CN104288160A - 闭花木酮的o-(哌嗪基)乙基衍生物在制备抗急性痛风药物中的应用 - Google Patents
闭花木酮的o-(哌嗪基)乙基衍生物在制备抗急性痛风药物中的应用 Download PDFInfo
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Abstract
本发明涉及有机合成和药物化学领域，具体涉及闭花木酮的O-(哌嗪基)乙基衍生物、制备方法及其在制备抗急性痛风药物上的用途。本发明合成了一个新的闭花木酮的O-(哌嗪基)乙基衍生物，并公开了其制备方法。药理学实验表明，本发明的闭花木酮的O-(哌嗪基)乙基衍生物具有抗急性痛风的作用，具有开发抗急性痛风药物的价值。
Description
技术领域
本发明涉及有机合成和药物化学领域，具体涉及闭花木酮的O-(哌嗪基)乙基衍生物、制备方法及其用途。
背景技术
痛风(gouty)，又称痛风性关节炎(gouty arthritis)，是体内嘌呤代谢紊乱所致的疾病，表现为血中尿酸过多，易于使尿酸盐(MSU)在关节等组织析出结晶。痛风的急性发作是由于沉积在关节的MSU引起中性粒细胞局部浸润和炎性反应。
西药在痛风急性发作期选用三种药：秋水仙碱、非甾体抗炎药和肾上腺皮质激素。秋水仙碱的作用机制是与中性粒细胞的微管蛋白结合，从而妨碍粒细胞的活动，抑制粒细胞浸润。非甾体抗炎药如吲哚美辛，抑制环氧酶(COX)活性而发挥抗炎作用，以及选择性COX2抑制药。它们虽然消炎止痛作用快，但毒副作用也相当明显，如秋水仙碱的有效剂量与产生腹泻等胃肠道症状的剂量相近，非甾体抗炎药在有活动性消化性溃疡、胃肠道出血情况下绝对禁用，而保泰松用药短至3周也可致严重的粒细胞减少症或再生障碍性贫血。
从天然产物中寻找化合物或先导化合物并进行结构修饰获得其衍生物，从而得到高效低毒的潜在药物最有重要价值。
本发明涉及的化合物闭花木酮Cleistanone是一个2011年发表(Van Trinh ThiThanh et al.,2011.Cleistanone:A Triterpenoid from Cleistanthus indochinensis witha New Carbon Skeleton.Volume 2011,Issue 22,pages 4108–4111,August 2011)的化合物，我们对化合物闭花木酮Cleistanone进行了结构修饰，获得了一个新的闭花木酮Cleistanone衍生物，并对其抗急性痛风活性进行了评价，其具有抗急性痛风活性。
发明内容
本发明公开了一个闭花木酮的O-(哌嗪基)乙基衍生物，其结构为：
本发明闭花木酮Cleistanone衍生物(III)可通过下面方法制备：
(1)闭花木酮Cleistanone(I)与1,2-二溴乙烷反应得到闭花木酮Cleistanone的O-溴乙基衍生物(II)；
(2)闭花木酮Cleistanone的O-溴乙基衍生物(II)与无水哌嗪发生取代反应制得闭花木酮Cleistanone衍生物(III)。
进一步的闭花木酮Cleistanone衍生物(III)的制备方法为：
(1)将440mg化合物闭花木酮Cleistanone(I)溶于10mL苯，向溶液中加入0.04g的四丁基溴化铵，3.760g的1,2-二溴乙烷和6mL的50％氢氧化钠溶液；混合物在25摄氏度搅拌24h；24h之后将反应液倒入冰水中，立即用二氯甲烷萃取两次，合并有机相溶液；然后对有机相溶液依次用水和饱和食盐水洗涤3次，再用无水硫酸钠干燥，最后减压浓缩去除溶剂得到产物粗品；产物粗品用硅胶柱层析纯化，流动相为：石油醚/丙酮＝100:1，v/v，收集黄色集中洗脱带即得到闭花木酮Cleistanone的O-溴乙基衍生物(II)的黄色固体。
(2)将273mg的闭花木酮Cleistanone的O-溴乙基衍生物(II)溶于30mL乙腈当中，向其中加入690mg的无水碳酸钾，168mg的碘化钾和3446mg的无水哌嗪，混合物加热回流3h；反应结束后将反应液倒入冰水中，用等量二氯甲烷萃取2次，合并有机相；依次用水和饱和食盐水洗涤合并之后的有机相，再用无水硫酸钠干燥，减压浓缩去除溶剂得到产物粗品；产物粗品用硅胶柱层析纯化，流动相为：石油醚/丙酮＝100:1.5，v/v，收集淡棕色集中洗脱带即得到闭花木酮Cleistanone衍生物(III)的淡棕色固体。
本发明公开的化合物可以制成药学上可接受的盐或药学上可接受的载体。
本发明用尿酸钠(MSU)致人血管内皮细胞(HUVEC)损伤的急性痛风模型评价显示，本发明化合物对MSU致HUVEC损伤具有保护作用，并抑制ICAM-1表达，可用于制备治疗急性痛风炎症药物。本发明的目的在于提供本发明化合物在医学中的新用途，具体地说是涉及本发明化合物在制备治疗急性痛风药物中的应用。
本发明的目的是通过以下的技术方案来实现的：
