U.S. patent application number 13/133357 was filed with the patent office on 2011-09-29 for labeling composition for intraocular tissue, labeling method of intraocular tissue, and screening method.
This patent application is currently assigned to Canon Kabushiki Kaisha. Invention is credited to Takeshi Miyazaki, Norihiro Nishimura, Yuhei Nishimura, Tsuyoshi Nomoto, Yasuhito Shimada, Taichi Shintou, Toshio Tanaka, Kohei Watanabe.
Application Number | 20110236310 13/133357 |
Document ID | / |
Family ID | 42287915 |
Filed Date | 2011-09-29 |
United States Patent
Application |
20110236310 |
Kind Code |
A1 |
Watanabe; Kohei ; et
al. |
September 29, 2011 |
LABELING COMPOSITION FOR INTRAOCULAR TISSUE, LABELING METHOD OF
INTRAOCULAR TISSUE, AND SCREENING METHOD
Abstract
The invention provides a labeling composition for an intraocular
tissue of a living individual, which specifically labels the
intraocular tissue without need of an invasive operation such as
exposure of an ocular tissue or injection of a staining agent into
the ocular tissue or a nerve tissue linking to the ocular tissue, a
method of noninvasively labeling an intraocular tissue of a living
individual, and a screening method using the labeling composition
for the intraocular tissues. The composition contains a compound
capable of labeling at least a photoreceptor cell layer of a
retina, wherein the compound is a staining compound having a
particular structure as a partial structure thereof.
Inventors: |
Watanabe; Kohei;
(Yokohama-shi, JP) ; Shintou; Taichi;
(Saitama-shi, JP) ; Nomoto; Tsuyoshi; (Tokyo,
JP) ; Miyazaki; Takeshi; (Yokohama-shi, JP) ;
Tanaka; Toshio; (Tsu-shi, JP) ; Nishimura; Yuhei;
(Tsu-shi, JP) ; Shimada; Yasuhito; (Tsu-shi,
JP) ; Nishimura; Norihiro; (Tsu-shi, JP) |
Assignee: |
Canon Kabushiki Kaisha
Tokyo
JP
|
Family ID: |
42287915 |
Appl. No.: |
13/133357 |
Filed: |
December 24, 2009 |
PCT Filed: |
December 24, 2009 |
PCT NO: |
PCT/JP2009/071865 |
371 Date: |
June 7, 2011 |
Current U.S.
Class: |
424/1.69 ;
424/1.65; 424/9.2; 424/9.6; 546/66; 546/76; 548/156; 548/301.7;
549/394; 552/241 |
Current CPC
Class: |
A61K 49/0041 20130101;
C07D 311/82 20130101; C07D 405/04 20130101; C07D 417/04 20130101;
C07D 221/18 20130101; C07D 497/10 20130101; C07D 209/70 20130101;
C07D 491/052 20130101; C07D 209/96 20130101; C07D 413/06 20130101;
A61K 49/0039 20130101; C07D 215/00 20130101; C07D 263/54 20130101;
C07D 417/06 20130101; G01N 33/582 20130101; G01N 2333/4603
20130101; A61K 49/0021 20130101; G01N 33/5088 20130101; C07D 277/36
20130101; C07D 491/12 20130101; C07D 405/06 20130101; C07D 221/14
20130101; A61K 49/006 20130101; C07D 417/14 20130101; C07D 413/04
20130101; C07D 277/64 20130101; C07D 277/34 20130101; C07D 311/86
20130101; A61K 49/0002 20130101 |
Class at
Publication: |
424/1.69 ;
546/76; 424/9.2; 424/9.6; 424/1.65; 548/301.7; 546/66; 552/241;
549/394; 548/156 |
International
Class: |
A61K 49/00 20060101
A61K049/00; C07D 221/18 20060101 C07D221/18; A61K 51/04 20060101
A61K051/04; C07D 491/14 20060101 C07D491/14; C09B 1/54 20060101
C09B001/54; C07D 311/82 20060101 C07D311/82; C07D 417/06 20060101
C07D417/06; A61K 51/08 20060101 A61K051/08 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 25, 2008 |
JP |
2008-330988 |
Claims
1. A labeling composition for an intraocular tissue of a living
individual, which comprises a compound capable of labeling at least
a photoreceptor cell layer of a retina, wherein the compound is a
staining compound having a structure represented by the following
general formula (I) or general formula (II) as a partial structure
thereof, ##STR00040## wherein in the general formula (I), R.sub.1
