WO2012129914A1 - 3-氨基-2-羟基-4-苯基-缬氨酰-异亮氨酸及其制备方法和应用 - Google Patents

3-氨基-2-羟基-4-苯基-缬氨酰-异亮氨酸及其制备方法和应用 Download PDF

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WO2012129914A1
WO2012129914A1 PCT/CN2011/082278 CN2011082278W WO2012129914A1 WO 2012129914 A1 WO2012129914 A1 WO 2012129914A1 CN 2011082278 W CN2011082278 W CN 2011082278W WO 2012129914 A1 WO2012129914 A1 WO 2012129914A1
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Prior art keywords
aminopeptidase
compound
preparation
inhibitor
hydroxy
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PCT/CN2011/082278
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English (en)
French (fr)
Inventor
饶敏
戈梅
罗敏玉
阮丽军
夏兴
朱丽
阮林高
康怀侠
杨天
王天娇
李秋爽
蒋含嫣
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上海来益生物药物研究开发中心有限责任公司
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Priority claimed from CN201110076555.0A external-priority patent/CN102631664B/zh
Priority claimed from CN201110076554.6A external-priority patent/CN102477067B/zh
Application filed by 上海来益生物药物研究开发中心有限责任公司 filed Critical 上海来益生物药物研究开发中心有限责任公司
Priority to US14/008,195 priority Critical patent/US20140018303A1/en
Publication of WO2012129914A1 publication Critical patent/WO2012129914A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
    • C07K5/0202—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing the structure -NH-X-X-C(=0)-, X being an optionally substituted carbon atom or a heteroatom, e.g. beta-amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants

Definitions

  • the present invention belongs to the field of chemistry, and more particularly to a novel compound, 3-amino-2-hydroxy-4-phenyl-prolyl-isoleucine, and a process for its preparation and use. Background technique
  • the applicant of this case carried out microbial separation from the soil samples collected from the natural environment, and obtained a new strain of Streptomyces parvus which can produce daptomycin. On July 20, 2010, the microbial strains in China.
  • the General Management Center of the Deposit Management Committee is officially deposited with the deposit number: CGMCC No. 4027.
  • the inventors of the present application have extensively and intensively studied to isolate a novel compound from the secondary metabolite of Streptomyces faecalis CGMCC No. 4027; further studies have shown that the compound has an effect of inhibiting aminopeptidase, Used as an aminopeptidase inhibitor.
  • a first object of the present invention is to provide a novel compound.
  • a second object of the present invention is to provide a process for the preparation of the compound.
  • a further object of the invention is to provide for the use of the compound.
  • a fourth object of the present invention is to provide a pharmaceutical composition of an aminopeptidase inhibitor containing the compound.
  • the structural formula of the compound provided by the present invention is as follows -. It is named: 3-amino-2-hydroxy-4-phenyl-4-pyryl-isoleucine (3-amino-2-hydroxy-4-pheny 1 but anoy l-va lyl-i so leuc ine ).
  • the method for producing the compound of the present invention is produced by fermenting Streptomyces sp. CGMCC No. 4027.
  • the compound has an action of inhibiting aminopeptidase and can be used for the preparation of an aminopeptidase inhibitor.
  • the aminopeptidase inhibitor is an aminopeptidase N inhibitor.
  • the aminopeptidase inhibitor can be used for preparing a medicament for treating a tumor, preparing a medicament for adjuvant treatment of a tumor, and preparing an immunopotentiating drug.
  • composition of the aminopeptidase inhibitor provided by the present invention contains the compound as an active ingredient.
  • the compositions also contain one or more pharmaceutically acceptable carriers or excipients.
  • the present invention not only provides a novel compound, but also, the compound can be used for the preparation of an aminopeptidase inhibitor drug, and is expected to control tumor invasion and metastasis and angiogenesis by inhibiting the activity of aminopeptidase N (APN), and Improve the body's immunity.
  • APN aminopeptidase N
  • Figure 1 is an HPLC analysis chart.
  • Figure 2 shows the positive ion high resolution mass spectrum.
  • Figure 3 shows the positive ion secondary mass spectrum.
  • Figure 4 is a hydrogen spectrum.
  • Figure 5 shows the carbon spectrum
  • Figure 6 is a H-H COSY spectrum.
