JPH06281581A - Detection and determination of superoxide radical - Google Patents

Abstract

PURPOSE:To make it possible to measure superoxide radical with more higher sensitivity by making the superoxide radical react with pyrido-pyridazine derivative or the salt thereof, and performing chemical light emission. CONSTITUTION:Superoxide-radical (02<->) and pyride-pyridazine derivative or the salt thereof expressed by the formula are made to react, and chemical light emission is generated. In the formula, R1 indicates hydrocarbon or hetero- ring group, which can be substituted, respectively. R2 indicates hydrogen, hydroxyl group, thyol and the like R3 indicates hydrogen atoms, oxygen atoms and hydroxyl group, which can be substituted X indicates oxygen atoms or sulfur atoms. As a sample, which is applied in this method, it is preferable to apply the sample in the measurement of 02<->, which is produced from, e.g. cells. As the cells producing 02<->, there are the leukocyte-based, vascular-system-based and nervous-system-based cells and the like. When this method is used, the specific light emitting quantity and background light emitting quantity are high, 02<-> can be detected with high sensitivity and the quantity can be determined.

Description

Detailed Description of the Invention

[0001]

The present invention relates to a superoxide radical ^- for the detection and quantification method of (O _2).

[0002]

BACKGROUND OF THE INVENTION superoxide radical (O ₂ ^-) is,
Granulocyte leukocytes such as neutrophils, eosinophils, basophils, monocytes,
Macrophages, mesangial cells, vascular smooth muscle cells,
Produced from various cells such as vascular endothelial cells, O ₂ ^- hydrogen peroxide from (H ₂ O _2), hydroxy radical (· O
H), hypochlorite (OCl ^-), singlet oxygen ⁽¹ O ₂₎
It is said that these reactive oxygen species damage tissues and cells and are involved in various diseases (Chemistry and Biology, Vol. 30 (No. 3), p. 184, (1992)). Conventionally, as a method for measuring O ₂ ^−, a spectroscopic method using cytochrome C, nitroblue tetrazolium, etc., a chemiluminescence method using luminol, a Cypridina luciferin derivative, etc., an electron spin resonance method, etc. are often used. Among them, the chemiluminescence method is widely used as a simple method capable of tracing the production of active oxygen in the same sample. However, luminol has a low sensitivity to O ₂ ⁻ and a Cypridina luciferin derivative has a high sensitivity to O ₂ ⁻ , but has a drawback that the background value is high due to the result of autoxidation.

[0003]

Therefore, if a luminescent substance having a high sensitivity to O ₂ ⁻ and a low background value is found, it becomes possible to measure O ₂ ⁻ by a more sensitive chemiluminescence method. result, for example, O ₂ from various cell ^-
It can be advantageously used to screen for drugs that regulate production.

[0004]

In view of the above situation, the present inventors have made various studies to establish a practical means for measuring O ₂ ⁻ , and have completed the present invention. That is, the present invention is (1) superoxide radical (O ₂ ^-) and the following general formula (I)