and R.sub.2 are, independently of each other, a hydrogen atom, an
alkyl group or an aryl group, R.sub.1 and R.sub.2 may bond to each
other to form a ring, and A is a structure represented by any one
of the following general formulae (III) to (IX) and (VIII')
##STR00041## wherein in the general formula (III), R.sub.6 is an
aryl group, R.sub.7, R.sub.8, R.sub.10 and R.sub.11 are,
independently of one another, a hydrogen atom, an alkyl group, an
aryl group or a halogen atom, and R.sub.9 is an ammonium salt group
having a counter anion, in the general formula (IV), R.sub.12 and
R.sub.13 are, independently of each other, a hydrogen atom, an
alkyl group, an aryl group or a heterocyclic group, and R.sub.14 to
R.sub.16 are, independently of one another, a hydrogen atom, an
alkyl group, an aryl group, a heterocyclic group, a halogen atom or
an amino group, in the general formula (V), R.sub.17 and R.sub.18
are, independently of each other, a hydrogen atom, an alkyl group
or an alkoxy group, R.sub.19 and R.sub.20 are, independently of
each other, a hydrogen atom, an alkyl group, an alkoxy group, a
hydroxyl group or a cyano group, R.sub.21 is a heterocyclic group
or --CH.dbd.C(R.sub.22)(R.sub.23), R.sub.22 and R.sub.23 are,
independently of each other, a hydrogen atom, a cyano group, a
heterocyclic group, a carboxyl group or a carboxylate group, and
R.sub.1 and R.sub.17, R.sub.2 and R.sub.18, and R.sub.22 and
R.sub.23 may, independently of one another, bond to each other to
form a ring, in the general formula (VI), R.sub.24 is a hydrogen
atom, an alkyl group, an aryl group or a heterocyclic group, in the
general formula (VII), R.sub.25 is an oxygen atom, a sulfur atom or
N(R.sub.27), R.sub.26 is a hydrogen atom, an alkyl group, an alkoxy
group or a sulfonic group, and R.sub.27 is a hydrogen atom, an
alkyl group or an aryl group, in the general formula (VIII),
R.sub.28 is a hydrogen atom, an alkyl group or an aryl group, in
the general formula (VIII'), R.sub.28' is a halogen atom, an alkoxy
group or an aryloxy group, and in the general formula (IX),
R.sub.29 is a hydrogen atom, an alkyl group or an aryl group; and
wherein A is also a structure represented by the following general
formula (X) ##STR00042## wherein in the general formula (X),
R.sub.31 and R.sub.32 are individually a sulfonic group or a salt
thereof, and R.sub.33 and R.sub.34 are individually a hydrogen
atom, an alkyl group, an alkoxy group or a halogen atom, with the
proviso that R.sub.1 and R.sub.2 in the general formula (I) bond to
each other to form one substituent group .dbd.N--R.sub.30, and
R.sub.30 is an aryl group or a heterocyclic group, further wherein
in the general formula (II), R.sub.3 and R.sub.4 are, independently
of each other, a hydrogen atom, an alkenyl group, a cyano group, a
carboxyl group, a carboxylate group, a sulfonic group, an acyl
group or a heterocyclic group, R.sub.3 and R.sub.4 may bond to each
other to form a ring, and R.sub.5 is a hydrogen atom, an alkyl
group, an aryl group or a heterocyclic group, with the proviso that
one of R.sub.3 and R.sub.4 is a hydrogen atom, and the other is a
heterocyclic group represented by the following general formula
(XI) ##STR00043## wherein in the general formula (XI), R.sub.35 is
an alkyl group or an aryl group, R.sub.36 to R.sub.39 are,
independently of one another, a hydrogen atom, an alkyl group, an
aryl group, an alkoxy group, a carboxyl group, a sulfonic group, a
heterocyclic group, an amino group or a halogen atom, R.sub.36 and
R.sub.37, R.sub.37 and R.sub.38, or R.sub.38 and R.sub.39 may bond