  • Figure 7 is a HMBC spectrum. detailed description
  • the strain used in the present invention is Streptomyces parvus, which has been deposited in the General Microbiology Center (CGMCC) of the China Microbial Culture Collection Management Committee in 2010, and has a deposit number of CGMCC No. 4027.
  • CGMCC General Microbiology Center
  • 4027 is selected from the group consisting of: dextrin, soluble starch, citrate, glycerin, glucose, mannitol, rhamnose, L-arabinose , cellobiose, glycogen, salicin, amygdalin, sodium propionate, sodium succinate, sodium acetate or sodium malate, the content is 1.0 to 8.0%;
  • the gas source of the fermentation medium is selected from the group consisting of: yeast powder, soybean cake powder, cottonseed cake powder or peanut cake powder, and the content is 0.5 to 5.0%;
  • the fermentation medium contains dextrin 3.5 to 4.0%, glucose 0.5 to 1.0%, soybean cake powder 2.0 to 2.5%, and ferrous ammonium sulfate 0.05 to 0.15%.
  • the fermentation medium contains dextrin 3.6%, glucose 0.8%, soybean cake powder 2.2%, and ferrous ammonium sulfate 0.1%.
  • Incline medium Gao's No. 1 medium.
  • Seed medium glucose (2%), glycerol (2%), soluble starch (6%), soy cake powder (5%), potassium dihydrogen phosphate (0.02%), magnesium sulfate heptahydrate (0.04%), the rest For water.
  • the initial pH is 7.3-7.5.
  • Fermentation medium soy cake powder (2.2%), dextrin (3.6%), glucose (0.8%), ammonium ferrous sulfate hexahydrate (Fe (NH 4 ) 2 (S0J 2 6H 2 0) (0.1%),
  • the rest is water.
  • the initial pH is 7.3-7.5.
  • Example 1 Preparation of the compound
  • the strain CGMCC No. 4027 was inoculated from the lyophilized tube onto the Takashi No. 1 slant medium, and cultured at 28 ° C - 30 ° C for four days, and then transferred to a seed medium which was autoclaved at 121 ° C for 30 min.
  • the flask was placed on a shaker for seed culture, wherein the shaker speed was 200-230 rpm, the temperature was 28-30 ° C, and the culture time was 46-50 h. After the seed culture was completed, it was transferred to a 5 L glass fermenter containing the sterilized fermentation medium.
  • the fermentation amount was 5%, fermentation was carried out in the fermenter, the stirring speed was 400-500 rpm, the temperature was controlled at 28-30 ° C, the aeration was 7-9 L/min, and the fermentation was terminated after 4-6 days of culture.
  • the fermentation broth obtained after fermentation was placed in a refrigerator at 4 ° C overnight to precipitate a protein.
  • the fermentation broth was then centrifuged at 4000 rpm for 30 min at 4 °C.
  • the lower layer precipitate is discarded, and the supernatant is adsorbed by a macroporous adsorption resin.
  • the macroporous adsorption resin may be adsorbed by XAD-1600, SP850, or JD-1, either statically or dynamically. After adsorption, elute with twice column volume of pure water, 10%, 30%, 50%, 75%, 100% ethanol, and concentrate the crude in 50% ethanol eluent.
  • the crude liquid was concentrated under reduced pressure at 40 ° C using a rotary evaporator, and then subjected to crudeness using a medium pressure system.
  • the conditions are as follows: The crude concentrate was diluted 10 times with 30% acetonitrile, centrifuged at 12000 rpm for 10 min, and the supernatant was the injection sample. The loading was 15-25 ml, and the medium pressure column (Model: Biotage Si 25+M 1603-2, USA) was used for separation. The column temperature was natural, and the mobile phase was 30% acetonitrile 500 ml, which was eluted isocratically. The flow rate was approximately 3-5 ml/min and the sample was concentrated in 200-400 ml of eluent.
  • the eluate was concentrated under reduced pressure at 40 °C using a rotary evaporator, and then prepared by DIONEXP 680 dual pump preparation liquid phase system or preparative Agilent 1100 DAD single pump liquid phase system.
  • the collected preparation liquid was steamed at 40 ° C. Concentrate to dryness to obtain the pure product of the title compound, which is obtained as a pale yellow powder, which is easily dissolved in methanol, ethanol, water, and acetonitrile. Do not dissolve in chloroform.
