WO2013060258A1

WO2013060258A1 - Clavatine a-c, preparation method thereof and pharmaceutical composition and use thereof

Info

Publication number: WO2013060258A1
Application number: PCT/CN2012/083360
Authority: WO
Inventors: 庾石山; 陈乃宏; 王晓婧; 苑玉和; 屈晶; 马双刚; 李勇
Original assignee: 中国医学科学院药物研究所
Priority date: 2011-10-24
Filing date: 2012-10-23
Publication date: 2013-05-02
Also published as: CN103059043A

Abstract

The present invention relates to 3 alkaloids (clavatine A-C) isolated from Lycopodium japonicum Thunb., a pharmaccodynamically acceptable salt thereof and a preparation method thereof, a pharmaceutical composition containing this class of compounds and the use of this class of compounds in preparing acetylcholinesterase inhibitor drugs, drugs for treating Alzheimer's disease and drugs for resisting diseases related to decline in memory and cognitive ability of middle-aged and elderly people.

Description

说明书伸筋草碱 A-C、其制法和其药物组合物与用途 Description: Astragalus A-C, its preparation method and its pharmaceutical composition and use

技术领域 Technical field

本发明属于药物技术领域，具体而言，本发明涉及一类石松生物碱化合物：伸筋草碱 A-C (1-3),其药效学上可接受的盐，其制备方法，含有这类化合物的药物组合物，以及这类化合物在制备乙酰胆碱酯酶抑制剂药物中、在制备治疗早老性痴呆症药物中和制备抗中老年记忆和认知能力减退疾病药物中的应用。 The present invention relates to the field of pharmaceutical technology, and in particular, the present invention relates to a class of stone pine alkaloid compounds: Streptococcus alkaloid AC (1-3), a pharmaceutically acceptable salt thereof, a preparation method thereof, and a compound containing the same Pharmaceutical compositions, and the use of such compounds in the preparation of acetylcholinesterase inhibitor drugs, in the preparation of a medicament for the treatment of Alzheimer's disease, and in the preparation of a medicament for the prevention of memory and cognitive decline in the elderly.

背景技术 Background technique

随着世界人口平均寿命的逐年增加，老年性痴呆症的发病率也随着迅速上升，老年痴呆症中有 50%-70%为阿尔兹海默氏病（AD) , 又称早老性痴呆症，患者的脑功能逐渐衰退，出现记忆力、抽象思维能力和语言表达能力减退、行动不便和其他功能性障碍，影响神经系统功能，不仅给患者带来痛苦，而且也给家庭和社会带来许多问题。 As the world's average life expectancy increases year by year, the incidence of Alzheimer's disease increases rapidly. Alzheimer's disease (AD) is also found in Alzheimer's disease (AD), also known as Alzheimer's disease. The patient's brain function is gradually declining, memory, abstract thinking ability and language expression decline, inconvenience and other functional disorders, affecting nervous system function, not only bring pain to patients, but also bring many problems to family and society. .

AD是一种以进行性认知和记忆力损伤为主的中枢神经***退行性疾病，在发达国家成为继心血管病和肿瘤之后引起死亡的主要疾病。治疗 AD的药物主要有胆碱能类药物、神经细胞代谢增强剂、抗氧化剂、钙离子拮抗剂、神经生长因子、 ***、抗炎药、抗 β淀粉样蛋白药物等。目前临床上治疗该病最成功的方法就是通过提高中枢神经*** 内胆碱能神经递质一乙酰胆碱的水平，改善 AD病人的症状。乙酰胆碱酯酶抑制剂（AChEI) 通过针对性地抑制乙酰胆碱酯酶（AChE) 对乙酰胆碱的水解作用而提高脑内的乙酰胆碱水平，成为治疗 AD的一线药物。临床上有效的药物如他克林、安理申、艾斯能、加兰他敏和石杉碱甲均为乙酰胆碱酯酶抑制剂。而著名的天然产物石杉碱甲是从中国特有蕨类植物千层塔（蛇足石杉， Huperzia serrata (Thunb) Trev.，石杉科（Huperziaceae ) ) 中提取的一种生物碱，为可逆性胆碱酯酶专一性抑制剂，对乙酰胆碱酯酶具有选择性抑制作用，而且毒性低，作用时间长，目前已被开发成治疗良性记忆障碍和早老性痴呆最为安全有效的药物之一，也是中国首创的一种世界级新药，其成功开发也使我们看到了从天然产物中寻找新型活性成分并进行新药研制的希望。 AD is a central nervous system degenerative disease characterized by progressive cognitive and memory impairment. It has become the leading cause of death after cardiovascular disease and cancer in developed countries. The main drugs for treating AD include cholinergic drugs, neuronal cell metabolism enhancers, antioxidants, calcium ion antagonists, nerve growth factors, estrogens, anti-inflammatory drugs, and anti-beta amyloid drugs. The most successful method for the treatment of this disease in the clinic is to improve the symptoms of AD patients by increasing the level of cholinergic neurotransmitter-acetylcholine in the central nervous system. Acetylcholinesterase inhibitor (AChEI) specifically inhibits acetylcholinesterase (AChE) The hydrolysis of acetylcholine increases the level of acetylcholine in the brain and becomes the first-line drug for the treatment of AD. Clinically effective drugs such as tacrine, alexin, isgen, galantamine and huperzine A are all acetylcholinesterase inhibitors. The famous natural product, huperzine A, is an alkaloid extracted from the Chinese fern phylum (Huperzia serrata (Thunb) Trev., Huperziaceae), which is reversible. A specific inhibitor of cholinesterase, which has a selective inhibitory effect on acetylcholinesterase, and has low toxicity and long duration of action. It has been developed as one of the safest and most effective drugs for the treatment of benign memory disorders and Alzheimer's disease. The successful development of a world-class new drug pioneered in China has also enabled us to see the hope of finding new active ingredients from natural products and developing new drugs.

