CN102516351A - Ursolic acid derivative with anti-cancer activity and preparation method thereof - Google Patents

Abstract

Translated fromChinese

本发明提供了一种熊果酸衍生物，所述熊果酸衍生物为N-[3β-乙酰氧基-熊果烷-12-烯-28-酰]-氨基乙二胺-叶酸，本发明对天然产物熊果酸进行结构改造，得N-[3β-乙酰氧基-熊果烷-12-烯-28-酰]-氨基乙二胺-叶酸（Ⅰ）、N-[3β-乙酰氧基-熊果烷-12-烯-28-酰]-氨基乙二胺（Ⅲ）和N-[3β-羟基-熊果烷-12-烯-28-酰]-氨基乙二胺（Ⅳ），体外细胞增殖抑制实验表明，化合物Ⅰ、Ⅲ和Ⅳ对所测肿瘤细胞株HepG2、BGC823、A-375、Hela等的增殖抑制作用优于熊果酸，而对人胚肺成纤维细胞HELF的毒性作用则低于熊果酸。其中化合物Ⅲ的体外抗肿瘤效果最好，化合物Ⅰ具有一定的靶向性。The invention provides a derivative of ursolic acid, the derivative of ursolic acid is N-[3β-acetoxy-arbutane-12-ene-28-yl]-aminoethylenediamine-folic acid. Invented to modify the structure of the natural product ursolic acid to obtain N-[3β-acetoxy-arbutane-12-en-28-yl]-aminoethylenediamine-folic acid (Ⅰ), N-[3β-acetyl Oxy-arbutane-12-ene-28-yl]-aminoethylenediamine (Ⅲ) and N-[3β-hydroxy-arbutane-12-ene-28-yl]-aminoethylenediamine (Ⅳ ), the in vitro cell proliferation inhibition experiments showed that compounds Ⅰ, Ⅲ, and Ⅳ had better inhibitory effects on the proliferation of tested tumor cell lines HepG2, BGC823, A-375, Hela, etc. than ursolic acid, while on human embryonic lung fibroblast HELF The toxic effect is lower than ursolic acid. Among them, compound Ⅲ has the best antitumor effect in vitro, and compound Ⅰ has certain targeting properties.

Description

Translated fromChinese

一种具有抗癌活性的熊果酸衍生物及其制备方法A kind of ursolic acid derivative with anticancer activity and preparation method thereof

技术领域technical field

本发明涉及一种熊果酸衍生物及其制备方法，具体说是一种具有抗肿瘤活性的熊果酸衍生物及其制备方法。The invention relates to an ursolic acid derivative and a preparation method thereof, in particular to an ursolic acid derivative with antitumor activity and a preparation method thereof.

背景技术Background technique

熊果酸(Ursolic acid，UA)，又名乌索酸、乌苏酸，属五环三萜类化合物，化学名(3β)-3-Hydroxyurs-12-en-28-oic acid，其相对分子量为456.68，分子式为C₃₀H₄₈O₃，它以游离或糖苷的形式，广泛分布于自然界中。药理学研究发现，熊果酸具有广泛的生物学效应，熊果酸具有多种生物学活性，如抗癌、抗HIV、抗肝损伤、抗疟、抗炎抑菌等，其中尤以抗癌活性最为显著，其抗癌谱广，不仅对多种致癌、促癌物有抵抗作用，而且对多种肿瘤细胞体内、外均有抑制作用。其副作用小，毒性低，显示出较大的临床应用潜力。Ursolic acid (Ursolic acid, UA), also known as ursolic acid, ursolic acid, belongs to pentacyclic triterpenoids, chemical name (3β)-3-Hydroxyurs-12-en-28-oic acid, its relative molecular weight It is 456.68, and its molecular formula is C₃₀ H₄₈ O₃ . It is widely distributed in nature in the form of free or glycoside. Pharmacological studies have found that ursolic acid has a wide range of biological effects, and ursolic acid has a variety of biological activities, such as anti-cancer, anti-HIV, anti-liver damage, anti-malarial, anti-inflammatory and antibacterial, etc., especially anti-cancer The activity is the most significant, and its anti-cancer spectrum is broad. It not only has resistance to various carcinogens and carcinogens, but also has inhibitory effects on various tumor cells in vivo and in vitro. It has few side effects and low toxicity, and shows great clinical application potential.

熊果酸的化学结构式为：The chemical structural formula of ursolic acid is:

Ⅴ。

V.

 the

目前，国内外对熊果酸的研究报到多集中在熊果酸的分离提取工艺和药理学研究方面，而对熊果酸的结构修饰方面研究较少，对于靶向性的研究更是为少。由于熊果酸的显著抗癌作用使得这一方面的研究较为深入。如林凤屏等的“熊果酸衍生物的合成、表征及其对癌细胞的抑制活性”（《应用化学》，2010, 27, 893-898）；Meng Y. Q.等的“The synthesis of ursolic acid derivatives with cytotoxic activity and the investigation of their preliminary mechanism of action”(Bioorg. Med. Chem. Lett. 2009, 17, 848–854)；Oxana B.等的“Betulin and ursolic acid synthetic derivatives as inhibitors of Papilloma virus”(Bioorg. Med. Chem. Lett. 2010, 20, 4088-4090)。多数研究者认为，熊果酸能通过化学预防、抗突变、细胞毒作用、抗肿瘤血管生成及诱导肿瘤细胞分化、凋亡等来抑制肿瘤的发生、入侵与转移。At present, most of the research reports on ursolic acid at home and abroad focus on the separation and extraction process and pharmacological research of ursolic acid, while there are few studies on the structural modification of ursolic acid, and even less research on targeting . Due to the significant anti-cancer effect of ursolic acid, the research in this area is more in-depth. For example, "Synthesis and Characterization of Ursolic Acid Derivatives and Their Inhibitory Activity on Cancer Cells" by Lin Fengping et al. ("Applied Chemistry", 2010, 27, 893-898); "The synthesis of ursolic acid derivatives" by Meng Y. Q. et al. acid derivatives with cytotoxic activity and the investigation of their preliminary mechanism of action” (Bioorg. Med. Chem. Lett. 2009, 17, 848–854); Oxana B. et al. ” (Bioorg. Med. Chem. Lett. 2010, 20, 4088-4090). Most researchers believe that ursolic acid can inhibit tumor occurrence, invasion and metastasis through chemoprevention, anti-mutation, cytotoxicity, anti-tumor angiogenesis and induction of tumor cell differentiation and apoptosis.