  • the liquid phase preparation conditions are as follows: Preparation column: YMC CombiPreP 0DS-A 5 ⁇ 20x250mm
  • the analytical analytical Ag i lent 1100 DAD dual pump liquid phase system prepared by the preparation of the liquid phase system was analyzed and the conditions were as follows -.
  • the target compound has a peak retention time of about 8.6 min under this condition, and two solvent peaks of about 3 min are removed, and the purity is > 95%.
  • the target compound After high-resolution mass spectrometry (as shown in Figure 2), the target compound has an accurate molecular weight of 407. 2522 and a molecular formula of C21H33N305.
  • the hydrogen spectrum Fig. 4
  • the carbon spectrum Fig. 5
  • the COSY spectrum Fig. 6
  • the BC spectrum Fig. 7
  • the compound obtained in Examples 1 to 2 was subjected to the method reported in J. Microbiol. Biotechnol., 1995, 5(1): 36-40 to give 3-amino-2-hydroxy-4-phenyl-prolyl - Isoleucine (3-amino-2-hydroxy-4-phenyl but anoyl-valyl-i so leucine ) was assayed for activity.
  • Bestatin is a typical APN inhibitor and has been used as a therapeutic drug for leukemia for many years. In this example, it was used as a control group.
  • the specific experimental procedure is as follows: adding a substrate (L-leucyl p-nitroaniline) to a 96-well plate, ⁇ lmg/ml in a 0. IM Tris-HCl, pH 7.0), different concentrations (0.86, 1.71, 2.57, 4.29, 8.57 g/ml ) Gradient test compound 20 ⁇ 1 (0. IM Tris-HCl, pH 7.0), 37 °C, 3min reaction, then add aminopeptidase N (L5006- 25UN, Sigma) solution 20 ⁇ 1 (2mU, 0. IM Tris-HCl, pH 7.0), 37 ° C, 30 min reaction, then the absorbance was measured at a wavelength of 405 nm.
  • a substrate L-leucyl p-nitroaniline
  • A is the absorbance measured by the reaction system without inhibitor
  • B is the absorbance measured by the reaction system containing different concentrations of the test compound, and is adjusted with 0.1 M Tris-HCl, pH 7.0 buffer, and the result is as follows.
  • Table 2 shows the inhibition rate of aminopeptidase N by the reference substance at 5 concentrations and the compound of the present invention.
  • the compound 3-amino-2-hydroxy-4-phenyl-prolyl-isoleucine isolated from the secondary metabolite of Streptomyces sp. CGMCC No. 4027 of the present invention has ammonia.
  • Peptidase N inhibits activity, and its inhibitory activity against aminopeptidase N is better than Bes tat in at the same concentration.
  • Aminopeptidase N is a membrane-bound exopeptidase containing zinc ions with a large amount of APN expression on the surface of tumor cells. It is closely related to the growth of primary and secondary tumors and the formation of blood vessels, and even promotes the process of proliferation and differentiation of tumor cells.
  • the active substance reduces the immunity of the body and weakens the ability of macrophages and NK cells to recognize and kill tumor cells.
  • APN has become an important target for anti-tumor and antiviral drugs, developing highly effective, low-toxic, highly selective APN inhibitors for the treatment of tumors.
  • Bes tat in is a compound having aminopeptidase inhibitory activity found in the culture solution of Streptomyces ol ivoret icul i. It was used as an APN inhibitor antitumor drug in 1987, for acute leukemia, Malignant melanoma and other significant effects.
  • the compound 3-amino-2-hydroxy-4-phenyl-prolyl-isoleucine has a better inhibitory activity against aminopeptidase N than Bes tat in, and can be used as An aminopeptidase inhibitor for the preparation of a therapeutic tumor, or for the treatment of a tumor, or for the enhancement of an immunological drug.
  • these drugs may comprise, in addition to a therapeutically effective amount of 3-amino-2-hydroxy-4-phenyl-prolyl-isoleucine, a pharmaceutically acceptable carrier.