伸筋草 (石松， Lycopodium japonicum Thunb. ) 为石松禾斗 (Lycopodiaceae )石松属植物，为我国常用传统中药，主要功效为祛风除湿、舒筋活络，用于关节酸痛，屈伸不利。民间及临床使用发现伴有增强心肌收缩力、兴奋平滑肌、引起肌肉收缩抽搐等胆碱能副反应，暗示其中可能含有对胆碱能***有作用的化学成分。本发明技术方案试图对伸筋草中的微量生物碱类成分进行深入研究，期望发现结构新颖，具有乙酰胆碱酯酶抑制作用的天然活性成分。 Lycopodium japonicum Thunb. is a Lycopodiaceae stone pine plant. It is a traditional Chinese medicine commonly used in China. Its main functions are phlegm dehumidification, sturdy muscles, and it is used for joint pain and flexion and extension. It is found in folk and clinical use that it has a cholinergic side reaction that enhances myocardial contractility, excitatory smooth muscle, and muscle contraction and convulsions, suggesting that it may contain chemical components that have an effect on the cholinergic system. The technical scheme of the present invention attempts to conduct intensive studies on trace alkaloids in Strutgrass, and it is expected to find a novel active ingredient having a novel structure and having an acetylcholinesterase inhibitory action.

发明内容 Summary of the invention

本发明要解决的技术问题是，提供一类新结构类型的化合物：伸筋草碱 A-C (l-3) 及其药效学上可接受的盐。

The technical problem to be solved by the present invention is to provide a new class of compounds of the type: Streptococcus grass AC (l-3) and its pharmacologically acceptable salts.

本发明的另一个方面涉及化合物伸筋草碱 A-C (1-3)的制备方法。本发明又一个方面又涉及一种药物组合物，其包括药物有效剂量的、作为活性成分的化合物伸筋草碱 A-C (1-3)及制药领域中常用的载体。 Another aspect of the invention relates to a process for the preparation of the compound ergic acid A-C (1-3). Still another aspect of the invention relates to a pharmaceutical composition comprising a pharmaceutically effective amount of the compound Asparticillin A-C (1-3) as an active ingredient and a carrier commonly used in the pharmaceutical field.

本发明再一个方面涉及化合物伸筋草碱 A-C (1-3)及其组合物在制备乙酰胆碱酯酶抑制剂药物中、在制备治疗早老性痴呆症药物中、和制备抗中老年记忆和认知能力减退疾病药物中的应用。 Still another aspect of the present invention relates to a compound of sylvestreine AC (1-3) and a composition thereof for use in the preparation of an acetylcholinesterase inhibitor drug, in the preparation of a medicament for treating Alzheimer's disease, and for preparing anti-middle-age memory and cognition The application of drugs with reduced ability.

本发明化合物的药物组合物可根据本领域公知的方法制备。用于此目的时，如果需要，可将本发明化合物与一种或多种固体或液体药物赋形剂和 /或辅剂结合，制成可作为药物使用的适当的施用形式或剂量形式。 Pharmaceutical compositions of the compounds of the invention can be prepared according to methods well known in the art. For this purpose, if desired, the compound of the present invention may be combined with one or more solid or liquid pharmaceutical excipients and/or adjuvants to provide a suitable administration form or dosage form for use as a medicament.

本发明化合物或含有它的药物组合物可以单位剂量形式给药，给药途径可为肠道或非肠道，如口服、静脉注射、肌肉注射、皮下注射、腹腔注射、鼻腔、口腔粘膜、目艮、肺和呼吸道、皮肤、 ***、直肠等，优选口服给药。 The compound of the present invention or a pharmaceutical composition containing the same may be administered in a unit dosage form, which may be enterally or parenterally, such as oral, intravenous, intramuscular, subcutaneous, intraperitoneal, nasal, oral mucosa, The sputum, lungs and respiratory tract, skin, vagina, rectum, etc., are preferably administered orally.

给药剂型可以是液体剂型、固体剂型或半固体剂型。液体剂型可以是溶液剂（包括真溶液和胶体溶液）、乳剂（包括 o/w型、 w/o型和复乳）、混悬剂、注射剂（包括水针剂、粉针剂和输液）、滴眼剂、滴鼻剂、洗剂和搽剂等。固体剂型可以是片剂（包括普通片、肠溶片、含片、分散片、咀嚼片、泡腾片、口腔崩解片）、胶囊剂（包括硬胶囊、软胶囊、肠溶胶囊）、颗粒剂、散剂、微丸、滴丸、栓剂、膜剂、贴片、气（粉）雾剂、喷雾剂等；半固体剂型可以是软膏剂、凝胶剂、糊剂等。 The dosage form can be a liquid dosage form, a solid dosage form or a semi-solid dosage form. Liquid dosage forms can be solutions (including true and colloidal solutions), emulsions (including o/w, w/o and double emulsions), suspensions, injections (including water injections, powder injections and infusions), eye drops Agents, nasal drops, lotions and tinctures. The solid dosage form can be a tablet (including ordinary tablets, enteric coated tablets, Tablets, dispersible tablets, chewable tablets, effervescent tablets, orally disintegrating tablets), capsules (including hard capsules, soft capsules, enteric capsules), granules, powders, pellets, dropping pills, suppositories, films, Patch, gas (powder) spray, spray, etc.; semi-solid dosage form may be ointment, gel, paste, and the like.

本发明化合物可以制成普通制剂、也可以是缓释制剂、控释制剂、靶向制剂及各种微粒给药***。 The compounds of the present invention can be formulated into common preparations, sustained release preparations, controlled release preparations, targeted preparations, and various microparticle delivery systems.

