WO2017215676A1 - 卡比多巴的药物组合物及其治疗肝癌的医药用途 - Google Patents

卡比多巴的药物组合物及其治疗肝癌的医药用途 Download PDF

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WO2017215676A1
WO2017215676A1 PCT/CN2017/097011 CN2017097011W WO2017215676A1 WO 2017215676 A1 WO2017215676 A1 WO 2017215676A1 CN 2017097011 W CN2017097011 W CN 2017097011W WO 2017215676 A1 WO2017215676 A1 WO 2017215676A1
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carbidopa
compound
liver cancer
ethanol
extract
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PCT/CN2017/097011
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French (fr)
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崔坤峰
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赵吉永
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
    • A61K31/343—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide condensed with a carbocyclic ring, e.g. coumaran, bufuralol, befunolol, clobenfurol, amiodarone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/23—Apiaceae or Umbelliferae (Carrot family), e.g. dill, chervil, coriander or cumin
    • A61K36/237—Notopterygium

Definitions

  • the invention belongs to the field of biomedicine and relates to a new use of carbidopa, in particular to a pharmaceutical composition of carbidopa and a medical use thereof for treating liver cancer.
  • Carbidopa is (S)- ⁇ -methyl- ⁇ -mercapto-3,4-dihydroxyphenylpropionic acid monohydrate.
  • Carbidopa has a strong peripheral dopa decarboxylase inhibitor. It is not easy to pass through the blood-brain barrier. When combined with levodopa, it only inhibits the activity of peripheral dopa decarboxylase, reduces the formation of dopamine in peripheral tissues, reduces peripheral adverse reactions, and increases the number of levodopa entering the central nervous system. The concentration of endolamine enhances the efficacy of levodopa and is therefore an important adjunct to levodopa. Carbidopa is not effective alone. Clinically, carbidopa and levodopa are usually formulated in a ratio of 1:10 or 1:4 to make a compound preparation.
  • It is an object of the present invention to provide a pharmaceutical composition of carbidopa comprising carbidopa and a novel natural product isolated from dried roots and rhizomes of clams, Carbidoo Ba and the natural product can synergistically treat liver cancer.
  • a pharmaceutical composition of carbidopa comprising carbidopa, compound (I) as described above, and pharmaceutically acceptable Accepted by the carrier.
  • the preparation method of the compound (I) as described above comprises the following steps: (a) pulverizing the dried roots and rhizomes of the cockroaches, extracting them by hot reflux with 65-85% ethanol, combining the extracts, and concentrating to an alcohol-free taste, followed by Extracted with petroleum ether, ethyl acetate and water-saturated n-butanol to obtain petroleum ether extract, ethyl acetate extract and n-butanol extract respectively; (b) macroporous resin for n-butanol extract in step (a) For impurity removal, first elute 8 column volumes with 6% ethanol, then elute 8 column volumes with 70% ethanol, collect 70% eluent, concentrate under reduced pressure to obtain 70% ethanol eluting concentrate; (c) (b) The 70% ethanol eluting concentrate was separated by normal phase silica gel, and sequentially eluted with a gradient of 50:1, 25:1, 15:1, and 5:1 by
  • step (d) component 4 is further separated by normal phase silica gel, and sequentially eluted with a dichloromethane-methanol gradient of 10:1, 5:1 and 2:1 by volume to obtain three components.
  • step (e) component 2 is separated by octadecylsilane-bonded reversed phase silica gel and eluted isocratically with 75% by volume aqueous methanol solution to collect 8 to 14 columns. Product eluate, the eluate was concentrated under reduced pressure to give compound (I).
  • step (a) the extract is extracted by hot reflux with 75% ethanol, and the extracts are combined.
  • the macroporous resin is an AB-8 type macroporous adsorption resin.
  • the pharmaceutical composition of carbidopa provided by the invention comprises carbidopa and a novel natural product isolated from dried roots and rhizomes of scorpion, when carbidopa and the natural product act alone. It has a therapeutic effect on liver cancer; when the two are combined, the therapeutic effect on liver cancer is further improved, and it can be developed into a drug for treating liver cancer.
