24-disubstituted cholesterol glycoside

Abstract

The present invention addresses the problem of providing a novel active ingredient that is capable of selectively inducing cell death of cancer cells in a nutritionally depleted state. Specifically disclosed is a 24-disubstituted cholesterol glycoside represented by the structural formula below. The 24-disubstituted cholesterol glycoside is a solution for the problem and is a novel active ingredient that is capable of selectively inducing cell death of cancer cells in a nutritionally depleted state.

Description

24-position disubstituted cholesterol glycoside

[0001] This invention relates to a 24-position disubstituted cholesterol glycoside.

[0002] In solid tumors, cancer cells promote angiogenesis within the tumor and proliferate by utilizing oxygen and nutrients supplied by the bloodstream. However, because their proliferation is rapid, angiogenesis is insufficient in the deeper parts of many tumors, leading to a state of nutrient starvation characterized by hypoxia and nutrient deficiency. However, it is known that some cancer cells can acquire resistance by adapting to the harsh environment of nutrient starvation, and maintain their proliferative capacity. In recent years, cancer treatment methods that inhibit the nutrient starvation tolerance of such cancer cells have attracted attention. Therefore, efforts are being made to find active ingredients that can selectively induce cell death in cancer cells under nutrient starvation conditions. For example, Patent Document 1 reports that kigamicin, found in actinomycetes, has the property of selectively inducing cell death in cancer cells under nutrient starvation conditions. However, the search for active ingredients that can selectively induce cell death in cancer cells under nutrient starvation conditions remains a significant undertaking.

[0003] Japanese Patent Publication No. 2004-137175

[0004] Therefore, the present invention aims to provide a novel active ingredient that can selectively induce cell death in cancer cells under a nutrient-starved state.

[0005] In view of the above points, the inventors conducted diligent studies and discovered a 24-position disubstituted cholesterol glycoside from Plumeria rubra, a deciduous tree belonging to the genus Plumeria in the family Apocynaceae, which can selectively induce cell death in cancer cells under nutrient starvation conditions.

[0006] Based on the above findings, the present invention is a 24-position disubstituted cholesterol glycoside represented by the following structural formula, as described in claim 1.

[0007]

[0008] Furthermore, as described in claim 2, the present invention is an antitumor agent comprising a 24-disubstituted cholesterol glycoside represented by the following structural formula or a pharmaceutically acceptable salt thereof as an active ingredient.

[0009]

[0010] Furthermore, as described in claim 3, the present invention is a method for obtaining a 24-position disubstituted cholesterol glycoside represented by the following structural formula by isolating and purifying it from Jasminum jasminoides.

[0011]

[0012] According to the present invention, it is possible to provide a novel active ingredient that can selectively induce cell death in cancer cells under a nutrient-starved state.

[0013] The 24-position disubstituted cholesterol glycoside of the present invention １ H-NMR and １３ This is a table showing the results (assignments) of C-NMR. This is a graph showing the selective cell death induction activity of the 24-position disubstituted cholesterol glycoside of the present invention against nutrient-starved HeLa cells.

[0014] The 24-disubstituted cholesterol glycoside of the present invention, 24-hydroperoxy-24-vinylcholesteryl glucoside, can be obtained, for example, by isolation and purification from Jasminum nigrum. When obtaining the 24-disubstituted cholesterol glycoside of the present invention from Jasminum nigrum, Jasminum nigrum may be grown naturally or cultivated by producers. The isolation and purification of the 24-disubstituted cholesterol glycoside of the present invention from Jasminum jasminoides can be carried out using general methods for isolating and purifying organic compounds contained in plants, such as extraction using water or organic solvents (such as monoalcohols exemplified by methanol, ethanol, and isopropanol, or polyhydric alcohols exemplified by 1,3-butylene glycol, ethers, hexane, ethyl acetate, acetonitrile, etc., which may include water), separation using ion exchange resins, nonionic adsorption resins, gel filtration chromatography, chromatography using adsorbents such as activated carbon, alumina, and silica gel, and high-performance liquid chromatography, as well as crystallization, vacuum concentration, and freeze-drying, either alone or in appropriate combinations. Furthermore, the 24-disubstituted cholesterol glycoside of the present invention can also be synthesized by organic synthesis chemical methods. Note that since the 24-disubstituted cholesterol glycoside of the present invention has multiple chiral carbons, various stereoisomers and optical isomers may exist, but all of them are included within the scope of the patent.

[0015] Examples of pharmaceutically acceptable salts of the 24-position disubstituted cholesterol glycoside of the present invention include ammonium salts, alkali metal salts such as sodium salts and potassium salts, alkaline earth metal salts such as calcium salts and magnesium salts, and amino acid salts such as arginine salts and lysine salts.

[0016] The 24-disubstituted cholesterol glycoside of the present invention can be administered orally or parenterally to humans (cancer patients) as a pharmaceutical (antinomatous agent), for example. Oral administration forms include tablets, granules, and capsules, while parenteral administration forms include injections. However, the formulation composition in these forms is not particularly limited, and commonly known forms can be used. The dosage of the 24-disubstituted cholesterol glycoside of the present invention can be appropriately determined based on the age, sex, and severity of symptoms of the target person. By administering an appropriate dosage, it is possible to expect an effect that selectively induces cell death in cancer cells under a nutrient-starved state. Furthermore, the 24-disubstituted cholesterol glycoside of the present invention may be consumed by adding an effective amount sufficient to exert an effect of selectively inducing cell death in cancer cells under a nutrient-starved state to various forms of food and beverages (including supplements). The 24-disubstituted cholesterol glycoside of the present invention can also be administered or consumed in the form of a solvent extract from Jasminum nigrum containing the 24-disubstituted cholesterol glycoside of the present invention.

