JP2000128893A - Dithiocarbamate compound and spin trapping agent for scavenging nitrogen monoxide containing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new compound useful as a spin trapping agent for scavenging nitrogen monoxide in vivo. SOLUTION: This (S)- or (R)-hydroxyproline derivative dithiocarbamate is shown by formula I (R is H, an alkyl or the like; A is a cation) and its iron complex is shown by formula II such as ammonium salt of (2S,4R)4- methoxymethyloxy-2-pyrrolidinecarboxylic acid dithiocarbamate. The compound of formula I is obtained by reacting N-benzyloxycarbonyl-(2S,4R)-4-hydroxy-2- pyrrolidinecarboxylic acid benzyl ester obtained by a routine procedure with chloromethyl methyl ether, etc. An obtained O-alkylamino acid derivative is catalytically reduced and N-benzyloxycarbonyl and benzyl ester are removed to give a hydroxyproline derivative of formula III. The compound of formula III is reacted with carbon disulfide in ammonia water. The iron complex of formula II is obtain by reacting the compound of formula I with iron sulfate.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a dithiocarbamate or an iron complex thereof, and a spin trapping agent containing the same for trapping nitric oxide (NO).

[0002]

BACKGROUND OF THE INVENTION Nitric oxide (NO) is a compound that is produced at various sites in the body and is involved in important biological reactions such as vascular smooth muscle relaxation, neurotransmission and excitatory cytotoxicity. It is chemically characterized as a low molecular compound (molecular weight 30) and a radical compound, and is known to react relatively quickly with oxygen molecules, etc., exhibit relatively high reactivity, and is unstable and short-lived in vivo. ing. For this reason, there is no definitive method for measuring NO in vivo at present, and the Griess method,
A chemiluminescence method, an electrode method and the like are used.

[0003] On the other hand, the ESR spin trapping method is one of the effective means for measuring unstable radical species. In this method, unstable radicals are reacted with a spin trapping agent to lead to a stable radical compound, and this ESR signal is measured. This is a method for indirectly measuring unstable radicals. However, in the report on the NO spin trapping method, only a limited number of spin trapping agents are used, and a water-soluble spin trapping agent considered to be applicable to biological systems is Fe (MGD) ₂ (iron- Only two types are known, N-methyl-D-glucamine dithiocarbamate complex) and Fe (DTCS) ₂ (iron-sarcosine dithiocarbamate complex). It is also known that pharmacokinetics is particularly likely to accumulate in the liver.

[0004] Therefore, it has been an issue to develop a spin trap agent having a different pharmacokinetics in exploring the generation site, behavior and role of nitric oxide in a living body. The invention of this application has been made in view of the state of the prior art as described above, and is intended to provide a novel spin trapping agent compound excellent in action as a spin trapping agent for capturing nitric oxide (NO) in a living body. The task is to provide.

[0005]

SUMMARY OF THE INVENTION In order to solve the above-mentioned problems, the invention of the present application firstly (S)-or (R) -hydroxyproline represented by the following formula (1): A derivative dithiocarbamate or an iron complex thereof represented by the formula [2] (where R is a hydrogen atom, an alkyl group, an aryl group,
An alkylalkyl group, an alkoxyalkyl group, and an oxygen-containing heterocyclic group or an alkyl substituent thereof, wherein A represents a cation).

[0006]

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[0007]

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[0008] Secondly, the invention of this application relates to an N-methyl- (S)-or (R) -serine derivative dithiocarbamate represented by the following formula [3] or a formula [4]: The iron complex (where R represents a hydrogen atom, an alkyl group, an aryl group, an aralkyl group, an alkoxyalkyl group, and an oxygen-containing heterocyclic group or an alkyl substituent thereof, and A represents (Indicating a cation).

[0009]

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[0010]

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Furthermore, the invention of this application thirdly provides a spin trapping agent for trapping nitric oxide, which comprises the dithiocarbamate or the iron complex thereof according to the first and second inventions.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION The invention of this application has the features as described above, and the embodiments will be described below. First, the formulas [1] [2] [3] [4]
In the dithiocarbamate and the iron complex thereof of the present invention represented by the following formula, the symbol R represents any one of a hydrogen atom, an alkyl group, an aryl group, an aralkyl group, an alkoxyalkyl group, and an oxygen-containing heterocyclic group or an alkyl substituent thereof. And examples thereof include a hydrogen atom, a methyl group, an ethyl group, a butyl group, a hexyl group, a cyclohexyl group, a benzyl group, a methylphenyl group, a methoxymethyl group, a methoxyethoxymethyl group, and a tetrahydropyranyl group. You. The symbol A may be a cation, for example, various kinds including an ammonium cation. Preferred are amine-based and ammonium-based cations.

