WO2017190421A1 - 一种新型罗汉果醇衍生物单体 - Google Patents

一种新型罗汉果醇衍生物单体 Download PDF

Info

Publication number
WO2017190421A1
WO2017190421A1 PCT/CN2016/089207 CN2016089207W WO2017190421A1 WO 2017190421 A1 WO2017190421 A1 WO 2017190421A1 CN 2016089207 W CN2016089207 W CN 2016089207W WO 2017190421 A1 WO2017190421 A1 WO 2017190421A1
Authority
WO
WIPO (PCT)
Prior art keywords
composition
tumors
cancer
dosage form
composition according
Prior art date
Application number
PCT/CN2016/089207
Other languages
English (en)
French (fr)
Inventor
余佩华
邓跃敏
钟牧源
Original Assignee
深圳以诺生物制药有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 深圳以诺生物制药有限公司 filed Critical 深圳以诺生物制药有限公司
Publication of WO2017190421A1 publication Critical patent/WO2017190421A1/zh

Links

Images

Classifications

    • 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
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/575—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J9/00—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

  • the invention relates to the field of medicine, in particular to a novel mogroside derivative monomer.
  • Cancer is the second largest disease in the world that afflicts human life. Mortality is second only to cardiovascular and cerebrovascular diseases and is one of the most important causes of human death.
  • IARC International Agency for Research on Cancer
  • the latest edition of the World Cancer Report led by the International Agency for Research on Cancer (IARC), the official cancer organization of the WHO organization, predicts that global cancer cases will proliferate rapidly, from 14 million in 2012 to 19 million in 2025 will reach 24 million by 2035.
  • the report also showed that nearly half of the world's new cancer cases occurred in Asia in 2012, most of which were in China, and China's new cancer cases ranked first.
  • China China added 3.07 million cancer patients and caused about 2.2 million deaths, accounting for 21.9% and 26.8% of the global total, respectively.
  • the WHO's data is slightly lower than China's own statistics. According to the 2012 data released by the National Cancer Registry, about 3.5 million new cancer cases are reported each year in China, and about 2.5 million people die.
  • the backbone of cancer drug therapy has poor effect on slow-proliferating solid tumors, small drug selectivity, and many toxic side effects and serious defects have become important limiting factors in clinical treatment.
  • the fourth mode after surgery, radiotherapy and chemotherapy is the biological treatment of tumors, which mainly regulates the body's own biological response through the action of tumor host defense mechanisms or biological agents, thereby inhibiting or eliminating tumors; Too many toxic side effects, but due to strict technical requirements and complicated processes, the price is high, and many cancer patients and their families are unbearable, affecting their popularity in the field of cancer treatment.
  • Natural anticancer drugs play an important role in inhibiting or killing tumor cells, adjusting immune function, improving symptoms and characteristics, and reducing the side effects of radiotherapy and chemotherapy. As a result, new natural plant therapies will become the fifth mode after surgery, radiation therapy, chemotherapy and biological therapy.
  • Mangosteen is a precious medicinal material belonging to the perennial vine of Cucurbitaceae. It is cool and sweet.
  • the non-sugar sweet saponin component of Luo Han Guo extract also known as Luo Han Guo total saponin, has been isolated and identified in the total saponin of Siraitia grosveni according to the number of saponin-linked glycosyl groups and the position of the saponin.
  • mogrosides II, III, IV, V, VI mogroside II, III, IV, V, VI
  • mogroside V-Mogroside The sweetness of V(1A) (shown in Figure 1) is 256-344 times that of sucrose, and the sweetness of mogroside IV-Mogroside IV (1B) (shown in Figure 1) is 126 times that of sucrose.
  • Mogrosides are two glucoside side chains consisting of four or fewer glucose units linked by a ⁇ -glycosidic bond to C-3 and C-24 of the sapogenin, Lo Hanol Mogrol (2) (as shown in Figure 2).
  • the linkage between the side chain glucose is ⁇ -1,6 and --l, 2 glycosidic bond.
  • mogroside can increase the utilization of glucose and fat and increase the sensitivity of insulin, but the active ingredients and mechanism of action of mogroside in hypoglycemic effect are not clear.
  • the highest content of mogroside V (1A) in Luo Han Guo was tested and found that it did not have AMPK.
  • the technical problem to be solved by the present invention is to provide a novel mogroside derivative monomer (4), which can be used as a pharmaceutical preparation or a health care product for tumors and/or cancers. Treatment and / or prevention.
  • the first aspect of the present invention provides a roentyl alcohol derivative monomer (4) having the following structure:
  • a second aspect of the invention provides a composition comprising the following structure
  • the composition is a pharmaceutical formulation further comprising a pharmaceutically acceptable diluent, carrier, excipient, adjuvant or vehicle.
  • the pharmaceutical preparation is in the form of an oral dosage form, an injection dosage form or a topical dosage form.
  • the oral dosage form is a tablet, a powder, a suspension, an emulsion, a capsule, a granule, a sugar-coated tablet, a pill, a liquid, an elixir, a syrup or a lemonade.
  • the injectable dosage form comprises a liquid, suspension or solution.
  • the topical dosage form comprises an ointment, a solid, a suspension, a liquid, an elixir, a powder, a paste, a suppository, an aerosol, a poultice, a spread, a lotion, an enema or an emulsion. .
  • the composition is for treating and/or preventing a tumor and/or cancer
  • the tumor and/or cancer described therein is selected from the group consisting of:
  • Malignant tumors including bladder, breast, colon, kidney, liver, lung, head and neck cancer, esophageal cancer, gallbladder, ovarian, pancreatic, gastric, cervical, thyroid, prostate and skin cancer;
  • Hematopoietic tumors of the lymphatic system including leukemia, acute lymphoblastic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy cell lymphoma , mantle cell lymphoma, myeloma, and Burkett's lymphoma;
  • Hematopoietic tumors of the myeloid system including acute and chronic myeloid leukemia, myelodysplastic syndrome, and promyelocytic leukemia;
  • Tumors of interstitial origin including fibrosarcoma and rhabdomyosarcoma;
  • Tumors of the central and peripheral nervous system including astrocytoma, fibrone, glioma, and schwannomas;
  • tumors include melanoma, seminoma, teratocarcinoma, osteosarcoma, exogenous pigmented neck tumor, thyroid follicular carcinoma, and Kaposi's sarcoma.
  • the composition is a health supplement and further comprises an acceptable carrier in an optional health care product.
  • the composition is used for immunomodulation, improving microcirculation, and/or improving quality of life.
  • rohansol derivative monomer (3) as used herein means 3 ⁇ -hydroxy-25-dehydroxy-24-oxomogol (3 ⁇ -hydroxy-25-Dehydroxy-24-oxomogrol) (structure is shown in Figure 3 As shown herein; the term “new romanol derivative monomer (4)” as used herein means 3 ⁇ -hydroxy-22,24-diene-24,25-dehydroxy-rohansol (3 ⁇ -hydroxy-22, 24) -diene-24, 25-dehydroxy-mogrol) (4) (Structural formula shown in Figure 4).
  • composition as used herein is intended to include a product comprising specified amounts of each of the specified ingredients, as well as any product that results, directly or indirectly, from the specified combination of the specified ingredients.
  • the topical dosage form includes an ointment, a solid, a suspension, a liquid, an elixir, a powder, a paste, a suppository, an aerosol, a poultice, a spread, a lotion, an enema or an emulsion.
  • the active compound is admixed under sterile conditions with apharmaceutically acceptable carrier and any of the required preservatives, buffers or propellants. Ophthalmic formulations, eye ointments, powders and solutions are also contemplated as being within the scope of the invention.
  • a therapeutically and/or prophylactically effective amount of a compound of the invention may be administered in pure form, or in the form of a pharmaceutically acceptable salt, ester or prodrug (in the presence of such forms) application.
  • the compound can be administered in a pharmaceutical composition comprising the compound of interest and one or more pharmaceutically acceptable excipients.
  • the phrase "therapeutically and/or prophylactically effective amount" of a compound of the invention refers to a sufficient amount of a compound to treat the disorder at a reasonable effect/risk ratio applicable to any medical treatment. It will be appreciated, however, that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment.
  • the specific therapeutically and/or prophylactically effective dosage level for any particular patient will depend on a number of factors, including the disorder being treated and the severity of the disorder; the activity of the particular compound employed; Specific composition; age, weight, general health, sex and diet of the patient; time of administration, route of administration and excretion rate of the particular compound employed; duration of treatment; use in combination or concurrent use with the particular compound employed Drugs; and similar factors well known in the medical field.
  • the dosage of the compound be started from a level lower than that required to achieve the desired therapeutic effect, and the dosage is gradually increased until the desired effect is obtained.
  • the invention also provides a medicament comprising a compound of the invention, optionally formulated with one or more non-toxic pharmaceutically acceptable diluents, carriers, excipients, adjuvants or vehicles.
  • a medicament comprising a compound of the invention, optionally formulated with one or more non-toxic pharmaceutically acceptable diluents, carriers, excipients, adjuvants or vehicles.
  • the pharmaceutical preparations may be specially formulated for oral administration in solid or liquid form for parenteral injection or for rectal administration.
  • composition of the present invention can be administered orally, rectally, parenterally, intra-, intravaginally, intraperitoneally, topically (e.g., by powder, ointment or drops), buccally to humans and other mammals, or as an oral spray. Or nasal spray.
  • parenteral refers to a mode of administration including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous, and intraarticular injections and infusions.
  • the invention provides a pharmaceutical composition comprising a component of the invention and a physiologically tolerable diluent.
  • the present invention includes one or more of the above compounds formulated in combination with one or more non-toxic physiologically tolerable or acceptable diluents, carriers, adjuvants or vehicles (collectively referred to herein as diluents)
  • diluents non-toxic physiologically tolerable or acceptable diluents, carriers, adjuvants or vehicles
  • compositions suitable for parenteral injection may include physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, and sterile powders for the preparation of sterile injectable solutions or dispersions.
  • suitable aqueous or nonaqueous vehicles, diluents, solvents or vehicles include water, ethanol, polyols (propylene glycol, polyethylene glycol, glycerol, etc.), vegetable oils (such as olive oil), injectable organic esters such as oleic acid. Ethyl esters and suitable mixtures thereof.
  • compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents.
  • adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents.
  • the action of microorganisms can be ensured by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid and the like.
  • isotonic agents for example, sugars, sodium chloride, and the like.
  • Prolonged absorption of the injectable pharmaceutical form can be brought about through the use of materials which delay absorption, such as aluminum monostearate and gelatin.
  • Suspensions may contain suspending agents in addition to the active compound, such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite , agar and tragacanth or a mixture of these substances.
  • suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite , agar and tragacanth or a mixture of these substances.
  • the rate of absorption of the drug depends on its rate of dissolution, which in turn may depend on crystal size and crystalline form.
  • delayed absorption of a pharmaceutical form for parenteral administration is accomplished by dissolving or suspending the drug in an oil vehicle.
  • Injectable depot formulations are prepared by forming microencapsule matrices of the drug in a biodegradable polymer such as polylactide-polyglycolide. The rate of drug release can be controlled based on the ratio of drug to polymer and the nature of the particular polymer employed. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Injectable depot formulations are also prepared by embedding the drug in liposomes or microemulsions which are compatible with body tissues.
  • the injectable preparation can be sterilized, for example, by filtration with a bacteriophage or by incorporating a sterilizing agent in the form of a sterile solid composition which can be dissolved or dispersed in sterile water or other sterilized form before use. Injectable medium.
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.
  • the active compound may be mixed with at least one inert pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or the following: a) filler or extender such as starch , lactose, sucrose, glucose, mannitol and silicic acid; b) binders such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and gum arabic; c) humectants such as glycerin; d) Decomposing agents such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate; e) solution retarders such as paraffin; f) absorption accelerators such as quaternary ammonium compounds; g) humectants such as whales Wax alcohol and glycerol monostearate;
  • Solid compositions of a similar type may be used as fillers in soft and hard capsules using such excipients as lactose and high molecular weight polyethylene glycols and the like.
  • the solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other materials known in the art of pharmaceutical preparations. These solid dosage forms may optionally contain opacifying agents and may be formulated such that they are only or preferentially released in a certain portion of the intestinal tract in a delayed manner. Examples of the embedding composition that can be used include high molecular substances and waxes. If appropriate, the active compounds may also be formulated in microencapsulated form with one or more of the above-mentioned excipients.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs.
  • the liquid dosage form may contain, in addition to the active compound, an inert diluent commonly used in the art, such as water or other solvents, solubilizers and emulsifiers such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate.
  • the oral compositions may contain, in addition to inert diluents, excipients such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and flavoring agents.
  • compositions for rectal or vaginal administration are preferably suppositories.
  • Suppositories can be prepared by admixing a compound of the present invention with a suitable non- irritating excipient or carrier such as cocoa butter, polyethylene glycol or suppository wax, which are solid at room temperature but liquid at body temperature, thus Melting in the rectal cavity or vaginal cavity releases the active compound.
  • the compounds of the invention may also be administered in the form of liposomes.
  • liposomes are typically prepared using phospholipids or other lipid materials. Liposomes are formed from single or multiple layers of hydrated liquid crystal dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolizable lipid capable of forming liposomes can be used.
  • a composition of the invention in liposome form in addition to In addition to the compound of the present invention, a stabilizer, a preservative, an excipient or the like may be contained.
  • Preferred lipids are natural and synthetic phospholipids and phosphatidylcholines (lecithins), which may be used alone or together. Methods of forming liposomes are well known in the art.
  • prodrug denotes a prodrug of a compound of the invention which is suitable for use in contact with human and lower animal tissues without undue toxicity, irritation, allergies within the scope of sound medical judgment.
  • the reaction, etc. is commensurate with a reasonable effect/risk ratio and is effective for its intended use, and where possible, represents the zwitterionic form of the compound of the invention.
  • Prodrugs of the invention can be rapidly converted in vivo to the parent compound of the above formula, for example by hydrolysis in blood.
  • the present invention provides a novel mogroside derivative monomer (4) which can be formulated into a medicament for treating and/or preventing different cancers and/or tumors, and for immunomodulating and/or improving microcirculation and / or improve the quality of life of health care products, focusing on the treatment of malignant tumors, has broad application prospects.
  • Fig. 1 is a schematic view showing the molecular structure of mogroside V(1A) and VI(1B).
  • Figure 2 is a schematic diagram showing the molecular structure of mogroside Mogrol (2).
  • Fig. 3 is a schematic view showing the molecular structure of the mogroside derivative monomer (3).
  • Fig. 4 is a schematic view showing the molecular structure of a novel mogroside derivative monomer (4).
  • Example 1 Preparation of mogroside derivative monomers (3) and (4) from 98% of the total saponins of Siraitia grosvenorum
  • Luo Han Guo extract 98% of Luo Han Guo total saponin, containing 55% Luo Han Guo ⁇ V, purchased from Guilin Rhein Biotechnology Co., Ltd.
  • the hydrochloric acid was adjusted to a pH of 3.0), stirred and dissolved in a reaction vessel, and then heated to 120 ° C for 2 hours. After cooling to room temperature at 25 ° C, the reaction solution was neutralized with a 1 M NaOH lye to a pH of 7. Evaporate the ethanol as much as possible at 90-95 ° C. Add the remaining concentrate to 200 mL of distilled water, stir well, cool, and let stand.
  • Example 2 Preparation of mogroside derivative monomers (3) and (4) from 95% of the total saponins of Siraitia grosvenor
  • Luo Han Guo extract 95% of Luo Han Guo total saponin, containing 45% Luo Han Guo ⁇ V, purchased from Guilin Rhein Biotechnology Co., Ltd.
  • methanol solution methanol and water volume ratio of 3:2
  • the sulfuric acid was adjusted to a pH of 2.8), stirred and dissolved in a reaction vessel, and then heated to 120 ° C for 2 hours. After cooling to room temperature, the reaction solution was neutralized with a 1 M KOH lye to a pH of 7 at 90- Methanol was distilled off as much as possible at 95 ° C. The remaining concentrate was added to 200 mL of distilled water, stirred thoroughly, cooled, and allowed to stand.
  • the upper aqueous layer was separated and discarded to obtain a brown extract; 100 mL, 50 mL, 50 mL of ethyl acetate were used respectively.
  • the coffee extract was extracted 3 times, and after 3 times of extracting, the mixture was concentrated at 100-120 ° C with a rotary evaporator, and the ethyl acetate was completely evaporated to obtain a monomer containing the mogroside derivative (3) and rohanol.
  • the light brown solid of the monomer (4) was 15.5 g; the light brown solid was loaded onto a silica gel column with methanol, eluted with a gradient of acetonitrile:water (19:1), and the different effluent was collected in order, and concentrated to give A. (6.0g), B (5.2g) two components; first collected A (6 The .0g) component was separated by a silica gel column (chloroform: ethanol/60:5 elution) to obtain a mannose alcohol derivative monomer (3) 3.9 g (HPLC>99%); HPLC, 13 C-NMR, ESESI detection The result was identical to the compound (3) in Example 1.
  • Luo Han Guo extract 90% of Luo Han Guo total saponin, containing 40% Luo Han Guo ⁇ V, purchased from Guilin Rhein Biotechnology Co., Ltd.
  • n-propanol the ratio of n-propanol to water was 2:3.
  • the remaining concentrate was added to 200 mL of distilled water, stirred thoroughly, cooled, and allowed to stand. The upper aqueous layer was separated and discarded to obtain a brown extract; 100 mL, 50 mL, respectively. Extracting the brown extract 3 times with 50 mL of ethyl acetate, combining the extracts three times, concentrating at 100-120 ° C with a rotary evaporator, and evaporating the ethyl acetate completely to obtain the monomer containing the rohanol derivative (3) And light brown solid of 14.1 g of the mogroside derivative monomer (4); the light brown solid was loaded onto a silica gel column with methanol/ethanol, and eluted with a gradient of acetonitrile:water (20:1).
  • Example 4 Preparation of mogroside derivative monomers (3) and (4) from an extract of 80% of total saponins of Siraitia grosvenorum
  • Luo Han Guo extract 80% of Luo Han Guo total saponin, containing 20% Luo Han Guo ⁇ V, purchased from Guilin Rhein Biotechnology Co., Ltd.
  • ethanol the volume ratio of ethanol to water is 1:1
  • the oxalic acid was adjusted to a pH of 2.6), dissolved in a reaction vessel, and then heated to 120 ° C for 2 hours. After cooling to room temperature, the reaction solution was neutralized to a pH of 1 M Na 2 CO 3 . 7. Evaporate the ethanol as much as possible at 90-95 ° C. Add the remaining concentrate to 200 mL of distilled water, stir well, cool, and let stand.
  • Example 5 Monocrotool derivative monomer (4) inhibiting cell proliferation experiment
  • Test drug the mogroside derivative monomer prepared in Example 1 (4);
  • Control drug paclitaxel (SELLECK; Cat. #S1150); ginsenoside monomer Rg3 (purchased from Shanghai Yuanye Biotechnology Co., Ltd., product number B21059).
  • Paclitaxel was added at a concentration ranging from a high concentration of 1 ⁇ M to a low concentration of 0.0014 ⁇ M. After test/control drug solution for 72 hours, use (Promega; Cat. #G7573) Luminescent cell viability assay to determine the percentage inhibition of proliferation of each cell of each cell in each cell line, and to plot the dose-effect relationship, and finally calculate the IC according to the curve in the figure 50 and the highest percent inhibition ( Emax ), as shown in Tables 1 and 2.
  • Table 1 Experimental results of inhibition of cell proliferation activity by the mogroside derivative monomer (4) and the control drug Rg3.
  • Table 2 Experimental results of the effectiveness of the mogroside derivative monomer (4) and the control drugs paclitaxel and Rg3 inhibiting cell proliferation.
  • the independent-sample T-test was used to determine whether the difference between the mean of the drug group and the control group was statistically significant (P ⁇ 0.05 or P ⁇ 0.01).
  • the average IC50 of the mogroside derivative monomer (4) of the present invention in 24 cell lines was statistically significant when compared with the average IC50 of the control drug ginsenoside monomer Rg3 in 24 cell lines (P ⁇ 0.05). At the same time P ⁇ 0.01).
  • the average maximum percent inhibition of the mogroside derivative monomer (4) of the present invention in 24 cell lines is compared with the average highest percent inhibition of the control drug paclitaxel and the control drug ginsenoside monomer Rg3 in 24 cell lines. The difference was statistically significant (P ⁇ 0.05 and P ⁇ 0.01).
  • the cellulose-inhibiting activity of the mogroside derivative monomer (4) of the present invention in all 24 cell lines is higher than that of the similar control drug ginsenoside monomer Rg3 (the composition of the mogroside derivative monomer (4)) 50 is less than the IC 50 of the ginsenoside monomer Rg3.
  • the mogroside derivative monomer (4) of the present invention is the most common for all 24 cell lines tested.
  • the high inhibition rate (effectiveness) is very close to 100%; while the control drugs paclitaxel and ginsenoside monomer Rg3 have nearly 100% inhibition rate on only 2 cell lines in the 24 cell lines tested. This indicates that the mogroin derivative monomer (4) is significantly more effective than the control drug, and its high anticancer effectiveness is applicable to a plurality of cancer types.

