JPH0859465A - Spin trapping agent - Google Patents

Abstract

PURPOSE: To provide a stable and easy-to-handle spin trapping agent which is useful in detection and determination of free radical in vivo according to the spin-trapping technology. CONSTITUTION: This spin trapping agent is represented by the formula (R is H OH, amino which may be substituted with lower alkyls, 2-oxy-1-pyridyl). This spin trapping agent can efficiently trap or capture unstable free radicals such as superoxide radical, hydroxy radical, methyl radical or hydrogen radical. The spin trapping agent of the formula can be produced, for example, by reaction of methyl acrylate with 2-nitropropane, then with ethyl formate, protecting the aldehyde group in the form of 1,3-dioxolane, reducing the ester group with lithium aluminum hydride, and treating the product with an acid to 5,5- dimethyl-3-hydroxymethyl-1-pyrroline-N-oxide.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a spin trap agent. More specifically, it relates to a spin trap agent useful for detecting and quantifying free radicals in vivo by a spin trap method.

[0002]

2. Description of the Related Art As a method for detecting an unstable free radical species using ESR, a high-speed flow method, a freezing method, a matrix separation method and the like are known. In all of these methods, unstable radical species are observed as they are at high speed, or under conditions where their reactivity is suppressed. On the other hand, there is known a method of identifying an unstable radical species by converting an unstable free radical species into a relatively stable radical species and measuring ESR. This method is called a spin trap method. In this method, a spin trapping agent that does not have unpaired electrons traps the unstable radical R · and produces a spin adduct with spins observable by ESR.

Nitroso compounds and nitrone compounds are used as typical spin trap agents. DMPO (5,5-dimethyl-1) is the most commonly used spin trap agent for trapping active oxygen radicals.
-pyrroline-N-oxide) is known. The spin adduct between this reagent and the superoxide radical has the characteristic of giving a specific and easily identifiable ESR spectrum. However, this substance is inconvenient to handle because it has a low melting point and is easily decomposed. Further, since it is inferior in lipophilicity and the reaction rate of radical scavenging is small, it was not possible to efficiently capture superoxide radicals in the living body.

Various derivatives have been proposed with DMPO as a basic compound in order to improve the above-mentioned drawbacks. For example,
A spin-trapping agent has been proposed in which a substituent is introduced at the 4-position of the pyrroline ring of DMPO (Magnetic Resonance and Medicine, 4, pp.17-2
0, 1993). However, even such compounds could not be said to have sufficient performance for capturing superoxide radicals in vivo.

[0005]

SUMMARY OF THE INVENTION An object of the present invention is to provide a spin trap agent which can efficiently capture unstable free radical species. More specifically, it is to provide a spin trap agent having sufficient performance for capturing unstable free radical species such as superoxide radicals in a living body. Another object of the present invention is to provide a spin trap agent which is stable and easy to handle.

[0006]

Means for Solving the Problems As a result of diligent efforts to solve the above problems, the present inventor was able to efficiently trap unstable free radical species by introducing a substituent at the 3-position of the pyrroline ring of DMPO. Found. Moreover, they have found that the above compounds are stable and easy to handle, and have completed the present invention.

That is, the present invention has the following formula:

Embedded image (Wherein R represents a hydrogen atom, a hydroxyl group, an amino group which may be substituted with a lower alkyl group, or a 2-oxy-1-pyridyl group). The present invention also provides a method for trapping an unstable free radical species using the above spin trap agent.

In the above formula, as the lower alkyl group represented by R, for example, a C ₁ -C ₄ linear, branched or cyclic lower alkyl group can be used. For example, methyl group, ethyl group, n-propyl group, iso-propyl group, cyclopropyl group, n-butyl group, sec-butyl group, tert-butyl group, cyclobutyl group and the like can be used. Of these,
It is preferable to use an n-butyl group. In addition, all kinds of isomers such as optically active compounds based on the carbon atom at the 3-position of the pyrroline ring of the compound represented by the above formula, salts, and arbitrary hydrates are all included in the spin trap agent of the present invention.