[Chemical 3] [In the formula, R ₁ represents an optionally substituted hydrocarbon group or a heterocyclic group, R ₂ represents hydrogen, a hydroxyl group, a thiol group, an amino group or a mono-substituted amino group, and R ₂ represents a mono-substituted amino group. In the case of a group, R ₂ together with R ₁ may form a ring. R ₃ is a hydrogen atom, an oxygen atom, an optionally substituted hydroxyl group, an optionally substituted amino group, an optionally substituted thiol group, a halogen atom,
A heterocyclic group, a nitro group, a cyano group, an optionally esterified or amidated carboxyl group, an azido group, a sulfo group or an organic sulfonyl group, provided that when R ₁ is an aliphatic group, R ₃ is hydrogen. Not an atom. X represents an oxygen atom or a sulfur atom], and a pyridopyridazine derivative or a salt thereof is reacted to generate chemiluminescence. As a sample to which the method of the present invention can be applied,
There is no particular limitation as long as it contains or generates O ₂ ⁻ . For example, it can be preferably applied to the measurement of O ₂ ⁻ produced from cells. Examples of cells that produce O ₂ ⁻ include leukocyte, vascular or nervous system cells and cell lines derived therefrom, but any cell or cell line that produces O ₂ ⁻ can be used. Good. Examples of leukocyte cells include granulocyte leukocytes such as neutrophils, eosinophils and basophils, monocytes, macrophages and the like, and examples of vascular cells include mesangium cells, vascular smooth muscle cells, vascular endothelium. Examples of the cells and the like include nerve cells and glial cells. The cell suspension is a culture medium of each cell [eg, RPMI (Canser Research 47 , 4771
-4775 (1987))] or Hanks solution [eg HBSS]
(Methods in Enzymology 186 , 585-591 (1990))], and the serum content is 0 to 20%, preferably 0 to 10%. Fetal bovine serum is preferred as the serum. The number of cells used for measurement may be any number as long as it is a number for obtaining a detectable amount of luminescence, but 10 ⁴ to 10 ⁸ cells, preferably 10 ⁵ to 10 ⁷ cells are used.

Specific examples of the hydrocarbon group of the substituent R ₁ in the pyridopyridazine derivative represented by the general formula (I) include, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec. -Butyl, tert
-A straight-chain or branched lower alkyl group having 1 to 6 carbon atoms such as butyl, n-pentyl, isopentyl, and hexyl; for example, 2 carbon atoms such as vinyl and allyl.
Alkenyl group having 3 to 3 carbon atoms; alkynyl group having 2 to 3 carbon atoms such as ethynyl and propargyl; aralkyl group having 7 to 12 carbon atoms such as benzyl and phenethyl; and 6 carbon atoms such as phenyl and naphthyl. ~ 1
An aryl group of 0 may be mentioned. Further, as the unsaturated or saturated 5- or 6-membered heterocyclic group containing at least one, preferably 1 to 4 heteroatoms of nitrogen, sulfur or oxygen, 2-pyridyl, 4-pyridyl, Two
-Thienyl, 2-furyl, imidazolyl, triazolyl, tetrazolyl, morpholino, piperazinyl, 4-thiazolyl, 2-amino-4-thiazolyl and the like. The substituent which these substituents R ₁ may have is represented by the formula:

[0006]

[Chemical 4]

[Wherein R ₄ is --CN, --hal, --OPO ₃ M
_{2, -OSO 3 M, -CO 2} R 5, -SR 6, -OR 6, -N
HR ₆ , A is a sulfur, oxygen, or nitrogen atom, and n is 1 to 4
Means an integer of, M is an alkali metal or hydrogen atom,
hal represents a halogen atom such as fluorine, chlorine, bromine and iodine. R ₅ is a hydrogen atom or a lower alkyl group such as methyl, ethyl or propyl, or a group forming an active imide ester capable of forming a complex such as maleimide, succinimide or 5-norbornene-2,3-carboximide group, R 5 ₆ is a hydrogen atom or a lower alkyl group such as methyl, ethyl or propyl, or a formula

[0008]

[Chemical 5]

[Wherein R ₇ represents a maleimide, succinimide or 5-norbornene-2,3-carboximide group] to form an active imide ester such as a succinic acid half ester having a complex-forming ability. [Showing a group].

Specific examples of the substituent of the mono-substituted amino group in the substituent R ₂ represented by the general formula (I) include, for example, R
The number of carbon atoms of as exemplified straight chain or branched in ₁ 1
Examples thereof include a lower alkyl group having 6 carbon atoms, an alkenyl group having 2 to 3 carbon atoms, an alkynyl group having 2 to 3 carbon atoms, an aralkyl group having 7 to 12 carbon atoms, and an aryl group having 6 to 10 carbon atoms.
Further, as a specific example of the compound in which R ₂ is a mono-substituted amino group which may form a ring together with the substituent R ₁ ,

[0011]

[Chemical 6]

[Wherein R ₃ and X have the same meanings as described above, and ring A is a nitrogen-containing compound which may have 1 or 2 unsaturated bonds such as imidazole, thiazole, pyrrole and pyridazine. A 5- to 7-membered ring or a benzene ring or a nitrogen-containing 5- to 7-membered ring which may be condensed with, for example, a hetero ring such as indole, benzofuran, or quinoline is shown. Ring A may be substituted with a lower alkyl group having 1 to 6 carbon atoms such as methyl, ethyl, and propyl].

