U.S. patent application number 11/757853 was filed with the patent office on 2008-05-08 for diaminorhodamine derivative.
This patent application is currently assigned to Tetsuo Nagano. Invention is credited to Kazuya KIKUCHI, Hirotatsu KOJIMA, Tetsuo NAGANO.
Application Number | 20080108141 11/757853 |
Document ID | / |
Family ID | 32508345 |
Filed Date | 2008-05-08 |
United States Patent
Application |
20080108141 |
Kind Code |
A1 |
NAGANO; Tetsuo ; et
al. |
May 8, 2008 |
DIAMINORHODAMINE DERIVATIVE
Abstract
A compound represented by the following formula (I) or (II):
##STR00001## wherein, in the formula (I), R.sup.1, R.sup.2,
R.sup.3, and R.sup.4 independently represent methyl group or ethyl
group; and in the formula (II), R.sup.5, R.sup.6, R.sup.7, and
R.sup.8 independently represent methyl group or ethyl group and X--
represents an anion, and a reagent for measurement of nitric oxide
which comprises said compound.
Inventors: |
NAGANO; Tetsuo; (Tokyo,
JP) ; KOJIMA; Hirotatsu; (Tokyo, JP) ;
KIKUCHI; Kazuya; (Kanagawa, JP) |
Correspondence
Address: |
GREENBLUM & BERNSTEIN, P.L.C.
1950 ROLAND CLARKE PLACE
RESTON
VA
20191
US
|
Assignee: |
Nagano; Tetsuo
Tokyo
JP
DAIICHI PURE CHEMICALS CO., LTD.
Tokyo
JP
|
Family ID: |
32508345 |
Appl. No.: |
11/757853 |
Filed: |
June 4, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10744007 |
Dec 24, 2003 |
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11757853 |
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09641350 |
Aug 18, 2000 |
6756231 |
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10744007 |
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Current U.S.
Class: |
436/8 ; 436/116;
548/256; 549/265 |
Current CPC
Class: |
C07D 311/90 20130101;
C07D 493/10 20130101; Y10T 436/141111 20150115; Y10T 436/10
20150115; Y10T 436/179228 20150115; C07D 491/20 20130101; G01N
21/6428 20130101; G01N 2021/6439 20130101; C07D 405/04 20130101;
Y10T 436/17 20150115; Y10T 436/177692 20150115 |
Class at
Publication: |
436/8 ; 436/116;
549/265; 548/256 |
International
Class: |
G01N 33/00 20060101
G01N033/00; G01N 37/00 20060101 G01N037/00; C07D 519/00 20060101
C07D519/00 |
Claims
1. A compound represented by the following formula (I)':
##STR00007## wherein R.sup.1, R.sup.2, R.sup.3, and R.sup.4
independently represent methyl group or ethyl group.
2. The compound of the formula (I)' according to claim 1 wherein
R.sup.1, R.sup.2, R.sup.3, and R.sup.4 are methyl groups.
3. A reagent composition for measurement of nitric oxide which
comprises a compound represented by the formula (I)' according to
claim 1.
4. A compound represented by the following formula (III)':
##STR00008## wherein R.sup.11, R.sup.12, R.sup.13, and R.sup.14
independently represent methyl group or ethyl group.
5. The compound of the formula (III) according to claim 4, wherein
R.sup.11, R.sup.12, R.sup.13, and R.sup.14 are methyl groups.
6. A method for measurement of nitric oxide, which comprises: (1) a
step of reacting a compound represented by the formula (I)'
according to claim 1 with nitric oxide, and (2) a step of detecting
a compound represented by the formula (III)' having the following
formula produced in (1). ##STR00009##
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
NO.sub.2.sup..quadrature. and NO.sub.3.sup..quadrature. as
oxidative degradation products of nitric oxide, and methods based
on direct measurement of nitric oxide. The direct methods have been
desired from viewpoints of detection and quantification of nitric
oxide under physiological conditions. However, any specific and
highly sensitive detection method that can be applied to in vitro
systems has not been developed so far.