The spin trap agent of the present invention can be easily produced by the method specifically and specifically described in Examples of the present specification. In addition, a person skilled in the art can produce the target compound by adding alterations or modifications based on these disclosures, if necessary. For example, 5,5-dimethyl-3-hydroxymethyl-1-pyrroline-N-oxide (3HMDMPO)
Synthesizes methyl 4-methyl-4-nitrovalerate from readily available methyl acrylate and 2-nitropropane and reacts with ethyl formate to give 2-methoxycarbonyl-4-
After conversion to methyl-4-nitro-1-pentanal, the aldehyde group was protected as 1,3-dioxolane, followed by reduction of the ester group with lithium aluminum hydride.
2- (1-hydroxymethyl-3-methyl-3-nitrobutyl)
It can be synthesized by producing -1,3-dioxolane, reducing it with zinc, and treating it with an acid.

Further, 3- (Nn-butylaminomethyl) -5,5-
Dimethyl-1-pyrroline-N-oxide (3BADMPO) is converted to tosylate by tosylating the hydroxyl group of 2- (1-hydroxymethyl-3-methyl-3-nitrobutyl) -1,3-dioxolane described above, It can be synthesized by reacting with n-butylamine, further reducing with zinc and treating with acid. Similarly, 3- (2-oxy-1-pyridylmethyl)-
5,5-dimethyl-1-pyrroline-N-oxide (3PMDMPO) also
It can be synthesized by reacting the above tosylate with 2-pyridine oxide, further reducing with zinc and treating with acid.

Further, 3,5,5-trimethyl-1-pyrroline-
N-oxide (3MDMPO) was synthesized from 2-nitropropane and methacrolein to 2,4-dimethyl-4-nitro-pentanal, and after converting the aldehyde group to 1,3-dioxolane,
It can be synthesized by reducing with zinc and treating with an acid.

The spin trap agent of the present invention comprises a superoxide radical (O _2- ^. ) And a hydroxy radical (OH ^. )
, Methyl radicals (CH ^_3.), The hydrogen radicals ^(H.) Can be captured unstable free radicals, such as is particularly useful for capturing the unstable radical in vivo. For example, Organic Synthetic Chemistry, 29 (4), pp.386-399, 1971, Can. J. Chem.,
60 (12), pp.1532-1541, 1982, J. Org. Chem., 51, p.
p.4298-4300, 1986 and Magnetic Resonance and Medicine, 4, pp.17
-20, 1993, etc. can be used to trap unstable radicals. The amount used is generally 0.1-0.2 M
It is a degree.

The spin trap agent of the present invention can trap various unstable radicals generated experimentally. For example, various radicals generated by the following method can be captured at room temperature, and the ESR spectrum thereof can be measured, for example, by an ESR device (JES-RE3X or JES-FR80) manufactured by JEOL. In this case, the cell can be a water-soluble cell, but if deoxidation is necessary, reverse U
What is connected to the character tube may be used.

[0014] O ₂ ^-:... Hypoxanthine (HPX) - method according to xanthine oxidase (XOD) OH: Fenton (Fenton) reaction or of H ₂ O ₂ irradiation CH _3: light irradiation H ₂ O _{2-DMSO ^{... H: [CH 3}} (CH 2) 3] 3 light irradiation SnH (in _{benzene) CH 2 OH: H 2 O} 2 -CH 3 OH light irradiation _{CH (CH 3) OH: H} 2 O 2 - light irradiation of _{C 2 H 5 OH (CH 3} ) 3 CO:. [(CH 3) 3 CO] light irradiation ₂ (in benzene)

