JPH04282391A - Ethanolamine derivative and production thereof - Google Patents

Abstract

PURPOSE:To obtain a new ethanolamine derivative suitable as a fluorescent substrate useful for high-sensitivity measurement of phospholipase A2, having high reaction sensitivity to phospholipase A2. CONSTITUTION:A compound shown by formula I (R<1> is aliphatic acyl; R<2> is aliphatic acyl replaced with 1-pyrenyl) or a salt thereof such as 1- triacontanoyl-2-[10-(1-pyrenyl)decanoyl]-sn-glycero-3-phosphoethanolam ine. The compound is obtained by subjecting a compound shown by formula II (R<3> is protective amino) or a salt thereof to amino-protecting group elimination reaction. The amino-protecting group elimination reaction, for example, is carried out by the hydrolysis method, reduction method, etc. The starting raw material shown by formula II is a new substance and, for example, is obtained by successively reacting a compound shown by formula III (X<1> is halogen) with a compound shown by formula R<1>-OH and a compound shown by formula R2OH, reacting the formed compound shown by formula IV with a compound shown by formula V (M is metal) and finally eliminating hydroxy-protecting group of the reaction product.

Description

[Detailed description of the invention]

0001

[Industrial Application Field] This invention relates to phospholipase A2
The present invention relates to a novel ethanolamine derivative that can be used as a useful substrate for measuring the activity of phospholipase A2, particularly as a fluorescent substrate for phospholipase A2.

0002

[Prior Art] Phospholipase A2 (hereinafter referred to as PLA2) is an acylated sn-glycero-3 at the 1 and 2 positions.
- An enzyme that hydrolyzes the 2-position acyl group in phospholipids. Therefore, the principle of the method for measuring the enzymatic activity of PLA2 is to use the above sn-glycero-3-phospholipid as a substrate and measure the fatty acid derived from the above-mentioned position 2, which is liberated by the enzymatic reaction. The mainstream method is to use a radioactive substrate whose acyl group is labeled with a radioactive isotope. However, this method requires the troublesome effort of separating the reaction products, and due to poor operability, there are problems in that short-term reactions cannot be tracked and measured over time. In contrast, a method has been developed that uses a fluorescent substrate represented by the following formula in which an acyl group having a pyrene skeleton is substituted at the 2-position.

0003

[C4]

In this method, the reaction raw material (substrate) is sn
-The fluorescence peak position derived from the 1-pyrenyl group in glycero-3-phospholipid (around 480 nm) is different from the fluorescence peak position derived from the pyrenyl group in the generated (free) fatty acid (around 380 nm and around 400 nm). Therefore, there is no need to take the trouble of separating reaction products, which is required when using a radioactive substrate, and it is therefore expected to be a useful method, such as being able to measure changes over time.

However, the measurement method using the above compound [C] has the drawback that the presence of albumin in the reaction solution increases the background and deteriorates the measurement accuracy. Therefore, when it is desired to measure blood PLA2 activity, This is extremely inconvenient when it is desired to measure PLA2 in a culture solution containing serum. Furthermore, it is known that the reactivity of certain types of PLA2 changes depending on the structure of the 3-position of the phospholipid that is the substrate. For example synovialPLA2
(PLA2 derived from joint cavity fluid) has low reactivity to commonly used phosphatidylcholine-type substrates [
Journal of Biochemistry Volume 105 (1)
989), pp. 395-399].

[0006]

[Problems to be Solved by the Invention] This invention was made in consideration of the above-mentioned circumstances, and it can further improve the reactivity with PLA2 and contribute to even more sensitive measurements. The purpose of this research is to provide new fluorescent substrates for various purposes.

[0007]

[Structure of the Invention] The fluorescent substrate of the present invention has the general formula

000
8]

It is an ethanolamine derivative or a salt thereof represented by the formula [wherein R1 means an aliphatic acyl group and R2 means an aliphatic acyl group substituted with a 1-pyrenyl group].