[0004] As typical methods, there are known, for example, the
chemiluminescence method utilizing the luminescence generated by
ozone oxidation of NO radicals (Palmer R. M., et al., Nature, 327,
pp. 524-526, 1987), a method determining absorption spectrum of
metHb which is produced by oxidation of oxyhemoglobin (O.sub.2Hb)
(Kelm M., et al., Circ. Res. 66, pp. 1561-1575, 1990), a method for
quantification utilizing the flow of electric current produced in
oxidation when electrodes are placed in a tissue (Shibuki K.,
Neurosci. Res. 9, pp. 69-76, 1990; Malinski, and T., Nature, 358,
pp. 676-678, 1992), the Griess reaction method (Green L. C., et
al., Anal. Biochem., 126, pp. 131-138, 1992) and so forth (as
reviews, see, "Approaches From The Newest Medicine [Saishin Igaku
Kara No Approach] 12, NO", Ed. by Noboru Toda, pp. 42-52, Section
3, Tetsuo Nagano, Measuring Method of NO, published by Medical View
Co., Ltd; Archer, S., FASEB J., 7, pp. 349-360, 1993).
[0005] The Griess reaction method achieves the detection by using
azo coupling of a diazonium salt compound and
naphthylethylenediamine in the presence of NO.sub.2.sup.- that is
produced by oxidation of a nitric oxide radical. Although this
method does not achieve direct measurement of nitric oxide
radicals, the method is advantageous since it requires no special
apparatuses or techniques. Moreover, this method also has a
characteristic feature that nitric oxide-related metabolites can be
quantified, since NO.sub.3.sup.- can be measured after being
reduced to NO.sub.2.sup.- with cadmium (Stainton M. P., Anal.
Chem., 46, p. 1616, 1974; Green L. C., et al., Anal. Biochem., 126,
pp. 131-138, 1982) or hydrazine (Sawicki, C. R. and Scaringelli, F.
P., Microchem. J., 16, pp. 657-672, 1971).
[0006] 2,3-Diaminonaphthalene is known as a reagent for measuring
nitric oxide by detecting NO.sub.2.sup.- in a similar manner to the
Griess reaction method. This reagent reacts with NO.sub.2.sup.-
under an acidic condition to form a fluorescent adduct,
naphthalenetriazole (chemical name: 1-[H]-naphtho[2,3-d]triazole,
Wiersma J. H., Anal. Lett., 3, pp. 123-132, 1970). The conditions
for the reaction of 2,3-diaminonaphthalene with NO.sub.2.sup.- have
been studied in detail. The reaction proceeds most quickly at pH 2
or lower and completes in approximately 5 minutes at room
temperature (Wiersma J. H., Anal. Lett., 3, pp. 123-132, 1970;
Sawicki, C. R., Anal. Lett., 4, pp. 761-775, 1971). The generated
adduct emits fluorescence most efficiently at pH 10 or higher
(Damiani, P. and Burini, G., Talanta, 8, pp. 649-652, 1986).
[0007] The method for measuring nitric oxide using the
2,3-diaminonaphthalene is characterized in that a detection limit
is about several tens nanomoles: and sensitivity is 50 to 100 times
higher than that of the Griess reaction method (Misko, T. P., Anal.
Biochem. 214, pp. 11-16, 1993). This method is also excellent since
it can be carried out conveniently without need of any special
apparatuses or techniques (as a review of the above methods, see,
DOJIN News, No. 74, Information Measurement Reagents for NO:
2,3-Diaminonaphthalene, published by Dojindo Laboratories Co.,
Ltd., 1995). However, this method does not utilizes nitric oxide,
per se, but its oxidation product NO.sub.2.sup.- 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 NO.sub.2.sup.- 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.
[0008] The inventors of the present invention conducted researches
to provide means for direct measurement of nitric oxide with high
sensitivity under a physiological condition. As a result, the
inventors found that 2,3-diaminonaphthalene or a derivative thereof
efficiently reacts with nitric oxide to give fluorescent
naphthalenetriazole or its derivative, even under a neutral
condition, in the presence of an oxygen source such as dissolved
oxygen or oxide compounds (for example, PTIO and its derivatives
such as carboxy-PTIO). Moreover, the inventors also found that a
method for measuring nitric oxide employing this reaction gave
extremely high detection sensitivity and achieved accurate
quantification of a trace amount of nitric oxide (see, the
specification of Japanese Patent Unexamined Publication (Kokai) No.
9-043153/1997).
[0009] 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.
[0010] The inventors of the present invention proposed two methods
for measurement of nitric oxide that successively solve these
problems.
