JPH03188088A - Novel lysophosphatidylserine - Google Patents

Abstract

NEW MATERIAL:Compounds of the formula (RCO is 10-25C acyl). EXAMPLE:0-(1-nonadecanoyl-sn-glycero-3-phospho)-N-(9- fluorenylmethyloxycarbonyl)-L-serine 9-fluorenylmethyl ester. USE:An inhibitor for Na<+>, Ka<+>-ATP ase. PREPARATION: From a glycerol derivative and a serine derivative, a phosphite ester is synthesized using the phosphomonochloromonoamidite method and the resultant phosphite ester is then treated with hydrogen peroxide to obtain a phosphoric diester. Methyl groups as protective groups for phosphoric acid and isopropylidene groups as protective groups for glycerol are eliminated therefrom and the resultant compound is subjected to condensation with an anhydride of a mixed acid prepared from various kinds of fatty acids and isobutyl chlorocarbonate, followed by elimination of protective groups for serines in the obtained condensate.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a novel zophosphatidylserine and its salts having an inhibitor of NB", K"-ATPase.

[Conventional technology]

Morphine is the main component of opium alkaloids contained in unripe poppy fruits, and is known for its excellent anesthetic and analgesic effects. Receptors that bind to morphine exist in living organisms, and enkephalins were discovered through a search for endogenous analgesic substances that specifically bind to these morphine receptors.

Digitalis is a cardiac glycoside obtained from a plant of the Physaceae family, which has cardiotonic, blood pressure increasing, and vasoconstricting effects, and is used as a therapeutic agent for heart failure. As in the case of enkephalins, endogenous digitalis-like active substances exist in living organisms, and in animal cells Na', K'''-, which are ion pumps integrated into cell membranes for the active transport of Na and Ni, are present. Regulating ATPage activity,
It is believed that it is involved in the onset of essential hypertension by regulating natriuric excretion, and various reports have been made regarding the search for its active substance. For example, Na” + K from bovine adrenal glands by Tamura et al.
” - Attempt to purify TPase inhibitor (M, Ta
Mura et al.

Bioche+++1stry, 27.4244 (
(1988)), an attempt by Goto et al. to purify a digitalis-like active substance present in dog plasma (8. Goto et al.
et al-+ Biochem, Biophys.

Res, Coimun, 152.322 (19
8B)). Attempt to purify endogenous digoxin-like active substances from human plasma by Dasgupta et al. (A, Da
Sgupta et al., Biochem.

Biophys, Res, Commun,, 14
8.623 (1987)). However, despite these attempts, the isolation of its active substance and detailed reports on its structure have not yet been made. Only Dasgupta et al. have developed an active substance, DLIS-2 (digoxin-like imm
M of unoreatjve 5ubstance-2)
The following estimated structure has been proposed by S/MS spectrum analysis. However, even in this formula, there are many unknown points regarding the position of the double bond in the acyl group portion of lysophosphatidylserine.

[Problem to be solved by the invention]

The object of the present invention is to provide a compound that has an excellent Na.+K'-ATPase inhibitory effect and has a structure closer to that of an endogenous digitalis-like active substance.

[Means to solve the problem]

The present inventors synthesized lysophosphatidylserine having various acyl groups, and found that lysophosphatidylserine having a specific acyl group has an inhibitory effect on Na', K'-ATPase.
They discovered that it has a Page inhibitory effect and completed the present invention. That is, the present invention is a novel lysophosphatidylserine represented by formula (1) % formula % and its salt.

In the formula, RCO represents an acyl group having 10 to 25 carbon atoms.

The lysophosphatidylserine of the present invention was obtained by introducing various acyl groups and searching for compounds having digitalis-like activity. That is, a phosphorous acid ester is prepared by the phosnimonochloromonoamidite method using a glycerol derivative and a serine derivative, and then treated with hydrogen peroxide to obtain a phosphoric acid diester. After removing the methyl group, which is a protective group for phosphoric acid, and the isopropylidene group, which is a protective group for glycerol, this phosphoric acid diester is condensed with a mixed acid anhydride prepared from various fatty acids and isobutyl chlorocarbonate. , can be produced by removing the protecting group of serine in the resulting condensate.