现代医学病理研究说明，痛风性关节炎是MSU引起中性粒细胞局部浸润和炎性反应，急性痛风产生的本质是中性粒细胞(PMN)-血管内皮细胞(HUVEC)黏连增强(Terkeltaub R，et al.The murine homolog of the interleukin-8receptorcxcr-2is essential for the occurrence of neutrophilic inflammation in airpouch model of acute urate crystal-induced gouty synovitis.ArthritisRheum,1998,41(5):900-909.)，HUVEC损伤，其分子生物学基础在于PMN与HUVEC表面黏附分子的相互作用(FujiwaraY,et al.Interleukin-8stimulatesleukocyte migration across amonolayer of cultured rabbit NF-α,IL-1β,IL-8,and IL-1rain Monosodium Urate Crystal induced rabbit arthritis.Labinvest,19998,78(5):559-569.)，其中细胞间黏附分子-1(ICAM-1)与粘附功能关系最密切，是急性痛风炎症的重要指标之一(张春，唐怡，刘军，等.痛风灵方对尿酸钠致大鼠模型关节软组织ICAM-1表达的影响，重庆医学，2002,31(12)：1211-1213.)。
因此，针对急性痛风MSU致HUVEC损伤，ICAM-1表达增多的病理特征，本发明用MSU致HUVEC损伤的体外急性痛风模型(杨妍华，尹莲，王明艳，等.尿酸钠诱导HUVEC损伤的急性痛风模型研究，中华中医药学刊，2010,28(3)：592-594.)，评价本发明化合物抗急性痛风炎症活性。MSU致人血管内皮细胞(HUVEC)损伤的急性痛风模型评价显示，本发明化合物对MSU致HUVEC损伤具有保护作用，并抑制ICAM-1表达。
有益效果
研究结果表明，用MSU致HUVEC损伤的急性痛风模型评价显示，本发明化合物可保护MSU致HUVEC损伤，减少细胞凋亡，提高细胞活性，抑制ICAM-1表达，具有抗急性痛风炎症的活性，本发明化合物可用于制备治疗急性痛风炎症药物。
以下通过实施例对本发明作进一步详细的说明，但本发明的保护范围不受具体实施例的任何限制，而是由权利要求加以限定。
具体实施方式
实施例1化合物闭花木酮Cleistanone的制备
化合物闭花木酮Cleistanone(I)的制备方法参照Van Trinh Thi Thanh等人发表的文献(Van Trinh Thi Thanh et al.,2011.Cleistanone:A Triterpenoid fromCleistanthus indochinensis with a New Carbon Skeleton.Volume 2011,Issue 22,pages 4108–4111,August 2011)的方法。
实施例2闭花木酮Cleistanone的O-溴乙基衍生物(II)的合成
将化合物I(440mg，1.00mmol)溶于10mL苯，向溶液中加入四丁基溴化铵(TBAB)(0.04g)，1,2-二溴乙烷(3.760g，20.00mmol)和6mL的50％氢氧化钠溶液。混合物在25摄氏度搅拌24h。24h之后将反应液倒入冰水中，立即用二氯甲烷萃取两次，合并有机相溶液。然后对有机相溶液依次用水和饱和食盐水洗涤3次，再用无水硫酸钠干燥，最后减压浓缩去除溶剂得到产物粗品。产物粗品用硅胶柱层析纯化(流动相为：石油醚/丙酮＝100:1，v/v)，收集黄色集中洗脱带即得到化合物II的黄色固体(344mg，63％)。
1H NMR(500MHz,DMSO-d6)δ5.04(s,1H),4.82(s,1H),3.94(d,J＝26.5Hz,1H),3.87(d,J＝26.5Hz,2H),3.57(s,2H),2.40(d,J＝14.0Hz,1H),2.39(d,J＝14.0Hz,1H),2.27(s,1H),2.21(s,1H),2.15(s,1H),1.82(s,1H),1.62(s,2H),1.57(d,J＝3.3Hz,1H),1.54(d,J＝3.3Hz,1H),1.50(d,J＝1.2Hz,1H),1.47(d,J＝1.2Hz,1H),1.39(d,J＝15.3Hz,2H),1.34(d,J＝15.3Hz,1H),1.26(dd,J＝32.6,13.7Hz,4H),1.13(d,J＝18.0Hz,2H),1.05(s,6H),0.98(s,1H),0.88(s,12H),0.78(s,3H),0.74(s,1H)。
13C NMR(125MHz,DMSO-d6)δ216.59(s),154.50(s),105.23(s),74.63(s),69.85(s),59.71(s),52.55(s),51.21(s),47.92(s),44.10(s),42.25(s),41.73(s),40.64(s),40.16(s),38.88(s),38.65(s),37.21(s),36.23(s),33.34(d,J＝1.1Hz),32.96(s),29.91(s),27.18(s),26.03(s),24.23(s),23.96(s),20.77(s),18.48(s),17.98(s),16.93(s)。
HRMS(ESI)m/z[M+H]+calcd for C32H52BrO2:547.3151；found 547.3159.