The compound represented by the above formula is useful as a spin trapping agent for trapping nitric oxide (NO).
Although various types of buffer systems are considered, a Tris-HCl buffer is most suitable from the viewpoint of a trapping effect. In the spin trap agent of the present invention, a spin trap agent in which a nitric oxide adduct is mainly detected in the liver and a spin trap agent that traps nitric oxide in blood without accumulating in the liver are found.

For the production of the NO spin trapping agent compound of the present invention, various methods including known means can be adopted. For example, the compounds of the formulas [1] and [2] can be produced by the following synthesis route. It is possible.

[0015]

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The reaction steps (a) to (d) are described as follows. Step (a) N-benzyloxycarbonyl- synthesized by a conventional method
(2S, 4R) -4-Hydroxy-2-pyrrolidinecarboxylic acid benzyl ester (5) was reacted with chloromethyl methyl ether in the presence of N, N-diethylaniline to give 6 (R
= -CH ₂ OCH ₃ ). Similarly, 6 (R = -CH ₂ OCH ₂ CH ₂ OCH ₃ ) by reacting with 2- (methoxyethoxymethyl) triethylammonium chloride, and 6 (R) by reacting dihydropyran with paratoluenesulfonic acid. R = TH
P) is synthesized. Step (b) The O-alkylamino acid derivative (6) is subjected to catalytic reduction using hydrogen-palladium carbon to remove the N-benzyloxycarbonyl group and the benzyl ester to synthesize the hydroxyproline derivative (7). Step (c) The hydroxyproline derivative (7) is reacted with carbon disulfide in aqueous ammonia to synthesize the corresponding dithiocarbamate ammonium salt (1). Step (d) The dithiocarbamate derivative (1) is reacted with, for example, iron sulfate in a Tris-HCl buffer (40 mM, pH 7.4) under a stream of argon to obtain an iron complex (2).

The compounds of the formulas [3] and [4] can be produced, for example, by the following synthetic route.

[0018]

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The reaction steps (a), (c) and (d) are the same as described above,
The reaction step (e) can be performed, for example, as follows. Step (e) N-methylbenzyloxycarbonyl-O-methoxymethyl-
Dissolve (S) -serine methyl ester (9) in methanol,
After hydrolyzing the methyl ester with an aqueous solution of sodium hydroxide, catalytic reduction is carried out with palladium carbon-hydrogen to remove the N-benzyloxycarbonyl group and remove the N-methyl
-O-methoxymethyl- (S) -serine (10) is obtained.

[0020]

Next, the present invention will be described in more detail with reference to Examples. In the following examples, the numbers of the synthesized compounds correspond to those in the above-mentioned reaction steps, and the abbreviations for the symbol R indicate the following.

[0021]