对于熊果酸结构修饰的研究主要有两个方向，一个是提高熊果酸的水溶性，另一个是提高熊果酸的抗癌活性。于舟等通过对熊果酸的3 位和28 位进行化学修饰, 制备熊果酸-三乙醇胺衍生物，对熊果酸的水溶有一定得提高（《药物生物技术》，2010, 17(5) : 382-385）；夏燕等提出自行设计的熊果酸A 环开环结构修饰路线成本较低，试剂低毒，为熊果酸结构修饰物的深入研究提供基础（《中草药》，2011，42(1)：34-37）；孟艳秋等以天然产物熊果酸为先导化合物, 经过氧化、酰化、酯化、水解等反应设计合成了16 个熊果酸衍生物，其中化合物7a等具有较好的抗癌活性（《药学学报》，2011, 46 (5): 556-560）。Jing-Wei Shao等对熊果酸3 位和28 位进行修饰，制备熊果酸-哌嗪及熊果酸-二乙醇胺衍生物，其抗癌活性得到显著提高（Eur. J. Med. Chem. 2011, 46 (7):2652-61）。尽管上述熊果酸结构修饰物显示了良好的应用前景，但是也存在靶向性不够明确，即缺乏癌细胞的靶向特异性的局限。因此为减少其对正常组织的毒性和增加对靶器官的亲和性，近几年人们将抗肿瘤药物制成靶向药物。其中以叶酸受体介导的肿瘤靶向药物受到广泛关注, 已成为肿瘤诊断和治疗研究的新靶点，具有广泛的应用前景。目前以熊果酸为先导化合物与叶酸的连接，此类化合物的合成及靶向性研究国内外均未见其报道。There are two main directions for the research on the structure modification of ursolic acid, one is to improve the water solubility of ursolic acid, and the other is to improve the anticancer activity of ursolic acid. Yu Zhou et al prepared ursolic acid-triethanolamine derivatives by chemically modifying the 3-position and 28-position of ursolic acid, which improved the water solubility of ursolic acid ("Pharmaceutical Biotechnology", 2010, 17(5 ) : 382-385); Xia Yan et al. proposed a self-designed ursolic acid A ring-opening structure modification route with low cost and low toxicity of reagents, which provided a basis for in-depth research on ursolic acid structure modifications ("Chinese Herbal Medicine", 2011 , 42(1):34-37); Meng Yanqiu et al. took the natural product ursolic acid as the lead compound, designed and synthesized 16 ursolic acid derivatives through oxidation, acylation, esterification, hydrolysis and other reactions, among which compound 7a etc. It has good anticancer activity ("Acta Pharmaceutica Sinica", 2011, 46 (5): 556-560). Jing-Wei Shao et al. modified the 3-position and 28-position of ursolic acid to prepare ursolic acid-piperazine and ursolic acid-diethanolamine derivatives, and their anticancer activity was significantly improved (Eur. J. Med. Chem. 2011, 46(7):2652-61). Although the above-mentioned ursolic acid structural modifiers have shown good application prospects, there is also the limitation that the targeting is not clear enough, that is, the lack of targeting specificity of cancer cells. Therefore, in order to reduce its toxicity to normal tissues and increase its affinity to target organs, anticancer drugs have been made into targeted drugs in recent years. Among them, the tumor-targeted drugs mediated by folate receptors have attracted extensive attention, and have become new targets for tumor diagnosis and treatment research, with broad application prospects. At present, ursolic acid is used as the lead compound to connect folic acid, and the synthesis and targeting research of such compounds have not been reported at home and abroad.

发明内容Contents of the invention

本发明的目的是以熊果酸为先导化合物，合成一种具有抗肿瘤活性的熊果酸衍生物及其制备方法。The object of the present invention is to take ursolic acid as a lead compound to synthesize a ursolic acid derivative with antitumor activity and a preparation method thereof.

本发明提供了一种熊果酸衍生物，所述熊果酸衍生物为N-[3β-乙酰氧基-熊果烷-12-烯-28-酰]-氨基乙二胺-叶酸，结构式如式I所示：The invention provides a derivative of ursolic acid, the derivative of ursolic acid is N-[3β-acetoxy-arbutane-12-en-28-yl]-aminoethylenediamine-folic acid, structural formula As shown in formula I:

  

   Ⅰ。

I.

其制备方法如下：Its preparation method is as follows:

（1） 熊果酸与醋酸酐反应，生成3-O-乙酰基熊果酸；3-O-乙酰基熊果酸的结构式如式II所示，(1) Ursolic acid reacts with acetic anhydride to generate 3-O-acetyl ursolic acid; the structural formula of 3-O-acetyl ursolic acid is shown in formula II,

      

      

                             Ⅱ；Ⅱ;

（2）3-O-乙酰基熊果酸与草酰氯反应，再与乙二胺反应得N-[3β-乙酰氧基-熊果烷-12-烯-28-酰]-氨基乙二胺，结构式如式 Ⅲ 所示，(2) 3-O-acetyl ursolic acid reacts with oxalyl chloride, and then reacts with ethylenediamine to obtain N-[3β-acetoxy-arbutane-12-ene-28-yl]-aminoethylenediamine , the structural formula is shown in formula Ⅲ,

    Ⅲ；

III;

（3）N-[3β-乙酰氧基-熊果烷-12-烯-28-酰]-氨基乙二胺与叶酸反应，得N-[3β-乙酰氧基-熊果烷-12-烯-28-酰]-氨基乙二胺-叶酸，结构式如式Ⅰ所示。(3) Reaction of N-[3β-acetoxy-arbutane-12-ene-28-yl]-aminoethylenediamine with folic acid to obtain N-[3β-acetoxy-arbutane-12-ene -28-acyl]-aminoethylenediamine-folic acid, the structural formula is shown in formula I.

所述制备方法具体步骤如下：The specific steps of the preparation method are as follows:

（1）1重量份的熊果酸搅拌溶解于40-50重量份的吡啶中，滴加入2-3重量份的醋酸酐，加入0.001-0.02重量份的DMAP，室温下搅拌16-18 h，反应结束后，用2 N HCl调节pH 3-4，蒸除溶剂，抽滤，水洗滤饼至中性，干燥，干燥后的产物干法上样，柱层析纯化，无水乙醇热溶，冷却重结晶，得白色粉末状的3-O-乙酰基熊果酸；(1) Stir and dissolve 1 part by weight of ursolic acid in 40-50 parts by weight of pyridine, dropwise add 2-3 parts by weight of acetic anhydride, add 0.001-0.02 parts by weight of DMAP, and stir at room temperature for 16-18 h, After the reaction, adjust the pH to 3-4 with 2 N HCl, distill off the solvent, filter with suction, wash the filter cake with water until neutral, dry, dry the dried product, purify it by column chromatography, dissolve it in absolute ethanol, Cooling and recrystallization to obtain white powdery 3-O-acetyl ursolic acid;