为了将单位给药剂型制成片剂，可以广泛使用本领域公知的各种赋形剂，包括稀释剂、黏合剂、润湿剂、崩解剂、润滑剂、助流剂。稀释剂可以是淀粉、糊精、蔗糖、葡萄糖、乳糖、甘露醇、山梨醇、木糖醇、微晶纤维素、硫酸钙、磷酸氢钙、碳酸钙等；湿润剂可以是水、乙醇、异丙醇等；粘合剂可以是淀粉桨、糊精、糖桨、蜂蜜、葡萄糖溶液、微晶纤维素、 ***胶桨、明胶桨、羧甲基纤维素钠、甲基纤维素、羟丙基甲基纤维素、乙基纤维素、丙烯酸树脂、卡波姆、聚乙烯吡咯垸酮、聚乙二丙醇等；崩解剂可以是干淀粉、微晶纤维素、低取代羟丙基纤维素、交联聚乙烯吡咯垸酮、交联羧甲基纤维素钠、羧甲基淀粉钠、碳酸氢钠与枸橼酸、碳酸钙、聚氧乙烯山梨糖醇脂肪酸酯、十二垸基磺酸钠；润滑剂和助流剂可以是滑石粉、二氧化硅、硬脂酸盐、酒石酸、液体石蜡、聚乙二醇等。 In order to prepare a unit dosage form into tablets, various excipients well known in the art, including diluents, binders, wetting agents, disintegrating agents, lubricants, and glidants, can be widely used. The diluent may be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc.; the humectant may be water, ethanol, or different Propyl alcohol, etc.; the binder may be starch paddle, dextrin, sugar paddle, honey, glucose solution, microcrystalline cellulose, gum arabic paddle, gelatin paddle, sodium carboxymethylcellulose, methylcellulose, hydroxypropyl Methyl cellulose, ethyl cellulose, acrylic resin, carbomer, polyvinylpyrrolidone, polyethylene glycol, etc.; disintegrant can be dry starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose , cross-linked polyvinylpyrrolidone, croscarmellose sodium, sodium carboxymethyl starch, sodium hydrogencarbonate and citric acid, calcium carbonate, polyoxyethylene sorbitan fatty acid ester, dodecylsulfonate Sodium; the lubricant and glidant may be talc, silica, stearate, tartaric acid, liquid paraffin, polyethylene glycol, and the like.

还可以将片剂进一步制成包衣片，例如糖包衣片、薄膜包衣片、肠溶包衣片，或双层片和多层片。 Tablets may also be further formulated into coated tablets, such as sugar coated tablets, film coated tablets, enteric coated tablets, or bilayer tablets and multilayer tablets.

为了将给药单元制成丸剂，可以广泛使用本领域公知的各种载体。关于载体的例子是，例如稀释剂与吸收剂，如葡萄糖、乳糖、淀粉、可可脂、氢化植物油、聚乙烯吡咯垸酮、月桂酸聚乙二醇甘油酯、高岭土、滑石粉等；粘合剂，如***胶、黄耆胶、明胶、乙醇、蜂蜜、液糖、米糊或面糊等；崩解剂，如琼脂粉、干燥淀粉、海藻酸盐、十二垸基磺酸钠、甲基纤维素、乙基纤维素等。 In order to prepare the administration unit into a pellet, various carriers known in the art can be widely used. Examples of carriers are, for example, diluents and absorbents such as glucose, lactose, and lakes. Powder, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, lauric acid polyethylene glycol glyceride, kaolin, talcum powder, etc.; binders such as acacia, tragacanth, gelatin, ethanol, honey, liquid sugar, Rice paste or batter; etc.; disintegrating agents, such as agar powder, dried starch, alginate, sodium dodecylsulfonate, methylcellulose, ethylcellulose, and the like.

为了将给药单元制成栓剂，可以广泛使用本领域公知的各种载体。关于载体的例子是，例如聚乙二醇、卵磷脂、可可脂、高级醇、高级醇的酯、明胶、半合成甘油酯等。 In order to prepare the drug delivery unit as a suppository, various carriers well known in the art can be widely used. Examples of the carrier are, for example, polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, semi-synthetic glycerides and the like.

为了将给药单元制成胶囊，将有效成分本发明化合物伸筋草碱 In order to encapsulate the drug delivery unit, the active ingredient of the present invention

A-C (1-3)与上述的各种载体混合，并将由此得到的混合物置于硬的明胶胶囊或软胶囊中。也可将有效成分本发明化合物制成微囊剂，混悬于水性介质中形成混悬剂，亦可装入硬胶囊中或制成注射剂应用。 A-C (1-3) is mixed with the various carriers described above, and the mixture thus obtained is placed in a hard gelatin capsule or soft capsule. The active ingredient of the compound of the present invention can also be formulated into a microcapsule, suspended in an aqueous medium to form a suspension, or can be enclosed in a hard capsule or used as an injection.

例如，将本发明化合物伸筋草碱 A-C (1-3)制成注射用制剂，如溶液剂、混悬剂溶液剂、乳剂、冻干粉针剂，这种制剂可以是含水或非水的，可含一种和 /或多种药效学上可接受的载体、稀释剂、粘合剂、润滑剂、防腐剂、表面活性剂或分散剂。如稀释剂可选自水、乙醇、聚乙二醇、 1, 3-丙二醇、乙氧基化的异硬脂醇、多氧化的异硬脂醇、聚氧乙烯山梨醇脂肪酸酯等。另外，为了制备等渗注射液，可以向注射用制剂中添加适量的氯化钠、葡萄糖或甘油，此外，还可以添加常规的助溶剂、缓冲剂、 pH调节剂等。这些辅料是本领域常用的。 For example, the compound of the present invention, ergic acid AC (1-3), is prepared as an injectable preparation, such as a solution, a suspension solution, an emulsion, a lyophilized powder injection, and the preparation may be aqueous or non-aqueous. One or more pharmaceutically acceptable carriers, diluents, binders, lubricants, preservatives, surfactants or dispersing agents may be included. For example, the diluent may be selected from the group consisting of water, ethanol, polyethylene glycol, 1, 3-propanediol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitan fatty acid ester, and the like. Further, in order to prepare an isotonic injection, an appropriate amount of sodium chloride, glucose or glycerin may be added to the preparation for injection, and a usual solubilizer, a buffer, a pH adjuster or the like may be added. These excipients are commonly used in the art.

