JPH04290899A - Cardenolide compound - Google Patents

Abstract

PURPOSE:To obtain a new cardenolide compound, composed of a glycoside, extracted and isolated from a herb medicine teen-ped grown in Thailand, having cardiotonic activity and useful as a therapeutic agent for improving cardiac insufficiency, arrhythmia, etc. CONSTITUTION:Extracting operation of teen-ped grown in Thailand is performed using methanol and the resultant extract solution is then concentrated under reduced pressure to provide a concentrate, which is subsequently distributed with n-hexane. The obtained methanol layer is separated and the methanol is distilled away under reduced pressure to distribute the residue with ethyl acetate and water. The aqueous layer is further distributed with n-butanol to afford an n-butanol fraction, which is subsequently fractionated by centrifugal liquid-liquid partition chromatography using 5:5:1:4 composition of chloroform : methanol : n-butanol : water of a distributing solution. Thereby, the objective 3-[(O-glucopyranosyl-(1 6)-O-glucopyranosyl-(1 4)-6-deoxy-3-O-methyl- glucopyranosyl)oxy]-7-ene-14-hydroxycard-20(22)-enolide expressed by the formula.

Description

[Detailed description of the invention]

0001

[Industrial Application Field] The present invention provides a novel 3-[(O-glucopyranosyl-(1→6)-) having cardiotonic activity.
O-glucopyranosyl-(1→4)-6-deoxy-3-O-methyl-glucopyranosyl)oxy]
-7-ene-14-hydroxy-5-cardo-2
0 (22)-enolide { 3-[( O-Gluc
opyranosyl- (1→6)- O-gluc
opyranosyl- (1→4)-6-deox
y-3-O- methl-glucopyra
nosyl)oxy]-7-ene-14-
hydroxy-5- card-20 (22)-
(hereinafter referred to as "cardenolide compound") and a method for producing the same.

0002

[Prior Art] Cardiac glycosides are mainly effective in treating congestive heart failure, and digitalis therapy is said to be the most effective for treating high blood pressure and certain arrhythmias (original author, W.
．． H. Lewis, M. P. F. Elvin Lewis;
Translated by Yasuo Otsuka and Sotetsu Cho, “Clinical Medicine and Medicinal Plants”
113 pages, (1985), Enterprise (
Co., Ltd.). The reason for this is that digitalis has the following effects: (1) enhancing myocardial contractility, (2) stimulating the vagus nerve, and (3) inhibiting atrioventricular nodal conduction. In other words, when used in the treatment of heart failure, it is thought to have an effect of improving hemodynamics by increasing cardiac output, and in the treatment of tachycardial atrial fibrillation, it is thought to contribute to bradycardia and atrioventricular conduction suppression. (Masatoshi Kawana, Eiichi Hosoda, Japan Medical Information, No. 3475, p. 3, (1990)). For this reason, more than 3 million heart patients in the United States regularly use the cardiac glycoside digoxin obtained from digitalis (original author, W.H. Lewis, M.P.
F. Translated by Elvin Lewis, Yasuo Otsuka, Sotetsu Cho,
"Clinical Medicine and Medicinal Plants", p. 113, (1985
), published by Enterprise Co., Ltd.).

[0003] Species with digitalis-like effects are widely distributed in the plant kingdom, and include Strophanthus gratu, a member of the Apocynaceae family.
Ouabain from the seeds of Nerium oleander, Oleandrin from the leaves of Nerium oleander, Thevetia neriifolia (Theve.
tia neriifolia) from tebetine, Convallaria majalis (Liliaceae)
ia majalis) from the rhizome of Tecomella nutulata (Tec.
Tecomine is extracted from the bark of Chei omella nudulata, a member of the Brassicaceae family.
Ranthus cheiri) from cheiranthin,
Adonis berungis (Ranunculaceae)
Adonitoxin and other substances have been obtained from the rhizomes and roots of P. verndiis (original author, W.H. Lewis, M.P.
．． F. Elvin Lewis; Translator, Yasuo Otsuka, Sotetsu Cho
, “Clinical Medicine and Medicinal Plants”, p. 114, (198
5 years) Published by Enterprise Co., Ltd.).

0004

[Problems to be Solved by the Invention] The present inventors have developed a herbal medicine called Teen-ped from Thailand.
mul Sapphakhun Ya Thai (M
edicinal Uses of Thai Dru
gs)”, ed. by Phol Phaetthan
esura, Samakhon Rongrien P
haet Boran (The Association
on of the School of Old-s
style medicine), Bangkok,
Thailand (1964, 1967 and 1
The present invention was completed based on the discovery that a cardenolide compound having strong cardiotonic activity is present in the extract of A. 969 )].

[0005]

Means for Solving the Problems The cardenolide compound having formula (I) of the present invention has the following structural formula.

[0006]

[Chemical formula 3]

The cardenolide compounds of formula (I) of the present invention have various isomers. In the above formula, all of these isomers and mixtures of these isomers are represented by a single formula. Therefore, the present invention includes all of these isomers and mixtures of these isomers.

