JPH10330346A - Linear chain nitron derivative, and medicine and reagent containing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject stable compound having an excellent spin trap ability, capable of rapidly binding it with active oxygen and a free radical even in vivo, high in safety to the living body and useful as a medicine and a reagent. SOLUTION: This compound is a nitron derivative of formula I Ar is a phenylene or pyridinylene; R<1> and R<3> are each a lower alkyl; R<2> is a lower alkylene; X is H, or CO(AO)1 OCH3 [A is a lower alkylene; (l) is 1-100); Y is H, or a group of formula II [R<4> and R<5> are each H or methyl; R<6> and R<7> are each H, or a lower alkyl; (m) and (n) are each 10-10000]; with the proviso that when X and Y are each H, Ar is pyridinylene], e.g. C-2-pyridyl-N-(2-hydroxy-1,1- dimethylethyl)nitron. A compound of formula III (Py is a pyridyl) in the compound of the formula I can be obtained, for example, by condensing pyridyl aldehyde with the nitroxy alcohol of formula IV.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a linear nitrone derivative and a drug containing the same as an active ingredient. More specifically, the present invention is capable of capturing active oxygen and free radicals in a living body. As an agent for preventing and treating various diseases caused by free radicals in the body, or as an ESR (El
A linear nitrone derivative that is useful as a reagent for non-invasively obtaining biological images by magnetic resonance methods such as ectron spin resonance (electron spin resonance) and detecting active oxygen and free radicals in collected biological tissues It also relates to a medicine and a reagent containing the same.

[0002]

2. Description of the Related Art Active oxygen can be defined as an oxygen species which has a short life span, is highly reactive, and is involved in various in vivo oxidation reactions. The range differs from the definition, but in a narrow sense, hydroxyl radical (.OH), superoxide (O ₂ ⁻ ), singlet oxygen ( ¹ O ₂ ), hydrogen peroxide (H
₂ O ₂ ). In a broad sense, a peroxy radical (LOO.) Or an alkoxy radical (L) derived from the reaction of the above-mentioned active species with a biological component (for example, unsaturated fatty acid L).
O.), and hypochlorite ion (Cl) generated from the reaction of H ₂ O ₂ with Cl ^{− with} myeloperoxidase or the like.
O ^-) is also active oxygen like. Hydroxyl radicals and superoxides are radicals. The radical is 1
It can be defined as an atom or molecule having one or more unpaired electrons. Hypochlorite ions and hydrogen peroxide are not themselves radicals, but may arise from certain radical reactions or cause other radical reactions.

[0003] Active oxygen and radicals are generally unstable, and their lifetime is 10 ^{-5 for} benzyl radicals in the gas phase.
It is ^{-10 -4} seconds, and 10 ^-3 to 10 ^-2 seconds for simple methyl radicals and hydroxyl radicals in atmospheric gas phase.

In recent years, active oxygen and free radicals showing various physiological activities in the living body have attracted attention in the fields of biology, medicine and pharmacy, and research has been advanced. The causes of active oxygen and free radicals generated in the living body are ultraviolet rays,
Radiation, air pollution, oxygen, lipid peroxidation, metal ions, ischemia / reperfusion, and the like. The resulting active oxygen and free radicals in the body cause various in vivo reactions such as peroxidation of lipids, denaturation of proteins, and decomposition of nucleic acids. Associated diseases include cerebral ischemia, heart disease,
Gastrointestinal diseases, cancer, aging, inflammation and the like. In this way, if the active oxygen and free radicals in vivo related to various diseases can be detected non-invasively from outside the body, the cause of various diseases can be investigated and it can be useful information medically.

In order to detect active oxygen and free radicals, a reaction reagent is added to the reaction system, and an indirect method for detecting changes in the absorbance and luminescence of the reaction system resulting therefrom and an ESR method for directly detecting unpaired electrons of free radicals. There is. Of these, ES
The sample by the R method can be measured as a solution or a solid, and it can be measured as an opaque or heterogeneous sample, which is extremely advantageous for detecting active oxygen in a living body. Since it is unstable and has a short life, it is difficult to directly measure by the ESR method. Also, such as singlet oxygen,
Active oxygen having no unpaired electron cannot be measured by ESR.

[0006] In order to overcome such disadvantages of the ESR method, a spin trap method has been developed. According to this method, as shown in the following formula, the trapping agent (T) reacts rapidly with short-lived active oxygen and free radicals (R.) and can be detected by ESR, and the stable and long-lived spin adduct (RT) can be detected.
・) Is used.