[0011] One of the methods utilizes a diaminofluorescein derivative
(hereafter also referred to as "DAF" in the specification, Japanese
Patent Unexamined Publication (Kokai) No. 10-226688/1998). This
method utilizing DAF is much excellent in reactivity with nitric
oxide and measurement sensitivity. The method enables measurement
of nitric oxide with excitation light of a long wavelength that
does not damage living tissues and cells, and accurate measurement
of nitric oxide existing in inside of cells for each individual
cell, which are characteristic features of the aforementioned
method. 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.
[0012] The second method utilizes a diaminorhodamine derivative
(hereafter also referred to as "DAR", International Patent
Publication WO99/01447). This method is also based on the
measurement of fluorescence of a triazole derivative (hereafter
also referred to as "DAR-T") which is produced by the reaction of
DAR with nitric oxide. The peak of the fluorescence spectrum of
DAR-T lies around 580 nm (excitation wavelength: 565 nm), while the
peak of the fluorescence spectrum of the aforementioned DAF-T is
observed around 515 nm (excitation wavelength: 495 nm).
Accordingly, measurements without being influenced by
autofluorescence of cells can be performed by using DAR.
Furthermore, since DAR-T can maintain a certain level or higher
intensity, of fluorescence in an acidic region as well as in a
basic or a neutral region, it enables measurement of nitric oxide
over a wide pH range. 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.
DISCLOSURE OF THE INVENTION
[0013] An object of the present invention is to provide a compound
useful for the measurement of nitric oxide. More specifically, the
object is to provide a compound which has higher fluorescence
intensity and whose fluorescent intensity is not fluctuated by pH,
which compound is based on the aforementioned DAR that can maintain
a certain level of fluorescence intensity in an acidic region as
well as in a basic and a neutral region without being influenced by
the autofluorescence. Another object of the present invention is to
provide a reagent for measurement of nitric oxide comprising a
compound having the aforementioned characteristics.
[0014] The inventors of the present invention conducted various
researches to achieve the foregoing objects. As a result, they
found that the rhodamine derivatives of the following structures
readily reacted with nitric oxide in a sample for measurement to
give triazole derivatives having high fluorescence intensity. They
also found that the triazole derivatives had a substantially
constant level of fluorescence intensity over a wide pH range from
basic to acidic condition, and that their fluorescence intensities
were improved. The present invention was achieved on the basis of
these findings.
[0015] The present invention thus provides compounds represented by
the following formula (I) or (II):
##STR00002##
wherein, in the formula (I), R.sup.1, R.sup.2, R.sup.3, and R.sup.4
independently represent methyl group or ethyl group; and in the
formula (II), R.sup.5, R.sup.6, R.sup.7, and R.sup.8 independently
represent methyl group or ethyl group and X-- represents an anion.
According to preferred embodiments of the present invention, there
are provided: the compounds of the formula (I) wherein R.sup.1,
R.sup.2, R.sup.3, and R.sup.4 are ethyl groups; the compounds of
the formula (I) wherein R.sup.1, R.sup.2, R.sup.3, and R.sup.4 are
methyl groups; the compounds of the formula (II) wherein R.sup.5,
R.sup.6, R.sup.7, and R.sup.8 are methyl groups; and the compounds
of the formula (II) wherein R.sup.5, R.sup.6, R.sup.7, and R.sup.8
are methyl groups and X-- is I--. According to another aspect of
the present invention, there are provided reagents for measurement
of nitric oxide which comprises a compound represented by the
aforementioned formula (I) or formula (II).
[0016] According to a further aspect of the present invention,
there are provided compounds represented by the following formula
(III) or (IV):
##STR00003##
wherein, in the formula (III), R.sup.11, R.sup.12, R.sup.13, and
R.sup.14 independently represent methyl group or ethyl group; and
in the formula (IV), R.sup.15, R.sup.16, R.sup.17, and R.sup.18
independently represent methyl group or ethyl group and Y--
represents an anion. According to preferred embodiments of the
aforementioned invention, there are provided: the compounds wherein
R.sup.11, R.sup.12, R.sup.13, and R.sup.14 are ethyl groups; the
compounds wherein R.sup.11, R.sup.12, R.sup.13 and R.sup.14 are
methyl groups; the aforementioned compounds wherein R.sup.15,
R.sup.16, R.sup.17 and R.sup.18 are methyl groups; and the
aforementioned compounds wherein R.sup.15, R.sup.16, R.sup.17 and
R.sup.18 are methyl groups and Y-- is I--.