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Example 1 3.3 g of N-benzyloxycarbonyl- (2S, 4R) -4-hydroxy-2-pyrrolidinecarboxylic acid benzyl ester (5, R = H) synthesized by a conventional method (8.
(4 mmol) was added with 20 ml of methylene chloride, and 6.3 g (42.2 mmol) of N, N-diethylaniline and 3.4 g (42.2 mmol) of chloromethyl methyl ether were further added. After stirring at room temperature for 22 hours, 500 ml of ethyl acetate was added,
Wash with 5% aqueous hydrochloric acid, water, saturated aqueous sodium bicarbonate, and water. After drying over anhydrous magnesium sulfate and evaporating the solvent, the residue obtained is purified by column chromatography (silica gel C-200, eluent; hexane: ethyl acetate = 1: 1) to give N-benzyloxycarbonyl. -(2S, 4R) -4
-Methoxymethyloxy-2-pyrrolidinecarboxylic acid benzyl ester (6, R = MOM) as an oily substance, 3.0 g (yield 88
%). Optical rotation; [α] _D ²⁰ -45.0 ゜ (c = 2, CHCl ₃ ) mass spectrometry; (m / z) 399 (M ⁺ ) ¹ H NMR (CDCl ₃ ) δ: 2.00-2.42 (2H, m, CH ₂ ), 3.29 (3
H, d, OCH ₃ ), 3.54-3.74 (2H, m, CH ₂ ), 4.27 (1H, br
s, CH ₂ ), 4.41-4.42 (1H, m, CH), 4.58 (2H, d, OCH
₂ O), 4.99 (2H, d, CH ₂ ), 5.09-5.22 (2H, m, CH), 7.
25 (10H, m, aromatic protons); ¹³ C NMR (CDCl ₃ )
δ: 35.86 (t), 52.22 (t), 55.34 (q), 57.79 (d), 66.
84 (d, t), 74.30 (t), 95.25 (t), 127.96 (d), 135.3
1 (s), 136.26 (s), 154.39 (s), 172.14 (s). (Example 2) N-benzyloxycarbonyl- (2S, 4R) -4
20 ml of acetonitrile was added to 2.0 g (5.6 mmol) of benzyl ester of 5-hydroxy-2-pyrrolidinecarboxylic acid (5), and 2.3 g (14 mmol) of 2- (methoxyethoxymethyl) triethylammonium chloride was further added. 7
After refluxing for an hour, add 200 ml of ethyl acetate and wash with water. The residue obtained by drying over anhydrous magnesium sulfate and evaporating the solvent is subjected to column chromatography (silica gel).
Purification with C-200, eluent; hexane: ethyl acetate = 1: 1) yields N-benzyloxycarbonyl- (2S, 4R) -4-methoxyethoxymethyloxy-2-pyrrolidinecarboxylic acid benzyl ester (6 , R = MEM) as an oily substance 2.1 g
(83% yield). Optical rotation: [a] _D ²⁰ -35.2 ゜ (c = 2.7, CHCl ₃ ) mass spectrometry; (m / z) 443 (M ⁺ ) ¹ H NMR (CDCl ₃ ) δ: 2.08 (1H, m, CH), 2.36 (1H, m, CH),
, 3.33 (3H, s, OCH ₃ ), 3.50 (2H, m, CH ₂ ), 3.57-3.68
(4H, m, 2xCH ₂ ), 4.32 (1H, m, CH), 4.44 (1H, m, CH),
4.68-5.20 (2H, s, OCH ₂ O), 4.98 (1H, d, J = 4 Hz, CH),
5.09-5.21 (2H, m, CH ₂ ), 7.16-7.30 (5H, m, aromatic p
rotons); ¹³ C NMR (CDCl ₃ ) δ: 35.85 and 36.85 (t),
52.10 and 52.42 (t), 57.72 and 57.95 (q), 58.84 (d),
66.65 and 66.77 (t), 66.98 and 67.04 (t), 71.49 (t), 7
3.90 and 74.92 (d), 94.16 and94.34 (t), 127.66, 12
7.75, 127.80, 127.86, 128.10, 128.25, 128.31, and
128.40, (aromatic carbons), 135.23 and 135.28 (s),
136.22 and 136.36 (s), 154.13 and 154.73 (s), 172.0
6 and 172.26 (s) (Example 3) N-benzyloxycarbonyl- (2S, 4R) -4
-To 2.0 g (5.6 mmol) of hydroxy-2-pyrrolidinecarboxylic acid benzyl ester (5) was added 10 ml of ethyl ether, and 1.3 g (15.5 mmol) of dihydropyran and 30 mg of paratoluenesulfonic acid were further added. After stirring at room temperature for 30 hours, 200 ml of ethyl acetate is added, and the mixture is washed with a saturated aqueous solution of sodium hydrogen carbonate and water. After drying over anhydrous magnesium sulfate and evaporating the solvent, the residue obtained is purified by column chromatography (silica gel C-200, eluent; hexane: ethyl acetate = 1: 1) to give N-benzyloxycarbonyl. 2.24 g (92% yield) of-(2S, 4R) -4-tetrahydropyranyloxy-2-pyrrolidinecarboxylic acid benzyl ester (6, R = THP) was obtained as an oil. Optical rotation; [α] _D ²⁰ -39.4 ゜ (c = 2, CHCl ₃ ) mass spectrometry; (m / z) 439 (M ⁺ ) ¹ H NMR (CDCl ₃ ) δ: 1.3-1.7 (6H, br s, 3xCH ₂ ), 1.9-
2.5 (2H, m, CH ₂ ), 3.3-3.8 (2H, m, CH ₂ ), 4.4 (2H,
br s, CH ₂ ), 4.5-4.7 (1H, m, CH), 4.95 (2H, d, C
H ₂ ), 5.2 (1H, br s, CH); ¹³ C NMR (CDCl ₃ ) δ: 19.30
(t), 25.25 (t), 30.67 (t), 36.46 (t), 52.37 (t),
57.95 (d), 62.49 (t), 66.91 (t), 67.01 (t), 7
3.75 (d), 97.71 (d), 128.23 (d), 135.38 (s), 136.
38 (s), 154.98 (s), 172.27 (s). (Example 4) N-benzyloxycarbonyl- (2S, 4R) -4-methoxymethyloxy-2-pyrrolidinecarboxylic acid obtained in Example 1 Acid benzyl ester (6, R = MOM) 3.0 g
(7.5 mmol) was added with 50 ml of ethanol, and 213 mg of 10% palladium-carbon was subjected to catalytic reduction with 1 atm of hydrogen gas at room temperature for 1 hour. The reaction solution is filtered and the solvent is distilled off under reduced pressure to obtain crystals. Recrystallized from ethanol-ethyl acetate-hexane to give 1.05 g of (2S, 4R) -4-methoxymethyloxy-2-pyrrolidinecarboxylic acid (7, R = MOM) as white crystals