此外，如需要，也可以向药物制剂中添加着色剂、防腐剂、香料、矫味剂、甜味剂或其它材料。 In addition, coloring agents, preservatives, perfumes, flavoring agents, sweeteners or other materials may also be added to the pharmaceutical preparations as needed.

为达到用药目的，增强治疗效果，本发明的药物或药物组合物可用任何公知的给药方法给药。 In order to achieve the purpose of administration, the therapeutic effect is enhanced, and the medicament or the pharmaceutical composition of the present invention can be Administration is by any known method of administration.

本发明化合物药物组合物的给药剂量取决于许多因素，例如所要预防或治疗疾病的性质和严重程度，患者或动物的性别、年龄、体重、性格及个体反应，给药途径、给药次数、治疗目的，因此本发明的治疗剂量可以有大范围的变化。一般来讲，本发明中药学成分的使用剂量是本领域技术人员公知的。可以根据本发明化合物组合物中最后的制剂中所含有的实际药物数量，加以适当的调整，以达到其治疗有效量的要求，完成本发明的预防或治疗目的。本发明化合物的每天的合适剂量范围为 0.001〜10 mg/Kg体重，分 2-4次服用。本发明的化合物或组合物可单独服用，或与其他治疗药物或对症药物合并使用并调整剂量。术语与简称 The dosage of the pharmaceutical composition of the compound of the present invention depends on a number of factors, such as the nature and severity of the disease to be prevented or treated, the sex, age, weight, personality and individual response of the patient or animal, the route of administration, the number of administrations, For therapeutic purposes, the therapeutic dose of the present invention can vary widely. In general, the dosage of the pharmaceutical ingredient of the present invention is well known to those skilled in the art. The prophylactic or therapeutic effect of the present invention can be accomplished by appropriately adjusting the amount of the actual drug contained in the final formulation of the compound composition of the present invention to achieve its therapeutically effective amount. The compound of the present invention is administered in a daily dose ranging from 0.001 to 10 mg/kg body weight in 2-4 divided doses. The compounds or compositions of this invention may be administered alone or in combination with other therapeutic or symptomatic agents and adjusted in dosage. Term and abbreviation

AChE 乙酰胆碱酯酶 AChE acetylcholinesterase

ESI-MS 电喷雾质谱 ESI-MS electrospray mass spectrometry

HMBC 异核多键相关（一种测定分子中远程氢碳连接关系的二维核磁共振谱） HMBC heteronuclear multi-bond correlation (a two-dimensional nuclear magnetic resonance spectrum for determining the relationship between long-range hydrogen and carbon in a molecule)

HPLC 高效液相色谱 HPLC high performance liquid chromatography

HRESI-MS 高分辨电喷雾质谱 HRESI-MS high resolution electrospray ionization mass spectrometry

HSQC 异核单量子相关（一种测定分子中氢碳直接相连关系的二维核磁共振谱） HSQC heteronuclear single quantum correlation (a two-dimensional nuclear magnetic resonance spectrum for determining the direct relationship between hydrogen and carbon in a molecule)

IC₅₀ 半数抑制剂量 IC ₅₀ half inhibitor

IR 红外光谱 IR infrared spectroscopy

NMR 核磁共振 NMR nuclear magnetic resonance

ROESY 旋转坐标系欧沃豪斯增益谱（一种测定分子中氢原子空间位置临近关系的二维核磁共振谱） ROESY Rotating Coordinate System, Ovhaus Spectrum (a two-dimensional nuclear magnetic resonance spectrum for determining the spatial relationship of hydrogen atoms in a molecule)

UV 紫外光谱附图说明 UV ultraviolet spectrum DRAWINGS

图 1 伸筋草的提取分离及伸筋草碱 A-C (1-3)的制备流程图图 2伸筋草碱 A-C (1-3)的 X-ray单晶衍射结果（分子椭球图）具体实施方式据文献报道，石松科植物主要含有石松生物碱、萜类、有机酸、黄酮和蒽醌等成分，而伸筋草作为一种常见的石松科植物，其生物碱类成分由于含量较低而没有进行深入彻底的研究。本发明人通过加大药材用量，针对该药材提取物的生物碱部位，通过正、反相色谱等分离手段得到具有乙酰胆碱酯酶抑制活性的三个活性化合物，并经多种波谱数据及 X-ray单晶衍射确证其化学结构为前人未报道过的具有新颖骨架的三个石松生物碱类化合物。下面的实施例及药物活性实验用来进一步说明本发明，但这并不意味着对本发明的任何限制。实施例 1伸筋草碱 A-C (1-3)的分离及结构表征 Fig.1 Extraction and separation of glutinous grass and preparation of acetaminophen AC (1-3)Fig.2 X-ray single crystal diffraction results of acetaminophen AC (1-3) (molecular ellipsoid) According to the literature, the plant of the genus Pinus sylvestris mainly contains components such as alkaloids, terpenoids, organic acids, flavonoids and strontium, and the grasses of the genus Struts are a common plant of the genus Pinus sylvestris. There is no thorough and thorough research. The inventors obtained three active compounds having acetylcholinesterase inhibitory activity by means of separation of positive and reversed phase chromatography by augmenting the amount of the medicinal material, and using various spectral data and X- Ray single crystal diffraction confirmed the chemical structure of three stone pine alkaloids with novel skeletons not previously reported. The following examples and pharmaceutically active experiments are intended to further illustrate the invention, but are not intended to limit the invention in any way. Example 1 Separation and Structural Characterization of Strenichoprine A-C (1-3)