EXAMPLES The present invention will now be described in more detail with reference to examples, but the present invention is not limited to these examples. The following were used as reagents. Dioxane: Hydrochloric acid was added to a commercially available product (manufactured by Wako Pure Chemical Industries, special grade), and the mixture was refluxed for 10 hours in a nitrogen atmosphere. Then, the mixture was shaken with solid sodium hydroxide, the dioxane layer was separated, sodium was added, refluxed for several hours, and purified by distillation. Diethyl ether: A commercially available product (manufactured by Kanto Kagaku Co., Ltd., first grade) was added with a small amount of hydroquinone and a sodium metal wire, refluxed for several hours, and then purified by distillation. Ethanol / methanol: Calcium oxide was added to a commercial product (manufactured by Wako Pure Chemical Industries, Ltd.), refluxed for several hours, and purified by distillation. Benzene: A commercially available product (manufactured by Wako Pure Chemical Industries, Ltd.) was washed with concentrated sulfuric acid several times to remove thiophene. When the concentrated sulfuric acid was not completely colored, it was washed with water, 1 N sodium hydroxide and water in this order, calcium chloride was added, the mixture was refluxed for several hours, and purified by distillation.

Pyridine: Sodium hydroxide was added to a commercial product (Wako Pure Chemical Industries, first grade), left overnight, and refluxed for several hours.
The one purified by distillation was used. Toluene: Commercially available product (special grade manufactured by Kanto Chemical Co., Ltd.) is concentrated sulfuric acid, water,
A 1 N aqueous sodium hydroxide solution and water were washed in this order, calcium chloride was added, the mixture was refluxed for several hours, and then purified by distillation. Tetrahydrofuran (THF): A metal wire was added to a commercial product (manufactured by Kanto Kagaku Co., Ltd.), refluxed for several hours, allowed to cool, and benzophenone was added and refluxed again. After the color changed from blue to purple, the one purified by distillation was used. As other reagents, commercial products were used as they were.

Example 1: 5,5-Dimethyl-3-hydroxymethyl
Synthesis of -1-pyrroline-N-oxide (3HMDMPO) Synthesis of methyl 4-methyl-4-nitrovalerate In a three-necked flask equipped with a thermometer, dropping funnel and cooling tube.
220 g (2.5 mol) of 2-nitropropane, 25 ml of purified dioxane and 15 ml of benzyltrimethylammonium hydroxide 40% aqueous solution (Triton B) were added and kept at 70 ° C. 215 g (2.5 mol) of methyl acrylate was added dropwise to this mixture over 15 minutes, and the mixture was stirred for 4 hours. Then, let it cool, make it weakly acidic (pH> 6) with dilute hydrochloric acid, and extract with ether.
It was dried over anhydrous magnesium sulfate. After concentration, it was distilled under reduced pressure to obtain light green methyl 4-methyl-4-nitrovalerate. Yield 89% (literature value 80-86%); bp 69-73 ℃ / (1 mmHg): Literature value (RB Moffett, Or
ganic Synthesis, Coll.Vol. 4, 652) 79 ℃ (1 mmHg)

Synthesis of 2-methoxycarbonyl-4-methyl-4-nitro-1-pentanal 300 ml in a three-necked flask equipped with a cooling tube and a dropping funnel.
Put the purified ether and 1.5 ml of purified ethanol,
To this, 14 g (0.6 mol) of metallic sodium wire was added and stirred for 2 hours and 30 minutes. Then 105 g (0.6 mol) of
Methyl 4-methyl-4-nitrovalerate, 44.5 g (0.6 mol)
A mixed solution of ethyl formate and 50 ml of purified ether was added dropwise, and the mixture was left for 24 hours. The above reaction was performed under a nitrogen atmosphere. Then, it was made weakly acidic (pH> 4) with acetic acid, extracted with ether, and dried over anhydrous magnesium sulfate. After concentration, it was distilled under reduced pressure to give pale yellow 2-methoxycarbonyl-4-methyl-4-
Nitro-1-pentanal was obtained. Yield 12%; bp 10
0 ° C (1 mmHg)