(80% yield). Melting point: 168-170 ° C (decomposition) Optical rotation: [α] _D ²⁰ -61.2 ゜ (c = 1.4, MeOH) Elemental analysis; Calculated C ₇ H ₁₃ NO ₄ (%) C, 47.99; H, 7.48; N , 8.00 Found (%) C, 47.71; H , 7.75; N, 7.66 1 H NMR (CD 3 0D) δ:. 2.03-2.50 (2H, m, CH 2), 3.30
(3H, s, CH ₃ O), 3.20-3.45 (2H, m, CH ₂ ), 4.20 (1H,
dd, J ₁ = 9.9 Hz, J ₂ = 7.8 Hz, CH), 4.42 (1H, br
s, CH), 4.61 (2H, s, OCH ₂ O), 4.89 (2.5H, br, NH);
¹³ C NMR (CD ₃ OD) δ: 36.87 (t), 52.44 (t), 56.03
(q), 60.91 (d), 76.78 (d), 96.51 (t), 173.19 (s). (Example 5) N-benzyloxycarbonyl- (2S, 4R) -4 obtained in Example 2 -Methoxyethoxymethyloxy-
(2S, 4R) -4-methoxyethoxymethyloxy-2-pyrrolidinecarboxylic acid (7, R = MEM) as white crystals from benzyl 2-pyrrolidinecarboxylate (6, R = MEM) in the same manner as in Example 4. (75% yield). Optical rotation; [α] _D ²⁰ -56.6 ゜ (c = 2, MeOH) Melting point: 130-133 ° C (decomposition) Elemental analysis; C ₉ H ₁₇ NO ₅ Calculated (%) C, 49.30; H, 7.82; N , 6.39 Found (%) C, 48.96; H , 7.98; N, 6.58 1 H NMR (D 2 O) δ:. 2.04 (H, m, CH), 2.44 (1H, m, CH),
3.25 (3H, s, OCH ₃ ), 3.38 (2H, m, CH ₂ ), 3.51 (2H, m, CH
₂ ), 3.64 (2H, m, CH, CH ₂ ), 4.15 (1H, m, CH), 4.47 (1H,
m, CH), 4.67 (2H, m, OCH ₂ O); ¹³ C NMR (D ₂ O, internal standard: dioxane δ = 67.4) δ: 36.11 (t), 51.82 (t),
58.89 (q), 60.73 (d), 67.78 (t), 71.84 (t), 76.52 (d),
94.48 (t), 174.98 (s). (Example 6) N-benzyloxycarbonyl- (2S, 4R) -4-tetrahydropyranyloxy-2 obtained in Example 3
-(2S, 4R) -4-tetrahydropyranyloxy-2-pyrrolidinecarboxylic acid (7, R = THP) as white crystals from benzyl pyrrolidinecarboxylate (6, R = THP) in the same manner as in Example 4. (81% yield). Optical rotation: [α] _D ²⁰ -3.1 ゜ (c = 1.2, MeOH) Melting point: 187-191 ° C (decomposition) Elemental analysis; Calculated C ₁₀ H ₁₇ NO ₄ (%) C, 55.80; H, 7.96; N , 6.51 Found (%) C, 55.52; H , 8.22; N, 6.29 1 H NMR (CD 3 0D) δ:. 1.4-2.0 (6H, m, 3xCH 2), 2.0-2.8
(2H, m, CH ₂ ), 3.3-4.0 (4H, m, 2xCH ₂ ), 4.2-4.5 (1
H, m, CH), 4.6 (1H, m, CH), 4.8 (1H, brs, CH). (Example 7) (2S, 4R) -4-methoxymethyloxy obtained in Example 4 -2-pyrrolidinecarboxylic acid (7, R = MOM) 1.2
3 g of concentrated aqueous ammonia was added to 5 g (7.14 mmol), and 0.5 ml of carbon disulfide was further added, followed by reaction at 0 ° C. for 4 hours.
After evaporating the solvent under reduced pressure, water (10 ml) was added and lyophilized (3 times), and ammonium salt of (2S, 4R) -4-methoxymethyloxy-2-pyrrolidinecarboxylic acid dithiocarbamate was used.
1.63 g (82% yield) of (1, R = MOM) was obtained as amorphous. Optical rotation; [α] _D ²⁰ +15.2 DEG _{(c = 2.2, H 2 O} ) Elemental _{analysis; C 8 H 19 N 3 O} 4 S 2 · 2H 2 O Calculated (%) C, 29.89; H , 7.21; N, 13.07 Found (%) C, 29.67; H, 7.58; N, 12.80. ¹ H NMR (D ₂ O) δ: 2.08-2.42 (2H, m, CH ₂ ), 3.27 (3H,
s, CH ₃ ), 3.84 (1H, dd, J ₁ = 13.2Hz, J ₂ = 4.5 Hz, C
H), 4.19 (1H, d, J = 13.2 Hz, CH), 4.32 (1H, m, C
H), 4.62 (2H, br s, CH 2); 13 C NMR (D 2 O, internal standard:
(Dioxane δ = 67.4) δ: 37.04 (t), 55.20 (q), 5
9.96 (d), 66.84 (t), 75.54 (d), 95.04 (t), 179.15
(s), 207.50 (s). (Example 8) (2S, 4R) -4-methoxyethoxymethyloxy-2-pyrrolidinecarboxylic acid (7, R = M) obtained in Example 5
EM) to give (2S, 4R) -4-methoxyethoxymethyloxy-2-pyrrolidinecarboxylic acid dithiocarbamate ammonium salt (1, R = MEM) as amorphous (yield 61%) in the same manner as in Example 7. Can be Optical rotation; [α] _D ²⁰ +8.9 DEG _{(c = 2.4, H 2 O} ) Elemental _{analysis; C 10 H 23 N 3 O} 5 S 2 · 2H 2 O Calculated (%) C, 32.87; H , 7.45; . N, 11.50 Found (%) C, 33.11; H , 7.55; N, 11.25 1 H NMR (D 2 O) δ: 2.06-2.21 (2H, m, CH 2), 3.30 (3
H, s, CH ₃ ), 3.58 (2H, br s, CH ₂ ), 3.69 (2H, br s,
CH ₂ ), 3.87-4.26 (2H, m, CH ₂ ), 4.39 (1H, br s, C
H), 4.73 (3H, brs, CH ₂ , CH); ¹³ C NMR (D ₂ O, internal standard: dioxane δ = 67.4) δ: 37.13 (t), 57.97
(q), 59.86 (d), 66.74 (t), 66.90 (t), 71.12 (t),
75.47 (d), 93.62 (t), 179.14 (s), 203.86 (s). (Example 9) (2S, 4R) -4-tetrahydropyranyloxy-2-pyrrolidinecarboxylic acid obtained in Example 6 Acid (7, R = TH
From (P) to ammonium salt of (2S, 4R) -4-tetrahydropyranyloxy-2-pyrrolidinecarboxylic acid dithiocarbamate (1, R = THP) in the same manner as in Example 7,
(78% yield). Optical rotation; [α] _D ²⁰ +5.1 c (c = 2.5, H ₂ O) Elemental analysis; C ₁₁ H ₂₃ N ₃ O ₄ S ₂ .H ₂ O Calculated value (%) C, 38.46; H, 7.34; . N, 12.23 Found (%) C, 38.26; H , 7.55; N, 12.13 1 H NMR (D 2 O) δ: 1.41-1.64 (3x2H, m, 3xCH 2), 2.12-
2.36 (2H, m, CH ₂ ), 3.51 (2H, m, CH ₂ ), 3.80-3.92 (2
H, m, CH ₂ ), 4.11 (1H, m, CH), 4.41 (1H, br s, CH),
4.65 (2H, br s, 2XCH); ¹³ C NMR (D ₂ O, internal standard: dioxane δ = 67.4) δ: 19.41 (t), 24.53 (t), 30.34
(t), 36.17 (t), 59.76 (t), 63.85 (t), 66.73 (d), 7