In compound (A), the aliphatic acyl group represented by R1 and the aliphatic acyl group represented by R2 are residues obtained by removing OH from the carboxy group in an aliphatic carboxylic acid, and generally have about 7 to 40 carbon atoms. Intermediate to higher fatty acid residues are preferred. Particularly representative ones among these include lauroyl, myristoyl, palmitoyl, steroyl, etc., as well as higher aliphatic acyl groups having 20 to 40 carbon atoms, such as eicosanoyl, Heneicosanoyl, docosanoyl, tricosanoyl, tetracosanoyl, pentacosanoyl, hexacosanoyl, heptacosanoyl, octacosanoyl, nonacosanoyl, triacontanoyl,
Linear higher aliphatic acyl such as hentriacontanoyl, dotriacontanoyl, tritriacontanoyl, tetracontanoyl and the like may be mentioned, but branched higher aliphatic acyl may also be used. In addition, the aliphatic acyl moiety in the group represented by R2 (i.e., the moiety excluding the 1-pyrenyl group) includes intermediate to higher fatty acid residues having about 7 to 20 carbon atoms;
Examples include linear aliphatic acyls such as heptanoyl, octanoyl, nonanoyl, decanoyl, lauroyl, undecanoyl, lauroyl, tridecanoyl, myristoyl, pentadecanoyl, palmitoyl, heptadecanoyl, stearoyl, nonadecanoyl, and eicosanoyl; There may be. The substitution position of the 1-pyrenyl group on these aliphatic acyls is not particularly limited, but it is generally at the terminal carbon, that is, ω
position is recommended.

Compound (A) can be expressed as a chemical substance as shown in chemical formulas 1, 3, and 5 above, and can also be expressed as an inner salt as shown in chemical formula 6 below, all of which are included in the present invention. Included within the range.

[0011]

[C6]

In addition to the above-mentioned inner salts, the salts of the ethanolamine derivatives used in the present invention include inorganic acid addition salts such as hydrochloride, hydrobromic acid, sulfate, and phosphate, acetate, benzenesulfonate, and trichloride. Organic acid addition salts such as fluoroacetate, maleate, tartrate, methanesulfonate, toluenesulfonate, alkali metal salts such as sodium salt and potassium salt, salts with inorganic bases such as ammonium salt,
Examples include salts with organic bases such as triethylamine salts, pyridine salts, and picoline salts, and salts with basic or acidic amino acids such as arginine salts and aspartate salts. Next, a method for producing the compound (A) of the present invention is shown in Chemical Formula 7.

[0013]

[C7] (In the formula, R1, R2, and R3 have the same meanings as before)

[0014]
Protecting groups for the protected amino group represented by R3 include conventional amino protecting groups, such as acyl as described below; (lower)alkyl; 1-methoxycarbonyl-1
-Lower alkoxycarbonyl such as propen-2-yl (
lower) alkylidene or its enamine tautomer;
Dimethylaminomethylene; di(lower) alkylaminomethylene such as phthaloyl, etc. are included. A particularly preferred amino group having such a protecting group is aroylamino such as phthalimide.

Suitable acyls herein include aliphatic acyls, aromatic acyls, heterocyclic acyls, and aliphatic acyls substituted with aromatic or heterocyclic groups. These acyls will be explained below.

First, the aliphatic acyl includes saturated or unsaturated, acyclic or cyclic ones, such as lower alkanoyl such as formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, isovaleryl, pivaloyl, hexanoyl; Lower alkanesulfonyl such as mesyl, ethanesulfonyl, propanesulfonyl; Lower alkoxycarbonyl such as methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl, tertiary butoxycarbonyl; Lower alkenoyl such as acryloyl, methacryloyl, crotonoyl; C3-C7 cycloalkane carbonyl; amidino, etc. are included.

Examples of the aromatic acyl include aroyl such as benzoyl, toluoyl and xyloyl; arenesulfonyl such as benzenesulfonyl and tosyl; and phthaloyl.

Examples of the heterocyclic acyl include heterocyclic carbonyls such as furoyl, thenoyl, nicotinoyl, isonicotinoyl, thiazolinecarbonyl, thiadiazolylcarbonyl, and tetrazolylcarbonyl.

Examples of the aliphatic acyl substituted with an aromatic group include alkanoyl (lower) alkanoyl such as phenyl (lower) alkanoyl such as phenyl acetyl, phenylpropionyl, phenylhexanoyl; benzyloxycarbonyl, phenylhexanoyl; Al(lower)alkoxycarbonyl such as phenyl(lower)alkoxycarbonyl such as tyloxycarbonyl; phenoxy(lower)alkanoyl such as phenoxyacetyl and phenoxypropionyl; and the like.