Abstract

本发明提供了一种新型的罗汉果醇衍生物单体(4)，可以被制成用于治疗和/或预防不同癌症和/或肿瘤的药物，也可以被制成用于免疫调节和/或改善微循环和/或提高生命质量的保健品，重点应用于恶性肿瘤的治疗，具有广阔的应用前景。

Description

一种新型罗汉果醇衍生物单体 技术领域
本发明涉及医药领域，具体涉及一种新型罗汉果醇衍生物单体。
背景技术
癌症是残害人类生命的世界第二大疾病，死亡率仅次于心脑血管疾病，是人类死亡的最主要因素之一。由世卫组织组织下属的的官方癌症机构国际癌症研究中心(IARC)负责的最新版《世界癌症报告》预测，全球癌症病例将呈现迅猛增殖态势，由2012年的1400万人，逐年迅增至2025年的1900万人，到2035年将达到2400万人。报告还显示，2012年全球新增癌症病例有近一半出现在亚洲，其中大部分在中国，中国新增癌症病例高居首位。2012年中国新增307万癌症患者并造成约220万人死亡，分別占全球总量的21.9％和26.8％。世卫的数据略低于中国自己的统计。全国肿瘤登记中心发布的2012年数据显示，中国每年新增癌症病例约350万，约有250万人因此死亡。
现今癌症的常用治疗方法主要有三种模式：手术、放疗和药物治疗，而选定哪个治疗方法则取决于肿瘤的位置、恶性程度、发展程度以及病人身体状态。三种模式中，手术的治疗方法，常因为癌细胞入侵蔓延到邻近组织或远端转移而效果有限；放疗的治疗方法，则受限于对体内其他正常组织造成的伤害；药物的治疗方法，对于晚期弥散性和转移性恶性肿瘤是最基本的治疗方法。过去的几十年里，着眼于直接杀伤肿瘤细胞的化疗虽有明显的发展和进步，成为 肿瘤药物治疗的中坚，但这一治疗模式对增殖缓慢的实体瘤效果差、药物选择性小、毒副反应多且严重的缺陷成为临床治疗中的重要限制因素。继手术、放疗和化疗之后的第四种模式是肿瘤的生物治疗，其主要是通过肿瘤宿主防御机制或生物制剂的作用来调节机体自身的生物学反应，从而抑制或消除肿瘤；生物治疗虽然没有太大毒副作用，但由于技术要求严、工艺复杂，因此价格高，众多癌症患者及家属难以承受，影响其在癌症治疗领域的普及。
由于存在上述各种限制，天然抗肿瘤药物的研发取得了越来越多的关注。天然抗癌药物无论是在抑制或杀伤肿瘤细胞、调整机体免疫功能、改善症状与特征和减轻放化疗毒副作用上，还是在肿瘤的病后调理上，均具有重要作用。由此，天然植物新疗法将成为继手术、放疗、化疗和生物疗法之后的第五种模式。
罗汉果是一种名贵药材，属于葫芦科多年生藤本植物，性凉味甘。罗汉果提取物中含非糖甜味的皂苷成分，又称罗汉果总皂苷，根据其中皂苷元连接糖基的数目以及连接位置的不同已经在罗汉果总皂苷中分离鉴定出罗汉果二糖苷、三糖苷、四糖苷、五糖苷、六糖苷等多种皂苷单体，分别命名为罗汉果甜苷II、III、IV、V、VI(Mogroside II、III、IV、V、VI)等，其中罗汉果甜苷V-Mogroside V(1A)(如图1所示)的甜度是蔗糖的256-344倍，罗汉果甜苷IV–Mogroside IV(1B)(如图1所示)的甜度为蔗糖的126倍，其热量为零，具有清热润肺镇咳、润肠通便之功效，对肥胖、便秘、糖尿病等具有防治作用。
罗汉果皂苷Mogrosides是两条由四个以下葡萄糖单位组成的葡萄糖苷侧链以β-糖苷键与皂苷元罗汉果醇Mogrol(2)(如图2所示)的C-3、C-24相连，在侧链葡萄糖之间的连接键为β-1,6和 β-l,2糖苷键。
许多研究表明，罗汉果皂苷能提高葡萄糖和脂肪的利用，增加胰岛素的敏感性，但罗汉果降血糖作用的有效成分和作用机制并不明确。中科院上海药物研究所胡立宏课题组和沈旭课题组对罗汉果的降血糖作用进行了研究，首先在HepG2细胞中，对罗汉果中含量最高的罗汉果甜苷V(1A)进行测试，发现它对AMPK没有活性；而罗汉果醇Mogrol(2)及其衍生物3α-羟基-25-脱羟基-24-酮-罗汉果醇(3α-hydroxy-25-dehydroxy-24-oxomogrol)(3)(如图3所示)却能够激活AMPK(Chen X.B.，et al；Bioorganic&Medicinal Chemistry 2011,19:5776)。目前，还没有任何专利和文献报道通过罗汉果醇(2)在特定条件下生成罗汉果醇衍生物单体(3)和罗汉果醇衍生物单体(4)(如图4所示)，也没有任何专利和文献报道存在有罗汉果醇衍生物单体(4)。
发明内容
本发明要解决的技术问题是提供了一种新型的罗汉果醇衍生物单体(4)，此罗汉果醇衍生物单体(4)可以制成药物制剂或保健品，用于肿瘤和/或癌症的治疗和/或预防。
本发明第一方面提供了一种罗汉果醇衍生物单体(4)，具有如下结构：
Figure PCTCN2016089207-appb-000001
本发明第二方面提供了一种组合物，包含如下结构的
Figure PCTCN2016089207-appb-000002
罗汉果醇衍生物单体(4)。
在一优选例中，所述组合物为药物制剂，所述药物制剂还包括药学可接受的稀释剂、载体、赋形剂、辅料或媒介物。
在一优选例中，所述药物制剂的剂型为口服剂型、注射剂型或局部用药剂型。
在一优选例中，所述口服剂型为片剂、粉剂、悬浊液、乳浊液、胶囊、颗粒剂、糖衣片、药丸、液体、醑剂、糖浆或柠檬水剂。
在一优选例中，所述注射剂型包括水剂、悬浊液或溶液。
在一优选例中，所述局部用药剂型包括软膏、固体、悬浊液、水剂、醑剂、粉剂、糊剂、栓剂、气溶胶、泥敷剂、涂抹剂、洗剂、灌肠剂或乳剂。
在一优选例中，所述组合物用于治疗和/或预防肿瘤和/或癌症；
其中所述的肿瘤和/或癌症选自：
恶性肿瘤，包括膀胱癌、乳腺癌、结肠癌、肾癌、肝癌、肺癌、头和颈癌、食管癌、胆囊癌、卵巢癌、胰腺癌、胃癌、子宫颈癌、甲状腺癌、前列腺癌和皮肤癌；
淋巴系统的造血肿瘤，包括白血病、急性淋巴细胞白血病、急性成淋巴细胞白血病、B-细胞淋巴癌、T-细胞淋巴癌、霍奇金淋巴癌、非-霍奇金淋巴癌、毛细胞淋巴癌、外套细胞淋巴瘤、骨髓瘤和Burkett′s氏淋巴癌；
骨髓系统的造血肿瘤，包括急性和慢性髓细胞性白血病、骨髓增生异常综合征和前髓细胞性白血病；
间质成因的肿瘤，包括纤维肉瘤和横纹肌肉瘤；
中枢和周围神经系统的肿瘤，包括星形细胞瘤、成纤维神经瘤、神经胶质瘤和神经鞘瘤；以及
其他肿瘤，包括黑素瘤、精原细胞瘤、畸胎癌、骨肉瘤、外生性色素颈瘤、甲状腺滤囊癌和卡波氏肉瘤。
在一优选例中，所述组合物为保健品，还包括任选的保健品中可接受的载体。
在一优选例中，所述组合物用于免疫调节、改善微循环和/或提高生命质量。
本发明使用的术语具有以下定义，除非另有描述：
