JPS63135862A - Benzo (c) pyrazolo (1:2-i a-) pyrazol-1,9-dione derivative and making thereof - Google Patents

Abstract

PURPOSE:To manufacture a benzo [c] pyrazolo [1:2-a] pyrazol-1,9-dione derivative as given by another specified formula, by making a compound as given by a specified formula react with a bromide reagent in a anhydride organic solvent. CONSTITUTION:A compound of the formula I is treated with a normal bromide reagent, namely, N-bromosuccinate imide, N-bromocaprolactam, N- bromophthalimide in an anhydride organic solvent to manufacture a compound of the formula II, namely, a benzo [c] pyrazolo [1:2-a] pyrazol-1,9-dione derivative.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides novel benzo(c)pyrazolo[1:2-α]
This invention relates to a p2zole-1,9-dione derivative, its production method, and a fluorescent reagent.

Conventionally, for the purpose of adding fluorescent molecules to thiol groups in proteins, peptides, and amino acid molecules, maleimide-type compounds such as N-acrinidylmaleimito and N-C1)-(2-benzimidazolyl)phenyl]maleimide have been used. reagents,
Dansylaciridine, ammonium 7-fluorobenzo-2-oxa-1,6-diacyl-4-sulfonate,
Monopromobima/(3-bromomethyl-2,
5,6-drimethylpyrazolo (1:2-α]pyrazole-1,7-dione) and the like have been used.

However, maleimide-type reagents have the disadvantage that the fluorescent label is unstable, and because they involve the opening of the maleimide ring and the ring-closing with other moieties after ring-opening, the chromatogram shows that the fluorescent label is unstable. The drawback is that it is difficult to obtain a peak, and dansyl acylidine and 7-fluoropene/-2-oxa-1,5-diazole-4-sulfone m7mmonium are reacted with thiol groups at 60°C. Since it requires harsh conditions of 1 hour, it has the disadvantage that it can lead to denaturation of proteins, etc., and cannot be used for biopolymers. Furthermore, conventional reagents such as monopromobimane have insufficient sensitivity.

As a result of various studies in order to solve the problems with the conventional reagents, the present inventor has developed a new benzene according to the present invention [
c] Pyrazolo[1:2-0]pyrazole-1,9-dione derivatives have been found to have excellent properties in various aspects as a fluorescent reagent for thiol compounds. The present invention is based on this knowledge.

That is, the present invention is based on the general formula (RI: [wherein R1 represents an alkyl group, phenyl group, halogen, or a hydrogen atom; The present invention provides a compound represented by:

The compound of general formula (I) has the general formula (transformed): [wherein represents an alkyl group, a phenyl group, a halogen, or a hydrogen atom, and R2 and R3 are an alkyl group, a methoxy group, and a nitro group, respectively. Or represents a hydrogen atom. ] It can be obtained by treating the compound represented by with a conventional brominating reagent in an anhydrous organic solvent. The brominating reagent is preferably used for N-bromosuccinimide, N-bromocaprolactam, and N-bromo 2-talimide. As the reaction solvent, organic solvents normally used in reactions using brominating reagents, such as methylene chloride, chloroform, benzene, and carbon tetrachloride, can be used. These organic solvents are used in anhydrous form.

In the above bromination reaction, it is desirable to use a peroxide such as benzoyl peroxide as a catalyst.

The compound of the above general formula (capital) is β-acetenol, -0
~A method of reacting carboxyphenylhydrazine with ethyl acetoacetate in dichloride trichloride (S.

Veibel and H, Lielund, T
etrahedron+ 1 + 201 (1957
)) or by reacting 3-indazolinone with ethyl acetoacetate and silicon tribromide in benzene (E., M., Koscrwerand et al.
B, pazhenchevsky, 102.4983
(1980)). However, in the method of S. Veibel et al., the corresponding raw material β
The disadvantage is that the synthesis of -acetyl-〇-carboxylic diphenylhydrazine derivatives requires more steps and that unstable intermediates must be handled during the process. The yield is 8% with this method.
It is too low and not practical.