（2）1重量份3-O-乙酰基熊果酸溶解于30-35重量份CH₂Cl₂中，逐滴加入1-2重量份的草酰氯，室温下搅拌反应12-24h，蒸除溶剂及反应产生的气体，得3-O-乙酰基UA酰氯中间体的粗品，直接进入下一步反应；在上述中间体的粗品中加入20-30重量份的CH₂Cl₂，三乙胺调节溶液pH 8-9，加入1-2重量份的乙二胺，室温下搅拌反应12-24h，反应结束后，反应液中加5-10重量份的水，以2N HCl调节pH3-4，过滤，水洗滤饼至滤液pH为中性，干燥；干燥后的产物干法上样，柱层析纯化，无水乙醇热溶，冷却重结晶，得白色粉末状N-[3β-乙酰氧基-熊果烷-12-烯-28-酰]-氨基乙二胺；(2) Dissolve 1 part by weight of 3-O-acetylursolic acid in 30-35 parts by weight of CH₂ Cl₂ , add 1-2 parts by weight of oxalyl chloride dropwise, stir at room temperature for 12-24 hours, and evaporate solvent and the gas generated by the reaction, the crude product of 3-O-acetyl UA acid chloride intermediate is obtained, and directly enters the next step reaction; 20-30 parts by weight of CH₂ Cl₂ are added to the crude product of the above intermediate, and triethylamine is adjusted The pH of the solution is 8-9, add 1-2 parts by weight of ethylenediamine, stir and react at room temperature for 12-24 hours, after the reaction is completed, add 5-10 parts by weight of water to the reaction solution, adjust the pH to 3-4 with 2N HCl, filter , wash the filter cake with water until the pH of the filtrate is neutral, and dry; the dried product is dry-loaded, purified by column chromatography, dissolved in absolute ethanol, cooled and recrystallized to obtain white powder N-[3β-acetoxy- Ursbutan-12-en-28-yl]-aminoethylenediamine;

（3）1重量份N-[3β-乙酰氧基-熊果烷-12-烯-28-酰]-氨基乙二胺溶解于20-30重量份的无水二甲基亚砜中，加入1-2重量份的叶酸，室温搅拌30-60min，然后加入1-2重量份的1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐、1-2重量份的N-羟基丁二酰亚胺，室温避光反应20-30 h；反应结束后，蒸除溶剂，然后加入5-10重量份的水，以2N HCl调节pH3-4，过滤，水洗滤饼至滤液pH为中性，干燥； 干燥后的产物干法上样，反相柱纯化，无水乙醇热溶，冷却重结晶，得黄色粉末状N-[3β-乙酰氧基-熊果烷-12-烯-28-酰]-氨基乙二胺-叶酸。(3) 1 part by weight of N-[3β-acetoxy-arbutane-12-en-28-yl]-aminoethylenediamine was dissolved in 20-30 parts by weight of anhydrous dimethyl sulfoxide, and added 1-2 parts by weight of folic acid, stirred at room temperature for 30-60min, then added 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 1-2 parts by weight Parts of N-hydroxysuccinimide, react at room temperature for 20-30 h in the dark; after the reaction, evaporate the solvent, then add 5-10 parts by weight of water, adjust the pH to 3-4 with 2N HCl, filter, wash and filter Cake until the pH of the filtrate is neutral, and dry; the dried product is dry-loaded, purified by reverse-phase column, dissolved in absolute ethanol, cooled and recrystallized, and N-[3β-acetoxy-urbutane is obtained in the form of yellow powder -12-en-28-yl]-aminoethylenediamine-folic acid.

上述步骤（1）所述柱层析纯化采用的流动相为石油醚:乙酸乙酯=3:1；步骤（2）所述柱层析纯化采用的流动相为石油醚:乙酸乙酯=1:5；步骤（3）所述反相柱纯化采用的流动相为甲醇：水= 8: 1。The mobile phase used in the column chromatography purification in the above step (1) is petroleum ether: ethyl acetate=3:1; the mobile phase used in the column chromatography purification in step (2) is petroleum ether: ethyl acetate=1 : 5; the mobile phase that the reverse-phase column purification described in step (3) adopts is methyl alcohol: water=8: 1.

本发明还提供了一种熊果酸衍生物，所述熊果酸衍生物为N-[3β-羟基-熊果烷-12-烯-28-酰]-氨基乙二胺，结构式如式Ⅳ所示：The present invention also provides a derivative of ursolic acid, the derivative of ursolic acid is N-[3β-hydroxy-arbutane-12-en-28-yl]-aminoethylenediamine, the structural formula is as follows: Shown:

     Ⅳ。

IV.

 the

其制备方法如下：Its preparation method is as follows:

（1） 熊果酸与醋酸酐反应，生成3-O-乙酰基熊果酸；3-O-乙酰基熊果酸的结构式如式II所示；(1) ursolic acid reacts with acetic anhydride to generate 3-O-acetyl ursolic acid; the structural formula of 3-O-acetyl ursolic acid is shown in formula II;

（2）3-O-乙酰基熊果酸与草酰氯反应，再与乙二胺反应得N-[3β-乙酰氧基-熊果烷-12-烯-28-酰]-氨基乙二胺，结构式如式Ⅲ所示；(2) 3-O-acetyl ursolic acid reacts with oxalyl chloride, and then reacts with ethylenediamine to obtain N-[3β-acetoxy-arbutane-12-ene-28-yl]-aminoethylenediamine , the structural formula is shown in formula III;

（3）N-[3β-乙酰氧基-熊果烷-12-烯-28-酰]-氨基乙二胺与NaOH水解反应，得到N-[3β-羟基-熊果烷-12-烯-28-酰]-氨基乙二胺，结构式如式Ⅳ所示。(3) Hydrolysis of N-[3β-acetoxy-arbutane-12-en-28-yl]-aminoethylenediamine with NaOH to obtain N-[3β-hydroxy-arbutane-12-ene- 28-acyl]-aminoethylenediamine, the structural formula is as shown in formula IV.

所述制备方法的具体步骤如下：The concrete steps of described preparation method are as follows:

（1）1重量份的熊果酸搅拌溶解于40-50重量份的吡啶中，滴加入2-3重量份的醋酸酐，加入少量的DMAP，室温下搅拌16-18 h，反应结束后，用2 N HCl调pH 3-4，蒸除溶剂，抽滤，水洗滤饼至中性，干燥，干燥后的产物干法上样，柱层析纯化，无水乙醇热溶，冷却重结晶，得白色粉末状的3-O-乙酰基熊果酸；(1) Stir and dissolve 1 part by weight of ursolic acid in 40-50 parts by weight of pyridine, dropwise add 2-3 parts by weight of acetic anhydride, add a small amount of DMAP, and stir at room temperature for 16-18 h. After the reaction, Use 2 N HCl to adjust the pH to 3-4, distill off the solvent, filter with suction, wash the filter cake with water until neutral, dry, dry the dried product, apply column chromatography, heat dissolve in absolute ethanol, cool and recrystallize, Obtain white powdery 3-O-acetyl ursolic acid;

（2）1重量份3-O-乙酰基熊果酸溶解于30-35重量份CH₂Cl₂中，分批次、逐滴加入1-2重量份的草酰氯，室温下搅拌反应12-24 h，蒸除溶剂及反应产生的气体，得3-O-乙酰基UA酰氯中间体的粗品，直接进入下一步反应；在上述中间体中加入20-30重量份的CH₂Cl₂，三乙胺调节溶液pH 8-9，加入1-2重量份的乙二胺，室温下搅拌反应12-24 h，反应结束后，反应液中加5-10重量份的水，以2N HCl调pH3-4，过滤，水洗滤饼至滤液pH为中性，干燥，干燥后的产物干法上样，柱层析纯化，无水乙醇热溶，冷却重结晶，得白色粉末状N-[3β-乙酰氧基-熊果烷-12-烯-28-酰]-氨基乙二胺；(2) Dissolve 1 part by weight of 3-O-acetylursolic acid in 30-35 parts by weight of CH₂ Cl₂ , add 1-2 parts by weight of oxalyl chloride dropwise in batches, and stir at room temperature to react 12- After 24 h, the solvent and the gas produced by the reaction were evaporated to obtain the crude product of the 3-O-acetyl UA acid chloride intermediate, which was directly entered into the next reaction; 20-30 parts by weight of CH₂ Cl₂ were added to the above intermediate, Adjust the pH of the solution to 8-9 with ethylamine, add 1-2 parts by weight of ethylenediamine, stir and react at room temperature for 12-24 h, after the reaction, add 5-10 parts by weight of water to the reaction solution, adjust the pH to 3 with 2N HCl -4, filter, wash the filter cake with water until the pH of the filtrate is neutral, dry, apply the dried product by dry method, purify by column chromatography, dissolve in absolute ethanol, cool and recrystallize, and obtain white powder N-[3β- Acetoxy-arbutane-12-en-28-yl]-aminoethylenediamine;