  • an excipient such as a solvent, diluent, etc., 3-amino-2-hydroxy-4-phenyl-prolyl-isoleucine, mixed with one or more pharmaceutically acceptable carriers or excipients
  • a pharmaceutical preparation for treating a tumor, or assisting in the treatment of a tumor, or enhancing immunity.

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Abstract

本发明涉及一种新的化合物——3-氨基-2-羟基-4-苯基-缬氨酰-异亮氨酸，其结构式如下：，该化合物由小链霉菌（Streptomycesparvus）CGMCCNo.4027发酵制得，对氨肽酶具有抑制作用，可用于制备氨肽酶抑制剂类药物。

Description

3 -氨基 -2-羟基 -4-苯基-缬氨酰 -异亮氨酸
及其制备方法和应用
技术领域
本发明属于化学领域， 具体地说， 是关于一种新的化合物 3-氨基 -2-羟 基 -4-苯基 -缬氨酰-异亮氨酸及其制备方法和应用。 背景技术
本案申请人从釆集自自然环境中的土壤样品中进行微生物分离， 得到了一株 可产达托霉素的新型小链霉菌 （Streptomyces parvus ), 于 2010年 7月 20曰在 中国微生物菌种保藏管理委员会普通微生物中心正式保藏， 保藏号为： CGMCC No. 4027。
目前对于小链霉菌 CGMCC No. 4027 的次级代谢产物的研究较少， 仅限于达 托霉素。 发明内容
本申请的发明人经过广泛而深入的研究， 在小链霉菌 CGMCC No. 4027的次级 代谢产物中分离出一种新的化合物； 进一步的研究证明， 该化合物具有抑制氨肽 酶的作用， 可用作氨肽酶抑制剂。
因此， 本发明的第一个目的在于提供一种新的化合物。
本发明的第二个目的在于提供该化合物的制备方法。
本发明的再一个目的在于提供该化合物的应用。
本发明的第四个目的在于提供一种含有该化合物的氨肽酶抑制剂药物组合 物。
本发明提供的化合物的结构式如下-.
Figure imgf000004_0001
命名为： 3-氨基 -2-羟基 -4-苯基 -缬氨酰-异亮氨酸（ 3-amino -2-hydroxy-4- pheny 1 but anoy l-va lyl-i so leuc ine )。
本发明的化合物的制备方法， 是通过发酵培养小链霉菌 CGMCC No. 4027而制 得。
根据本发明，所述化合物具有抑制氨肽酶的作用，可用于制备氨肽酶抑制剂。 根据一个优选实施例， 所述氨肽酶抑制剂为氨肽酶 N抑制剂。
根据本发明， 所述氨肽酶抑制剂可用于制备治疗肿瘤的药物， 制备辅助治疗 肿瘤的药物， 以及制备免疫增强药物。
本发明提供的氨肽酶抑制剂药物组合物， 含有作为活性成分的所述化合物。 此外， 所述组合物还含有一种或多种药学上可接受的载体或赋形剂。
本发明不仅提供了一种新的化合物， 而且， 该化合物可用于制备氨肽酶抑制 剂类药物， 有望通过抑制氨肽酶 N ( APN )的活性， 控制肿瘤的侵袭和转移以及血 管生成， 并提高机体的免疫力。 附图说明
图 1为 HPLC分析图。
图 2为正离子高分辨质谱图。
图 3为正离子二级质谱图。
图 4为氢谱谱图。
图 5为碳谱谱图。
图 6为 H- H COSY谱图。 图 7为 HMBC谱谱图。 具体实施方式