to each other to form a ring, X.sup.- is an anionic group, and
Q.sub.1 is a sulfur atom, oxygen atom, --C(R.sub.40)(R.sub.41)--,
--CH.dbd.CH-- or --N(R.sub.42)--, in which R.sub.40 to R.sub.42 are
individually a hydrogen atom, an alkyl group or an aryl group, and
R.sub.40 and R.sub.41 may bond to each other to form a ring, and
wherein the ring formed by bonding R.sub.3 and R.sub.4 in the
general formula (II) to each other is represented by any one of the
following general formulae (XII), (XIII) and (XV) ##STR00044##
wherein in the general formula (XII), R.sub.43 is a hydrogen atom,
an alkyl group, an aryl group or a heterocyclic group, and R.sub.44
is an alkyl group, an aryl group, a carboxyl group, a carboxylate
group, a hydroxyl group or an amino group, in the general formula
(XIII), Q.sub.2 is an oxygen atom, a sulfur atom or
--N(R.sub.52)--, R.sub.45 is a hydrogen atom, an alkyl group, an
aryl group or a heterocyclic group, R.sub.46 is a sulfur atom, an
oxygen atom, .dbd.NR.sub.53, a heterocyclic group or a
dicyanomethylene group, and R.sub.52 and R.sub.53 are individually
a hydrogen atom, an alkyl group, an aryl group or a heterocyclic
group, and in the general formula (XV), R.sub.49 and R.sub.50 are,
independently of each other, a hydrogen atom, an alkyl group or an
aryl group, and R.sub.51 is an oxygen atom or a sulfur atom, and B
is a structure represented by any one of the following general
formulae (XVI) to (XVIV) and (XVII') ##STR00045## wherein in the
general formula (XVI), R.sub.54 is a hydrogen atom, an alkyl group,
an aralkyl group, an alkenyl group, an aryl group, a heterocyclic
group or an acyl group, R.sub.55 to R.sub.58 are, independently of
one another, a hydrogen atom, an alkyl group, an aryl group, a
carboxyl group, a carboxylate group or an acyl group, R.sub.55 and
R.sub.57 may bond to each other to form a ring, and R.sub.59 is a
hydrogen atom, an alkyl group, an alkoxy group or a halogen atom,
in the general formula (XVII), R.sub.60 is a hydrogen atom, an
alkyl group or an aryl group, R.sub.61 to R.sub.64 are,
independently of one another, a hydrogen atom, an alkyl group, an
aryl group, an alkoxy group, a carboxyl group, a sulfonic group, a
heterocyclic group, an amino group or a halogen atom, R.sub.61 and
R.sub.62, R.sub.62 and R.sub.63, or R.sub.63 and R.sub.64 may bond
to each other to form a ring, and Q is a sulfur atom, an oxygen
atom, --C(R.sub.65)(R.sub.66)-- or --CH.dbd.CH--, in which R.sub.65
and R.sub.66 bond to each other to form a ring, in the general
formula (XVII'), R.sub.60' is a hydrogen atom, an alkyl group or an
aryl group, Q.sub.3' is a sulfur atom or an oxygen atom, and dotted
lines in the general formula (XVII') represent the case where a
benzene ring is present or absent, in the general formula (XVIII),
R.sub.68 and R.sub.69 are, independently of each other, a hydrogen
atom, an alkyl group or an aryl group, and R.sub.70 is an oxygen
atom or a sulfur atom, and in the general formula (XVIV), R.sub.71
is an alkyl group, further wherein B and R.sub.5 may bond to each
other to form a benzothiophen-3-one ring, and at that time, R.sub.3
and R.sub.4 bond to each other to form a benzothiophen-3-one
ring.
2. The labeling composition for the intraocular tissue according to
claim 1, wherein the intraocular tissue is a retinal tissue.
3. The labeling composition for the intraocular tissue according to
claim 1, wherein the compound selectively labels at least one cell
selected from the group consisting of rod cells, plural kinds of
cone cells different in sensitivity to a wavelength region, and a
part of the cell bodies of these cells.