The cardenolide compound of formula (I) of the present invention can be obtained by extraction from the crude drug Teen Ped. Extraction is performed according to methods used for general animals and plants. That is, the herbal drug Teen Ped is cut into small pieces, water, alcohols such as methanol and ethanol, organic acid esters such as ethyl acetate, and halogenated hydrocarbons such as methylene chloride and chloroform. aromatic hydrocarbons such as benzene, toluene,
Alternatively, extract with a mixed solvent of these and concentrate the obtained extract to an appropriate amount. Next, water is added to the obtained residue as necessary, and the aqueous solution is extracted with a low polar organic solvent that separates into two phases from the aqueous solution, such as n-hexane or petroleum ether, and transferred to an organic solvent. The fat-soluble components are removed, and then the alcohol-water layer is distilled off. The residue obtained in this way is dissolved in water, extracted with a low polar organic solvent that separates into two phases with water, such as chloroform, ether, and ethyl acetate, and the organic solvent layer is distilled off under reduced pressure. A fraction containing a cardenolide compound having formula (I) is obtained. The fraction thus obtained can be further purified by various types of countercurrent partition chromatography using partitioning. For example, centrifugal liquid chromatography (K.Hos
Tettman et al. , “Preparati
ve Chromatography Techniq
ues”, Springer Verlag, Be
Rlin, 1986), a solvent system that separates into two phases, such as a solvent system obtained by appropriately mixing a polar solvent such as water or a lower alcohol with a low polar organic solvent such as chloroform or ethyl acetate, is used as a stationary phase. It can be used as a mobile phase for effective purification. Alternatively, partition column chromatography using cellulose such as Avicel, Sephadex LH 20 (manufactured by Pharmacia), Diaion (manufactured by Mitsubishi Kasei Corporation), or silica gel, alumina, magnesium-silica gel-based Florisil, etc. It can also be purified by adsorption chromatography using a carrier. The cardenolide compound having the formula (I) of the present invention thus obtained is further subjected to reverse phase chromatography using octadecyl or octylated silica gel as a carrier, and
Purification can also be carried out using a solvent mixed with water, acetonitrile, or the like as a developing solvent. Alternatively, it can be purified by an extraction purification method that utilizes the difference in the distribution ratio of the target compound and the mixed impurities to the solvent. By repeating the above purification operations alone or in appropriate combinations, a pure product of the target compound can be obtained.

[0009]

[Action] The cardenolide compound of formula (I) of the present invention has cardiotonic activity and is used to improve heart failure and treat arrhythmia. Its administration forms include oral or subcutaneous injection in the form of tablets, capsules, powders, granules, etc., either by itself or mixed with appropriate pharmaceutically acceptable carriers, excipients, and diluents according to conventional methods; It can be administered parenterally, such as by intravenous injection or intramuscular injection. The dosage varies depending on the target disease, route of administration, frequency of administration, etc., but for adults, for example, it is preferable to administer 1 mg to 1000 mg per day, once or in divided doses, depending on the symptoms.

0010

EXAMPLES Next, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited thereto. Example 1. 3β-[(O-β-D-glucopyranosyl-(1→6)-O-β-D-glucopyranosyl-(1→4)-6-deoxy-3-O-methyl-α-L-glucopyranosyl)oxy] -7-ene-14-hydroxy-5β-caldo-20 (
22)-enolide

[0011]

[C4]

[0012] Teens purchased at the Bangkok market in Thailand.
Extraction operation was performed using 40 liters of methanol for 10 kg of PED. This extract was concentrated to about 1/4 under reduced pressure, and the concentrated solution was partitioned between 5 liters of n-hexane. The methanol layer was separated and methanol was distilled off under reduced pressure. The resulting residue was mixed with 5 liters of ethyl acetate and 5 liters of ethyl acetate.
liter of water, and the aqueous layer was further partitioned with n-butanol. Of the obtained n-butanol fraction (82 g)
47 g was fractionated using centrifugal liquid-liquid partition chromatography. That is, the composition of the distributed liquid is chloroform: methanol: n-butanol: water = 5: 5
: 1:4, the rotation speed was 400 rpm, and the flow rate was 24 ml/min. The activity of the obtained fraction was measured using the contraction force of guinea pig papillary muscles as an index, and the active fraction was determined as 5.6.
g was obtained. This fraction was transferred to a reverse phase Rover column (
Manufactured by Merck, RP-18, φ37 mm x 440
mm) and subjected to reverse phase partition chromatography. 50% aqueous methanol was used as the solvent. The active fractions were collected, the solvent was distilled off, and further separated and purified using high performance liquid chromatography. In other words, the reverse phase Senshu-Pack ODS-5251-S (
(manufactured by Senshu Kagaku Co., Ltd.), methanol: water = 4
5:55, flow rate 6ml/min, detection wavelength 2
When fractionated under 20 nm conditions, the target compound 30
mg was obtained.