Examples of the aliphatic acyl substituted with a heterocyclic group include thienyl acetyl, imidazolylacetyl, furylacetyl, tetrazolylacetyl, thiazolyl acetyl, thiadiazolyl acetyl, thienylpropionyl,
Includes thiadiazolylpropionyl, etc.

These acyl groups further contain one or two
may be substituted with one or more suitable groups. Suitable substituents include lower alkyl such as methyl, ethyl, propyl, isopropyl, butyl, pentyl, hexyl; halogen such as chlorine, bromine, iodine, fluorine; methoxy, ethoxy, propoxy, isopropoxy, butoxy, pentyloxy. , lower alkoxy such as hexyloxy; methylthio, ethylthio, propylthio, isopropylthio,
Lower alkylthio such as butylthio, pentylthio, hexylthio; nitro, etc. are included; suitable acyls having such substituents include mono (or di or tri) acyl such as chloroacetyl, bromoacetyl, dichloroacetyl, trifluoroacetyl; Halo(lower)alkanoyl; mono(or di- or tri)halogen(lower)alkoxycarbonyl such as chloromethoxycarbonyl, dichloromethoxycarbonyl, 2,2,2-trichloroethoxycarbonyl; nitrobenzyloxycarbonyl, chlorobenzyloxycarbonyl, methoxy Includes nitro (or halo or lower alkoxy) phenyl (lower) alkoxycarbonyl such as benzyloxycarbonyl.

[0022] In addition to the hydrolysis method and the reduction method, the amino-protecting group elimination reaction carried out here can be carried out using an iminohalogenating agent and then iminoetherification for the compound [B] whose amino-protecting group is acyl. The reaction can be carried out by a conventional method, such as a method in which the product is hydrolyzed, if necessary, after the reaction with an agent. Hydrolysis includes methods using acids, bases, hydrazine, and the like. These methods are appropriately selected depending on the type of protecting group to be removed.

Among the above-mentioned methods, hydrolysis with acids is one of the most common methods, for example, hydrolysis using acids such as substituted or unsubstituted alkoxycarbonyl groups such as tertiary pentyloxycarbonyl, formyl, acetyl Protecting groups such as lower alkanoyl groups such as cycloalkoxycarbonyl groups, substituted or unsubstituted aralkoxycarbonyl groups, aralkyl groups such as trityl, substituted phenylthio groups, substituted aralkylidene groups, substituted alkylidene groups, substituted cycloalkylidene groups, etc. This can be said to be a preferable method for desorption.

Examples of the acids used include organic and inorganic acids such as formic acid, trifluoroacetic acid, benzenesulfonic acid, p-toluenesulfonic acid, and hydrochloric acid. Among these, formic acid, trifluoroacetic acid, hydrochloric acid, etc. Particularly preferred are those which can be easily removed from the reaction mixture by conventional methods such as vacuum distillation.

These acids are appropriately selected depending on the type of amino protecting group to be removed. When hydrolyzing using an acid in this elimination reaction, it can be carried out either in the absence of a solvent or in the presence of a solvent. Suitable solvents include water, hydrophilic organic solvents, and mixed solvents thereof.

The elimination reaction using trifluoroacetic acid may be carried out in the presence of anisole. Hydrolysis using hydrazine is commonly applied for the removal of amino protecting groups of the succinyl and phthaloyl types, for example.

Examples of the base used in the hydrolysis using a base include alkali metal hydroxides such as sodium hydroxide and potassium hydroxide, alkaline earth metal hydroxides such as magnesium hydroxide and calcium hydroxide, sodium carbonate,
Alkali metal carbonates such as potassium carbonate, alkaline earth metal carbonates such as magnesium carbonate and calcium carbonate, alkali metal hydrogen carbonates such as sodium hydrogen carbonate and potassium hydrogen carbonate, alkali metal acetates such as sodium acetate and potassium acetate, calcium phosphate, phosphoric acid Inorganic bases such as alkaline earth metal phosphates such as magnesium, alkali metal hydrogen phosphates such as disodium hydrogen phosphate and dipotassium hydrogen phosphate, trialkylamines such as trimethylamine and triethylamine, picoline, N-methylpyrrolidine, and N-methylmorpholine. , 1,5-diazabicyclo[4,3,0]non-5-ene, 1,4-diazabicyclo[2,2,2]octane, 1,5-diazabicyclo[5,4,0]undecene-5, etc. Examples include organic bases. Hydrolysis using a base is usually carried out in water, a hydrophilic organic solvent, or a mixed solvent thereof.