  • step (a) pulverize the dried roots and rhizomes (3 kg), extract them by hot reflux with 75% ethanol (20 L ⁇ 3 times), combine the extracts, concentrate to an alcohol-free taste (4 L), and then use petroleum ether ( 4L ⁇ 3 times), ethyl acetate (4L ⁇ 3 times) and water-saturated n-butanol (4L ⁇ 3 times) were extracted to obtain petroleum ether extract, ethyl acetate extract and n-butanol extract respectively;
  • the n-butanol extract in step (a) is decontaminated with AB-8 macroporous resin, first eluted with 6% ethanol for 8 column volumes, then with 70% ethanol for 8 column volumes, and 70% eluted.
  • step (c) the 70% ethanol eluting concentrate in step (b) is separated by normal phase silica gel in a volume ratio of 50:1 (8 column volumes), 25:1 (8 column volumes), 15:1 (8 column volumes) and 5:1 (10 column volumes) of dichloromethane-methanol gradient elution Up to 4 components;
  • step (d) component 4 is further separated by normal phase silica gel in a volume ratio of 10:1 (8 column volumes), 5:1 (10 column volumes) and 2 :1 (5 column volumes) of a dichloromethane-methanol gradient elution to give 3 components;
  • component 2 in step (d) separated by octadecylsilane-bonded reversed phase silica gel, using volume
  • the aqueous solution of 75% was eluted isocratically, and 8 to 14 column volumes of the eluate were collected.
  • the eluate was concentrated under reduced pressure to give compound (I)
  • the 1756 cm -1 absorption band in the infrared spectrum and the 236 nm absorption band in the UV spectrum indicate that the compound contains ⁇ , ⁇ . -Unsaturated lactone structure
  • the 1715 cm -1 and 1680 cm -1 absorption bands in the infrared spectrum indicate the presence of keto groups and double bond fragments in the structure.
  • 13 C-NMR, DEPT and HSQC spectra show 22 carbon signals, including six One methyl group, three methylene groups, eight methine groups (two oxygenated carbons and two olefin carbons), and five quaternary carbons (three carbonyl carbons and two olefin carbons), combined with the above functional structure
  • the number of unsaturation indicates that the compound is bicyclic.
  • C-5', H-3' and C-2', C-4' and C-1" and H 3 -2" and C-1" related signals can construct another part of the fragment, and this part of the structure exists
  • the acetyl group is attached to C-3' through an oxygen atom.
  • H-3, H 2 -5 and H-6 and C-15 related signals in the HMBC spectrum suggest a lactone ring between C-6 and C-15. Structure.
  • H-8 In the NOESY spectrum, assuming H-8 is in the ⁇ configuration, the correlation signal between H-8 and H-10 indicates that H-10 is also in the ⁇ configuration, therefore, O-3'-O-acetyl-2' - The methylbutanone group should be in the alpha configuration.
  • H-9b and H-8 have related signals, so H-9a and H-2a and H-9a and H-5a related signals can judge H-2a, H. Both -5a and H-9a are in the alpha configuration, and H-2a and H-5a-related signals indicate that the ⁇ 3(4) double bond is in the E configuration.
  • the compound can be basically determined as shown below, and the stereo configuration is further determined by the ECD test, and the theoretical value is basically consistent with the experimental value.
  • the carbon atom number is as follows:
  • the laboratory meets the DB44/61-94 clean grade standard.
  • the human hepatoma cell line Bel-7402 is from the Department of Microbiology and Immunology, Guangzhou Medical College. Nude mice (BALB/C-nu/nu) were provided by the Experimental Animal Center of Southern Medical University. Males, aged 4-6 weeks, weighing 20-25 g, averaged (23.28 ⁇ 1.62) were housed in SPF environment.
  • Carbidopa was purchased from the China National Institute for the Control of Pharmaceutical and Biological Products. The compound (I) was prepared by itself, and the preparation method is shown in Example 1. Carbidopa, compound (I) and its composition were first sonicated with a small amount of DMSO, and then diluted with purified water to dissolve the gavage.