伸筋草全草约 100Kg，切成小段，用 95%EtOH (乙醇， 1200L) 回流提取 3次，第一次 3小时，后两次每次 2小时。提取液减压浓缩得到浸膏 18Kg (；记作 A),将浸膏溶于 3%酒石酸水溶液（pH=2， 150L), 滤去不溶物 (记作 E， 5Kg)后再用乙酸乙酯 (3次，每次 150L)萃取。萃取后的酸水用饱和碳酸氢钠溶液 (18L)调 pH至 10，氯仿萃取至氯仿层无生物碱反应为止（4 次，每次 150L) , 氯仿层浓缩得总生物碱 135g (记作 B )。总生物碱经碱性硅胶柱（200-300目， pH 8-9) 色谱，用石油醚：丙酮 =20: 1， 10:1， 5:1， 1:1， 0:1，丙酮：甲醇 =20:1， 5: 1， 0: 1洗脱，每个梯度洗脱 18000ml, 每 1000ml接收 1个流份，各流份减压回收溶剂后经 TLC (石油醚：丙酮：二乙胺 =1:1:0.1 ) 检识，根据主斑点予以合并，得到 B1~B9九个组分。组分 B3(8.7g)经硅胶柱Stretch the whole grass about 100Kg, cut into small pieces, and extract it with 3 times with 95% EtOH (ethanol, 1200L), the first 3 hours, the last 2 times each time 2 hours. The extract was concentrated under reduced pressure to obtain 18 kg of an extract (referred to as A), and the extract was dissolved in a 3% aqueous solution of tartaric acid (pH = 2, 150 L), and the insoluble matter (denoted as E, 5 Kg) was filtered off and then ethyl acetate was used. (3 times, 150L each time) extraction. The extracted acid water was adjusted to pH 10 with saturated sodium bicarbonate solution (18 L), extracted with chloroform until the chloroform layer was reacted without alkaloids (4 times, 150 L each time), and the chloroform layer was concentrated to give a total alkaloid 135 g (recorded as B). ). The total alkaloid is chromatographed on an alkaline silica gel column (200-300 mesh, pH 8-9). With petroleum ether: acetone = 20: 1, 10:1, 5:1, 1:1, 0:1, acetone: methanol = 20:1, 5: 1, 0: 1 elution, 18000ml per gradient elution 1 stream is received every 1000ml, and the solvent is recovered under reduced pressure, and then detected by TLC (petroleum ether: acetone: diethylamine = 1:1:0.1), and combined according to the main spots to obtain nine B1~B9 Component. Component B3 (8.7g) via silica gel column

(200-300目， pH 6-7)色谱，采用石油醚：乙酸乙酯：二乙胺 =10:1:0.1， 8: 1:0.1， 6:1 :0.1， 4:1:0.1， 2:1:0.1， 1:1:0.1 梯度洗脱，每个梯度洗脱 1500ml, 每 100ml接收 1个流份，各流份回收溶剂后经 TLC (石油醚：乙酸乙酯：二乙胺 =1 :1:0.1 )检识合并得到 Β3-1~Β3-9九个组分， B3-3组分 (2.65g)再经凝胶 Sephadex LH-20 (二氯甲垸: 甲醇 =1:1 ) 纯化及制备型 HPLC (Shimadzu LC-6AD型制备液相仪， YMC-pack ODS-A色谱柱（250x20mm， 5μηι) , 洗脱条件：乙腈 /醋酸铵缓冲液(200-300 mesh, pH 6-7) chromatography using petroleum ether: ethyl acetate: diethylamine = 10:1:0.1, 8: 1:0.1, 6:1:0.1, 4:1:0.1, 2 :1:0.1, 1:1:0.1 Gradient elution, 1500ml per gradient elution, 1 fraction per 100ml, solvent recovery after TLC (petroleum ether: ethyl acetate: diethylamine=1) :1:0.1) The combination of Β3-1~Β3-9 was obtained by the combination, and the B3-3 component (2.65g) was passed through the gel Sephadex LH-20 (dichloromethane: methanol = 1:1) Purification and preparative HPLC (Shimadzu LC-6AD preparative liquid chromatography, YMC-pack ODS-A column (250x20mm, 5μηι), elution conditions: acetonitrile/ammonium acetate buffer

(0.04mol/L醋酸铵 +0.1%醋酸） =15/85， 5ml/min, t_R=27min)分离得到化合物 3 (33mg)；组分 B7 (34.0g)经硅胶柱 (200-300目， pH 6-7) 色谱，采用石油醚：乙酸乙酯：二乙胺 =8:1:0.1， 5: 1:0.1， 3:1:0.1， 1 :1:0.1， 1:2:0.2梯度洗脱，每个梯度洗脱 6000ml，每 500ml接收 1个流份，各流份回收溶剂后经 TLC (石油醚：乙酸乙酯：二乙胺 =1:2:0.1 ) 检识合并得到 B7-1~B7-13十三个组分， B7-9 (2.10g) 组分再经硅胶柱(0.04mol / L ammonium acetate + 0.1% acetic acid) = 15/85, 5ml / min, t R = 27min) isolated compound 3 (33mg); component B7 (34.0g) by silica gel column (200-300 mesh, pH 6-7) Chromatography using petroleum ether: ethyl acetate: diethylamine = 8:1:0.1, 5: 1:0.1, 3:1:0.1, 1 :1:0.1, 1:2:0.2 gradient wash Take off, elute 6000ml per gradient, receive 1 fraction per 500ml, recover the solvent in each fraction, and check and combine with TLC (petroleum ether: ethyl acetate: diethylamine = 1:2:0.1) to obtain B7-1. ~B7-13 thirteen components, B7-9 (2.10g) components and then passed through the silica gel column