5.24 (d), 98.95 (d), 179.12 (s), 214.73 (s). (Example 10) (2S, 4R) -4-methoxymethyloxy-2-pyrrolidinecarboxylic acid obtained in Example 7 To 270 mg (0.95 mmol) of dithiocarbamate (1, R = MOM) was added 4 ml of 40 mM Tris-HCl buffer (pH 7.4), and further, a 40 mM Tris-HCl buffer of ferrous sulfate (heptahydrate) (pH 7.
4) Add 4 ml of the solution (27 mg / ml) and react for 0.5 hours at room temperature.
A (2S, 4R) -4-methoxymethyloxy-2-pyrrolidinecarboxylic acid dithiocarbamate-iron complex (2, R = MOM) is synthesized. Normally, this solution is used as it is as a spin trap agent, but when this solution is added to 10 times volume of acetonitrile, precipitation occurs. (2S, 4R)-
4-methoxymethyloxy-2-pyrrolidinecarboxylic acid dithiocarbamate-iron complex (2: Fe (DTCMP) ₂ ; R = MOM) can be isolated (yield 90%). Optical rotation: [α] _D ²⁰ -145 ゜ (c = 0.04, 40 mM Tris-HCl buffer (pH 7.4)) UV (40 mM Tris-HCl buffer (pH 7.4)) λ (nm): 371.2 (ε7.17 × 10 ³ ), 263.2 (ε2.07x10 ³ ), 2
13.2 (ε9.02x10 ³ ), 283.6 (ε1.11x10 ⁴ ). IR (nujol) cm ^-1 2673, 1712, 1574, 1234, 1151, 1103,
1043, 914 (2S, 4R) -4-tetrahydropyranyloxy-2-
Pyrrolidinecarboxylic acid dithiocarbamate-iron complex (2:
Fe (DTCEP) ₂ ; R = MEM)), and (2S, 4R) -4-tetrahydropyranyloxy-2-pyrrolidinecarboxylic acid dithiocarbamate-iron complex (2: Fe (DTCTP) ₂ ; R = THP) obtain. (Example 11) N-methylbenzyloxycarbonyl-
N-methylbenzyloxycarbonyl-O-methoxymethyl- (S) -serine methyl ester (9) is obtained from (S) -serine methyl ester (8) in the same manner as in Example 1 (yield: 85).
%). Optical rotation: [α] _D ²⁰ -18.8 ゜ (c = 1.7, CHCl ₃ ) Mass spectrometry; (m / z) 311 (M ⁺ ) ¹ H NMR (CDCl ₃ ) δ: 2.97 (3H, s, NCH ₃ ) , 3.31 (3H, s,
OCH ₃ ), 3.62-3.71 (3H, m, 2xCH), 3.80-3.94 (2H, m, 2x
CH), 4.53-4.65 (2H, m , CH 2), 4.73-5.07 (1H, m, CH),
5.12 (2H, s, OCH ₂ Ph), 7.24-7.34 (5H, m, aromatic pr
otons); ¹³ CNMR (CDCl ₃ ) d: 31.83 (q), 52.12 (q), 5
5.32 (t), 58.93 (d), 65.25 (t), 67.38 (t), 96.36 (t),
127.69, 127.79, and 127.90, (aromatic carbons), 13
6.33 and 136.46 (s), 156.72 (s), 169.77 (s). (Example 12) N-methylbenzyloxycarbonyl-O-methoxymethyl- (S) -serine methyl ester obtained in Example 11 ( 9) 1.0 g (3.2 mmol) of methanol
Dissolve in 10 ml and add 2N aqueous sodium hydroxide solution 4
Add ml and react at room temperature for 4 hours. After methanol was distilled off under reduced pressure, the reaction solution was acidified with 5% hydrochloric acid and extracted with ethyl acetate. The ethyl acetate layer was washed with water, dried over anhydrous magnesium sulfate, and the solvent was distilled off to obtain 780 mg (yield: 82%) of the corresponding carboxylic acid as an oil. Next, the N-benzyloxycarbonyl group was removed by catalytic reduction in the same manner as in Example 5 to give N-methyl-O-methoxymethyl- (S) -serine (1
0) is obtained as white crystals (71% yield from 9). Optical rotation: [α] _D ²⁰ -1.5 ゜ (c = 2, MeOH) Melting point: 210-215 ° C (decomposition) Elemental analysis; Calculated C ₆ H ₁₃ NO ₄ (%) C, 44.16; H, 8.03; N , 8.58 Found (%) C, 43.99; H , 8.28; N, 8.30 1 H NMR (D 2 O) δ:. 2.64 (3H, s, NCH 3), 3.28 (3H, s,
OCH ₃ ), 3.63-3.76 (3H, m, CH ₂ , CH), 3.50-3.65 (1H, m,
CH), 3.70-3.95 (2H, m, CH ₂ ), 4.20-4.40 (2H, m, 2xC
H), 4.60 (2H, s, OCH ₂ O), 7.16-7.50 (15H, m, aromatic
¹³ C NMR (D ₂ O, internal standard: dioxane δ = 67.
4) δ: 32.4 (q), 59.55 (d), 63.549 (q), 69.20 (t), 96.
82 (t), 172.10 (s). (Example 13) N-methyl-O- obtained in Example 12
The ammonium salt of N-methyl-O-methoxymethyl- (S) -serine dithiocarbamate (3, R = MOM) was converted from methoxymethyl- (S) -serine (10) as amorphous in the same manner as in Example 7 (collected). Rate 26%). Optical rotation; [α] _D ²⁰ -4.5 ゜ (c = 1.6, MeOH) Elemental analysis; C ₇ H ₁₉ N ₃ O ₄ S ₂ · 1.5H ₂ O Calculated (%) C, 27.99; H, 7.38; N , 13.99. Found (%) C, 27.82; H, 7.68; N, 13.77. ¹ H NMR (D ₂ O) δ: 3.27 (3H, s, CH ₃ ), 3.28 (3H, s, C
H ₃ ), 3.65-4.03 (2H, m, CH ₂ ), 4.50-4.80 (2H, m, OCH ₂
O), 6.51 (1H, brs, CH); ¹³ C NMR (D ₂ O, internal standard:
(Dioxane δ = 67.4) δ: 32.53 (q), 59.07 (q), 6
3.54 (dt), 71.70 (d), 102.50 (t), 176.05 (s), 211.
(Example 14) N-methyl- (S) -serine dithiocarbamate (3, R = H) was prepared in the same manner as in Example 7 from N-methyl- (S) -serine (10, R = H). H) is obtained as amorphous (yield 82
%). Optical rotation; [α] _D ²⁰ +21.2 ゜ (c = 3.1, H ₂ O) Elemental analysis; C ₅ H ₁₅ N ₃ O ₃ S ₂ .H ₂ O Calculated value (%) C, 24.28; H, 6.93; . N, 16.99 Found (%) C, 23.95; H , 7.28; N, 17.25 1 H NMR (D 2 O) δ: 3.26 (3H, s, CH 3), 3.78-4.00 (2H,
m, CH ₂ ), 6.38 (1H, t, CH); ¹³ C NMR (D ₂ O, internal standard: dioxane δ = 67.4) δ: 38.78 (q), 60.71 (t),
69.33 (d), 175.40 (s), 211.77 (s). (Example 15) In the same manner as in Example 10, the following serine derivative dithiocarbamate iron complex (4) is obtained.