As the optionally substituted hydroxyl group represented by the substituent R ₃ in the general formula (I), for example, a hydroxyl group,
Examples thereof include an alkoxy group, an allyloxy group and an aralkyloxy group. As the alkyl group of the alkoxy group, R ₁
Linear or branched chain carbon number 1 to 6 as exemplified in
Is lower alkyl, aryl groups such as phenyl and naphthyl having 6 to 10 carbon atoms as aryl groups, and aralkyloxy groups having aralkyl groups having 7 to 12 carbon atoms such as benzyl and phenethyl. Groups. Examples of the optionally substituted amino group represented by R ₃ include an amino group, a mono-substituted amino group and a di-substituted amino group. As the substituent of the mono-substituted amino group, for example, a linear or branched lower alkyl group having 1 to 6 carbon atoms as exemplified for R ₁ ; for example, carbon such as cyclopropyl, cyclopentyl and cyclohexyl A cycloalkyl group of the number 3 to 6;
C2-C3 alkenyl groups such as vinyl and allyl;
For example, ethynyl, propargyl, etc.
An alkynyl group; for example, an aralkyl group having 7 to 12 carbon atoms such as benzyl and phenethyl; for example, phenyl,
An aryl group having 6 to 10 carbon atoms such as naphthyl can be mentioned. As the substituents in the di-substituted amino group, the same substituents as in the above-mentioned mono-substituted amino group are used, or different from each other.

Examples of the optionally substituted thiol group represented by R ₃ include a thiol group, an alkylthio group,
Examples thereof include an arylthio group and an aralkylthio group. Examples of the alkyl group of the alkylthio group include linear or branched lower alkyl having 1 to 6 carbon atoms as exemplified for R ₁ , and examples of the aryl group of the arylthio group include:
Phenyl and naphthyl having 6 to 10 carbon atoms and examples of the aralkyl group of the aralkylthio group include 7 carbon atoms.
.About.12 benzyl and phenethyl.

Examples of the halogen atom represented by R ₃ include iodine, bromine, chlorine and fluorine. The heterocyclic group represented by R _3, include heterocyclic groups mentioned for R _1. Examples of the esterified or amidated carboxyl group represented by R ₃ include, for example,
Examples thereof include a carboxyl group, a carbamoyl group and an alkoxycarbonyl group. Examples of the alkyl group in the alkoxycarbonyl group as the esterified carboxyl group represented by R ₃ include the alkyl groups mentioned for R ₁ . The organic sulfonyl group represented by R ₃ includes an alkylsulfonyl group and an arylsulfonyl group. Examples of the alkyl group in the alkylsulfonyl group and the aryl group in the arylsulfonyl group include the alkyl group and aryl group described for R ₁ . An aryl group, especially a phenyl group,
It may be substituted with a lower alkyl group such as methyl or ethyl. The carbonyl or thiocarbonyl group of the pyridopyridazine ring represented by the general formula (I) can be easily enolized to form a salt with a monovalent or divalent cation.

[0016] The monovalent cation, ammonium, ions and alkali metal, for example, ammonia, _{C. 1 to 4} monoalkyl amines such as methylamine, ethylamine, butylamine, dimethylamine, diethylamine, such as dibutylamine-di ( _{C. 1 to 4)} alkylamine, trimethylamine, such as triethylamine _{(C. 1 to 4)} alkylamine, ammonium ions or lithium, such as pyridinium or hydrazinium, potassium, and alkali metal such as sodium.
Examples of the divalent cation include alkaline earth metals such as calcium and magnesium.