The reductive elimination method includes, for example, haloalkoxycarbonyl groups such as trichloroethoxycarbonyl,
It is generally applied to the removal of substituted or unsubstituted aralkoxycarbonyl groups such as benzyloxycarbonyl, and protecting groups such as 2-pyridylmethoxycarbonyl. Suitable reduction methods include, for example, reduction with an alkali metal borohydride such as sodium borohydride, metals such as tin, zinc, iron, or metal salt compounds of these metals (chromous chloride, chromium acetate, etc.). (monochrome, etc.) in combination with organic or inorganic acids such as acetic acid, propionic acid, hydrochloric acid, and catalytic reduction. Suitable catalysts are conventional, such as Raney nickel, platinum oxide, palladium on carbon, and the like.

Among the protective groups, the acyl group can generally be removed by hydrolysis. In particular, halogen-substituted alkoxycarbonyl and 8-quinolyloxycarbonyl groups include copper,
Usually eliminated by treatment with heavy metals such as zinc.

The elimination of the acyl group is carried out using an iminohalogenating agent (
This can be carried out by treating with phosphorus oxychloride, etc.) and an iminoetherifying agent (lower alkanols such as methanol, ethanol, etc.), and then hydrolyzing if necessary.

The reaction temperature is not particularly limited and is appropriately selected depending on the type of amino protecting group mentioned above, the type of elimination method, etc., but the reaction temperature can be carried out under cooling at room temperature or at a mild temperature such as slightly warmed. be done.

The raw material [B] of the present invention is a new material and is produced according to the production process shown in Chemical Formula 8.

[0033]

[Image Omitted] (In the formula, R1, R2, and R3 have the same meanings as before, X1 is a halogen, M is a metal atom, and R5 is a hydroxy protecting group)

[
Examples of the halogen represented by X1 include fluorine, chlorine, bromine, and iodine, and the metal represented by M may be any metal as long as it reacts with the halogen to form a metal halide. Typical examples include alkali metals such as potassium and sodium, alkaline earth metals such as calcium, magnesium, and barium, as well as silver.

Suitable hydroxy protecting groups for R5 include silyl ether residues substituted with triorganic groups such as trimethylsilyl, tertiary-butyldimethylsilyl, etc., such as tertiary-butyl, benzyl, p-nitrobenzyl, trityl, Acyclic ether residues such as methylthiomethyl, methoxymethyl, β-methoxyethoxymethyl, e.g. 2-
Cyclic ether residues such as tetrahydropyranyl, 4-methoxy-4-tetrahydropyranyl, ester residues such as acetyl, chloroacetyl, etc., such as β,β,β-trichloroethoxycarbonyl, β-trimethylsilylethoxycarbonyl, etc. Mention may be made of the conventional hydroxy protecting groups used in the field of organic chemistry, such as esterified carboxy groups and the like.

Production method 1 Compound [G] is produced by reacting compound [E] with compound [F] or a reactive derivative thereof at the carboxy group.

Examples of reactive derivatives of carboxy groups include acid halides, acid anhydrides, active amides, and active esters. Suitable examples include acid chloride, acid azide, dialkyl phosphoric mixed acid anhydride, phenyl phosphoric acid mixed acid anhydride, diphenyl phosphoric acid mixed acid anhydride, dibenzyl phosphoric acid mixed acid anhydride, halogenated phosphoric acid mixed acid anhydride, etc. Phosphoric acid mixed acid anhydride, dialkyl phosphorous acid mixed acid anhydride, sulfite mixed acid anhydride, thiosulfuric acid mixed acid anhydride, sulfuric acid mixed acid anhydride, alkyl carbonic acid mixed acid anhydride, aliphatic carboxylic acid (e.g. acetic acid, pivalic acid) , pentanoic acid, isopentanoic acid, 2-ethylbutyric acid, trichloroacetic acid, etc.), mixed acid anhydrides such as aromatic carboxylic acids (e.g. benzoic acid, etc.) mixed acid anhydrides; symmetrical acid anhydrides; imidazole, Active amides with 4-substituted imidazoles, dimethylpyrazoles, triazoles, tetrazoles, etc.; cyanomethyl esters, methoxymethyl esters, dimethyliminomethyl

[0038]

[Chemical formula 9]

Ester, vinyl ester, propargyl ester, p-nitrophenyl ester, 2,4-dinitrophenyl ester, trichlorophenyl ester,
Pentachlorophenyl ester, mesylphenyl ester, phenylazophenyl ester, phenylthioester, p-nitrophenylthioester, p-cresylthioester, carboxymethylthioester, pyranyl ester, pyridyl ester, piperidyl ester,
Active esters such as 8-quinolyl thioester; or
N,N-dimethylhydroxylamine-1-hydroxy-2-(1H)-pyridone, N-hydroxysuccinimide, N-hydroxyphthalimide, 1-hydroxy-
Examples include esters with N-hydroxy compounds such as 6-chloro-1H-benzotriazole, and these reactive derivatives are appropriately selected depending on the type of acyl moiety in the acylating agent used.