Furthermore, the lysophosphatidylserine of the present invention is purified according to conventional methods. Examples include preparative f311i chromatography, silica gel chromatography, high performance liquid chromatography, and affinity chromatography.

The lysophosphatidylserine of the present invention can be prepared using various alkali metal and alkali +::metallic salts, sodium salts, calcium salts, Lys salts, etc. according to conventional methods. Furthermore, salts can also be obtained by treatment with inorganic or organic acids. These salts include:
Inorganic acid salts such as hydrochloride, hydrobromide, hydroiodide, sulfate, phosphate and acetate, trifluoroacetate, citrate, maleate, fumarate, tartrate, Examples include organic acid salts such as lactate, methanesulfonate, and p-toluenesulfonate.

The present invention's lysophosphatidylserine or its salt exhibits an excellent Na", K"-ATPage inhibitory effect, and has the same or similar structure to endogenous digitalis-like active substances, so it is more effective than highly toxic cardiac glycosides. It can be used as a cardiotonic agent with few side effects, a blood pressure regulator, and a natriuric excretion regulator.

The dosage of the lysophosphatidylserine or its salt of the present invention varies depending on the type of compound, administration method, patient's symptoms, age, etc., but is usually 1 time to 11,000 hours.
r, preferably 0.01 to 10 cm, 1 to 3 times per day. The lysophosphatidylserine or salt thereof of the present invention is usually administered in the form of a preparation prepared by mixing it with a pharmaceutical carrier. As the pharmaceutical carrier, a substance that is commonly used in the pharmaceutical field and does not react with the present invention's lysophosphatidylserine or its salt is used.

Dosage forms include tablets, capsules, granules, powders, syrups, suspensions, suppositories, ointments, creams, gels, patches, inhalants, injections, and the like. These formulations are prepared according to conventional methods.

In addition, in the case of a liquid preparation, it may be in the form of being dissolved or suspended in water or other suitable medium before use. In addition, tablets and granules may be coated by well-known methods. In the case of an injection, the lysophosphatidylserine of the present invention or a salt thereof is prepared by dissolving it in water, but it may be dissolved in physiological saline or glucose solution if necessary.
A buffer or preservative may also be added.

These preparations contain 0.2% or more, preferably 0.5 to 70%, of the present invention's lysophosphatidylserine or its salt.
It can be contained in a proportion of These formulations also
It may also contain other ingredients of therapeutic value.

EXAMPLES The present invention will be explained in more detail with reference to Examples below.

Example 1 Synthesis of O-(1-nonadecanoyl-5n-glycero-3r~monophos9'?)N(9-fluorenylmethyloxycarbonyl)-L-serine 9-fluorenylmethyl ester 7r) A, O-(sn-glycero-3-hostea)-N-(
9-Fluorenylmethoxycarbonyl)-L-serine
Synthesis of 9-fluorenylmethyl ester (synthesis of phosphoric acid diester) (11N-fluorenylmethyloxycarbonyl-0-
tert-Butyl-serine 9-fluorenylmethyl ester N-fluorenylmethyloxycarbonyl-0ter
t-Butyl-cyanoserine (5.09g, 13.3mmo
l) of THF (25 mj) under cooling in a salt-ice bath.
-Florenylmethanol (2.74g, 14.Om
mol), 4-dimethylaminopyridine (487 mg
.

3.99 mmol), DCC (2.74 g, I
After sequentially adding 3.3 tamol), the mixture was stirred for 2 hours while cooling with water, and then stirred for 3 hours at room temperature. After filtering off the insoluble matter,
Concentrate under reduced pressure, dissolve the residue in ethyl acetate and add saturated brine for 10 minutes.
% citric acid aqueous solution, saturated brine, saturated sodium bicarbonate solution, and saturated brine, and dried over magnesium sulfate. After concentration under reduced pressure, the residue was subjected to silica gel column chromatography (2
00g, purified with benzene-ethyl acetate 30:1) to give a colorless oil.

