CN1300163C - 3-羟基色原酮糖苷及其制法和用途 - Google Patents
3-羟基色原酮糖苷及其制法和用途 Download PDFInfo
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Abstract
一类3-羟基色原酮糖苷化合物,其特征是它有如下结构通式:R1=H或AcR2=H、OH或OAc本发明的3-羟基色原酮糖苷化合物对于混合淋巴细胞反应有明显的抑制作用,对于经2,4,6-三硝基氯苯和绵羊红细胞活化的脾细胞有一定诱导其凋亡的活性,对于PCl-CS模型小鼠脾细胞分泌NO也有显著的抑制作用。这些结果表明,本发明的3-羟基色原酮糖苷化合物可以用于制备免疫抑制剂或治疗包括器官移植、类风湿性关节炎、系统性红斑狼疮、慢性肾炎等自身免疫性疾病以及变态反应性疾病等各种免疫性疾病的药物。
Description
技术领域
本发明涉及色原酮类糖苷。
背景技术
色酮类糖苷是一类广泛存在于自然界的天然有机化合物,它们具有一系列重要的生理活性,例如:4,6-二羟基-3-L鼠李吡喃糖基色酮(Eucryphin)色酮类糖苷在整体疾病模型上或细胞模型上显示出选择性的免疫抑制效应,且无现有药物常见的毒副作用。
然而,迄今为止,合成色酮类糖苷的有效方法未见报道,大多数色酮类糖苷主要依靠天然提取。
发明内容
一类3-羟基色原酮糖苷化合物,它有如下结构通式:
R1=H或Ac
R2=H、OH或OAc
一种上述3-羟基色原酮糖苷化合物的制法,它由下列步骤组成:
步骤一、将3-羟基色原酮与2,3,4-三乙酰基-1-氧-L-鼠李吡喃糖基三氯乙亚胺或2,3,4,6-四乙酰基-1-氧-D-葡萄吡喃糖基三氯乙亚胺混合,经真空干燥后,在混合物中加入无水二氯甲烷,体系经液氮-乙醇浴冷却至-42~-78℃,在氮气保护下滴入三氟甲磺酸三甲硅酯,加毕,去冷浴,反应体系逐渐回到室温,用薄层层析监测反应完成后,体系中加入三乙胺终止反应,减压蒸去溶剂后得棕黄色粘稠状液体,经柱层析分离纯化,得白色固体,
步骤二、将步骤一得的白色固体溶于甲醇,通入氨气至饱和,室温下放置,薄层层析检测体系中已无原料,减压蒸去溶剂,得橙黄色至橙红色固体或粘稠状液体,柱层析分离纯化,得白色固体,即为本发明的3-羟基色原酮糖苷化合物。
本发明的3-羟基色原酮糖苷化合物对于混合淋巴细胞反应有明显的抑制作用,对于经2,4,6-三硝基氯苯和绵羊红细胞活化的脾细胞有一定诱导其凋亡的活性,对于PCl-CS模型小鼠脾细胞分泌NO也有显著的抑制作用。这些结果表明,本发明的3-羟基色原酮糖苷化合物可以用于制备免疫抑制剂或治疗包括器官移植、类风湿性关节炎、系统性红斑狼疮、慢性肾炎等自身免疫性疾病以及变态反应性疾病等各种免疫性疾病的药物。
具体实施方式
实施例一、3-L-鼠李吡喃糖基色原酮(VI)的制备
1.3-羟基-4-色原酮二甲基缩醛(II)的制备:
色原酮(I)(2.96g,0.02mol)与100ml绝对无水甲醇混合,冷却至0℃,滴入氢氧化钾(3.36g,0.06mol)与50ml甲醇的混合溶液,溶液呈棕黄色,搅拌下分批加入(分4~5份)碘苯二醋酸盐(C6H5IO(Ac)2,7.0g,0.022mol),时间约5分钟,之后,混合物在0℃下搅拌1小时,撤去冰浴,室温下搅拌过夜。旋转蒸发仪脱去甲醇后,体系中加入100ml水及少量碳酸钾中和,用乙醚萃取5次,每次约40ml。合并有机相,无水硫酸镁干燥,过滤除去干燥剂后,溶液浓缩至约60ml,冰盐浴冷却得无色固体,经乙醚重结晶得无色针状晶体2.83g,m.p.:70~72℃,收率67.30%。
1H-NMR(300MHz,DMSO-D6):
7.50(d,1H,J=7.68Hz) 7.23(t,1H,J=8.00Hz) 6.89(t,1H,J=7.47Hz) 6.80(d,1H,J=8.20Hz) 5.08(bs,1H) 4.25(m,2H) 4.00(m,1H) 3.35(s,3H) 3.03(s,3H)
13C-NMR(75MHz,DMSO-D6):
154.40 130.20 128.97 120.73 119.54 116.75 96.13 69.42 64.08 63.98 48.9248.72
2.3-羟基-4-色原酮(III)的制备:
3-羟基-4-色原酮二甲基缩醛(II)(1.05g,0.005mol)与30ml乙醇混合,体系中加入10ml 3N盐酸,室温下搅拌30分钟后,加入50ml水,继续搅拌15分钟,加入固体碳酸钾中和,用乙醚萃取4次,每次20ml,合并乙醚相,经无水硫酸钠干燥后,脱去溶剂得淡黄色固体,经乙醚重结晶得白色针状固体0.746g,收率90.93%,m.p.:58~59℃(文献值:m.p.:61~62℃)