This reaction is usually carried out using water, acetone, dioxane, isopropyl ether, acetonitrile, chloroform, methylene chloride, ethylene chloride, tetrahydrofuran, ethyl acetate, N,N-dimethylformamide, pyridine or any other common compound that does not adversely affect the reaction. The reaction is carried out in a solvent such as an organic solvent, and these conventional solvents can also be used in combination with water.

When compound [F] is used in the form of a free acid or its salt in this reaction, it is advantageous to carry out the reaction in the presence of a condensing agent. Examples of the condensing agent include acetic anhydride, N,N'-dicyclohexylcarbodiimide, N-cyclohexyl-N'-morpholinoethylcarbodiimide, N-cyclohexyl-N'-(4-diethylaminocyclohexyl)carbodiimide, and N,N'-diethylcarbodiimide. , N,N'-diisopropylcarbodiimide, N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide, N,N'-carbonylbis(2-methylimidazole), pentamethyleneketene-
N-cyclohexylimine, diphenylketene-N-cyclohexylimine, ethoxyacetylene, 1-alkoxy-1-chloroethylene, phosphorous acid trialkyl ester, polyphosphoric acid ethyl ester, polyphosphoric acid isopropyl ester, phosphorus oxychloride, phosphorus trichloride, chloride Thionyl, oxazolyl chloride, triphenylphosphine, 2-ethyl-7-hydroxybenzisoxazolium salt, 2-
Ethyl-5-(m-sulfophenyl)isoxazolium hydroxide inner salt, 1-(p-chlorobenzenesulfonyloxy)-6-chloro-1H-benzotriazole, dimethylformamide and thionyl chloride, phosgene or phosphorus oxychloride Examples include the so-called Vilsmeier reagent manufactured by E.P. et al.

This reaction may also be carried out in the presence of an inorganic or organic base such as an alkali metal bicarbonate, tri-lower alkylamine, pyridine, N-lower alkylmorpholine, N,N-di-lower alkylbenzylamine. Although the reaction temperature is not particularly limited, it is usually carried out under cooling or at room temperature.

Production method 2 Compound [I] is produced by reacting compound [G] with compound [H] or its reactive derivative at the carboxy group. For the meaning of the reactive derivative, reaction conditions, etc., refer to the corresponding section of Production Method 1.

Production method 3 Compound [K] is produced by reacting compound [I] with compound [J]. This reaction proceeds with the dehalogenation metal. The reaction is carried out in benzene, chloroform, methylene chloride, tetrahydrofuran, or other common organic solvents that do not adversely affect the reaction. Although the reaction temperature is not limited, it is usually carried out at heating or the reflux temperature of the solvent.

Production method 4 Compound [B] can be obtained by subjecting compound [K] to an elimination reaction of the hydroxy protecting group. As for the reaction method,
Conventional methods such as hydrolysis, reduction, etc. may be mentioned.

(i) Hydrolysis Hydrolysis is preferably carried out in the presence of an acid. Suitable acids include inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, etc.
Examples include organic acids such as formic acid, trifluoroacetic acid, propionic acid, methanesulfonic acid, benzelsulfonic acid, and p-toluenesulfonic acid, and acidic ion exchange resins. When organic acids such as trifluoroacetic acid and p-toluenesulfonic acid are used in this reaction, it is preferred to carry out the reaction in the presence of a cation scavenger, such as, for example, anisole.

Furthermore, boron trifluoride, instead of the above acid,
Boron trifluoride-ether addition compound, aluminum trichloride, antimony pentachloride, ferric chloride, tin chloride,
Lewis acids such as titanium tetrachloride, zinc chloride, barium iodide, etc. can also be used in this reaction, with barium iodide being particularly preferred. When using Lewis acids, the reaction is preferably carried out in the presence of a cation scavenger, such as, for example, anisole.

