JPH07149772A - Activity promoter of protein phosphorylated enzyme c - Google Patents

Abstract

PURPOSE:To obtain the active promoter useful for preventing and treating hypertension and hyperglycemia, having low toxicity and strong activating ability of protein phosphorylated enzyme C, comprising a compound having a specific structure as an active ingredient. CONSTITUTION:This activity promoter comprises a 1-0-acylglycerol-2,3phosphate of formula I [R is a 1-30C straight-chain or branched alkyl or a 2-30C straight- chain or branched alkenyl; M is H, an alkaline (earth) metal or a (substituted) ammoniuml as an active ingredient. For example, Na salt of 1-0-[(9S, 10R)-9,10- methanohexadecanoyl]-Sn-glycerol-2,3-phosphate may be cited as the compound of formula I.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention is a protein phosphatase C which is universally present in various tissues in the living body and is considered to be essentially involved in intracellular signal transduction and cell cycle control. The present invention relates to an activity promoter (hereinafter abbreviated as PKC).

[0002]

2. Description of the Related Art PKC is an extremely important enzyme that is ubiquitously present in the body and plays a central role in intracellular signal transduction and cell cycle control. It is also associated with cancer, immunity, inflammation, hypertension, and memory. It is believed to be related. Also, PKC is classified into many subtypes,
Many unclear points remain, such as the relationship between the subtypes and the physiological behaviors described above, or the activation mechanism.
Diacylglycerol (hereinafter abbreviated as DG), phorbol ester, and the like are well known as substances that promote the activity of PKC, and are used as important reagents for biochemical research. However, these are oncogenic promoters themselves, and there is a demand for the discovery of new activity promoters with low toxicity aiming at the development of pharmaceuticals related to PKC.

[0003]

DISCLOSURE OF THE INVENTION An object of the present invention is to provide a PKC activity-promoting agent having low toxicity and strong PKC activation ability.

[0004]

[Means for Solving the Problems] For the above reasons, the inventors of the present invention have made extensive studies on a substance that promotes the activity of PKC, and as a result, 1-O-acylglycerol-2,3, which has low toxicity, has been investigated.
-We have found that phosphate strongly enhances the activity of PKC and completed the present invention.

That is, the present invention has the general formula

[0006]

[Chemical 2]

(In the formula, R is a linear or branched alkyl group having 1 to 30 carbon atoms, a linear or branched alkenyl group having 2 to 30 carbon atoms, or a linear chain having 2 to 30 carbon atoms. Or, it represents a branched alkynyl group, and the alkyl group, alkenyl group or alkynyl group thereof may contain a cycloalkane ring or an aromatic ring, and M is a hydrogen atom, an alkali metal atom or an alkaline earth metal atom, or a substituted or The present invention relates to a PKC activity enhancer containing a compound represented by an unsubstituted ammonium group) as an active ingredient.

As the substituent R in the above formula, a methyl group,
Alkyl group such as ethyl group, propyl group, hexyl group, decyl group, pentadecyl group, octadecyl group, allyl group, butenyl group, octenyl group, decenyl group, dodecadienyl group, alkenyl group such as hexadecatrienyl group, ethynyl group, Examples thereof include alkynyl groups such as propynyl group and pentadecynyl group. Examples of the cycloalkane ring include a cyclopropane ring, a cyclobutane ring, a cyclopentane ring, a cyclohexane ring, a cyclohexene ring and a cycloheptene ring, and examples of the aromatic ring include a benzene ring, a naphthalene ring, a pyridine ring, a furan ring and a thiophene ring. be able to. Therefore, examples of the alkyl group containing a cycloalkane ring include a cyclopropylmethyl group, a cyclohexylethyl group, and 8,9-methanopentadecyl group, and examples of an alkyl group containing an aromatic ring include a benzyl group, a phenethyl group, and p-pentylphenyl group. An octyl group etc. can be illustrated. The above cycloalkane ring may contain one or more heteroatoms, and examples thereof include an oxirane ring, an oxetane ring, a tetrahydrofuran ring, an N-methylpyrrolidine ring and the like. Examples of the alkali metal atom of M include sodium and potassium, examples of the alkaline earth metal atom include magnesium and calcium, and examples of the substituted ammonium group include butylammonium group, triethylammonium group, and tetramethylammonium group. And the like.

