立体选择性合成α-细辛脑的工艺研究.docx

0 要

α-细辛脑是中药石菖蒲中提取出的有效成分。通过对大量文献调查研究，作 者发现 α-细辛脑具有多方面的作用。自上世纪 60 年代起，国内外对 α-细辛脑即 进行了广泛的研究，已证实其在止咳、祛痰、镇静、抗惊厥等方面具有广泛的生 物学活性。而中草药资源有限，关于 α-细辛脑的立体选择性合成问题越来越受到 关注。在此基础上，本文对立体选择性合成 α-细辛脑的反应条件进行了深入考察。

本文以 1,2,4-三甲氧基苯为原料，经 Vilsmeier 反应制得 2,4,5-三甲氧基苯甲 醛。首先研究了利用 Wittig 反应合成 α-细辛脑，结果表明产率和立体选择性都较 低。然后研究了 Grignard 反应合成 α-细辛脑，利用 2,4,5-三甲氧基苯甲醛与乙基 溴化镁反应制得相应的中间体醇，考察了该醇脱水时不同脱水剂对产率和立体选 择性的影响。结果表明采用硫酸铜催化，甲苯恒沸脱水，合成 α-细辛脑（2-((E)-1-丙烯基)-1,4,5-三甲氧基苯），立体选择性 93.3%，总产率 80.26%（以醛计）。

由 α-细辛脑的合成路线可知，在合成的 α-细辛脑中伴有少量的其顺反异构体 β-细辛脑，其结构与 α-细辛脑相似，但 β-细辛脑具有很强的毒性。根据中国药典

0 2000 年版）要求，在合成的 α-细辛脑中 β-细辛脑的含量应小于 1%。作者分别 采用了 HPLC 法和 GC 法对 α-细辛脑的含量进行了测定。对比发现，GC 法较 HPLC 法简便迅速。测定结果表明 α-细辛脑的含量符合药典要求。

该方法为 α-细辛脑的工业生产提供了一种高效简便的方法。

关键词：α-细辛脑、无水硫酸铜、立体选择性、合成、HPLC 法、GC 法

author has found that

Abstract

α-Asarone is the effective ingredient taked out from Acorus gramineus soland. The

α-Asarone is very useful in different fields after reading a number

of literatures. Internal and overseas scientists have studied on α-Asarone in the early 1960s. It is found that α-Asarone play an essential role in relieving a cough, removing the phlegm, calmless, resisting convulsion. Because Chinese traditional medicine is limited, studies on stereoselective synthesis of α-Asarone have been paid more and more attention to. This paper will study the condition of stereoselective synthesis of α-Asarone.

2, 4, 5-trimethoxybenzaldehyde was synthesized from 1, 2, 4-trimethoxybenzene by the Vilsmeier reaction. First, synthesis of α-Asarone by Wittig reaction was studied, the result showed that the yield and stereoselectivity to synthesize α-Asarone is lower. Then synthesis of α-Asarone by Grignard reaction was studied. The intermediate aromatic alcohol was prepared by the reaction of magnesium ethyl bromide with 2,4,5-trimethoxybenzaldehyde, the catalysis of different dehydrant was evaluated for the yield and stereoselectivity to synthesize α-Asarone. The results showed that α-Asarone was stereoselectively obtained with dominant E-stereoselectivity(93.3%) under catalysis of cupric sulfate in dry toluene, the yield was 80.2 % (based on the aromatic aldehyde).

From the synthetic route of α-Asarone, a few β-Asarone will be maked with α-Asarone. β-Asarone whose structure like α-Asarone has strong toxicity. The content of β-Asarone shouldn’t achieve 1% according to the requirement of Chinese pharmacopoeia (2000). The content of α-Asarone was mensurated by the method of HPLC and GC. Comparing the two methods GC is more simple and convenient than HPLC. The result showed that the content of α-Asarone reached the requirement of pharmacopoeia.

A new simple and efficient proces for the industrial production of α-Asarone was provided.