Yield 6.29g (84.3%) 0 Elemental analysis value: C, 71
,91;H,6,91;N, 1.96%.

CzbH8IsNOs ・Calculated value as 2CJsOz: C, 71,61, H, 6,97; N, 1.90%
.

(2) N-9-fluorenylmethyloxycarbonyl-L-serine Compound synthesized with 9-fluorenylmethyl ester (1) (6.29g + 11.2m
mol), 90% TFA (19.2 mj!, 0.
224 ml Iol) was added and left at room temperature for 2 hours. After concentration under reduced pressure, 90% TFA (19.2 ml, 0.2 ml) was added again.
224 mn+ol) was added and left at room temperature for 2 hours. After concentration under reduced pressure, the residue was dissolved in 90% ethanol by heating, and the precipitated crystals were recrystallized from ethyl acetate-hexane. Yield 5.45 g (96.3%). Elemental analysis value: C, 73,
16iH, 5,62; N, 2.59%.

Calculated value as C5JzJOs・HlO: 30 at room temperature
The mixture was stirred for a minute and immediately distilled under reduced pressure, with a boiling point of 65-66°C (1
, 3wmHg) were obtained. Yield (410-(C(S)
) -2,2-dimethyl-1,3-dioxolane-4-
methyl) methylphosphonaline]-N-(9-fluorenylmethy57 perfect carbonyl)-L~serine A suspension of 11.1 g of the compound synthesized with 9-fluorenyl methyl ester (2) in methylene chloride (o, 3 Mm ) to diisopropylethylamine (
10.5m 4! ), and then the compound 5.31m&' synthesized in (3) was added under water cooling. Stir at room temperature for 15 minutes, add ethyl acetate, 5% sodium bicarbonate,
Next, after washing with water, the mixture was concentrated under reduced pressure, and acetonitrile was added to the residue to give a 0.19M solution. After that, a 0.5M tetrazole-acetonitrile solution was added at room temperature, and the mixture was stirred for 1 hour. After concentrating under reduced pressure and distilling off acetonitrile, 2.85 ml of 30% hydrogen peroxide solution was added.
Stirred for 15 minutes.

Ethyl acetate was added to this, and the mixture was washed with a 5% aqueous sodium bisulfite solution and saturated brine. After concentration under reduced pressure, the remaining oil was subjected to medium pressure silica gel column chromatography (25
The resulting oil was lyophilized with dioxane to obtain a colorless amorphous powder. Yield 4.20 g (yield 76.6%) 0 Elemental analysis values: C, 65,03; H, 5,88; N, 1.85%.

C5qHa. Nor. Calculated value as P.1/2C4HsO□: C, 64,99; H, 5,85; N, 1.
85%.

(5) 0-(1,2-isopropylidene-5n-glyceride, core-3-hosty)-N-(9-fluorenylmethyloxycarbonyl)-L-serine 9-fluorenylmethyl ester sodium 2-butanone (
Sodium iodide (8,3■,
55μ+wol) was added and heated to 80°C for 5 hours. The residue was subjected to medium pressure silica gel column chromatography (9 g).

It was purified with chloroform-methanol (5:1) and lyophilized with dioxane to obtain a colorless amorphous powder.

C,60,21;)(,5,5Q;N,2.0
0%.

C3JsJO+oPNa' Calculated value as 2HzO: C, 60, 24, H, 5, 45; N, 1.85%
.

Ha(6) O-(sn-glycero-3-phos 5#)
N-(9-fluorenylmethyloxycarbonyl)L-
Compound synthesized with serine 9-fluorenyl methyl ester sodium salt (5) (21,4■, 29.7μs+
95% trifluoroacetic acid (1.25 ml) was added to a methylene chloride solution (25 ml) of oJ) and left at room temperature for 15 minutes. Concentrate under reduced pressure without heating, and add benzene to this and concentrate under reduced pressure for 2 times. Excess trifluoroacetic acid was distilled off repeatedly. The residue was lyophilized with dioxane to obtain a colorless amorphous powder. Yield: 1119.9cm (100%) 9
Elemental analysis value: C, 57,46, H, 5,10; N,
1.97%.