[0007]

Embedded image

That is, a compound that quickly reacts with active oxygen or radicals and generates a spin adduct that is sufficiently stable for ESR measurement is added to the measurement system as a spin trapping agent. Can be measured.

Therefore, the compound used as the spin trapping agent is capable of rapidly reacting with the above-mentioned (1) active oxygen and free radicals, (2) producing a sufficiently stable spin adduct, and (3) In terms of handling, it was necessary to satisfy the conditions such as being chemically stable itself and (4) low toxicity.

As a trapping agent which has been mainly used for the study of free radicals in vivo, DMPO (5,5-dimethyl-1-pyrroline-1-oxide), which is a cyclic nitrone, and PBN, which is a straight chain, are used as nitrones. (C-phenyl-N
-Tert-butyl nitrone), DBN in nitroso system
It was nothing but BS (sodium 3,5-dibromo-4-nitrosobenzenesulfonate), and none of them could be said to sufficiently satisfy the above conditions [for example, Pharmacia, 28,
1347-1352 (1992)].

That is, for example, PBN is a commonly used spin trapping agent along with DMPO, and has been studied for its effect of reducing damage after cardiac or cerebral ischemia-reperfusion. However, (i) its solubility in water is low. It is difficult to use to handle biological components,
(Ii) There are drawbacks such as low spin trapping ability.

[0012]

[Problems to be Solved by the Invention] Presently, prevention and treatment of adult diseases such as cerebral ischemia, heart disease, digestive system disease, cancer and inflammation,
There is a strong demand for prevention of aging and the like, and it has been demanded to find a drug capable of effectively capturing active oxygen and free radicals, which are the causes thereof, and to effectively prevent / treat the above-mentioned diseases.

Further, as described above, if there is a compound that rapidly binds to active oxygen and free radicals in the living body and has sufficient stability, it is used as a spin trap agent in the ESR method which is a non-invasive measurement method. It can be useful for diagnosis of pathologies involving active oxygen and free radicals such as cancer, ischemia and inflammation based on the biological spectrum and biological images of free radicals and free radicals in vivo. Is required.

Therefore, an object of the present invention is to provide a compound which is rapidly bound to active oxygen and free radicals not only in an in vitro system but also in a living body, is stable, and has high safety to the living body, and which is useful as a drug or a reagent. Especially.

[0015]

The inventors of the present invention have conducted extensive studies to solve the above problems, and as a result, the following general formula (1)
Found that the nitrone derivative represented by the formula (1) has excellent spin trapping ability, satisfies the above conditions, and is useful as a medicine, a clinical test reagent, and a reagent for detecting active oxygen and free radicals, and completed the present invention. I came to.

That is, the present invention provides the following general formula (1):

[0017]

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[In the formula, Ar represents a phenylene group or a pyridinylene group, R ¹ and R ³ are the same or different and represent a lower alkyl group, R ² represents a lower alkylene group, X represents a hydrogen atom or —CO ( AO) _l OCH ₃ (where A represents a lower alkylene group and l represents a number of 1 to 100), and Y represents a hydrogen atom or a formula

[0019]

Embedded image

(Where R ⁴ and R ^{5 each} represent a hydrogen atom or a methyl group, R ⁶ and R ^{7 each} represent a hydrogen atom or a lower alkyl group, and m and n each represent a number of 10 to 10,000. ) Shows the group shown by. However, when X and Y are hydrogen atoms, Ar is a pyridinylene group. ] The nitrone derivative represented by this is provided.

The present invention also provides the above nitrone derivative (1)
And a clinical test reagent comprising the following as an active ingredient:

The present invention also relates to the above nitrone derivative (1)
The present invention provides an active oxygen or free radical scavenger containing as an active ingredient.

The present invention further provides an agent for detecting active oxygen or free radicals comprising the above nitrone derivative as an active ingredient.

[0024]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, a nitrone derivative is shown.
In the general formula (1), R¹, R³, R⁶And R⁷Low indicated by
As the lower alkyl group, a straight or branched chain having 1 to 4 carbon atoms
Alkyl group of, specifically, methyl group, ethyl
Group, n-propyl group, isopropyl group, n-butyl group, etc.
Among these, a methyl group is particularly preferable. Ma
R ²And a lower alkylene group represented by A,
Examples include linear or branched alkylene groups having 1 to 4 carbon atoms.
Specifically, methylene group, ethylene group, propylene
Group, trimethylene group, butylene group, tetramethylene group, etc.
Of which R is²As a methylene group,
A is particularly preferably an ethylene group.