  • the cells were diluted with the RPM11640 medium of calf serum having a volume fraction of 0.1 to the tumor-containing 1 ⁇ 10 10 L -1 , and 0.2 mL was inoculated subcutaneously into the left anterior superior armpit of the nude mouse.
  • mice inoculated with liver cancer cell lines were randomly divided into normal control group, carbidopa group (10 mg ⁇ kg -1 ), compound (I) group (10 mg ⁇ kg -1 ), carbidopa and compound.
  • Composition group [5 mg ⁇ kg -1 carbidopa + 5 mg ⁇ kg -1 compound (I)], a total of 4 groups. All nude mice were intragastrically administered from the third day of inoculation of the hepatoma cell line, and the normal control group was intragastrically administered with the same amount of normal saline once every other day for 20 times. One week after stopping the drug, the neck was sacrificed: the tumor mass was completely removed, and the tumor mass was examined. Calculate the tumor inhibition rate according to the following formula:
  • Tumor inhibition rate (average tumor mass of the control group - mean tumor mass of the administration group) / mean tumor mass of the control group ⁇ 100%.
  • Two-step staining After the specimen was fixed by formaldehyde, serial sectioning was carried out for 3um: dewaxing of paraffin sections to water: high pressure boiled antigen for 3 min, H2O2 with volume fraction of 0.03 was incubated for 10 min at room temperature, and rabbit anti-human factor VIII was added dropwise.
  • Antibody 4 degrees Celsius, add 1:200 biotinylated goat anti-rabbit IgG, incubate for 30 min at 37 ° C, diaminobenzidine for 5 min, replace the primary antibody with 0.01 mol / L phosphate buffer as a blank control. Hemangiomas were used as positive controls.
  • the microvessel density is determined by the method of Clinica limportance of the determination of tumor angiogenesis in breast carcinoma: much more than a new prognostictool.
  • the microvascular staining was examined under low power field: the color of the neovascular endothelial stain was brownish yellow, and the field of view of the interstitial blood vessel of the cancer nest was taken. Under the 200-fold field of view, it was counted by three observers according to the double-blind method. 3 fields of view: observe single cells and cell bundles stained brown, and use this as a blood vessel: blood vessels and intraluminal red blood cells are not counted. If there are muscle vessels or lumens >50, they should be excluded. The average of the human counts was taken as the microvessel density of this example.
  • nude mice The quality of life and drug side effects of nude mice were evaluated by observation of the mental state, activity status, response to stimulation, body mass loss, appetite, urine and stool.
  • the subject group applied SPSS11.0 software for statistical processing.
  • the tumor inhibition rate and microvessel density were compared by F analysis. P ⁇ 0.05 was considered statistically significant.
  • nude mice survived in the carbidopa and compound (I) compositions: 5 survived in the carbidopa and compound (I) groups, and only 4 survived in the normal control group. All dead nude mice have been dissected to rule out the possibility of death due to improper experimental operation. The death of nude mice is caused by the deterioration of the tumor itself or the side effects of drugs.
  • Carbidopa and compound (I) composition group nude mice mental state, activity status, response to stimulation, body mass There was no obvious abnormality in the decrease, appetite and urine. The second was compound (I), the carbidopa group again, and the normal control group.
  • the carbidopa and the compound (I) composition group had a stronger tumor inhibition effect, which showed a decrease in microvessel density (P ⁇ 0.01); compared with the normal control group, the carbidopa group, the compound (I) The microvessel density decreased (P ⁇ 0.05); the formation of tumor blood vessels in nude mice was inhibited.
  • the results are shown in Table 1.
  • carbidopa, compound (I), carbidopa and compound (I) compositions can significantly inhibit the growth of liver cancer xenografts, and the inhibition of carbidopa and compound (I) compositions The effect is better than that of carbidopa and compound (I), and it can be developed into a drug for preventing and treating liver cancer.