Cs5H! Calculated values as JO+oPNa 38zO: C, 57,14; H, 5,34, N, 1.90%.

400 MHz 'H-NMR (DMSO-d6) δ7
．． 8B (4H, d, J=7.7Hz).

7.72 (4N, +++), 7.41 (4H, br
,t,,J=8)1z), 7.30(4H,m).

5.35 (1B, br), 4.80 (IH, br)
, 4.2~4-4-4 (7H9+4.03(2)1
．． m), 3.71 (2B, m), 3.52 (2
H, m) nonadecanoic acid (10,8■, 36.2μn+o
Add triethylamine (5.0 μl, 36 μmol) and isobutyl chlorocarbonate (4.7 μl, 36 μmol) to a DMF (1 ml) solution of 1) under stirring while cooling in a salt-ice bath.
Stir for 15 minutes. This was added to the mixture of phosphoric acid diester (19.9 µ, 30.2 µ5 ol) synthesized in A and 4-dimethylaminopyridine (1,3 µ, 11 µ + 5 ol), and the mixture was stirred at room temperature for 3 hours. Concentrate under reduced pressure,
The remaining oil was purified by medium pressure silica gel column chromatography (6 g, chloroform-methanol 5:1) to obtain a colorless amorphous powder. Yield 7.0■400M1lz
'H-NMR (DMSO-di) 67.87 (4B, d, J=7
．． 5Hz), 7.71 (4H, br.

t), 7.40 (48, br, t), 7.29 (4
8, br, q), 5.80 (IH, br.

s), 4.2-4.4 (7H, m), 4.0 (48
, m), 3.73 (3H, m).

2.22 (2B, t, J=7.6)1z), 1.46
(2H, brt), 1.1-1.2 (30H, m),
0.84 (3H, t, J=7.0Hz).

Example 2 Synthesis of O-(1-arachito+Rusn-glycero-3-sho\phos#)9-N-(9-fluorenylmethyloxycarbonyl)-L-serine 9-fluorenylmethyl ester Commercially available arachidonic acid and Example 1.
It was condensed with the phosphoric acid diester synthesized in A to obtain the title compound 6.2.

Example 3 O-(1-nonadeca-5,8,11,14-tetrine 7T''X noyl-5n-glycero-3-phos#i)-N-(9-
fluorenylmethyloxycarbonyl) L-serine 9
-Synthesis of fluorenyl methyl ester Nonadeca-5,8,11,14-tetrayic acid was synthesized as follows.

(1) 2-hebutynol magnesium ribbon (7,92g, 0.326m
A solution of ethyl bromide (28.5 ml, 0.381 mol) in ether (25 ml) was added dropwise over 2 hours to a 125 mL solution of ethyl bromide (28.5 ml, 0.381 mol) under water cooling and stirring. A solution of 1-hexyne (35.0 ml, 0.305 mol) in ether (40 mj) was added dropwise to this mixture over 1 hour and 30 minutes with stirring.

Further, after stirring at room temperature for 1 hour, formaldehyde was blown into the mixture for 3 hours while stirring while cooling with water. After adding this to cold 2N sulfuric acid, extraction with ether was performed, and the ether layer was washed with saturated brine. After drying with magnesium sulfate and concentrating under reduced pressure, the mixture was distilled under reduced pressure. Yield 25.3 g (73.8%). boiling point 55-
At 57°C/0.5 flHg….

(2) 2-bromoheptine The compound synthesized in (1) (54.8 g) was stirred at room temperature.

0.489 mol) and pyridine (1,98mff1.
Phosphorus tribromide (16.7 mIt, 0.176 mol) was added to a solution of ether (380 mj)
) was added dropwise over 1 hour.