Hydrolysis is usually carried out using water, methanol, ethanol, propanol, tertiary butyl alcohol, tetrahydrofuran, N,N-dimethylformamide, N,N
- carried out in the presence or absence of customary solvents or mixtures thereof which do not adversely affect the reaction, such as dimethylacetamide, dioxane, dichloromethane; furthermore, the acids mentioned above may also be used as solvents if they are liquid; can. The reaction temperature for this hydrolysis is not particularly limited, but the reaction is usually carried out under cooling or at a temperature that is slightly elevated.

(ii) Reduction Reduction is carried out by conventional methods including chemical reduction and catalytic reduction. Suitable reducing agents used for chemical reduction include, for example, tin,
Metals such as zinc and iron or metal compounds such as chromium chloride and chromium acetate, and formic acid, acetic acid, propionic acid,
It is a combination with an organic or inorganic acid such as trifluoroacetic acid, p-toluenesulfonic acid, hydrochloric acid, or hydrobromic acid.

Suitable catalysts used in the catalytic reduction are platinum catalysts such as platinum plates, platinum sponges, platinum black, colloidal platinum, platinum oxide, platinum wire, such as palladium sponges, palladium black, palladium oxide, palladium-carbon. , colloidal palladium, palladium-barium sulfate, palladium-
Palladium catalysts such as barium carbonate, nickel catalysts such as reduced nickel, nickel oxide, Raney nickel, cobalt catalysts such as reduced cobalt, Raney cobalt, etc.
Examples include conventional catalysts such as iron catalysts such as reduced iron and Raney iron, copper catalysts such as reduced copper, Raney copper, Ullmann copper, and the like.

This reduction is usually carried out in a conventional solvent such as water, methanol, ethanol, propanol, N,N-dimethylformamide, or a mixture thereof, which does not adversely affect the reaction. If the acids used in the chemical reduction are liquids, they can also be used as solvents. Still further, suitable solvents for use in the catalytic reduction include those mentioned above and other conventional solvents such as diethyl ether, dioxane, tetrahydrofuran, etc., or mixtures thereof. The reaction temperature for this reduction is not particularly limited, but the reaction is usually carried out under cooling or heating.

[0052]

[Example] Production Example 1 Triacontanic acid (6.02g) in dry benzene (10g)
0ml) Add oxalyl chloride (11ml) to the suspension.
After adding, the reaction solution was heated under reflux for 16 hours. After concentrating this reaction solution under reduced pressure, dry benzene (500 ml) was added to the residue.
) and further concentrated under reduced pressure. After vacuum drying the residue,
Dissolved in dry benzene (100ml). Separately add this solution to dry chloroform (40 ml) and dry pyridine (
It was added dropwise to a solution in which sn-glycerol-3-iodohydrin [E] (4.04 g) had been dissolved in 10 ml of water. After stirring the reaction solution at room temperature for 18 hours in the dark, ether (500 ml) and water (100 ml) were added to the mixture, and then cooled 0.5 N hydrochloric acid was added to adjust the pH to 7. The organic layer was washed with an aqueous sodium thiosulfate solution and water, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was subjected to silica gel column chromatography and eluted with chloroform/n-hexane (5/1). The eluate was concentrated under reduced pressure and 5.5 g of 1-triacontinol-sn-glycerol-3-iodohydrin [G]
I got it. NMR (CDCl3) δ 0.88 (t, J=6.5
Hz, 3H), 1.2-1.7 (m, 54H), 2.3
5 (t, J = 7.5Hz, 2H), 3.29 (m, 2H
), 3.85 (m, 1H), 4.20 (d, J=5Hz
, 2H) Mass (EC) 509 (M+-1)