The present inventor has developed a compound of the general formula @) [wherein R2 and R3 each represent an alkyl group, a methoxy group, a nitro group, or a hydrogen atom. 5-indazolinone rust conductor represented by the general formula (n), R in the absence of a solvent.

[In the formula, R1 represents an alkyl group, a phenyl group, a halogen, or a hydrogen atom, and R4 represents an alkyl group. ] It has been found that the compound of the general formula (2) can be obtained in high yield by reacting with β-[ester] and a syntrihalide.

Therefore, the present invention provides a compound of the general formula (II) in a high yield by reacting the compound of the general formula (II) with a trihalide and a compound of the general formula (2) in the absence of a solvent. The present invention also provides a method for producing the compound (conversion).

Specific examples of the amount of each reactant used, reaction time, reaction temperature, etc. in each of the above reactions will be explained in Examples below, but these various conditions can be changed as appropriate.

The novel compound according to the present invention has the following properties.

1. Reacts with thiol group under mild conditions to give a single reaction product.

2. The reaction is selective and sensitive, and trace amounts of thiol groups can be detected with a sensitivity ten to several hundred times higher than that of conventional reagents.

6. Not only can low-molecular-weight thiol compounds be quantitatively analyzed, but also thiol groups in proteins, which are biopolymers, can be detected.

4. The fluorescence produced by bonding with thiol groups is in the visible region with a long wavelength of 450 nm or more, and can be clearly distinguished from the fluorescence of tryptophan and tyrosine in protein backbones. Taking the glutathione reaction product as an example, in the case of excitation light of 405 nm, the quantum yield in water is Q, 55, and the maximum value of fluorescence is 48037 FI.

5. The solubility of the reagent in water is better than that of other fluorescent dyes.

Therefore, the novel compound represented by the general formula (RI) according to the present invention can be used as a fluorescent reagent for detecting a thiol group.

Thus, the present invention provides a fluorescent reagent for detecting a thiol group, which contains a compound of general formula (1) as an active ingredient.

This active ingredient can be used alone, but
Preferably, the reagent is dissolved in an aprotic organic solvent. Since the presence of a buffer solution in the compound of general formula (1) brings about good results in the reaction with the test sample, it is preferable to add a buffer solution to the reagent.

Specific examples of the aprotic organic solvent include acetonitrile, acetone, dihydrofuran, dioxane, etc. Among these, acetonitrile is most suitable. The type, concentration, and pH of the buffer are not particularly limited, but α01-O, IM, 9-phosphate buffer (pH 7)
, 5-al) are available at i&M.

The fluorescent reagent according to the invention specifically reacts with thiol groups. It is desirable to use the fluorescent reagent in an excess amount relative to the test sample to cause a sufficient reaction. Specific examples of these will be explained in Examples below.

In measuring the actual elimination, the sample is reacted with the test sample in advance, subjected to high performance liquid chromatography, and the fluorescence of the resulting disulfide is detected with a fluorophotometer. When using a glutathione test sample, the detection sensitivity was found to be 10 fmol (S/N = 3) using a reverse phase system of high performance liquid chromatography.

The detection limit of conventional reagents is 10100f#-10ptntr
1, it can be seen that the fluorescent reagent according to the present invention has a sensitivity ten to several hundred times higher. Further, the fluorescent wavelength of the reaction product was 480 nm (maximum excitation wavelength 405 wm) in the case of glutathione. The type and size of the column, the composition of the mobile phase, the flow rate, etc. used in the examples are merely examples, and these various conditions can be arbitrarily selected for implementation.

The present invention will be explained in further detail by the following examples.