In the general formula (1), one of X and Y is preferably a hydrogen atom. When X and Y are both hydrogen atoms, Ar is a pyridinylene group, and the compound is particularly preferable. That is, in the general formula (1), the following formulas (1A), (1B) and (1
It is a compound represented by C), and among these, the compound of the formula (1A) is particularly preferable.

[0026]

Embedded image

[In the formula, Py represents a pyridyl group, and R ¹ ,
R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , Ar, A, l,
m and n are the same as described above)

The compound of the formula (1A) can be produced, for example, according to the following reaction formula.

[0029]

Embedded image

Wherein Py, R ¹ , R ² and R ³ are the same as above.

That is, the compound (1A) is obtained by condensing the pyridyl aldehyde (2) and the nitroxy alcohol (3).

This condensation reaction may be carried out at a temperature of about 0 to 180 ° C. for about 3 to 50 hours in the presence of zinc and acetic acid.

The compound of formula (1B) can be produced, for example, according to the following reaction formula.

[0034]

Embedded image

[Wherein R ¹ , R ² , R ³ , Ar, A and l are the same as above]

That is, the compound (1B) is obtained by reacting the polyalkylene glycol monomethyl ether (4) with the aldehyde acid (5) to obtain a compound (6), and then reacting the compound with a nitroxy alcohol (3). can get.

The reaction between the compound (4) and the compound (5) is
What is necessary is just to carry out under conditions of usual ester synthesis reaction, for example, It is preferable to carry out in the presence of a condensing agent such as dicyclohexylcarbodiimide. The condensation reaction between the compound (6) and the compound (3) may be performed in the same manner as in the reaction for obtaining the compound (1A).

The compound of formula (1C) is produced, for example, according to the following reaction formula.

[0039]

[Chemical 9]

[Wherein R ¹ , R ² , R ³ , R ⁴ , R ⁵ ,
R ⁶ , R ⁷ , Ar, m and n are the same as described above.

That is, the compound (1C) is obtained by condensing the acrylamide-nitroalkyl acrylate copolymer (8) and the aldehyde (7). This condensation reaction may be performed in the same manner as the reaction for obtaining the compound (1A).

The nitrone derivative (1) of the present invention can be isolated from the reaction mixture by recrystallization, chromatography and the like.

The nitrone derivative (1) obtained as described above can be used as a medicine and a reagent after being purified if necessary and then formulated into a preparation by a conventional method. More specifically, the compound of the present invention, as an active oxygen or free radical scavenger, diseases associated with these, for example, prevention of diseases such as cerebral ischemia, heart disease, digestive system disease, cancer, aging, inflammation, etc. It can be used for therapeutic agents. For this purpose, it may be orally or parenterally administered, and known pharmaceutically acceptable carriers, for example, lactose, sucrose, crystalline cellulose, talc, stearic acid, lecithin, sodium chloride, inositol and the like. And a liquid carrier such as syrup, glycerin, olive oil, ethanol, benzyl alcohol, propylene glycol, and water can be used.

The compound of the present invention can be used as a clinical test reagent for non-invasively obtaining a biological image by a magnetic resonance method represented by ESR or detecting active oxygen and free radicals in a collected biological tissue. it can.

The reason that the compound of the present invention binds to active oxygen and free radicals in a living body and has sufficient stability is because the compounds of the present invention capture active oxygen and free radicals by a reaction represented by the following formula. It is presumed.

[0046]

[Chemical 10]

[In the formula, R ¹ , R ² , R ³ , Ar, X and Y are the same as the above]

The compound of the present invention binds to active oxygen and free radicals in a living body as described above and stably retains the same. Therefore, it can be used as a detecting agent for imaging active oxygen and free radicals in a living body by the ESR method. It is useful.

That is, the ESR method is one of the magnetic resonance methods and targets unpaired electrons of atoms and molecules, but free radicals such as active oxygen and transition elements have unpaired electrons.
Both are measurement objects of ESR. Recently, as the relationship between active oxygen and physiological functions has been clarified, ESR has been actively used for identification of active oxygen and analysis of its redox reaction.

However, microwaves in the X-band (approximately 9.5 GHz) have been used in ESR so far, but the dielectric loss due to water is large, and it is impossible to measure a large volume sample such as a living body. Was. However, recently, the L-band (1
An ESR device using microwaves (hereinafter referred to as GHz or less) has been developed, and non-invasive measurement of free radicals in a sample having a high water content or a biological sample has become possible [for example, Pharmacia, 27, 710-715 (1991)]. See], the possibility of measuring very large volumes of biological samples such as humans has been opened.