Key Words: α-Asarone、cupric sulfate、stereoselectivity、Synthesis、HPLC、

GC

0 录

第 1 章 绪 论···································································································· 1

1.1 α-细辛脑简介 ·································································································· 1

1.2 α-细辛脑的研究进展············································································ 2

1.2.1 体内代谢································································································ 2

1.2.2 药理作用································································································ 4

1.2.3 临床应用································································································ 5

1.3 本论文的选题背景及目的 ·············································································· 9 第 2 章 2,4,5-三甲氧基苯甲醛的合成及利用 Wittig 反应合成 α-细辛脑 ········· 11

2.1 引言·············································································································· 11

2.2 2,4,5-三甲氧基苯甲醛的合成········································································ 12

2.2.1 合成方法研究························································································· 11

2.2.2 实验部分 ······························································································· 14

2.2.3 小结 ······································································································· 11

2.3 利用 Wittig 反应合成 α-细辛脑···································································· 15

2.3.1 合成方法研究························································································· 12

2.3.2 实验部分 ······························································································· 12

2.4 小结·············································································································· 17

第 3 章 1-(2,4,5-三甲氧基苯)-1-丙醇的合成及合成 α-细辛脑的脱水剂选择 ··· 18

3.1 引言·············································································································· 18

3.2 仪器与药品··································································································· 19

3.2.1 THF 的精制 ···························································································· 19

3.2.2 溴乙烷的精制 ························································································ 20

3.2.3 无水甲苯的精制 ···················································································· 20

3.3 1-(2,4,5-三甲氧基苯)-1-丙醇的合成······························································ 20

3.3.1 合成方法研究 ························································································ 20

3.3.2 实验部分 ······························································································· 20

3.3 合成 α-细辛脑的脱水剂选择········································································ 21

3.4.1 以对甲苯磺酸（p-TsOH）为脱水剂 ····················································· 21

3.4.2 以 20 %的硫酸溶液为脱水剂 ································································ 21

3.4.3 以无水氯化钙为脱水剂········································································· 22

3.4.4 以硫酸氢钾为脱水剂············································································· 22

3.4.5 以无水硫酸铜为脱水剂········································································· 22

3.5 结果与讨论··································································································· 22

3.6 α-细辛脑及中间体醇、原料醛的 1HNMR 谱 ··············································· 24 第 4 章 α-细辛脑的含量分析············································································ 27
4.1 引言·············································································································· 27

4.2 HPLC 法测定 α-细辛脑的含量······································································ 27

4.2.1 HPLC 的类型 ·························································································· 30

4.2.2 HPLC 法在药物分析中的应用································································ 30 4.2.3 HP1050A-高效液相色谱仪的检测条件 ·················································· 30 4.2.4 HPLC 法测定 α-细辛脑的含量 ······························································· 30

4.3 GC 法测定 α-细辛脑的含量 ·········································································· 30

4.3.1 GC 法在药物分析中的应用 ···································································· 31

4.3.2 GC7890Ⅱ气相色谱仪的检测条件·························································· 32

4.3.3 GC 法测定 α-细辛脑的含量···································································· 32

4.4 小结·············································································································· 39 第 5 章 总结与展望 ·························································································· 40
5.1 总结·············································································································· 40

5.2 展望·············································································································· 40 参考文献············································································································· 42 附录 A 攻读学位期间所发表的学术论文目录··················································· 47

0 谢··············································································································· 48