本文所用的术语“罗汉果醇衍生物单体(3)”意指3α-羟基-25-脱羟基-24-酮-罗汉果醇(3α-hydroxy-25-Dehydroxy-24-oxomogrol)(结构式如图3所示)；本文所用的术语“新型罗汉果醇衍生物单体(4)”意指3α-羟基-22、24-二烯-24、25-脱羟基-罗汉果醇(3α-hydroxy-22、24-diene-24、25-dehydroxy-mogrol)(4)(结构式如图4所示)。
本文所用的术语“组合物”意指包括包含指定量的各指定成分的产品，以及直接或间接从指定量的各指定成分的组合产生的任何产品。所述局部用药剂型包括软膏、固体、悬浊液、水剂、醑剂、粉剂、糊剂、栓剂、气溶胶、泥敷剂、涂抹剂、洗剂、灌肠剂或乳剂。。在无菌条件下将活性化合物与药学可接受的载体和任何所需的防腐剂、缓冲剂或推进剂混合。眼用制剂、眼软膏剂、散剂和溶液剂也被考虑在本发明范围内。
当用于上述治疗或其他治疗时，治疗和/或预防有效量的一种本发明化合物可以以纯形式应用，或者以药学可接受的盐、酯或前药形式(在存在这些形式的情况下)应用。或者，所述化合物可以以含有该目的化合物与一种或多种药学可接受的赋形剂的药物组合物给药。词语“治疗和/或预防有效量”的本发明化合物指以适用于任何医学治疗的合理效果/风险比治疗障碍的足够量的化合物。但应认识到，本发明化合物和组合物的总日用量须由主诊医师在可靠的医学判断范围内作出决定。对于任何具体的患者，具体的治疗和/或预防有效剂量水平须根据多种因素而定，所述因素包括所治疗的障碍和该障碍的严重程度；所采用的具体化合物的活性；所采用的具体组合物；患者的年龄、体重、一般健康状况、性别和饮食；所采用的具体化合物的给药时间、给药途径和排泄率；治疗持续时间；与所采用的具体化合物组合使用或同时使用的药物；及医疗领域公知的类似因素。例如，本领域的做法是，化合物的剂量从低于为得到所需治疗效果而要求的水平开始，逐渐增加剂量，直到得到所需的效果。
本发明还提供包含任选地与一种或多种无毒药学可接受的稀释剂、载体、赋形剂、辅料或媒介物配制在一起的本发明化合物的药 物制剂。所述药物制剂可特别专门配制成以固体或液体形式供口服给药、供胃肠外注射或供直肠给药。
本发明的药物组合物可通过口服、直肠、胃肠外、池内、阴道内、腹膜内、局部(如通过散剂、软膏剂或滴剂)、口颊给予人类和其他哺乳动物，或者作为口腔喷雾剂或鼻腔喷雾剂给予。本文所用术语“胃肠外”指包括静脉内、肌肉内、腹膜内、胸骨内、皮下和关节内注射和输液的给药方式。
在另一个方面，本发明提供包含本发明成分和生理可耐受稀释剂的药物组合物。本发明包括一种或多种上述化合物，其与一种或多种无毒生理可耐受或可接受的稀释剂、载体、辅料或媒介物(本文将它们统称为稀释剂)一起配制成组合物，以供胃肠外注射、鼻内传递、以固体或液体形式口服给药、直肠或局部给药等等。
适合于胃肠外注射的组合物可包括生理上可接受的无菌含水或非水溶液剂、分散剂、混悬剂或乳剂，及供重构成无菌可注射溶液剂或分散剂的无菌散剂。合适的含水或非水载体、稀释剂、溶剂或媒介物的实例包括水、乙醇、多元醇(丙二醇、聚乙二醇、甘油等)、植物油(如橄榄油)、可注射有机酯如油酸乙酯及它们的合适混合物。
这些组合物也可含有辅料，如防腐剂、湿润剂、乳化剂和分散剂。通过各种抗细菌剂和抗真菌剂，例如尼泊金酯类、三氯叔丁醇、苯酚、山梨酸等，可确保防止微生物的作用。还期望包括等渗剂，例如糖类、氯化钠等。通过使用能延迟吸收的物质，例如单硬脂酸铝和明胶，可达到可注射药物形式的延长吸收。
混悬剂中除活性化合物外还可含有悬浮剂，例如乙氧基化异十八醇、聚氧乙烯山梨醇和聚氧乙烯失水山梨糖醇酯、微晶纤维素、偏氢氧化铝、膨润土、琼脂和黄蓍胶或者这些物质的混合物等。
在一些情况下，为延长药物的作用，期望减慢皮下或肌内注射药物的吸收。这可通过使用水溶性差的晶体或无定形物质的液体混悬剂来实现。这样，药物的吸收速度取决于其溶解速度，而溶解速度又可取决于晶体大小和晶型。或者，胃肠外给药的药物形式的延迟吸收通过将该药物溶解于或悬浮于油媒介物中来实现。
可注射贮库制剂形式可通过在生物可降解聚合物如聚丙交酯-聚乙交酯(polylactide-polyglycolide)中形成药物的微胶囊基质来制备。可根据药物与聚合物之比和所采用的具体聚合物的性质，对药物释放速度加以控制。其他生物可降解聚合物的实例包括聚原酸酯类(poly(orthoesters))和聚酐类(poly(anhydrides))。可注射贮库制剂也可通过将药物包埋于能与身体组织相容的脂质体或微乳中来制备。
可注射制剂可例如通过用滤菌器过滤或通过掺入无菌固体组合物形式的灭菌剂来灭菌，所述固体组合物可在临用前溶解或分散于无菌水或其他无菌可注射介质。
供口服给药的固体剂型包括胶囊剂、片剂、丸剂、散剂和颗粒剂。在此类固体剂型中，活性化合物可与至少一种惰性的药学可接受的赋形剂或载体如柠檬酸钠或磷酸二钙和/或以下物质混合：a)填充剂或增量剂如淀粉、乳糖、蔗糖、葡萄糖、甘露糖醇和硅酸；b)粘合剂如羧甲基纤维素、海藻酸盐、明胶、聚乙烯吡咯烷酮、蔗糖和阿拉伯树胶；c)保湿剂如甘油；d)崩解剂如琼脂、碳酸钙、马铃薯或木薯淀粉、海藻酸、某些硅酸盐和碳酸钠；e)溶液阻滞剂如石蜡；f)吸收加速剂如季铵化合物；g)湿润剂如鲸蜡醇和甘油单硬脂酸酯；h)吸附剂如高岭土和膨润土以及i)润滑剂如滑石粉、硬脂酸钙、硬脂酸镁、固体聚乙二醇、十二烷基硫酸钠和它们的混合物。在胶囊剂、片剂和丸剂的情况下，所述剂型中也可包含缓冲剂。
相似类型的固体组合物使用赋形剂例如乳糖及高分子量聚乙二醇等，也可用作软胶囊和硬胶囊中的填充物。
片剂、糖衣丸剂(dragees)、胶囊剂、丸剂和颗粒剂的固体剂型可与包衣和壳料如肠溶衣材和医药制剂领域公知的其他衣材一起制备。这些固体剂型可任选含有遮光剂，且其组成还可使其只是或优先地在肠道的某个部位任选以延迟方式释放活性成分。可以使用的包埋组合物的实例包括高分子物质和蜡类。如果适合，活性化合物也可与一种或多种上述赋形剂配成微囊形式。
供口服给药的液体剂型包括药学可接受的乳剂、溶液剂、混悬剂、糖浆剂和酏剂。液体剂型除含有活性化合物外还可含有本领域常用的惰性稀释剂，例如水或其他溶剂，增溶剂和乳化剂例如乙醇、异丙醇、碳酸乙酯、乙酸乙酯、苄醇、苯甲酸苄酯、丙二醇、1，3-丁二醇、二甲基甲酰胺、油类(特别是棉籽油、花生油、玉米油、胚芽油、橄榄油、蓖麻油和芝麻油)、甘油、四氢糠醇(tetrahydrofurfuryl alcohol)、聚乙二醇和脱水山梨糖醇的脂肪酸酯及它们的混合物。口服组合物除包含惰性稀释剂外还可包含辅料，例如湿润剂、乳化和悬浮剂、甜味剂、矫味剂和香味剂。
供直肠或阴道给药的组合物优选是栓剂。栓剂可通过将本发明化合物与合适的非刺激性赋形剂或载体例如可可脂、聚乙二醇或栓剂蜡混合来制备，它们在室温下为固体，但在体温下则为液体，因此可在直肠腔或阴道腔内熔化而释放出活性化合物。