2- (1-methoxycarbonyl-3-methyl-3-
Nitrobutyl) -1,3-dioxolane synthesis in eggplant flask equipped with Dean Stark trap
3.5 g (56 mmol) ethylene glycol, 10 g (49 mmo
l) 2-methoxycarbonyl-4-methyl-4-nitro-1-
The mixture was refluxed by adding pentanal, 0.1 g (0.53 mmol) of p-toluenesulfonic acid and 70 ml of purified benzene. As the reaction progresses, water begins to collect in the trap, about 0.9 ml
The reflux was stopped when the water in the pool had accumulated. After cooling, it was washed with an aqueous solution of sodium hydrogen carbonate until the gas disappeared, and dried over anhydrous magnesium sulfate. After concentration, distillation under reduced pressure gave colorless 2- (1-methoxycarbonyl-3-methyl-3-nitrobutyl) -1,3-dioxolane. Yield 66%; bp 1
20 ° C (0.9 mmHg)

Synthesis of 2- (1-hydroxymethyl-3-methyl-3-nitrobutyl) -1,3-dioxolane In a three-necked flask equipped with a condenser, 20 g (80 mmol) of 2-
(1-methoxycarbonyl-3-methyl-3-nitrobutyl)
-1,3-Dioxolane (3) and 250 ml of purified ether were added and stirred. Next, while cooling the three-necked flask with ice water, 3 g (80 mmol) of lithium aluminum hydride was added little by little, and after 1 hour, the mixture was placed in an oil bath and refluxed for 24 hours. The reaction was performed under a nitrogen atmosphere. Then, water was added little by little to completely crush unreacted lithium aluminum hydride, and the mixture was stirred for 2 hours. After stirring, the reaction product was filtered off, the solid was thoroughly washed with ether, combined with the filtrate, and dried over anhydrous magnesium sulfate. After concentration, distillation under reduced pressure gave colorless 2- (1-hydroxymethyl-3-methyl-3-nitrobutyl) -1,3-dioxolane. Yield 50%; bp
136-140 ℃ (0.8 mmHg)

Synthesis of 5,5-dimethyl-3-hydroxymethyl-1-pyrroline-N-oxide (3HMDMPO) A three-necked flask equipped with a thermometer was charged with 2.4 g (46 mmol) of ammonium chloride and 44 ml of water. It was stirred. Then 1
0 g (46 mmol) of 2- (1-hydroxymethyl-3-methyl-3
-Nitrobutyl) -1,3-dioxolane was added, and ice water
It was kept at ℃. With thorough stirring, 10 g (0.153 mol) of zinc powder whose surface was polished with dilute hydrochloric acid was gradually added. Although the reaction temperature gradually increased when the reaction started, it was controlled so as not to rise above 15 ° C. 1 more after adding zinc powder
The stirring was continued for 5 to 30 minutes, and unreacted zinc powder and salt were removed by suction filtration. The reaction was performed under a nitrogen atmosphere. About precipitation
After washing several times with warm water at 60 ° C, the filtrate and the mixture were acidified (pH> 2) with concentrated hydrochloric acid, and left overnight without light shielding.

After the filtrate was left standing, it was heated to 70 ° C. and stirred for 40 minutes. After allowing to cool, it is alkaline (pH <1
And was concentrated under reduced pressure. After almost removing water, the residue was extracted with chloroform and dried over anhydrous magnesium sulfate. Then, after concentration, column chromatography (developing solvent: water) using activated carbon was performed. The obtained product was further subjected to column chromatography (developing solvent: acetone → chloroform / methanol) using silica gel.
= 10/1), a colorless target product was obtained. Yield 33
% IR (NaCl) ν cm ^-1 ; 3360, 1583 ¹ H-NMR (CDCl ₃ ) δ ppm: 1.40 (s, 3H), 1.43 (s, 3H), 1.
90 (dd, 1H), 2.22 (dd, 1H), 3.10 (m, 1H), 3.53 (m, 1H), 3.
75 (m, 1H), 6.95 (d, 1H) ¹³ C-NMR (CDCl ₃ ) δ ppm: 25.8, 26.1, 36.5, 40.7, 6
2.5, 74.1, 137.1