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Abstract

提供一种卡比多巴的药物组合物及其治疗肝癌的医药用途，该卡比多巴的药物组合物中含有卡比多巴和一种从羌活的干燥根及根茎中分离得到的结构新颖的天然产物化合物(Ⅰ)，卡比多巴和该天然产物单独作用时，对肝癌具有治疗效果；二者联合作用时，对肝癌的治疗效果进一步提高，可以开发成治疗肝癌的药物。

Description

卡比多巴的药物组合物及其治疗肝癌的医药用途 技术领域
本发明属于生物医药领域，涉及卡比多巴的新用途，具体涉及一种卡比多巴的药物组合物及其治疗肝癌的医药用途。
背景技术
卡比多巴为(S)-α-甲基-α-肼基-3,4-二羟基苯丙酸一水合物。
卡比多巴具有较强的外周多巴脱羧酶抑制剂。不易透过血脑屏障，与左旋多巴合用时，仅抑制外周多巴脱羧酶的活性，减少多巴胺在外周组织的生成,减轻其外周不良反应，进而使进入中枢的左旋多巴增多，提高脑内多巴胺的浓度，增强左旋多巴的疗效，所以是左旋多巴的重要辅助用药。卡比多巴单用无效，临床上通常将卡比多巴与左旋多巴按1：10或1:4比例配伍制成复方制剂。
迄今为止，尚未见卡比多巴及其药物组合物与肝癌的相关性报道。
发明内容
本发明的目的在于提供一种卡比多巴的药物组合物，该药物组合物中含有卡比多巴和一种从羌活的干燥根及根茎中分离得到的结构新颖的天然产物，卡比多巴和该天然产物可以协同治疗肝癌。
本发明的上述目的是通过下面的技术方案得以实现的：
一种具有下述结构式的化合物(Ⅰ)，
Figure PCTCN2017097011-appb-000001
一种卡比多巴的药物组合物，包括卡比多巴、如上所述的化合物(Ⅰ)和药学上可以接 受的载体。
如上所述的化合物(Ⅰ)的制备方法，包含以下操作步骤：(a)将羌活的干燥根及根茎粉碎，用65～85％乙醇热回流提取，合并提取液，浓缩至无醇味，依次用石油醚、乙酸乙酯和水饱和的正丁醇萃取，分别得到石油醚萃取物、乙酸乙酯萃取物和正丁醇萃取物；(b)步骤(a)中正丁醇萃取物用大孔树脂除杂，先用6％乙醇洗脱8个柱体积，再用70％乙醇洗脱8个柱体积，收集70％洗脱液，减压浓缩得70％乙醇洗脱浓缩物；(c)步骤(b)中70％乙醇洗脱浓缩物用正相硅胶分离，依次用体积比为50:1、25:1、15:1和5:1的二氯甲烷-甲醇梯度洗脱得到4个组分；(d)步骤(c)中组分4用正相硅胶进一步分离，依次用体积比为10:1、5:1和2:1的二氯甲烷-甲醇梯度洗脱得到3个组分；(e)步骤(d)中组分2用十八烷基硅烷键合的反相硅胶分离，用体积百分浓度为75％的甲醇水溶液等度洗脱，收集8～14个柱体积洗脱液，洗脱液减压浓缩得到化合物(Ⅰ)。
进一步地，步骤(a)中用75％乙醇热回流提取，合并提取液。
进一步地，所述大孔树脂为AB-8型大孔吸附树脂。
如上所述的化合物(Ⅰ)在制备治疗肝癌的药物中的应用。
如上所述的卡比多巴的药物组合物在制备治疗肝癌的药物中的应用。
本发明的优点：
本发明提供的卡比多巴的药物组合物中含有卡比多巴和一种从羌活的干燥根及根茎中分离得到的结构新颖的天然产物，卡比多巴和该天然产物单独作用时，对肝癌具有治疗效果；二者联合作用时，对肝癌的治疗效果进一步提高，可以开发成治疗肝癌的药物。
具体实施方式
下面结合实施例进一步说明本发明的实质性内容，但并不以此限定本发明保护范围。尽管参照较佳实施例对本发明作了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的实质和范围。
实施例1：化合物(Ⅰ)分离制备及结构确证
分离方法：(a)将羌活的干燥根及根茎(3kg)粉碎，用75％乙醇热回流提取(20L×3次)，合并提取液，浓缩至无醇味(4L)，依次用石油醚(4L×3次)、乙酸乙酯(4L×3次)和水饱和的正丁醇(4L×3次)萃取，分别得到石油醚萃取物、乙酸乙酯萃取物和正丁醇萃取物；(b)步骤(a)中正丁醇萃取物用AB-8型大孔树脂除杂，先用6％乙醇洗脱8个柱体积，再用70％乙醇洗脱8个柱体积，收集70％洗脱液，减压浓缩得70％乙醇洗脱浓缩物；(c)步骤(b)中70％乙醇洗脱浓缩物用正相硅胶分离，依次用体积比为50:1(8个柱体积)、25:1(8个柱体积)、15:1(8个柱体积)和5:1(10个柱体积)的二氯甲烷-甲醇梯度洗脱得 到4个组分；(d)步骤(c)中组分4用正相硅胶进一步分离，依次用体积比为10:1(8个柱体积)、5:1(10个柱体积)和2:1(5个柱体积)的二氯甲烷-甲醇梯度洗脱得到3个组分；(e)步骤(d)中组分2用十八烷基硅烷键合的反相硅胶分离，用体积百分浓度为75％的甲醇水溶液等度洗脱，收集8～14个柱体积洗脱液，洗脱液减压浓缩得到化合物(Ⅰ)(375mg，HPLC归一化纯度大于98％)。
