRF3通路的关键组分蛋白有重要影响,抑制下游炎症通路激活。
实施例3
化合物CX24抑制TLR3活化的炎症因子表达
1.实验步骤
(1)腹腔巨噬细胞以每孔(8×105个)细胞接种到6孔细胞培养板,每孔接种体积2mL。37℃孵育2h使细胞贴壁;弃去不贴壁的细胞,RPMI培养基洗2遍,加入新鲜RPMI培养基(含10%FBS 和1%P/S)
(2)腹腔巨噬细胞经不同浓度的化合物CX24(0.1μM和1μM) 提前处理半小时,再用Poly I:C(10μg/mL)刺激1h。弃去细胞培养基,PBS洗3次,每次1mL;
(3)向细胞中加入TRIzol试剂500μL,室温裂解10min,收集裂解液于无酶离心管中;加入200μL的氯仿,剧烈震荡摇匀15s,室温静置3min后,4℃、12000rmp离心15min;小心吸取上层水相至另一新的无酶1.5mL离心管中;向离心管中加入等体积的异丙醇,上下颠倒混匀,室温静置15min,4℃、12000rmp离心10min;弃去上清,加入80%乙醇1mL(配制:无水乙醇80mL+DEPC 水20mL),用移液枪轻轻吹打,使沉淀悬浮,4℃、12000rmp离心10min;弃去上清,离心管倒置于生物安全柜中干燥10min,可见RNA沉淀变成透明薄膜;加入20μL DEPC水,用移液枪小心吹匀,使RNA完全溶解;RNA样品保存-80℃保存备用;
(4)RNA定量和检测:设DEPC水作为空白对照,使用核酸蛋白浓度测定仪测定样品RNA浓度,记录RNA浓度和OD(260/280);
(5)逆转录
2.实验结果
化合物CX24亦能抑制细胞I型干扰素的分泌,能够呈剂量依赖显著抑制IFNα和IFNβ的表达,如图2所示。
实施例4
化合物CX24抑制动脉粥样硬化
1.实验步骤
(1)西方饮食喂养的ApoE-/-小鼠的动脉粥样硬化模型的构建
a实验动物:6-8周龄的SPF级ApoE-/-小鼠(雄性,C57BL/6J 背景)购买于江苏集萃药康生物科技有限公司,普通饲料适应性喂养一周;
b实验动物分组及给药:将体重配对的ApoE-/-小鼠随机分成两组,每组8只,分别为对照组(对照溶剂柠檬酸-磷酸氢二钠缓冲液 pH 7.0处理)、化合物CX24治疗组(5mg/kg化合物CX24处理,化合物CX24溶解于柠檬酸-磷酸氢二钠缓冲液pH 7.0);所有小鼠给予西方饮食饲料(含20%脂肪,1.25%胆固醇)连续喂养12周;其中在第5-12周,化合物CX24处理组隔天腹腔注射给予药物处理,同时对照组给予等体积对照溶剂处理;12周后收集样本进行分析;
c饲养条件:所有动物均饲养于南方医科大学SPF级实验动物部。于22℃和12h光照/12h黑夜的条件下饲养,小鼠自由饮水和采食。
(2)主动脉大体的油红O染色
新鲜解剖的小鼠整条主动脉,置于4%多聚甲醛中固定48h以上;在体式显微镜下,小心剔除主动脉外周的结缔组织和脂肪组织;用维纳斯剪小心纵向破开主动脉,并用钢针将主动脉平铺固定在黑色橡胶平皿上;PBS洗3次以弃去漂浮的杂质,每次2min;弃去PBS,加入油红O染色工作液覆盖主动脉,避光浸染60min;弃去油红O 染色液,用70%乙醇分化3次,每次10s,至动脉显白色,PBS洗 3次;转移至显微镜下观察主动脉斑块分布情况和并拍照;组织中脂质斑块呈橘红色,用image J软件计算油红染色脂质斑块面积占整条主动脉大体面积的比值。
(3)主动脉的根部的冰冻切片制备和油红O染色
主动脉根部的冰冻切片制备
a.固定:新鲜心脏组织保留0.5cm的主动脉弓,置于4%多聚甲醛中固定24h;
b.组织脱水:将组织放于20%蔗糖溶液中脱水沉底后,又转移至 30%蔗糖溶液脱水沉底;
c.OCT包埋:脱水后的组织用吸水纸稍擦干表面水分后,用切刀修平主动脉切面切面,并将切面朝下垂直放于包埋台上,周围滴加 OCT包埋剂包埋组织;将包埋台转移至-25℃预冷的冷冰冻切片机内速冻台上进行速冻,至OCT变白变硬;