Example 2: 3- (Nn-butylaminomethyl) -5,5-
Synthesis of dimethyl-1-pyrroline-N-oxide (3BADMPO) Synthesis of 2- (1-tosyloxymethyl-3-methyl-3-nitrobutyl) -1,3-dioxolane 11.0 g (50 mmol) of 2 in a round-bottomed flask -(1-Hydroxymethyl-3-methyl-3-nitrobutyl) -1,3-dioxolane, 100 ml of purified ether, 7.95 g (0.1 mol) of pyridine and 10.53 g (55 mmol) of tosyl chloride were added, and the mixture was stirred at room temperature. so
It was stirred for 24 hours. Then, the reaction product was filtered off, the solid was washed well with ether, dried over anhydrous magnesium sulfate,
Concentration gave 2- (1-tosyloxymethyl-3-methyl-3-nitrobutyl) -1,3-dioxolane as pale yellow crystals (yield: quantitative).

2- (1-Nn-butylaminomethyl-3-methyl
Synthesis of 3-nitrobutyl) -1,3-dioxolane In a round-bottomed flask equipped with a cooling tube, 15 g (40 mmol) of 2-
(1-tosyloxymethyl-3-methyl-3-nitrobutyl)-
1,3-dioxolane, 150 ml purified toluene and 3.2 g
(44 mmol) of n-butylamine was added, and the mixture was refluxed for 6 hours.
Then, the reaction solution is poured into water, extracted with benzene, dried over anhydrous magnesium sulfate, and then concentrated to give colorless 2- (1-Nn-butylaminomethyl-3-methyl-3-nitrobutyl) -1,3- Dioxolane was obtained. Crude yield: 70%

Synthesis of 3- (Nn-butylaminomethyl) -5,5-dimethyl-1-pyrroline-N-oxide (3BADMPO) 1.28 g (24 mmol) ammonium chloride and 23 in a three-necked flask equipped with a thermometer. ml water was added and stirred. Next, 6.58 g (24 mmol) of 2- (1-Nn-butylaminomethyl)
-3-Methyl-3-nitrobutyl) -1,3-dioxolane was added, and the mixture was kept at 10 ° C with ice water. While stirring well, 5.21 g (80 mmol) of zinc powder whose surface was polished with dilute hydrochloric acid was gradually added. When the reaction starts, the reaction temperature rises gradually, but 15 ℃
I tried not to go above. After the addition of the zinc powder was completed, stirring was continued for another 15 to 30 minutes, and unreacted zinc powder and salt were removed by suction filtration. The reaction was performed under a nitrogen atmosphere. The precipitate was washed several times with warm water at 60 ° C., and then combined with the filtrate to be acidified (pH> 2) with concentrated hydrochloric acid, and allowed to stand overnight in the dark.

After the filtrate was left standing, it was heated to 70 ° C. and stirred for 40 minutes. After allowing to cool, it is alkaline (pH <1
And was concentrated under reduced pressure. After almost removing water, the residue was extracted with chloroform and dried over anhydrous magnesium sulfate. Then, after concentration, column chromatography (developing solvent: water) using activated carbon was performed. The obtained product was further subjected to column chromatography using activated alumina (developing solvent: chloroform / methanol [10/1]).
Then, a colorless target product was obtained. Yield 11% IR (NaCl) νcm ^-1 ; 3300, 1587 ¹ H-NMR (CDCl ₃ ) δ ppm: 0.9 (t, 3H), 1.30-1.53 (m, 10
H), 1.83 (dd, 1H), 2.34 (dd, 1H), 2.43 (brds, 1H), 2.66
(t, 2H), 2.75 (dd, 2H), 3.12 (m, 1H), 6.92 (d, 1H) ¹³ C-NMR (D ₂ O) δ ppm: 2.42, 9.01, 13.58, 14.71, 1
9.79, 27.30, 27.66, 37.64, 39.90, 63.96, 132.51