本发明化合物也可以脂质体形式给药。如本领域所公知，脂质体通常用磷脂或其他脂类物质制得。脂质体由分散于含水介质中的单层或多层水化液晶所形成。任何能够形成脂质体的无毒、生理上可接受和可代谢的脂质均可使用。脂质体形式的本发明组合物除含 有本发明化合物外，还可含有稳定剂、防腐剂、赋形剂等。优选的脂类是天然和合成的磷脂和磷脂酰胆碱(卵磷脂)，它们可单独或者一起使用。形成脂质体的方法是本领域公知的。
本文所用的术语“药学可接受的前药”代表本发明化合物的前药，其在可靠的医学判断范围内适合用于与人类和低等动物的组织接触而不出现过度的毒性、刺激、过敏反应等，与合理的效果/风险比相称且对其预定用途有效，在可能的情况下还代表本发明化合物的两性离子形式。本发明的前药可例如通过在血液中水解而在体内快速转化成上式的母体化合物。
本发明提供了一种新型的罗汉果醇衍生物单体(4)，可以被制成用于治疗和/或预防不同癌症和/或肿瘤的药物，以及用于免疫调节和/或改善微循环和/或提高生命质量的保健品，重点应用于恶性肿瘤的治疗，具有广阔的应用前景。
附图说明
图1为罗汉果甜苷V(1A)、VI(1B)的分子结构示意图。
图2为罗汉果醇Mogrol(2)的分子结构示意图。
图3为罗汉果醇衍生物单体(3)的分子结构示意图。
图4为新型罗汉果醇衍生物单体(4)的分子结构示意图。
具体实施方式
除非特殊说明，本发明所用术语具有本发明所属领域中的一般含义。
下面参考具体实施例，对本发明进行说明，需要说明的是，这些实施例仅仅是说明性的，而不能理解为对本发明的限制。实施例 中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1：从罗汉果总皂苷98％的提取物制备罗汉果醇衍生物单体(3)和(4)
将罗汉果提取物50g(罗汉果总皂苷98％，含55％罗汉果甙V，购买于桂林莱茵生物科技股份有限公司)加入250mL的乙醇的水溶液(乙醇和水的体积比为1:1)(预先用盐酸调节至PH值为3.0)，在反应釜中搅拌溶解，然后升温至120℃下加热2小时，冷却至室温25℃后，反应液用1M的NaOH碱液中和至PH值为7，在90-95℃下将乙醇尽量蒸出，剩下的浓缩液加入200mL蒸馏水，充分搅拌后冷却、静置，将上面的水层分离弃去，得到咖啡色浸膏；分别用100mL、50mL、50mL乙酸乙酯萃取咖啡色浸膏3次，合并3次萃取液后，在100-120℃下用旋转蒸发仪浓缩，把乙酸乙酯完全蒸干，即得到含罗汉果醇衍生物单体(3)和罗汉果醇衍生物单体(4)的浅咖啡色固体18.2g；用乙醇将浅咖啡色固体载入硅胶柱，用乙腈：水(体积比20:1)梯度洗脱，按先后顺序收集不同的流出液，浓缩后得到A(6.8g)、B(5.6g)两组分；先收集得到的A(6.8g)组分再用硅胶柱分离(氯仿：乙醇/60:5洗脱)，得到罗汉果醇衍生物单体(3)4.1g(HPLC>99％)。ESIMS m/z 503(458+45)[M+HCOO-]-(calcd for C31H51O5)；13C-NMR(600MHz，MeOD)δppm:29.1(C-1),31.7(C-2),79.4(C-3),42.8(C-4),144.1(C-5),120.7(C-6),25.2(C-7),44.8(C-8),41.1(C-9),35.4(C-10),77.7(C-11),30.6(C-12),48.3(C-13),49.6(C-14),34.9(C-15),27.5(C-16),50.7(C- 17),17.2(C-18),26.6(C-19),41.1(C-20),19.0(C-21),37.8(C-22),40.3(C-23),218.2(C-24),35.4(C-25),18.8(C-26),18.8(C-27),26.4(C-28),19.9(C-29),19.3(C-30)；与文献报道的3a-羟基-25-脱羟基-24-羰基-罗汉果醇(3a-hydroxy-25-Dehydroxy-24-oxomogrol)碳谱数据比较，二者基本一致(Chen X.B.，et al；Bioorganic&Medicinal Chemistry 2011,19:5776)。
后收集得到的B(5.6g)组分再用硅胶柱分离(石油醚：乙酸乙酯/5:2洗脱)，得到罗汉果醇衍生物单体(4)。3.2g(HPLC>99％)。ESIMS m/z 441[M+H]+(calcd for C30H49O2)；13C-NMR(600MHz，MeOD)δppm:23.5(C-1),29.5(C-2),87.8(C-3),44.2(C-4),144.1(C-5),121.8(C-6),25.5(C-7),40.9(C-8),40.8(C-9),51.3(C-10),71.9(C-11),41.5(C-12),48.2(C-13),52.2(C-14),35.3(C-15),24.9(C-16),54.3(C-17),15.2(C-18),18.6(C-19),40.2(C-20),20.3(C-21),137.4(C-22),128.8(C-23),125.4(C-24),143.3(C-25),19.7(C-26),25.7(C-27),21.7(C-28),21.7(C-29),18.6(C-30)。
实施例2：从罗汉果总皂苷95％的提取物制备罗汉果醇衍生物单体(3)和(4)
将罗汉果提取物50g(罗汉果总皂苷95％，含45％罗汉果甙V，从桂林莱茵生物科技股份有限公司购买)加入250mL的甲醇的水溶液(甲醇和水的体积比为3:2)(预先用硫酸调节至PH值为2.8)，在反应釜中搅拌溶解，然后升温至120℃下加热2小时，冷却至室温后，反应液用1M的KOH碱液中和至PH值为7，在90-95℃下将甲醇尽量蒸出，剩下的浓缩液加入200mL蒸馏水，充分搅拌后冷却、静 置，将上面的水层分离弃去，得到咖啡色浸膏；分别用100mL、50mL、50mL乙酸乙酯萃取咖啡色浸膏3次，合并3次萃取液后，在100-120℃下用旋转蒸发仪浓缩，把乙酸乙酯完全蒸干，即得到含罗汉果醇衍生物单体(3)和罗汉果醇衍生物单体(4)的浅咖啡色固体15.5g；用甲醇将浅咖啡色固体载入硅胶柱，用乙腈：水(19:1)梯度洗脱，按先后顺序收集不同的流出液，浓缩后得到A(6.0g)、B(5.2g)两组分；先收集得到的A(6.0g)组分再用硅胶柱分离(氯仿：乙醇/60:5洗脱)，得到罗汉果醇衍生物单体(3)3.9g(HPLC>99％)；HPLC、13C-NMR、ESIMS检测结果和实施例1中的化合物(3)一致。后收集得到的B(5.2g)组分再用硅胶柱分离(石油醚：乙酸乙酯/3:1洗脱)，得到罗汉果醇衍生物单体(4)。3.0g(HPLC>99％)；HPLC、13C-NMR、ESIMS检测结果和实施例1中的罗汉果醇衍生物单体(4)一致。
