JPS5818374A - Eicosapentaenoic acid tocopherol ester - Google Patents

Abstract

NEW MATERIAL:The compound of formula (both R1 and R2 are methyl, or one of R1 and R2 is H and the other is methyl). USE:Remedy and preventive for arteriosclerosis, cardiac infarction, cerebral infarction, diabetes, menopausal disorder, etc. PROCESS:The compound of formula is prepared by esterifying tocopherol with eicosapentaenoic acid either by using a condensation agent such as dicyclohexyl carbodiimide, trifluoroacetic anhydride, etc., or by using a reactive derivative of eicosapentaenoic acid such as halide, anhydride, active ester, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a novel eicosapentaenoic acid tocopherol ester, more specifically, the following formula (1), (wherein R and R2 are both methyl groups, or one is a hydrogen atom and the other is (indicates a methyl group), which is well absorbed from the gastrointestinal tract, and is sometimes abbreviated as high surface platelet aggregation).

It is already known that eicosapentaenoic acid (hereinafter sometimes abbreviated as EPA) and its esters, ami-P, etc. are effective in treating and preventing thrombotic diseases such as myocardial infarction and cerebral infarction ( JP-A-55-15444).

However, KPA is a fatty acid with five unsaturated bonds, and EiPA, its esters, and glycerides have the property of forming peroxides extremely easily, so if they are administered to living organisms, they may be harmful to living organisms. It is predicted that lipid peroxides will increase.

Therefore, the present inventors synthesized and investigated various EP'A derivatives in order to overcome this deficiency of KPA. In particular, tocopherol is a powerful antioxidant that prevents the production of lipid peroxides in living organisms, stabilizes biological membranes, and has an activating effect on the gonads, promotion of peripheral circulation, and succinimosis. It acts on enzymes such as nick oxidase, has an effect on substance metabolism, suppresses platelet aggregation, and is currently widely used to treat adult diseases such as menopause, arteriosclerosis, venous thrombosis, and diabetes. There is. Focusing on the effects of tocopherol, we found that it is absorbed extremely well from the gastrointestinal tract compared to a mixture of EPA, tocof, and tocopherol.
Compared to EP8, the production of lipid peroxides is slightly lower, and
The present invention was completed based on the discovery that it has an extremely excellent effect on inhibiting blood clotting and platelet coagulation.

Therefore, the present invention provides an EPA that has an excellent inhibitory effect on yuzu platelet aggregation, and is also effective in treating and preventing a wide range of adult diseases such as arteriosclerosis, myocardial infarction, cerebral infarction, diabetes, and menopausal disorders.
) provides cofferol ester.

KPA of the invention) Copherol ester Id, e.g.
:,] It is produced by esterifying 1cPA and tocopherol using a conventional esterification method.

It is separated from the raw material KPA, marine oil, and fat.
Not necessarily pure F! , it doesn't have to be something. tocopherol, α-tocopherol, β-tocopherol, γ-tocopherol or a mixture thereof
Either body, 71 body, or 04 body can be used.

Esterification methods include methods using condensation side such as dicyclohexylcarbodiimir (DCC) and trifluoroacetic anhydride, methods using reactive derivatives such as EPA no-ride, anhydride, and active ester. can be used to advantage.

The results of testing the pharmacological action of the EiPA) cophyerol ester of the present invention thus obtained are as follows.

1. Absorption of BPAl and copherol esters (3 Experiments 1 Uptake of KPA into the intestinal tract: The rat air septum was removed and inverted, then 500η of it was taken and sliced into rings at approximately 5-inch intervals to prepare inverted intestinal specimens. This was mixed with 19- isotonic nutrient solution (Krepe Sourinkel).
bs-Rlnger) buffer) and added K.
PA-d/! , -α-tocopherol ester or 1jJ
11 nt of iliPA ethyl ester - gum arabic suspension was added, and oxygen (5% -Co,
Incubations were carried out for 30 and 60 minutes in air flow.

The final concentration of FiPA-d4-α-tocopherol ester or 1BPA ethyl ester in the incubation was adjusted to 1/- in terms of EPA. After incubation, the intestinal segment was removed, washed with physiological saline, and mixed with chloroform-methanol (2:
1) Add 10% of the mixed solution and homogenize with a Mr. Botter homogenizer to extract fat. Boil the extract at 300 O
After centrifugation at rpm for 10 minutes, the supernatant was taken and dried under reduced pressure.