（3）称取1-2重量份的N-[3β-乙酰氧基-熊果烷-12-烯-28-酰]-氨基乙二胺，溶于20-30重量份的CH₃OH-THF中，所述CH₃OH与THF的体积比为1：1.5-2.5；加入4-8重量份的4N NaOH，室温下搅拌反应3-5h，向反应液中加入5-10重量份的水，2 N HCl调节pH 3-4，减压蒸除溶剂，水洗沉淀至中性，干燥；干燥后的产物干法上样，柱层析纯化，无水乙醇热溶，冷却重结晶，得N-[3β-羟基-熊果烷-12-烯-28-酰]-氨基乙二胺。(3) Weigh 1-2 parts by weight of N-[3β-acetoxy-arbutane-12-en-28-yl]-aminoethylenediamine, dissolve in 20-30 parts by weight of CH₃ OH- In THF, the volume ratio of CH₃ OH to THF is 1:1.5-2.5; add 4-8 parts by weight of 4N NaOH, stir and react at room temperature for 3-5 hours, and add 5-10 parts by weight of water to the reaction solution , 2 N HCl to adjust the pH to 3-4, evaporate the solvent under reduced pressure, wash the precipitate with water until neutral, and dry; the dried product is dry-loaded, purified by column chromatography, dissolved in absolute ethanol, cooled and recrystallized to obtain N -[3β-Hydroxy-arbutan-12-en-28-yl]-aminoethylenediamine.

上述步骤（1）所述柱层析纯化采用的流动相为石油醚:乙酸乙酯=3:1，步骤（2）、（3）所述柱层析纯化采用的流动相为石油醚:乙酸乙酯=1:5。The mobile phase used in the column chromatography purification of the above steps (1) is petroleum ether: ethyl acetate=3:1, and the mobile phase used in the column chromatography purification of steps (2) and (3) is petroleum ether: acetic acid Ethyl ester=1:5.

所述乙二胺用丙二胺、丁二胺、戊二胺或己二胺代替，得到一系列对应的熊果酸衍生物。The ethylenediamine is replaced with propylenediamine, butylenediamine, pentamethylenediamine or hexamethylenediamine to obtain a series of corresponding ursolic acid derivatives.

上述制备的熊果酸衍生物具有抗肿瘤活性，用于抗肿瘤药物的制备。The ursolic acid derivatives prepared above have antitumor activity and are used for the preparation of antitumor drugs.

以下为N-[3β-乙酰氧基-熊果烷-12-烯-28-酰]-氨基乙二胺-叶酸（化合物Ⅰ）、N-[3β-乙酰氧基-熊果烷-12-烯-28-酰]-氨基乙二胺（化合物Ⅲ）以及N-[3β-羟基-熊果烷-12-烯-28-酰]-氨基乙二胺（化合物Ⅳ）的合成路线：The following are N-[3β-acetoxy-arbutane-12-en-28-yl]-aminoethylenediamine-folate (compound Ⅰ), N-[3β-acetoxy-arbutane-12- Synthetic routes of en-28-yl]-aminoethylenediamine (compound Ⅲ) and N-[3β-hydroxy-arbutane-12-en-28-yl]-aminoethylenediamine (compound Ⅳ):

本发明的优点在于，本发明对天然产物熊果酸进行结构改造，得N-[3β-乙酰氧基-熊果烷-12-烯-28-酰]-氨基乙二胺-叶酸（Ⅰ）、N-[3β-乙酰氧基-熊果烷-12-烯-28-酰]-氨基乙二胺（Ⅲ）和N-[3β-羟基-熊果烷-12-烯-28-酰]-氨基乙二胺（Ⅳ），体外细胞增殖抑制实验表明，化合物Ⅰ、Ⅲ和Ⅳ对所测肿瘤细胞株HepG2、BGC823、A-375、Hela等的增殖抑制作用优于熊果酸，而对人胚肺成纤维细胞HELF的毒性作用则低于熊果酸。其中化合物Ⅲ的体外抗肿瘤效果最好，化合物Ⅰ具有一定的靶向性。The advantage of the present invention is that the structure of the natural product ursolic acid is modified to obtain N-[3β-acetoxy-arbutane-12-en-28-yl]-aminoethylenediamine-folic acid (I) , N-[3β-acetoxy-arbutane-12-en-28-yl]-aminoethylenediamine (Ⅲ) and N-[3β-hydroxy-arbutane-12-en-28-yl] -Aminoethylenediamine (Ⅳ), the in vitro cell proliferation inhibition experiments showed that compounds Ⅰ, Ⅲ, and Ⅳ had better inhibitory effects on the proliferation of tested tumor cell lines HepG2, BGC823, A-375, Hela, etc. than ursolic acid, while on The toxicity of HELF in human embryonic lung fibroblasts was lower than that of ursolic acid. Among them, compound Ⅲ has the best antitumor effect in vitro, and compound Ⅰ has certain targeting properties.

具体实施方式Detailed ways

以下为本发明的具体实施例子以及对比实施例可以进一步清楚地了解本发明，但是它们不是对本发明的限定：The following are specific implementation examples and comparative examples of the present invention and can further clearly understand the present invention, but they are not limitation of the present invention:

实施例1Example 1

3-O乙酰基熊果酸（化合物Ⅱ）的制备：Preparation of 3-O acetyl ursolic acid (compound Ⅱ):

称取0.7062 g 熊果酸，加入30 mL吡啶，搅拌溶解后滴加入1.8 mL醋酸酐，加入少量的DMAP，室温下搅拌16 h，反应结束后，用2 N HCl调pH 3-4，蒸除溶剂，抽滤，水洗滤饼至中性，常压100℃干燥，干燥后的产物干法上样，柱层析纯化（石油醚（60-90℃）：乙酸乙酯=3:1），无水乙醇热溶，冷却重结晶，得3-O乙酰基熊果酸。Weigh 0.7062 g of ursolic acid, add 30 mL of pyridine, stir to dissolve, add 1.8 mL of acetic anhydride dropwise, add a small amount of DMAP, stir at room temperature for 16 h, after the reaction, adjust the pH to 3-4 with 2 N HCl, evaporate Solvent, suction filtration, washing filter cake with water until neutral, drying at normal pressure 100°C, dry loading of the dried product, column chromatography purification (petroleum ether (60-90°C): ethyl acetate = 3:1), Dissolve in absolute ethanol, cool and recrystallize to obtain 3-Oacetyl ursolic acid.

