JPH10338693A - Isatin derivative - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new derivative comprising a specific isatin derivative, useful as a criterion, etc. for highly sensitive ready determination of isatin in vivo, used for study of state of stress, development of an antidepressant agent, diagnosis of the state of the stress and depression, etc. SOLUTION: This isatin derivative is a new isatin derivative of formula I (R1 and R2 are each an alkoxy group), [e.g. 8,9-dimethoxy-5H-indolo(3,2-b)- quinoxalin-11-oxide] and used for quantitative determination of in vivo isatin(2,3- diketoindoline), which is an endogenous inhibiting material of a monoamine oxidase activity, increasing when being stressed. The compound is obtained by reacting 4,5-disubstituted-1,2-diaminobenzene with isatin dissolved in a small amount of ethanol. The determination of the isatin is carried out by reacting the isatin in a sample with a compound of formula II to derivatize the isatin to a compound of formula I and measuring the amount of the isatin by using fluorescence of the obtained compound as criterion.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel isatin derivative and a method for quantifying isatin using the isatin derivative.

[0002]

2. Description of the Related Art Isatin (2,3-diketoindoline)
Is one of endogenous monoamine oxidase (MAO) activity inhibitors that increase during stress [Minamikatsu et al .; Clinical Pharmacology, Vol.23, No.1, p.311-312 (1992), etc.]. Inhibition of in vivo MAO activity by isatin accumulates physiologically active amines such as catecholamines and serotonin in the brain and peripheral tissues, and induces physiological changes such as antidepressant action. 2. Description of the Related Art Isatin in a living body has been quantified in research, development of antidepressant drugs, diagnosis of stress conditions / depression, and the like. The method of isatin quantification currently used is the method of Naoya Hamagami et al. [Abstracts of the 22nd Pharmaceutical Activity Symposium, p.291-297 (P-11)]
And Glover et al. [J. Neurochem., 51 (2), p.
6-659 (1988)]. The method of Hamakami et al. Is a method of quantifying isatin without derivatizing isatin using high performance liquid chromatography (HPLC) using ultraviolet absorption at 240 nm as an index. Also, the method of Glover et al. Oximes isatin and separates it with a silica gel column.
This is a method of reacting with N- (tert-butyldimethylsilyl) trifluoroacetamide to form tert-butyldimethylsilyl (TBDMS), and quantifying the volatile isatin derivative of the reaction product by gas chromatography-mass spectrometry (GC-MS).

[0003]

Although the method of Hamakami et al. Is a simple quantitative method, the disadvantage is that the tautomerism (lactam type and lactim type) of isatin causes broadening of the peak and lowers the resolution. In addition to 240n
The ultraviolet absorption of m has a problem that it has poor specificity and insufficient sensitivity because there are many compounds having ultraviolet absorption in the vicinity of m. Although the method of Glover et al. Is capable of accurate quantification, it involves an extraction operation in addition to a two-step derivatization reaction, and is extremely complicated and requires a long time for analysis. And it is not practical. Furthermore, GC-MS equipment used for analysis is very expensive, is not always provided in a laboratory, and has a problem in terms of analysis cost. The present inventors have intensively studied a more practical isatin quantification method in order to solve the above problems, and have found a novel fluorescent derivative of isatin. According to the present invention, for example, when quantifying isatin in a sample such as a brain extract containing other multicomponents, blood, and urine, isatin is used as an index using the fluorescence of the isatin derivative of the present invention as an index using an HPLC system. Specific quantification is possible, and very sensitive, simple and practical quantification of isatin is possible.

[0004]

An object of the present invention is to provide a novel isatin derivative.

[0005]

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel isatin derivative represented by the following general formula (I) and a method for quantitatively determining isatin using the isatin derivative.

Embedded image [Wherein, R ₁ and R ₂ are the same or different and each represents an alkoxyl group. ]

In the above general formula (I), R represents a linear or branched carbon number such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentoxy, isopentoxy, neopentoxy, etc. Represents 1 to 5 alkoxyl groups.

The isatin derivative of the present invention can be produced, for example, by the following method. The following general formula (II):

Embedded image [Wherein, R ₁ and R ₂ are the same or different and each represents an alkoxyl group. The compound represented by the general formula (I) is dissolved by dissolving the compound represented by the formula (I) together with isatin to be reacted in a water-soluble organic solvent such as water or alcohol or a suitable solvent which does not inhibit the reaction such as a mixed solvent thereof, and subjecting it to a condensation reaction. Can be produced. The reaction solvent is preferably an acidic aqueous solution in order to suppress by-products. During the condensation reaction, a suitable antioxidant such as 2-mercaptoethanol may be added for the purpose of preventing the compound represented by the general formula (II) from being oxidized. The condensation reaction is carried out, for example, when using 4,5-dimethoxy-1,2-diaminobenzene (DDB) at room temperature for about 2 hours.
The condensation reaction is completed in a short time, but a longer reaction time is required when an antioxidant such as 2-mercaptoethanol is added. The reaction temperature is 37 to suppress by-products.
The reaction is preferably carried out at a temperature of not more than ℃, particularly preferably at room temperature.

