WO2017220042A2 - 氨力农的药物组合物及其在高血压治疗中的应用 - Google Patents

氨力农的药物组合物及其在高血压治疗中的应用 Download PDF

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WO2017220042A2
WO2017220042A2 PCT/CN2017/097767 CN2017097767W WO2017220042A2 WO 2017220042 A2 WO2017220042 A2 WO 2017220042A2 CN 2017097767 W CN2017097767 W CN 2017097767W WO 2017220042 A2 WO2017220042 A2 WO 2017220042A2
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amrinone
compound
ethanol
eluted
pharmaceutical composition
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WO2017220042A3 (zh
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崔坤峰
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赵吉永
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    • 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
    • A61K31/365—Lactones
    • 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
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/444—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring heteroatom, e.g. amrinone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • 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)
    • A61K36/88—Liliopsida (monocotyledons)

Definitions

  • the invention belongs to the field of biomedicine and relates to a new use of amrinone, in particular to a pharmaceutical composition of amrinone and its application in the treatment of hypertension.
  • Ammonium also known as aminopyridinone, ampicillin, ampicillin, aminopyridone, comonone, annoxol, cardiotonin, lactate ketone, is a new type of non- ⁇ , non
  • the catecholamines cardiotonic drugs are effective both orally and intravenously, and have positive inotropic effects and vasodilating effects, which can increase myocardial contractility, increase cardiac output, reduce pre- and post-loading of the heart, and reduce left ventricular filling pressure. Improve left ventricular function and increase cardiac index, but have no significant effect on mean arterial pressure and heart rate.
  • Renal vascular hypertension is secondary to many cardiovascular diseases, such as atherosclerosis, renal artery stenosis, etc., and its incidence increases with age.
  • the renin-angiotensin system is closely related to the development of this type of hypertension.
  • the renin-angiotensin system is composed of renin, angiotensinogen, angiotensin I, angiotensin II, and angiotensin converting enzyme.
  • angiotensin II exerts physiological and pathological effects on the cardiovascular system by stimulating angiotensin II type I receptor. After renal artery stenosis, RAS is activated and angiotensin II is increased.
  • angiotensin II activates the NADH/ADPH oxidative pathway, promotes the production of superoxide anion, oxidatively inactivates the vasodilator EDRF/NO, thereby attenuating the vasodilator effect, leading to a further increase in peripheral resistance.
  • angiotensin II in plasma plays an important role in the early stage of hypertension, and in the middle and late stages of hypertension, local RAS activation may play a more important role.
  • elevated blood pressure is not the only factor leading to cardiovascular remodeling.
  • Angiotensin II also has the property of growth factors, which is induced by acting on cell membrane receptors and activating intracellular signal transduction kinases (such as MAPK).
  • MAPK intracellular signal transduction kinases
  • Overexpression of the proto-oncogene c-fos and c-myc genes promotes the proliferation of vascular smooth muscle cells and changes in the cell matrix, leading to the rearrangement of vascular wall cells, causing the centripetal remodeling of the vessel wall, and the decrease in vessel diameter.
  • Vascular remodeling has different vascular wall structures in different hypertensive rats.
  • the vascular remodeling index is higher (90%), and the vessel wall is not thickened.
  • Vascular smooth muscle cells have less proliferation or hypertrophy, while lumen diameter and area are reduced, tube wall thickness to tube diameter ratio and tube wall area and lumen area ratio increase; in secondary hypertensive rats, vascular remodeling index Lower (75%), thickened blood vessel wall, proliferation or hypertrophy of vascular smooth muscle cells, diameter and area of lumen reduced, ratio of wall thickness to diameter, wall area and lumen area ratio increase.
  • the object of the present invention is to provide a pharmaceutical composition of amrinone containing amrinone and a novel natural product isolated from herbs, which can be synergistically treated with amrinone and the natural product. Renal vascular hypertension.
  • a pharmaceutical composition of amrinone comprising amrinone, compound (I) as described above, and a pharmaceutically acceptable carrier.
  • the preparation method of the compound (I) as described above comprises the following steps: (a) pulverizing the dried dried rhizome, extracting it by hot reflux with 75-85% ethanol, combining the extracts, concentrating to an alcohol-free taste, and sequentially using petroleum.