性状：白色粉末；产率：86.17%。Properties: white powder; yield: 86.17%.

 IR数据（KBr，cm^-1）及其归属：υ：3293（O-H伸缩振动），2927(C-H伸缩振动)，1736（s，C=O伸缩振动），1370（UA A区特征吸收），1245 (s，C-O-C不对称伸缩振动) cm^-1。IR data (KBr, cm^-1 ) and their assignments: υ: 3293 (OH stretching vibration), 2927 (CH stretching vibration), 1736 (s, C=O stretching vibration), 1370 (UA A region characteristic absorption), 1245 (s, COC asymmetric stretching vibration) cm^-1 .

 ESI-MS：m/z 497.5 [M-H]⁺，521.5[M+Na] ⁺。ESI-MS : m/z 497.5 [MH]⁺ , 521.5 [M+Na]⁺ .

1H NMR（400MHz，CDCl₃）δ：5.23（t，J=2.8Hz，1H，H-12），4.49（t，J=7.2Hz, 1H，H-3），2.17（d，J=10.8Hz, 1H，H-18），2.04（s，3 H，CH₃COO），1.06（s，3 H，CH₃），0.97（s，3H, CH₃），0.94（s，3 H，CH₃），0.92（s，3H，CH₃），0.82（d, J=6.4Hz, 3 H，CH₃），0.78（s，3 H，CH₃），0.76（s，3 H，CH₃）。1H NMR (400MHz, CDCl₃ ) δ: 5.23 (t,J =2.8Hz, 1H, H-12), 4.49 (t,J =7.2Hz, 1H, H-3), 2.17 (d,J =10.8Hz , 1H, H-18), 2.04 (s, 3 H, CH₃ COO), 1.06 (s, 3 H, CH₃ ), 0.97 (s, 3H, CH₃ ), 0.94 (s, 3 H, CH₃ ), 0.92 (s, 3H, CH₃ ), 0.82 (d,J =6.4Hz, 3 H, CH₃ ), 0.78 (s, 3 H, CH₃ ), 0.76 (s, 3 H, CH₃ ).

实施例2Example 2

N-[3β-乙酰氧基-熊果烷-12-烯-28-酰]-氨基乙二胺（化合物Ⅲ）的制备：Preparation of N-[3β-acetoxy-arbutane-12-en-28-yl]-aminoethylenediamine (compound Ⅲ):

称取0.1954 g 3-O乙酰基熊果酸溶解于20 mL CH₂Cl₂中，分批次、逐滴加入草酰氯0.19 mL，室温下搅拌反应24 h，蒸除溶剂及反应产生的气体，得3-O-乙酰基UA酰氯中间体的粗品，直接进入下一步反应。在上述中间体中加入20 mL CH₂Cl₂，三乙胺调节溶液pH 8-9，加入乙二胺0.19 mL，室温下搅拌反应3 h，反应结束后，反应液中加入10 mL蒸馏水，以2N HCl调pH3-4，过滤，水洗滤饼至滤液pH为中性，干燥。柱层析纯化（氯仿: 丙酮(v: v)=1:5），无水乙醇热溶，冷却重结晶，得N-[3β-乙酰氧基-熊果烷-12-烯-28-酰]-氨基乙二胺。Weigh 0.1954 g of 3-O-acetyl ursolic acid and dissolve it in 20 mL of CH₂ Cl₂ , add 0.19 mL of oxalyl chloride dropwise in batches, stir and react at room temperature for 24 h, evaporate the solvent and the gas generated by the reaction, The crude product of 3-O-acetyl UA acid chloride intermediate was obtained, which was directly used for the next reaction. Add 20 mL of CH₂ Cl₂ to the above intermediate, adjust the pH of the solution to 8-9 with triethylamine, add 0.19 mL of ethylenediamine, and stir for 3 h at room temperature. After the reaction, add 10 mL of distilled water to the reaction solution to Adjust the pH to 3-4 with 2N HCl, filter, wash the filter cake with water until the pH of the filtrate is neutral, and dry. Purified by column chromatography (chloroform: acetone (v: v)=1:5), dissolved in absolute ethanol, cooled and recrystallized to obtain N-[3β-acetoxy-arbutane-12-en-28-yl ]-Aminoethylenediamine.

性状：白色粉末；产率：78.2%。Properties: white powder; yield: 78.2%.

 IR数据(KBr，cm^-1)及其归属：υ：3395（NH₂伸缩振动），2927（C-H伸缩振动），1735（s，C=O伸缩振动），1640（ s，C=O伸缩振动，酰胺Ⅰ峰）,1526（N-H弯曲振动，酰胺Ⅱ峰），1370（UA A区特征吸收），1246（C-O-C伸缩振动）。 IR data (KBr, cm^-1 ) and their assignments: υ: 3395 (NH₂ stretching vibration), 2927 (CH stretching vibration), 1735 (s, C=O stretching vibration), 1640 (s, C=O stretching vibration , amide I peak), 1526 (NH bending vibration, amide II peak), 1370 (UA A region characteristic absorption), 1246 (COC stretching vibration).

 ESI-MS：m/z 541.5 [M-H]⁺。ESI-MS : m/z 541.5 [MH]⁺ .

 1H NMR（400MHz, CDCl₃）δ： 5.34（t, J=2.8 Hz, 1 H, H-12）, 4.49 (t, J = 6.0 Hz, 1 H, H-3), 3.47 (m, J=5.6 Hz, 2H，NHCH₂), 3.16 (m, J=5.2 Hz, 2H, CH₂NH₂), 2.91 (t, J=5.6 Hz, 2H, NH₂), 2.17 (d, 1 H, J=3.6 Hz, H-18), 2.05 (s, 3H, CH₃COO), 1.25 (s, 3 H, CH₃), 1.09 (s, 3 H, CH₃), 0.97-0.93 (m, 6H, 2×CH₃), 0.89-0.85 (m, 9H, 3×CH₃), 0.78 (s, 1 H CH₃).1H NMR (400MHz, CDCl₃ ) δ: 5.34 (t, J=2.8 Hz, 1 H, H-12), 4.49 (t, J = 6.0 Hz, 1 H, H-3), 3.47 (m, J= 5.6 Hz, 2H, NHCH₂ ), 3.16 (m, J=5.2 Hz, 2H,CH₂ NH₂ ), 2.91 (t, J=5.6 Hz, 2H, NH₂ ), 2.17 (d, 1 H , J=3.6 Hz, H-18), 2.05 (s, 3H, CH₃ COO), 1.25 (s, 3 H, CH₃ ), 1.09 (s, 3 H, CH₃ ), 0.97-0.93 (m, 6H, 2×CH₃ ), 0.89-0.85 (m, 9H, 3×CH₃ ), 0.78 (s, 1 H CH₃ ).

