JP3032599B2 - Long chain carboxylic maleimide - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a long-chain carboxylic maleimide. The long-chain carboxylic maleimide provided by the present invention is useful as a chemical modifier for extending the half-life in blood of superoxide dismutase, which has attracted attention as a medicament.

[0002]

2. Description of the Related Art Conventionally, superoxide dismutase (hereinafter sometimes abbreviated as SOD) has been used for animals,
It is widely known in plants, microorganisms, and other living organisms, and is known as an enzyme that decomposes superoxide harmful to living organisms.
Examination of use as anti-inflammatory agent [Pharmacia, 17, 41
1-412 (1981) and current therapeutic
Research (Current Therapeutic Research), 16, 706
Page (1974)], study of the protective effect of myocardium during valvular heart surgery (“Clinical Study of Free Radicals, Vol. 5”, 113-1).
(See page 19, The Nippon Medical Hall, published on November 30, 1990). However, when SOD is administered intravenously, the molecular weight of SOD (approximately 32,000) is reduced by the filtration limit value of renal glomeruli (approximately the molecular weight). 50,000), it is rapidly excreted from the blood into the urine and metabolized, and its half-life in blood is said to be only 4-6 minutes. Modification with natural high molecular compounds or organic synthetic compounds has been attempted for the main purpose of prolonging the blood half-life.

[0003] SO modified with natural polymers such as phycol, inulin, dextran sulfate, dextran, heparin, hyaluronic acid, glycosaminoglycan and the like.
D is known [Procedures of the
National Academy of Sciences of United States of America (Proc. Natl. A
cad. Sci. USA), 77, 1159 (1980);
826 publication; JP-A-2-231075; JP-A-2-23107
No. 6; Japanese Unexamined Patent Publication No. 2-231077;
Japanese Patent Application Laid-Open No. 2-273176], and the natural polymer compounds have a wide range of molecular weights, so that the molecular weights of these modified SODs are not constant. Rat albumin-modified SODs are also known [Agents and Actions, 10, 231
P. (1980)], this modified SOD is antigenic.

SOD modified with a synthetic polymer compound such as polyalkylene glycol such as polyethylene glycol
It is also known [Research Communications
In Chemical Pathology & Pharmacolology (Res. Commun. Chem. Pathol. Pharmacol.), Vol. 29,
113 (1980); see JP-A-61-249388], and synthetic high molecular compounds have a distribution in molecular weight similar to natural high molecular compounds.

[0005] Partial half-esterified styrene-maleic anhydride copolymer (hereinafter abbreviated as SMA)
Modification of OD is also being studied [see Japanese Patent Application Laid-Open No. 1-104164]. A derivative obtained by modifying SOD with SMA (hereinafter, abbreviated as SMA-SOD) is
It has the property of reversibly binding and dissociating with serum proteins mainly composed of albumin in the blood, prolonging the half-life in the blood, and containing carboxylic acid in SMA, which has become acidic due to inflammation. It also has the ability to be integrated. Further, by using dicarboxylic acid, two molecules of S
A crosslinked derivative having a narrow molecular weight distribution to which OD is bound is also known [see JP-A-1-95775], but this derivative cannot be expected to accumulate in inflammatory sites.

[0006]

In any of the above-mentioned derivatives, SOD is modified via an amino group. Human SOD has 22 or 24 amino groups per molecule, and bovine SOD has 20 amino groups per molecule. As described above, SOD has a large number of amino groups, and it is extremely difficult to specify an amino group that serves as a binding site in a SOD molecule during modification. Considering that it is preferable that the active ingredient of a drug is a compound having a single chemical structure, it is desirable that a modified SOD provided for pharmaceutical use has a modified position in the SOD molecule specified. .

From this viewpoint, a derivative obtained by modifying SOD via a mercapto group with SMA having a functional group reactive with the mercapto group (hereinafter referred to as SMI)
-SOD) has also been created [Japanese Patent Application Laid-Open No. 1-257.
No. 479]. This derivative is equivalent to the above-mentioned SMA-SOD in terms of blood lifetime, residual enzyme activity, and accumulation in acidic sites, and the modification position in the SOD molecule is specified. Although it is specified specifically, it is complicated, and there remains a problem to be improved from the viewpoint of the above-mentioned active ingredient of a medicine. Therefore, in terms of blood life, residual enzyme activity, and accumulation in acidic sites, S
Equivalent to MA-SOD and SMI-SOD, and S
As in MI-SOD, the modification position in the SOD molecule has been specified, and the appearance of a modified SOD to which a modifier having a clear structure is bound has been desired.

Accordingly, an object of the present invention is to provide a novel SOD derivative having a significantly prolonged blood half-life in comparison with SOD and having a property of accumulating in an acidic site. To provide a long-chain carboxylic maleimide which is a novel chemical modifier having a binding property to a mercapto group.

[0009]

According to the present invention, the above object is achieved by the following general formula (I)

[0010]

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[0011] (wherein, W is one or more oxygen atom, a sulfur atom or -N, (R) - (wherein, R represents a lower alkyl group) in the main may be interrupted by a group represented Number of chain atoms
Represents a long-chain hydrocarbon group of 17 to 23 ) (hereinafter, abbreviated as long-chain carboxylic maleimide (I)).

In the long-chain carboxylic maleimide (I), examples of the divalent long-chain hydrocarbon group represented by W include the following groups.