[0017] According to a still further aspect of the present
invention, there are provided methods for measurement of nitric
oxide, which comprise step (1) in which a compound represented by
the aforementioned formula (I) or (II) is reacted with nitric
oxide, and step (2) in which a compound represented by the formula
(III) or (IV) produced in the step (1) is detected.
BRIEF EXPLANATION OF THE DRAWINGS
[0018] FIG. 1 shows change in fluorescence intensity of a compound
of the present invention (DAR-MT) and DAR-1T and DAR-2T disclosed
in WO99/01447 with pH fluctuation. In the figure, .smallcircle.
indicates the results for DAR-1T, .quadrature. for DAR-2T, and for
DAR-MT.
[0019] FIG. 2 shows change in fluorescence intensity with change in
nitric oxide concentration measured by using compounds of the
present invention (DAR-M and DAR-4M) and the compounds disclosed in
WO99/0.1447 (DAR-1 and DAR-2). In the figure, .smallcircle.
indicates the results for DAR-1, .DELTA. for DAR-2, X for DAR-M,
and for DAR-4M.
[0020] FIG. 3 shows results of imaging of intracellular nitric
oxide performed by using a compound of the present invention
(DAR-4M AM). The results are shown with averaged change in relative
fluorescence intensity of arbitrary seven cells based on the
fluorescence intensity at the time of the start of the measurement.
In the figure, (a) shows that the fluorescence intensity was
increased by stimulation with bradykinin, and (b) shows that the
fluorescence intensity was not increased in the presence of a
nitric oxide synthetase inhibitor (1 mM L-NAME).
BEST MODE FOR CARRYING OUT THE INVENTION
[0021] In the aforementioned formula (II), the kind of the anion
represented by X-- is not particularly limited. For example,
halogen ions such as chlorine ion, bromine ion and iodine ion,
inorganic ions such as sulfate ion, nitrate ion and perchlorate ion
as well as organic acid ions such as methanesulfonate ion,
p-toluenesulfonate ion, oxalate ion and citrate ion may be used.
Preferably, iodine ion may be used.
[0022] In the formula (I), a compound wherein R.sup.1, R.sup.2,
R.sup.3, and R.sup.4 all represent ethyl group, or wherein R.sup.1,
R.sup.2, R.sup.3, and R.sup.4 all represent methyl group is
preferred, and a compound wherein R.sup.1, R.sup.2, R.sup.3, and
R.sup.4 all represent methyl group is particularly preferred. In
the formula (II), a compound wherein R.sup.5, R.sup.6, R.sup.7, and
R.sup.8 are methyl groups is preferred, and a compound wherein
R.sup.6, R.sup.7, and R.sup.8 are methyl groups and X-- is I-- is
most preferred.
[0023] In the formula (III), a compound wherein R.sup.11, R.sup.12,
R.sup.13, and R.sup.14 all represent ethyl group, or wherein
R.sup.11, R.sup.12, R.sup.13, and R.sup.14 all represent methyl
group is preferred, and a compound wherein R.sup.11, R.sup.12,
R.sup.13, and R.sup.14 all represent methyl group is particularly
preferred. In the formula (IV), a compound wherein R.sup.15,
R.sup.16, R.sup.17, and R.sup.18 are methyl groups is preferred,
and a compound wherein R.sup.15, R.sup.16, R.sup.17, and R.sup.18
are methyl groups and Y-- is I-- is most preferred.
[0024] Methods for preparing typical compounds falling within the
compounds of the present invention will be shown in the following
schemes. However, the methods for preparing the compounds of the
present invention are not limited to the following methods (in the
schemes, AM represents acetoxymethyl group, and DIEA represents
diisopropylethylamine). It will be understood by those skilled in
the art that any compounds represented by the formulas (I) and (II)
or the formulas (III) and (IV) can readily be prepared by referring
to the general explanation in the following schemes and specific
description in the examples. The method for preparing
diaminorhodamine is disclosed in WO99/01447 and a specific method
for preparing DAR-1 is also described therein. Therefore, any
compounds falling within the formulas (I) through (IV) can be
readily prepared by combination of known preparation methods
available to those skilled in the art, the methods disclosed in the
above publication, and the methods described in the examples of the
present specification.