实施例3Example 3

N-[3β-羟基-熊果烷-12-烯-28-酰]-氨基乙二胺（化合物Ⅳ）的制备：Preparation of N-[3β-hydroxy-arbutane-12-ene-28-acyl]-aminoethylenediamine (compound Ⅳ):

称取N-[3β-乙酰氧基-熊果烷-12-烯-28-酰]-氨基乙二胺0.5859g，溶于20 ml CH₃OH-THF（1：1.5 v：v）中，加入2.0ml 4N NaOH，室温下搅拌反应3.5 h，反应完全，向混合物中加入适量的水，2 N HCl调pH 3-4，减压蒸除溶剂，水洗沉淀至中性，柱层析纯化（氯仿: 丙酮 =1:5），无水乙醇热溶，冷却重结晶，得N-[3β-羟基-熊果烷-12-烯-28-酰]-氨基乙二胺。Weigh 0.5859 g of N-[3β-acetoxy-arbutan-12-en-28-yl]-aminoethylenediamine, dissolve it in 20 ml CH₃ OH-THF (1:1.5 v:v), Add 2.0ml 4N NaOH, stir and react at room temperature for 3.5 h, the reaction is complete, add an appropriate amount of water to the mixture, adjust the pH to 3-4 with 2 N HCl, evaporate the solvent under reduced pressure, wash the precipitate with water until neutral, and purify by column chromatography ( Chloroform: acetone = 1:5), dissolved in absolute ethanol, cooled and recrystallized to obtain N-[3β-hydroxy-arbutane-12-en-28-yl]-aminoethylenediamine.

性状：白色粉末；产率：85.7%。Properties: white powder; yield: 85.7%.

 IR数据(KBr，cm-1)及其归属：υ：3384（O-H伸缩振动），2926（C-H伸缩振动），1636（ s，C=O伸缩振动，酰胺Ⅰ峰）,1528（N-H弯曲振动，酰胺Ⅱ峰），1378（UA  A区特征吸收）。IR data (KBr, cm-1) and its attribution: υ: 3384 (OH stretching vibration), 2926 (CH stretching vibration), 1636 (s, C=O stretching vibration, amide I peak), 1528 (NH bending vibration, Amide II peak), 1378 (characteristic absorption of UA A region).

 ESI-MS：m/z 499.1 [M-H]⁺。ESI-MS : m/z 499.1 [MH]⁺ .

 1H NMR（400MHz, CDCl₃）δ： 5.31（t, J=2.8 Hz, 1 H, H-12）, 4.32 (m, J = 5.6 Hz, 1 H, H-3), 3.49 (m, J=6.0 Hz, 2H，NHCH₂), 3.17 (m, J=5.2 Hz, 2H, CH₂NH₂), 2.92 (t, J=5.2 Hz, 2H, NH₂), 2.17 (d, 1 H, J=4.8 Hz, H-18), 1.24 (s, 3 H, CH₃), 1.10 (s, 3 H, CH₃), 0.97-0.93 (m, 6H, 2×CH₃), 0.89-0.85 (m, 9H, 3×CH₃), 0.78 (s, 1 H CH₃).1H NMR (400MHz, CDCl₃ ) δ: 5.31 (t, J=2.8 Hz, 1 H, H-12), 4.32 (m, J = 5.6 Hz, 1 H, H-3), 3.49 (m, J= 6.0 Hz, 2H, NHCH₂ ), 3.17 (m, J=5.2 Hz, 2H,_{CH 2}NH₂ ), 2.92 (t, J=5.2 Hz, 2H, NH₂ ), 2.17 (d, 1 H , J=4.8 Hz, H-18), 1.24 (s, 3 H, CH₃ ), 1.10 (s, 3 H, CH₃ ), 0.97-0.93 (m, 6H, 2×CH₃ ), 0.89-0.85 (m, 9H, 3×CH₃ ), 0.78 (s, 1 H CH₃ ).

实施例4Example 4

N-[3β-乙酰氧-熊果烷-12-烯-28-酰]-氨基乙二胺-叶酸（化合物Ⅰ）的制备：Preparation of N-[3β-acetoxy-arbutane-12-en-28-yl]-aminoethylenediamine-folic acid (compound Ⅰ):

称取N-[3β-乙酰氧基-熊果烷-12-烯-28-酰]-氨基乙二胺0.203g溶解于20ml DMSO中，加入0.219g叶酸，室温搅拌30 min，然后加入0.09 g EDC（1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐）、0.082g NHS（N-羟基丁二酰亚胺），室温避光反应20 h。反应结束后，用油泵蒸除溶剂，然后加入20ml水，以2N HCl调节pH3-4，过滤，水洗滤饼至滤液pH为中性，干燥；干燥后的产物干法上样，反相柱纯化（甲醇：水=8:1），无水乙醇热溶，冷却重结晶，制得N-[3β-乙酰氧基-熊果烷-12-烯-28-酰]-氨基乙二胺-叶酸。Weigh 0.203g of N-[3β-acetoxy-arbutane-12-en-28-yl]-aminoethylenediamine and dissolve it in 20ml of DMSO, add 0.219g of folic acid, stir at room temperature for 30 minutes, then add 0.09g EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride), 0.082g NHS (N-hydroxysuccinimide), react at room temperature for 20 h in the dark. After the reaction, remove the solvent with an oil pump, then add 20ml of water, adjust the pH to 3-4 with 2N HCl, filter, wash the filter cake with water until the pH of the filtrate is neutral, and dry; the dried product is dry-loaded and purified by reverse-phase column (Methanol: water = 8:1), dissolved in absolute ethanol, cooled and recrystallized to obtain N-[3β-acetoxy-arbutane-12-en-28-yl]-aminoethylenediamine-folic acid .

性状：橙黄色粉末；产率：49.5%。Properties: Orange-yellow powder; Yield: 49.5%.

 IR数据(KBr，cm-1)及其归属：υ：3328（NH₂伸缩振动），2928（C-H伸缩振动），1733（s，C=O伸缩振动），1640（s，C=O伸缩振动，酰胺Ⅰ峰）, 1516（N-H弯曲振动，酰胺Ⅱ峰），1371（UA  A区特征吸收），1245（C-O-C伸缩振动）。IR data (KBr, cm-1) and its attribution: υ: 3328 (NH₂ stretching vibration), 2928 (CH stretching vibration), 1733 (s, C=O stretching vibration), 1640 (s, C=O stretching vibration , amide I peak), 1516 (NH bending vibration, amide II peak), 1371 (UA A region characteristic absorption), 1245 (COC stretching vibration).