(200-300目， pH 6-7)色谱，采用二氯甲垸：甲醇 =60: 1， 40:1， 20:1， 10: 1， 5:1梯度洗脱，经 TLC (二氯甲垸：甲醇 =10:1 ) 检识合并得到 B7-9a~B7-9g七个组分， B7-9c (0.21g)由制备型 TLC(二氯甲垸：甲醇 =20: 1)得到化合物 2 (30mg)_; B7-10 ( 1.34g)组分经硅胶柱 (200-300 目， pH 6-7) 色谱，采用二氯甲垸：甲醇 =1:0， 40:1， 30:1， 20:1 梯度洗脱，经 TLC (二氯甲垸：甲醇 =10:1)检识合并得到 B7-10a~B7-10e 五个组分， B7-10a组分放置过程中有针状结晶析出，用丙酮洗涤得白色结晶即化合物 1 (40mg)。提取分离的流程图如附图 1所示。 (200-300 mesh, pH 6-7) chromatography using dichloromethane: methanol = 60: 1, 40:1, 20:1, 10: 1, 5:1 gradient elution, TLC (dichloroform)垸: Methanol = 10:1) Detected and combined to obtain seven components of B7-9a~B7-9g, B7-9c (0.21g) was obtained from preparative TLC (dichloromethane: methanol = 20: 1) to obtain compound 2 (30mg) _; B7-10 ( 1.34g) fraction was chromatographed on silica gel column (200-300 mesh, pH 6-7) using dichloromethane: methanol = 1:0, 40:1, 30:1, 20 :1 ladder Degree elution, TLC (methylene chloride: methanol = 10:1) detected and combined to obtain five components B7-10a~B7-10e, B7-10a components during the process of needle crystal precipitation, with acetone The white crystal was washed as Compound 1 (40 mg). The flow chart of the extraction separation is shown in Fig. 1.

伸筋草碱 A-C 1-3)的结构式及鉴定方法 Structural formula and identification method of glutinine A-C 1-3)

伸筋草碱 A-C (1-3)的波谱数据和理化性质

Spectral data and physicochemical properties of erectine AC (1-3)

1. 伸筋草碱 A (1) 1. Stretching grass A (1)

无色透明结晶， mp. 223-225 °C; [a]² _D°: +130.6 （c 0.05 MeOH); UV (MeOH): λ _max=203, 300 nm; IR: v_max=3371, 2967, 2928, 1736, 1697, 1488 1454 1415 1378, 1257, 1138, 910, 856 612, 587, 544cm—¹; (+) ESI-MS: m/z =292.1 [M+H]+; HR-ESI-MS: m/z =292.1545 [M+H]⁺ (计算为 C₁₆H₂₁N0₄); 1HNMR和 ¹³CNMR 数据见表 1 Colorless, transparent crystals, mp. 223-225 °C; [A] ² _D °: +130.6 (c 0.05 MeOH); UV (MeOH): λ _max = 203, 300 nm; IR: v _max = 3371, 2967, 2928, 1736, 1697, 1488 1454 1415 1378, 1257, 1138, 910, 856 612, 587, 544cm— ¹ ; (+) ESI-MS: m/z =292.1 [M+H]+; HR-ESI-MS : m/z =292.1545 [M+H] ⁺ (calculated as C ₁₆ H ₂₁ N0 ₄ ); 1HNMR and ¹³ CNMR data are shown in Table 1.

2. 伸筋草碱 B (2) 2. Stretching grass B (2)

无色透明结晶， mp. 175-177°C; [a]² _D°: -17.4 （c 0.05 MeOH); UV (MeOH): λ =202 259, 300 nm; IR: v =3460 3319, 2921, 2850, 1700, 1646, 1465, 1416, 1279, 1095 1080, 848 787, 581 547cm"¹; (+) ESI-MS: m/z =294.2 [M+H]+; HR-ESI-MS: m/z =294.1703 [M+H]⁺ (计算为 C₁₆H₂₃N0₄); 1HNMR和 ¹³CNMR 数据见表 1 Colorless transparent crystals, mp. 175-177°C; [ _D ] ² _D °: -17.4 (c 0.05 MeOH); UV (MeOH): λ = 202 259, 300 nm; IR: v = 3460 3319, 2921, 2850, 1700, 1646, 1465, 1416, 1279, 1095 1080, 848 787, 581 547cm"¹; (+) ESI-MS: m/z =294.2 [M+H]+; HR-ESI-MS: m/ z = 294.1703 [M+H] ⁺ (calculated as C ₁₆ H ₂₃ N0 ₄ ); 1H NMR and ¹³ C NMR data are shown in Table 1.

3. 伸筋草碱 C (3) 3. Stretching grass C (3)

无色透明结晶， mp. 147-149°C; [a]² _D°: -139.6° (c 0.05 CHC1₃); UV (CHC1₃): 入 _max=241nm: IR: v_max=2981 2956, 2938, 2773, 1726, 1657, 1442, 1384, 1262, 1203, 1159, 1121 , 1069, 885cm—¹; (+) ESI-MS: m/z =274.2 [M+H]+; HR-ESI-MS : m/z =274.1809 [M+H]⁺ (计算为 C₁₇H₂₃N0₂ ) ; 1H NMR禾卩 ¹³C NMR数据见表 1。表 1. 化合物 1-3的 ^ NMR ( 500MHz ) 和 ¹³C NMR (125MHz)数据 (t5 in ppm, J in Hz )Colorless transparent crystal, mp. 147-149 ° C; [a] ² _D °: -139.6 ° (c 0.05 CHC1 ₃ ); UV (CHC1 ₃ ): into _max = 241 nm: IR: v _max = 2981 2956, 2938 , 2773, 1726, 1657, 1442, 1384, 1262, 1203, 1159, 1121, 1069, 885cm— ¹ ; (+) ESI-MS: m/z =274.2 [M+H]+; HR-ESI-MS: m/z =274.1809 [M+H] ⁺ (calculated as C ₁₇ H ₂₃ N0 ₂ ) ; 1H NMR and ¹³ C NMR data are shown in Table 1. Table 1. NMR (500MHz) and ¹³ C NMR (125MHz) data for compound 1-3 (t5 in ppm, J in Hz)

NO. l^a 2^a 3^b NO. l ^a 2 ^a 3 ^b

¾ ¾ Sc ¾ 3⁄4 3⁄4 Sc 3⁄4

la 2.98 (lH, ddd, 12.0, 12.0, 5.0) 48.7 2.98 (1H, m) 48.4 2.16 (1H, m) 52.6 lb 3.06 (1H, ddd, 12.0, 8.0, 4.5) 2.98 (1H, m) 2.74 (1H, m) La 2.98 (lH, ddd, 12.0, 12.0, 5.0) 48.7 2.98 (1H, m) 48.4 2.16 (1H, m) 52.6 lb 3.06 (1H, ddd, 12.0, 8.0, 4.5) 2.98 (1H, m) 2.74 (1H , m)