##STR00004## ##STR00005##
[0025] The compounds of the present invention may exist as hydrates
or solvates, and it should be understood that these substances also
fall within the scope of the present invention. Furthermore, it is
known that rhodamine derivatives may form a lactone ring and exist
as compounds in a free form. It will be readily understood by those
skilled in the art that the compounds of the present invention
represented by the formulas (I) and (III) may exist in structural
isomers forming the lactone ring. Those structural isomers also
fall within the scope of the present invention (it will also be
readily understood by those skilled in the art that a quaternary
amino group does not exist in the aforementioned compounds). The
compounds of the present invention forming a lactone ring are shown
by the following formulas (I)' and (III)' (these compounds
correspond to those represented by the formulas (I) and (III),
respectively, and R.sup.1 to R.sup.4 and R.sup.11 to R.sup.14 have
the same meanings as those defined above).
##STR00006##
[0026] In the aforementioned formulas (I) and (III), as well as in
the aforementioned schemes, only the compounds that do not form a
lactone ring are shown for the sake of convenience. In these
compounds, the positive charge on the quaternary amino group is
neutralized by the carboxyl anion to form an intramolecular
zwitterion. Therefore, they do not have an anion represented by X--
or Y-- that serves as a counter ion.
[0027] The compounds of the present invention represented by the
formula (I) or (II) have a property that they efficiently react
with nitric oxide under a neutral condition to give a compound of
the formula (III) or (IV) in a good yield. The compounds
represented by the formula (I) or (II) themselves do not emit
substantial fluorescence when they are irradiated by excitation
light at about 565 nm under a neutral condition, whilst the
compounds of the formula (III) or (IV) emit extremely strong
fluorescence under the same condition (emission: 580 nm).
Therefore, nitric oxide in living tissues or cells can be measured
by allowing the compounds represented by the formula (I) or (II) to
be uptaken into the living tissues or cells and to react with
nitric oxide to generate fluorescent compounds of the formula (III)
or (IV), and then measuring fluorescence of the resulting
compounds. The compounds of the formula (III) or (IV) produced as
described above maintain substantially constant fluorescence
intensity over a region of from weakly basic condition of about pH
9 to acidic condition of about pH 4, and they show more superior
fluorescence intensity than that of DAR-1T or DAR-2T specifically
disclosed in WO99/01447.
[0028] Therefore, the method for measuring nitric oxide provided by
the present invention thus comprises steps of reacting a compound
represented by the formula (I) or (II) with nitric oxide to form a
compound of the formula (III) or (IV), respectively, and then
measuring the fluorescence of the compound of the formula (III) of
(IV). In the present specification, the term "measurement" should
be construed in its broadest sense including measurement for
various purposes such as detection, and quantitative or qualitative
determinations. The aforementioned reaction can preferably be
performed under a neutral condition, for example, within a range of
pH 6.0 to 8.0, preferably pH 6.5 to 7.8, more preferably pH 6.8 to
7.6. However, the measurement of nitric oxide according to the
present invention is not limited to measurement under a neutral
condition. The fluorescence intensity of the compounds of the
formula (III) or (IV) is constant over a wide pH range, and hence
accurate measurement can be conducted even when measurement is
performed for a sample in which pH fluctuation is expected during
the measurement, for example, a tissue of a patient with an
ischemic disease.
[0029] The compounds of the formula (II) readily pass through
lipophilic cytoplasmic membranes and are uptaken into the cells,
and then the ester is hydrolyzed to give compounds of the formula
(I) having carboxyl group. Since the compounds of the formula (I)
are highly hydrophilic, they are not easily excreted from the
inside of the cells. Therefore, the compounds of the formula (II)
per se are useful as an agent for the measurement.
[0030] The measurement of fluorescence can be performed by a known
conventional fluorescence measuring method (see, for example,
publications such as Wiersma, J. H., Anal. Lett., 3, pp. 123-132,
1970; Sawicki, C. R., Anal. Lett., 4, pp. 761-775, 1971; Damiani,
P. and Burini, G., Talanta, 8, pp. 649-652, 1986; Misko, T. P.,
Anal. Biochem. 214, pp. 11-16, 1993 and so forth). In the method
for measurement of nitric oxide according to the present invention,
for example, it is preferred that light of about 565 nm is
irradiated as excitation light, and fluorescence at about 580 nm is
measured. By using lights having such wavelengths, efficient
spectrometry can be performed by means of a fluorescence filter of
an ordinarily used fluorescence microscope, and measurement with
high sensitivity can be attained without using a special
filter.