Thereafter, after heating under reflux for 1 hour and 30 minutes, the mixture was allowed to cool to room temperature, added to ice, and washed sequentially with a saturated aqueous sodium bicarbonate solution, a 5% aqueous sodium thiosulfate solution, IN hydrochloric acid, and saturated brine, followed by sulfuric acid. Dry with magnesium. After concentration under reduced pressure, it was distilled under reduced pressure.

Yield 85.6 g (100%). Boiling point 43-45℃/
1.8 m Hg.

(3) 2,5-decadiinol magnesium ribbon (6,17 g, 0.258 m
Ethyl bromide (21.1 m It , 0.2
83 mol) of THF (25 ml) was added dropwise over 20 minutes. After stirring at room temperature for 1 hour, it was cooled on ice and mixed with propargyl alcohol (7.51ml!, 0.12ml) with stirring.
A solution of 7.6 ml of T'HF (9 mol) was added dropwise over 1 hour. After the dropwise addition was completed, the mixture was incubated at room temperature for 30 minutes and at 50°C for 30 minutes.
After stirring for minutes, copper chloride (1) (273 ng, 2
．． 76 mmol), the compound (14,
1g+80.6mmol) of THF (15ml)
After adding the solution and heating under reflux for a total of 8 hours, copper(I) chloride (1
52■, 1.54 mmo)) was added thereto, and the mixture was further heated under reflux for 6 hours. After cooling, the mixture was added to cold 2N hydrochloric acid, extracted with ether, and washed with a saturated aqueous sodium hydrogen carbonate solution and saturated brine. After drying with magnesium sulfate, it was concentrated under reduced pressure and then distilled under reduced pressure. Yield 8.86g, (73.2%)
. Boiling point 101-103 °C/1-2 vnx Hg
*(4) Compound synthesized with 2,5-bromodecadiine (3) (8,00g, 53.3w
8.19 g ('71.8%) of the title compound was obtained by carrying out the same operation as in the synthesis of (2) using a + ol).

Boiling point 95-96°C/1.2 mHg.

TH of T512.5.8-bromotridecatriine magnesium ribbon (2,87 g, 0.120 mol)
Add ethyl bromide to the F (25mj) solution under a nitrogen stream.9
．． 82 ml, 0.132 mol) of THF
(12 ml) solution was added dropwise over 25 minutes while stirring with water cooling.

After stirring at room temperature for 45 minutes, propargyl alcohol (3,49 m It , 60.0 ms +
ol) in THF (3.5 ml) was added dropwise over 30 minutes. After stirring at room temperature for 2 hours, copper chloride (1) (12
7■.

1,28 ma+ol), the compound synthesized in (4) (
7.99g.

Add 37.5 meal) of THF (7mf),
The mixture was heated under reflux for 6 hours. Copper(I) chloride (71,
0 ■.

0.718 mmol) was added and heated under reflux for a total of 10 hours.

This was added to cold 2N hydrochloric acid, extracted with ether, and 5%
It was washed with an aqueous sodium bisulfite solution, a saturated aqueous sodium bicarbonate solution, and a saturated saline solution. After drying over magnesium sulfate, it was concentrated under reduced pressure, and the residue was recrystallized from hexane to obtain a pale yellow powder. Pyridine (152, cl) was added to a solution of this in ether (30 ml).

After adding phosphorus tribromide (1,28 ml, 13.5 mmol) over 20 minutes while stirring at room temperature, the mixture was heated under reflux for 2 hours. This was transferred to ice, extracted with ether, and then washed with a saturated aqueous sodium bicarbonate solution and saturated brine. After drying with magnesium sulfate, it was concentrated under reduced pressure to obtain a brown liquid. Yield 6.76g (7
1.8%).

(6) Sodium 5-hexanoic acid hydroxide (3.61g, 90.3mmo
l) in water-methanol (1: 1.24 mj solution),
5-hexynenitrile (5,00g, 53.7++
uaol) was added thereto, and the mixture was heated under reflux for a total of 15 hours in a nitrogen stream.