以下结合具体实施例， 对本发明做进一步说明。 应理解， 以下实施例仅用于 说明本发明而非用于限制本发明的范围。 本发明所用的菌种为小链霉菌 （Streptomyces parvus ), 已于 2010年 Ί月 20 曰保藏于中国微生物菌种保藏管理委员会普通微生物中心 （CGMCC), 保藏号 CGMCC No.4027。 根据本发明， 小链霉菌 （ Streptomyces parvus ) CGMCC No.4027的发酵培养 基的碳源选自： 糊精、 可溶性淀粉、 柠檬酸盐、 甘油、 葡萄糖、 甘露醇、 鼠李糖、 L-阿拉伯糖、 纤维二糖、 糖原、 水杨苷、 苦杏仁苷、 丙酸钠、 琥珀酸钠、 乙酸钠 或苹果酸钠， 含量为 1.0~8.0%;
发酵培养基的氣源选自： 酵母粉、 黄豆饼粉、 棉籽饼粉或花生饼粉， 含量为 0.5 ~ 5.0%;
根据本发明， 所述发酵培养基含有糊精 3.5 ~ 4.0%, 葡萄糖 0.5~1.0%, 黄 豆饼粉 2.0~2.5%, 硫酸亚铁铵 0.05~0.15%。 优选的， 所述发酵培养基含有糊 精 3.6%, 葡萄糖 0.8%, 黄豆饼粉 2.2%, 硫酸亚铁铵 0.1%。 以下实施例中所述斜面培养基、 所述种子培养基、 所述发酵培养基的配方 ( w/v ) 如下：
斜面培养基： 高氏一号培养基。 种子培养基： 葡萄糖（2%)、 甘油（2%)、 可溶性淀粉（6%)、 黄豆饼粉（5%)、 磷酸二氢钾 （0.02%)、 七水合硫酸镁（ 0.04%), 其余为水。 初始 pH 为 7.3-7.5。 发酵培养基： 黄豆饼粉（2.2%)、 糊精 （3.6%)、 葡萄糖 （0.8%)、 六水合硫 酸亚铁铵 （ Fe (NH4) 2 (S0J 2 6H20) ( 0.1% ), 其余为水。 初始 pH为 7.3- 7.5。 实施例 1、 化合物的制备
1.1、 发酵
将菌株 CGMCC No.4027从冻干管中接种到高氏一号斜面培养基上，于 28°C-30 °C培养四天后， 转入装有经 121°C高压灭菌 30min的种子培养基的三角瓶中， 置 于摇床进行种子培养， 其中， 摇床转速为 200-230rpm, 温度为 28-30°C, 培养时 间为 46-50h。 种子培养结束后再转入装有已灭菌的发酵培养基的 5L玻璃发酵罐 内。 转种量为 5%, 在发酵罐内进行发酵， 搅拌转速为 400-500rpm, 温度控制在 28-30°C, 通气量为 7- 9L/min, 培养 4- 6天后结束发酵。
1.2、 分离提取
将经发酵后得到的发酵液置于 4°C冰箱中过夜， 沉淀蛋白。 之后将发酵液于 4°C、 4000rpm离心 30min。 弃下层沉淀， 上清液用大孔吸附树脂吸附， 大孔吸附 树脂可以釆用 XAD-1600, SP850, 或 JD-1, 于静态或动态进行吸附皆可。 吸附后 用两倍柱体积的纯水， 10%, 30%, 50%, 75%, 100%乙醇依次洗脱， 粗品集中在 50% 乙醇洗脱液中。粗品液用旋转蒸发仪于 40°C减压浓缩，再釆用中压系统进行粗制。 条件如下： 粗品浓缩液用 30%乙腈稀释 10倍， 12000rpm离心 lOmin, 上清为进样 样品。 上样量为 15- 25ml, 釆用中压柱 (型号: Biotage Si 25+M 1603-2, USA) 进行分离， 柱温自然， 流动相为 30%乙腈 500ml, 等度洗脱。 流速约 3-5ml/min, 样品集中于 200-400ml的洗脱液中。洗脱液用旋转蒸发仪于 40°C减压浓缩后釆用 DIONEXP680双元泵制备液相系统或制备型 Agilent 1100 DAD单泵液相系统进行 制备， 收集到的制备液于 40°C旋蒸浓缩至干获得目标化合物纯品， 得到的纯品为 淡黄色粉末， 易溶于甲醇， 乙醇， 水， 乙腈。 不溶于氯仿。 液相制备条件如下： 制备柱： YMC CombiPreP 0DS-A 5μΐιι 20x250mm
柱温： 自然 检测器： DAD ( 210nm, 223nm, 254nm, 280nm )
流动相 (体积比）: 25%乙腈 + 75%含 0. 1%TFA的水溶液
流速 ·· 8ml /min
进样量： 60- 700μ1
等度洗脱 实施例 2、 化合物的结构鉴定
将经制备液相系统制备收集的纯品上分析型 Ag i lent 1100 DAD双元泵液相 系统进行分析， 条件如下-.