Example 1 2.5-dimethyl-benzo(C)pyrazolo[1:2-α
]Synthesis of virazole-1,9-dione 3-indazolinone (2P) was converted into ethyl 2-methylacetoacetate (415')
The mixture was mixed and stirred, and to this was added silicon tribromide (8?), and after heating and stirring at 100° C. for 1 hour, the reaction product was pressurized into ice water and extracted with methylene chloride. After washing the extract with water and drying,
The product was recrystallized from acetonitrile under reduced pressure to obtain yellow needle-like crystals (1,95').

Based on the following physical properties, the obtained compound was confirmed to be the title compound.

Melting point: 224-226°C IR(KBr) vmax: 1745 and 167
0m-' (C=O)MSrn/e: 214
CM”) NMR (CDCls): 1.85 (
,9,3H), 2.50(11,3H), 6.95
7.95° C.m, 4H) Each compound represented by the general formula (1) is produced according to this example using the corresponding 3-indazolinone spinner and β-keto acid ester derivative as raw materials.

Example 2 Synthesis of 5-bromomethyl-2-methyl-benz[c]pyrazolo[1:2-α]pyrazole-1,9-dione 2,6-dimethyl-penzo[c)pyrazolo(1: 2-α]pyrazole-1,9-
Dione (3y-) and N-bromo amber imide (2,6
F) was stirred and mixed in 40 ml of methylene chloride, and to this,
3! After adding benzoyl oxide (SMP) A, stir at room temperature for 1 hour. The reaction solution was washed with water, dried, concentrated under reduced pressure, and recrystallized from ethyl acetate to give yellow needle-like crystals (41
) was obtained.

Based on the following physical properties, the obtained compound was confirmed to be the title compound.

Melting point: 192-194℃ UV (CH3CN): 243im (t 1940
0), 300℃m (t7100), 595℃m (
ε6600) IRCKBr) I/mW: 1750
and 1670 cm-” (C=0) MS nv/e
: 292(M”) and 213(M+-Br)NM
R(CDCls): 1.93 (8,3H), 4.
40 (st2H) + 7.00-7.90 (?FL,
4f() Each compound represented by the general formula (1) is the corresponding benzo(C)pyrazolo(1:2-a,)pyrazole-
It is manufactured according to this example using a 1,9-dione rust conductor as a raw material.

Ability 1 Example 3 Solutions of each thiol compound listed in Table 1 below (α1m
M) α05M for 1 m/m4) Phosphate buffer (pH=8.
1) 1 ml and 6-bromomethyl-2-methyl-benzCC]hirazolo (1:
2a,) Add 1 me of pyrazole-1,9-dione acetonitrile solution (1 mM) and incubate for 10 min at 20°C.
Allowed to react for minutes. After the reaction was completed, the mixture was passed through a C11nEhtt cartridge to remove excess reagents, and the relative fluorescence intensity was measured using a spectrofluorophotometer at an excitation wavelength of 4051 m and a fluorescence wavelength of 480 nm. Table 1 shows the results. As seen in Table 1, it can be seen that the compound according to the present invention reacts with various thiol compounds to give a highly luminescent product, and is suitable for highly sensitive detection of thiol compounds.

Table 1 Actual h1 value when set to 100 Example 4 Glutathione, N-aceterome-cysteine, L-cysteine, L-cysteine ethyl ether chloride (each 1-6μf) was used as the test sample, 0.025M
, dissolved in 100 μJ of 0 phosphate buffer (pH = a1).
Add 100 μl of a 1 mM acetonitrile solution of pyrazolo (1:2-cL) pyrazole-1,9-dione and
C. for 10 minutes, and the fluorescence was measured after the reaction was completed.

In the measurement, the generated disulfide was separated by high performance liquid chromatography, and the excitation wavelength was 405 nmK and the fluorescence wavelength was 480 nm. This was carried out by measuring the fluorescence of Lm, which was further determined using an integrating needle.

