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Diaminorhodamine derivative

a technology of diaminorhodamine and derivative, applied in the field of diaminorhodamine derivative, can solve the problems of inability to use physiological conditions for detection and quantification of nitric oxide, inability to develop specific and highly sensitive detection methods that can be applied to in vitro systems, and inability to achieve physiological conditions. detection and quantification methods, the effect of constant fluorescence intensity and improved fluorescence intensities

Inactive Publication Date: 2008-05-08
SEKISUI MEDICAL CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The rhodamine derivatives enable accurate and sensitive measurement of nitric oxide with high fluorescence intensity, unaffected by pH fluctuations and autofluorescence, enabling precise detection and quantification even in physiological conditions.

Problems solved by technology

However, any specific and highly sensitive detection method that can be applied to in vitro systems has not been developed so far.
However, this method does not utilizes nitric oxide, per se, but its oxidation product NO2− as the reaction species, and accordingly, the method is rather indirect as compared to the direct methods for measuring nitric oxide.
In addition, since the reaction of 2,3-diaminonaphthalene and NO2− is performed under a strongly acidic condition (pH 2 or lower), it has a problem that the method cannot be used for detection and quantification of nitric oxide under a physiological condition.
However, the aforementioned method utilizing 2,3-diaminonaphthalene needs irradiation by excitation light of a short wavelength such as about 370 to 390 nm for the detection of fluorescence, and accordingly, cells and tissues in a measurement system may possibly be damaged.
The method also has a problem in that strong autofluorescence of cells may affect the measurement.
Moreover, the fluorescent triazole compound produced from 2,3-diaminonaphthalene does not necessarily have sufficient fluorescence intensity, and for this reason, it is difficult to accurately measure fluorescence in individual cells by using conventional fluorescence microscopes.
There is also a problem in that 2,3-diaminonaphthalene itself has a relatively simple chemical structure and is not suitable as a fundamental structure for various chemical modification so as to be localized inside of cells.
However, since a part of fluorescence wavelength range of the triazole derivatives (hereafter also referred to as “DAF-T”) that are produced by the reaction of DAF with nitric oxide overlaps with the autofluorescence range of cells, the method may sometimes fail to accurately measure nitric oxide in certain types of samples.
Further, since the fluorescence of DAF-T may be attenuated from weakly acidic to acidic region, a problem also arises in that accurate measurement over a wide pH range cannot be conducted.
However, in the method utilizing DAR, fluorescence intensity of some DAR-T may sometimes slightly fluctuate depending on pH, and a problem arises that accurate measurement cannot be performed when a sample is measured whose pH is possibly fluctuate during measurement, e.g., a tissue of a patient with an ischemic disease.
There is also a problem that DAR-T has lower fluorescence intensity as compared to the class of DAF-T.

Method used

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Examples

Experimental program
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Effect test

example 1

Preparation of 3,6-bis diethylamino)-9-[3-amino-4-(N-methylamino)-2-carboxyphenyl]xanthylium intramolecular salt (DAR-M)

[0034]DAR-1 [3,6-bis(diethylamino)-9-[3,4-diamino-2-carboxyphenyl]xanthylium intramolecular salt] prepared by the method disclosed in WO99 / 01447 (231 mg) was dissolved in 10 ml of ethanol and added with 50 μl of methyl iodide (1.7 equivalents based on DAR-1), and the temperature of the reaction mixture was raised to 80° C. While the degrees of disappearance of the starting material and production of a dimethyl compound were observed for every hour using thin layer chromatography (TLC), the mixture was added with additional 50 μl of methyl iodide. The reaction was terminated when the desired compound was produced. The product was purified by silica gel column chromatography and preparative TLC to obtain the title compound (25 mg, 11%).

[0035]m.p. 150-154° C.

[0036]1H-NMR (300 MHz, CDCl3) δ 1.13 (12H, t, J=7.0), 2.86 (3H, s), 3.33 (8H, q, J=7.0), 6.37-6.43 (5H, m), 6.7...

example 2

Preparation of 3,6-bis(diethylamino)-9-(4-carboxy-1-methylbenzotriazol-5-yl)xanthylium intramolecular salt (DAR-MT)

[0038]DAR-M (8.0 mg) obtained in Example 1 was dissolved in methanol and the solution was bubbled with nitric oxide, and then the solvent was evaporated. The residue was purified by preparative TLC to obtain DAR-MT.

[0039]m.p. 155-160° C.

[0040]1H-NMR (300 MHz, CDCl3) δ 1.12 (12H, t, J=7.1), 3.32 (8H, q, J=7.1), 4.37 (3H, s), 6.31 (2H, dd, J=9.0, 2.5), 6.43 (2H, d, J=2.5), 6.58 (2H, d, J=9.0), 7.26 (1H, d, J=8.6), 7.83 (1H, d, J=8.6)

[0041]FAB-MS 498 (M++1)

example 3

Preparation of 3,6-bis(dimethylamino)-9-[3-amino-4-(N-methylamino)-2-carboxyphenyl]xanthylium intramolecular salt (DAR-4M)

[0042]In the same manner as in Example 1, the title compound was obtained (140 mg, 24.5%) from 553 mg of DAR-4 [3,6-bis(dimethylamino)-9-[3,4-diamino-2-carboxyphenyl]xanthylium intramolecular salt] which was prepared by the method disclosed in WO99 / 01447.

[0043]m.p. 219° C.

[0044]1H-NMR (300 MHz, CDCl3) δ 2.92 (3H, s), 2.97 (12H, s), 4.99 (2H, br), 6.40 (2H, dd, J=8.6, 2.6), 6.47 (2H, d, J=2.6); 6.47 (2H, d, J=8.0), 6.75 (2H, d, J=8.6), 6.85 (1H, d, J=8.0)

[0045]FAB-MS 431 (M++1)

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Abstract

A compound represented by the following formula (I) or (II):wherein, in the formula (I), R1, R2, R3, and R4 independently represent methyl group or ethyl group; and in the formula (II), R5, R6, R7, and R8 independently represent methyl group or ethyl group and X— represents an anion, and a reagent for measurement of nitric oxide which comprises said compound.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application is a continuation of application Ser. No. 10 / 744,007, filed on Dec. 24, 2003, which is a divisional of application Ser. No. 09 / 641,350, filed Aug. 18, 2000, which are hereby incorporated by reference in their entireties.TECHNICAL FIELD[0002]The present invention relates to a rhodamine derivative useful as a reagent for measurement of nitric oxide, and a reagent for nitric oxide measurement that comprises said compound.BACKGROUND ART[0003]Nitric oxide (NO) is an unstable radical species of a short life, and has been elucidated to have important functions as a physiological active substance in a living body (Chemistry Today [Gendai Kagaku], April, 1994, Special Edition; Pharmacia, May, 1997, Special Edition). Methods for measuring nitric oxide are roughly classified into indirect methods, which measure NO2□ and NO3□ as oxidative degradation products of nitric oxide, and methods based on direct measurement of nitric oxide. T...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): G01N33/00G01N37/00C07D519/00C07D311/90C07D405/04G01N21/64
CPCC07D311/90C07D405/04C07D491/20C07D493/10G01N21/6428Y10T436/17Y10T436/177692Y10T436/179228Y10T436/10Y10T436/141111G01N2021/6439
Inventor NAGANOKOJIMA, HIROTATSUKIKUCHI, KAZUYA
Owner SEKISUI MEDICAL CO LTD