Physical and chemical properties of the target compound 1. Properties of substance: colorless powder, neutral. 2. Specific optical rotation: [α]D -61.4° (c = 1
．． 09, methanol) 3. Molecular weight: 856 (measured by FAB-MS method)4. Ultraviolet absorption spectrum: λmax nm (l
og ε) 215 (4.25) (in methanol) 5
．． 13C-Nuclear Magnetic Resonance Spectrum: 13C-Nuclear Magnetic Resonance Spectrum (67.8 MHz) measured in δ ppm heavy methanol using tetramethylsilane as an internal standard.
) is as follows. 16.6(q), 18.3(q), 22.9(t), 2
5.2(q), 28.2(t), 28.8(t), 29
．． 7(t), 31.6(t), 32.7(t), 35.
0(d), 35.1(s), 36.1(d), 40.1
(t), 40.7(t), 51.7(d), 52.1(
s), 61.3(q), 62.8(t), 68.0(d
), 70.6(t), 71.6(d), 72.0(d)
, 74.2(d), 75.2(d), 75.3(d),
75.3(t), 75.6(d), 77.0(d), 7
7.9(d), 78.0(d), 78.0(d), 81
．． 4(d), 85.4(d), 86.2(s), 99.
2(d), 104.4(d), 105.1(d), 11
7.8(d), 119.0(d), 139.9(s),
177.2 (s), 178.3 (s) 6.1 H-nuclear magnetic resonance spectrum: 1 H-nuclear magnetic resonance spectrum measured in δ ppm heavy methanol using tetramethylsilane as an internal standard. (270 MHz) is as follows. 0.83 (3H, s), 0.88 (3H, s), 1.2
4 (3H, d, J = 6Hz), 1.4-2.6 (18H
, m), 2.91 (1H, dd, J=6 and 9Hz)
, 3.1-3.9 (28H, m), 4.14 (1H, d
, J=10Hz), 4.38 (1H, d, J=8Hz)
, 4.62 (1H, d, J = 8Hz), 4.75 (1H
, d, J = 4Hz), 4.93 (1H, dd, J = 1 and 18Hz), 5.07 (1H, dd, J = 1 and 18Hz), 5.84 (1H, d, J = 6Hz) ,5.
92 (1H, bs.)7. Solubility: Soluble in methanol and ethanol. Relatively soluble in water. Slightly soluble in n-hexane and chloroform. 8. High performance liquid chromatography: Separation column; Senshu Pack ODS-5251-
S (particle size, 5 microns, column size, φ20 m
m x 250 mm (manufactured by Senshu Kagaku Co., Ltd.)
) Mobile phase: methanol:water = 45:55 Flow rate: 6 ml/ml Detection wavelength: UV 220 nm Column temperature: 25°C, elution time: 103.8
A peak of the target compound could be observed at the minute position.

[0014]

[Effect of the invention] Example 1. A cardiotonic activity test was conducted on the cardenolide compound obtained using guinea pig papillary muscle. Test example 1. Male heart-tray guinea pig (300-450
g) The right atrium was excised and immediately insufflated with 95% oxygen and 5% carbon dioxide at 37°.
Krebs-Henseleit solution of C.
The cells were suspended in an organ bath filled with 20 ml of seleit solution. The composition of Klebes-Henseleit liquid is as follows. Sodium chloride 118.0
mM potassium chloride
4.8 mM calcium chloride
2.5 mM potassium dihydrogen phosphate
1.0mM magnesium sulfate
1.2mM sodium bicarbonate
27.2mM glucose
The contractile force of 11.1 mM was measured using a contractile force measuring device (trade name: FD-pickup; manufactured by Nihon Kohden Co., Ltd.). The test drug was dissolved in dimethyl sulfoxide (DMSO) and administered cumulatively to measure the activity. The results are shown in the table. −−−−−−−−−−−−−−−−−−−−−−−−−
−−−−−−−−−− Concentration g/ml
10-6 3×10-6
10-5 3×10-5
−−−−−−−−−−−−−−−−−−−−−−−−
−−−−−−−−−− Contraction force CF (%)
5 19
88 135
Heart rate BR (%) -1
1 20
40 ----------
−−−−−−−−−−−−−−−−−−−−−−−−−
--- From the table, the cardenolide compound having formula (I) of the present invention exhibits excellent cardiotonic activity.

Claims (3)

[Claims] Claim 1: 3-[( O-glucopyranosyl- (1
→6)- O-glucopyranosyl- (1→4)-6-
(deoxy-3-O-methyl-glucopyranosyl)
oxy]-7-ene-14-hydroxy-5-caldo-20 (22)-enolide. [Claim 2] The compound represented by claim 1 is 3β-[( O-β-D- glucopyranosyl- (1→6)- O-β-D- glucopyranosyl- (1→ 4)-6-deoxy-3-O-methyl-α-L-glucopyranosyl)oxy]-7-ene-14-hydroxy-5β-caldo-20 (
22) - Enolide. 3. A method for producing a cardenolide compound, which comprises isolating the cardenolide compound according to claim 1 or claim 2 from the crude drug Teen Ped produced in Thailand.