That is, N-methyl- (S) -serine dithiocarbamate-iron complex (4: Fe (MSD) ₂ ; R = H), and N
-Methyl-O-methoxymethyl- (S) -serine dithiocarbamate-iron complex (4: Fe (MMSD) ₂ ; R = MOM) is obtained.

[0024]

[Test Example] These compounds (Formula 2, R = MOM, MEM, THP; Formula
4, R = H, MOM), the measurement results of nitric oxide scavenging ability using ESR and the measurement results of nitric oxide generated in the mouse body are as follows. (Test Example 1) Nitrogen monoxide trapping ability of (S) -hydroxyproline derivative dithiocarbamate-iron complex (2) and N-methyl-L-serine dithiocarbamate-iron complex (4) One of each complex in an aqueous solution The nitric oxide trapping ability was examined using the ESR spin trapping method. An aqueous solution of nitric oxide was prepared by blowing nitric oxide gas into a 40 mM Tris-HCl buffer (pH 7.4) under anaerobic conditions. The concentration was determined by the grease method. Various concentrations of nitric oxide aqueous solution
0.5 mM hydroxyamino acid derivative dithiocarbamate
-In addition to the iron complex (2: R = MOM, THP, MEM 4: R = H, MOM), the generated nitric oxide adduct was measured by X-band ESR. FIG. 1 shows the results using 2 (R = MOM, THP) and 4 (R = H). In each of the complexes, an adduct formation curve was given in proportion to the concentration of nitric oxide. Was found to be less efficient and trapped nitric oxide efficiently. (Test Example 2) Effect of Buffer on Nitric Oxide Capture of (S) -Hydroxyproline Derivative Dithiocarbamate-Iron Complex (2) We examined using the method.
40 mM Tris-HCl buffer, 10 mM phosphate saline, 40
1 mM mM Hepes-KOH buffer
1 ml of each buffer solution of the (S) -hydroxyproline derivative dithiocarbamate-iron complex (Formula 2, R = MOM) was prepared,
The reagent 3- [2-hydroxy-1- (1-methylethyl) -2-nitrosohydrazino] -1- which spontaneously releases nitric oxide
0.02 ml of 50 mM weakly basic aqueous solution of propaneamine (NOC5)
The nitric oxide adduct formed after 20 minutes was added to X-band E
Measured by SR. As shown in FIG. 2, the ability to capture nitric oxide varies depending on the type of buffer used for preparation, and it was found that the use of a Tris-HCl buffer most efficiently captures nitric oxide. (Test Example 3) In Vivo ESR Measurement of Iron Complex-Nitric Oxide Adduct Nitrogen monoxide gas was blown into an iron complex solution (50 mM) of DTCMP (Formula 1, R = MOM) under anaerobic conditions. Obtained. Fe (DTC
MP) ₂ (Equation 2, R = MOM) was administered intravenously to mice (200 μl of a 50 mM solution), and the ESR spectrum of the abdomen was changed to L
As shown in Fig. 3, the signal of the nitric oxide adduct was detected for more than 30 minutes, and the nitric oxide adduct was found to be relatively stable in the mouse body, as shown in Fig. 3. did. Further, Fe (DTCMP) ₂ nitric oxide adduct had a nitric oxide adduct equivalent signal intensity Fe (DTCS) ₂ that have been reported so far. (Test Example 4) Spin trapping lipopolysaccharide of nitric oxide (LPS, E. coli 026: B6) induced by LPS (lipopolysaccharide) in mice was intravenously administered (20 mg / Kg)
Six hours after administration to a mouse (ICR (female, 5 weeks old) or ddY (female, 5 weeks old)), the synthesized Fe (MSD) ₂ (Formula 4, R = H) solution was intravenously administered (0.42 mM / 2 hours later, L-band ESR
Was used to non-invasively measure nitric oxide generated in the living body of mice. As shown in FIG. 4, the signal of the nitric oxide adduct was efficiently detected.