Example 3: 2- (1-Pyridineoxy-4-methyl-
Synthesis of 4-nitro) pentyl 1,3-dioxolane In a 25 ml round bottom flask, 2- (1-tosyloxymethyl-
3-Methyl-3-nitrobutyl) -1,3-dioxolane 1 g
(0.3 mmol) and sodium 2-pyridoxide 0.36 g
(0.3 mmol) was added and this was dissolved in 10 ml of toluene.
After stirring at room temperature for 1 hour under a nitrogen stream, the mixture was reacted at 100 ° C for 24 hours. After the reaction, the mixture was poured into 200 ml of water and extracted with benzene. The organic layer is dried over magnesium sulfate, concentrated under reduced pressure, and the residue is purified by an alumina column (eluent: benzene) to give 2- (1-pyridineoxy-4-methyl-4-nitro) pentyl 1,3-dioxolane. Got Yield 63% IR (KBr) νcm ^-1 ; 1662

Synthesis of 3- (2-oxy-1-pyridylmethyl) -5,5-dimethyl-1-pyrroline-N-oxide (3PMDMPO) In a 25 ml flask equipped with a nitrogen inlet tube, thermometer and stirrer.
Add 1.5 g (5.1 mmol) of 1,3-dioxolane, add 5 ml of ethanol and 5 ml of water, and stir 1
Cooled to 0 ° C. After adding 0.28 g of ammonium chloride, 1.33 g of zinc whose surface was polished with dilute hydrochloric acid was slowly added so that the reaction temperature did not exceed 15 ° C. After stirring for 1 hour while maintaining the reaction temperature, the precipitate was filtered off. The precipitate was washed well with hot water at 70 ° C., the aqueous layer was made acidic (pH = 1) with concentrated hydrochloric acid, and then stored overnight in the dark. After that, the aqueous layer was stirred at 70 ° C. for 1 hour, neutralized with sodium hydrogen carbonate and concentrated under reduced pressure, the residue was extracted with chloroform, dried and the chloroform was evaporated under reduced pressure, and the residue was subjected to silica gel column chromatography ( The target product was purified with an eluent: methanol / chloroform = 1:10). Yield 22
% IR (NaCl) ν cm ^-1 ; 1660, 1583 ¹ H-NMR (CDCl ₃ ) δ ppm: 1.38 (s, 3H), 1.43 (s, 3H), 1.
92 (dd, 1H), 2.34 (dd, 1H), 3.51 (m, 1H), 4.01 (dd, 1H),
4.12 (dd, 1H), 6.23 (t, 1H), 6.54 (d, 1H), 6.73 (d, 1H),
7.31-7.45 (m, 2H) ¹³ C-NMR (CDCl ₃ ) δ ppm: 25.59, 26.43, 37.05, 38.3
1, 52.49, 74.14, 106.47, 121.29, 132.22, 137.64, 1
40.02, 162.57