2a 1.78 (1H, m) 30.9 2.09 (1H, m) 31.8 1.48 (1H, m) 20.9 2a 1.78 (1H, m) 30.9 2.09 (1H, m) 31.8 1.48 (1H, m) 20.9

2b 2.00 (1H, m) 2.13 (1H, m) 1.62 (1H, m) 2b 2.00 (1H, m) 2.13 (1H, m) 1.62 (1H, m)

3 5.59 (lH, d, 6.0) 87.9 5.95 (lH, d, 5.0) 85.4 1.62 (2H, m) 25.9 3 5.59 (lH, d, 6.0) 87.9 5.95 (lH, d, 5.0) 85.4 1.62 (2H, m) 25.9

4 75.0 71.8 63.14 75.0 71.8 63.1

5 215.9 4.38 (1H, brs (o)) 80.9 219.85 215.9 4.38 (1H, brs (o)) 80.9 219.8

6a 2.39 (1H, m) 43.6 4.38 (1H, brs (o)) 71.3 2.24 (1H, m) 43.86a 2.39 (1H, m) 43.6 4.38 (1H, brs (o)) 71.3 2.24 (1H, m) 43.8

6b 3.47 (lH, dd, 18.0, 14.5) 2.66 (1H, m) 6b 3.47 (lH, dd, 18.0, 14.5) 2.66 (1H, m)

7 2.55 (1H, br ddd, 14.5, 6.5, 6.5) 41.3 2.43 (1H, m) 52.8 2.68 (1H, m) 39.5 7 2.55 (1H, br ddd, 14.5, 6.5, 6.5) 41.3 2.43 (1H, m) 52.8 2.68 (1H, m) 39.5

8a 2.02 (1H, m) 34.7 1.58 (1H, ddd, 13.0, 13.0, 13.0) 33.7 2.00 (lH, dd, 19.0, 8.0) 34.98a 2.02 (1H, m) 34.7 1.58 (1H, ddd, 13.0, 13.0, 13.0) 33.7 2.00 (lH, dd, 19.0, 8.0) 34.9

8b 2.14 (1H, dd, 15.0, 6.5) 1.95 (1H, m) 2.43 (lH, dd, 19.0, 6.0)8b 2.14 (1H, dd, 15.0, 6.5) 1.95 (1H, m) 2.43 (lH, dd, 19.0, 6.0)

9 4.10 (lH, d, 8.5) 78.9 3.72 (lH, d, 8.0) 77.7 2.61 (lH, m (o)) 71.69 4.10 (lH, d, 8.5) 78.9 3.72 (lH, d, 8.0) 77.7 2.61 (lH, m (o)) 71.6

10a 1.82 (1H, m) 24.2 1.67 (1H, dd, 14.5, 6.0) 23.5 1.58 (1H, m) 27.710a 1.82 (1H, m) 24.2 1.67 (1H, dd, 14.5, 6.0) 23.5 1.58 (1H, m) 27.7

10b 2.10 (1H, m) 1.85 (1H, m) 2.16 (1H, m) 10b 2.10 (1H, m) 1.85 (1H, m) 2.16 (1H, m)

1 1a 2.03 (1H, m) 34.0 1.95 (1H, m) 39.7 1.56 (1H, m) 34.2 l ib 2.39 (1H, m) 1.95 (1H, m) 2.93 (1H, m) 1 1a 2.03 (1H, m) 34.0 1.95 (1H, m) 39.7 1.56 (1H, m) 34.2 l ib 2.39 (1H, m) 1.95 (1H, m) 2.93 (1H, m)

12 66.6 63.7 60.0 12 66.6 63.7 60.0

13 210.5 212.0 199.513 210.5 212.0 199.5

14a 2.61 (1H, dd, 1 1.5, 2.0) 53.7 1.95 (1H, m) 46.9 6.04 (1H, s) 127.814a 2.61 (1H, dd, 1 1.5, 2.0) 53.7 1.95 (1H, m) 46.9 6.04 (1H, s) 127.8

14b 2.90 (1H, d, 1 1.5) 2.74 (1H, ddd, 16.5, 9.0, 1.5) 14b 2.90 (1H, d, 1 1.5) 2.74 (1H, ddd, 16.5, 9.0, 1.5)

15 77.5 2.04 (1H, m) 29.0 157.6 15 77.5 2.04 (1H, m) 29.0 157.6

16 1.44 (3H, s) 32.2 0.86 (3H, d, 6.5) 23.3 1.92 (3H, s) 24.016 1.44 (3H, s) 32.2 0.86 (3H, d, 6.5) 23.3 1.92 (3H, s) 24.0

5-OH 6.53 (1H, brs) 5-OH 6.53 (1H, brs)

6-OH 5.59 (lH, d, 6.5) 6-OH 5.59 (lH, d, 6.5)

iV-CH₃ 2.27 (3H, s) 43.9 a氘代吡啶 b氘代氯仿伸筋草碱 A-C (1-3)的 X-ray单晶衍射结果（分子椭球图）见附图 2。药理实验 iV-CH ₃ 2.27 (3H, s) 43.9 a deuterated pyridine b deuterated chloroform erectin AC (1-3) X-ray single crystal diffraction results (molecular ellipsoid) See Figure 2. Pharmacological experiment

试验例 1伸筋草碱 A-C (1-3)的 AChE抑制活性测定 Test Example 1 Determination of AChE Inhibitory Activity of Strenicin A-C (1-3)

实验原理： Experimental principle:

乙酰胆碱酯酶（acetylcholinesterase, AChE ) 水解乙酰胆碱生成胆碱及乙酸，胆碱可以与巯基显色剂反应生成 TNB (；对称三硝基苯， Sym-Trinitrobenzene) 黄色化合物，根据颜色深浅进行比色定量，水解产物胆碱的数量可以反映胆碱酯酶的活力。实验材料及所需仪器： Acetylcholinesterase (AChE) hydrolyzes acetylcholine to produce choline and acetic acid. Choline can react with sulfhydryl chromogenic reagent to form TNB (symmetric trinitrobenzene, Sym-Trinitrobenzene) yellow compound, which is colorimetrically quantified according to color depth. The amount of hydrolysate choline can reflect the activity of cholinesterase. Experimental materials and instruments required:

酶标仪，水浴箱，微量移液器，乙酰胆碱酯酶测定试剂盒（南京建成生物工程研究所），试剂组成包括：试剂一： l mol/L标准应用液；试剂二：底物；试剂三：显色剂储备液；试剂四：抑制剂；试剂五：透明剂；试剂六：稳定剂；试剂七：生理盐水。 Microplate reader, water bath, micropipette, acetylcholinesterase assay kit (Nanjing Institute of Bioengineering), reagent composition includes: reagent one: l mol / L standard application solution; reagent two: substrate; test Agent three: developer solution; reagent four: inhibitor; reagent five: transparent agent; reagent six: stabilizer; reagent seven: normal saline.

操作步骤： Steps:

混匀，放置 15分钟， 412nm下酶标仪测定吸光度。抑制率 =[1- (测定管 OD值-对照管 OD值 M标准管 OD值-空白管 OD值)] X 100% 选用石杉碱甲为阳性对照，其浓度为其 IC_5Q浓度 8.2x lO—⁸mol/L，在化合物与标准应用液的混合液中化合物的终浓度分别为 10—⁵， 10"⁶, 10—⁷ mol/L。

Mix and place for 15 minutes, and measure the absorbance at 412 nm. Inhibition rate = [1 - (measurement tube OD value - control tube OD value M standard tube OD value - blank tube OD value)] X 100% Select Huperzine A as a positive control, its concentration is its IC _5Q concentration 8.2x lO - ⁸ mol / L, the final concentration of the compound in a mixture of the compound in the standard solution were applied ^{^{10- 5, 10 "6, 10-}} 7 mol / L.

测定结果: 从上述药理活性研究结果来看，伸筋草 A-C(l-3) 均具有显著的乙酰胆碱酯酶抑制活性，其活性与阳性对照药石杉碱甲（HupA) 相当或更强，是很有价值的新颖结构化合物。序号抑制率 (%) 石杉碱甲 10- ⁸ 50.98 ±12.3As a result of the above pharmacological activity studies, AC(l-3) has significant acetylcholinesterase inhibitory activity, and its activity is equivalent to or stronger than the positive control substance Huperlin A (HupA). A valuable novel structural compound. No. inhibition rate (%) huperzine 10- ⁸ 50.98 ± 12.3

1 10-⁵ 66.19±5.51 10- ⁵ 66.19±5.5

10-⁷ 59.4±5.810- ⁷ 59.4±5.8

2 10-⁵ 60.79 ±6.52 10- ⁵ 60.79 ±6.5

10-⁷ 55.35±7.510- ⁷ 55.35±7.5

3 10-⁵ 60.59±3.7

3 10- ⁵ 60.59±3.7

Claims

1. 如下式 1、式 2和式 3所示的伸筋草碱 A-C及其药效学上可接受的盐 1. Stretch oxalate A-C and its pharmacologically acceptable salt as shown in the following formula 1, formula 2 and formula 3

2. 一种药物组合物，其特征在于，含有有效剂量的如式 1-3所示的伸筋草碱 A-C 中至少一种或其药效学上可接受的盐，以及药效学上可以接受的载体。 A pharmaceutical composition comprising an effective amount of at least one of the stalk base AC as shown in Formula 1-3 or a pharmaceutically acceptable salt thereof, and pharmacologically acceptable Accepted carrier.

3. 根据权利要求 2 的药物组合物，其特征在于，所述的药物组合物选自片剂、胶囊、丸剂、注射剂。 The pharmaceutical composition according to claim 2, wherein the pharmaceutical composition is selected from the group consisting of a tablet, a capsule, a pill, and an injection.

4. 根据权利要求 2 的药物组合物，其特征在于，所述的药物组合物选自微粒给药***。 4. Pharmaceutical composition according to claim 2, characterized in that the pharmaceutical composition is selected from a microparticle delivery system.

5. 根据权利要求 2 的药物组合物，其特征在于，所述的药物组合物选自缓释制剂、控释制剂。 The pharmaceutical composition according to claim 2, wherein the pharmaceutical composition is selected from the group consisting of sustained release preparations and controlled release preparations.

6. 权利要求 1化合物伸筋草碱 A-C的制备方法，其主要来源于蕨类植物伸筋草 ( Lycopodium japonicum Thunb. ) 的全草。 6. The method for preparing a compound of the glutinosa A-C according to claim 1, which is mainly derived from the whole grass of the genus Lycopodium japonicum Thunb.

7. 权利要求 1化合物伸筋草碱 A-C及其药效学上可接受的盐在制备乙酰胆碱酯酶抑制剂药物中的应用。 7. The use of the compound of claim 1 for the treatment of an acetylcholinesterase inhibitor drug, A-C, and a pharmacologically acceptable salt thereof.

8. 权利要求 1化合物伸筋草碱 A-C及其药效学上可接受的盐在制备治疗早老性痴呆症药物中的应用。权利要求 1化合物伸筋草碱 A-C及其药效学上可接受的盐在制备抗中老年记忆和认知能力减退疾病药物中的应用 8. The use of the compound of claim 1 and the pharmacologically acceptable salt thereof for the preparation of a medicament for the treatment of Alzheimer's disease. The use of the compound of claim 1 and the pharmacologically acceptable salt thereof for the preparation of a medicament for preventing memory and cognitive decline in middle-aged and elderly people