1H-NMR(300MHz,DMSO-D6):
7.79(d,1H,J=7.83Hz) 7.53(m,1H) 7.08(m,1H) 7.03(d,1H,J=8.36Hz))4.50(m,1H) 4.25(m,1H) 4.48(m,1H)
13C-NMR(75MHz,DMSO-D6):
193.78 161.91 136.81 127.60 122.30 120.46 118.41 71.62 69.33 69.23
3.3-(2’3,4-三乙酰基-L-鼠李吡喃糖基)色原酮(V)的制备:
3-羟基色原酮(III)(2.455g,14.970mmol)和2,3,4-三乙酰基-1-氧-α-L-鼠李吡喃糖基三氯乙亚胺(IV)(6.697g,15.413mmol)混合,经真空干燥2h后,混合物中加入200ml无水CH2Cl2,体系经液氮-乙醇浴冷却至-42℃,N2保护下滴入三氟甲磺酸三甲硅酯355μl,加毕,去冷浴,反应体系逐渐回到室温,总反应时间约需3h,薄层层析(展开剂为石油醚∶乙酸乙酯=3∶2)监测反应结束后,体系中加入三乙胺终止反应,旋转蒸发仪脱去溶剂后得棕黄色粘稠状液体,经柱层析分离纯化(淋洗剂为:石油醚∶乙酸乙酯=3∶1))得白色固体4.656g,m.p.:48~50℃,收率71.33%。化合物经1H-NMR、13C-NMR、IR及元素分析进行了结构验证,数据如下:
1H-NMR(300MHz,CDCl3):
7.89(d,1H,J=7.88Hz) 7.48(t,1H,J=7.78) 7.05(t,1H,J=7.50Hz)6.98(d,1H,J=8.37Hz) 5.33~5.29(m,2H) 5.06(t,1H,J=9.57Hz) 4.95(bs,1H)4.49~4.38(m,3H) 4.27~4.21(m,1H) 2.14(s,3H) 2.03(s,3H) 1.97(s,3H)1.10(d,3H,J=6.24Hz)
13C-NMR(75MHz,CDCl3):
189.56 170.46 170.39 170.21 161.56 136.70 127.95 122.28 120.31 118.2097.20 74.13 71.15 70.08 69.35 69.10 67.50 21 16 21 12 21 01 17.47
IR(KBr);cm-1
2986.35,2938.81,1749.42,1702.85,1607.74,1579.83,1479.26,1466.95,1371.50,1325.63,1288.13,1222.53,1146.72,1095.90,1048.31,982.85,937.69,909.02,835.76,763.80
元素分析结果:计算值:C(%):57.80 H(%):5.50
实测值:C(%):57.72 H(%):5.72
4.3-L-鼠李吡喃糖基色原酮(VI)的制备:
化合物(V)(0.383g,0.878mmol)与CH3OH 25ml混合后,通入NH3至饱和,室温下放置48h后,薄层层析(展开剂为石油醚∶乙酸乙酯=1∶5)监测体系中已无原料,旋转蒸发仪脱去溶剂得橙黄色粘稠状液体,经柱层析分离纯化得白色固体0.159g,m.p.=61~63℃,收率58.46%。化合物经1H-NMR、13C-NMR进行了结构验证,结果表明结构正确,数据如下:
1H-NMR(300MHz,MeOH-D4):
7.84(dd,1H,J=1.58,7.88Hz) 7.55(t×d,1H,J=1.58,8.53Hz) 7.07(t,1H,J=7.57Hz)7.01(d,1H,J=8.40Hz) 5.11(d,0.6H,J=1.28Hz) 4.97(d,0.4H,J=1.2(Hz)4.61~4.37(m,3H) 3.94~3.92(m,0.6H) 3.87~3.86(m,0.4H) 3.75~3.62(m,2H)3.46~3.31(m,1H) 1.32(d,2H,J=6.20Hz) 1.16(d,1H,J=6.20Hz)
13C-NMR(300MHz,MeOH-D4):
191.20 190.39 161.92 161.75 136.58 136.45 127.28 127.20 121.82 121.70120.17 119.95 117.89 117.84 101.00 99.69 73.54 72.74 72.70 72.46 71.1671.06 70.99 70.88 70.84 70.08 69.72 69.47 69.17 17.07 16.74