  • step (b) n-butanol extract in step (a) is removed by macroporous resin First, elute 8 column volumes with 25% ethanol, then elute 12 column volumes with 70% ethanol, collect 70% eluent, concentrate under reduced pressure to obtain 70% ethanol eluting concentrate; (c) step (b) The 70% ethanol eluting concentrate was separated by normal phase silica gel, and successively eluted with a gradient of methylene chloride-methanol of 85:1, 45:1, 25:1 and 15:1 to obtain 4 components; (d) In step (c), component 4 is further separated by normal phase silica gel, and sequentially eluted with a dichloromethane-methanol gradient of 20:1, 15:1 and 1:1 by volume to obtain three components; e) In step (d), component
  • step (a) is carried out by hot reflux extraction with 80% ethanol, and the extracts are combined.
  • the macroporous resin is a D101 type macroporous adsorption resin.
  • the pharmaceutical composition of the amrinone provided by the invention comprises amrinone and a novel natural product separated from the dried rhizome of the dried stem.
  • the amrinone and the natural product act alone, the renal blood vessel is high. Blood pressure has a therapeutic effect; When the two are combined, the therapeutic effect on renal vascular hypertension is further improved, and a drug for treating renal vascular hypertension can be developed.
  • the present invention has outstanding substantial features and significant advances over the prior art.
  • the concentrate was concentrated by pressure to obtain 70% ethanol;
  • 70% ethanol eluted concentrate in step (b) was separated by normal phase silica gel in a volume ratio of 85:1 (10 column volumes), 45:1. (8 column volumes), 25:1 (10 column volumes) and 15:1 (8 column volumes) of dichloromethane-methanol gradient elution to give 4 components;
  • Group (c) Part 4 was further separated by normal phase silica gel in a volume ratio of 20:1 (10 column volumes), 15:1 (8 column volumes) and 1:1 (6 column volumes) of dichloromethane-methanol gradient.
  • 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 an ⁇ , ⁇ -unsaturated lactone structure
  • the 1715 cm -1 and 1624 cm -1 absorption bands in the infrared spectrum indicate the presence of a ketone group in the structure.
  • 13 C-NMR, DEPT and HSQC spectra show 20 carbon signals, including five methyl groups, two methylene groups, nine methine groups (one oxygenated carbon and four olefin carbons), and four seasons.
  • the -1 and H-2, H 2 -5 and H-6 and C-4 related signals can construct a gemasane type sesquiterpene skeleton.
  • the H-3' and C-2' and C-4' related signals in the HMBC spectrum can construct 2'-methyl-2'-butenone-based fragments, while the HMBC spectrum H-10 and C-1' related signals indicate This fragment is attached to the position of the geminane-type sesquiterpene skeleton.
  • the HMBC spectra H-3, H 2 -5 and H-6 and C-15 related signals suggest the presence of a lactone ring structure between C-6 and C-15.
  • the correlation signal between H-8 and H-10 indicates that H-10 is also in the ⁇ configuration. Therefore, the 2'-methyl-2'-butenone group is Alpha configuration.
  • Ammonia is 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.
  • Angiotensin II and renin activity assay kits were purchased from the Institute of Radioimmunology of the PLA General Hospital; the rest were commercially available analytically pure.
  • High-speed centrifuge made in France
  • electrophoresis system produced by Beijing Liuyi Instrument
  • rat tail pressure tester HX-1 type
  • physiological record two-way instrument is produced by Chengdu Instrument Factory.
  • Rats were divided into 6 groups: normal control group, sham operation group, model control group, amrinone group (20 mg ⁇ kg -1 ), compound (I) group (20 mg ⁇ kg -1 ), amrinone and Compound (I) composition group [10 mg ⁇ kg -1 amrinone + 10 mg ⁇ kg -1 compound (I)].
  • the carotid cannula was connected to the baroreceptor and the mean arterial pressure was measured by a physiological two-channel instrument. Then, 4 ml of blood was taken and placed in a test tube containing aprotinin, centrifuged at 4° C. for 3000 r ⁇ min -1 for 10 min, and stored for plasma. Stand by at -20 °C. Frozen plasma samples were quickly thawed in a flowing cold water bath and sampled into an ice water bath. The test tube group number is used, and the sample loading step is performed by referring to the kit program description table. The radioactive label was 125 IAng II.