实施例3：从罗汉果总皂苷90％的提取物制备罗汉果醇衍生物单体(3)和(4)
将罗汉果提取物50g(罗汉果总皂苷90％，含40％罗汉果甙V，从桂林莱茵生物科技股份有限公司购买)加入250mL的正丙醇的水溶液(正丙醇和水的体积比为2:3)(预先用甲酸调节至PH值为3.2，在反应釜中搅拌溶解，然后升温至120℃下加热2小时，冷却至室温后，反应液用1M的NaOH碱液中和至PH值为7，在90-95℃下将正丙醇尽量蒸出，剩下的浓缩液加入200mL蒸馏水，充分搅拌后冷却、静置，将上面的水层分离弃去，得到咖啡色浸膏；分别用100mL、50mL、50mL乙酸乙酯萃取咖啡色浸膏3次，合并3次萃取液后，在100-120℃下用旋转蒸发仪浓缩，把乙酸乙酯完全蒸干， 即得到含罗汉果醇衍生物单体(3)和罗汉果醇衍生物单体(4)的浅咖啡色固体14.2g；用甲醇/乙醇将浅咖啡色固体载入硅胶柱，用乙腈：水(20:1)梯度洗脱，按先后顺序收集不同的流出液，浓缩后得到A(5.4g)、B(4.3g)两组分；先收集得到的A(5.4g)组分再用硅胶柱分离(氯仿：乙醇/60:5洗脱)，得到罗汉果醇衍生物单体(3)3.5g(HPLC>99％)；HPLC、13C-NMR、ESIMS检测结果和实施例1中的化合物(3)一致。后收集得到的B(4.3g)组分再用硅胶柱分离(石油醚：乙酸乙酯/6:4洗脱)，得到罗汉果醇衍生物单体(4)2.8g(HPLC>99％)；HPLC、13C-NMR、ESIMS检测结果和实施例1中的罗汉果醇衍生物单体(4)一致。
实施例4：从罗汉果总皂苷80％的提取物制备罗汉果醇衍生物单体(3)和(4)
将罗汉果提取物50g(罗汉果总皂苷80％，含20％罗汉果甙V，从桂林莱茵生物科技股份有限公司购买)加入250mL的乙醇的水溶液(乙醇和水的体积比为1:1)(预先用乙二酸调节至PH值为2.6)，在反应釜中搅拌溶解，然后升温至120℃下加热2小时，冷却至室温后，反应液用1M的Na2CO3碱液中和至PH值为7，在90-95℃下将乙醇尽量蒸出，剩下的浓缩液加入200mL蒸馏水，充分搅拌后冷却、静置，将上面的水层分离弃去，得到咖啡色浸膏；分别用100mL、50mL、50mL乙酸乙酯萃取咖啡色浸膏3次，合并3次萃取液后，在100-120℃下用旋转蒸发仪浓缩，把乙酸乙酯完全蒸干，即得到含罗汉果醇衍生物单体(3)和罗汉果醇衍生物单体(4)的浅咖啡色固体8.8g；用甲醇/乙醇将浅咖啡色固体载入硅胶柱，用乙腈：水(95:5)梯度洗脱，按先后顺序收集不同的流出液，浓缩后得到A (3.8g)、B(3.0g)两组分；先收集得到的A(3.8g)组分再用硅胶柱分离(氯仿：乙醇/60:5洗脱)，得到罗汉果醇衍生物单体(3)2.1g(HPLC>99％)；HPLC、13C-NMR、ESIMS检测结果和实施例1中的化合物(3)一致。后收集得到的B(3.0g)组分再用硅胶柱分离(石油醚：乙酸乙酯/4:1洗脱)，得到罗汉果醇衍生物单体(4)1.8g(HPLC>99％)；HPLC、13C-NMR、ESIMS检测结果和实施例1中的罗汉果醇衍生物单体(4)一致。
实施例5：罗汉果醇衍生物单体(4)抑制细胞增殖实验
实验样品：
测试药物：实施例1制备得到的罗汉果醇衍生物单体(4)；
对照药物：紫杉醇(SELLECK；Cat.#S1150)；人参皂苷单体Rg3(自上海源叶生物科技有限公司购买，商品货号为B21059)。
实验步骤：
以24种细胞系(包括23种肿瘤细胞系和1种人脐静脉内皮细胞系)为实验用细胞系，取对数生长期细胞(3x104/mL至2.5x105/mL)，以每孔100μL接种在96孔板内，每个细胞系用一个96孔板；然后，除对照药物紫杉醇以外，以高浓度150μM至低浓度2μM取7个对数递减浓度(每个浓度设两个复孔)，分别加入测试药物溶液和对照药物溶液500nL(测试药物溶液或对照药物溶液配制：分别采用测试药物或对照药物溶于0.5％的DMSO溶液)。紫杉醇的加入浓度为由高浓度1μM至低浓度0.0014μM的7个三倍递减浓度。经测试/对照药物溶液作用72小时后，用
Figure PCTCN2016089207-appb-000003
(Promega；Cat.#G7573)发光细胞活力检测法求出各细胞系中的每种药物的每个浓度对该系细胞的增殖抑制百分率，并绘制量效关系图，最后根据图中曲线测算IC50 和最高抑制百分率(Emax)，如表1和表2所示。
表1：罗汉果醇衍生物单体(4)以及对照药Rg3抑制细胞增殖活性实验检测结果。
Figure PCTCN2016089207-appb-000004
表2：罗汉果醇衍生物单体(4)以及对照药紫杉醇、Rg3抑制细胞增殖有效性实验检测结果。
Figure PCTCN2016089207-appb-000005
Figure PCTCN2016089207-appb-000006
使用SPSS Statistics软件(提供者：IBM corporation,软件版本:XL fit 21)提供统计学分析。
用假设方差不相等的独立样本检验(independent-sample T-test)来检测药物组与对照组平均值的差异是否呈现统计显著(P<0.05或P<0.01)。
分析结果：
1.本发明罗汉果醇衍生物单体(4)在24个细胞系中的平均IC50与对照药物人参皂苷单体Rg3在24个细胞系中的平均IC50比较时，差异呈现统计显著(P<0.05同时P<0.01)。
2.本发明罗汉果醇衍生物单体(4)在24个细胞系中的平均最高抑制百分率与对照药物紫杉醇以及对照药物人参皂苷单体Rg3在24个细胞系中的平均最高抑制百分率比较时，差异呈现统计显著(P<0.05同时P<0.01)。
结论：
1.本发明罗汉果醇衍生物单体(4)在所有24种所测细胞系中的抑制细胞增殖活性均高于同类对照药物人参皂苷单体Rg3(罗汉果醇衍生物单体(4)的IC50小于人参皂苷单体Rg3的IC50)。
2.在所有被测细胞系中，本发明罗汉果醇衍生物单体(4)在人脐血静脉内皮细胞系HUVEC中具有最高抑制细胞增殖活性(IC50＝10.72μM)，表现出其强大的抗肿瘤血管生成潜力；罗汉果醇衍生物单体(3)在结直肠腺癌细胞系HCT-8中抑制细胞增殖活性排在其次(IC50＝14.83μM)。
3.本发明罗汉果醇衍生物单体(4)对所有24种被测细胞系的最 高抑制率(有效性)都非常接近100％；而对照药紫杉醇和人参皂苷单体Rg3在已测的24种细胞系中，均只对2种细胞系有近100％的抑制率。这表明罗汉果醇衍生物单体(4)的有效性明显高于对照药物，且其高抗癌有效性适用于多类癌种。