 ESI-MS：m/z 963.3 [M-H]⁺。ESI-MS : m/z 963.3 [MH]⁺ .

 the

¹H NMR（400MHz, CDCl₃）δ：7.73-7.70 (m, J=3.6 Hz, 2 H, 2×Ar-H ), 7.55-7.52 (m，J=3.2 Hz, 2 H，2×Ar-H），5.30 (t, J=3.2 Hz, 1 H, H-12), 4.49 (m, J = 7.2 Hz, 1 H, CHCOOH), 4.31 (d，J=6.0 Hz, 2 H，Ar-NH-CH₂), 4.22 (t, J = 5.6 Hz, 1 H, H-3), 3.48 (m, J=7.2 Hz, 2 H，CH₂NH), 2.89 (t, J=2.0 Hz, 2 H, CH₂CHCOOH), 2.62 (m, 9 H, 3×CH₃), 2.17 (d, 1 H, J=4.0 Hz, H-18), 2.05 (s, 3 H, CH₃COO), 1.25 (s, 3 H, CH₃), 1.09 (s, 3 H, CH₃), 0.98-0.94 (m, 6H, 2×CH₃), 0.89-0.85 (m, 9H, 3×CH₃), 0.76 (s, 1 H CH₃).¹H NMR (400MHz, CDCl₃ ) δ: 7.73-7.70 (m, J=3.6 Hz, 2 H, 2×Ar-H ), 7.55-7.52 (m, J=3.2 Hz, 2 H, 2×Ar- H), 5.30 (t, J=3.2 Hz, 1 H, H-12), 4.49 (m, J = 7.2 Hz, 1 H,CH COOH), 4.31 (d, J=6.0 Hz, 2 H, Ar -NH-CH₂ ), 4.22 (t, J = 5.6 Hz, 1 H, H-3), 3.48 (m, J=7.2 Hz, 2 H,CH₂ NH), 2.89 (t, J=2.0 Hz, 2 H,CH₂ CHCOOH), 2.62 (m, 9 H, 3×CH₃ ), 2.17 (d, 1 H, J=4.0 Hz, H-18), 2.05 (s, 3 H, CH₃ COO), 1.25 (s, 3 H, CH₃ ), 1.09 (s, 3 H, CH₃ ), 0.98-0.94 (m, 6H, 2×CH₃ ), 0.89-0.85 (m, 9H, 3×CH₃ ), 0.76 (s, 1 H CH₃ ).

实施例5Example 5

熊果酸（UA）及其衍生物（化合物Ⅰ、Ⅱ、Ⅲ、Ⅳ）的体外抗癌活性--MTT法检测药物对细胞的增殖抑制作用In Vitro Anticancer Activity of Ursolic Acid (UA) and Its Derivatives (Compounds Ⅰ, Ⅱ, Ⅲ, Ⅳ)--MTT Assay to Detect the Inhibitory Effect of Drugs on Cell Proliferation

取处于对数生长期状态良好的细胞一瓶，胰蛋白酶消化，制成5×10⁴个/mL的细胞悬液。

Take a bottle of cells in good logarithmic growth phase, digest with trypsin, and make a cell suspension of 5×10⁴ cells/mL.

细胞悬液移入96孔板，每孔100 μL，周围一圈用PBS填充，置37℃, 5% CO₂培养箱中培养24 h。

The cell suspension was transferred into a 96-well plate, 100 μL per well, filled with PBS around the circle, and cultured in a 37°C, 5%_CO2 incubator for 24 h.

移去旧培养基，加入受试衍生物（用培养基将受试衍生物存储液稀释，设定不同作用浓度: 5、10、15、20、25、30 μ mol/L），每孔100μL，另设空白对照组、UA对照组（UA 60 μmol/L或20μ mol/L）和紫杉醇对照组（60 μmol/L或20μ mol/L），每组设6个复孔。药物作用24 h后，吸弃含药培养基，于每孔中加入无血清、无酚红1640培养基100 μ L，再加入MTT溶液10 μL，继续孵育4 h，终止培养。

Remove the old medium, add the test derivative (dilute the test derivative stock solution with the medium, set different action concentrations: 5, 10, 15, 20, 25, 30 μmol/L), 100μL per well In addition, a blank control group, a UA control group (UA 60 μmol/L or 20 μmol/L) and a paclitaxel control group (60 μmol/L or 20 μmol/L) were set up, with 6 replicate wells in each group. After 24 h of drug action, the drug-containing medium was discarded, 100 μL of serum-free and phenol red-free 1640 medium was added to each well, and 10 μL of MTT solution was added, and the incubation was continued for 4 h before the culture was terminated.

小心吸弃96孔板孔内上清液，每孔加入150 μL DMSO，振荡10 min，于490 nm波长处在酶标仪上测定各孔光吸收值(OD值)，计算细胞的增殖抑制率：抑制率（%）=（1-用药组平均OD值÷空白对照组平均OD值）×100%，应用SPSS16.0软件进行数据处理并计算癌细胞增殖的半数抑制浓度（IC₅₀），结果见表1。

Carefully discard the supernatant in the wells of the 96-well plate, add 150 μL DMSO to each well, shake for 10 min, measure the optical absorption value (OD value) of each well on a microplate reader at a wavelength of 490 nm, and calculate the cell proliferation inhibition rate : Inhibition rate (%)=(1-Average OD value of medication group÷Average OD value of blank control group)×100%, using SPSS16.0 software for data processing and calculation of half inhibitory concentration (IC₅₀ ) of cancer cell proliferation, the result See Table 1.

如表1结果所示，所合成的UA衍生物对不同的肿瘤细胞均具有一定的增殖抑制作用，其中化合物Ⅰ、Ⅲ和Ⅳ的抑制作用要优于UA，而且它们对正常细胞的毒副作用也低于UA。其中，化合物Ⅰ的抗癌活性数据可以看出，对叶酸受体高表达的Hela细胞的IC₅₀明显低于其他癌细胞（HepG2、BGC、A-375），说明该化合物具有一定的靶向性；与其他的化合物相比较，化合物Ⅰ对正常细胞（HELF细胞）的IC₅₀值很高，说明化合物Ⅰ具有低毒性。总之，化合物Ⅰ、Ⅲ和Ⅳ具有较好的抗癌活性潜力。As shown in the results in Table 1, the synthesized UA derivatives have a certain inhibitory effect on the proliferation of different tumor cells, and the inhibitory effects of compounds Ⅰ, Ⅲ and Ⅳ are better than those of UA, and their toxic side effects on normal cells are also lower. lower than UA. Among them, the anticancer activity data of compound Ⅰ can be seen that the IC₅₀ of Hela cells with high expression of folate receptors is significantly lower than that of other cancer cells (HepG2, BGC, A-375), indicating that the compound has certain targeting ; Compared with other compounds, the IC₅₀ value of compound Ⅰ to normal cells (HELF cells) is very high, indicating that compound Ⅰ has low toxicity. In conclusion, compounds Ⅰ, Ⅲ and Ⅳ have good anticancer activity potential.

表1 熊果酸衍生物对不同肿瘤细胞的增殖抑制作用Table 1 Inhibitory effects of ursolic acid derivatives on the proliferation of different tumor cells

 mean（±SD fold, n≥6）,compared with UA ,*P<0.05;**P<0.01mean (±SD fold, n≥6), compared with UA , *P <0.05; **P <0.01

Claims (8)

1. ursolic acid verivate, it is characterized in that: said ursolic acid verivate is N-[3 β-acetoxyl group-black bearberry alkane-12-alkene-28-acyl]-amino quadrol-folic acid, and structural formula is suc as formula shown in the I:

Ⅰ。

2. the preparation method of a ursolic acid verivate as claimed in claim 1 is characterized in that:

(1) ursolic acid and acetic anhydride reaction generates 3-O-ethanoyl ursolic acid; The structural formula of 3-O-ethanoyl ursolic acid is suc as formula shown in the II,

Ⅱ；

(2) 3-O-ethanoyl ursolic acid and oxalyl chloride reaction gets N-[3 β-acetoxyl group-black bearberry alkane-12-alkene-28-acyl]-amino quadrol with reacting ethylenediamine again, and structural formula is suc as formula shown in the III,

Ⅲ；

(3) N-[3 β-acetoxyl group-black bearberry alkane-12-alkene-28-acyl]-amino quadrol and folic acid reaction gets N-[3 β-acetoxyl group-black bearberry alkane-12-alkene-28-acyl]-amino quadrol-folic acid, and structural formula is suc as formula shown in the I.