4. The labeling composition for the intraocular tissue according to
claim 1, which contains the compound at a concentration of 0.001
ng/mL or more and 100 .mu.g/mL or less.
5. The labeling composition for the intraocular tissue according to
claim 1, wherein the compound is a fluorescent compound.
6. A labeling composition for labeling an intraocular tissue of a
living individual, which comprises a compound capable of labeling
at least a photoreceptor cell layer of the retina.
7. The labeling composition for the intraocular tissue according to
claim 1, wherein the compound is administered without causing a
surgical damage to an ocular tissue or a nerve tissue linking to
the ocular tissue to label the intraocular tissue.
8. A staining agent for an intraocular tissue, which comprises the
labeling composition for the intraocular tissue according to claim
1.
9. A diagnostic composition comprising, as an active agent, the
labeling composition for the intraocular tissue according to claim
1 or a staining agent for the intraocular tissue, which comprises
the labeling composition for the intraocular tissue.
10. A staining method for an ocular tissue, which comprises a step
of bringing the labeling composition for the intraocular tissue
according to claim 1 or a staining agent for the intraocular
tissue, which comprises the labeling composition for the
intraocular tissue, into contact with the ocular tissue.
11. A multiple labeling method for an ocular tissue, which
comprises using plural kinds of labeling compositions including at
least one of the labeling composition for the intraocular tissue
according to claim 1 and a staining agent for the intraocular
tissue, which comprises the labeling composition for the
intraocular tissue.
12. The multiple labeling method according to claim 11, wherein at
least one of the plural kinds of labeling compositions contains at
least one antibody.
13. An imaging method for an intraocular tissue by conducting
labeling with the labeling method according to claim 10, which
comprises a step of irradiating the intraocular tissue with
excitation light to observe fluorescence.
14. An evaluating method for a condition of a retina, which
comprises using the imaging method of the intraocular tissue
according to claim 13.
15. A screening method comprising using a model animal labeled with
the labeling composition for the intraocular tissue according to
claim 1.
16. A screening method for a disease curing medicine or disease
preventing medicine, which comprises the steps of administering a
test substance to a model animal, administering the labeling
composition for the intraocular tissue according to claim 1 to the
model animal, and checking a labeled condition caused by the
labeling composition for the intraocular tissue with respect to the
intraocular tissue of the model animal.
17. The screening method according to claim 15, wherein the model
animal is Zebrafish.
18. The screening method according to claim 15, wherein the model
animal is mouse or rat.
Description
TECHNICAL FIELD
[0001] The present invention relates a labeling composition for an
intraocular tissue, which is used for noninvasively labeling an
intraocular tissue of a living sample, a labeling method of the
intraocular tissue, and a screening method.
BACKGROUND ART
[0002] In recent years, there has been a tendency to increase the
number of patients suffering ophthalmic diseases with the progress
of population aging. The progress of amblyopia by an ophthalmic
disease leads to blindness in the worst case, and the patient is
markedly impaired in the quality of life. Therefore, it is required
to detect the disease in its early stage and take a measure to cure
the disease or retard the progress thereof. Typical diseases
forming a cause of halfway blindness include diabetic retinopathy,
macular degeneration diseases and glaucoma, and the fundus retina
is concerned with many of them. For diagnoses of these diseases,
diagnoses by evaluation of visual function by inspection of visual
field, and morphological evaluation of the fundus retina using a
fundus camera, an optical coherence tomograph (OCT) and a scanning
laser ophthalmoscope are conducted.
[0003] In a retinopathic disease, reduction of visual functions is
caused by disorder of neurocytes. In recent years, it has become
possible to visualize the condition of a layer structure of a
reticular tissue by an OCT technique, and many new findings
relating to a living body tissue of a retinal lesion have been
obtained. However, a current OCT image is low in resolution, and so
it has been unable to visualize the morphology of cells forming the
retina.