插图索引

图 1.1 α-细辛脑的药时曲线 3

图 2.1 利用乙烯化反应合成 α-细辛脑的反应路线 11

图 2.2 利用 Wittig 反应合成 α-细辛脑的反应路线 12

图 2.3 利用 Grignard 反应合成 α-细辛脑的反应路线 12

图 2.4 利用 Vilsmeier 反应合成 2,4,5-三甲氧基苯的反应机理 13

图 2.5 利用 Wittig 反应合成 α-细辛脑的反应机理 16

图 3.1 Grignard 试剂在乙醚中形成稳定的溶剂化物示意图 18

图 3.2 THF 精制的实验装置图 19

图 3.3 α-细辛脑的 1HNMR 谱图 24

图 3.4 中间体醇的 1HNMR 谱图 25

图 3.5 原料醛的 1HNMR 谱图 26

插・\索引

表 1.1 α-细辛脑生物利用度参数 3

表 2.1 三氯氧磷的用量对产物收率的影响 14

表 3.1 不同脱水剂的脱水情况 23

表 4.1 GC 法测定 α-细辛脑的含量对照表 33

第 1 章 绪 论

自上世纪 60 年代起，国内外对 α-细辛脑进行了广泛的研究，已证实其有很 强的药理活性，具止咳、祛痰、平喘、镇静、解痉以及抗惊厥作用。对肺炎双球 菌、金黄色葡萄球菌和大肠杆菌的生长也有不同程度抑制作用。提取的单体临床 应用于肺炎、哮喘、癫痫大发作等收到良好效果[1]。目前国内有四家细辛脑注射 液生产厂家，市场需求量较大，它的身上蕴藏着巨大的商业价值。

1.1 α-细辛脑简介

α-细辛脑（α-Asarone），又名 α-细辛醚，化学名为 2-((E)-1-丙烯基)-1,4,5-

三甲氧基苯，分子式为 C12H16O3，分子量为 208.25 ，结构式为：

OCH3

OCH3

H3CO

H

H

CH3

其顺反异构体为 β-细辛脑，结构式为：

OCH3

OCH3

H3CO

HCH3

H

α-细辛脑呈微黄色针状结晶，熔点 61 ℃，不溶于水，溶于乙醚，乙醇，氯

0 ，脂溶性极强，它和其异构体 β- 细辛脑是天南星科植物石菖蒲（Acorus gramineus soland ）的主要有效成份之一，主要存在于中药石菖蒲（Acorus gramineus soland）等植物的挥发油中。α-细辛脑口服吸收迅速，15 分钟可达峰， 血浆蛋白结合率为 61 %，可迅速分布于肝、肾、胆汁及心、脑、肺、脾等脏器， 绝对生物利用度极低，胶囊为 2.75 %，片剂为 5.56 %，半衰期长为 3~4 小时， 个体差异大，表观分布容积为 126 L/㎏。

α-细辛脑具有镇咳、祛痰、平喘作用，临床用于治疗慢性支气管炎，慢性阻 塞性肺病。具有镇静抗惊作用，临床用于抗癫痫，是初发癫痫患者首选药之一。

除此之外还有降压、降脂[2]、利胆作用。通过对 α-细辛脑的毒理学研究发现 α-细辛脑小鼠口服 LD60 为 417.6 mg/㎏，腹腔注射 LD50 为 310 mg/㎏，实验表明 α-

0 1 -

细辛脑对鼠伤寒沙门氏菌 TA98 有致突变作用[3]。当剂量大于 182.5 mg/㎏是产生 致畸作用[4]。

1.2 α-细辛脑的研究进展

0 10多年来α-细辛脑的研究不断深入，在基础药理、体内代谢、临床应用， 及在毒理学方面的研究有很大进展。以下为α-细辛脑在体内代谢、药理作用、临 床应用和毒理学方面的研究进展状况。

1.2.1 体内代谢

1.2.1.1 血药浓度测定

杨巷菁等[5] 在1987年报道了用气相色谱法测定兔血液中α-细辛脑浓度的方 法，采用FID检测器，最低检出浓度为400 ng·ml-1。Tsai等[6]1992年用HPLC-UV

法将兔血清中的最低检出浓度提高到100 ng·ml-1。但是仍不能满足人体内药动学 研究的要求，因为α-细辛脑进入人体后浓度极低，达1~100 ng·ml-1。1994年刘宗 河等[7]首次报道了测定人血中α-细辛脑的方法，其主要是依据α-细辛脑化学结构 在苯环上接有3个氧基和1个丙烯基，在紫外光的照射下可发射出极强的荧光，故 可用荧光检测器检测，最后使血清最低检出浓度达到1~200 ng·ml-1。而后，又有 改进此法的报道，检出最低浓度为0.2 ng·ml-1，取血量减少到0.25 ml，更适合药 动学和生物利用度的研究[8] 。