The compound represented by the general formula (II) is, for example, hydrochloric acid, sulfuric acid, nitric acid, hydrobromic acid, phosphoric acid, perchloric acid, thiocyanic acid, boric acid, formic acid, acetic acid, haloacetate, propionate Acids with acids, glycolic acid, citric acid, tartaric acid, succinic acid, gluconic acid, lactic acid, malonic acid, fumaric acid, anthranilic acid, benzoic acid, cinnamic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, sulfanilic acid, etc. And addition salts. These salts can be produced from the compound represented by the general formula (II) by a known method, or can be mutually converted.

The isatin derivative of the present invention represented by the general formula (I) and the compound represented by the general formula (II) include hydrates and complex compounds thereof.

By using the isatin derivative of the present invention as an index, isatin can be quantified more simply and specifically than the conventional method. For example, the isatin derivative of the present invention represented by the general formula (I) produced by adding the compound represented by the general formula (II) to a sample whose isatin content is unknown and reacting with the isatin in the sample Can be quantified by using an analytical instrument such as an HPLC system using the fluorescence of the above as an index. For example, when isatin in a sample is quantified using DDB, the fluorescence (Ex340 nm, Em415) of Compound 1 described below generated by the reaction between DDB and isatin
nm) as an index.

Preferred embodiments of the present invention are described below. (1) An isatin derivative represented by the general formula (I), wherein R ₁ and R ₂ are methoxy groups. (2) A method for quantifying isatin by reacting isatin with the compound represented by the general formula (II) or a salt thereof and derivatizing the compound to the compound represented by the general formula (I) for measurement. A method for quantifying isatin using the fluorescence of the derivative as an index. (3) The method for quantifying isatin according to (2), wherein an antioxidant is added during the reaction. (4) The method for quantifying isatin according to (3), wherein the antioxidant is 2-mercaptoethanol. (5) The method for quantifying isatin according to (2) to (4), wherein the isatin derivative represented by the general formula (I) is quantified by an HPLC system. (6) The method for quantifying isatin according to (2) to (5), wherein the isatin derivative represented by the general formula (I), wherein R ₁ and R ₂ are methoxy groups, is used as an index.

[0012]

EXAMPLES Examples of the present invention will be shown below, but the present invention is not limited by these examples. In the following Examples, the melting point was measured by placing a sample in a glass capillary and using a melting point measuring device (MP-21, manufactured by YAMATO). The fluorescence spectrum was 650-40 manufactured by Hitachi.
It was measured using a mold. B for measuring UV absorption spectrum
ECKMAN DU-650 type was used. The mass spectrum was measured by ionization using the Hitachi M-80B type by the EI method. Nuclear magnetic resonance (NMR) spectra were dissolved sample of DMSO-d ₆ (containing 0.05% of tetramethylsilane as an internal standard), Burker Co. AR
It measured using X-500.

Example (1) Synthesis of isatin derivative 29 dissolved in 482 mg of 4,5-dimethoxy-1,2-diaminobenzene (DDB) and a small amount of ethanol
4 mg of isatin was dissolved in 50 mL of 0.5N hydrochloric acid. After stirring overnight at room temperature, the resulting precipitate was collected by filtration.
The precipitate was dried, dissolved in ethyl acetate, applied to a silica gel column (10 g) and eluted with ethyl acetate. The eluate was dried and recrystallized from ethanol to obtain 372 mg of 8,9-dimethoxy-5H-indolo (3,2-b) -quinoxaline 11-oxide (compound 1) as colorless needles. Melting point: 333-334 ° C (decomposition) Elemental analysis: as C ₁₆ H ₁₃ N ₃ O ₃ Theoretical value: (C = 65.08, H = 4.44, N = 14.23); Measurement value: (C = 65.
25, H = 4.37, N = 14.03) λ _max (EtOH): 233, 338, 352 (sh) Mass: 295 (M +), 280 (M + -Me), 252 (M + -COMe), 237 (M +
-COMe-Me) ¹ H-NMR (DMSO-d ₆ ): 3.88 (3H, s, -OMe), 3.89 (3H, s, -OMe),
7.35 (1H, t), 7.39 (1H, d), 7.44 (1H, s), 7.60 (1H, t),
7.89 (1H, s), 8.24 (1H, d), 11.91 (1H, s, -NH) 13 C-NMR (DMSO-d 6): 55.85 (q), 55.92 (q), 97.49 (d), 10
1.51 (d), 112.04 (s), 115.80 (d), 123.26 (d), 123.65
(d), 124.00 (s), 131.30 (d), 136.34 (s), 137.24 (s), 1
46.00 (s), 146.35 (s), 147.09 (s), 148.18 (s)