[0004] As a visualization technique of an ocular tissue, a method
of imaging and staining the ocular tissue using a staining compound
has heretofore been known. For example, there is a method of
visualizing a choroidal vessel and a retinal vessel using a
fluorescent pigment such as indocyanine green or fluorescein
(Ophthalmologic Photograph, 23, pp. 3-10 (2007)). As a staining
method of the cornea, a method of using rose Bengal or fluorescein
is also known (EYE CONTACT LENS, 34, pages 61 to 64 (2008)).
[0005] On the other hand, as a visualization technique of
intraocular tissues such as a vitreous body, retina and optic
nerve, a method of administering a stain composition to a vitreous
cavity mainly at surgical operation is disclosed. For example,
there is a composition for staining a retinal membrane in a
vitreous retina surgery (Japanese Patent No. 3469198). It is also
disclosed that a transmission substance injected into the vitreous
body remains in the retina (Japanese Patent Application Laid-Open
No. 2007-262078). Besides, it is reported that in order to image
retinal ganglion cells of a rat, a retrograde axonal transport
tracer is injected into a superior colliculus of the midbrain
linking to a retinal ganglion to observe it with a laser
ophthalmoscope (Invest Ophthalmol Vis Sci, 47(10), p. 4198 (2006)).
As described above, it has been necessary to directly administer
the stain composition to the inside of an eye for visualizing the
intraocular tissue.
[0006] Incidentally, a drug to act on the retina or retinal nerve
tissue is administered by intravenous injection or orally. However,
the quantity of the drug transferred to the retina is extremely
small. On the other hand, drug administration to the vitreous
cavity enables a large quantity of the drug transferred to the
retinal tissue compared with the intravenous injection or oral
administration. However, an advanced technique is required, and a
burden on a patient is also great, and so it is extremely difficult
to use it except for the case of surgery or curing, and such
administration has been unable to be used for the morphological or
functional evaluation of the fundus retina.
DISCLOSURE OF THE INVENTION
[0007] It is an object of the present invention to provide a
labeling composition for an intraocular tissue, which can simply
label an intraocular tissue of a living individual. In particular,
the present invention provides a labeling composition, which can
label the intraocular tissue in vivo. Another object of the present
invention is to provide a method of noninvasively labeling an
intraocular tissue of a living individual without need of an
invasive operation such as exposure of an ocular tissue or needling
into the ocular tissue or a nerve tissue linking to the ocular
tissue. A further object of the present invention is to provide a
screening method using the labeling composition for the intraocular
tissue.
[0008] The present inventors have carried out an extensive
investigation for achieving the above objects. As a result, a group
of compounds, which provide new use of labeling an intraocular
tissue of a living individual, has been found. In other words, the
group of compounds, which can label the intraocular tissue of the
living individual, has been found to succeed in providing a
labeling composition for an intraocular tissue.
[0009] The present inventors have also established a method for
noninvasively labeling the intraocular tissue. The present
inventors have further developed an imaging method of the
intraocular tissue using the labeling composition for the
intraocular tissue according to the present invention, a screening
method using the labeling composition for the intraocular tissue,
which can label the intraocular tissue, and a diagnostic drug for
the intraocular tissue, thus leading to completion of the present
invention.
[0010] As shown in the following Comparative Examples, compounds
for staining an intraocular tissue, which have been conventionally
known, cannot stain an intraocular tissue of a living individual
according to a conventional method.
[0011] The present invention is as follows.