(CH ₂ ) ₁₇ , (CH ₂ ) ₁₈ , (CH ₂ )
_{19, (CH 2) 20,} (CH 2) 21, (CH 2) 8 CH =
CH (CH ₂ ) ₇ , (CH ₂ ) ₉ CH = CH (CH ₂ ) ₇ ,
(CH ₂ ) ₁₀ CH = CH (CH ₂ ) ₇ , (CH ₂ ) ₁₁ CH
ＣＨCH (CH ₂ ) ₇ , (CH ₂ ) ₁₂ CH ＝ CH (CH ₂ ) ₇
, (CH ₂ ) ₈ CH = CH (CH ₂ ) ₈ , (CH ₂ ) ₈ CH
ＣＨCH (CH ₂ ) ₉ , (CH ₂ ) ₈ CH ＝ CH (CH ₂ ) ₁₀
, (CH ₂ ) ₈ CH = CH (CH ₂ ) ₁₁ , (CH ₂ ) ₅ C
H = CHCH ₂ CH = CH (CH ₂ ) ₇ , (CH ₂ ) ₆ CH
= CHCH ₂ CH = CH (CH ₂ ) ₇ , (CH ₂ ) ₇ CH =
CHCH ₂ CH = CH (CH ₂ ) ₇ , (CH ₂ ) ₈ CH = C
HCH ₂ CH = CH (CH ₂ ) ₇ , (CH ₂ ) ₉ CH = CH
_{CH 2 CH = CH (CH 2} ) 7, (CH 2) 2 -O- (CH
₂ ) ₁₅ , (CH ₂ ) ₄ -O- (CH ₂ ) ₁₆ , (CH ₂ ) _{2-
O- (CH 2) 17, (} CH 2) 4 -O- (CH 2) 18,
_{(CH 2) 2 -O- (CH} 2) 19, (CH 2) 4 -O- (C
_{H 2) 13, (CH 2} ) 4 -O- (CH 2) 14, (CH 2) 4
—O— (CH ₂ ) ₁₅ , (CH ₂ ) ₄ —O— (CH ₂ ) ₁₆ ,
_{(CH 2) 4 -O- (CH} 2) 17, (CH 2) 6 -O- (C
_{H 2) 11, (CH 2} ) 6 -O- (CH 2) 12, (CH 2) 6
—O— (CH ₂ ) ₁₃ , (CH ₂ ) ₆ —O— (CH ₂ ) ₁₄ ,
_{(CH 2) 6 -O- (CH} 2) 15, (CH 2) 8 -O- (C
_{H 2) 9, (CH 2} ) 8 -O- (CH 2) 10, (CH 2) 8
—O— (CH ₂ ) ₁₁ , (CH ₂ ) ₈ —O— (CH ₂ ) ₁₂ ,
_{(CH 2) 8 -O- (CH} 2) 13, (CH 2) 10 -O-
_{(CH 2) 7, (CH} 2) 10 -O- (CH 2) 9, (C
_{H 2) 10 -O- (CH 2} ) 11, (CH 2) 12 -O- (CH
_{2) 5, (CH 2)} 12 -O- (CH 2) 7, (CH 2) 12 -
_{O- (CH 2) 9, (} CH 2) 2 -O- (CH 2) 5 CH =
_{CH (CH 2) 7, (} CH 2) 4 -O- (CH 2) 5 CH =
_{CH (CH 2) 7, (} CH 2) 6 -O- (CH 2) 5 CH =
_{CH (CH 2) 7, (} CH 2) 2 -S- (CH 2) 15,
_{(CH 2) 2 -S- (CH} 2) 16, (CH 2) 2 -S- (C
_{H 2) 17, (CH 2} ) 2 -S- (CH 2) 18, (CH 2) 2
—S— (CH ₂ ) ₁₉ , (CH ₂ ) ₄ —S— (CH ₂ ) ₁₃ ,
(CH ₂ ) ₄ —S— (CH ₂ ) ₁₄ , (CH ₂ ) ₄ —S— (C
_{H 2) 15, (CH 2} ) 4 -S- (CH 2) 16, (CH 2) 4
—S— (CH ₂ ) ₁₇ , (CH ₂ ) ₆ —S— (CH ₂ ) ₁₁ ,
_{(CH 2) 6 -S- (CH} 2) 12, (CH 2) 6 -S- (C
_{H 2) 13, (CH 2} ) 6 -S- (CH 2) 14, (CH 2) 6
—S— (CH ₂ ) ₁₅ , (CH ₂ ) ₈ —S— (CH ₂ ) ₉ ,
_{(CH 2) 8 -S- (CH} 2) 10, (CH 2) 8 -S- (C
_{H 2) 11, (CH 2} ) 8 -S- (CH 2) 12, (CH 2) 8
—S— (CH ₂ ) ₁₃ , (CH ₂ ) ₁₀ —S— (CH ₂ ) ₇ ,
_{(CH 2) 10 -S- (CH} 2) 9, (CH 2) 10 -S-
_{(CH 2) 11, (CH} 2) 12 -S- (CH 2) 5, (CH
_{2) 12 -S- (CH 2)} 7, (CH 2) 12 -S- (CH 2)
_{9, (CH 2) 2 -N} (CH 3) - (CH 2) 14, (C
_{H 2) 4 -N (CH 3} ) - (CH 2) 14, (CH 2) 6 -N
_{(CH 3) - (CH 2} ) 14, (CH 2) 2 -N (C 2 H 5)
_{- (CH 2) 14, (} CH 2) 4 -N (C 2 H 5) - (C
_{H 2) 14, (CH 2} ) 6 -N (C 2 H 5) - (CH 2) 14,
(CH ₂ ) ₂ —O— (CH ₂ ) ₂ —O— (CH ₂ ) ₁₂ , (C
_{H 2) 4 -O- (CH 2} ) 2 -O- (CH 2) 12, (C
_{H 2) 6 -O- (CH 2} ) 2 -O- (CH 2) 12, (C
H ₂ ) ₂ —O— (CH ₂ ) ₂ —O— (CH ₂ ) ₁₄ , (C
_{H 2) 2 -O- (CH 2} ) 2 -O- (CH 2) 16, (C
_{H 2) 2 -S-S- (} CH 2) 14, (CH 2) 2 -S-S-
_{(CH 2) 16, (CH} 2) 2 -S-S- (CH 2) 18,
_{(CH 2) 4 -S-S-} (CH 2) 12, (CH 2) 4 -S-
_{S- (CH 2) 14, (} CH 2) 4 -S-S- (CH 2)
_{16, (CH 2) 6 -S} -S- (CH 2) 10, (CH 2) 6
-SS- (CH ₂ ) ₁₂ , (CH ₂ ) ₆ -SS- (CH
₂ ) ₁₄ , (CH ₂ ) ₈ -SS- (CH ₂ ) ₈ , (CH ₂ )
₈ -SS- (CH ₂ ) ₁₀ , (CH ₂ ) ₈ -SS- (C
H ₂ ) ₁₂

The long-chain carboxylic maleimide (I) has the following general formula (II)

H ₂ N—W—CO ₂ H (II)

Wherein W is as defined above (hereinafter abbreviated as aminocarboxylic acid (II)) with maleic anhydride to obtain General formula (III)

[0017]

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(Wherein W is as defined above) (hereinafter abbreviated as amidodicarboxylic acid (III)), and then the amide dicarboxylic acid (III) is subjected to a ring closing treatment. [E-
Org. Synth., 41, 93 (1
961) and `` New Experimental Chemistry Course 14-II '', 1145-1147
Page (see Maruzen Co., Ltd., published in 1977)], but it can also be synthesized by a partially modified method of this method.

The aminocarboxylic acid (II) is synthesized using the Gabriel method known as a method for synthesizing amino acids (see Berichte, Ber. 22, p. 426 (1889)) as a key reaction. It is convenient to carry out according to a known method. That is, the following general formula (IV)

HO-W-CO ₂ R ¹ (IV)

Wherein W is as defined above, and R
¹ represents a lower alkyl group), an oxycarboxylic acid ester represented by (hereinafter abbreviated as oxycarboxylic acid ester (IV)) is treated with a sulfonic acid halide such as p-toluenesulfonyl chloride and methanesulfonyl chloride; The following general formula (V) obtained

[0022]

Embedded image

Wherein W and R ¹ are as defined above, and R ² represents a lower alkyl group or an aryl group which may be substituted with a lower alkyl group. And the sulfonic acid ester (V)
Is abbreviated to Gabriel's method, and a phthalimide alkali metal salt is first reacted to obtain the following general formula (VI)

[0024]

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(Wherein W and R ¹ are as defined above) (hereinafter referred to as phthalimide carboxylic acid ester (VI)
) And treated with hydrazine to decompose the phthalimide group, and then hydrolyze the generated aminocarboxylic acid ester under alkaline conditions to obtain aminocarboxylic acid (II).