IR(KBr);cm-1
3416.04,2933.58,1698.48,1608.07,1579.65,1478.90,1286.82,1217.82,1147.82,1099.28,1046.49,980.93,941.42,835.59,760.22
元素分析结果:计算值:C(%):58.06 H(%):5.81
实测值:C(%):58.00 H(%):5.87
实施例四、3-D-葡萄吡喃糖基色原酮(IX)的制备
1.3-(2,3,4,6-四乙酰基-D-葡萄吡喃糖基)色原酮(VIII)的制备:
3-羟基色原酮(III)(1.853g,11.299mmol)和2,3,4,6-四乙酰基-1-氧-D-葡萄吡喃糖基三氯乙亚胺(VII)(5.505g,11.178mmol)混合,经真空干燥2h后,混合物中加入200ml无水CH2Cl2,体系经液氮-乙醇浴冷却至-78℃,N2保护下滴入三氟甲磺酸三甲硅酯250μl,加毕,去冷浴,反应体系逐渐回到室温,总反应时间约需3h,薄层层析(展开剂为石油醚∶乙酸乙酯=3∶2)监测反应结束后,体系中加入三乙胺终止反应,旋转蒸发仪脱去溶剂后得棕黄色粘稠状液体,经柱层析分离纯化(淋洗剂为石油醚∶乙酸乙酯=3∶1)得白色固体3.697g,m.p.:132~135℃,收率66.24%。化合物经1H-NMR、13C-NMR进行了结构验证,结果表明其结构正确,数据如下:
1H-NMR(300MHz,CDCl3):
7.88dd,1H,J=1.69,7.88Hz) 7.51(t×d,1H,J=1.69,7.86Hz) 7.06(t,1H,J=7.52Hz)7.00(d,1H,J=8.31Hz) 5.24(t,1H,J=9.42Hz) 5.13~4.99(m,2H)4.92~4.89(m,1H) 4.58~4.46(m,3H) 4.31~4.14(m,2H) 3.77~3.72(m,1H)2.12(s,3H) 2.04(s,3H) 2.01(s,3H)1.85s,3H)
13C-NMR(300MHz,CDCl3):
189.95 170.96 170.46 169.86 169.78 161.67 136.80 127.78 122.14 119.84118.24 100.37 73.77 72.87 71.39 71.36 70.01 68.74 62.19 21.09 20.93 20.66
IR(KBr);cm-1
2954.59,2900.47,1757.31,1737.02,1688.30,1605.37,1478.19,1466.47,1380.48,1327.47,1293.77,1236.39,1217.27,1153.05,1122.09,1079.61,1037.58,9 4.70,914.09,779.16
元素分析结果:计算值:C(%):55.87 H(%):5.26
实测值:C(%):55.80 H(%):5.41
2.3-D-葡萄吡喃糖基色原酮(IX)的制备:
化合物(XIII)(0.918g,1.858mmol)与CH3OH 25ml混合后,通入NH3至饱和,室温下放置48h后,薄层层析(展开剂为石油醚∶乙酸乙酯=1∶5)监测体系中已无原料,旋转蒸发仪脱去溶剂得橙红色粘稠状固体,柱层析分离纯化得白色固体0.432g,m.p.:54~56℃,收率71.30%。化合物经1H-NMR、13C-NMR进行了结构验证,结果表明结构正确,数据如下:
1H-NMR(300MHz,MeOH-D4):
7.83(d,1H,J=7.84Hz) 7.54(t,1H,J=7.68Hz) 7.06(t,1H,J=7.49Hz)6.99(d,1H,J=8.37Hz) 4.81~4.72(m,1.5H) 4.68~4.23(m,2.5H)3.94~3.83(m,1H) 3.73~3.66(m,1H) 3.46~3.26(m,4H)
13C-NMR(300MHz,MeOH-D4):
192.07 192.01 162.09 161.97 136.78 136.71 127.34 121.83 120.16 119.93117.90 103.75 102.02 77.17 77.13 76.75 75.05 74.14 73.48 70.50 70.4370.27 69.25 61.71 61.66
IR(KBr);cm-1