结构确证：无色结晶，HR-ESI-MS显示[M+H]+为m/z 377.2289，结合核磁特征可得分子式为C22H32O5，不饱和度为7。核磁共振氢谱数据δH(ppm，pyridine-d5，500MHz)：H-1(6.46，dd，J＝4.8，10.6Hz)，H-2a(3.39，m)，H-2b(3.10，m)，H-3(6.85，m)，H-5a(3.23，d，J＝15.6Hz)，H-5b(2.98，dd，J＝15.6，9.3Hz)，H-6(5.06，dd，J＝9.3，1.4Hz)，H-7(1.87，m)，H-8(1.46，m)，H-9a(2.13，dd，J＝16.1，2.6Hz)，H-9b(1.67，dd，J＝16.1，7.3Hz)，H-11(1.80，m)，H-12(0.93，d，J＝5.7Hz)，H-13(1.13，d，J＝5.7Hz)，H-14(1.19，d，J＝6.8Hz)，H-2’(2.82，m)，H-3’(5.26，m)，H-4’(1.23，d，J＝6.3Hz)，H-5’(1.09，d，J＝7.1Hz)，H-2”(1.97，s)；核磁共振碳谱数据δC(ppm，pyridine-d5，125MHz)：132.4(CH，1-C)，29.6(CH2，2-C)131.7(CH，3-C)，132.4(C，4-C)，28.3(CH2，5-C)，77.4(CH，6-C)，52.7(CH，7-C)，22.4(CH，8-C)，32.5(CH2，9-C)，158.6(C，10-C)，26.4(CH，11-C)，22.1(CH3，12-C)，23.8(CH3，13-C)，18.4(CH3，14-C)，171.2(C，15-C)，204.6(C，1’-C)，47.6(CH，2’-C)，72.4(CH，3’-C)，17.3(CH3，4’-C)，12.7(CH3，5’-C)，170.3(C，1”-C)，21.4(CH3，2”-C)。红外波谱中的1756cm-1吸收带与UV谱中的236nm吸收带表明该化合物含有α,β-不饱和内酯结构，红外波谱中的1715cm-1与1680cm-1吸收带表明结构中存在酮基与双键片段。13C-NMR、DEPT和HSQC谱中显示有22个碳信号，包括六个甲基，三个亚甲基，八个次甲基(两个连氧碳和两个烯烃碳)，以及五个季碳(三个羰基碳和两个烯烃碳)，以上功能结构再结合不饱和数表明该化合物为双环结构。1H-NMR谱结合HSQC谱显示五个甲基质子信号δH 0.93(3H，d，J＝5.7Hz)、1.13(3H，d，J＝5.7Hz)、1.19(3H，d，J＝6.8Hz)、1.23(3H，d，J＝6.3Hz)、1.09(3H，d，J＝7.1Hz)，一个乙酰基甲基质子信号δH 1.97(3H，s)，两个烯烃质子信号δH 6.46(1H，dd，J＝4.8，10.6Hz)与6.85(1H，m)，两个连氧次甲基质子信号δH 5.06(1H，dd，J＝9.3，1.4Hz)与5.26(1H，m)。1H-1H COSY谱中存在H-1/H2-2/H-3、H2-5/H-6/H-7/H-8/H2-9、H-7/H-11/H3-12、H-11/H3-13、以及H-8/H3-14相关信号，结合HMBC谱中显示的H2-2和H2-9与C-1，H2-2、H2-5和H-6与C-4相关信号可以构建吉马烷型倍半萜骨架。而1H-1H COSY谱中H3-5’/H-2’/H-3’/H3-4’相关信号与HMBC谱中H-2’与C-1’、C-3’和C-5’，H-3’与C-2’、C-4’和C-1”以及H3-2”与C-1”相关信号可以构建另一部分片段，并且这一部分结构中存在的 乙酰基通过氧原子与C-3’相连。另外HMBC谱中H-3、H2-5和H-6与C-15相关信号暗示C-6与C-15之间存在一个内酯环结构。NOESY谱中，假设H-8为β构型，那么H-8与H-10的相关信号表明H-10也为β构型，因此，O-3’-O-乙酰基-2’-甲基丁酮基应该为α构型。此外H-9b与H-8存在相关信号，所以H-9a与H-2a以及H-9a与H-5a相关信号可判断判断H-2a、H-5a、H-9a皆为α构型，同时H-2a与H-5a相关信号表明Δ3(4)双键为E构型。综合氢谱、碳谱、HMBC谱和NOESY谱，以及文献关于相关类型核磁数据，可基本确定该化合物如下所示，立体构型进一步通过ECD试验确定，理论值与实验值基本一致。碳原子编号如下：
Figure PCTCN2017097011-appb-000002
实施例2：对肝癌的治疗作用
1、材料和方法
1.1细胞及动物
实验室符合DB44/61-94清洁级标准。人肝癌细胞株Bel-7402来源于广州医学院微生物免疫学教研室。裸鼠(BALB/C-nu/nu)由南方医科大学实验动物中心提供，雄性，鼠龄4-6周，体质量20-25g，平均(23.28±1.62)饲养于SPF环境中。