  • sensitivity is 10 ng ⁇ L -1 ; the curve range is 25-800 ng ⁇ L -1 ; precision is: intra-assay variation (CVw) ⁇ 5%, inter-assay variation (CVb) ⁇ 10%.
  • angiotensin I It is determined by the rate at which angiotensin I is produced per unit time. Take two copies of plasma, one directly reacted with antisera, determine the concentration of angiotensin I (control tube), and the other is incubated at 37 ° C for a certain period of time, and then react with antiserum to determine the angiotensin I Concentration (measurement tube). The concentration of the measuring tube minus the concentration of the control tube, and then the incubation time, is the rate of production of angiotensin I per unit time, which is renin activity.
  • the data is expressed as x ⁇ s.
  • Data analysis was performed using analysis of variance and Newman-Keuls multiple comparison t test.
  • the plasma angiotensin II level in the model control group was significantly increased (P ⁇ 0.01). Compared with the model control group, the plasma angiotensin II level was significantly higher in the amrinone and compound (I) composition groups. The decrease was (P ⁇ 0.01). Compared with the model control group, the plasma angiotensin II level was decreased in the amrinone group and the compound (I) group (P ⁇ 0.05).
  • the plasma renin activity of the model control group was increased in the hypertension group (P ⁇ 0.05).
  • the plasma renin activity of the amoxicillin and compound (I) combination group was compared.
  • Table 1 shows changes in plasma angiotensin II and plasma renin activity in hypertensive rat models
  • Increased renin-angiotensin system (RAS) activity is one of the important mechanisms of elevated blood pressure, and angiotensin II (Ang II) is directly related to elevated blood pressure.
  • RAS angiotensin II
  • Ang II angiotensin II
  • the angiotensin II of the circulatory system plays a role by promoting the secretion of aldosterone, affecting the balance of water and electrolytes, and increasing vascular tone.
  • angiotensin II with increased local tissue (heart, brain, blood vessels) and other growth factors stimulate centripetal vascular remodeling in the cardiovascular wall is the pathological basis for maintaining blood pressure.
  • Inhibition of angiotensin II concentration and renin activity in a hypertensive rat model can control the further development of hypertension.