[0017] The present invention will be described in detail below with reference to examples, but the present invention is not limited to the following description.

[0018] Example 1: (1) Isolation and purification of the 24-disubstituted cholesteryl glycoside of the present invention from Indoskea monophylla Using the selective cell death-inducing activity against HeLa cells in a nutrient-starved state as an index, the 24-disubstituted cholesteryl glycoside of the present invention was obtained from the flowers of Indoskea monophylla. Specifically, 80% ethanol was added to a dried (commercially available) powder of the flowers of Indoskea monophylla in a 15-fold amount and refluxed to obtain an extract. After removing ethanol under reduced pressure, the extract was freeze-dried to obtain a dried product (80% ethanol extract of the flowers of Indoskea monophylla). 166 g of the obtained dried product was dissolved in 17.4 L of ethyl acetate, and then 5.8 L of water was added for liquid separation treatment. The ethyl acetate layer was recovered and concentrated to dryness. 56.6 g of the obtained dried solid was dissolved in 4.2 L of 90% methanol, and then 14.2 L of hexane was added for liquid separation treatment. The 90% methanol layer was recovered and concentrated to dryness. 25.9 g of the obtained dried solid dissolved in methanol was eluted with a methanol / aqueous solvent by ODS column chromatography, and the 100% methanol elution fraction was purified by high performance liquid chromatography (methanol / aqueous solvent) using an ODS column to isolate 12.2 mg of the 24-disubstituted cholesteryl glycoside of the present invention, which is the active ingredient, as a white powder. The molecular weight (606.4) and molecular formula (C ３５ H ５８ O ８ ) of the 24-disubstituted cholesteryl glycoside of the present invention were determined by HR-ESI-MS measurement. The chemical structure was determined from various NMR ( １ H-NMR, １３ C-NMR, DEPT, COSY, NOESY, HMQC, HMBC) spectra. Figures 1 shows the １ H-NMR and １３ C-NMR results (assignments).

[0019] The selective cell death-inducing activity against HeLa cells in a nutrient-starved state used as an index was evaluated based on a comparison of the effects of the sample on HeLa cells cultured at high density (cultured in a nutrient-starved environment by densely populating the cells) and at low density. Specifically, HeLa cells (RIKEN Cell Bank) were seeded in a 96-well plate at 1.0×10 ５Cell seeding and high-density culture, with 0.5 × 10 per well. ４ Cells were seeded and cultured at low density. Cell seeding was performed using 200 μL of culture medium per well (3.8 g of AccuDia (trade name) D-MEM medium 2 (Shimadzu) dissolved in 400 mL of pure water, with the addition of 4 mL of 10% sodium bicarbonate solution, 4 mL of 200 mmol / L L-glutamic acid solution, 4 mL of antibiotic-antimicrobial agent mixture, and 40 mL of fetal bovine serum). Cell culture was performed at 37°C and 5% CO2. ２ The experiment was conducted under specific conditions. After 24 hours, the sample was added to the wells to the predetermined concentration (non-liquid samples were dissolved in methanol before addition, and liquid samples were dried and then dissolved in methanol before addition). After a further 72 hours of incubation, cell viability was measured using the MTT method. The effects of the sample on cells cultured at high and low density were compared, and the sample was evaluated based on whether or not it had selective cell death induction activity against cells cultured at high density. Cell viability was measured using the MTT method by adding 30 μL of 5% MTT to the well and culturing for 4 hours to generate a formazan precipitate. After removing the medium, the formazan precipitate was dissolved in 150 μL of DMSO, and the absorbance at 540 nm was measured. The validity of this evaluation method was confirmed by using rotenone, which is known to have selective cell death induction activity against cancer cells in a nutrient-starved state, as a positive control.

[0020] (2) Selective cell death induction activity of the 24-disubstituted cholesterol glycoside of the present invention on nutrient-starved HeLa cells Figure 2 shows the effect of the 24-disubstituted cholesterol glycoside of the present invention on HeLa cells cultured at high density and low density (the vertical axis is the relative ratio with the cell viability when methanol alone was added to the well set to 100%). As is clear from Figure 2, the 24-disubstituted cholesterol glycoside of the present invention had a concentration-dependent selective cell death induction activity on cells cultured at high density.

[0021] Formulation Example 1: Tablets comprising the following components were manufactured by a known method to selectively induce cell death in cancer cells under nutrient starvation conditions. The components are: 24-disubstituted cholesterol glycoside of the present invention; 1 Lactose; 80 Magnesium stearate; 19

[0022] Formulation Example 2: Capsules A capsule containing a composition having the effect of selectively inducing cell death in cancer cells under nutrient starvation conditions, consisting of the following components, was manufactured by a known method (unit: wt%). 24-disubstituted cholesterol glycoside of the present invention 5 Lactose 40 Potato starch 50 Hydroxypropyl methylcellulose 3 Magnesium stearate 2

[0023] Formulation Example 3: A biscuit having the effect of selectively inducing cell death in cancer cells under nutrient starvation conditions, consisting of the following components, was manufactured by a known method (unit: wt%). 24-position disubstituted cholesterol glycoside of the present invention 1 Cake flour 32 Whole egg 16 Butter 16 Sugar 24 Water 10 Baking powder 1

[0024] The present invention has industrial applicability in that it can provide a novel active ingredient that can selectively induce cell death in cancer cells under nutrient starvation conditions.

Claims

1. A 24-position disubstituted cholesterol glycoside represented by the following structural formula.

2. An antitumor agent comprising a 24-disubstituted cholesterol glycoside represented by the following structural formula or a pharmaceutically acceptable salt thereof as an active ingredient.

3. A method for obtaining a 24-position disubstituted cholesterol glycoside represented by the following structural formula by isolating and purifying it from Jasminum jasminoides.

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