1.2试剂与样品
卡比多巴购自中国药品生物制品检定所。化合物(Ⅰ)自制，制备方法见实施例1。卡比多巴、化合物(Ⅰ)及其组合物先用少量DMSO超声溶解，再用纯化水溶解稀释灌胃。
1.3小鼠分组及模型制备
移植瘤裸鼠动物模型复制：将冻存于液氮罐中肝癌细胞株Bel-7402取出，复苏，体外培养于RPM11640中，加入体积分数为0.1的小牛血清，青霉素10万单位/L，链霉素100mg/L，单层培养于CO2恒温孵育箱内，细胞为上皮样贴壁生长，每两三天传代1次，取对数生长期 细胞用于实验。用体积分数为0.1的小牛血清的RPM11640培养液稀释细胞至含瘤1×1010L-1，在裸鼠左前上肢腋窝皮下接种0.2mL。
分组：24只接种肝癌细胞株的裸鼠单纯随机分成正常对照组、卡比多巴组(10mg·kg-1)、化合物(Ⅰ)组(10mg·kg-1)、卡比多巴与化合物(Ⅰ)组合物组【5mg·kg-1卡比多巴+5mg·kg-1化合物(Ⅰ)】，共4组。所有裸鼠自接种肝癌细胞株第3天起开始灌胃给药，正常对照组为等量生理盐水灌胃，隔日1次，灌胃20次。停药1周后，脱颈处死：完整取出瘤块，检测瘤体质量。按以下公式计算肿瘤抑制率：
肿瘤抑制率＝(对照组平均瘤质量-给药组平均瘤质量)/对照组平均瘤质量×100％。
1.4微血管密度的测定
采用二步法染色：标本经甲醛固定后，进行3um连续切片：将石蜡切片脱蜡至水：高压煮沸抗原修复3min，体积分数为0.03的H2O2室温孵育10min，滴加兔抗人第Ⅷ因子相关抗体，4摄氏度过液，滴加1：200生物素标记的羊抗兔IgG，37摄氏度孵育30min，二氨基联苯胺显色5min，以0.01mol/L磷酸盐缓冲液代替一抗作为空白对照，以血管瘤为阳性对照。微血管密度的测定参照Clinica limportance of the determination of tumor angiogenesisin breast carcinoma：much more than a new prognostictool的方法。在低倍视野下检查微血管染色情况：以新生血管内皮染为棕黄色为阳性判定标准，取癌巢间质血管最多的视野，在200倍视野下，按双盲法由3位观察者分别计数3个视野：观察染成棕色的单个细胞和细胞丛，并以此作为一个血管：血管腔和腔内红细胞不作为计数，如有肌层的血管或管腔>50着者应排除，取3人计数平均值作为该例的微血管密度。
1.5裸鼠生存质量评价
通过裸鼠精神状态、活动状况、对刺激的反应、体质量下降幅度、食欲和尿、便6项指标的观察,评价裸鼠生存质量和药物毒副反应。
1.6统计学分析
由本课题组应用SPSS11.0软件进行统计学处理，肿瘤抑制率、微血管密度均值采用F分析进行组间比较，P<0.05为具有统计学意义。
2、实验结果
2.1对肝癌移植瘤裸鼠生存质量的影响
治疗方案结束时：卡比多巴与化合物(Ⅰ)组合物组6只裸鼠均存活：卡比多巴组和化合物(Ⅰ)组有5只存活，正常对照组只有4只存活。所有死亡裸鼠均已解剖排除了由于实验操作不当致死的可能，裸鼠死亡是由肿瘤本身恶化或药物毒副反应所致。
卡比多巴与化合物(Ⅰ)组合物组裸鼠精神状态、活动状况、对刺激的反应、体质量下 降幅度、食欲和尿便等未见明显异常：其次为化合物(Ⅰ)组，再次为卡比多巴组，最次为正常对照组
2.2对裸鼠肝癌移植瘤的抑制作用
与正常对照组比较，卡比多巴与化合物(Ⅰ)组合物组对肿瘤抑制作用明显较强，表现为平均瘤质量减少(P＜0.01)，肿瘤抑制率较高(P＜0.01)；与正常对照组比较，卡比多巴组、化合物(Ⅰ)组平均瘤质量减少(P＜0.05)，肿瘤抑制率较高(P＜0.05)；裸鼠肝癌移植瘤的形成得到抑制。结果见表1。
2.3对肿瘤血管形成的影响
与正常对照组比较，卡比多巴与化合物(Ⅰ)组合物组对肿瘤抑制作用明显较强，表现为微血管密度降低(P＜0.01)；与正常对照组比较，卡比多巴组、化合物(Ⅰ)组微血管密度降低(P＜0.05)；裸鼠肿瘤血管的形成得到抑制。结果见表1。
表1 对裸鼠肝癌移植瘤和肿瘤血管形成的影响
Figure PCTCN2017097011-appb-000003
上述结果表明，卡比多巴、化合物(Ⅰ)、卡比多巴与化合物(Ⅰ)组合物均能显著抑制肝癌移植瘤的生长，而且，卡比多巴与化合物(Ⅰ)组合物的抑制效果比卡比多巴、化合物(Ⅰ)单独抑制效果更好，可以开发成防治肝癌的药物。
上述实施例的作用在于说明本发明的实质性内容，但并不以此限定本发明的保护范围。本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的实质和保护范围。