Production Example 2 Pyrenedecanoic acid (3.68g) was mixed with dry benzene (400g).
ml) Oxalyl chloride (10 ml) to the suspension
After adding, the reaction solution was heated under reflux for 16 hours. After concentrating this reaction solution under reduced pressure, dry benzene (400 ml) was added to the residue.
and further concentrated under reduced pressure. The residue was dried in vacuo and then dissolved in dry benzene (100 ml). This solution,
Separately, add dry chloroform (40 ml) and dry pyridine (
It was added dropwise to a solution in which 1-triacontainol-sn-glycerol-3-iodohydrin [G] (5.3 g) had been dissolved in 10 ml of water. After stirring the reaction solution at room temperature for 16 hours in the dark, ether (500 ml) was added to the mixture.
and water (100 ml), then cooled to 0.5
The pH was adjusted to 7 by adding N hydrochloric acid. The organic layer was washed with an aqueous sodium thiosulfate solution and an aqueous sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was subjected to silica gel column chromatography and eluted with chloroform/n-hexane (5/1). The eluate was concentrated under reduced pressure to obtain 5.2 g of 1-triacontainol-2-
[10-(1-pyrenyl)decanoyl]-sn-glycerol-3-iodohydrin [I] was obtained. NMR (CDCl3) δ 0.88 (t, J=6.5
Hz, 3H), 1.2-2.0 (m, 68H), 2.3
0 and 2.33 (t, t, J=7.5Hz, 4H
), 3.2-3.4 (m, 4H), 4.1-4.4 (m
, 2H), 4.9-5.1 (m, 1H), 7.8-8.
3 (m, 9H) Mass (FAB) 990.5 (M+)

Production Example 3 Thoroughly dried 1-triacontinol-2-
[10-(1-pyrenyl)decanoyl]-sn-glycerol-3-iodohydrin [I] (1.9 g) and silver salt of benzyl 2-phthalimidoethyl phosphate [J] (2.0 g) were mixed with dry benzene (100 ml).
The reaction solution was heated under reflux for 4 hours in the dark. The reaction solution was filtered and the residue was washed with benzene. After concentrating the furnace liquid under reduced pressure, it was subjected to silica gel column chromatography and eluted with ether/n-hexane (3/2). The eluate was concentrated under reduced pressure to obtain 1.9 g of benzyl 2-phthalimidoethyl 1-triacontanoyl-2-[10-(1-pyrenyl)decanoyl]-sn-glycero-3-phosphate [K]. NMR (CDCl3) δ 0.88 (t, J=6.5
Hz, 3H), 1.1-1.9 (m, 68H), 2.2
7 (t, J=7.5Hz, 4H), 3.32 (t, J=
8Hz, 2H), 3.9-4.3 (m, 8H), 4.9
7-5.03(dd, J=2Hz and 8.5Hz
, 2H), 5.1-5.2 (m, 1H), 7.31 (s
, 5H), 7.6-8.3 (m, 13H) Mass (F
AB) 1223.7 (M+)

Production Example 4 Benzyl 2 dissolved in dry benzene (10 ml)
-phthalimidoethyl 1-triacontanoyl-2-[
10-(1-pyrenyl)decanoyl]-sn-glycero-3-phosphate [K] (1.7 g) and dry barium iodide (2.0 g) were mixed with dry acetone (90 ml).
The mixture was mixed and heated under reflux for 2.5 hours. After the reaction solution was concentrated under reduced pressure, chloroform (100 ml) and 3N hydrochloric acid (50 ml) were added to the residue and mixed thoroughly. The organic layers were collected and concentrated under reduced pressure, and the residue was subjected to silica gel column chromatography using chloroform/methanol (20/1
) was eluted. The eluate was concentrated under reduced pressure to give 1.2 g of 2-phthalimidoethyl 1-triacontanoyl-2-[
10-(1-pyrenyl)decanoyl]-sn-glycero-3-phosphate [H] was obtained. NMR (CDCl3) δ 0.87 (t, J=7Hz
, 3H), 1.0-1.9 (m, 68H), 2.1-2
．． 3 (m, 4H), 3.26 (t, J=7.5Hz, 2
H), 3.8-4.4 (br, 8H), 5.1-5.3
(br, 1H), 7.4-8.3 (m, 13H) Mas
s (FAB) 1133.9 (M+)

Example 1 2-phthalimidoethyl 1-triacontanoyl-2-[10-(1
-pyrenyl)decanoyl]-sn-glycero-3-phosphate [B] (200 mg) and dry ethanol (1
5 ml) and hydrazine hydrate (40 μl) were added, and the mixture was heated under reflux for 2 hours. After the reaction solution was concentrated under reduced pressure, the residue was dried in vacuo. The residue was subjected to silica gel column chromatography using chloroform/methanol/acetic acid/water (430/50/10/1
It was eluted at 0). The eluate was concentrated under reduced pressure to obtain 0.76 g of 1-triacontinol-2-[10-(1-pyrenyl)decanoyl]-sn-glycero-3-phosphoethanolamine [A]. NMR (CDCl3) δ 0.87 (t, J=6Hz
, 3H), 1.0-2.0 (m, 68H), 2.1-2
．． 4 (m, 4H), 30-33 (m, 4H), 3.7-
4.4 (br, 6H), 5.1-5.3 (br, 1H)
,7.7-8.3(m,9H)Mass(FAB)10
04.6 (M+)

(Test Example) For the purpose of comparing the case where the compound [A] produced in Example 1 was used as the fluorescent substrate and the case where the known compound [C] was used as the fluorescent substrate, PL was conducted.
The reactivity to A2 was investigated.