After cooling, unreacted nitrile was extracted with ethanol, and the water-cooled lower aqueous layer was adjusted to pH 4 with 5N sulfuric acid, extracted with ethyl acetate, and washed with saturated brine. After drying with magnesium sulfate, it was concentrated under reduced pressure and distilled under reduced pressure. Yield 5.47 g (9
1.0%). Boiling point 75-76°C/1.5 mmHg.

(7) Magnesium nonadeca-5,8,11,14-tetoraic acid ribbon (3,10μ, 127mmol
) to a THF (45 ml solution) was added water-cooled stirred ethyl bromide (1
0.9m 1. 147 mmol) of THF (17,
5 mJ) solution was added dropwise over 20 minutes. After stirring at room temperature for 30 minutes, the compound synthesized in (6) (7.13 g, 6
A solution of 3.7 + mol) of THF (6.5 mJ) was added dropwise over 15 minutes while stirring while cooling with water. After stirring at room temperature for 15 minutes, the mixture was heated to dissolve the precipitated solid, and the mixture was further stirred at room temperature for 1 hour. , copper(I) cyanide (286■, 3
．． After heating and stirring at 50° C. for 5 minutes, the compound synthesized in (5) (8,00 g, 31.
A solution of 8 mmol) in THF (10.9 mJl) was added thereto, heated under reflux for 5 hours, and then stirred at room temperature for 4 days. This was added to cold 2N hydrochloric acid, extracted with ethyl acetate, and washed with saturated brine. After drying with magnesium sulfate, petroleum ether (300 mF) was added to the oil obtained by concentration under reduced pressure, and the mixture was stirred ultrasonically for 10 minutes.The insoluble pale yellow solid obtained by this treatment was collected by filtration and 3.31 g. , 37.1%),
The mother liquor was concentrated under reduced pressure and subjected to medium pressure silica gel column chromatography (silica gel 200 g, benzene-ethyl acetate 5
Petroleum ether (100 ml) was added to the resulting oily substance, and the insoluble pale yellow solid was collected by filtration (
1.54g.

17.3%). Yield needle 4.85 g (54.4%).

270 MHz 'H-NMR (CDCI!s) 6
3.15 (6H, m), 2.48 (2H, L, J=6
．． 8Hz>, 2.26 (2H, tt, J=6.8Hz
, 2.2Hz).

2.17 (2H, tt, J=6.8Hz, 2.2Hz)
, 1.82 (2H, quintet.

J=6.8Hz), 1.5-1.3 (5H, a+),
0.89 (3H, t, J=6.8Hz).

Nonadeca-5,8°11 obtained in the same manner as in Example 1
．． 14-tetrayic acid and Example 1. It was condensed with the phosphoric acid diester synthesized in A to obtain the title compound 5.7.

Example 4 O-(1-nonadeca-5,8,11,14-tetraene+r% noyl-5n-glycero-3-phos≠clear)-N-(9-
Fluorenylmethyloxycarbonyl)-L-serine
Synthesis of 9-fluorenyl methyl ester Nonadeca-5,8,11,14-tetraenoic acid was synthesized as follows.

Quinoline (56,4μIl) with 5% palladium/barium sulfate (Ald, 20572-9) (61,7■)
was added, diluted with 99% ethanol (9 mJ), and stirred at room temperature under a hydrogen stream for 50 minutes until no more hydrogen was absorbed. To this, nonadeca-5 obtained in Example 3,
8,11,14-tetrayic acid (100■0.354
mmol) of 99% ethanol (11) was added thereto, and the mixture was stirred at room temperature under a hydrogen stream. After absorbing the calculated amount of hydrogen, the mixture was further stirred for 30 minutes. After filtering off the catalyst, it was concentrated under reduced pressure, ethyl acetate was added thereto, washed with IN hydrochloric acid and saturated brine, and dried over magnesium sulfate. After concentration under reduced pressure, the Rfo, 5 fraction was extracted with ethyl acetate using preparative silica gel thin layer chromatography (2 sheets, developed with benzene-ethyl acetate 3:1) and concentrated under reduced pressure to obtain a colorless liquid. Definitely, color magazine; Otsu 4-1. Revenue! 95.5■ (92.7%), 67
．． 5MHz, +3C-NMR (CDC1,) δ
180.02.130.42゜129.08. 12
8.74. 128.59. 128.25. 128
．． 12°127.87. 127.5B, 33.4
3. 31.84.29.2B, 27.15°26
．． 48. 25.65. 24.52. 22.35.
14.00.