1.2.1.2 体内代谢过程

α-细辛脑口服吸收迅速，15 min即可达到血浓度高峰，血浆蛋白结合率61%， 并迅速分布于肝、肾、胆汁及心、脑、肺、脾等脏器。其中肝、肾浓度接近于血 浆浓度，其余依次递减，部分由胆汁排泄后，仍经肠肝再吸收，最后主要随小便 排出，小部分被肝脏代谢[10]。主要代谢物为2,4,5-三甲氧丙烯酸(肉桂酸) [9]。

1.2.1.3 药动学

新西兰兔静脉给予不同剂量5，10，20 mg·kg-1后，α-细辛脑表现为二室模型， 分布容积没有改变，但清除率却成比例减少为1.28，0.99和0.69 L·h-1·kg-1，而t1/ 2β 却成比例增加为0.45，0.79和1.27 h。故认为α-细辛脑在兔体内的药动学属非线性 [18]。α-细辛脑在人体内的药动学研究，首先由杨正鸿等[10]于1993年报道。在10 名健康志愿者单剂量口服合成α-细辛脑片剂120 mg后，测得t1/ 2β为3.5 h，达峰时

( tmax) 0.91 h，峰浓度(cmax) 120 ng·ml–1，药时曲线下面积(AUC) 149 ng·h·ml-1，吸

- 2 -

收速度常数( Ka) 4.1 h-1，口服显二室模型。该药在人体内吸收快，分布广泛，但 存在较大的个体差异。

1.2.1.4 生物利用度

杨正鸿[11]对12名健康受试者单剂量用药后，通过HPLC法测定血药浓度，测 定了α-细辛脑片剂和胶囊剂在人体中的生物利用度。12名受试者的平均血药浓度 -时间曲线见附图。附表列出了α-细辛脑片剂和胶囊剂的绝对生物利用度, 分别为 5.56%和2.73%。可见α-细辛脑的绝对生物利用度极低。α-细辛脑是脂溶性很强的 药物，水溶性差可能是其生物利用度低的重要原因。故有必要对其生产工艺和处 方组成做一改进。关于α-细辛脑体外溶出速率与体内生物利用度的相关性正待进 一步研究中。

0 1.1 α-细辛脑药时曲线

○-○注射剂(16 mg)；△-△片剂(120 mg)；●-●胶囊剂(120 mg)

0 1.1 α-细辛脑生物利用度参数

剂型
AUC0~∞
F*
TLag
Tmax
Cmax

(ng·h/ml)
(%)
(h)
(h)
(ng/ml)

注射剂
396±250

片剂
135±118
5.5±4.9
0.18±0.04
0.88±0.31
54±55
胶囊剂
72±70
2.7±2.2
0.21±0.02
0.80±0.31
31±40

α-细辛脑片剂的绝对生物利用度明显高于胶囊剂(P < 0.05) , 胶囊剂与片剂 的相对生物利用度为49.1%。究其原因, 可能是生产过程中α-细辛脑的粒度、辅 料及工艺不同造成。α-细辛脑的吸收迅速, 口服后滞留时间均较小, 达峰时较快。 静脉给药平均药时曲线最佳拟合为三室模型(权重选择1/C2) , 口服给药最佳拟合 则为二室模型(权重选择1/C2)。实验证明α-细辛脑片剂和胶囊剂的绝对生物利用 度不仅低, 而且个体差异很大, 有待进一步研究。生物利用度低的产品，其吸收

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更易受生理等因素变化的影响, 往往出现个体差异大的情况。 通过对12名健康受试者单剂量交叉给予人工合成的α-细辛脑胶囊剂120 mg、

片剂120 mg和针剂16 mg后，HPLC-荧光法测定血药浓度。结果发现胶囊剂和片 剂的绝对生物利用度极低，这可能与α-细辛脑是脂溶性极强的化合物，且熔点低 有关。说明制成生物利用度高的固体制剂还有一定困难。