The reaction between the oxycarboxylic acid ester (IV) and the sulfonic acid halide is usually carried out in the presence of a base represented by, for example, pyridine, triethylamine, etc., in the presence of a suitable solvent such as methylene chloride, ether or the like. The reaction is carried out in the absence of -20 ° C to + 50 ° C.

The reaction between the sulfonic acid ester (V) and the alkali metal phthalimide is usually carried out in the presence or absence of a polar high-boiling solvent, but is conveniently carried out in N, N-dimethylformamide. . That is, potassium phthalimide is dissolved in N, N-dimethylformamide, and the resulting solution is added at room temperature to 153 ° C., preferably 80 to 1 ° C.
This is done by adding the sulfonic acid ester (V) at a temperature in the range of 20 ° C.

The phthalimide carboxylic acid ester (VI) obtained as described above is converted into an aminocarboxylic acid (II) by treating the phthalimide carboxylic acid ester (VI) with hydrazine to decompose the phthalimide group. The hydrolysis is carried out by hydrolyzing the aminocarboxylic acid ester formed under alkaline conditions. That is, the phthalimide carboxylic acid ester (VI) is, for example, ethanol-
Dissolved in a suitable solvent such as methanol, methanol and the like, and hydrazine was added to the obtained solution and stirred to remove the generated phthalhydrazide. Alternatively, the reaction is carried out by stirring in a solution of potassium hydroxide in hydrated methanol, hydrated ethanol or hydrated isopropanol. The amount of hydrazine to be used is preferably about 1.0 to 20 molar equivalents, more preferably 1.0 to 10 molar equivalents, to phthalimide carboxylic acid ester (VI). In order for the reaction to proceed smoothly, it is preferable to employ a reaction temperature in the range of 0 ° C. to the boiling point of the solvent, and more preferably a temperature in the range of 25 ° C. to the boiling point of the solvent. . The time required for the completion of the reaction depends on the temperature conditions employed at this time, but is usually in the range of about 10 minutes to 3 days.

The phthalhydrazide formed by the above-mentioned reaction is obtained by precipitating the reaction mixture at a pH of about 1 to 4 using a mineral acid such as hydrochloric acid or sulfuric acid, preferably at room temperature or under ice-cooling. It is convenient to remove by filtration. After performing such an operation, the solvent is distilled off, and the remaining aminocarboxylic acid ester is subjected to a hydrolysis reaction under alkaline conditions. This hydrolysis reaction
It is preferably carried out at a temperature ranging from 0 ° C. to the boiling point of the solvent, more preferably at a temperature ranging from 25 ° C. to the boiling point of the solvent. The time required for the completion of the reaction varies depending on the temperature conditions employed, but is usually in the range of about 10 minutes to 3 days. After the reaction, the aminocarboxylic acid (II) is isolated from the reaction mixture by neutralizing the reaction mixture with a mineral acid such as hydrochloric acid, sulfuric acid or the like, preferably at a temperature ranging from room temperature to ice cooling. The resulting precipitate is obtained by filtration and recrystallization.

The reaction between aminocarboxylic acid (II) and maleic anhydride is preferably carried out in a solvent. Suitable solvents include hydrated methanol, hydrated ethanol,
A hydrated alcohol such as hydrated isopropanol may be used. In order for the reaction to proceed smoothly, the pH of the reaction solution should be 6
It is preferable to keep the above. At a pH of 6 or less, the solubility of the aminocarboxylic acid (II) decreases, and the reaction does not easily proceed. The maleic anhydride is generally used in a proportion of 1 to 300 mol, preferably 1 to 50 mol, per 1 mol of the aminocarboxylic acid (II). In a preferred embodiment, the reaction is carried out by adding the maleic anhydride to the solution or dispersion of the aminocarboxylic acid (II) all at once or gradually in small portions. The reaction temperature preferably ranges from 0 ° C. to the boiling point of the solvent, and more preferably ranges from 20 ° C. to 60 ° C. In order to confirm the progress of the reaction, it is convenient to follow the disappearance of the raw materials by thin layer chromatography. After the reaction, the amidodicarboxylic acid (III) is isolated from the reaction mixture by using a mineral acid such as hydrochloric acid or sulfuric acid, preferably at a temperature ranging from room temperature to ice cooling. It is convenient to make it acidic by about 4 and filter the resulting precipitate.

The long-chain maleimide (I) can be easily derived by heating the amide dicarboxylic acid (III) together with acetic anhydride in the presence of sodium acetate, for example, in the usual manner, followed by dehydration.

SOD and long chain carboxylic maleimide (I)
And in an aqueous solution having a pH of 6 to 10,
The following general formula

[0033]

Embedded image

(Wherein [SOD] represents a residue obtained by removing two mercapto groups from superoxide dismutase, and W represents one or more oxygen atoms, sulfur atoms, or -N
(R) —wherein R represents a divalent long-chain hydrocarbon group having 17 to 23 main chain atoms which may be interrupted by a group represented by the following formula: Superoxide dismutase derivatives (hereinafter, abbreviated as SOD derivatives) can be produced.

SOD and long-chain carboxylic maleimide (I)
Is usually carried out in an aqueous solution of a salt such as tris (hydroxymethyl) aminomethane hydrochloride, sodium carbonate, sodium bicarbonate, sodium acetate and sodium phosphate.
This is carried out by dissolving the OD and adding the powdered long-chain carboxylic maleimide (I) or a long-chain carboxylic maleimide (I) dissolved in an organic solvent such as dimethyl sulfoxide to the resulting solution. During the reaction, the pH of the solution was 6
It is necessary that the temperature be maintained in the range of from 10 to 10, preferably in the range of from 8 to 10. When the pH is lower than 6, the solubility of the long-chain carboxylic maleimide (I) decreases, and the reaction hardly proceeds. On the other hand, when the pH is higher than 10, the long-chain carboxylic maleimide (I) may react with the amino group of SOD. The reaction temperature is preferably lower than room temperature. The reaction time varies depending on the method of adding the long-chain carboxylic maleimide (I), but is usually 10 minutes to 2 days. The amount of the long-chain carboxylic maleimide (I) used is about 2.0 per mole of SOD.
In the range of ~ 30 moles.

The reaction solution obtained in this manner contains SOD
Derivatives and unreacted SOD and long-chain carboxylic maleimide (I) are present. Such a reaction solution is filtered, the filtrate is subjected to gel filtration, and the resulting eluate containing the SOD derivative is hydrolyzed as necessary. After subjecting to BIC column chromatography, ion exchange column chromatography, etc., the filtrate is concentrated by ultrafiltration and then freeze-dried to obtain a solid SOD derivative.

By the above reaction, the mercapto group of the SOD and the maleimide ring of the long-chain carboxylic maleimide (I) are bonded by an addition reaction to produce an SOD derivative.