EPA in this lipid fraction was quantified by the following method.

That is, 5% -KOH (ethanol-
water (9:1)) solution 5m/! The mixture was heated under reflux for 45 minutes, and 10 mm of water and 5 mm of hexane were added thereto, transferred to a separating funnel, and the hexane layer was removed. 6N-HO in the aqueous layer
1 to bring the pH to 1.0 or lower, and then add 55 hexane to this.
tnI! , and internal standard substance (022+.Fatty acid 31■
150-hexane) 0.3 fnt and shaken.
Fatty acids were extracted with hexane. This extraction operation is done in 3
The extracted hexane layers were combined and washed with water. Washing with water was repeated until the aqueous layer became neutral, and the hexane layer was dehydrated with anhydrous sulfuric acid (IJ), then transferred to a small vial and dried under a stream of fluorine. 14%B in this vial
F3-methanol-benzene (35:35:30) solution 0.27! was added, the mixture was sealed tightly, and the mixture was heated at 100°C for 10 minutes to effect methyl esterification. After cooling this for 10 minutes, add 0.2 mg of water and 0.4 mg of hexane.
After shaking for a minute, the hexane layer was injected into a gas chromatograph to quantify EPA methyl ester.

A DEGS A Om capillary column was used for the gas chromatograph. The column temperature was 18,000''C-◇ The results are shown in Table 1.As shown in Table 1, when the incubation was carried out for 30 minutes, and when it was incubated for 60 minutes, -at
-α-Tocopherol ester showed higher absorption rate than EPA ethyl ester.

Table (11) Experiment 2] 1 [iP'A blood concentration: 1 group of 5 rabbits, 2 groups of 10 Japanese white rabbits in total were used, and they were fasted for 24 hours. A-d
i-α-tocopherol ester, and one other group includes E
Blood concentrations of EPA were compared when PA ethyl ester and dt-α-tocopherol mixtures were administered. The dose of each drug was 500+Iui/Kr in terms of EPA, and the drug was forcibly administered orally. Blood was collected from the ear vein using a heparinized syringe immediately before administration and 1.6.5.7.10.24 hours after administration.

This blood was centrifuged at 300 Orpm for 10 minutes to separate plasma, which was separated by 0.5 m/! 8 m7 of chloroform-methanol (2:1) mixture! was added and shaken for 10 minutes to extract lipids. The amount of BPA in this lipid was quantified using the same method as in Experiment 1, and 1! ! The PA blood concentration is expressed in μfAnl.

The results are shown in Figure 4. As shown in Figure 4, blood FiPAJ levels are higher when IPA is administered as tocopherol ester than when administered as a mixture of BPA ethyl ester and tocopherol;
FiPA-dt-α-tocopherol ester is EPA
It became clear that absorption was better than ethyl ester.
c.

■ Effect experiment on platelet aggregation 6 Wistar male rats were used in each group with 5 rats, total of 15 rats in 3 groups.
The effect of BPA-dt-α-tocopherol ester on platelet aggregation was investigated using F! A comparison was made with the case of PA ethyl ester.

Among these, one group received KPA-dt-α-tocopherol ester, and the other group received 7 mg of HaFiPA ethyl ester, which was 100 mfKq per day in terms of KPA.
The drug was administered orally by force. The remaining group served as a control group, to which physiological saline was administered. Drugs once a day
Administration was continued for several months, and blood was collected after one month and the platelet aggregation ability was compared using a collagen probe.

Platelet aggregation ability was measured by the following method. Section 3.8 - Sodium citrate 0.54 Blood was collected from the abdominal vena cava of the rat using a syringe for 4.5 days. This is 103 Orp
The upper layer was collected as platelet-rich plasma (PRP), and the remaining lower layer was centrifuged at 300 m
The mixture was centrifuged in Orpm for 10 minutes, and the upper layer was used as platelet-poor plasma (PPP). Platelet aggregation ability was measured using a commonly used platelet aggregation ability according to the method of Porno and O'Prien. That is, a constant concentration of an aggregation-inducing substance (collagen) was added to PRP, kept at 67°C, stirred, and the increase in light transmittance due to aggregation was recorded using an attached recording device.