On the other hand, in order to examine the pharmacokinetics of the iron complex, LPS
Six hours after administration, various iron complexes (Fe (DTCMP) ₂ (Formula 2, R
= MOM), Fe (DTCTP) ₂ (Equation 2, R = THP), and Fe (MSD)
₂ (Equation 4, R = H)) was administered (0.42 mM / Kg), and 30 minutes later, blood was collected in a heparin-treated capillary and X-band E
Nitric oxide adduct was measured by SR. A liver homogenate was prepared and measured in the same manner. As shown in FIG. 5, a nitric oxide adduct was observed. Note that this signal is from ^NG -monomethyl- (S) -arginine ((S) -NMM), an inhibitor of nitric oxide synthase (NOS), an enzyme that induces nitric oxide.
Since no signal was observed when A) was administered, it was confirmed that this signal was derived from nitric oxide. Table 1 shows relative signal intensities in blood and liver, and signal intensity ratios in liver homogenate and blood. FIG. 6 illustrates this signal intensity ratio. As in the case of Fe (MGD) ₂ which has been known so far, Fe (DTCTP) ₂ and Fe (MSD) ₂
Nitric oxide adduct was mainly detected in the liver. Especially the nitric oxide adduct of Fe (MSD) ₂ gave a strong signal intensity. On the other hand, Fe (DTCMP) ₂ was hardly detected in the liver, and was found to have the characteristic of almost capturing nitric oxide generated in blood.