The compound of the present invention can be used for ES
It is considered that in the ESR method using the R device, active oxygen and free radicals of the human body are non-invasively measured as a detection agent, and useful information on diseases or symptoms caused by active oxygen and free radicals can be provided.

[0052]

EXAMPLES The present invention will be described more specifically with reference to the following examples, but the present invention is not limited to these examples.

Example 1 Synthesis of C-2-pyridyl-N- (2-hydroxy-1,1-dimethylethyl) nitrone In a conical flask equipped with a thermometer, a dropping funnel and an inlet tube, 4.27 g (0. 0403 mol), 2
9.59 g of -methyl-2-nitro-1-propanol
(0.0806 mol), 7.91 g of zinc (0.121 g)
mol) and 300 ml of ethanol, and kept at 5 ° C. Thereafter, 14.5 g (0.242 mol) of acetic acid was added to 1
Drop over a period of time and allow to react for 50 hours. After the reaction,
The solid is filtered off, the filtrate is concentrated, neutralized with saturated aqueous sodium hydrogen carbonate solution, extracted with ether and the organic layer is dried over sodium sulfate. Then, the mixture was concentrated, the residue was adsorbed on silica gel, purified by column chromatography (silica-ethyl acetate), and then recrystallized (ethyl acetate-n-hexane) to give C-2-pyridyl-N- (2- Hydroxy-1,1-dimethylethyl) nitrone (yield 32
%).

Melting point: 105-106 ° C IR: 3410, 3200, 2930, 1570, 1460, 1395, 1370, 128
0, 1130, 1060,810 (cm ^-1 ) ¹ H-NMR: 1.61 (s, 6H), 3.82 (s, 2H), 4.65 (brs, 1H),
7.22, 7.26, 7.31 (m, 1H), 7.68, 7.70, 7.73 (m, 1H),
7.82 (m, 1H), 8.59, 8.60 (m, 1H), 8.99, 9.02 (m, 1H) Elemental analysis Calculated value: C; 61.51%, H; 7.27%, N; 1
4.42% measured values: C; 61.62%, H; 7.16%, N; 1
4.36%

Example 2 Poly (methacrylamide) -CO- [2-methyl-2-
Synthesis of (N-benzylidene-N-oxide) aminopropyl acrylate]} (1) Synthesis of 2-methyl-2-nitropropyl acrylate 2-methyl-2 was placed in a three-necked flask equipped with an inlet tube, a dropping funnel, and a calcium chloride tube. -Nitro-2-propanol 3
0 g (0.28 mol), triethylamine 31.1 g
(0.308 mol) and 150 ml of purified tetrahydrofuran (THF), and brought to −10 ° C. with ice and salt.
Stir. After that, 25.3 g of acrylic acid chloride
(0.28 mol) and 150 ml of purified THF are added dropwise and stirred at room temperature overnight. Then, the solid is filtered and
It was washed with HF, concentrated, and purified by column chromatography (silica ethyl acetate: n-hexane = 1: 5) to obtain 2-methyl-2-nitropropyl acrylate (88.6% yield).

(2) Polymerization As a monomer having a nitro group in the side chain, 2-methyl-2
-Nitropropyl acrylate (MNA) and water-soluble N, N'-dimethylacrylamide (DMA), α, α 'azobisisobutyronitrile (AIB) as an initiator
N) and various solvents were put in a brown polymerization tube and polymerization was carried out at 60 ° C. for 5 hours. After completion of the polymerization, the reaction product was poured into diethyl ether, the precipitated polymer was separated by filtration, dried under reduced pressure, and the yield was determined by a gravimetric method. The composition ratio of the obtained copolymer is 1H-
It was calculated from the area of the peak derived from the ester of the MNA unit and the peak derived from the amide of the DMA unit by NMR. The solvent used in the polymerization reaction is toluene, tetrahydrofuran (THF), acetonitrile, dimethylformamide (DMF), dimethylsulfoxide (DMS).
Any of O) may be used, but toluene is particularly suitable. In order to obtain a water-soluble polymer having a nitro group, the charge ratio of DMA to MNA may be changed from 1: 1 to 1: 5,
In particular, 1: 5 is optimal.

(3) Poly (methacrylamide) -CO-
Synthesis of [2-methyl-2- (N-benzylidene-N-oxide) aminopropyl acrylate]} For the MNA unit of the copolymer obtained in (2), 1
An equivalent amount of benzaldehyde, 2 equivalents of zinc and 50 ml of ethanol are put into a brown reaction tube and stirred at 5 ° C. under a nitrogen stream. Then, 4 equivalents of acetic acid are added and the reaction is carried out for 50 hours. After the reaction, the solid is filtered off, and the filtrate is concentrated and poured into purified diethyl ether. The deposited precipitate was filtered and dried to obtain a polymer.