Example 1 was carried out in the same manner as in Example 1. It was condensed with the phosphoric acid diester synthesized in A to obtain the title compound 7.0.

Example 5 The inhibitory activity of lysophosphatidylserine of the present invention against Na'', K''-ATPase was evaluated in the following manner.

First, prepare the reagents (11 to (5)).

(11 enzyme substrates: Na(1! (140mM), M
Cl0.52g (14mM).

Naz ・ATP ・3) 1zO (3mM), MgC
ji! 0.48g (5-) for 500mj! 50mM of
Dissolve in 7 ml of Tris-salt buffer. (0,48u
(corresponds to nit/7+nj2) (3) Inhibitor solution: 0.04 x 10-'M~4
A solution with a concentration of X10-5M is prepared in the same buffer.

(4) Molybdic acid/sulfuric acid mixture: 0.67N sulfuric acid
．． Dissolve 333χ ammonium molybdate.

(5) Reducing reagent: 2×10 mixture of 0.2 g of 1-amino-2-naphthol-4-sulfonic acid, 1.2 g of sodium sulfite, and 1.2 g of sodium bisulfite.
．． 1mj! Dissolve in water in the proportion of

(The inhibitor refers to the compound described in Example 4.) Using these reagents, the residual rate of ATPase activity was determined by the following procedure.

stimulant substrate Q, lml, inhibitor Q, lml, Tris-HCl buffer 0. ' Take 2 m j2 in a test tube,
Enzyme Q, l m I! was added and reacted at 37°C for 60ml. (Bx) Also, those that do not contain any inhibitors (Bx)
o) are reacted at the same time to achieve 100% enzyme activity. Blank (Bk) contains no enzyme and is reacted in the same manner.

0.5 m j of the aforementioned enzyme reaction solution! Add 1.5 ml of a molybdic acid/sulfuric acid mixture that has been ice-cooled in advance. May stop the reaction. If insoluble matter precipitates, 30
Centrifuge at 3000 r, p, m-min.

Transfer Lml of supernatant to another test tube and add reducing reagent 0.0 to this.
Add 5 ml and leave for 15-30 rain, then 660
Colorimetric molybdenum blue formed in nm. Calculate the residual activity using the following formula.

Residual activity (%) −(Bx−Bk)/(Do−8k)
X100 Through such operations, the residual activity of ATPase at various concentrations of the test substance was determined, and the IC6° shown in the table below was determined.

1, Co Q, 0X
From the results of 10-'M or more, it is clear that the lysophosphatidylserine of the present invention has excellent Na'', K''-ATPase inhibitory activity.

〔Effect of the invention〕

The glue zophosphatidylserine of the present invention has excellent sodium content.

It has K''-ATPase inhibitory activity and is useful as a cardiotonic agent and a blood pressure regulator.

Claims (2)

[Claims] (1) General formula (I) ▲There are mathematical formulas, chemical formulas, tables, etc.▼(I) Lysophosphatidylserine or a salt thereof. In the formula, RCO represents an acyl group having 10 to 25 carbon atoms. (2) RCO is a nonadecanoyl group, nonadeca-5,8,
11,14-tetrinenoyl group, nonadeca-5,8,
The lysophosphatidylserine according to claim 1, which is a 11,14-tetraennoyl group or an arachidonyl group.