1.2.2 药理作用

1.2.2.1 抗癌活性

胡伯渊等[12]通过观察人癌细胞的形态学改变、活细胞染色计数和测定细胞 生长率等方法，进行了α-细辛脑对人癌细胞株抗癌活性的定量研究。发现在α-细辛脑的作用下，SGC-7901，Detroit-6和Hela人癌细胞发生变形，最终导致脱落、 破坏、溶解且生长稀疏，繁殖受到抑制，而环磷酰胺对照组的作用远较α-细辛脑 弱。α-细辛脑对SGC和D6细胞产生圆缩和脱落的时间可随剂量加大而缩短，与对 照组相比有明显差别。测得对SGC 细胞株有效抑制浓度(ED50)接近于25 µg·ml -1。 对SGC细胞株的生长抑制，比环磷酰胺对照组强。在细胞接种48 h后，才将α-细 辛脑(25 µg·ml-1) 加入到生长液中，6 d后的活细胞染色计数为0.07 万·ml-1，而环 磷酰胺(100 µg·ml-1)则为7.0 万·ml-1。如果在细胞接种同时加入α-细辛脑，则6 d 后的细胞染色计数可降到零，而环磷酰胺仍有2.47 万·ml-1。研究显示了α-细辛 脑对SGC，D6和Hela等人癌细胞株有一定抗癌活性。

1.2.2.2 降血脂作用

α-细辛脑是肝内HMG-CoA还原酶的抑制剂，Pazed等[13]将成年雄鼠按80 mg·kg-1 的α-细辛脑口服用药8 d后，可降血浆中胆固醇38 %。α-细辛脑的治疗主 要是影响血浆中LDL，而HDL不受影响，结果是LDL∕HDL的比值降低74 %。此 结果说明α-细辛脑可抑制脂类的合成和降血脂作用。

Hernandez等[14]用成年鼠的肝细胞进行体外试验，于培养物中加入10 mg·ml-1 的α-细辛脑1~2周后，发现各种脂类明显减少。三酰甘油减少80 %~97 %，胆固 醇酯减少50 %~92 %，胆固醇减少64 %~70 %，磷脂减少70 %~87 %，磷酸盐脱 氢酶和苹果酸酶(由甘油脂类和脂肪酸合成的标记酶)的活性也同样分别减少22 %~50 %和30 %~70 %。此结果说明α-细辛脑可明显地抑制脂类的合成和分泌，具 有降血脂作用。Chamorro等[25]也用成年鼠肝细胞试验，同样显示了上述的结果。 并且作了动物体内的研究，对高血胆固醇症模型雄性鼠，每日口服80 mg·kg-1，7 d后胆固醇和三酰甘油分别减少57.3 %和42.5 %。

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1.2.2.3 利胆作用

α-细辛脑能刺激高胆固醇大鼠的胆汁运输，增加胆盐、磷脂和胆固醇的分泌， 它还能降低胆囊中胆固醇的水平，这是由于α-细辛脑对胆结石细胞溶解酶的影响

[13]

。

经肠道给予1/5的LD50剂量(约136 mg·kg-1)的α-细辛脑后，大鼠的胆汁引流量 可显著升高，作用较去氢胆酸持久，但没有去氢胆酸的胆汁后抑制现象。另一项 研究也表明α-细辛脑可减少80.0 %的大田鼠体内的胆石[15]。

1.2.3 临床应用

1.2.3.1 止咳祛痰

实验证明α-细辛脑通过增强气管纤毛运动，且能减少纤毛与粘液之间的粘附， 引起分泌物增加,使浓痰变稀,减低痰液粘滞，易于咳出；同时对咳嗽中枢也有较 强的抑制作用；从而达到显著的祛痰、镇咳目的，现已被临床实验所证实[16]。

刘露等[17]对有咳嗽、咳痰等症状的50例患儿在常规治疗的基础上辅以α-细辛脑 注射液治疗，用量为0.5 ~1.0 mg·kg-1·d-1，稀释于5 %的葡萄糖注射液中,最大量不 超过24 mg·d-1稀释于5 %的葡萄糖注射液中使之成为浓度为0.01%~0.02%，静脉 滴注，其治疗组有效率达83.3%。陈国水等[18]抽取112例喘息性疾病，按1∶1随 机分组，其中治疗组56例，对照组56例，用量为0.5~1.0 mg·kg-1 ·d-1 稀释于5%或 10%的葡萄糖注射液中，最大量不超过24 mg·d-1稀释于5 %的葡萄糖注射液, 使之 成为浓度为0.01% ~ 0.02%，静脉滴注，治疗组的平均住院天数为6.5 d，而对照 组为9 d，明显缩短，有显著差异(P<0.05)。