The long-chain carboxylic maleimide (I) of the present invention
Has one maleimide group in one molecule, and SOD
Has usually two free mercapto groups that can react with human SOD. Therefore, by the above-mentioned reaction and post-reaction treatment, an SOD derivative in which two molecules of long-chain carboxylic maleimide (I) are bonded to one human SOD molecule can be obtained. The carboxyl group of the SOD derivative may form an alkali metal salt or an ammonium salt by this reaction and the treatment after the reaction. There is no inconvenience to use as.

The SOD derivative has a characteristic that it has a significantly longer half-life in blood compared to SOD, and has good transferability to an anti-inflammatory site due to its accumulation in acidic sites. .

As the SOD used as a raw material, those contained in living organisms such as animals (humans, cows, etc.), plants, microorganisms, etc. are separated and obtained from each living organism by known methods, And those obtained using engineering techniques. The chemical structure (coordination metal, molecular weight, amino acid sequence, etc.) of SOD has been elucidated considerably, and SOD is composed of Fe-coordinate SOD, Mn-coordinate S
It is classified into OD, Cu-Zn coordinated SOD, and the like, and has a molecular weight of 30,000 to 80,000 depending on the existing living tissue. The amino acid sequence of SOD also differs slightly depending on the living tissue in which it is present ["SOD and Reactive Oxygen Regulators" Chapter 2 SOD (by Yoshihiko Oyanagi, Nippon Medical Center, November 1989)
6th)). Human Cu-Zn coordinated SOD has a molecular weight of 32,000 and has two reactive free mercapto groups. This human SOD is obtained, for example, by subjecting human blood to heat treatment, ion exchange, and gel filtration sequentially, and by using genetic engineering techniques.

Long-chain carboxylic maleimide (I) of the present invention
Has a fatty acid moiety. Therefore, the SOD derivative to which the long-chain carboxylic maleimide (I) is bound has reversible binding to serum proteins and biological membranes, thereby extending the half-life in blood, The transferability to is improved.

In the long-chain carboxylic maleimide (I), the number of main chain atoms of the long-chain hydrocarbon group represented by W is 9 to 29.
And more preferably 17 to 23. An SOD derivative obtained by reacting a long-chain carboxylic maleimide (I) having less than 9 main chain atoms with SOD is not preferred because it has poor binding ability to serum proteins. The long-chain carboxylic maleimide (I) having more than 29 main chain atoms has poor solubility in an aqueous solution having a pH of 6 to 10, and it becomes difficult to bond the long-chain carboxylic maleimide (I) to SOD. .

The SOD derivative has excellent anti-ulcer and anti-arrhythmic effects, as is apparent from the results of Test Examples 2 and 3 described below. SOD derivatives also have pharmacological actions such as anti-inflammatory action, anti-ischemic injury action, and anti-cerebral edema action.

In addition, SOD derivatives have been confirmed to have low toxicity in toxicity tests.

From the above results, the SOD derivative is effective against various diseases in which active oxygen radicals are involved, and particularly, anti-inflammatory agent, anti-ulcer agent, therapeutic agent for ischemic disease, therapeutic agent for cerebral edema, paraquat It can be used as a therapeutic. The SOD derivative is also useful as a drug for reducing the side effects of anticancer drugs caused by active oxygen radicals. Further, the SOD derivative is also useful as a remedy for skin diseases such as burns, trauma and various dermatitis.

The dosage of the SOD derivative varies depending on the disease, the severity of the patient, the tolerability of the drug, and the like.
In the range of 0.1 to 500 mg per day, preferably 0.5 to 500 mg
It may be in the range of 100 mg and should be administered in single or divided doses. For administration, any form suitable for the administration route can be used.

The SOD derivative can be prepared for administration using any conventional formulation method. Pharmaceutical compositions containing at least one SOD derivative are prepared by conventional means using pharmaceutically acceptable additives such as optional pharmaceutical carriers and excipients.

When the pharmaceutical composition is an oral preparation, the preparation is preferably provided in a form suitable for absorption from the digestive tract. Tablets and capsules for oral administration are in unit dosage form and binders such as syrup, acacia, gelatin, sorbitol, tragacanth, polyvinylpyrrolidone and the like; excipients such as lactose, corn starch, calcium phosphate, sorbit, glycine And the like. Lubricants such as magnesium stearate, talc, polyethylene glycol, silica and the like; may contain conventional excipients such as disintegrants such as sodium lauryl sulfate. Tablets may be coated in a manner well known in the art. Oral liquid preparations may be aqueous or oily suspensions, solutions, syrups, elixirs, etc., or may be dry products which are redissolved in water or other suitable vehicle before use. Good.
Such liquid preparations may contain commonly used additives such as suspending agents such as sorbit syrup, methylcellulo-
Water, glucose / sugar syrup, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminum stearate gel, hydrogenated edible fat and the like; emulsifier,
For example, lecithin, sorbitan monooleate, gum arabic and the like; water-insoluble vehicles such as almond oil, fractionated coconut oil, oily esters, propylene glyco-
Preservatives such as methyl p-hydroxybenzoate, propyl p-hydroxybenzoate, sorbic acid and the like.

When preparing an injection, the SOD derivative is dissolved in a solvent such as physiological saline or glucose solution for injection, and the concentration is adjusted to 2 to 20 mg of the SOD derivative / 2 to 10 ml of the solvent. Subcutaneous, intramuscular and intravenous injections are prepared according to the method. During preparation, a pH adjuster, a buffer, a stabilizer, a preservative, a solubilizing agent, and the like can be added to the aqueous solution as needed.

The above-mentioned pharmaceutical composition can contain the SOD derivative generally at a concentration of about 0.01 to 50% by weight, preferably about 0.1 to 20% by weight, depending on the form and the like.

[0051]

The present invention will be specifically described below with reference to examples. Note that the present invention is not limited by these examples. ¹ H-NMR was measured using tetramethylsilane as an internal standard, and IR was measured by the KBr tablet method.