Table 2 shows the aggregation rates when the final concentration of collagen was 2 and 4 μf//me. As is clear from Table 2, the aggregation rate in the drug administration group was lower than that in the control group, indicating that EP'A had an inhibitory effect on platelet aggregation. Furthermore,
wpp, -dt-α-tocopherol ester,! :E
In comparison with PA ethyl ester, KPA-dt-α-
It has become clear that tocopherol ester has a stronger inhibitory effect on platelet aggregation.

Table 2 L-lipid peroxide formation experiment 4 Immediately after blood collection in experiment 6, the liver and kidneys of the animals were removed, and the amount of lipid peroxide in the organs was measured.

Lipid peroxide is malondialdehyde produced by peroxidation of fat, as described by Yagi et al. (``Vitamin'', Volume 49,
403 Negative, 1957) by measuring the fluorescence caused by the thiobarbylic acid reactant.

The results are shown in Table-3. As shown in Table 3 (
The K P A -dt-(χ-tocopherol ester administration group produced a smaller amount of malondialdehyde in the tissues than the other groups, and EPA dt-α-tocopherol ester was found to be effective against lipid peroxide formation. It has been suggested that it has a protective effect.

Table-3 Next, an example will be given and explained.

Example 1 5.8,11°14.17 in a brown 200-volume flask
- 5.43 f of eicosapentaenoic acid was added, and 20 ml of methylene chloride was added thereto and stirred to dissolve it. To this was dissolved in 60 me of methylene chloride, 7.73 r of 9dt-α-tocopherol was added, and further DOO4,1[
] and 4-dimethylaminopyridine 0.658'I
The mixture was allowed to react for 24 hours in a ventilated air stream under agitation. After the reaction, the reaction solution was poured into a column packed with 300v of Keyvirchill, and column chroma +-4 was performed using benzene as an eluent.
t (yield 76%) was obtained.

Elemental analysis value calculated value (ch): 082.29. Hlo, 99 actual value (%
):082.23. Hlo, 94 Infrared absorption spectrum: Figure 1 1.30-NMR spectrum: Figure 2 IH-NMR spectrum: Figure 3 MS dual molecular weight peak 714 Solubility: Insoluble in water, soluble in ethanol, benzene, hexane Condition: Pale yellow oil Example 2 5.8.1'l in a brown 300fnl flask.

14. Add 2.25 r of 17-eicosapentaenoic acid and add 50 mj of benzene! was added and stirred to dissolve. To this was added 5.85% of trifluoroacetic anhydride under stirring, and the mixture was reacted for 40 minutes at room temperature in a stream of 9 atoms, and then 3.85% of at-α-tocopherol dissolved in benzene 50% was added.
611 was added thereto, and the reaction was further continued for 24 hours with stirring. After the reaction, in order to neutralize and hydrolyze trifluoroacetic acid and the by-product tocopherol trifluoroacetate, 1% was added to the reaction solution.
0% carbon dioxide) was added to the mixture at 0'O and stirred. The benzene layer was taken, washed with water, the benzene was distilled off under reduced pressure, and a small amount of benzene was added to the residue.
502 into a column packed with hexane-ether (
9:1) to obtain 5,8,11°14.17-eicosapentaenoic acid/dt-α-tocopherol ester 2.73f (yield 51%).

Elemental analysis value divided value (chi) +082.29. Hlo, 99 Actual value (chi): C82, 25, Hlo, 95

[Brief explanation of the drawing]

Figure 1 is the infrared absorption spectrum of fd eicosapentaenoic acid/dt-α-tocopherol ester, Figure 2 is the same as 1
39-N MR spectrum, Figure 6 shows the same IH-N
MR spectra are shown, and FIG. 4 shows a comparison of blood concentrations when eicosapentaenoic acid dt~α-tocopherol ester and eicosapentaenoic acid ethyl ester were orally administered. Above (15)

Claims (1)

[Claims] 1. Eicosapentaenoic acid represented by the following formula (1) (in which R1 and R3 are both methyl groups, or one is a hydrogen atom and the other is a methyl group) Tocopherol ester.