Among the compounds represented by the general formula (I), the following general formula (II)

[Chemical 7] [In the formula, R ₁ represents an optionally substituted phenyl group, R ₂
Represents a hydrogen atom or an amino group, R ₃ represents a hydrogen atom, an oxygen atom, an optionally substituted hydroxyl group, a halogen atom, or a heterocyclic group], and a pyridopyridazine derivative represented by Preferably, in the general formula (II), R ₁ is a phenyl group, R ₂ is an amino group, and R ₃ is a halogen atom (preferably chlorine).

Examples of the pyridopyridazine derivative represented by the general formula (I) or a salt thereof in the present invention include EP Publication No. 0491477 and Pharmaceutical Journal, Vol. 92, No. 1333.
The thing described in the page (1972) is mentioned.

[0019] The present invention method, O ₂ in the sample ^- is carried out by causing the coexistence is not chemiluminescence and pyridopyridazine derivative or a salt thereof the general formula (I). The pyridopyridazine derivative of the general formula (I) or a salt thereof which is a luminescent substrate is used as a solution at the time of use, for example, by dissolving it in a buffer solution. The buffer solution can be used for diluting the luminescent substrate. Any of them may be used, and examples thereof include a phosphate buffer solution, a borate buffer solution, a carbonate buffer solution, and a Tris buffer solution. Further, as the pH of the buffer solution, it is possible to select near neutrality, for example, pH 7 to 8. The reaction temperature is preferably in the range of 0 to 60 ° C, particularly 20 to 37 ° C. The substrate concentration used for measurement is 10 μM
The range of 10 mM, especially 300 μM to 1 mM is desirable.

When measuring cell-derived O ₂ ⁻ , chemiluminescence is caused by coexisting a cell suspension prepared to have the above-mentioned concentration with a luminescent substrate, and the luminescence is measured immediately. At this time, if necessary, a stimulant that induces O ₂ ⁻ production in the mixed solution, for example, neutrophils, fMLP (N-
formyl-L-methionyl-L-leucyl-L-phenylalani
ne), C5a (anaphylatoxin), PAF (platelet activating factor) or the like can be added. Luminescence can be measured with a commercially available or self-made measuring device (for example, a photon counter equipped with a highly sensitive photomultiplier tube). That is, the measurement may be started immediately after mixing the cell suspension and the luminescent substrate, or after mixing and adding the stimulant, and measuring the luminescence amount for several minutes to several tens of minutes. By using the method as described above, it becomes possible to quantify the O ₂ ⁻ produced from the cells. Therefore, when it is desired to screen a drug that regulates the production of O ₂ ⁻ from cells, the target drug and target cells are allowed to coexist and the O
₂ ^- by detecting quantifying the present invention method, it can be very effectively utilized as a highly sensitive evaluation of the target drug.

[0021]

EXAMPLES The present invention will be described in more detail with reference to the following Reference Examples and Examples, but these do not limit the scope of the present invention. Reference Example 1 Ethyl 3-amino-3-ethoxy acrylate Ethyl cyanoacetate 226 g (2 mol), ethanol 101 g
(2.2 mol), 100 g of dry diethyl ether under ice cooling,
93.5 g (1.3 eq) of hydrochloric acid gas was blown thereinto and left overnight.
The precipitated colorless prism crystals were collected by filtration using diethyl ether (hydrochloride). Yield 352.5 g (90.3%). Melting point 1
03-105 ° C. NMR (DMSO-d ₆ ) δ: 1.23 (3H, t, J = 7.2)
Hz), 1.37 (3H, t, J = 7.2Hz), 3.94 (2
H, br.), 4.15 (2H, q, J = 7.2Hz), 4.51
(2H, q, J = 6.8Hz). The crystals obtained were added to an ice-cooled 200 g (1.2 eq) aqueous solution of sodium hydrogencarbonate and extracted with diethyl ether. The organic layer was washed with water and saturated brine, dried over magnesium sulfate, the solvent was distilled off, and the residue was purified by vacuum distillation. Yield 253.8 g (79.7%). boiling point
_1.8 78 ° C. IR (neat) ν: 2980, 1660, 1610, 154
0, 1160, 1070 cm ^-1 .