分析用柱： 菲罗门 C18柱 5μηι 4. 6 x250腿
柱温： 30°C 检测器： VWD 210nm
流动相 (体积比）: 25%乙腈 + 75%含 0. 1%TFA的水溶液
流速： lml /min 进样量： 5μ1
等度洗脱。 如图 1所示，目标化合物在此条件下出峰保留时间在 8. 6min左右，除去 3min 左右的两个溶剂峰， 纯度〉 95%。 经高分辨质谱检测（图谱如图 2所示）， 目标化合物精确分子量为 407. 2522 , 分子式为 C21H33N305。 进一步， 经二维质谱（图 3 )检测， 结合目标化合物的氢 谱（图 4 )、 碳谱（图 5 )及 COSY谱 （图 6 )、 丽 BC谱（图 7 )进行结构分析， 核 磁数据如表 1所示。 表 1、 丽 R核磁数据
基团结构 1HNMR 13CNMR HMBC
qc 174.4
2- ■CH 4. 29 (d， J=6.0) 58.2 Qc,C3,C4,C6
3- ■CH 1. 84 (m) 38.2
lie 1. 47 (m)
4- ■CH2 26.2
1.21 (m)
5- ■CH3 0. 87 (dd, J=6.8， 14.4) 16.0 C2,C3,C4
6- ■CH3 0. 84 (dd, J=6.8, 14.4) 11.8 C2,C3,C4 qc 173.5
1' -CH 4. 19 (d， J=8.0) 60.1 Qc,C3'
Val V -CH 2. 10 (dd, J=5.2, 13.2) 31.9
4' -CH3 0. 97 (t, J=6.8) 19.6 C2',C3'
5' -CH3 0. 95 (t, J=6.8) 18.8 C2',C3' qc 173.2
1
T '-CH 4. 09 (d， J=3.6) 69.9 Qc 、 o
V '-CH 3. 72 (m) 56.3
3. 07 (dd, J=7.8， 13.5)
4' '-CH2 36.5
2.89 (dd, J=7.8， 13.5)
AHPA 4' ' a-qc 136.6
4' 'b-CH 7. 25 (m) 130.5
4' 'c-CH 7. 35 (m) 130.1
4' 'd-CH 7. 24 (m) 128.6
4' 'e-CH 7. 35 (m) 130.1
4' 'f-CH 7. 25 (m) 130.5 对其结构进行分析， 确定了所有碳原子和氢原子的归属， 得到了小链霉菌的 次级代谢产物的结构如下：
Figure imgf000009_0001
经检索， 现有技术中未发现有报道过相同结构的化合物， 可见， 本发明获得 的上述化合物是一个新的化合物。 实施例 3、 化合物的活性分析
本实施例参照文献 J. Microbiol. Biotechnol. , 1995, 5 (1)： 36- 40报道的 方法对实施例 1 ~2 得到的化合物 3-氨基 -2-羟基 -4-苯基 -缬氨酰-异亮氨酸 ( 3-amino-2-hydroxy-4-phenyl but anoyl-valyl-i so leucine )进行活性分析。
Bestatin是典型的 APN抑制剂， 作为白血病治疗药物已经上巿多年， 在本实 施例中作为对照组。
具体实验步骤为：在 96孔板中加入底物（L-亮氨酰对硝基苯胺） ΙΟΟμΚ lmg/ml in a 0. IM Tris-HCl, pH 7.0 ) , 不同浓度 （ 0.86, 1.71, 2.57, 4.29, 8.57 g/ml ) 梯度的待测化合物 20μ1 ( 0. IM Tris-HCl, pH 7.0), 37 °C, 3min反应， 再加入 氨肽酶 N(L5006- 25UN, Sigma )溶液 20μ1 ( 2mU, 0. IM Tris-HCl, pH 7.0 ) , 37 °C, 30min反应， 后于 405nm波长处测定吸收值。
按照如下公式计算抑制率：
抑制率 = ( A-B) /A*100%