1-O-acylglycerol according to the present invention
Specific examples of 2,3-phosphate include the following formula [I]

[0010]

[Chemical 3]

PHYLPA represented by (K. Murakami-Murofu
shi et al., J. Biol. Chem., 267, 21512 (1992).) and related substances, and the like. These compounds are described in the literature (S. Kobayashi et al., Tetrahe).
dron Letters, 34, 4047 (1993).).

[0012]

[Examples] Examples showing the activity of 1-O-acylglycerol-2,3-phosphate according to the present invention for promoting PKC activity are shown below, but the present invention is not limited to these examples. is not. The structural formula of 1-O-acylglycerol-2,3-phosphate according to the present invention used in the following examples is shown below.

[0013]

[Chemical 4]

Example 1 20 mM Tris-HCl buffer (pH 7.5), 5 mM Mg (OAc) ₂ , 50
μM CaCl ₂ , 50 μg / mL MBP _4-14 (I. Yasuda et al., Bio
Chem. Biophys. Res. commun., 166, 1220 (1990).), 1
0 μg / mL leupeptin, each concentration of 1-O-[(9S, 10R) -9,10-
Methanohexadecanoyl] -sn-glycerol 2,3-phosphate sodium salt (PHYLPA), 10 μL purified cPKCα
(D. Schaap et al., J. Biol. Chem., 265, 7301 (199
0), and DJ Burns et al., J. Biol. Chem., 265,
40 μL of a solution containing 12044 (1990). Was treated at 0 ° C. for 1 hour. Add 10 μL ATP solution (20 μM ATP, 0.5 μC
i [g- ³² P] ATP was included), and the mixture was reacted at 30 ° C. for 10 minutes. Take 40 μL of reaction solution and filter paper (Whatman, P81, 2x2cm)
And the filter paper was washed 4 times with a 75 mM phosphoric acid solution to remove free [ ³² P] ATP. The filter paper was dried and the Cherenkov line was measured with a liquid scintillation counter. From the radioactivity, the activation ability of PHYLPA, a test compound, for cPKCα was determined. Table 1 shows the cPKCα activating ability of the test compound as a relative value when the test compound was not added.

[0015]

[Table 1]

Example 2 As a test compound, 3-O-[(9S, 10R) -9,10-methanohexadecanoyl] -sn-glycerol 1,2-phosphate sodium salt ((9S, 10R) -D- Example 1 except that PHYLPA) was used
In the same manner as described above, the ability of (9S, 10R) -D-PHYLPA to activate cPKCα was determined. The results are shown in Table 2.

[0017]

[Table 2]

Example 3 1-O-[(9R, 10S) -as a test compound
9,10-Methanohexadecanoyl] -sn-glycerol 2,3-
(9R, 10S) -L-PHYLP was prepared in the same manner as in Example 1 except that the sodium salt of phosphate ((9R, 10S) -L-PHYLPA) was used.
The ability of A to activate cPKCα was determined. The results are shown in Table 3.

[0019]

[Table 3]

Example 4 As a test compound, 3-O-[(9R, 10S) -9,10-methanohexadecanoyl] -sn-glycerol 1,2-phosphate sodium salt ((9R, 10S) -D- Example 1 except that PHYLPA) was used
In the same manner as above, the ability of (9R, 10S) -D-PHYLPA to activate cPKCα was determined. The results are shown in Table 4.

[0021]

[Table 4]

Example 5 Example 1 was repeated except that 1-O-hexadecanoylglycerol 2,3-phosphate sodium salt (Pal-PHYLPA) was used as a test compound.
The activation ability of Pal-PHYLPA for cPKCα was determined. The results are shown in Table 5.

[0023]

[Table 5]

Reference Example 1 The activation ability of DG to cPKCα was determined in the same manner as in Example 1 except that diacylglycerol (DG) was used as the test compound. The results are shown in Table 6.