Claims (7)

  1. 一种具有下述结构式的化合物(Ⅰ)，
    Figure PCTCN2017097011-appb-100001
  2. 一种卡比多巴的药物组合物，其特征在于：包括卡比多巴、如权利要求1所述的化合物(Ⅰ)和药学上可以接受的载体。
  3. 权利要求1所述的化合物(Ⅰ)的制备方法，其特征在于，包含以下操作步骤：(a)将羌活的干燥根及根茎粉碎，用65～85％乙醇热回流提取，合并提取液，浓缩至无醇味，依次用石油醚、乙酸乙酯和水饱和的正丁醇萃取，分别得到石油醚萃取物、乙酸乙酯萃取物和正丁醇萃取物；(b)步骤(a)中正丁醇萃取物用大孔树脂除杂，先用6％乙醇洗脱8个柱体积，再用70％乙醇洗脱8个柱体积，收集70％洗脱液，减压浓缩得70％乙醇洗脱浓缩物；(c)步骤(b)中70％乙醇洗脱浓缩物用正相硅胶分离，依次用体积比为50:1、25:1、15:1和5:1的二氯甲烷-甲醇梯度洗脱得到4个组分；(d)步骤(c)中组分4用正相硅胶进一步分离，依次用体积比为10:1、5:1和2:1的二氯甲烷-甲醇梯度洗脱得到3个组分；(e)步骤(d)中组分2用十八烷基硅烷键合的反相硅胶分离，用体积百分浓度为75％的甲醇水溶液等度洗脱，收集8～14个柱体积洗脱液，洗脱液减压浓缩得到化合物(Ⅰ)。
  4. 根据权利要求3所述的化合物(Ⅰ)的制备方法，其特征在于：步骤(a)中用75％乙醇热回流提取，合并提取液。
  5. 根据权利要求3所述的化合物(Ⅰ)的制备方法，其特征在于：所述大孔树脂为AB-8型大孔吸附树脂。
  6. 权利要求1所述的化合物(Ⅰ)在制备治疗肝癌的药物中的应用。
  7. 权利要求2所述的卡比多巴的药物组合物在制备治疗肝癌的药物中的应用。
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114890870A (zh) * 2022-06-07 2022-08-12 新疆维吾尔自治区中药民族药研究所 大苞荆芥提取物中松香烷二萜及其制备方法和应用
CN115974695A (zh) * 2022-12-13 2023-04-18 沈阳药科大学 珊瑚树中降vibsane二萜类化合物的制备方法及其应用

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105777672A (zh) * 2016-03-16 2016-07-20 郭根松 一种苯磺酸氨氯地平的药物组合物及其心室重塑作用
CN106083771A (zh) * 2016-06-13 2016-11-09 崔坤峰 卡比多巴的药物组合物及其治疗肝癌的医药用途
CN106045835A (zh) * 2016-06-23 2016-10-26 崔坤峰 盐酸苄丝肼的药物组合物及其降血糖的医药用途

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002020505A1 (fr) * 2000-05-12 2002-03-14 Institute Of Materia Medica, Chinese Academy Of Medical Sciences Medicaments permettant de prevenir ou de traiter la maladie d'alzheimer contenant un derive de dihydroagafuran
CN101190258A (zh) * 2007-05-30 2008-06-04 北京星昊医药股份有限公司 富含小白菊内酯的总倍半萜内酯提取物及其制备方法和应用
WO2012083953A1 (en) * 2010-12-22 2012-06-28 Leo Pharma A/S Ingenol-3-acylates iii and ingenol-3-carbamates
CN104628680A (zh) * 2015-01-23 2015-05-20 中国人民解放军第二军医大学 一种具有抗菌活性的倍半萜内酯类化合物及其应用
CN105777672A (zh) * 2016-03-16 2016-07-20 郭根松 一种苯磺酸氨氯地平的药物组合物及其心室重塑作用
CN105801527A (zh) * 2016-03-31 2016-07-27 河南省洛正制药厂 豨桐胶囊的质量控制方法
CN105820144A (zh) * 2016-04-23 2016-08-03 吴珺 一种头孢地尼的药物组合物及其医药用途
CN106083771A (zh) * 2016-06-13 2016-11-09 崔坤峰 卡比多巴的药物组合物及其治疗肝癌的医药用途