[0012] The present invention provides a labeling composition for an
intraocular tissue of a living individual, which comprises a
compound capable of labeling at least a photoreceptor cell layer of
a retina, wherein the compound is a staining compound having a
structure represented by the following general formula (I) or
general formula (II) as a partial structure thereof,
##STR00001##
wherein in the general formula (I), R.sub.1 and R.sub.2 are,
independently of each other, a hydrogen atom, an alkyl group or an
aryl group, R.sub.1 and R.sub.2 may bond to each other to form a
ring, and A is a structure represented by any one of the following
general formulae (III) to (IX) and (VIII')
##STR00002##
wherein in the general formula (III), R.sub.6 is an aryl group,
R.sub.7, R.sub.8, R.sub.10 and R.sub.11 are, independently of one
another, a hydrogen atom, an alkyl group, an aryl group or a
halogen atom, and R.sub.g is an ammonium salt group having a
counter anion, in the general formula (IV), R.sub.12 and R.sub.13
are, independently of each other, a hydrogen atom, an alkyl group,
an aryl group or a heterocyclic group, and R.sub.14 to R.sub.16
are, independently of one another, a hydrogen atom, an alkyl group,
an aryl group, a heterocyclic group, a halogen atom or an amino
group, in the general formula (V), R.sub.17 and R.sub.18 are,
independently of each other, a hydrogen atom, an alkyl group or an
alkoxy group, R.sub.19 and R.sub.20 are, independently of each
other, a hydrogen atom, an alkyl group, an alkoxy group, a hydroxyl
group or a cyano group, R.sub.21 is a heterocyclic group or
--CH.dbd.C(R.sub.22)(R.sub.23), R.sub.22 and R.sub.23 are,
independently of each other, a hydrogen atom, a cyano group, a
heterocyclic group, a carboxyl group or a carboxylate group, and
R.sub.1 and R.sub.17, R.sub.2 and R.sub.18, and R.sub.22 and
R.sub.23 may, independently of one another, bond to each other to
form a ring, in the general formula (VI), R.sub.24 is a hydrogen
atom, an alkyl group, an aryl group or a heterocyclic group, in the
general formula (VII), R.sub.25 is an oxygen atom, a sulfur atom or
N(R.sub.27), R.sub.26 is a hydrogen atom, an alkyl group, an alkoxy
group or a sulfonic group, and R.sub.27 is a hydrogen atom, an
alkyl group or an aryl group, in the general formula (VIII),
R.sub.28 is a hydrogen atom, an alkyl group or an aryl group, in
the general formula (VIII'), R.sub.28' is a halogen atom, an alkoxy
group or an aryloxy group, and in the general formula (IX),
R.sub.29 is a hydrogen atom, an alkyl group or an aryl group; and
wherein A is also a structure represented by the following general
formula (X)
##STR00003##
wherein in the general formula (X), R.sub.31 and R.sub.32 are
individually a sulfonic group or a salt thereof, and R.sub.33 and
R.sub.34 are individually a hydrogen atom, an alkyl group, an
alkoxy group or a halogen atom, with the proviso that R.sub.1 and
R.sub.2 in the general formula (I) bond to each other to form one
substituent group .dbd.N--R.sub.30, and R.sub.30 is an aryl group
or a heterocyclic group, further wherein in the general formula
(II), R.sub.3 and R.sub.4 are, independently of each other, a
hydrogen atom, an alkenyl group, a cyano group, a carboxyl group, a
carboxylate group, a sulfonic group, an acyl group or a
heterocyclic group, R.sub.3 and R.sub.4 may bond to each other to
form a ring, and R.sub.5 is a hydrogen atom, an alkyl group, an
aryl group or a heterocyclic group, with the proviso that one of
R.sub.3 and R.sub.4 is a hydrogen atom, and the other is a
heterocyclic group represented by the following general formula
(XI)
##STR00004##
wherein in the general formula (XI), R.sub.35 is an alkyl group or
an aryl group, R.sub.36 to R.sub.39 are, independently of one
another, a hydrogen atom, an alkyl group, an aryl group, an alkoxy
group, a carboxyl group, a sulfonic group, a heterocyclic group, an
amino group or a halogen atom, R.sub.36 and R.sub.37, R.sub.37 and
R.sub.38, or R.sub.38 and R.sub.39 may bond to each other to form a
ring, X.sup.- is an anionic group, and Q.sub.1 is a sulfur atom,
oxygen atom, --C(R.sub.40)(R.sub.41)--, --CH.dbd.CH-- or
--N(R.sub.42)--, in which R.sub.40 to R.sub.42 are individually a
hydrogen atom, an alkyl group or an aryl group, and R.sub.40 and