  • the amrinone and compound (I) composition provided by the invention can significantly reduce angiotensin II concentration and renin activity in a hypertensive rat model, and the composition is superior to amrinone or compound (I) alone. Effect.
  • the amrinone and compound (I) compositions can be developed into drugs for the treatment of renal vascular hypertension.

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Abstract

本发明公开了氨力农的药物组合物及其在高血压治疗中的应用，本发明提供的氨力农的药物组合物中含有氨力农和一种从射干的干燥根茎中分离得到的结构新颖的天然产物化合物(Ⅰ)，氨力农和该天然产物化合物(Ⅰ)单独作用时，对肾血管性高血压具有治疗作用；二者联合作用时，对肾血管性高血压的治疗效果进一步提高，可以开发成治疗肾血管性高血压的药物，与现有技术相比具有突出的实质性特点和显著的进步。

Description

氨力农的药物组合物及其在高血压治疗中的应用 技术领域
本发明属于生物医药领域，涉及氨力农的新用途，具体涉及氨力农的药物组合物及其在高血压治疗中的应用。
背景技术
氨力农又名氨基双吡酮、氨联吡啶酮、氨双吡酮、氨吡酮、安联酮、安诺可、强心隆、乳酸氨利酮，是一种新型的非甙、非儿茶酚胺类强心药，口服和静注均有效，兼有正性肌力作用和血管扩张作用，能增加心肌收缩力，增加心排血量，降低心脏前、后负荷，降低左心室充盈压，改善左心室功能，增加心脏指数，但对平均动脉压和心率无明显影响。
肾血管性高血压继发于许多心血管疾病，如动脉粥样硬化、肾动脉狭窄等，其发生率随着年龄的增长而增加。肾素-血管紧张素系统与此型高血压的发生发展密切相关。肾素-血管紧张素系统由肾素、血管紧张素原、血管紧张素Ⅰ、血管紧张素Ⅱ及血管紧张素转化酶构成。其中血管紧张素Ⅱ通过激动血管紧张素Ⅱ的Ⅰ型受体而对心血管系统产生生理和病理作用。肾动脉狭窄后，RAS被激活，血管紧张素Ⅱ生成增加，它除激活血管紧张素受体强烈收缩血管外，还通过刺激肾上腺髓质释放儿茶酚胺，以及刺激其它缩血管物质的合成与释放(如诱导内皮素合成)来增强缩血管效应，致使外周阻力增加，血压升高。此外，血管紧张素Ⅱ激活组织NADH/ADPH氧化途径，促进超氧阴离子产生，氧化灭活舒血管因子EDRF/NO，进而减弱舒血管效应，导致外周阻力进一步升高。近年研究认为，血浆中血管紧张素Ⅱ在高血压早期起重要作用，而在高血压中、晚期，组织局部RAS激活可能起的作用更大。目前观点认为，血压升高并不是导致心血管重构的唯一因素，血管紧张素Ⅱ还具有生长因子的特性，它通过作用于细胞膜受体，激活细胞内信号转导激酶(如MAPK)来诱导原癌基因c-fos和c-myc基因的过量表达，促进血管平滑肌细胞的增殖和细胞基质的改变，导致血管壁细胞的重新排列而引起血管壁向心性重构，血管管径变小阻力增加，进一步促进高血压的发生发展血管重构在不同的高血压血管壁结构表现不同，在遗传性自发性高血压大鼠，血管重构指数较高(90％)，血管壁并不增厚，血管平滑肌细胞有较少增殖或肥大，而管腔直径和面积缩小，管壁厚度与管径之比和管壁面积和管腔面积比值增加；在继发性高血压大鼠，血管重构指数较低(75％)，血管壁增厚，血管平滑肌细胞增殖或肥大，管腔直径和面积缩小，管壁厚度与管径之比和管壁面积和管腔面积比值增加。
迄今为止，尚未见氨力农的药物组合物与肾血管性高血压的相关性报道。
发明内容
本发明的目的在于提供一种氨力农的药物组合物，该药物组合物中含有氨力农和一种从草本中分离得到的结构新颖的天然产物，氨力农和该天然产物可以协同治疗肾血管性高血压。
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 PCTCN2017097767-appb-000001
一种氨力农的药物组合物，包括氨力农、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)将射干的干燥根茎粉碎，用75～85％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)中正丁醇取物用大孔树脂除杂，先用25％乙醇洗脱8个柱体积，再用70％乙醇洗脱12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85:1、45:1、25:1和15:1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20:1、15:1和1:1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72％的甲醇水溶液等度洗脱，收集10～16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)用80％乙醇热回流提取，合并提取液。
进一步地，所述大孔树脂为D101型大孔吸附树脂。
如上所述的化合物(Ⅰ)在制备治疗肾血管性高血压的药物中的应用。
如上所述的氨力农的药物组合物在制备治疗肾血管性高血压的药物中的应用。
本发明的优点：
本发明提供的氨力农的药物组合物中含有氨力农和一种从射干的干燥根茎中分离得到的结构新颖的天然产物，氨力农和该天然产物单独作用时，对肾血管性高血压具有治疗作用； 二者联合作用时，对肾血管性高血压的治疗效果进一步提高，可以开发成治疗肾血管性高血压的药物。本发明与现有技术相比具有突出的实质性特点和显著的进步。