[0031] When particularly high sensitivity measurement is required,
the measurement of nitric oxide may be performed in the presence of
an oxygen source. As the oxygen source, for example, oxygen, ozone,
oxide compounds and so forth may be used. Dissolved oxygen can
generally be used as the oxygen source, and if necessary, oxygen
gas may be introduced into a reaction system, or a reagent for
generating oxygen (for example, hydrogen peroxide and the like) may
be added. The oxide compounds are not particularly limited so long
as they have an oxide bond from which oxygen atom is readily
released, for example, N--O, S--O, and P--O. For example, PTIO
(2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, Maeda, H.,
et al., J. Leuk. Biol., 56, pp. 588-592, 1994; Akaike T., et al.,
Biochemistry, 32, pp. 827-832, 1993) or its derivatives
(carboxy-PTIO which is formed by introducing carboxyl group into
the para-position of the phenyl group of PTIO and the like),
triphenylphosphine oxide, triethylamine oxide and so forth can be
used.
[0032] Among the aforementioned oxide compounds, PTIO and its
derivatives (for example, carboxy-PTIO and the like) are
particularly preferred compounds, and they are readily obtained by
those skilled in the art (listed in Tokyo Chemical Industry Co.,
Ltd., Organic Chemicals Catalog, 32, 1994 and so forth). The oxide
compounds, per se, may be used as a reaction reagent, or those
encapsulated in liposomes or similar materials can also be used. An
amount of the oxygen source is not particularly limited, and the
amount may preferably be at least about 1 .mu.M or more, more
preferably about 10 to 30 .mu.M, and most preferably about 10 to 20
.mu.M based on nitric oxide to be measured. In the measurement for
biological samples and so forth, the oxygen source may preferably
be added to a sample in an amount of about 10 to 20 .mu.M. However,
a necessary amount of the oxygen source may generally be supplied
by dissolved oxygen. The reaction may be performed within a
temperature range of 10 to 40.degree. C.
EXAMPLES
[0033] The present invention will be more specifically explained
with reference to the following examples. However, the scope of the
present invention is not limited to the following examples. In the
examples, the descriptions such as "DAR-4M" correspond to those of
the compounds shown in the above schemes.
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 .mu.l
of methyl iodide (1.7 equivalents based on DAR-1), and the
temperature of the reaction mixture was raised to 80.degree. 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 .mu.l 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.degree. C.
[0036] .sup.1H-NMR (300 MHz, CDCl.sub.3) .delta. 1.13 (12H, t,
J=7.0), 2.86 (3H, s), 3.33 (8H, q, J=7.0), 6.37-6.43 (5H, m), 6.75
(1H, d, J=7.9), 6.81 (2H, d, J=9.0)
[0037] FAB-MS 487 (M.sup.++1)
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.degree. C.
[0040] .sup.1H-NMR (300 MHz, CDCl.sub.3) .delta. 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.sup.++1)
Example 3
Preparation of
3,6-bis(dimethylamino)-9-[3-amino-4-(N-methylamino)-2-carboxyphenyl]xanth-
ylium 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.degree. C.
[0044] .sup.1H-NMR (300 MHz, CDCl.sub.3) .delta. 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.sup.++1)
Example 4
Preparation of 3,6-bis(dim
ethylamino)-9-(4-carboxy-1-methylbenzotriazol-5-yl)xanthylium
intramolecular salt (DAR-4MT)
[0046] In the same manner as in Example 2, the title compound was
obtained (18.5 mg, 72%) from DAR-4M (25 mg) obtained in Example
3.
[0047] m.p. 242-245.degree. C.
[0048] .sup.1H-NMR (300 MHz, CDCl.sub.3) .delta. 2.98 (12H, s),
4.41 (3H, s), 6.36 (2H, dd, J=9.0, 2.6), 6.50 (2H, d, J=2.6), 6.62
(2H, d, J=9.0), 7.24 (1H, d, J=8.5), 7.77 (1H, d, J=8.5)
[0049] FAB-MS 442 (M.sup.++1)
Example 5
Preparation of
3,6-bis(dimethylamino)-9-[2-acetoxymethoxycarbonyl-3-amino-4-(N-methylami-
no)phenyl]xanthylium iodide (DAR-4M AM)
[0050] 3,6-Bis(dim
ethylamino)-9-(3-amino-2-carboxy-4-nitrophenyl)xanthylium
intramolecular salt (2.44 g) prepared by the method disclosed in
WO99/01447 was dissolved in 120 ml of dichloroethane, and the
solution was added with 1 ml of diisopropylethylamine. The mixture
was further added with acetoxymethyl bromide (0.55 ml), and stirred
at room temperature. The progress of the reaction was monitored
every day by TLC, and additional diisopropylethylamine and
acetoxymethyl bromide were added in amounts half of those used for
the first addition. After 4 days, the reaction mixture was added
with dichloroethane and washed with water, and the solvent was
evaporated. The residue was purified by silica gel column
chromatography to obtain
3,6-bis(dimethylamino)-9-(2-acetoxymethoxycarbonyl-3-amino-4-nitrophenyl)-
xanthylium bromide (0.445 g, 13.6%).