1.2.3.2 解痉平喘

α-细辛脑能对抗组胺、乙酰胆碱、5-羟色胺所致气道平滑肌收缩，可松弛支 气管平滑肌，减轻粘膜充血和水肿，缓解下呼吸道阻塞，是临床喘憋症状得以减 轻[19] 。姚德胜等[20]研究认为其解痉、平喘的机制可能是通过抑制T细胞的增殖 与活化，抑制气道嗜酸性粒细胞炎症反应来抑制速发型哮喘反应。田欣等[21]对 116例急性气管炎患儿进行观察，随机分为两组，治疗组63例，对照组53例，两 组在年龄、性别、病程和临床症状上均无显著性差异(P>0.05)。对照组给予青霉 素类药物静脉滴注，治疗组则在对照组的基础上，加用α-细辛脑注射掖0.5 ~ 1.0 mg·kg-1·d-1用5 % GS稀释成0.01% ~ 0.02%的溶液静脉滴注，喘息较重的患儿，静

脉给药8 h后，再给α-细辛脑注射液加入2 ml生理盐水中雾化吸入一次，两组疗程 均为7 d。治疗组经α-细辛脑注射液佐治后,临床症状和体症减轻。陶付营等[22]对

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α-细辛脑辅助治疗支气管哮喘的急性发作进行观察，支气管急性发作病例46例， 分为治疗组21例，对照组25例，对照组常规治疗、吸氧、输液、抗生素等应用治 疗，而治疗组在此基础上加用α-细辛脑注射液。1次16 ~ 24 mg，用5 %或10 %葡 萄糖注射液稀释成0.01 % ~ 0.02 %的溶液静点，结果治疗组显效76.19 %，总有 效率95.24 %，对照组显效20 %，总有效率64 %，治疗组明显优于对照组的疗效。 严健等[23]将188例喘息性支气管炎患儿随机分为对照组62例，治疗组126例，对 照组按常规治疗，治疗组在常治疗的基础上家用α-细辛脑0.15 ~ 1.0 mg·kg-1·d-1， 每日1次，连用3 ~ 9 d，结果治疗治疗组在咳嗽、喘息及肺部哮鸣音吸收方面均 优于常规治疗组，病程也明显缩短(P < 0. 01)。α-细辛脑可拮抗气管致痉剂组胺 和5-羟色胺引起的支气管收缩，能减少动物咳嗽次数[24]，魏楷等[25]在部分慢性 阻塞性肺病(COPD)患者中进行了试用，选定60例病例，随机分为三组α-细辛脑 组：20例用α-细辛脑16 mg溶于100 ml NS中静脉滴注，每日2次，对照组：20例 用氨茶碱0.25 g溶于5 %葡萄糖250 ml静脉滴每日1次，联用组：20例每日用α-细 辛脑32 mg分两次静滴，并用氨茶碱0.25 g静滴。所以α-细辛脑与氨茶碱联合使用 有明显的协同作用，有良好的解痉平喘作用。

1.2.3.3 抗菌消炎

本品可抑制杀灭肺炎球菌、金黄色葡萄球菌等，可用于治疗成人、儿童细菌 性肺炎和肺部感染，临床实验表明α-细辛脑与青霉素的抗菌消炎作用具有可比 性，其祛痰作用远远超过青霉素，只有退热作用次于青霉素，并且不存在过敏实 验的麻烦和过敏实验的危险性[26]。王维实等[26]应用细辛脑片剂治疗148例，以60 mg tidpo连服14 ~ 21 d为一个疗程。对照组：应用野马追风片治疗69例，以4片tid po。治疗组控制率61.1 %，总有效率85.8 %，而对照组控制率为27.5 %总有效率 60.9 %，从数据上看明显差异。细辛脑注射液