Example 4: 3,5,5-Trimethyl-1-pyrroline -N-
Synthesis of oxide (3MDMPO) Synthesis of 2,4-dimethyl-4-nitro-pentanal 250 ml of purified methanol was placed in a three-necked flask equipped with a thermometer and a cooling tube, and 7 g of metallic sodium was added little by little to it. Keep at 10 ° C. Then 202 g (2.3 mol) of 2-
Nitropropane was added dropwise to bring the reaction system to -15 ° C. In addition, 25 ml (0.3 mol) of methacrolein and 50.5 g (0.
A mixture of 6 mol) of 2-nitropropane was added dropwise over 1 hour, and the mixture was stirred for 1 hour. Then, the mixture was returned to room temperature, neutralized with acetic acid (pH> 4), and dried over anhydrous sodium sulfate.
After the desiccant was filtered off and concentrated, it was distilled under reduced pressure to give a pale yellow 2,
4-Dimethyl-4-nitro-pentanal was obtained. Yield 79%; bp 70 ℃ -75 ℃ (2mmHg) ¹ H-NMR (CDCl ₃ ) δ, ppm 1.1-1.2 (d, 3H), 1.8-2.0 (d, 2H),
1.5-1.6 (s, 3H), 2.4-2.6 (m, 1H), 1.6-1.7 (s, 3H), 9.6
(s, 1H)

2- (1,3-dimethyl-3-nitropentyl)-
Synthesis of 1,3-dioxolane In an eggplant flask equipped with a Dean Stark trap
13.4 g (0.2 mol) ethylene glycol, 34.4 g (0.
2 mol) of 2,4-dimethyl-4-nitro-pentanal, 1.5
g (8.5 mol) of p-toluenesulfonic acid and 250 ml of purified benzene were added and the mixture was refluxed. As the reaction proceeded, water began to accumulate in the trap, and the reflux was stopped when about 3.9 ml (0.2 mol) of water had accumulated. After cooling, the organic layer was washed with an aqueous solution of sodium bicarbonate until the gas disappeared, and dried over anhydrous sodium sulfate. After the desiccant was filtered off and concentrated, it was distilled under reduced pressure to give brown 2- (1,3-dimethyl-3-nitropentyl) -1,3-
Dioxolane was obtained. Yield 91%; bp 90-96 ℃ (2 mmHg) ¹ H-NMR (CDCl ₃ ) δ, ppm 0.9-1.0 (d, 3H), 2.1-2.2 (m, 1H),
1.5-1.6 (s, 6H), 3.7-4.0 (m, 4H), 1.8-1.9 (d, 2H), 4.6-
4.7 (d, 1H)

Synthesis of 3,5,5-trimethyl-1-pyrroline-N-oxide (3MDMPO) In a three-necked flask equipped with a thermometer, 11 g (0.21 mol) of ammonium chloride and 197 ml of water were added and stirred. Next, 42 g (0.21 mol) of 2- (1,3-dimethyl-3-nitropentyl) -1,3-dioxolane was added, and the mixture was kept at 10 ° C with ice water. With thorough stirring, 45 g (0.69 mol) of zinc powder whose surface was polished with dilute hydrochloric acid was gradually added. Although the reaction temperature gradually increased when the reaction started, it was controlled so as not to rise above 15 ° C. After the addition of the zinc powder was completed, stirring was continued for another 30 minutes, and unreacted zinc powder and salt were removed by suction filtration. The precipitate was washed several times with warm water at 60 ° C., and then combined with the filtrate to be acidified (pH> 2) with concentrated hydrochloric acid, and allowed to stand overnight in the dark.

After the filtrate was left standing, it was heated to 70 ° C. and stirred for 40 minutes. After cooling down, it is alkaline (pH> 1
And was concentrated under reduced pressure. After almost removing water, the residue was washed with chloroform and suction filtered. Anhydrous sodium sulfate was added to the filtrate and dried, then concentrated and distilled under reduced pressure. Then, column chromatography using silica gel (developing solvent: acetone → chloroform / methanol [10/1] → methanol) was performed. The target product was obtained from the outlet of chloroform / methanol [10/1]. Furthermore, column chromatography using activated carbon (developing solvent: water)
The obtained target product was subjected to column chromatography again using silica gel with chloroform / methanol [10/1] as a developing solvent to obtain a colorless target product. Yield 41%; bp 77 ° C (2mmHg) IR (NaCl) νcm ^-1 ; 1578 ¹ H-NMR (CDCl ₃ ) δ ppm: 1.20 (d, 3H), 1.41 (s, 3H), 1.
45 (s, 3H), 1.67 (dd, 1H), 2.39 (dd, 1H), 2.98 (m, 1H), 6.7
2 (d, 1H) ¹³ C-NMR (CDCl ₃ ) δ ppm: 19.0, 25.4, 26.7, 31.8, 4
3.1, 74.2, 136.9