[0051] .sup.1H-NMR (300 MHz, CDCl.sub.3) .delta. 1.75 (3H, s), 3.40
(12H, s), 5.41 (2H, s), 6.56 (1H, d, J=8.6), 6.97 (2H, d, J=2.4),
7.07 (2H, dd, J=9.5, 2.4), 7.18 (2H, d, J=9.5), 8.45 (2H, br), 8.54
(1H, d, J=8.6)
[0052] The aforementioned
3,6-bis(dimethylamino)-9-(2-acetoxymethoxycarbonyl-3-amino-4-nitrophenyl)-
xanthylium bromide (443 mg) was dissolved in a mixed solvent of
ethanol (100 ml) and dichloromethane (100 ml), and the solution was
added with 0.25 g of 10% Pd/C catalyst. The mixture was stirred at
room temperature under a hydrogen atmosphere for 2 hours. The
reaction mixture was filtered, and the solvent was evaporated. The
residue was purified by silica gel column chromatography to obtain
3,6-bis(dimethylamino)-9-(2-acetoxymethoxycarbonyl-3,4-diaminophenyl)xant-
hylium bromide (214 mg, 50.9%).
[0053] .sup.1H-NMR (300 MHz, CDCl.sub.3) .delta. 1.69 (3H, s), 3.33
(12H, s), 4.81 (2H, br), 5.32 (2H, s), 6.29 (1H, d, J=7.9), 6.47
(2H, br), 6.78 (2H, d, J=2.6), 6.91 (2H, dd, J=9.5, 2.6), 6.94 (2H,
d, J=7.9), 7.45 (2H, d, J=9.5)
[0054] From the above 3,6-bis(dim
ethylamino)-9-(2-acetoxymethoxycarbonyl-3,4-diaminophenyl)xanthylium
bromide (133 mg),
3,6-bis(dimethylamino)-9-[2-acetoxymethoxycarbonyl-3-amino-4-(N-methylami-
no)phenyl]xanthylium iodide was obtained (40.5 mg, 27.5%) in the
same manner as in Example 1.
[0055] m.p. 177-178.degree. C.
[0056] .sup.1H-NMR (300 MHz, CDCl.sub.3) .delta. 1.69 (3H, s), 2.99
(2H, d, J=4.6), 3.36 (12H, s), 5.06 (1H, q, J=4.6), 5.33 (2H, s),
6.34 (2H, s), 6.46 (1H, d, J=7.9), 6.69 (1H, d, J=7.9), 6.79 (2H,
d, J=2.6), 6.91 (2H, dd, J=9.5, 2.6), 7.48 (2H, d, J=9.5)
[0057] FAB-MS 503 (M.sup.+)
Example 6
Preparation of
3,6-bis(diethylamino)-9-[2-acetoxymethoxycarbonyl-3-amino-4-(N-methylamin-
o)phenyl]xanthylium iodide (DAR-M AM)
[0058] In the same manner as in Example 5, the title compound was
obtained from
3,6-bis(diethylamino)-9-(3-amino-2-carboxy-4-nitrophenyl)xanthylium
intramolecular salt which was prepared by the method disclosed in
WO99/01447.
[0059] m.p. 147-150.degree. C.
[0060] .sup.1H-NMR (300 MHz, CDCl.sub.3) .delta. 1.34 (12H, t,
J=7.0), 1.67 (3H, s), 2.98 (3H, s), 3.63 (8H, q, J=7.0), 5.36 (2H,
s), 6.42 (1H, d, J=8.0), 6.66 (1H, d, J=8.0), 6.75 (2H, d, J=2.4),
6.84 (2H, dd, J=9.3, 2.4), 7.43 (2H, d, J=9.3)
[0061] FAB-MS 559 (M.sup.+)
Example 7
Change in Fluorescence Intensity of the Compounds of the Present
Invention by pH Fluctuation
[0062] The triazole derivatives DAR-1T and DAR-2T prepared by the
method disclosed in WO99/01447 and DAR-MT prepared in Example 2
were each dissolved at 1 .mu.M in 0.1 M sodium phosphate buffer
prepared at various pHs, and fluorescence intensity of each sample
was measured. The excitation wavelength/emission wavelength were as
follows: DAR-1T; 565/580, DAR-2T; 565/575 and DAR-MT; 565/575 (m).