Example 1 Synthesis of 18-tosyloxyoctadecanoic acid methyl ester
Growth 18 - hydroxy octadecanoic acid methyl ester (4.5
g, 14.3 mmol) in 50 ml of methylene chloride, pyridine (4.53 g, 57.2 mmol) was added to the resulting solution, and then p-toluenesulfonyl chloride (5.46 g,
28.6 mmol) was gradually added over 1 hour, and the mixture was reacted in a refrigerator for 16 hours. The reaction solution was washed sequentially with 10% hydrochloric acid, water, a saturated aqueous solution of sodium bicarbonate, water and saturated saline, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The residue was separated and purified by silica gel column chromatography (developing solution: benzene), and showed the following physical properties.
8-Tosyloxyoctadecanoic acid methyl ester (5.6
9 g, 85%). mp 67.5 to 68.5 ° C ¹ H-NMR (CDCl ₃ , 270 MHz): δ 1.09 to 1.42 (m, 26
H), 1.53 to 1.72 (m, 4H), 2.30 (t, 2H), 2.45 (
s, 3H), 3.68 (s, 3H), 4.03 (t, 2H), 7.35 (d, 2H)
), 7.79 (d, 2H)

Example 2 Synthesis of 18-phthalimidooctadecanoic acid methyl ester
Formation of potassium phthalimide (2.96 g, 16.0 mmol) and dry
Mix 100 ml of N, N-dimethylformamide with 110 ml
C. and add 18-tosyloxyoctadecanoic acid methyl ester (5.0 g, 10.7 mmol) in N, N
-Add 80 ml of dimethylformamide solution dropwise,
For 2 hours. Pour the reaction mixture into ice water and add
After stirring for 30 minutes, the precipitate was collected by filtration and dissolved in chloroform. The obtained solution is washed successively with water and saturated saline, dried over anhydrous magnesium sulfate, and then separated and purified by silica gel column chromatography [developing solution: a mixture of benzene and methylene chloride (volume ratio: 2: 1)]. Thus, 18-phthalimidooctadecanoic acid methyl ester (4.15 g, 88%) having the following physical data was obtained. mp 82 to 83 ° C ¹ H-NMR (CDCl ₃ , 270 MHz): δ 1.09 to 1.42 (m, 26
H), 1.53 to 1.76 (m, 4H), 2.30 (t, 2H), 3.66,
3.67 (s, t, 5H), 7.71 (m, 2H), 7.84 (m, 2H)

Example 3 Synthesis of 18 -aminooctadecanoic acid 18-phthalimidooctadecanoic acid methyl ester (2.
0 g, 4.51 mmol), 30 ml of ethanol and 80% hydrazine hydrate (0.42 ml, 6.76 mmol) in 9
Heated to reflux for an hour. Add 6N hydrochloric acid (11.3 ml, 67.6
mmol), and the mixture was heated under reflux for an additional 1 hour. Insolubles were removed by filtration, and the filtrate was concentrated under reduced pressure. Etano in residue
30 ml and 1N aqueous sodium hydroxide solution 18.1 ml
Was added and the mixture was refluxed for 18 hours. 6N the reaction solution under ice cooling
Neutralize with hydrochloric acid, collect the precipitate by filtration and recrystallize (ethanol-
Acetic acid-water) to give 18-aminooctadecanoic acid (800 mg, 59%) having the following physical data. mp 172 to 174 ° C IR (cm ^-1 ): 2920, 2850, 1640, 1535, 1470, 1400 FD-MS (m / z): [M + H] ⁺ 300

Example 4 Synthesis of N- (17-carboxyheptadecyl) maleamic acid
Preparation of 18-aminooctadecanoic acid (400 mg, 1.33 mmol) in 50 ml of ethanol and 1N aqueous sodium hydroxide
Dissolve in a 25 ml mixture at 40 ° C and maintain the temperature of the resulting solution at 40 ° C with maleic anhydride (1.97g, 20.0g).
mmol) was added slowly over 2 hours. Reaction mixture 3
After stirring for 0 minutes, the mixture was acidified with hydrochloric acid under ice-cooling and centrifuged. The precipitate was filtered, sufficiently washed with water, and dried under reduced pressure to obtain N- (17-carboxyheptadecyl) maleamic acid (436 mg, 88%) having the following physical data. mp 144 to 147.5 ° C IR (cm ^-1 ): 3305, 2920, 2850, 1710, 1630, 1585,
1470, 1400, 1280,1250, 1230, 1215, 1195, 1180 FD-MS (m / z): [M + H] ⁺ 398

Example 5 Synthesis of 18-maleimidooctadecanoic acid N- (17-carboxyheptadecyl) maleamic acid (4
00 mg, 1.01 mmol), 2.83 ml of acetic anhydride and 41.0 mg, 0.50 mmol of sodium acetate anhydride.
The reaction was carried out at a temperature of 1 hour. The reaction was allowed to cool, poured on ice, stirred for 1 hour, and extracted with chloroform. The organic layer is washed successively with water and saturated saline, dried over anhydrous magnesium sulfate, concentrated under reduced pressure, and then subjected to silica gel column chromatography [developing solution: a mixture of benzene and chloroform (volume ratio: 1: 1)]. 18-maleimidooctadecanoic acid (172 mg, 45
%). mp 101-103 ° C ¹ H-NMR (CDCl ₃ , 270 MHz): δ 1.14-1.40 (m, 26
H), 1.48 to 1.72 (m, 4H), 2.35 (t, 2H), 3.50 (
t, 2H), 6.68 (s, 2H) IR (cm- ¹ ): 2920, 2850, 1710, 1470, 1450, 1410,
840, 700 FD-MS (m / z): [M + H] ⁺ 380

Example 6 Synthesis of 20-tosyloxyeicosanoic acid methyl ester 20-hydroxyeicosanoic acid methyl ester (8.0 g,
23.4 mmol) was dissolved in 90 ml of methylene chloride, pyridine (7.39 g, 93.4 mmol) was added to the obtained solution, and then p-toluenesulfonyl chloride (8.90 g,
46.7 mmol) was gradually added over 1 hour, and the mixture was reacted in a refrigerator for 14 hours. After completion of the reaction, post-treatment was carried out in the same manner as in Example 1, and the following physical property values were obtained.
-Tosyloxyeicosanoic acid methyl ester (10.57
g, 91%). mp 72.5 to 73.5 ° C ¹ H-NMR (CDCl ₃ , 270 MHz): δ 1.10 to 1.38 (m, 30
H), 1.52 to 1.72 (m, 4H), 2.30 (t, 2H), 2.45 (
s, 3H), 3.67 (s, 3H), 4.02 (t, 2H), 7.34 (d, 2H)
), 7.78 (d, 2H)

Example 7 Synthesis of 20-phthalimidoeicosanoic acid methyl ester Potassium phthalimide (5.59 g, 30.2 mmol) and drying
Mix with 200 ml of N, N-dimethylformamide
At 20 ° C, and a solution of 20-tosyloxyeicosanoic acid methyl ester (10.0 g, 20.1 mmol) in N, N-dimethylformamide (300 ml) was added dropwise thereto.
Allowed to react for hours. After completion of the reaction, the same post-treatment as in Example 2 was performed to obtain 20-phthalimidoeicosanoic acid methyl ester (8.97 g, 95%) having the following physical properties. mp 86.5 to 87.5 ° C ¹ H-NMR (CDCl ₃ , 270 MHz): δ 1.08 to 1.50 (m, 30
H), 1.55 to 1.81 (m, 4H), 2.30 (t, 2H), 3.66,
3.67 (s, t, 5H), 7.71 (m, 2H), 7.84 (m, 2H)