Reference Example 2 2-Amino-3,4-diethoxycarbonyl-6-phenylpyridine hydrochloride 1.11 g (5 mmol) ethylbenzoylpyruvate, 1.75 g ethyl 3-amino-3-ethoxyacrylate
(2.2 eq) was heated at 100 ° C. for 1.5 hours. After distilling off the low boiling point substance under reduced pressure, 2 ml of 10% hydrochloric acid was added to the reaction solution,
The generated crystals were collected by filtration using diethyl ether. Recrystallization from ethyl acetate gave pale yellow needle crystals. Yield 0.7g
(39.8%). Melting point 78-81, 145-148 [deg.] C (double melting point). NMR (CDCl ₃ ) δ: 1.26 (3H, t, J = 7.5H
z), 1.30 (3H, t, J = 7.5Hz), 4.27 (2H,
q, J = 7.5Hz), 4.28 (2H, q, J = 7.5Hz),
7.17 (1H, s), 7.43 (3H, m), 8.05 (2H,
m). IR (KBr) ν: 1740, 1700, 1650, 130
0 cm ^-1 . Calcd _{(C 17 H 18 N 2 O} 4 · HCl) C: 58.2
1; H: 5.46; N: 7.99. Measured value C: 58.45;
H: 5.48; N: 8.04.

Reference Example 3 3,4-diethoxycarbonyl-6-phenyl-2-pyridone 2-amino-3,4-diethoxycarbonyl-6-phenylpyridine hydrochloride (3.50 g, 10 mmol) was added to 2% hydrochloric acid. 90 ml and dioxane 60 ml were dissolved, and sodium nitrite 0.83 g (1.2 eq) was dissolved in water 3 ml and added dropwise. It was stirred at room temperature for 4 hours and left overnight. The solvent was distilled off, and the precipitated crystals were collected by filtration with water. Recrystallization from ethanol gave pale yellow prism crystals. Yield 1.9 g (60.3%). Melting point 143-144 [deg.] C. NMR (CDCl ₃ ) δ: 1.33 (3H, t, J = 7.2H
z), 1.36 (3H, t, J = 7.2Hz), 4.37 (4H,
q, J = 7.2Hz), 6.95 (1H, s), 7.47 (3H,
m), 7.79 (2H, m). IR (KBr) ν: 2900-30
00, 1740, 1635, 1615, 1245 cm ^-1 . Elemental analysis: Calculated value (C ₁₇ H ₁₇ NO ₅ ) C: 64.75; H: 5.
43; N: 4.44. Measured C: 64.82; H: 5.55;
N: 4.46.

Reference Example 4 3,4-diethoxycarbonyl-5-nitro-6-phenyl-2-pyridone 5.0 g of 3,4-diethoxycarbonyl-6-phenyl-2-pyridone was suspended in 13 ml of acetic anhydride. After cooling to -10 ° C, 1.32 ml (2.0 eq) of fuming nitric acid was added dropwise over 1 hour, and the mixture was stirred for 45 minutes as it was. Add 40 ml of water and stir at room temperature,
I left it overnight. The generated crystals were collected by filtration and dried. Yield 4.
61 g (80.7%). Recrystallization from ethanol gave pale yellow needle crystals. Melting point 172-173 [deg.] C. NMR (CDCl ₃ ) δ (ppm): 1.37 (3H, t, J = 7.1)
Hz), 1.38 (3H, t, J = 7.1Hz), 4.40 (2
H, q, J = 7.1Hz), 4.42 (2H, q, J = 7.1H)
z), 7.52 (5H, m). IR (KBr) ν: 3450, 1740, 1650, 152
5, 1345, 1285 cm ^-1 . Calcd _{(C 17 H 16 N 2 O} 7) C: 56.67; H: 4.
48; N: 7.77. Measured C: 56.96; H: 4.56;
N: 7.60.