¹ H-NMR: 0.58-0.67 (m), 1.11-2.59 (m), 2.
77-2.96 (m), 3.93-5.02 (m), 7.25-7.62 (m), 7.94-8.01
(m), 8.22-8.36 (m)

Example 3 Synthesis of C-monomethoxypoly (ethylene glycol) carbonyl phenyl-N- (2-hydroxy-1,1-dimethylethyl) nitrone (1) Preparation of para-formyl monomethoxypoly (ethylene glycol) benzoate Synthetic Nitrogen introduction tube and calcium chloride tube equipped with Erlenmeyer flask 1.2g terephthalaldehyde acid (0.008mo
l), 35 ml of dimethyl sulfoxide was added, and after dissolution,
Carbodiimidazole 1.56g (0.0096mo
l) was added little by little, and then 5 ml of dimethyl sulfoxide
Monomethoxy polyethylene glycol 3.3 diluted with
Add 6 g (0.0096 mol) in small portions and stir at 80 ° C. overnight. After the reaction was completed, dimethylsulfoxide was distilled off, the mixture was extracted with benzene, dried over sodium sulfate, and concentrated to obtain para-formyl monomethoxypoly (ethylene glycol) benzoate (yield 54%).

(2) Synthesis of C-monomethoxypoly (ethylene glycol) carbonyl phenyl-N- (2-hydroxy-1,1-dimethylethyl) nitrone Para-formyl monomethoxypoly (ethylene glycol) benzoate was added to a brown reaction tube. 1g (2.07mm
ol), 2-methyl-2-nitro-1-propanol 0.49 g (4.1 m4 mol), zinc 0.406 g
(6.21 mmol) and 50 ml of ethanol are stirred at 5 ° C. under a nitrogen stream. Then, 0.75 g of acetic acid (12.4
1 mmol) is slowly added dropwise and the mixture is stirred for 48 hours. After the reaction, the solid was filtered, the filtrate was concentrated, and C-monomethoxypoly (ethylene glycol) carbonylphenyl-N-
(2-Hydroxy-1,1-dimethylethyl) nitrone (yield 42%) was obtained.

IR: 3430, 2880, 1720, 1545, 1470, 141
0, 1360, 1275, 860, 775 ¹ H-NMR: 1.58 (s), 3.33-3.98 (m), 4.50-4.60 (brs), 4.
90 (s), 7.88-8.17 (m), 8.28-8.39 (m) Calculated molecular weight: 559 (n = 7)

Example 4 C-2-pyridyl-N- (2-hydroxy-1,1-dimethylethyl) nitrone (sample A) which is a compound of the present invention and C-phenyl-N-ter which is a known compound
Radical scavenging test of t-butyl nitrone (PBN) Using a hydroxyl radical generating system utilizing the Fenton reaction, the active oxygen scavenging ability of the specimen and PBN was investigated. The principle is as follows. The generated radicals have a short life and cannot be directly measured by the ESR method. However, if a compound acting as a spin trapping agent is present in the system and reacts with a radical to form a stable and long-lived spin adduct, this can be measured by the ESR method. Utilizing this principle, it was examined as follows whether the compound of the present invention has spin trapping ability and thus radical scavenging ability.

[Measurement system for hydroxyl radical generation by Fenton reaction] Iron (II) sulfate heptahydrate and diethylenetriamine-N, N, N ', N ", N" -pentaacetic acid (DTP
A) 70 μl of mixed solution (1.5 mM), 70 μl of specimen A solution (1.5 mM) diluted with phosphate buffered saline, or PBN solution (1.5 mM) diluted with phosphate buffered saline
70 μl and 70 μl of a hydrogen peroxide solution (15 mM) diluted with a phosphate buffered saline were placed in a micro test tube in order, and the time was simultaneously measured with the addition of the hydrogen peroxide solution, followed by thorough stirring. It was sucked into a hematocrit tube (manufactured by Terumo, Brain Glass), and after 30 seconds, a magnetic field sweep was started. [Comparative measurement system (excluding hydroxyl radical generation system)]
Specimen A solution diluted with phosphate buffered saline (1.5 m
M) PB diluted with 70 μl or phosphate buffered saline
In a micro test tube, 70 μl of N solution (1.5 mM) and 140 μl of phosphate buffered saline were placed in this order. At the same time as adding the phosphate buffered saline, timing was started, and the mixture was stirred well. It was sucked into a hematocrit tube (made by Terumo, Brain glass), and after 30 seconds, a magnetic field sweep was started.