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)将射干的干燥根茎(2kg)粉碎，用80％乙醇热回流提取(15L×3次)，合并提取液，浓缩至无醇味(3L)，依次用石油醚(3L×3次)、乙酸乙酯(3L×3次)和水饱和的正丁醇(3L×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)中乙酸乙酯萃取物用D101型大孔树脂除杂，先用25％乙醇洗脱8个柱体积，再用70％乙醇洗脱10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85:1(10个柱体积)、45:1(8个柱体积)、25:1(10个柱体积)和15:1(8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20:1(10个柱体积)、15:1(8个柱体积)和1:1(6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72％的甲醇水溶液等度洗脱，收集10～16个柱体积洗脱液，洗脱液减压浓缩得到化合物(Ⅰ)(385mg，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 317.2079，结合核磁特征可得分子式为C20H28O3，不饱和度为7。核磁共振氢谱数据δH(ppm，pyridine-d5，500MHz)：H-1(6.04，dd，J＝6.7，12.1Hz)，H-2(6.52，dd，J＝12.1，9.5Hz)，H-3(7.13，d，J＝9.5Hz)，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(1.53，m)，H-9a(2.02，m)，H-9b(1.61，m)，H-10(1.96，m)，H-11(1.77，m)，H-12(0.85，d，J＝5.9Hz)，H-13(1.13，d，J＝6.8Hz)，H-14(1.20，d，J＝6.8Hz)，H-3’(7.01，m)，H-4’(1.65，d，J＝7.3Hz)，H-5’(1.84，s)；核磁共振碳谱数据δC(ppm，pyridine-d5，125MHz)：133.2(CH，1-C)，127.4(CH，2-C)135.3(CH，3-C)，122.5(C，4-C)，28.3(CH2，5-C)，77.4(CH，6-C)，52.7(CH，7-C)，27.7(CH，8-C)，35.2(CH2，9-C)，42.8(CH，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)，200.1(C，1’-C)，129.3(C，2’-C)，138.2(CH，3’-C)，14.5(CH3，4’-C)，13.1(CH3，5’-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1624cm-1吸收带表明结构中存在酮基与α,β-不饱共轭体系。13C-NMR、DEPT和HSQC谱中显示有20个碳信号，包括五个甲基，两个亚甲基，九个次甲基(一个连氧碳和四个烯烃碳)，以及四个季碳(两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 0.85(3H，d，J＝5.9Hz)、1.13(3H，d，J＝6.8Hz)、1.20(3H，d，J＝6.8Hz)、1.65(3H，d，J＝7.3Hz)、1.84(3H，s)，两组亚甲基质子信号δH 3.23(1H，d，J＝15.6Hz)与2.98(1H，dd，J＝15.6，9.3Hz)、2.02(1H，m)与1.61(1H，m)，三个共轭烯烃质子信号δH 6.04(1H，dd，J＝6.7，12.1Hz)、6.52(1H，dd，J＝12.1，9.5Hz)与7.13(1H，d，J＝9.5Hz)，一个烯烃质子信号δH 7.01(1H，m)，一个连氧次甲基质子信号δH 5.06(1H，dd，J＝9.3，1.4Hz)，四个次甲基质子信号δH 1.87(1H，m)、1.53(1H，m)、1.96(1H，m)、1.77(1H，m)。1H-1H COSY谱中存在H-1/H-2/H-3、H2-5/H-6/H-7/H-8/H2-9/H-10、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H-2、H2-9和H-10与C-1以及H-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。HMBC谱中H-3’与C-2’和C-4’相关信号可以构建2’-甲基-2’-丁烯酮基片段，同时HMBC谱H-10与C-1’相关信号表明这一片段与吉马烷型倍半萜骨架连接的位置。另外HMBC谱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，2’-甲基-2’-丁烯酮基为α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。该化合物化学式及碳原子编号如下：
Figure PCTCN2017097767-appb-000002
实施例2：药理作用
1、材料与方法
1.1动物
SD大鼠，体重200～250g，购于中南大学湘雅医学院动物部。实验前在本实验室喂养1wk，测定其基础血压。
1.2试剂与样品