[0025]

[Table 6]

Example 6 20 mM Tris-HCl buffer (pH 7.5), 5 mM Mg (OAc) ₂ , 50
μg / mL MBP _4-14 (I. Yasuda et al., Biochem. Biophy
s. Res. commun., 166, 1220 (1990).), 10 μg / mL leupeptin, each concentration of 1-O-[(9S, 10R) -9,10-methanohexadecanoyl] -sn-glycerol 2, Sodium salt of 3-phosphate (PHYLPA), 10 μL purified nPKCδ (D. Schaap
et al., J. Biol. Chem., 265, 7301 (1990), and D.
J. Burnset al., J. Biol. Chem., 265, 12044 (199
40 μL of a solution containing 0).) Was treated at 0 ° C. for 1 hour. Add 10 μL ATP solution (20 μM ATP, 0.5 μCi [g- ³² P] AT) to the reaction solution.
(Including P) was added and reacted at 30 ° C. for 10 minutes. Reaction solution 4
Take 0 μL and adsorb to filter paper (Whatman, P81, 2x2cm),
The filter paper was washed 4 times with a 75 mM phosphoric acid solution to remove free [ ³² P] ATP. The filter paper was dried and the Cherenkov line was measured with a liquid scintillation counter. From the radioactivity, the activation ability of PHYLPA, a test compound, for cPKCα was determined. Table 7 shows the nPKCδ activating ability of the test compound as a relative value when the test compound was not added.

[0027]

[Table 7]

Example 7 As a test compound, 3-O-[(9S, 10R) -9,10-methanohexadecanoyl] -sn-glycerol 1,2-phosphate sodium salt ((9S, 10R) -D- Example 6 except using PHYLPA)
Similarly to the above, the activation ability of (9S, 10R) -D-PHYLPA for nPKCδ was determined. The results are shown in Table 8.

[0029]

[Table 8]

Example 8 As a test compound, 1-O-[(9R, 10S) -9,10-methanohexadecanoyl] -sn-glycerol sodium salt of 2,3-phosphate ((9R, 10S) -L- Example 6 except using PHYLPA)
Similarly, the activation ability of (9R, 10S) -L-PHYLPA for nPKCδ was determined. The results are shown in Table 9.

[0031]

[Table 9]

Example 9 As a test compound, 3-O-[(9R, 10S) -9,10-methanohexadecanoyl] -sn-glycerol 1,2-phosphate sodium salt ((9R, 10S) -D- Example 6 except using PHYLPA)
Similarly, the activation ability of (9R, 10S) -D-PHYLPA for nPKCδ was determined. The results are shown in Table 10.

[0033]

[Table 10]

Example 10 1-O-hexadecanoylglycerol as a test compound
NPKC of Pal-PHYLPA was obtained in the same manner as in Example 6 except that sodium salt of 2,3-phosphate (Pal-PHYLPA) was used.
The activation ability for δ was determined. The results are shown in Table 11.

[0035]

[Table 11]

Reference Example 2 The activation ability of DG for nPKCδ was determined in the same manner as in Example 6 except that diacylglycerol (DG) was used as the test compound. The results are shown in Table 12.

[0037]

[Table 12]

[0038]

[Effects of the Invention] 1-O-acylglycerol-2,3-phosphate has no adverse effect on cell survival at low concentrations (several μg / mL) (K. Murakami-Murofushi).
et al., J. Biol. Chem., 267, 21512 (1992).), DG
Since these substances activate PKC as strongly as or higher than that, it is considered that these substances are useful for the prevention or treatment of hypertension, hyperglycemia, and dementia, as well as analysis of PKC-mediated signal transduction systems and various It can be used as a useful drug in the physiological experimental system.

Claims (1)

[Claims] 1. A general formula: (In the formula, R represents a linear or branched alkyl group having 1 to 30 carbon atoms or a linear or branched alkenyl group having 2 to 30 carbon atoms, and the alkyl group or alkenyl group is a cycloalkane ring. Alternatively, it may contain an aromatic ring, and M represents a hydrogen atom, an alkali metal atom or an alkaline earth metal atom, or a substituted or unsubstituted ammonium group). Activity promoter.