CN102268054A - 一种β-胞嘧啶核苷-5’-三磷酸精氨酸衍生酯及其制备方法和应用 - Google Patents
一种β-胞嘧啶核苷-5’-三磷酸精氨酸衍生酯及其制备方法和应用 Download PDFInfo
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Abstract
本发明属于药物化学领域，特别涉及一种β-胞嘧啶核苷-5′-三磷酸精氨酸酯；本发明还提供了该类化合物的合成方法以及其在治疗由急性脑梗塞引发的逆转脑细胞由缺血向坏死的病变过程，治疗和改善脑细胞功能。
Description
【技术领域】
本发明属于药物化学领域，特别涉及一种β-胞嘧啶核苷-5’-三磷酸精氨酸酯；本发明还提供了该类化合物的合成方法以及其在治疗由急性脑梗塞引发的逆转脑细胞由缺血向坏死的病变过程，治疗和改善脑细胞功能。
【背景技术】
本发明化合物涉及主要病种如下；
一、缺血性脑部疾病
脑血管疾病是由各种脑血管病变所引起的脑部疾病。根据病理性质可分为缺血性脑卒中和出血性脑卒中，前者又称为脑梗死，另外血管性痴呆(vascular dementia，VD)及认知障碍(vascular mild cognitive impairment，VaMCI)也属于脑血管疾病的范畴。
急性脑梗塞(ACI)是神经系统常见病多发病，致死率和致残率高，脑梗塞是脑血管严重狭窄或闭塞，导致脑血流阻断而使脑组织发生缺血坏死和软化。约占全部急性脑血管病的50％-60％。引起脑梗塞的原因较多，主要的是脑血管阻塞及脑部血液循环障碍2种。脑血管急性闭塞后，最初4h-6h缺血区逐渐出现脑水肿，12h后脑细胞开始坏死，但梗塞区与正常脑组织尚难区别。24h后至第5天，脑水肿达到高峰，侧支循环开始建立。从第2周开始，脑水肿逐渐减轻，但梗塞区组织坏死及液化更明显。虽已建立较充分的侧支循环，但可有部分病人在血栓溶解、血管再通的同时，梗塞区的血管壁因缺血性损伤通透性增高，可形成出血性梗塞。
二、重度颅脑损伤
随着交通事故、建筑事故、自然灾害的不断发生，颅脑损伤已成为危及公共生命和健康的重大问题，占各种死亡原因的第4位，其中交通事故占65％左右，全世界每年死亡人数约70万人，尤其青壮年患者发生率高，伴有严重休克和迅速死亡，早期积极有效救治可减少死亡率和伤残率。是神经外科常见病，是严重影响生存质量的疾病，特别是重度颅脑损伤(severe·TBI)其伤残率和死亡率占各部位损伤首位。
三、脑血管病性痴呆
因脑血管病引起的智能障碍，叫作脑血管病性痴呆。这些病人不仅有肢体的瘫痪、语言功能障碍，而且有精神方面的异常，治疗和护理都比较困难，给家庭和社会造成沉重的负担。脑血管病性痴呆发病率很高。我国人口正向老龄化发展，随着平均年龄的增长，痴呆的病人逐渐增多，据统计，在痴呆的病人中，脑血管病性痴呆占30％，老年性痴呆占30％，混合性痴呆占30％，10％是其他类型痴呆，比如煤气中毒后痴呆、脑炎后遗症。
【发明内容】
本发明的目的在于提供一种β-胞嘧啶核苷-5’-三磷酸精氨酸衍生酯，具有下式结构：
本发明的又一目的在于提供上述β-胞嘧啶核苷-5’-三磷酸精氨酸衍生酯的合成方法：
本发明的又一目的在于提供上述β-胞嘧啶核苷-5’-三磷酸精氨酸衍生酯在制备治疗脑血管疾病药物中的应用。
上述应用中的脑血管疾病优选缺血性脑部疾病。
上述应用中的脑血管疾病优选重度颅脑损伤。
上述应用中的脑血管疾病优选痴呆。
上述应用中的脑血管疾病药物优选脑卒中。
上述应用中的脑血管性痴呆优选阿尔茨海默病。
基于此发现，本发明用于治疗和改善脑细胞功能的组合物、方法和用途，包括实施有效量的盐、酯、或相关衍生物都属于本发明涵盖的发明实质和范畴。
综上所述，本发明具有以下特征：
(a)β-胞嘧啶核苷-5’-三磷酸精氨酸酯的β-胞嘧啶核苷-5”-三磷酸胞苷，其可药用前药、衍生物和对应的精氨酸盐。
(b)体外药物作用机制(mechanism of action)研究表明，机体细胞内的5’-三磷酸酯部分显示出对RNA聚合酶的抑制和诱导作用。
(c)制备β-胞嘧啶核苷-5’-三磷酸精氨酸酯的方法。
(d)本发明的化合物通过机体细胞的代谢转化具有活性的化合物。