[ESR spectrum measurement conditions] ESR was measured under the following conditions. Measuring device: Electron spin resonance device [JES-FR3
0 (JEOL)] External magnetic field: 337 ± 5 mT Magnetic field modulation width: 0.1 mT Response time: 0.1 seconds Microwave output: 20 mW Magnetic field sweep time: 2 minutes / 10 mT Measurement temperature: Room temperature

[Results] FIG. 1 [bottom] shows the ESR spectrum of the sample A solution in the hydroxyl radical generating system. FIG. 1 shows the ESR spectrum of the system containing the sample A and not containing the hydroxyl radical generating reagent (comparative measurement system).
Shown above. P in hydroxyl radical generation system
The ESR spectrum of the BN solution is shown in FIG. 2 [bottom]. PB
The ESR spectrum of the system containing N and not containing the hydroxyl radical generating reagent (comparative measurement system) is shown in FIG. 2 [top]. As is clear from this result, in the system containing the analyte A and the hydroxyl radical generating system, a strong signal was obtained because the hydroxyl radical and the analyte A generate a stable spin adduct. In the system containing no hydroxyl radical-generating reagent (comparative measurement system), no hydroxyl radical was generated, so no signal was observed. or,
A stronger spectrum was obtained for sample A than for PBN.

From the above, the compound of the present invention C-
It was confirmed that 2-pyridyl-N- (2-hydroxy-1,1-dimethylethyl) nitrone scavenged a short-lived hydroxyl radical and that it had a higher hydroxyl radical detection sensitivity than PBN.

Example 5 Poly {methacrylamido-CO- [2-methyl-2-
Radical scavenging test of (N-benzylidene-N-oxide) aminopropyl acrylate]} (specimen B) An active oxygen scavenging ability of the specimen B using a hydroxyl radical generating system and a 2-cyano-propyl radical generating system utilizing the Fenton reaction. I checked.

[Hydroxyl Radical Generating System by Fenton Reaction] Iron (II) sulfate heptahydrate and diethylenetriamine-N, N, N ′, N ″, N ″ -pentaacetic acid (DTPA)
20 μl of the mixed solution (0.9 mM) and the sample B solution (0.0
2 g / ml) and 200 μl of hydrogen peroxide (15 g
mM) 20 μl in this order in a micro test tube,
The time was started at the same time when the hydrogen peroxide solution was added, and the mixture was stirred well. Hematocrit tube (made by Terumo, Brain glass)
It was sucked up into the magnetic field and the magnetic field sweep was started after 60 seconds.

[2-Cyano-propyl radical generating system]
In a brown reaction tube, α, α-azobisisobutyronitrile 0.
1 g, 0.1 g of sample B, and 5 ml of purified dimethyl sulfoxide (DMSO) were added, and the mixture was allowed to react at 70 ° C. for 1 hour.

[Measurement conditions of ESR spectrum] ESR was measured under the following conditions. Measuring device: Electron spin resonance device [JES-FR3
0 (JEOL)] External magnetic field: 335 ± 5 mT Magnetic field modulation width: 0.1 mT Time constant: 0.1 seconds Microwave output: 16 mW Magnetic field sweep time: 2 minutes / 10 mT Measurement temperature: Room temperature

[Results] FIG. 3 shows the ESR spectrum of the sample B solution in the hydroxyl radical generating system. 2-
FIG. 4 shows an ESR spectrum of the sample B solution in the cyano-propyl radical generating system. As is clear from this result, in the system including the hydroxyl radical-generating system and the 2-cyano-propyl radical-generating system, the hydroxyl radical and the 2-cyano-propyl radical are not contained in the analyte B.
And a strong signal was obtained to generate a stable spin adduct.

From the above, the compound of the present invention, poly {methacrylamide-CO- [2-methyl-2- (N
It was confirmed that -benzylidene-N-oxide) aminopropyl acrylate]} traps short-lived hydroxyl radicals and 2-cyano-propyl radicals.

Example 6 Radical scavenging test of C-monomethoxypoly (ethylene glycol) carbonyl phenyl-N- (2-hydroxy-1,1-dimethylethyl) nitrone (specimen C) Superoxide by hypoxanthine-xanthine oxidase system The active oxygen scavenging ability of the sample C was examined using a generation system, a hydroxyl radical generation system by Fenton reaction, a 2-cyano-propyl radical generation system, and a benzoyl radical generation system.