Claims (10)

  1. 一种罗汉果醇衍生物单体(4)，具有如下结构：
    Figure PCTCN2016089207-appb-100001
  2. 一种组合物，其特征在于，包含如下结构的
    Figure PCTCN2016089207-appb-100002
    罗汉果醇衍生物单体(4)。
  3. 根据权利要求2所述的组合物，其特征在于，所述组合物为药物制剂，所述药物制剂还包括药学可接受的稀释剂、载体、赋形剂、辅料或媒介物。
  4. 根据权利要求3所述的组合物，其特征在于，所述药物制剂的剂型为口服剂型、注射剂型或局部用药剂型。
  5. 根据权利要求4所述的组合物，其特征在于，所述口服剂型为片剂、粉剂、悬浊液、乳浊液、胶囊、颗粒剂、糖衣片、药丸、液体、醑剂、糖浆或柠檬水剂。
  6. 根据权利要求4所述的组合物，其特征在于，所述注射剂型包括水剂、悬浊液或溶液。
  7. 根据权利要求4所述的组合物，其特征在于，所述局部用药剂型包括软膏、固体、悬浊液、水剂、醑剂、粉剂、糊剂、栓剂、气溶胶、泥敷剂、涂抹剂、洗剂、灌肠剂或乳剂。
  8. 根据权利要求7所述的组合物，其特征在于，所述组合物用于治疗和/或预防肿瘤和/或癌症；
    其中所述的肿瘤和/或癌症选自：
    恶性肿瘤，包括膀胱癌、乳腺癌、结肠癌、肾癌、肝癌、肺癌、头和颈癌、食管癌、胆囊癌、卵巢癌、胰腺癌、胃癌、子宫颈癌、甲状腺癌、前列腺癌和皮肤癌；
    淋巴系统的造血肿瘤，包括白血病、急性淋巴细胞白血病、急性成淋巴细胞白血病、B-细胞淋巴癌、T-细胞淋巴癌、霍奇金淋巴癌、非-霍奇金淋巴癌、毛细胞淋巴癌、外套细胞淋巴瘤、骨髓瘤和Burkett′s氏淋巴癌；
    骨髓系统的造血肿瘤，包括急性和慢性髓细胞性白血病、骨髓增生异常综合征和前髓细胞性白血病；
    间质成因的肿瘤，包括纤维肉瘤和横纹肌肉瘤；
    中枢和周围神经系统的肿瘤，包括星形细胞瘤、成纤维神经瘤、神经胶质瘤和神经鞘瘤；以及
    其他肿瘤，包括黑素瘤、精原细胞瘤、畸胎癌、骨肉瘤、外生性色素颈瘤、甲状腺滤囊癌和卡波氏肉瘤。
  9. 根据权利要求2所述的组合物，其特征在于，所述组合物为保健品，还包括任选的保健品中可接受的载体。
  10. 根据权利要求9所述的组合物，其特征在于，所述组合物用于免疫调节、改善微循环和/或提高生命质量。
PCT/CN2016/089207 2016-05-06 2016-07-07 一种新型罗汉果醇衍生物单体 WO2017190421A1 (zh)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
CN201610321210.XA CN105777837A (zh) 2016-05-06 2016-05-06 一种新型罗汉果醇衍生物单体
CN201610321210.X 2016-05-06
CN201610522398.4 2016-07-04
CN201610522398.4A CN106083972B (zh) 2016-05-06 2016-07-04 一种罗汉果醇衍生物单体