[0026]

[Table 1]

[0027]

As described above in detail, according to the invention of this application, a novel spin trapping agent for trapping NO in vivo and a compound thereof are provided.

[Brief description of the drawings]

FIG. 1 is a diagram illustrating the NO trapping ability of an iron complex of the present invention.

FIG. 2 is a diagram illustrating the influence of a buffer when trapping NO in an iron complex.

FIG. 3 shows an NO adduct of an iron complex of the present invention in mice.
It is the figure which illustrated the in vivo ESR spectrum.

[Fig. 4] In-vivo of NO adduct in LPS-treated mice
o A diagram illustrating an ESR spectrum.

FIG. 5: X-ba of NO adduct in LSP-treated mouse blood
FIG. 5 is a diagram illustrating an nd ESR spectrum.

FIG. 6 is a graph showing the ratio of NO adduct in liver and blood of LSP-treated mice.

 ────────────────────────────────────────────────── ─── Continued on the front page (72) Inventor Hidehiko Nakagawa 5-5-38-403 Inage Beach, Mihama-ku, Chiba-shi, Chiba F-term (reference) 4C069 AA15 BD02 4C085 HH17 JJ02 KA07 KB56 LL07 4H006 AA01 AA03 AB20 TN30 TN50 TN60 4H050 AA01 AA03 AB20 AB90 WB15 WB21

Claims (3)

[Claims] (1) A compound represented by the following formula (1):
(R) -hydroxyproline derivative dithiocarbamate or an iron complex thereof represented by the formula [2] (where R is a hydrogen atom, an alkyl group, an aryl group, an aralkyl group, an alkoxyalkyl group, and an oxygen-containing heterocyclic group) Or a group selected from the group of the alkyl substituents thereof, and A represents a cation). Embedded image Embedded image 2. N-methyl (S)-represented by the following formula [3]:
Or (R) -serine derivative dithiocarbamate or an iron complex thereof represented by the formula [4] (where R is a hydrogen atom,
A group selected from the group consisting of an alkyl group, an aryl group, an aralkyl group, an alkoxyalkyl group, and an oxygen-containing heterocyclic group or an alkyl substituent thereof, and A represents a cation). Embedded image Embedded image 3. A spin trapping agent for trapping nitric oxide, comprising the dithiocarbamate of claim 1 or 2 or an iron complex thereof.