其中 A为不含抑制剂的反应体系测得的吸收度， B为含有不同浓度待测化合 物的反应体系测得的吸收度， 用 0.1M Tris-HCl, pH 7.0的缓冲液调零， 结果如 表 2所示， 为 5个浓度下对照品与本发明的化合物对氨肽酶 N的抑制率。 表 2、 氨肽酶 N的抑制率
Figure imgf000010_0001
由表 2的结果可见， 本发明的从小链霉菌 CGMCC No. 4027的次级代谢产物中 分离出的化合物 3-氨基 -2-羟基 -4-苯基-缬氨酰-异亮氨酸具有氨肽酶 N抑制活 性， 且在相同浓度下对氨肽酶 N的抑制活性比 Bes tat in好。 氨肽酶 N ( APN )是一种含锌离子的膜结合型外肽酶， 在肿瘤细胞表面有大量 的 APN表达。 与原发肿瘤和继发肿瘤的生长以及血管的生成密切相关， 甚至对肿 瘤细胞的增殖分化过程也起促进作用。 对恶性肿瘤细胞的侵袭和转移过程起重要 作用， 可使细胞基质 （ECM ) 的主要成分降解， 促进肿瘤细胞的生长和转移， 另 外， 它还能够表达于抗原递呈细胞表面， 降解多种免疫活性物质， 使机体免疫力 下降， 削弱巨噬细胞和 N K细胞对肿瘤细胞的识别和杀伤能力。
因此，通过抑制 APN的活性可以对肿瘤的侵袭和转移以及血管生成进行干预， 同时增强粒细胞的趋化性， 提高机体的免疫力。 APN 已经成为抗肿瘤抗病毒药物 发挥作用的一个重要靶点， 开发高效低毒、 高选择性的 APN抑制剂， 可用于治疗 肿瘤。
Bes tat in是从橄榄网状链霉菌（ Streptomyces o l ivoret icul i ) 的培养液中 发现的具有氨肽酶抑制活性的化合物， 作为 APN抑制剂类抗肿瘤药物于 1987年 上巿， 对急性白血病、 恶性黑色素瘤等有显著疗效。
由本实施例的实验结果可知， 与 Bes tat in相比， 化合物 3-氨基 -2-羟基 -4- 苯基-缬氨酰 -异亮氨酸对氨肽酶 N的抑制活性更好， 可作为氨肽酶抑制剂， 用于 制备治疗肿瘤、 或辅助治疗肿瘤、 或增强免疫药物。 进一步地， 本领域技术人员可知， 这些药物中除了包含治疗有效量的 3-氨基 -2-羟基 -4-苯基-缬氨酰-异亮氨酸外， 还可以包含药学上可接受的载体或赋形 剂、 如溶剂、 稀释剂等， 3-氨基 -2-羟基 -4-苯基 -缬氨酰-异亮氨酸与一种或多种 药学上可接受的载体或赋形剂混合， 从而形成药物制剂， 用于治疗肿瘤、 或辅助 治疗肿瘤、 或增强免疫。

Claims

杈利要求书
1、 一 ：
Figure imgf000012_0001
2、 权利要求 1的化合物的制备方法， 其特征在于， 通过发酵培养小链霉菌 ( St reptomyces parvus ) CGMCC No. 4027而制得。
3、 权利要求 1的化合物在制备氨肽酶抑制剂中的应用。
4、 如权利要求 3 所述的应用， 其特征在于， 所述氨肽酶抑制剂为氨肽酶 N 抑制剂。
5、 如权利要求 3或 4所述的应用， 其特征在于， 所述氨肽酶抑制剂用于制 备治疗肿瘤的药物。
6、 如权利要求 3或 4所述的应用， 其特征在于， 所述氨肽酶抑制剂用于制 备辅助治疗肿瘤的药物。
7、 如权利要求 3或 4所述的应用， 其特征在于， 所述氨肽酶抑制剂用于制 备免疫增强药物。
8、 一种氨肽酶抑制剂药物组合物， 其特征在于， 所述药物组合物含有作为 活性成分的权利要求 1所述的化合物。
9、 如权利要求 8 所述的药物组合物， 其特征在于， 所述组合物还含有一种 或多种药学上可接受的载体或赋形剂。
10、 如权利要求 8或 9所述的药物组合物， 其特征在于， 所述氨肽酶抑制剂 为氨肽酶 N抑制剂。
PCT/CN2011/082278 2010-11-19 2011-11-16 3-氨基-2-羟基-4-苯基-缬氨酰-异亮氨酸及其制备方法和应用 WO2012129914A1 (zh)

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