Publications (1)

Publication Number Publication Date
WO2017190421A1 true WO2017190421A1 (zh) 2017-11-09

Family

ID=56378939

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2016/089207 WO2017190421A1 (zh) 2016-05-06 2016-07-07 一种新型罗汉果醇衍生物单体

Country Status (2)

Country Link
CN (2) CN105777837A (zh)
WO (1) WO2017190421A1 (zh)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106188205B (zh) * 2016-05-06 2017-08-25 深圳以诺生物制药有限公司 一种罗汉果醇衍生物单体及其组合物的用途
CN105832748B (zh) * 2016-05-06 2019-06-07 深圳以诺生物制药有限公司 一种从罗汉果总皂苷中制备罗汉果醇衍生物的方法
CN111349133B (zh) * 2020-03-27 2021-03-12 湖南华诚生物资源股份有限公司 一种乙酰化罗汉果醇及其制备方法和用途

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014086842A1 (en) * 2012-12-04 2014-06-12 Evolva Sa Methods and materials for biosynthesis of mogroside compounds
CN105832748A (zh) * 2016-05-06 2016-08-10 深圳以诺生物制药有限公司 一种从罗汉果总皂苷中制备罗汉果醇新型衍生物的方法
CN106188205A (zh) * 2016-05-06 2016-12-07 深圳以诺生物制药有限公司 一种新型罗汉果醇衍生物单体及其组合物的用途

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101033244B (zh) * 2007-04-05 2010-05-19 上海交通大学 罗汉果醇的提纯制备方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014086842A1 (en) * 2012-12-04 2014-06-12 Evolva Sa Methods and materials for biosynthesis of mogroside compounds
CN105832748A (zh) * 2016-05-06 2016-08-10 深圳以诺生物制药有限公司 一种从罗汉果总皂苷中制备罗汉果醇新型衍生物的方法
CN106188205A (zh) * 2016-05-06 2016-12-07 深圳以诺生物制药有限公司 一种新型罗汉果醇衍生物单体及其组合物的用途

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
YANG, XIUWEI ET AL.: "Biotransformation of mogroside III by human intestinal bacteria", JOURNAL OF PEKING UNIVERSITY(HEALTH SCIENCES, vol. 39, no. 6, 31 December 2007 (2007-12-31), pages 657 - 662, XP055438095 *

Also Published As

Publication number Publication date
CN105777837A (zh) 2016-07-20
CN106083972B (zh) 2017-09-01
CN106083972A (zh) 2016-11-09

Similar Documents

Publication Publication Date Title
WO2017133468A1 (zh) 白头翁皂苷类化合物作为ev71病毒抑制剂的用途
CN109575099B (zh) 达玛烷皂苷元衍生物及其制备方法和应用
CN107986951B (zh) 新型拓扑异构酶i抑制剂及其药物组合物与其制备方法及应用
WO2017190422A1 (zh) 一种从罗汉果总皂苷中制备罗汉果醇新型衍生物的方法
US20110160152A1 (en) Extracts of aquilaria hulls and use thereof in the treatment of cancer
WO2017190421A1 (zh) 一种新型罗汉果醇衍生物单体
WO2011044751A1 (zh) 芒果叶苷及其制备方法与应用
KR20150136001A (ko) Ｅｒｋ 신호활성 억제제를 유효성분으로 함유하는 암 줄기세포 성장 억제용 조성물
US20090318400A1 (en) Method for inhibiting tumor growth with dehydrosulphurenic acid extracted from antrodia cinnamomea
WO2017190420A1 (zh) 一种罗汉果醇衍生物单体及其组合物的用途
CA2961747C (en) Composition for preventing or treating cervical cancer including gypenoside lxxv
CN101322714A (zh) 抗单纯疱疹病毒ⅰ型的药物组合物及其应用
WO2017190419A1 (zh) 一种罗汉果醇衍生物单体及其组合物的用途
WO2022134657A1 (zh) 一类抗前列腺癌的瑞香烷型二萜及其制备方法
WO2015192758A1 (zh) 瓦草五环三萜皂苷类化合物抗肿瘤的药物用途
CN106220701A (zh) 三萜化合物及其制备方法与应用
CN102516344A (zh) 一种具有抗肿瘤活性的化合物及其制备方法和应用
CN108659089B (zh) 一种具有抗氧化作用的甾醇化合物及其在制备药物中的应用
CN102225147A (zh) 改善胰腺功能的药物和食品或饮料
CN105777854A (zh) 硫酸依替米星的药物组合物及其在生物医药中的应用
KR101745504B1 (ko) 잠분으로부터 항암 활성을 갖는 보미폴리올 및 스티그마스테롤을 분리하는 방법
CN114891058A (zh) 胡黄连葫芦烷型皂苷提取物及其在制备治疗便秘药物中的应用
WO2019119675A1 (zh) 甘薯谷甾醇糖苷饱和脂肪酸酯、提取物及其制备方法与应用
WO2006124720A1 (en) Multidrug resistant anticancer anthracyclines
CN101212979B (zh) 内脏脂肪蓄积抑制剂

Legal Events

Date Code Title Description
NENP Non-entry into the national phase

Ref country code: DE

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 16900943

Country of ref document: EP

Kind code of ref document: A1

122 Ep: pct application non-entry in european phase

Ref document number: 16900943

Country of ref document: EP

Kind code of ref document: A1