The high performance liquid chromatograph equipment and measurement conditions used were as follows: Equipment: Hitachi High Performance Liquid Chromatograph 63B-50 type Detector: Hitachi Spectrofluorophotometer 650-10 S type column:
LiChrosorb RP 18 (10μm)
4 wp 10%/fnin Flow rate: 1 ml/fnin An example of the measurement results is shown in FIG. As seen in FIG. 1, various thiol compounds were detected with high sensitivity and clearly separated. In addition, in Example 4, as an example of the detection limit, 10 fmol (S/
'N = 3) was observed.

[Brief explanation of the drawing]

FIG. 1 shows a high-performance liquid chromatogram obtained in Example 4 using a spectrofluorophotometer. In the figure, 1.2 and 3.4 indicate the following test samples. 1, : Glutathione 2, : N-Acetyl-L-7 Stain 6, : L-Cystine 4, : L-Cystine ethyl ester Figure 1 Time (min)

Claims (1)

[Claims] 1) General formula (I): ▲There are mathematical formulas, chemical formulas, tables, etc.▼ [In the formula, R_1 represents an alkyl group, phenyl group, halogen atom, or hydrogen atom, and R_2 and R_3 are Each represents an alkyl group, a methoxy group, an etro group or a hydrogen atom. ] A benzo[c]pyrazolo[1:2-α]pyrazole-1,9-dione compound represented by: 2) Whether R_1 is an alkyl group having 1-4 carbon atoms,
or a hydrogen atom, the compound according to claim 1. 3) The compound according to claim 2, wherein R_2 and R_3 are both hydrogen atoms. 4) General formula (I): ▲ Numerical formulas, chemical formulas, tables, etc. , represents a nitro group or a hydrogen atom. ] A fluorescent reagent for detecting a theol group, which contains a benzo[c]pyrazolo[1:2-a]pyrazole-1,9-dione derivative represented by the following as an active ingredient. 5) Benzo[c]pyrazolo[1:2-a] where R_1 is an alkyl group having 1-4 carbon atoms or a hydrogen atom
The fluorescent reagent according to claim 4, which contains a pyrazole-1,9-dione derivative as an active ingredient. 6) Benzo [c] where R_2 and R_3 are both hydrogen atoms
The fluorescent reagent according to claim 5, which contains a pyrazolo[1:2-α]pyrazole-1,9-dione derivative as an active ingredient. 7) General formula (M: ▲There are mathematical formulas, chemical formulas, tables, etc.▼ [In the formula, R_1 represents an alkyl group, phenyl group, halogen atom, or hydrogen atom, and R_2 and R_3 represent an alkyl group and a methoxy group, respectively. , a nitro group, or a hydrogen atom.] is characterized by reacting a benzo[c]pyrazolo[1:2-α]pyrazole-1,9-dione derivative represented by the following with a brominating reagent in an anhydrous organic solvent. General formula (I): ▲ There are mathematical formulas, chemical formulas, tables, etc. 8) General formula (II): ▲ Numerical formula, chemical formula, table, etc. ▼ [In the formula, R_1 represents an alkyl group, phenyl group, halogen atom, or hydrogen atom, and R_4 represents an alkyl group. ] In the absence of a solvent, β-keto acid ester represented by phosphorus trihalide and general formula (III): ▲ mathematical formula, chemical formula,
There are tables, etc. ▼ [In the formula, R_2 and R_3 are an alkyl group,
Represents a methoxy group, a nitro group, or a hydrogen atom. ] General formula (IV) characterized by reacting with a 3-indazolinone derivative represented by: ▲ Numerical formulas, chemical formulas, tables, etc. . ] A method for producing a benzo[c]pyrazolo[1:2-α]pyrazole-1,9-dione derivative represented by: 9) The manufacturing method according to each of claims 7 to 8, wherein the brominating reagent is N-bromosuccinimide, N-bromocaprolactam, or N-bromophthalimide. 10) The production method according to claim 9, wherein a peroxide is used as a catalyst in the reaction with the brominating reagent.