[0014] (2) according to isatin derivatives DDB quantitative 12.05mg of isatin was added 2-mercaptoethanol 490μL was dissolved in 0.5N hydrochloric acid 10mL the DDB reagent 0.5mL, prepared, 5 × 10 ^-14 ~ 5x
Isatin aqueous solutions prepared at various concentrations of 10 ⁻¹⁰ M.
5 mL each was added, and the mixture was stirred at 50 ° C. for 3 hours. 10 μL of each reaction solution was measured using an HPLC system, and the fluorescence (Ex340 nm, Em415n) of Compound 1 was measured.
Isatin in the sample was quantified using m) as an index. In addition, HP
The LC system uses 900 series manufactured by JASCO Corporation,
FP-920 was used as a fluorescence detector. Column is YMC
J'sphere ODS-M80 (4.6 × 1
(50 mm) and eluted with a 65% methanol / 50 mM phosphate buffer solution (pH = 6.5, flow rate 0.8 mL / min). One example of the result is shown in FIG.

FIG.

(3) Quantification of Isatin in Rat Brain Extract [Watkins et al., Neurochem. Int., 17, p.321]
-323 (1990)]. First, the rat brain extract was directly measured by an HPLC system, but no fluorescence peak appeared. Next, a DDB reagent was added to the rat brain extract to cause a reaction, and the reaction product was separated by an HPLC system using fluorescence as an index. As a result, a strong fluorescence peak was observed near the fluorescence peak of Compound 1 described above. As a result of comparing the physical property values of the collected fluorescent compound and Compound 1, the two were in good agreement with each other, indicating that they were the same compound. Furthermore, when a standard isatin was added to the rat brain extract and reacted with a DDB reagent and measured, it was confirmed that the height of the fluorescence peak was increased corresponding to the amount of isatin added to the standard. In addition, the results of quantifying isatin using the compound of the present invention were in good agreement with the results of quantifying isatin by the above-described method of Glover et al. Using GC-MS, which requires complicated operations.

[0016]

As described above, the reaction required for carrying out the measurement method of the present invention is only one step of the derivatization reaction with a simple operation, and can be quantitatively determined by an HPLC system, so that it is very simple and practical. Is a typical method. As for the sensitivity, as shown in FIG. 1, the detection limit of the isatin derivative in the measurement method of the present invention was 5 × 10 ⁻¹³ mole, which was extremely high. Also, good linearity was observed up to 1 × 10 ⁻⁹ mole between the fluorescence intensity of the isatin derivative of the present invention and the isatin concentration, and the coefficient of variation was 1.26% (2.5 × 10
At the time of the ^-12 mole reaction, n = 9) and the reproducibility was also good. Further, as shown in Example (3), accurate measurement was possible even when measuring a sample containing other multicomponents such as a brain extract. Therefore, by quantifying the isatin derivative of the present invention as an index, the above-mentioned various problems of the prior art can be overcome, and the quantification of isatin can be performed with high sensitivity, simply, and practically. For example, even when quantifying isatin in a sample such as a brain extract containing other multiple components, blood, urine, etc., specifically quantifying isatin using the fluorescence of the isatin derivative of the present invention as an index using an HPLC system Thus, a very sensitive, simple and practical method for quantifying isatin is provided.

[Brief description of the drawings]

FIG. 1 is an example of a calibration curve for isatin quantification using the fluorescence of the isatin derivative of the present invention as an index.

Continuation of the front page (51) Int.Cl. ⁶ Identification symbol FI G01N 33/52 G01N 33/52 C (72) Inventor Tsutomu Yamamoto 442-1, Kawakitayama, Kinashiri, Kashiguna-cho, Hyogo Japan Nippon Organ Pharmaceutical Co., Ltd. Activity Science Institute

Claims (2)

[Reference number] PC-266 [Claims] 1. An isatin derivative represented by the following general formula (I). Embedded image [Wherein, R ₁ and R ₂ are the same or different and each represents an alkoxyl group. ] 2. A method for quantifying isatin by reacting isatin with a compound represented by the following general formula (II) or a salt thereof, derivatizing the compound according to claim 1, and measuring the derivative. Embedded image [Wherein, R ₁ and R ₂ are the same or different and each represents an alkoxyl group. ]