d.组织冰冻切片:包埋台固定在切片机上,先粗切修切平整组织面后,进行连续切片,切片厚度6μm,收集出现三个心脏瓣膜腔的切片,附在粘附型载玻片上,转移到-20℃冰箱保存待用;
主动脉根部切片的油红O染色:切片从冰箱中取出,室温放置 30min以平衡至室温;切片用PBS洗3次以去掉包埋液,每次5min; 60%异丙醇浸洗2min;油红O染色室温染色30min;60%异丙醇调色1s;自来水冲洗5min;苏木素染细胞核2min;自来水冲洗5 min;吸干切片周围水分,用70%甘油封片;显微镜下观察脂质分布情况并拍照;组织中脂质斑块呈橘红色,细胞核呈蓝色;image J 软件计算油红染色脂质斑块面积与整个切面主动脉瓣腔面面积的比值。
2.实验结果
如图3所示,收集处理后小鼠的整条主动脉和心脏,通过油红O 染色分析药物处理对主动脉斑块形成的影响,染色结果显示,空白溶剂对照组小鼠经过12周高脂高胆固醇饲料喂养后,其主动脉内壁形成明显的斑块,不规则分布于主动脉弓、腹主动脉和髂主动脉分支处,斑块面积通过定量分析结果显示,占整条主动脉面积的7.62±0.68%;与对照组相比,化合物CX24治疗组小鼠主动脉斑块面积减少,占整条主动脉面积的3.87±0.15%,抑制率为49.2%,具有显著差异。主动脉根部处于心脏内,是主动脉位于心脏左心室口连接升主动脉之间的管状结构,对心脏进行石蜡切片,切片通过油红O染色,分析主动脉根部斑块形成情况,染色结果显示,空白溶剂对照组小鼠主动脉根部瓣膜有大量斑块、脂质含量和坏死脂质核心堆积,通过定量分析,占比整个主动脉根部管腔面积5.28±0.68%;与对照组相比,化合物 CX24治疗组显著降低主动脉根部瓣膜脂质成分堆积,占比2.23± 0.26%,抑制率为57.7%,具有显著差异。这一结果表明化合物CX24 处理动脉粥样硬化模型小鼠,能够减少小鼠主动脉和主动脉根部的脂质堆积和斑块形成,表现出良好的体内抗动脉粥样硬化效果。
对于本领域的技术人员来说,可以根据以上的技术方案和构思,给出各种相应的改变,而所有的这些改变,都应该包括在本发明权利要求的保护范围之内。
Claims (4)
1.一种NSC69187的合成方法,其特征在于,所述合成方法包括:
A 5-甲基吲哚、2,5-己二酮和对甲苯磺酸一水合物溶解于无水乙醇中,80℃回流3h;乙醇旋干,反应物加硅胶粉拌匀、干燥后,转移至索氏提取器中,正己烷69℃回流、提取;收集提取瓶中的液体,旋干得到白色粉末状固体化合物CX1-a;
B N-甲基甲酰苯胺、三氯氧磷和甲苯混合于洁净干燥的反应瓶中,室温反应半小时;将化合物CX1-a溶解于甲苯中,并逐滴加入反应瓶混合物中,升温至105℃,反应4h;甲苯旋干,超声条件下,加入乙醇溶解不溶固体;硅胶柱纯化,得到淡黄色固体粉末化合物CX1-b;
C将化合物CX1-b与氨基乙醛二乙缩醛混合于洁净干燥的反应瓶中,升温至115℃,反应3h;反应液冷却至室温,加入无水乙醇和硼氢化钠,室温搅拌1h;旋干乙醇,使用饱和氯化钠溶液和乙酸乙酯萃取,收集有机相,旋干乙酸乙酯;硅胶柱纯化(洗脱液为乙酸乙酯/石油醚/三乙胺,得到淡黄色油状液体化合物CX1-c;
D将化合物CX1-c、对甲苯磺酰氯、碳酸钠、水和四氢呋喃混合,室温反应2h;乙酸乙酯和水萃取,收集有机相,旋干;反应物溶于1,4-二氧六环中,边搅拌边滴入6M盐酸溶液,室温反应过夜;加入二氯甲烷和水萃取,收集水相;往水相中加入边搅拌边滴加1M氢氧化钠溶液,调pH至8.0,有大量固体析出,过滤,收集滤渣,得到黄色NSC69187粗产物;硅胶柱纯化,得到黄色固体化合物NSC69187;其中,化合物NSC69187的结构式为:
4.一种含有如权利要求2获得的NSC69187衍生化合物的抑制剂,其特征在于,所述抑制剂含有所述化合物CX24。
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