The results are shown in FIG. 1. In the figure, .smallcircle.
indicates the results for DAR-1T, .quadrature. for DAR-2T, and for
DAR-MT. DAR-MT of the present invention was found to have constant
fluorescence intensity in the range of pH from 4 to 11.
Example 8
Measurement of Nitric Oxide Using the Compounds of the Present
Invention
[0063] Nitric oxide was measured by using DAR-M obtained in Example
1 and DAR-4M obtained in Example 3. For comparison of measurement
sensitivity, DAR-1 and DAR-2 disclosed in WO99/01447 were used as
reference compounds. Each compound was dissolved at 10 .mu.M in 0.1
M sodium phosphate buffer at 37.degree. C. The solution was added
with nitric oxide, and change in fluorescence intensity with
variation of nitric oxide concentration was measured. The nitric
oxide used was prepared by bubbling nitric oxide in 0.1 M sodium
phosphate buffer (pH 7.4) after argon substitution, and measured by
the HRP method (Kikuchi, K. et al., Biol. Pharm. Bull., 19, pp.
649-651, 1996). Excitation wavelength/emission wavelength were as
follows: DAR-1; 565/580, DAR-2; 565/575, DAR-M; 565/580 and DAR-4M;
560/575 (nm). The results of sensitivity comparison using a
calibration curve are shown in FIG. 2. In the figure, .smallcircle.
indicates the results for DAR-1, A for DAR-2, X for DAR-M, and for
DAR-4M. DAR-M and DAR-4M of the present invention were found to
have 1.4 to 1.7 times higher sensitivity than DAR-1 and DAR-2.
Example 9
Imaging of Nitric Oxide in Cells
[0064] Bovine aorta endothelial cells were cultured on an imaging
dish. The culture broth was changed to DMEM medium (supplemented
with 0.2% DMSO) containing DAR-4M AM (10 .mu.M) prepared in Example
5, and incubated at 37.degree. C. for 30 minutes to load DAR-4M AM
on the cells. The cells were washed, and the culture broth was
changed again to DMEM medium not containing DAR-4M AM. After
incubation at 37.degree. C. for 1 hour, imaging was performed. The
cells were examined with a fluorescence microscope equipped with an
excitation filter (520 to 550 nm), a dichromic mirror (565 nm), and
a barrier filter (580 mm, long pass). The measurement was performed
in a warm box kept at 37.degree. C. Imaging interval was set to be
10 seconds, and images captured by a cooled charge-coupled device
camera were recorded and analyzed by an image analyzer. The results
are shown in FIG. 3. The results in the figure are shown with
averaged change of relative fluorescence intensity of arbitrary
seven cells based on the fluorescence intensity at the time of the
start of the measurement. The fluorescence intensity was increased
by stimulation with bradykinin (FIG. 3(A)), but the fluorescence
intensity was not increased in the presence of a nitric oxide
synthetase inhibitor (1 mM L-NAME) (FIG. 3(B)). These results
clearly indicate that DAR-4M AM was taken into the cells, and
DAR-4M produced in the cells specifically reacted with nitric oxide
in the cells. These results demonstrate that intracellular nitric
oxide can be specifically measured with high sensitivity at
individual cell level.
INDUSTRIAL APPLICABILITY
[0065] The compounds of the present invention are useful as
reagents for measurement of nitric oxide. The compounds of the
formula (I) or (II) of the present invention have a property to
efficiently react with nitric oxide to give fluorescent compounds
of the formula (III) or (IV). The compounds of the formula (III) or
(IV) are characterized in that (1) they have high fluorescence
intensity, (2) they are not influenced by autofluorescence, and (3)
they have no fluorescence intensity fluctuation over a wide pH
range. It is known that intracellular pH may fluctuate when
endothelial cells are stimulated by bradykinin (Fleming, I., et
al., Cir. Res., 74, pp. 1220-1226, 1994). In particular, said
compounds are featured by successful accurate measurement of nitric
oxide when a sample with expected pH fluctuation is measured.
* * * * *