3. the preparation method of ursolic acid verivate according to claim 2 is characterized in that: said preparing method's concrete steps are following:

The ursolic acid stirring and dissolving of (1) 1 weight part is added dropwise to the acetic anhydride of 2-3 weight part in the pyridine of 40-50 weight part, add the DMAP of 0.001-0.02 weight part, stirs 16-18 h under the room temperature; After reaction finishes, regulate pH 3-4 with 2 N HCl, steaming desolventizes, suction filtration; The washing filter cake is extremely neutral, drying, appearance on the dried product dry method, column chromatography purification; The absolute ethyl alcohol thermosol cools off recrystallization, gets the 3-O-ethanoyl ursolic acid of white powder;

(2) 1 weight part 3-O-ethanoyl ursolic acid are dissolved in 30-35 weight part CH₂Cl₂In, dropwise add the oxalyl chloride of 1-2 weight part, stirring reaction 12-24h under the room temperature steams the gas that desolventizes and react generation, the bullion of 3-O-ethanoyl UA acyl chlorides midbody, directly get into next step reaction; The CH that in the bullion of above-mentioned midbody, adds the 20-30 weight part₂Cl₂, triethylamine regulator solution pH 8-9 adds the quadrol of 1-2 weight part, stirring reaction 12-24h under the room temperature after reaction finishes, adds the water of 5-10 weight part in the reaction solution, regulate pH3-4 with 2N HCl, filter, the washing filter cake to the pH that filtrates for neutral, drying; Appearance on the dried product dry method, column chromatography purification, the absolute ethyl alcohol thermosol, the cooling recrystallization gets white powder N-[3 β-acetoxyl group-black bearberry alkane-12-alkene-28-acyl]-amino quadrol;

(3) 1 weight part N-[3 β-acetoxyl group-black bearberry alkane-12-alkene-28-acyl]-amino quadrol is dissolved in the anhydrous dimethyl sulphoxide of 20-30 weight part; The folic acid that adds the 1-2 weight part; Stirring at room 30-60min; Add 1-(3-the dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride of 1-2 weight part, the N-maloyl imines of 1-2 weight part then, room temperature lucifuge reaction 20-30 h; After reacting end, steaming desolventizes, and adds the water of 5-10 weight part then, regulates pH3-4 with 2N HCl, filters, and the washing filter cake is a neutrality to the pH that filtrates, drying; Appearance on the dried product dry method, the reversed-phase column purifying, the absolute ethyl alcohol thermosol, the cooling recrystallization gets yellow powder shape N-[3 β-acetoxyl group-black bearberry alkane-12-alkene-28-acyl]-amino quadrol-folic acid.

4. ursolic acid verivate, it is characterized in that: said ursolic acid verivate is N-[3 beta-hydroxies-black bearberry alkane-12-alkene-28-acyl]-amino quadrol, and structural formula is suc as formula shown in the IV:

Ⅳ。

5. the preparation method of a ursolic acid verivate as claimed in claim 4 is characterized in that:

(1) ursolic acid and acetic anhydride reaction generates 3-O-ethanoyl ursolic acid; The structural formula of 3-O-ethanoyl ursolic acid is suc as formula shown in the II;

(2) 3-O-ethanoyl ursolic acid and oxalyl chloride reaction gets N-[3 β-acetoxyl group-black bearberry alkane-12-alkene-28-acyl]-amino quadrol with reacting ethylenediamine again, and structural formula is suc as formula shown in the III;

(3) N-[3 β-acetoxyl group-black bearberry alkane-12-alkene-28-acyl]-amino quadrol and NaOH hydrolysis reaction obtains N-[3 beta-hydroxies-black bearberry alkane-12-alkene-28-acyl]-amino quadrol, and structural formula is suc as formula shown in the IV.

6. the preparation method of ursolic acid verivate according to claim 5 is characterized in that: said preparing method's concrete steps are following:

The ursolic acid stirring and dissolving of (1) 1 weight part is added dropwise to the acetic anhydride of 2-3 weight part in the pyridine of 40-50 weight part, add a spot of DMAP, stirs 16-18 h under the room temperature; After reaction finishes, transfer pH 3-4 with 2 N HCl, steaming desolventizes, suction filtration; The washing filter cake is extremely neutral, drying, appearance on the dried product dry method, column chromatography purification; The absolute ethyl alcohol thermosol cools off recrystallization, gets the 3-O-ethanoyl ursolic acid of white powder;

(2) 1 weight part 3-O-ethanoyl ursolic acid are dissolved in 30-35 weight part CH₂Cl₂In, in batches, dropwise add the oxalyl chloride of 1-2 weight part, stirring reaction 12-24 h under the room temperature steams the gas that desolventizes and react generation, the bullion of 3-O-ethanoyl UA acyl chlorides midbody, directly get into next step reaction; The CH that in above-mentioned midbody, adds the 20-30 weight part₂Cl₂, triethylamine regulator solution pH 8-9, the quadrol of adding 1-2 weight part, stirring reaction 12-24 h under the room temperature; After reaction finishes, add the water of 5-10 weight part in the reaction solution, transfer pH3-4, filter with 2N HCl; The washing filter cake is neutrality to the pH that filtrates, drying, appearance on the dried product dry method, column chromatography purification; The absolute ethyl alcohol thermosol, the cooling recrystallization gets white powder N-[3 β-acetoxyl group-black bearberry alkane-12-alkene-28-acyl]-amino quadrol;

(3) take by weighing N-[3 β-acetoxyl group-black bearberry alkane-12-alkene-28-acyl]-amino quadrol of 1-2 weight part, be dissolved in the CH of 20-30 weight part₃Among the OH-THF, said CH₃The volume ratio of OH and THF is 1:1.5-2.5; The 4N NaOH that adds the 4-8 weight part, stirring reaction 3-5h under the room temperature, the water of adding 5-10 weight part in reaction solution, 2 N HCl regulate pH 3-4, remove solvent under reduced pressure, and water washing and precipitating is to neutrality, drying; Appearance on the dried product dry method, column chromatography purification, the absolute ethyl alcohol thermosol, the cooling recrystallization gets N-[3 beta-hydroxies-black bearberry alkane-12-alkene-28-acyl]-amino quadrol.

7. one kind like claim 2 or 5 described ursolic acid verivates, it is characterized in that: said quadrol replaces with tn, tetramethylenediamine, pentamethylene diamine or hexanediamine, obtains the ursolic acid verivate of a series of correspondences.

8. one kind like claim 1 or 4 described ursolic acid verivates or by the application of ursolic acid verivate in the preparation antitumor drug of claim 2,3,5 or 6 said preparing methods' preparations.