实施例3闭花木酮Cleistanone的O-(哌嗪基)乙基衍生物(III)的合成
将化合物II(273mg，0.5mmol)溶于30mL乙腈当中，向其中加入无水碳酸钾(690mg，5.0mmol)，碘化钾(168mg，1.0mmol)和无水哌嗪(3446mg，40mmol)，混合物加热回流3h。反应结束后将反应液倒入冰水中，用等量二氯甲烷萃取2次，合并有机相。依次用水和饱和食盐水洗涤合并之后的有机相，再用无水硫酸钠干燥，减压浓缩去除溶剂得到产物粗品。产物粗品用硅胶柱层析纯化(流动相为：石油醚/丙酮＝100:1.5，v/v)，收集淡棕色集中洗脱带即得到化合物III的淡棕色固体(196.9mg,71％)。
1H NMR(500MHz,DMSO-d6)δ4.63(s,1H),4.53(s,1H),4.45(s,1H),3.56(s,2H),2.68(s,4H),2.58(d,J＝11.6Hz,3H),2.35(d,J＝15.0Hz,5H),2.26(d,J＝14.8Hz,2H),2.20(s,1H),1.89(s,2H),1.81(s,1H),1.63(d,J＝13.0Hz,3H),1.56(s,1H),1.49(d,J＝8.4Hz,2H),1.36(dd,J＝32.4,21.1Hz,3H),1.28(d,J＝3.3Hz,2H),1.23(d,J＝11.0Hz,2H),1.09(s,1H),1.04(s,6H),0.96(d,J＝3.0Hz,13H),0.86(s,3H),0.78(s,1H).
13C NMR(125MHz,DMSO-d6)δ216.60(s),154.52(s),105.26(s),74.66(s),66.84(s),59.80(s),54.86(s),54.35(s),52.60(s),51.21(s),47.93(s),45.78(s),44.13(s),42.31(s),41.82(s),40.61(s),40.10(s),38.79(s),38.58(s),37.21(s),36.22(s),33.27(s),32.92(s),29.84(s),27.13(s),26.03(s),24.22(s),23.89(s),20.73(s),18.41(s),17.93(s),16.93(s).
HRMS(ESI):m/z[M+H]+calcd for C36H61N2O2:553.4733；found:553.4729。
实施例4化合物III抗急性痛风炎症实验
1、溶液配制
5g尿酸加1000ml蒸馏水煮沸，加5％NaOH溶液调PH 7.4，搅拌，冷却析晶制成尿酸钠结晶(MSU)。将制好的MSU 10mg高压灭菌，加不含血清的DMEM培养液10ml，研磨配成1mg/ml的DMEM溶液。实验时，此溶液再加DMEM培养液配成不同浓度DMEM的MSU溶液。
化合物III 2.5mg，用二甲基亚砜(DMSO)溶解，DMSO终浓度<0.02％，再加无血清的DMEM培养液，配制成浓度25ug/ml。
2、血管内皮细胞的体外培养
人脐静脉血管内皮细胞HUVEC株由南京中医药大学基础医学院提供，细胞经支原体检测，无支原体污染，细胞经0.25％胰蛋白酶消化，含10％小牛血清的DMEM培养液中和，离心(1000r/min×6min)，去上清液，加含10％小牛血清的DMEM培养液，移入细胞培养瓶中，放37℃、5％CO2培养箱中传代培养。
3、对MSU刺激HUVEC活力的影响
HUVEC在培养瓶中培养，待生长至70％～80％融合时，以0.25％胰蛋白酶消化、离心，10％小牛血清DMEM培养液洗涤3次，用10％小牛血清DMEM培养液调成4×104/ml细胞悬液，植入96孔板(每孔200ul)，培养24小时后轻吸出原培养液，进行以下实验，每组各8孔，具体分组及加液如下：对照组(200ulDMEM培养液)、模型组(100ug/ml MSU溶液)、干预组(100ug/ml MSU溶液+25ug/ml化合物III)，加液后继续放37℃、5％CO2培养箱中培养24小时，收集上清液，剩余的HUVEC用于测定细胞活性，每孔再加5mg/ml MTT液20ul，继续放37℃、5％CO2培养箱中培养4小时后，弃MTT液，加入二甲基亚砜200ul溶解，震荡，于酶标仪读取吸光度值，波长490nm。
数据统计学处理，细胞活力(％)＝实验组吸光度值/对照组吸光度值×100％，结果见表1。
与对照组相比，模型组细胞活力显著减小(P<0.01，P<0.05)，化合物III干预后细胞活力显著提高(P<0.01，P<0.05)，并强于对照组。
表1化合物III对MSU刺激的血管内皮细胞活力的影响
注：与模型组相比，**P<0.01；与对照组相比，△△P<0.01
4对ICAM-1表达影响