Example 8 Synthesis of 20 -aminoeicosanoic acid 20-phthalimidoeicosanoic acid methyl ester (5.0
g, 10.6 mmol), a mixture of 100 ml of ethanol and 80% hydrazine hydrate (1.0 ml, 15.9 mmol) was heated to reflux for 9 hours. 26.5 ml of 6N hydrochloric acid was added to the reaction solution, and the mixture was further heated under reflux for 1 hour, and the insoluble matter was separated by filtration. The filtrate was concentrated under reduced pressure. 70 ml of ethanol and
42.4 ml of a 1N aqueous sodium hydroxide solution was added, and the mixture was heated under reflux for 16 hours. The reaction solution was neutralized with 6N hydrochloric acid under ice cooling, and the precipitate was collected by filtration and recrystallized (ethanol-acetic acid-water) to give 20-aminoeicosanoic acid (
2.50 g, 72%). mp 172.5 to 174 ° C FD-MS (m / z): [M + H] ⁺ 328 IR (cm ^-1 ): 2920, 2850, 1640, 1535, 1470, 1400

Example 9 Synthesis of N- (19-carboxynonadecyl) maleamic acid 20-aminoeicosanoic acid (500 mg, 1.53 mmol) was dissolved in 85 ml of ethanol and a 1N aqueous solution of sodium hydroxide 4
Dissolve the mixture at 0 ° C at 40 ° C and maintain the temperature of the resulting solution at 40 ° C with maleic anhydride (3.75 g, 38.3 g).
mmol) was added slowly over 3 hours. During this time, the pH of the reaction solution was kept at 8 to 10. After stirring the reaction solution for 30 minutes, post-treatment was carried out in the same manner as in Example 4 to give N- (19-carboxynonadecyl) having the following physical data.
Maleamic acid (538 mg, 83%) was obtained. mp 146 to 148 ° C FD-MS (m / z): [M + H] ⁺ 426 IR (cm ^-1 ): 3305, 2920, 2850, 1710, 1630, 1570,
1470, 1405, 1275,1245, 1225, 1210, 1195, 1180

Example 10 Synthesis of 20-maleimidoeicosanoic acid N- (19-carboxynonadecyl) maleamic acid (500
mg, 1.22 mmol), 3.44 ml of acetic anhydride and sodium acetate (50.1 mg, 0.61 mmol) at 100 ° C.
For 1 hour. After completion of the reaction, post-treatment was carried out in the same manner as in Example 5, and the following physical property values were obtained.
Maleimide eicosanoic acid (230 mg, 46%) was obtained. mp 104 to 105.5 ° C ¹ H-NMR (CDCl ₃ , 270 MHz): δ 1.17 to 1.40 (m, 30
H), 1.50-1.69 (m, 4H), 2.34 (t, 2H), 3.51 (
t, 2H), 6.68 (s, 2H) FD-MS (m / z): [M + H] ⁺ 408 IR (cm- ¹ ): 2920, 2850, 1710, 1470, 1455, 1410,
840, 700

Example 11 Synthesis of methyl 22-tosyloxidecosanoate 22-hydroxydocosanoic acid methyl ester (1.70 g,
4.59 mmol) in 15 ml of pyridine and 12 ml of methylene chloride.
Dissolve in 0 ml of the mixed solvent.
-Toluenesulfonyl chloride (1.75 g, 9.18 mmol)
Was slowly added over 1 hour, and the mixture was stirred in a refrigerator for 14 hours. Next, p-toluenesulfonyl chloride (1.75 g, 9.18 mmol) was added to the reaction solution, and the mixture was further reacted for 24 hours. After completion of the reaction, post-treatment was carried out in the same manner as in Example 1 to obtain methyl 22-tosyloxidecosanoate (2.28 g, 75%) having the following physical data. mp 74-76 ° C ¹ H-NMR (CDCl ₃ , 270 MHz): δ 1.08-1.40 (m, 34
H), 1.53 to 1.72 (m, 4H), 2.30 (t, 2H), 2.45 (
s, 3H), 3.67 (s, 3H), 4.02 (t, 2H), 7.35 (d, 2H)
), 7.80 (d, 2H)

Example 12 Synthesis of 22-Methyl phthalimidodocosanoate Potassium phthalimide (1.16 g, 6.29 mmol) and drying
Mix with 40 ml of N, N-dimethylformamide 110
The mixture was heated to 20 ° C., and a solution of 22-tosyloxidecosanoic acid methyl ester (2.20 g, 4.19 mmol) in 70 ml of N, N-dimethylformamide was added dropwise thereto, followed by reaction at 110 ° C. for 2 hours. After completion of the reaction, post-treatment was carried out in the same manner as in Example 2 to obtain methyl 22-phthalimidodocosanoate (1.57 g, 95%) having the following physical data. mp 88.5-90.5 ° C ¹ H-NMR (CDCl ₃ , 270 MHz): δ 1.12-1.41 (m, 34
H), 1.54 to 1.75 (m, 4H), 2.30 (t, 2H), 3.66,
3.67 (s, t, 5H), 7.71 (m, 2H), 7.84 (m, 2H)

Example 13 Synthesis of 22- aminodocosanoic acid Methyl 22-phthalimidodocosanoate (1.5 g,
3.0 mmol), a mixture of ethanol (40 ml) and 80% hydrazine hydrate (0.28 ml, 4.50 mmol) was heated to reflux for 10 hours. To the reaction solution was added 11.3 ml of 6N hydrochloric acid, and the mixture was further heated under reflux for 1 hour. The filtrate was concentrated under reduced pressure. Add 30 ml of ethanol to the residue and 1
To the mixture was added 15 ml of an aqueous solution of N sodium hydroxide, and the mixture was heated under reflux for 19 hours. The reaction solution was neutralized with 6N hydrochloric acid under ice cooling, and the precipitate was collected by filtration and recrystallized (ethanol-acetic acid-water) to give 22-aminodocosanoic acid (827 mg, 77
%). mp 166 to 168 ° C FD-MS (m / z): [M + H] ⁺ 356 IR (cm ^-1 ): 2920, 2850, 1640, 1535, 1470, 1400

Example 14 N- (21-Carboxyhenicosyl) maleamic acid
Synthesis 22-Aminodocosanoic acid (400 mg, 1.12 mmol) was added to 100 mL of ethanol and 50 mL of 1N aqueous sodium hydroxide solution.
Dissolve at 40 ° C in 1 ml of the mixture and raise the temperature of the resulting solution.
While maintaining the temperature at 40 ° C, maleic anhydride (4.39 g, 44.8 mmo
l) was added slowly over 5 hours. During this time, the pH of the reaction solution was kept in the range of 8-10. After stirring the reaction solution for 30 minutes, post-treatment was carried out in the same manner as in Example 4 to obtain N- (21-carboxyheneicosyl) maleamic acid (538 mg, 83%) having the following physical data. mp 140 to 143 ° C FD-MS (m / z): [M + H] ⁺ 454 IR (cm ^-1 ): 3305, 2920, 2850, 1710, 1630, 1570,
1470, 1405, 1275,1245, 1225, 1210, 1195, 1180