Substrate (liposome) solution synthesis The concentration of compound (A) used as a substrate was determined by UV absorption using Compound [C] manufactured by SIGMA, a commercially available pyrene-labeled substrate, as a standard. 500 μl of ethanol and 2 μl of triethylamine were added to 130 μg of each dried substrate, and dissolved by applying ultrasound for about 1 hour. Immediately add the substrate solution to the reaction buffer (50mM Tris-HCl pH
8.3, 100mM-NaCl, 1mM-EDTA)
The solution was vigorously flowed into the solution to prepare a 1 μM solution. A substrate solution was prepared by adding 1M calcium chloride to the solution to give a final concentration of 7mM.

As the enzyme solution PLA2, recombinant human synovial PLA
2 was used. This cDNA was expressed in Chinese hamster ovary (CHO) cells, and PLA2 in the culture supernatant was purified by reverse-phase HPLC after heparin affinity column chromatography. The purified enzyme showed a single band on polyacrylamide gel electrophoresis containing SDS. The purified enzyme solution was diluted with a serum-free animal cell culture medium (Dulbecco's modified Eagle's minimal essential medium) to obtain a diluted enzyme solution.

Reaction: 0.9 ml of substrate solution and about 0.1 ml of sample solution without enzyme were placed in a cuvette and warmed at 37° C. for 5 minutes. 0.1 ml of the diluted enzyme solution was added to this, and after mixing quickly with a stirring bar, the fluorescence intensity was measured while incubating at 37°C.

Fluorescence measurement: Using a Hitachi fluorometer 650-10S, recording was performed with a Yokogawa Electric pen recorder 3066 (50 mV/cm). E
The xcitation wavelength and emission wavelength are 345 nm and 380 nm, respectively (both slits are 4 nm).
). Fluorescence intensity was measured 30 seconds, 60 seconds, 90 seconds, and 120 seconds after the start of the reaction. The amount of change (mm/min) during each elapsed time was plotted in FIG. 1 as the reaction rate.

Results As shown in Figure 1, when the compound [A] of the present invention was used as a substrate, highly sensitive measurement results were obtained even in the low concentration range of PLA2, and there was also a good relationship between the PLA2 concentration and the reaction rate. A linear correlation is observed. On the other hand, in the case of compound [C], which is a known substrate, the sensitivity is low in the low concentration region of PLA2,
It can be seen that the reactivity of PLA2 towards compound [C] is extremely low.

[0063]

Effects of the Invention The compound (A) provided by the present invention is novel and reacts with human synovial PLA2 with extremely high sensitivity. In addition, if there is a risk that albumin may be included in the sample and the background will be high, increasing the number of carbon atoms in the aliphatic acyl moiety represented by R1 in compound (A) will reduce the generated pyrenyl group-containing fatty acid. Since the separation of the components is facilitated, obstacles to obtaining correct measurements are eliminated.

[Brief explanation of the drawing]

FIG. 1 is a graph showing the reactivity of synthetic substrates and PLA2.

Claims (2)

[Claims] 1. An ethanolamine derivative or a salt thereof represented by the general formula: [Formula R1 is an aliphatic acyl group, and R2 is an aliphatic acyl group substituted with a 1-pyrenyl group]. [Claim 2] General formula [Formula 2] [In the formula, R1 is an aliphatic acyl group, R2 is an aliphatic acyl group substituted with a 1-pyrenyl group, and R3 is a protected amino group] By subjecting compounds or their salts to an amino-protecting group elimination reaction, compounds of the general formula [Formula 3] [wherein R1 is an aliphatic acyl group and R2 is an aliphatic acyl group substituted with a 1-pyrenyl group] can be obtained. A method for producing an ethanolamine derivative or a salt thereof, the method comprising obtaining an ethanolamine derivative or a salt thereof.