Test Example: Spin Trapping Experiment Known Method (Can. J. Chem., 60 (12), pp.1532-1541, 1
982 and magnetic resonance and medicine, 4, pp.17-20, 1993) was used to trap various radicals generated at room temperature using the spin trap agent of the present invention, and an ESR measurement device (JES, JES-
The spectrum was measured by RE3X or JES-FR80). Each radical was generated by the following method. O ₂ ^-:... The process according HPX-XOD OH: Fenton reaction _{H: [CH 3 (CH 2} ) 3] 3 light irradiation cell SnH (in benzene) has been used as water-soluble only, the SnH In this case, deoxygenation was necessary, and therefore, an inverted U-shaped tube was used.

H of the spin adduct of each spin trap agent
The fsc and ESR spectra are shown in Figures 1-4. 1 and 2 are diagrams showing ESR spectra of spin adducts of 3HMDMPO and 3MDMPO with superoxide radicals, respectively. In the figure, (A) (B) (C) are after 1 minute,
The spectra after 4.5 minutes and after 8 minutes are shown. 3 (A) and 3 (B) are diagrams showing ESR spectra of spin adducts of 3HMDMPO, 3MDMPO and hydroxyl radical, respectively. FIG. 4 is a diagram showing an ESR spectrum of a spin adduct of 3PMDMPO and hydrogen radicals.

From these results, the spin label of the present invention showed an increase in the line width, which is considered to be due to the diastereomer of the spin adduct, but had an ESR similar to that of DMPO.
The spectra are given and it is clear that they are useful for trapping unstable free radicals in vivo.

[0035]

The spin trap agent of the present invention is characterized by being stable and easy to handle, and is useful because it can efficiently trap unstable free radicals.

[Brief description of drawings]

FIG. 1 is a diagram showing an ESR spectrum of a spin adduct of 3HMDMPO and a superoxide radical. In the figure, (A), (B), and (C) show the spectra after 1 minute, 4.5 minutes, and 8 minutes, respectively.

FIG. 2 is a diagram showing an ESR spectrum of a spin adduct of 3MDMPO and a superoxide radical. In the figure, (A) (B) (C) show the spectra after 1 minute, 4.5 minutes, and 8 minutes, respectively.

FIG. 3 is a diagram showing an ESR spectrum of a spin adduct of 3HMDMPO, 3MDMPO, and a hydroxyl radical. In the figure, (A) shows the ESR spectrum of the spin adduct between 3HMDMPO and hydroxyl radical, and (B) shows the 3MDM.
It is a figure which shows the ESR spectrum of the spin adduct of PO and a hydroxyl radical.

FIG. 4 is a diagram showing an ESR spectrum of a spin adduct of 3PMDMPO and hydrogen radicals.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Hiroaki Oya 4-3-4 Tokamachi, Yamagata City Yamagata Prefecture Lions Mansion Tokamachi Room 404 (72) Inventor Hitoshi Kamata 6-15-24 Kamikizaki, Urawa City, Saitama Prefecture

Claims (4)

[Claims] 1. The following formula: (In the formula, R represents a hydrogen atom, a hydroxyl group, an amino group which may be substituted with a lower alkyl group, or a 2-oxy-1-pyridyl group). 2. The spin trap agent according to claim 1, wherein R is a hydrogen atom, a hydroxyl group, an n-butylamino group, or a 2-oxy-1-pyridyl group. 3. A method for trapping an unstable free radical species using the spin trap agent according to claim 1. 4. The method according to claim 3, wherein unstable free radical species in the living body are trapped.