将处于对数生长期的HUVEC用0.25％的胰蛋白酶消化，轻轻吹打，制成细胞悬液，调整细胞密度为5×109/L，接种于细胞培养瓶中。待细胞长满后(约24h)，弃去上清液，分为下列组：对照组、模型组(100ug/ml MSU溶液)、化合物III组(100ug/ml MSU溶液+25ug/ml化合物III)，继续培养24小时，PBS收集细胞，离心去上清，加入CD54单克隆抗体，30min后，PBS洗涤，重悬细胞，应用流式细胞仪检测其阳性细胞百分率(n＝10000)，重复3次，结果见表2。
表2化合物III对MSU刺激的血管内皮细胞ICAM-1表达的影响
注：与模型组相比，**P<0.01；与对照组相比，△△P<0.01
结果显示，空白组HUVEC几乎无ICAM-1表达，模型组ICAM-1的表达最高，与模型组相比，化合物III对ICAM-1的表达有较强的抑制作用。
结论：用MSU致HUVEC损伤的急性痛风模型评价显示，化合物III可保护MSU致HUVEC损伤，减少细胞凋亡，提高细胞活性，抑制ICAM-1表达，具有抗急性痛风炎症的活性，化合物III可用于制备治疗急性痛风炎症药物。
实施例5本发明所涉及化合物III片剂的制备
取20克化合物III或者其药学上可接受的盐当中的一种，加入制备片剂的常规辅料180克，混匀，常规压片机制成1000片。
实施例6本发明所涉及化合物III胶囊的制备
取20克化合物III或者其药学上可接受的盐当中的一种，加入制备胶囊剂的常规辅料如淀粉180克，混匀，装胶囊制成1000粒。
Claims (4)
1.一种具有式III所示结构的闭花木酮的O-(哌嗪基)乙基衍生物(III)及其药学上可接受的盐在治疗急性痛风药物中的应用：
2.如权利要求1所述的一种具有式III所示结构的闭花木酮的O-(哌嗪基)乙基衍生物(III)及其药学上可接受的盐在治疗急性痛风药物中的应用，其特征为：所述急性痛风是由尿酸钠诱导的。
3.如权利要求1或2所述的一种具有式III所示结构的闭花木酮的O-(哌嗪基)乙基衍生物(III)及其药学上可接受的盐在治疗急性痛风药物中的应用，其特征为：闭花木酮的O-(哌嗪基)乙基衍生物(III)减少HUVEC细胞的损伤，提高HUVEC细胞的活性。
4.如权利要求1或2所述的一种具有式III所示结构的闭花木酮的O-(哌嗪基)乙基衍生物(III)及其药学上可接受的盐在治疗急性痛风药物中的应用，其特征为：闭花木酮的O-(哌嗪基)乙基衍生物(III)减少急性痛风过程中ICAM-1的表达。
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| CN104825470A (zh) * | 2015-04-29 | 2015-08-12 | 南京广康协生物医药技术有限公司 | 闭花木酮的o-(苯并咪唑基)乙基衍生物在制备抗急性痛风药物中的应用 |
| CN105250302A (zh) * | 2015-10-14 | 2016-01-20 | 常州南京大学高新技术研究院 | 组合物及其在抗急性痛风药物中的应用 |
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| CN104825470A (zh) * | 2015-04-29 | 2015-08-12 | 南京广康协生物医药技术有限公司 | 闭花木酮的o-(苯并咪唑基)乙基衍生物在制备抗急性痛风药物中的应用 |
| CN105250302A (zh) * | 2015-10-14 | 2016-01-20 | 常州南京大学高新技术研究院 | 组合物及其在抗急性痛风药物中的应用 |
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Inventor after: Le Xiu Inventor before: Zhou Ruixiu |
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Effective date of registration: 20170621 Address after: The science city of Guangzhou high tech Development Zone 510660 Guangdong Nanxiang Road No. 68 No. 303 on the third floor (1) Patentee after: Guangzhou towards the Biological Technology Co., Ltd. Address before: 266000 Licang, Qingdao, No. nine East water road, No. 320, No. Patentee before: Gu Yukui |
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