Reference Example 5 2-Chloro-3,4-diethoxycarbonyl-5-nitro-6-phenylpyridine 3,4-diethoxycarbonyl-5-nitro-6-phenyl-2-pyridone 2 g per 1.0 g. 1.3 ml (3.37 eq) of phenylphosphonic acid chloride was added and heated at 150 ° C. for 2.5 hours. Water was added to the reaction solution, and the mixture was extracted with ethyl acetate. The organic layer was washed with water and saturated saline and then dried using magnesium sulfate. The solvent was distilled off and the residue was purified by a silica gel column (eluting solvent: ethyl acetate-hexane 1: 2) to obtain an oily product. Yield 950 mg (90.5%). NMR (CDCl ₃ ) δ: 1.35 (3H, t, J = 7.2H
z), 1.42 (3H, t, J = 7.2Hz), 4.44 (4H,
q, J = 7.2 Hz), 7.52 (5 Hz, m). IR (KBr) ν: 2990, 1750, 1580, 1550
cm ^-1 .

Reference Example 6 3-Amino-6-chloro-4,5-diethoxycarbonyl-2-phenylpyridine 2-chloro-3,4-diethoxycarbonyl-5-nitro-6-phenylpyridine 400 mg, reduced iron 520 mg,
4 ml of ethanol was heated to 60 ° C and 2.3 ml of concentrated hydrochloric acid was added to 30 ml.
Dropped in minutes. The mixture was neutralized with saturated aqueous sodium hydrogen carbonate under ice cooling and extracted with dichloromethane. After drying the organic layer, the solvent was distilled off to obtain crystals. Yield 337 mg (91.5
%). Recrystallization from dichloromethane-hexane gave pale yellow prism crystals. Melting point 93-94 [deg.] C. NMR (CDCl ₃ ) δ (ppm): 1.37 (3H, t, J = 7.0)
Hz), 1.43 (3H, t, J = 7.0Hz), 4.37 (2
H, q, J = 7.0Hz), 4.43 (2H, q, J = 7.0H)
z), 5.93 (2H, m), 7.48-7.57 (5H, m). IR (KBr) ν: 3510, 3400, 1760, 172
5, 1615, 1265 cm ^-1 . Elemental analysis: Calculated value (C ₁₇ H ₁₇ N ₂ O ₄ Cl) C: 58.54;
H: 4.91; N: 8.03. Measured C: 58.47; H: 4.
87; N: 7.97.

Reference Example 7 8-amino-5-chloro-7-phenylpyrido [3,4-
d] Pyridazine-1,4 (2H, 3H) dione (L-012) 3-amino-6-chloro-4,5-diethoxycarbonyl-2-phenylpyridine (55 mg) obtained in Reference Example 6 was added to hydrazine monohydrate. 1 ml of the product was added and heated at 100 ° C. for 25 minutes under a nitrogen stream. Water was added to the reaction solution under ice cooling, neutralized with hydrochloric acid to pH 5, and the resulting yellow powdery crystals were collected by filtration. Yield 20 mg (43.9%). Melting point 300 ° C or higher. _{NMR (DMSO-d 6) δ} (ppm): 6.29 (2H, s), 7.
51-7.69 (5H, m). IR (KBr) ν: 3470,3
050, 1645, 1585 cm ^-1 . Calcd _{(C 13 H 9 N 4 O} 2 Cl) C: 54.09; H:
3.14; N: 19.41. Measured C: 54.18; H: 3.
07; N: 19.61.

Reference Example 8 8-amino-5-chloro-7-phenylpyrido [3,4-
d] Pyridazine-1,4 (2H, 3H) dione sodium salt
(L-012 sodium salt) 8-amino-5-chloro-7-phenylpyrido [3,4-
1.50 g of d] pyridazine-1,4 (2H, 3H) dione (L-012) was added to 50 ml of 1N aqueous sodium hydroxide solution while stirring. The pale yellow crystals that had once dissolved and were then filtered out were filtered, washed with a small amount of 1N aqueous sodium hydroxide solution, then with ethanol, and dried to obtain 1.48 g of the desired compound. mp> 300 ° C NMR (DMSO-d ₆ ) δ (ppm): 7.35 to 7.55 (3H,
m), 7.65 to 7.70 (2H, m), 7.40 to 8.20 (2
H, br.), 10.5 (1H, br.s). IR (KBr) υ: 1650,1550,1455,870c
m ⁻¹ . Elemental analysis: Calculated value (C ₁₃ H ₈ N ₄ O ₂ ClNa): C: 50.2
6; H: 2.60; N: 18.03. Found: C: 49.91; H:
2.35; N: 17.63.