[Superoxide generation system by hypoxanthine-xanthine oxidase system] Hypoxanthine solution (2 mM) 50 μl, phosphate buffered saline 50 μl
1, 50 μl of sample C solution (0.01 M), and 50 xanthine oxidase solution (0.4 unit / ml)
The samples were placed in a micro test tube in the order of μl, and the time measurement was started at the same time as the addition of the xanthine oxidase solution. A hematocrit tube (made by Terumo, Brain glass) was sucked up, and after 60 seconds, a magnetic field sweep was started.

[Hydroxyl Radical Generating System by Fenton Reaction] Iron (II) sulfate heptahydrate and diethylenetriamine-N, N, N ′, N ″, N ″ -pentaacetic acid (DTPA)
50 μl of the mixed solution (0.9 mM) and the sample C solution (0.0
1M) 50 μl and hydrogen peroxide solution (15 mM) 5
0 μl was taken in this order in a micro test tube, and hydrogen peroxide solution was added, and at the same time, the time measurement was started, and the mixture was well stirred. It was sucked into a hematocrit tube (manufactured by Terumo, Brain Glass), and a magnetic field sweep was started 60 seconds later.

[2-Cyano-propyl radical generating system]
Place α, α-azobisisobutyronitrile in a brown reaction tube.
1 g, sample C 0.114 g, and purified DMSO3m
1 was put thereinto, reacted at 70 ° C. for 1 hour, and sucked into a hematocrit tube (made by Terumo, Brain glass) to start a magnetic field sweep.

[Benzoyl Radical Generation System] In a brown reaction tube, 0.15 g of benzoyl peroxide, 0.114 g of sample C,
Then, 3 ml of purified DMSO was put therein, reacted at 70 ° C. for 1 hour, and sucked into a hematocrit tube (made by Terumo, Brain glass) to start magnetic field sweep.

[Measurement Conditions of ESR Spectrum] ESR was measured under the following conditions. Measuring device: Electron spin resonance device [JES-FR3
0 (JEOL)] External magnetic field: 335 ± 5 mT Magnetic field modulation width: 0.1 mT Time constant: 0.1 sec Microwave output: 4 mW Magnetic field sweep time: 2 minutes / 10 mT Measurement temperature: Room temperature

[Results] FIG. 5 shows the ESR spectrum of the sample C in the superoxide generation system using the hypoxanthine-xanthine oxidase system. The ESR spectrum of the sample C in the hydroxyl radical generation system utilizing the Fenton reaction is shown in FIG. FIG. 7 shows the ESR spectrum of the sample C in the 2-cyano-propyl radical generating system. FIG. 8 shows an ESR spectrum of the sample C in the benzoyl radical generating system.

As apparent from the results, in the system including the superoxide generating system, the hydroxyl radical generating system, the 2-cyano-propyl radical generating system and the benzoyl radical generating system, the superoxide, hydroxyl radical, 2-cyano-propyl A strong signal was obtained because the radical and the benzoyl radical formed a stable spin adduct with analyte C.

From the above, the compound of the present invention, C-
Monomethoxy poly (ethylene glycol) carbonyl phenyl-N- (2-hydroxy-1,1-dimethylethyl) nitrone is capable of capturing short-lived superoxide, hydroxyl radical, 2-cyano-propyl radical and benzoyl radical. Was confirmed.

Example 7 Partition ratio between 1-octanol phase and aqueous phase In order to investigate the improvement in solubility of the compound of the present invention in water, the partition ratio between 1-octanol phase and aqueous phase was determined by changing the existing spin trapping agents (PBN and DMPO). I checked with. A solution (1 mM) of each spin trap agent was prepared, 1 ml of each solution and 1 ml of 1-octanol were vigorously stirred, and then centrifuged for 5 minutes in a centrifuge (3,000 rpm).
After separation into an organic phase and an aqueous phase, a certain amount of each phase was taken, the amount of the spin trapping agent was measured by NMR, the concentration of each phase was determined, and the distribution ratio (P) was determined by the following equation.

[0083]

(Equation 1)

[0084]

[Table 1]

[Results] The smaller the distribution ratio is, the higher the solubility in water is. However, while the PBN is 21, the compound of the present invention (the compound of Example 1) is 0.6, showing a remarkable improvement. It was seen.