I 活性化合物，其生理学上可接受的酯和前药
通过选择性试验，5’-羟基被5’-羰基-R代替，其中R是磷酸酯(包括单磷酸酯、二磷酸酯、三磷酸酯或稳定的磷酸酯前药)，其在体内可以作为药用离去基团，这些离去基团在体内给药后能够提供R独立为H或磷酸盐的化合物。
II 活性化合物的合成
制备了胞嘧啶核苷-5’-三磷酸二钠，在其1‘P上发生精氨酸双取代成酯，其过程中会出现类似物的中间体，也可用于作为治疗由急性脑梗塞引发的逆转脑细胞由缺血向坏死的病变过程。
β-胞嘧啶核苷-5’-三磷酸精氨酸酯
III选择性合成
步骤1：化合物1的合成：制备β-胞嘧啶核苷-5’-三磷酸二钠
配备带有电磁搅拌器、数显式温度计和氮气保护气装置的2500ml三口圆底烧瓶一个。向2500ml三口圆底烧瓶加入磁力搅拌转子后，加入1000ml的室温的去离子水，向水面下通入经过过滤的氮气30分钟，然后加入β-胞嘧啶核苷-5”-三磷酸并搅拌使溶液澄清，分3次在10分钟内加入氢氧化钠溶液(0.1mol/L)，并对混合溶液进行激烈地搅拌，反应温度由室温温度上升到35℃左右，经过约30分钟的反应，液面逐渐出现白色悬浮液，反应液的pH保持在7.2～7.8左右，保持以上反应条件，白色悬浮液层逐渐加厚，使反应持续20小时以上即可终止反应，快速过滤得近无色滤液，为化合物1。
化合物1
步骤2：化合物2的合成：制备β-胞嘧啶核苷-5”-三磷酸精氨酸酯
向化合物1中通入经过过滤后的氮气，通气持续60分钟以上，然后向化合物1的溶液中逐滴加入精氨酸溶液(0.5mol/L)，(加入时注意滴加位置尽量靠近搅拌漩 涡的中心)。反应液先缓慢浑浊约10分钟左右，继续通入氮气保持至滴加结束后1个小时左右，整个过程的搅拌匀速且不得停止。该步反应终点为反应液的pH值达到6.5以下时，即可停止反应，将反应液移出三口圆底烧瓶，备用，为化合物2。光谱数据：13C-NMR(D2O)：25.03，28.77，41.64，55.45，65.52，64.59，69.24，，74.46，83.43，83.52，89.50，95.62，144.03，149.49，157.85，159.90，175.38。MS-FAB分子离子峰：810【M+Na】+
附图说明
图1：本发明化合物的结构式
图2本发明化合物的红外结构确认图谱
具体实施方式
实施例1-β-胞嘧啶核苷-5’-三磷酸精氨酸酯的紫外分析
采用紫外-可见分光光度法分析测定上述化合物，取化合物1配制供试品溶液，在280±2nm的波长处有最大吸收；
表1：最大吸收波长位置和吸收度比值范围：
取化合物2配制供试品溶液，在250±2nm的波长处有最大吸收；
表2：最大吸收波长位置和吸收度比值范围：
实施例2-β-胞嘧啶核苷-5’-三磷酸精氨酸酯的HPLC分析：
检测波长为280nm，以0.02mol/L磷酸盐缓冲液∶甲醇(75∶25)为流动相，理论塔板数不低于2000，分离度大于1.5，流速为1.0ml/mim，运行时间20分钟，进样体积为10μl/次，各次操作间隔10分钟达到平衡。
表1：化合物1的保留时间
紫外检测器的检测波长为272nm，色谱柱采用C18，3.9×150mmID，4μm的颗粒尺寸，以0.02mol/L磷酸盐缓冲液∶甲醇(75∶25)为流动相，理论塔板数不低于2000，分离度大于1.5，流速为1.00ml/mim，运行时间35分钟，进样体积为10μl/次，柱温控制在25℃，各次操作间隔10分钟达到平衡。
表2：化合物2的保留时间
实施例3-β-胞嘧啶核苷-5’-三磷酸精氨酸酯的药理学
β-胞嘧啶核苷-5’-三磷酸精氨酸酯可促进细胞内磷脂、脂肪以及核酸和蛋白质的合成代谢，调节细胞生物膜性结构的合成于改建，增强血管细胞的活性及再生和修复能力，提高细胞的抗损能力，促进神经细胞的物质、能量代谢，支持神经元存活，延缓神经细胞的衰老及血管硬化，并能通过血脑屏障，直接进入神经细胞中，影响呼吸链，改善脑细胞缺血、缺氧状况。
用于治疗多种原因引起的神经系统疾病及血管硬化症，参与机体膜性结构合成与构建的胞嘧啶核苷-5”-三磷酸胞苷在细胞内可被分解代谢成一磷酸胞苷，然后在酶的作用下逐步降解成二氧化碳和水，并释放能量。
VI化合物的体外药理活性