Example 1 Eosinophil cell line (EoL-1)
Measurement of O ₂ ⁻ produced by EoL-1 cells is based on Saito et al. [Blood, 66 , 1233-12.
40, (1985)], a human eosinophilic cell line established from a patient with acute eosinophilic leukemia. EoL-
GIT containing 1 cell containing 5% fetal calf serum (FCS)
Prepared 5 × 10 ⁵ cells / ml with medium (Nippon Pharmaceutical Co.) and
FN-γ was added to 100 U / ml and cultured for 3 days. The medium is replaced with the same medium containing IFN-γ,
The cells were differentiated by culturing for 3 days. 5x the differentiated EoL-1 cells in GIT medium containing 5% FCS.
Suspend to a concentration of 10 ⁶ cells / ml and measure at 4 ℃
It was left still. After incubating 200 μl of this cell suspension at 37 ° C. for 5 minutes, the pyridopyridazine derivative L-012 obtained in Reference Example 7 or 8 was added to a concentration of 500 μM, and after 2 minutes, a stimulant was added to emit light. Measurement was started. When stimulated with fMLP100 nM, luminescence was observed immediately after stimulation and reached a peak within 1 minute and then immediately decreased. This emission is due to SOD (superoxide dis
mutase) is suppressed in a concentration-dependent manner by the addition of
Was almost completely suppressed by the addition of 1 ml / ml (Fig. 1). C5
a, fM even when stimulated by platelet activating factor (PAF)
As in the case of LP stimulation, it was found that luminescence was promptly observed after stimulation and suppressed by the addition of SOD ([FIG. 2] and [FIG. 3]).

[0030] Comparison O ₂ of Example 2 luminescent substrate ^- luminol, which is frequently used as a luminescent substrate, and the pyridopyridazine derivative L-012 Cypridina luciferin derivative (MCLA), was examined each optimal concentration. [Table 1] and [Table 2] show fMLP10 ^-7
The results of stimulating EoL-1 cells with M and detecting O ₂ ⁻ with various concentrations of luminescent substrate are shown below. MCLA is 10 ⁻⁶ M, L-012 is 5 × 10 ⁻⁴ M, It was found that luminol had the highest specific luminescence amount / background luminescence amount (S / N) ratio in the case of 1.25 × 10 ⁻³ M. Using these optimal concentrations of luminescent substrate, various concentrations of fM
When EoL-1 cells were stimulated with LP, C5a, PAF, etc. and compared, in all cases, the best detection sensitivity was exhibited when L-012 was used as a luminescent substrate ([FIG. 4], [FIG. 5]). And [Fig. 6]). Similar results were obtained when the cells were changed from EoL-1 cells to guinea pig eosinophils.

[Table 1]

[Table 2]

[0031]

According to the method of the present invention, the specific luminescence amount / background luminescence amount (S / N) ratio is higher than that of the conventional method, so that O ₂ ⁻ can be detected and quantified with high sensitivity. Therefore, since it can be applied to, for example, a method of detecting and quantifying O ₂ ⁻ derived from cells with extremely high sensitivity, it can be advantageously used for screening drugs that regulate O ₂ ⁻ production in various cells.

[Brief description of drawings]

FIG. 1 shows an O ₂ ⁻ production pattern and inhibition by SOD when E ₀ L-1 cells were stimulated with fMLP.

FIG. 2 O ₂ when E ₀ L-1 cells were stimulated with C5a
^- shows the inhibition by production patterns and SOD of.