3420.44,2882.85,1698.43,1606.76,1579.62,1479.25,1288.31,1239.23,1218.40,1163.26,1075.39,1036.42,940.31,895.36,837.48,759.82
元素分析结果:计算值:C(%):55.21 H(%):5.52
实测值:C(%):55.29 H(%):5.81
实施例三:药理试验
一、本发明的色原酮苷类化合物对单向混合淋巴细胞反应(sMLR)的影响
取BALB/c及C57BL/6小鼠,按常规无菌制备脾细胞。即无菌取出小鼠脾脏,置于冷的5ml Hank’s液中,小心挤压获得脾细胞悬液,300g离心5min后,用0.17M Tris-0.75% NH4Cl去除红细胞,再用冷的RPMI-1640(GIBCO)培养液(含100U/ml青霉素、100U/ml链霉素和10%灭活无菌的新生牛血清)洗涤两次。将来自C57BL/6的脾细胞用1×10-6g/ml的丝裂霉素C处理1h后,用冷的RPMI-1640洗两遍,配成2×106cells/ml的细胞悬液。BALB/c小鼠的脾细胞配成2×107cells/ml的细胞悬液。将来自BALB/c小鼠的脾细胞50μl(1×106)与丝裂霉素C处理的C57BL/6的脾细胞50μl(1×105)混合培养96h,用MTT法检测细胞增殖。即在终止培养前4 h向细胞中加入20μl MTF(5mg/ml)培养基溶液,培养结束后,300g离心10min,弃上清,加入200μl二甲亚砜,振荡,使沉淀完全溶解,540nm处测吸光度。刺激指数(SI)按下面公式计算:
SI=(ODC57+BALB/c-ODC57 alone)/ODBALB/c alone
结果如表1所示,落新妇苷(astilbin)和化合物VI均在高浓度显著抑制了混合淋巴细胞的增殖,而化合物IX表现出一定的抑制趋势。其中,以化合物VI的作用最强,astilbin次之,化合物IX的作用较弱。
表1 药物对sMLR的影响
Control | 10e-7 | 10e-6 | 10e-5 | |
astilbinVIIX | 2.87±0.37 | 2.07±0.642.48±1.062.36±0.62 | 2.04±0.671.90±0.632.11±0.44 | 1.47±0.53*1.27±0.44*2.01±0.60 |
*P<0.05,**P<0.01vs control.药物浓度为g/ml.
二、本发明的黄酮苷类化合物对2,4,6-三硝基氯苯(PCl)所致小鼠耳肿胀及绵羊红细胞所致小鼠足肿胀模型中脾细胞凋亡的影响
2,4,6-三硝基氯苯(PCl)所致小鼠耳肿胀(PCl-CS):小鼠剃去腹毛,涂1% PCl的无水乙醇溶液100μl致敏,5d后用1% PCl的橄榄油溶液30μl涂于右耳两面进行攻击,6h后无菌取脾细胞,与不同浓度药物于37℃共孵。
绵羊红细胞所致小鼠足肿胀(SRBC-DTH):在无菌的条件下将SRBC用生理盐水洗三次,洗涤完毕后配成2.5×108/ml的生理盐水溶液,在小鼠左后足趾皮下注射该溶液40μl致敏,5d后在右后足趾皮下注射浓度为2.5×109/ml SRBC的生理盐水溶液40μl进行攻击,6h后无菌取脾细胞,与不同浓度药物于37℃共孵。
PI染色用流式细胞仪测细胞凋亡:常规制备正常及各种不同活化状态的小鼠脾细胞,与不同浓度药物于37℃共孵12h,离心收获细胞,PBS洗两次,加入PI(100μg/ml)100μl,RNAse(100μg/ml)1μl,Triton X-100(1%)1μl,室温下避光作用40min,流式细胞仪检测细胞凋亡,CELLQuest软件分析subG1峰比例(细胞凋亡百分率)。
结果如表2所示,与对照组相比,astilbin和化合物VI均明显诱导了来自2,4,6-三硝基氯苯和绵羊红细胞活化的脾细胞的凋亡,其中astilbin的作用最强,化合物VI作用与astilbin相当。
表2 药物诱导来自2,4,6-三硝基氯苯和绵羊红细胞活化的脾细胞的凋亡
细胞凋亡百分率(%) | PCl-CS | SRBC-DTH |
ControlAstilbinVI | 10.923.119.5 | 20.232.632.0 |
三、本发明的黄酮苷类化合物对2,4,6-三硝基氯苯所致小鼠耳肿胀(PCl-CS)模型小鼠脾细胞分泌NO水平的影响
细胞上清中NO的测定采用Griess法。待测上清与Griess液(1%磺胺,0.1%N-1-萘乙二胺盐酸盐,2.5%磷酸)等体积混合,室温放置10min,检测540nm处的吸光度。以亚硝酸钠为标准品,做标准曲线。