Example 15 Synthesis of 22-maleimidodocosanoic acid N- (21-carboxyheneicosyl) maleamic acid
(450 mg, 0.99 mmol), a mixture of 2.8 ml of acetic anhydride and sodium acetate (40.7 mg, 0.50 mmol) in 10
The reaction was performed at 0 ° C for 1 hour. After completion of the reaction, post-treatment was carried out in the same manner as in Example 5, and the following physical property values were obtained.
0-Maleimidoeicosanoic acid (170 mg, 39%) was obtained. mp 105.5 to 106.5 ° C ¹ H-NMR (CDCl ₃ , 270 MHz): δ 1.12 to 1.42 (m, 34
H), 1.50 to 1.71 (m, 4H), 2.34 (t, 2H), 3.51 (
t, 2H), 6.68 (s, 2H) FD-MS (m / z): [M + H] ⁺ 436 IR (cm- ¹ ): 2920, 2850, 1710, 1475, 1450, 1415,
840, 700

Reference Example 1 Reaction of 18-maleimidooctadecanoic acid with SOD
Preliminary Study on Synthesis of SOD Derivatives 1 mg (31 nmol) of human SOD was added to 0.9 ml of 0.1 M tris (hydroxymethyl) aminomethane-hydrochloric acid buffer (pH 9)
Prepare 7 samples of the solution obtained by dissolving in
The solutions obtained by dissolving the amounts of 18-maleimidooctadecanoic acid obtained in Example 5 in 0.1 ml of dimethyl sulfoxide, respectively, were added to the above SOD solution with stirring, respectively. After stirring in a refrigerator for two nights, the reaction solution was subjected to electrophoresis. The obtained electrophoretogram is schematically shown in FIG. In FIG. 1, (a), (b), (c), (d),
(E), (f) and (g) are each relative to SOD
The molar ratio of 18-maleimidooctadecanoic acid is 0, 1,
The schematic diagram of the electropherogram of the reaction solution obtained in the case of 2, 5, 10, 20 and 50 is shown.

[0068]

[Table 1]

In the table, the molar ratio is 18 to SOD.
-Represents the molar ratio of maleimide octadecanoic acid, weight is 1
8-Represents the weight of maleimide octadecanoic acid.

As is apparent from FIG. 1, SOD and 18-
The reaction product of maleimide octadecanoic acid was almost the same in each reaction with a molar ratio of 2 or more, indicating that all the reactive functional groups in the SOD had reacted in the reaction with a molar ratio of 2 or more.

The bands (1), (2) and (3) in FIG. 1 are the SOD channels used in the above preliminary study, respectively.
Bands (4), (5) and (6) correspond to diisomers, and one 18-maleimidooctadecanoic acid is the SOD charge isomer corresponding to bands (1), (2) and (3), respectively. Corresponds to the bound SOD derivative. Bands (7), (8) and (9) are SOD derivatives in which two 18-maleimidooctadecanoic acids are bonded to the SOD charge isomers corresponding to bands (1), (2) and (3), respectively. Corresponding.

18-maleimidooctadecanoic acid and SOD
Of SOD derivative by reaction with 1.12 ml of human SOD aqueous solution (88.95 mg / ml) and 1.68 ml of water
And 0.8 ml of 0.5 M Tris (hydroxymethyl) aminomethane-hydrochloric acid buffer (pH 9) were added.
The solution obtained by dissolving 4.7 mg of 18-maleimidooctadecanoic acid obtained in the above in 0.4 ml of dimethyl sulfoxide was gradually added with stirring. After stirring overnight at room temperature and filtering, the reaction solution was separated by Sephadex G-25.
: Eluent: 10 mM ammonium bicarbonate aqueous solution] using a carrier (trade name, manufactured by Pharmacia) as a carrier, and a polymer fraction was collected. This fraction is used as it is for DEAE-
Sepharose (DEAE-Sepharose Fast Flow: trade name,
Ion-exchange chromatography using Pharmacia) as a carrier [eluent: 10 mM Tris-HCl buffer
(pH 8) and 0.075M sodium chloride aqueous solution]
And fractions containing the SOD derivative were collected. This fraction was subjected to gel filtration [eluent: 10 mM aqueous ammonium bicarbonate] using Sephadex G-25 (trade name, manufactured by Pharmacia) as a carrier, and desalted. The fraction is freeze-dried and SOD derivative 52mg
I got In addition, free SH was obtained from the obtained SOD derivative.
No groups were detected.

The respective electropherograms of the used SOD and the obtained SOD derivative are schematically shown in FIG. 2 (a) and (b). Each band in (b) is shown in FIG.
Bands (7), (8) and (9), respectively. From this and the quantitative results of the SH group, the obtained SOD was
The derivative was identified as one in which 18-maleimidooctadecanoic acid molecules were bonded to two SH groups of SOD, respectively. FIG. 3 shows an infrared absorption spectrum of the obtained SOD derivative.

Reference Example 2 By reaction of 20-maleimidoeicosanoic acid with SOD
Synthesis of SOD derivatives Human SOD aqueous solution (88.95mg / ml) 1.12ml in water 1.68ml
And 0.8 ml of a 0.5 M aqueous solution of tris (hydroxymethyl) aminomethane.
-A solution obtained by dissolving 5.1 mg of maleimide eicosanoic acid in 0.4 ml of dimethyl sulfoxide was gradually added with stirring. After stirring overnight at room temperature and filtering, the reaction solution was subjected to gel filtration using Sephadex G-25 as a carrier [eluent: 10 mM aqueous solution of ammonium bicarbonate] to collect a polymer fraction. This fraction is used as is for ion exchange chromatography using DEAE-Sepharose as a carrier.
[Eluent: 10 mM Tris-HCl buffer (pH 8) and
0.10M aqueous solution of sodium chloride]
The fraction containing the derivative was collected. This fraction was subjected to gel filtration using Sephadex G-25 as a carrier [eluent: 10 mM
[Aqueous ammonium bicarbonate solution], desalted, and then the polymer fraction was freeze-dried to obtain 33 mg of the SOD derivative. No free SH group was detected from the obtained SOD derivative.

The respective electropherograms of the used SOD and the obtained SOD derivative are schematically shown in FIGS. 4 (a) and 4 (b). Hereinafter, in the same manner as in Reference Example 1,
The resulting SOD derivative has 20-
It was identified that the maleimide eicosanoic acid molecules were bonded respectively. FIG. 5 shows an infrared absorption spectrum of the obtained SOD derivative.

Reference Example 3 S by reaction of 22-maleimidodocosanoic acid with SOD
Synthesis of OD derivative Human SOD aqueous solution (88.95mg / ml) 1.12ml in water 4.88ml
And 4.0 ml of 0.5 M tris (hydroxymethyl) aminomethane-hydrochloric acid buffer (pH 9) were added.
The solution obtained by dissolving 13.6 mg of the 22-maleimidodocosanoic acid obtained in the above in 10 ml of dimethylsulfoxide was gradually added with stirring. After stirring overnight at room temperature and filtering, the reaction solution is subjected to gel filtration using Sephadex G-25 as a carrier [eluent: 10 mM aqueous ammonium bicarbonate]
And the high molecular fraction was collected. DEA
Ion exchange chromatography using E-Sepharose as a carrier [eluent: 10 mM Tris-HCl buffer (pH
8) and a mixture of 0.15 M aqueous sodium chloride solution] to collect a fraction containing the SOD derivative. This fraction was subjected to gel filtration using Sephadex G-25 as a carrier [eluent: 10 mM aqueous solution of ammonium bicarbonate], desalted, and the polymer fraction was lyophilized to give the SOD derivative 42m
g. It should be noted that free S
No H group was detected.