(a)化合物1的体外细胞药理学：体内转化为活性三磷酸酯的磷酸化测定：为 了确定化合物1的细胞代谢过程，选取原发性人肝细胞(primary human hepatocyte)培养物中获得HepG2细胞，每隔3天更换一次介质，细胞每间隔一周进行一次亲代分株传代培养，当融合性的HepG2细胞种植到密度为2.5×106细胞/孔的6-孔板上，用介质进行3次连续的洗涤而分离粘着的单层后，将细胞在37℃的5％的CO2环境中保持，在选定的时刻，采用磷酸盐缓冲液冲洗细胞3次后，用甲醇培养液培养细胞过夜，然后用20μl甲醇在冰水浴中提取1小时，合并提取物，在温和的过滤空气中干燥，并储存在-20℃用于分析测定磷酸酯化的程度(见下表1)。
表1化合物1对磷酸酯化的程度的作用
通常认为药物在临床使用过程中，进入体内细胞后，磷酸化程度越高，机体各种脂质细胞膜结构透过率就越高，药物参与机体循环和起效就越快。上述测定数据显示化合物1的体外活性较高。
(b)化合物2的体外代谢：对mCyd在人血浆中的稳定性进行了研究，在0，21或37℃温育，并在达到10小时的不同时刻对研究样品进行分析(见下图)。在37℃，化合物2有效转变为mCyd，在10小时后仅有2％的输入化合物2剩余，化合物2在人血浆的体外半衰期为1.81小时，并在37℃温育后，没有检测到化合物2的脱磷酸基发生。同时在人血浆的所有三种细胞类型中观察到少量β-胞嘧啶核苷-5’-三磷酸精氨酸酯的化合物原型，约为相应应转化TP量的10％以下。同步，在HepG2原发性人肝细胞和MDBK牛肾细胞中均未检测出β-胞嘧啶核苷-5’-三磷酸精氨酸酯的化合物原型及任何单磷酸酯代谢物。检测显示β-胞嘧啶核苷-5’-三磷酸精氨酸酯能够很好进入细胞代谢。
(c)化合物2的抗脑缺血研究：实验研究表明，β-胞嘧啶核苷-5’-三磷酸精氨酸酯小剂量(2mg/kg)和中剂量(5mg/kg)和大剂量(10mg/kg)均可明显延长结扎两侧颈总动脉造成急性脑缺血小鼠的存活时间，与生理盐水组比较，有统计学意义(P＜0.01)。提示该化合物对小鼠急性脑缺血具有保护作用。
实施例4-β-胞嘧啶核苷-5’-三磷酸精氨酸酯的影响因素试验：
(a)强光照射试验
将β-胞嘧啶核苷-5’-三磷酸精氨酸酯(下简称本供试品)置4500lx±500lx光照强度下，照射样品10天。分别于第5天和第10天取样，按稳定性重点考察项目进行检测。结果见表1。
表1：强光照射试验稳定性考察结果
结果表明：本品经光照后各项指标均无明显改变，对光有一定的稳定性。
(b)高温试验
将试验样品模拟市售包装，在60℃和条件下放置10天，分别于第5天和第10天取样，按稳定性重点考察项目进行检测。结果见表2。
表2：高温试验稳定性考察结果
以上结果表明：本品在60℃条件下试验10天，结果表明该品含量明显下降，其它指标无明显变化；在40℃条件下试验10天，结果表明该品含量无明显下降，其它指标也无明显变化，说明本品对60℃不稳定，不宜高温保存。
Claims (8)
1.一种β-胞嘧啶核苷-5’-三磷酸精氨酸衍生酯，具有下式结构：
2.权利要求1所述β-胞嘧啶核苷-5’-三磷酸精氨酸衍生酯的合成方法，其特征在于采用以下步骤：
3.权利要求1所述β-胞嘧啶核苷-5’-三磷酸精氨酸衍生酯在制备治疗脑血管疾病药物中的应用。
4.根据权利要求3所述应用，其特征在于所述脑血管疾病为缺血性脑部疾病。
5.根据权利要求3所述应用，其特征在于所述脑血管疾病为重度颅脑损伤。
6.根据权利要求3所述应用，其特征在于所述脑血管疾病为痴呆。
7.根据权利要求3所述应用，其特征在于所述脑血管疾病药物为脑卒中。
8.根据权利要求6所述应用，其特征在于所述脑血管性痴呆为阿尔茨海默病。
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| CN103044509B (zh) * | 2013-01-05 | 2015-04-01 | 宁辉 | γ-胞嘧啶核苷-5’-三磷酸二钠结晶化合物、其制备方法及其药物组合物 |
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