造PCl-CS小鼠模型,取攻击后6h的脾细胞,调成5×105cells/孔种于96孔板,在不同浓度药物及空白培养基溶液的共存下置37℃,5%CO2,培养72h,300g离心10min后,吸取上清用Griess反应检测培养上清中NO水平。
结果如表3所示,PCl-CS模型小鼠脾细胞较正常小鼠脾细胞释放了更多的NO,astilbin及化合物VI均显著抑制了脾细胞释放NO的水平。
表3 药物对PCl-CS小鼠脾细胞分泌NO的影响
NO(uM) | Normal | Control | 10e-6 | 10e-5 | 10e-4 |
astilbinVIIX | 17.5±8.6 | 89.9±8.7 | 70.4±13.277.3±7.684.2±3.9 | 55.3±5.0*60.9±3.8*71.0±4.7 | 48.4±12.6**46.5±2.9**77.3±5.8 |
*P<0.05,**P<0.01vs control.药物浓度为g/ml。
综上所述,本发明的3-羟基色原酮苷类化合物VI对于混合淋巴细胞反应有明显的抑制作用,对于经2,4,6-三硝基氯苯和绵羊红细胞活化的脾细胞有一定诱导其凋亡的活性,对于PCl-CS模型小鼠脾细胞分泌NO亦有显著的抑制作用。这些结果表明,3-羟基色原酮苷类化合物VI可作为免疫抑制剂,有可能用于治疗包括器官移植、类风湿性关节炎、系统性红斑狼疮、慢性肾炎等自身免疫性疾病以及变态反应性疾病等各种免疫性疾病。
Claims (3)
2.一种权利要求1所述的3-羟基色原酮糖苷化合物的制法,其特征是它由下列步骤组成:
步骤一、将3-羟基色原酮与2,3,4-三乙酰基-1-氧-L-鼠李吡喃糖基三氯乙亚胺或2,3,4,6-四乙酰基-1-氧-D-葡萄吡喃糖基三氯乙亚胺混合,经真空干燥后,在混合物中加入无水二氯甲烷,体系经液氮-乙醇浴冷却至-42~-78℃,在氮气保护下滴入三氟甲磺酸三甲硅酯,加毕,去冷浴,反应体系逐渐回到室温,用薄层层析监测反应完成后,体系中加入三乙胺终止反应,减压蒸去溶剂后得棕黄色粘稠状液体,经柱层析分离纯化,得白色固体,
步骤二、将步骤一得的白色固体溶于甲醇,通入氨气至饱和,室温下放置,薄层层析检测体系中已无原料,减压蒸去溶剂,得橙黄色至橙红色固体或粘稠状液体,柱层析分离纯化,得白色固体,即为本发明的3-羟基色原酮糖苷化合物。
3.权利要求1所述的3-羟基色原酮糖苷化合物在制备免疫抑制剂或治疗各种免疫性疾病的药物中的应用。
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US4085116A (en) * | 1975-04-11 | 1978-04-18 | Takeda Chemical Industries, Ltd. | Novel chromone derivatives |
CN1141921A (zh) * | 1995-04-24 | 1997-02-05 | 西巴-盖尔基股份公司 | 色酮衍生物 |
CN1418195A (zh) * | 2000-01-24 | 2003-05-14 | 基纳西亚股份有限公司 | 用于治疗的吗啉代基取代的化合物 |
JP2004077456A (ja) * | 2002-06-18 | 2004-03-11 | Masahiro Sakanaka | ジンセノサイドRb1様物質のスクリーニング方法 |
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US4085116A (en) * | 1975-04-11 | 1978-04-18 | Takeda Chemical Industries, Ltd. | Novel chromone derivatives |
CN1141921A (zh) * | 1995-04-24 | 1997-02-05 | 西巴-盖尔基股份公司 | 色酮衍生物 |
CN1418195A (zh) * | 2000-01-24 | 2003-05-14 | 基纳西亚股份有限公司 | 用于治疗的吗啉代基取代的化合物 |
JP2004077456A (ja) * | 2002-06-18 | 2004-03-11 | Masahiro Sakanaka | ジンセノサイドRb1様物質のスクリーニング方法 |
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