The respective electropherograms of the used SOD and the obtained SOD derivative are schematically shown in FIGS. 6 (a) and 6 (b). Hereinafter, in the same manner as in Reference Example 1,
The obtained SOD derivative was substituted with two SH groups of SOD.
The maleimide docosanoic acid molecules were identified as being bonded to each other. FIG. 7 shows an infrared absorption spectrum of the obtained SOD derivative.

Test Example 1 Blood concentration of SOD derivatives Rats (Wistar male, 7%) under pentobarbital anesthesia
A cannulation was performed from the femoral vein at the age of about 200 g, and 0.2 ml of a heparin solution (1000 U / ml) was injected. A solution (10 mg / ml) obtained by dissolving SOD or SOD derivative in physiological saline was used for each rat.
0.2 ml was injected through the femoral vein. Blood was collected at intervals of 0.2 ml each time, and the blood concentration was measured by measuring the SOD activity in plasma. FIG. 8 shows the change over time in the blood concentration of SOD and the blood concentration of the SOD derivative.

Test Example 2 SOD derivative against acute gastric mucosal lesion (gastric ulcer) in rat
After the SD male rats (body weight 200 g) were fasted overnight, 6 rats per group were restrained by placing them in a stress cage. Loaded. After 6 hours, the rats were withdrawn from the water, killed by blood loss, and the stomach was removed. The tissue was fixed by injecting 1% formalin into the gastric lumen. After fixation, the length of the linear ulcer on the mucosal surface was measured, and the total was defined as the ulcer index.

The control group was treated with 0.5 ml of physiological saline, and the test group was treated with 2 mg / rat of the SOD derivative obtained in Reference Example 1 as 0.2 ml of physiological saline solution for 5 minutes. Before intravenous administration. The results are shown in the table below.

[0081]

[Table 2]

As is evident from Table 2, the test group showed SO
A marked anti-ulcer effect of the D derivative was observed.

Test Example 3 Effect of SOD derivative on rat coronary reperfusion arrhythmia
Fruit rats (Wistar male, weight 200-230 g) were fasted overnight and then pentobarbital (50 mg / kg body weight)
Was anesthetized by intraperitoneal administration. Anesthetized rats are tracheostomized and respirated by a respirator (1).
Ventilation volume: 1.5ml / 100g body weight, ventilation frequency: 60 ventilation / min
), The heart was exposed by longitudinal sternotomy. The coronary artery was occluded by aspirating the left anterior descending coronary artery approximately 3 mm from the circumflex branch. Occlusion-reperfusion operation
Twice at 30-minute intervals, 15 minutes after the first reperfusion, the drug was injected into the femoral vein of the rat at 5 mg / kg body weight (total dose of 0.
1 ml). As a drug, physiological saline was used for the control group, and a prepared solution of the SOD derivative obtained in Reference Example 2 in physiological saline was used for the test group. During each reperfusion, the electrocardiogram (Lead II) was continuously monitored for 30 minutes to examine ventricular extrasystole (PVC), ventricular tachycardia (VT) and ventricular fibrillation (Vf). The results are shown in the table below.

[0084]

[Table 3]

As is clear from Table 3, in the test group, SO
A remarkable antiarrhythmic effect of the D derivative was observed.

[0086]

According to the present invention, there is provided a long-chain carboxylic maleimide (I) which has a significantly longer half-life in blood compared to SOD and which provides an SOD derivative having a single chemical structure. Is done. The SOD derivative has a reversible interaction with a serum protein, and can be easily transferred to a focal spot. In addition, the SOD derivative has an excellent anti-ulcer effect, and further has a pharmacological effect such as an anti-inflammatory effect, an anti-arrhythmic effect, an anti-ischemic injury effect, and an anti-cerebral edema effect.

[Brief description of the drawings]

FIG. 1 is a schematic diagram showing the results of electrophoresis, wherein (a)
(B), (c), (d), (e), (f) and (g)
In Reference Example 1, the molar ratio of 18-maleimidooctadecanoic acid to SOD is 0, 1, 2, 5, 10, 2
The schematic diagram of the electrophoretogram of the reaction solution obtained in the cases of 0 and 50 is shown.

FIGS. 2A and 2B are schematic diagrams showing the results of electrophoresis. FIG. 2A is a schematic diagram of the electrophoretic diagram of SOD used in Reference Example 1, and FIG. 2B is a schematic diagram of the SOD derivative obtained in Reference Example 1. The schematic diagram of an electrophoretogram is shown.

FIG. 3 is a view showing an infrared absorption spectrum of the SOD derivative obtained in Reference Example 1.

4A and 4B are schematic diagrams showing the results of electrophoresis. FIG. 4A is a schematic diagram of an electrophoretic diagram of SOD used in Reference Example 2, and FIG. The schematic diagram of an electrophoretogram is shown.

FIG. 5 is a view showing an infrared absorption spectrum of the SOD derivative obtained in Reference Example 2.

6A and 6B are schematic diagrams showing the results of electrophoresis. FIG. 6A is a schematic diagram of an electrophoretic diagram of SOD used in Reference Example 3, and FIG. 6B is a schematic diagram of the SOD derivative obtained in Reference Example 3. The schematic diagram of an electrophoretogram is shown.

FIG. 7 is a view showing an infrared absorption spectrum of the SOD derivative obtained in Reference Example 3.

FIG. 8 is a graph showing the change over time in blood concentration measured in Test Example 1, wherein (1), (2), (3) and (4) are SOD, and the SOD derivative obtained in Reference Example 1, respectively. , The SOD derivative obtained in Reference Example 2 and the SOD derivative obtained in Reference Example 3.
5 shows the time-dependent change in the blood concentration of the OD derivative.

──────────────────────────────────────────────────続 き Continuation of the front page (56) References JP-A-62-25137 (JP, A) JP-A-62-195387 (JP, A) Biochem. Soc. Trans. , Vol. 11, No. 6, p. 753-754 (1983) Biol. Membr. , Vol. 10, p. 1019-1025 (1987) Med Chem. , Vol18, No. 10, p. 1004-1010 (1975) (58) Fields investigated (Int. Cl. ⁷ , DB name) C07D 207/452 A61K 37/50 CA (STN) CAOLD (STN) REGISTRY (STN)

Claims (1)

(57) [Claims] 1. A compound represented by the following general formula (I): (Wherein W is one or more oxygen atoms, sulfur atoms, or-
The number of main chain atoms which may be interrupted by a group represented by N (R)-(wherein R represents a lower alkyl group) is 17 to 23;
Long chain carboxylic acid maleimide represented by the divalent long chain hydrocarbon group).