[0086]

The compound of the present invention captures active oxygen and free radicals, and the spin adduct formed has sufficient stability. And, active oxygen and free radicals generated in the living body cause cerebral ischemia, heart disease, digestive disease,
Since it is considered to be a cause of diseases such as cancer, aging and inflammation, the compound of the present invention can be used as an agent for preventing or treating these diseases. Further, by utilizing the compound of the present invention, active oxygen and free radicals can be detected by a magnetic resonance method such as ESR method which is a non-invasive measurement method. It can be used as a medicament for diagnosing pathological conditions involving radicals and a reagent for detecting active oxygen and free radicals present in collected biological tissues and the like, and medically useful information can be obtained.

[Brief description of the drawings]

FIG. 1 [Top] C-2-pyridyl-N- (2
1 is a drawing showing an ESR spectrum of a system in which (hydroxy-1,1-dimethylethyl) nitrone is present but a hydroxyl radical generating system is not present. [Bottom] Fig. 6 is a drawing showing an ESR spectrum in a hydroxyl radical generating system in which C-2-pyridyl-N- (2-hydroxy-1,1-dimethylethyl) nitrone is present as a sample.

FIG. 2 [Top] C-phenyl-N-tert as a sample
3 is a drawing showing an ESR spectrum of a system in which -butyl nitrone is present but a hydroxyl radical generating system is not present. [Bottom] is a drawing showing an ESR spectrum in a hydroxyl radical generating system in which C-phenyl-N-tert-butyl nitrone is present as a specimen.

FIG. 3 shows poly (methacrylamide) -CO-
It is a drawing which shows the ESR spectrum in the hydroxyl radical generating system in which [2-methyl-2- (N-benzylidene-N-oxide) aminopropyl acrylate]} is present.

FIG. 4 shows a sample of poly {methacrylamide-CO-
[2-methyl-2- (N-benzylidene-N-oxide) aminopropyl acrylate]} is present
It is a figure which shows the ESR spectrum in a cyano-propyl radical generation system.

FIG. 5 is a drawing showing an ESR spectrum in a superoxide generating system in which C-monomethoxypoly (ethylene glycol) carbonyl phenyl-N- (2-hydroxy-1,1-dimethylethyl) nitrone is present as a sample.

FIG. 6 is a drawing showing an ESR spectrum in a hydroxyl radical generating system in which C-monomethoxypoly (ethylene glycol) carbonyl phenyl-N- (2-hydroxy-1,1-dimethylethyl) nitrone is present as a sample.

FIG. 7 shows an ESR spectrum in a 2-cyano-propyl radical generating system in which C-monomethoxypoly (ethylene glycol) carbonyl phenyl-N- (2-hydroxy-1,1-dimethylethyl) nitrone is present as a sample. It is a drawing.

FIG. 8 is a drawing showing an ESR spectrum in a benzoyl radical generating system in which C-monomethoxypoly (ethylene glycol) carbonyl phenyl-N- (2-hydroxy-1,1-dimethylethyl) nitrone is present as a sample.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Hiroshi Iwadate 1-43 Omachi, Yonezawa-shi, Yamagata Pref. Makoto 3-39 Josai 1-3, Yonezawa City, Yamagata Prefecture

Claims (7)

[Claims] 1. The following formula (1) [In the formula, Ar represents a phenylene group or a pyridinylene group, R ¹ and R ³ are the same or different and each represents a lower alkyl group, R ² represents a lower alkylene group, and X represents a hydrogen atom or -CO (AO) _l. OCH ₃ (wherein A represents a lower alkylene group, 1 represents a number of 1 to 100), Y represents a hydrogen atom or a compound represented by the formula: (Where R ⁴ and R ⁵ represent a hydrogen atom or a methyl group, R ⁶ and R ⁷ represent a hydrogen atom or a lower alkyl group, and m and n each represent a number of 10 to 10,000) Represents a group to be However, when X and Y are hydrogen atoms, Ar is a pyridinylene group. A nitrone derivative represented by the formula: 2. The nitrone derivative according to claim 1, wherein X and Y are hydrogen atoms and Ar is a pyridinylene group. 3. A medicament comprising the nitrone derivative according to claim 1 as an active ingredient. 4. The medicine according to claim 3, which is a preventive or therapeutic agent for cerebral ischemia, heart disease, digestive system disease, cancer, aging or inflammation. 5. An active oxygen or free radical scavenger containing the nitrone derivative according to claim 1 or 2 as an active ingredient. 6. A clinical test reagent containing the nitrone derivative according to claim 1 or 2 as an active ingredient. 7. An agent for detecting active oxygen or free radicals, comprising the nitrone derivative according to claim 1 as an active ingredient.