Family Cites Families (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105801590A (zh) * 2016-03-22 2016-07-27 李晨露 一种阿普唑仑的药物组合物及其抗炎镇痛作用
CN105732650A (zh) * 2016-03-22 2016-07-06 李晨露 一种醋氨苯砜的药物组合物及其在骨科方面的应用
CN105796570A (zh) * 2016-03-22 2016-07-27 李晨露 一种苄氟噻嗪的药物组合物及其对肝癌的治疗作用
CN105801664A (zh) * 2016-03-22 2016-07-27 李晨露 一种盐酸氮卓斯汀的药物组合物及其医药用途
CN105753879A (zh) * 2016-03-22 2016-07-13 李晨露 一种盐酸阿扑吗啡的药物组合物及其心室重塑作用
CN105712957A (zh) * 2016-03-22 2016-06-29 李晨露 一种阿立哌唑的药物组合物及其防治糖尿病的用途
CN105837656A (zh) * 2016-04-01 2016-08-10 胡文杰 一种环阿尔廷型三萜烯及其医药用途
CN105837532A (zh) * 2016-04-20 2016-08-10 宋晓梅 一种苯溴马隆的药物组合物及其在生物医药中的应用
CN105820053A (zh) * 2016-04-23 2016-08-03 黄亦琼 头孢氨苄的药物组合物及其在生物医药中的应用
CN105669457A (zh) * 2016-04-23 2016-06-15 吴珺 氯噻酮的药物组合物及其在生物医药中的应用
CN105777695A (zh) * 2016-04-28 2016-07-20 薛丽云 盐酸依米丁的药物组合物及其在生物医药中的应用
CN105777854A (zh) * 2016-04-28 2016-07-20 周俭 硫酸依替米星的药物组合物及其在生物医药中的应用

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002020505A1 (fr) * 2000-05-12 2002-03-14 Institute Of Materia Medica, Chinese Academy Of Medical Sciences Medicaments permettant de prevenir ou de traiter la maladie d'alzheimer contenant un derive de dihydroagafuran
CN101190258A (zh) * 2007-05-30 2008-06-04 北京星昊医药股份有限公司 富含小白菊内酯的总倍半萜内酯提取物及其制备方法和应用
WO2012083953A1 (en) * 2010-12-22 2012-06-28 Leo Pharma A/S Ingenol-3-acylates iii and ingenol-3-carbamates
CN104628680A (zh) * 2015-01-23 2015-05-20 中国人民解放军第二军医大学 一种具有抗菌活性的倍半萜内酯类化合物及其应用
CN105777672A (zh) * 2016-03-16 2016-07-20 郭根松 一种苯磺酸氨氯地平的药物组合物及其心室重塑作用
CN105801527A (zh) * 2016-03-31 2016-07-27 河南省洛正制药厂 豨桐胶囊的质量控制方法
CN105820144A (zh) * 2016-04-23 2016-08-03 吴珺 一种头孢地尼的药物组合物及其医药用途
CN106083771A (zh) * 2016-06-13 2016-11-09 崔坤峰 卡比多巴的药物组合物及其治疗肝癌的医药用途

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LAI, YONGJI ET AL.: "Scapiformolactones A-I: Germacrane Sesquiterpenoids with an Unusual DELTA3-15, 6-Lactone Moiety from Salvia Scapiformis", PHYTOCHEMISTRY, vol. 96, 1 November 2013 (2013-11-01), pages 378 - 388 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114890870A (zh) * 2022-06-07 2022-08-12 新疆维吾尔自治区中药民族药研究所 大苞荆芥提取物中松香烷二萜及其制备方法和应用
CN115974695A (zh) * 2022-12-13 2023-04-18 沈阳药科大学 珊瑚树中降vibsane二萜类化合物的制备方法及其应用
CN115974695B (zh) * 2022-12-13 2024-05-03 沈阳药科大学 珊瑚树中降vibsane二萜类化合物的制备方法及其应用

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