FIG. 3 O ₂ when E ₀ L-1 cells were stimulated with PAF
^- shows the inhibition by production patterns and SOD of.

FIG. 4 shows the detection sensitivity of O ₂ ⁻ induced by various luminescent substrates when E ₀ L-1 cells were stimulated with fMLP.

FIG. 5 shows the detection sensitivity of O ₂ ⁻ induced by various luminescent substrates when E ₀ L-1 cells were stimulated with C5a.

FIG. 6 shows the detection sensitivity of O ₂ ⁻ induced by various luminescent substrates when E ₀ L-1 cells were stimulated with PAF.

[Explanation of symbols]

In FIG. 1, the symbols with Δ in black, Δ and ● indicate SOD 5 U / ml, SOD 300 U / ml and SOD control (0 U / ml), respectively. The marks marked with black in [Fig. 2], △ and ● are SOD 5U / ml and SOD, respectively.
300 U / ml and SOD control (0 U / ml) are shown. Marks in which [Delta] is black, [Delta] and ● in Fig. 3 indicate SOD 5U / ml, SOD 300U / ml, and SOD control (0U / ml), respectively. In FIG. 4, black, black and white marks indicate L-012, luminol and MCLA, respectively. The marks marked with black, ○ and △ in [Fig. 5] are L-012, luminol and MCL, respectively.
A is shown. In FIG. 6, black, black and white marks indicate L-012, MCLA and luminol, respectively.

Claims (8)

[Claims] 1. A superoxide radical (O ₂ ^-) and the following general formula (I) ## STR1 ## [In the formula, R ₁ represents an optionally substituted hydrocarbon group or a heterocyclic group, R ₂ represents hydrogen, a hydroxyl group, a thiol group, an amino group or a mono-substituted amino group, and R ₂ represents a mono-substituted amino group. In the case of a group, R ₂ together with R ₁ may form a ring. R ₃ is a hydrogen atom, an oxygen atom, an optionally substituted hydroxyl group, an optionally substituted amino group, an optionally substituted thiol group, a halogen atom,
A heterocyclic group, a nitro group, a cyano group, an optionally esterified or amidated carboxyl group, an azido group, a sulfo group or an organic sulfonyl group, provided that when R ₁ is an aliphatic group, R ₃ is hydrogen. Not an atom. X represents an oxygen atom or a sulfur atom], and chemiluminescence is caused by reacting with a pyridopyridazine derivative or a salt thereof. 2. The detection / quantification method according to claim 1, wherein X in the pyridopyridazine derivative represented by the general formula (I) or a salt thereof is an oxygen atom. 3. The detection / quantification method according to claim 1, wherein R ₁ in the pyridopyridazine derivative represented by the general formula (I) or a salt thereof is an optionally substituted phenyl group. 4. The detection / quantification method according to claim 1, wherein R ₂ in the pyridopyridazine derivative represented by the general formula (I) or a salt thereof is an amino group. 5. A pyridopyridazine derivative represented by the general formula (I) is represented by the following general formula (II): [In the formula, R ₁ represents an optionally substituted phenyl group, R ₂
Is a hydrogen atom or an amino group, and R ₃ is a hydrogen atom, an oxygen atom, an optionally substituted hydroxyl group, a halogen atom, or a heterocyclic group], which is a pyridopyridazine derivative. -Quantitative method. 6. In the pyridopyridazine derivative represented by the general formula (II), R ₁ is a phenyl group, R ₂ is an amino group, R ₃
Is a halogen atom, The detection / quantification method according to claim 5. 7. The detection / quantification method according to claim 1, wherein the superoxide radical is derived from leukocyte, vascular or nervous cells or cell lines thereof. 8. A cell such as a neutrophil, an eosinophil, a basophil, a monocyte, a macrophage, a mesangial cell, a vascular smooth muscle cell, a vascular endothelial cell, a nerve cell and a glial cell, or a cell thereof containing superoxide radical. It is derived from a cell line.
Detection and quantification method described.