氨力农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。血管紧张素Ⅱ、肾素活性检测试剂盒购于解放军总院放射免疫研究所；其余均为市售分析纯。
1.3仪器
高速离心机(法国产)；电泳系统(北京六一仪器产)；鼠尾测压仪(HX-1型)购于中南大学湘雅医学院；生理记录二道仪由成都仪器厂生产。
1.4大鼠分组及模型制备
大鼠腹腔注射戊巴比妥钠(60mg·kg-1)麻醉后，从腹部左侧切口，分离左侧肾动脉，用直径为0.3mm的银夹夹住肾动脉使其狭窄后，放回腹腔，脏器恢复原位，关闭腹腔。假手术组只实行相同的手术程序，而不结扎肾动脉。待术后3d，动物恢复后，从尾动脉测定收缩压，连续观察血压的变化。高血压模型以术后4wk，选取血压维持在160mmHg(21.3kPa)以上动物用于实验。大鼠分为6组，分别为正常对照组、假手术组、模型对照组、氨力农组(20mg·kg-1)、化合物(Ⅰ)组(20mg·kg-1)、氨力农与化合物(Ⅰ)组合物组【10mg·kg-1氨力农+10mg·kg-1化合物(Ⅰ)】。
1.5血管紧张素Ⅱ浓度测定
实验结束时，颈动脉插管连接压力感受器于生理二道仪测定平均动脉压，然后取血4ml置于含有抑肽酶的试管中，于4℃离心3000r·min-1，10min，取血浆保存于-20℃备用。取冰冻的血浆标本在流动的冷水浴中快速融化，取样放入冰水浴中。试管分组编号，加样步骤参照试剂盒程序说明表进行。放射性标记物为125IAngⅡ。技术特性指标为：灵敏度为10ng·L-1；曲线范围为25～800ng·L-1；精密度为：批内变异(CVw)<5％，批间变异(CVb)<10％。
1.6肾素活性测定
通过单位时间内生成血管紧张素Ⅰ的速率来确定。取双份血浆，一份直接与抗血清反应，测定其血管紧张素Ⅰ的浓度(对照管)，另一份在37℃温育一定时间，再与抗血清反应，测定其血管紧张素Ⅰ的浓度(测定管)。测定管的浓度减去对照管的浓度，再除于被温育时间，则为单位时间内血管紧张素Ⅰ的产生速率，即为肾素活性。
1.7统计学方法
数据资料用x±s表示。数据分析采用方差分析及Newman-Keuls多重比较t检验。
2、实验结果
2.1对高血压大鼠模型血浆血管紧张素Ⅱ的变化
与正常对照组比较，模型对照组大鼠血浆血管紧张素Ⅱ水平显著升高(P＜0.01)；与模型对照组比较，氨力农与化合物(Ⅰ)组合物组血浆血管紧张素Ⅱ水平显著降低(P<0.01)；与模型对照组比较，氨力农组、化合物(Ⅰ)组血浆血管紧张素Ⅱ水平降低(P＜0.05)。
结果见表1。
2.2对高血压大鼠模型血浆肾素活性的变化
与正常对照组比较，模型对照组大鼠血浆肾素活性在高血压组升高(P<0.05)；与模型对照组比较，氨力农与化合物(Ⅰ)组合物组大鼠血浆肾素活性明显降低(P<0.01)；与模型对照组比较，氨力农组、化合物(Ⅰ)组血浆肾素活性降低(P<0.05)。
结果见表1。
表1对高血压大鼠模型血浆血管紧张素Ⅱ和血浆肾素活性的变化
组别 血管紧张素Ⅱ浓度(ng·L-1) 肾素活性(ng·L-1)
正常对照组 74 12
假手术组 70 13
模型对照组 120 26
氨力农组 85 17
化合物(Ⅰ)组 82 18
氨力农与化合物(Ⅰ)组合物组 70 12
肾素-血管紧张素系统(RAS)活性增高是造成血压升高的重要机制之一，其中血管紧张素Ⅱ(AngⅡ)与血压升高有直接关系。在肾动脉狭窄后，肾血流量减少促使肾素释放增多，血浆中血管紧张素原生成血管紧张素Ⅱ增多。循环系统的血管紧张素Ⅱ通过促进醛固酮的分泌影响水电解质平衡，增加血管张力等因素而发挥作用。随着血压的持续长时间升高，局部组织(心、脑、血管)增多的血管紧张素Ⅱ与其它生长因子刺激心血管壁发生向心性血管重构是维持血压升高的病理基础。抑制高血压大鼠模型的血管紧张素Ⅱ浓度和肾素活性可以控制高血压病情的进一步发展。本发明提供的氨力农与化合物(Ⅰ)组合物可以显著降低高血压大鼠模型的血管紧张素Ⅱ浓度和肾素活性，且组合物的效果优于氨力农或化合物(Ⅰ)单独的作用效果。氨力农与化合物(Ⅰ)组合物可以开发成治疗肾血管性高血压的药物。
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 PCTCN2017097767-appb-100001
  2. 一种氨力农的药物组合物，其特征在于：包括氨力农、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)将射干的干燥根茎粉碎，用75～85％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)中正丁醇取物用大孔树脂除杂，先用25％乙醇洗脱8个柱体积，再用70％乙醇洗脱12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85:1、45:1、25:1和15:1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20:1、15:1和1:1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72％的甲醇水溶液等度洗脱，收集10～16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)用80％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D101型大孔吸附树脂。
  6. 权利要求1所述的化合物(Ⅰ)在制备治疗肾血管性高血压的药物中的应用。
  7. 权利要求2所述的氨力农的药物组合物在制备治疗肾血管性高血压的药物中的应用。
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