R.sub.41 may bond to each other to form a ring, and wherein the
ring formed by bonding R.sub.3 and R.sub.4 in the general formula
(II) to each other is represented by any one of the following
general formulae (XII), (XIII) and (XV)
##STR00005##
wherein in the general formula (XII), R.sub.43 is a hydrogen atom,
an alkyl group, an aryl group or a heterocyclic group, and R.sub.44
is an alkyl group, an aryl group, a carboxyl group, a carboxylate
group, a hydroxyl group or an amino group, in the general formula
(XIII), Q.sub.2 is an oxygen atom, a sulfur atom or
--N(R.sub.52)--, R.sub.45 is a hydrogen atom, an alkyl group, an
aryl group or a heterocyclic group, R.sub.46 is a sulfur atom, an
oxygen atom, .dbd.NR.sub.53, a heterocyclic group or a
dicyanomethylene group, and R.sub.52 and R.sub.53 are individually
a hydrogen atom, an alkyl group, an aryl group or a heterocyclic
group, and in the general formula (XV), R.sub.49 and R.sub.50 are,
independently of each other, a hydrogen atom, an alkyl group or an
aryl group, and R.sub.51 is an oxygen atom or a sulfur atom, and B
is a structure represented by any one of the following general
formulae (XVI) to (XVIV) and (XVII')
##STR00006##
wherein in the general formula (XVI), R.sub.54 is a hydrogen atom,
an alkyl group, an aralkyl group, an alkenyl group, an aryl group,
a heterocyclic group or an acyl group, R.sub.55 to R.sub.58 are,
independently of one another, a hydrogen atom, an alkyl group, an
aryl group, a carboxyl group, a carboxylate group or an acyl group,
R.sub.55 and R.sub.57 may bond to each other to form a ring, and
R.sub.59 is a hydrogen atom, an alkyl group, an alkoxy group or a
halogen atom, in the general formula (XVII), R.sub.60 is a hydrogen
atom, an alkyl group or an aryl group, R.sub.61 to R.sub.64 are,
independently of one another, a hydrogen atom, an alkyl group, an
aryl group, an alkoxy group, a carboxyl group, a sulfonic group, a
heterocyclic group, an amino group or a halogen atom, R.sub.61 and
R.sub.62, R.sub.62 and R.sub.63, or R.sub.63 and R.sub.64 may bond
to each other to form a ring, and Q.sub.3 is a sulfur atom, an
oxygen atom, --C(R.sub.65)(R.sub.66)-- or --CH.dbd.CH--, in which
R.sub.65 and R.sub.66 bond to each other to form a ring, in the
general formula (XVII'), R.sub.60' is a hydrogen atom, an alkyl
group or an aryl group, Q.sub.3' is a sulfur atom or an oxygen
atom, and dotted lines in the general formula (XVII') represent the
case where a benzene ring is present or absent, in the general
formula (XVIII), R.sub.68 and R.sub.69 are, independently of each
other, a hydrogen atom, an alkyl group or an aryl group, and
R.sub.70 is an oxygen atom or a sulfur atom, and in the general
formula (XVIV), R.sub.71 is an alkyl group, further wherein B and
R.sub.5 may bond to each other to form a benzothiophen-3-one ring,
and at that time, R.sub.3 and R.sub.4 bond to each other to form a
benzothiophen-3-one ring.
[0013] The present invention also provides a labeling composition
for labeling an intraocular tissue of a living individual, which
comprises a compound capable of labeling at least a photoreceptor
cell layer of the retina.
[0014] The present invention further provides a staining agent for
an intraocular tissue, which comprises the above-described labeling
composition for the intraocular tissue.
[0015] The present invention still further provides a diagnostic
composition comprising, as an active agent, the above-described
labeling composition or staining agent for the intraocular
tissue.
[0016] The present invention yet still further provides a staining
method of an ocular tissue, which comprises a step of bringing the
above-described labeling composition or staining agent for the
intraocular tissue into contact with the ocular tissue.
[0017] The present invention yet still further provides a multiple
staining method of an ocular tissue, which comprises using plural
kinds of labeling compositions including at least one of the
above-described labeling composition and staining agent for the
intraocular tissue.
[0018] The present invention yet still further provides an imaging
method of an intraocular tissue, which comprises a step of
irradiating the intraocular tissue with excitation light to observe
fluorescence, and comprises staining by the above-described
staining method.
[0019] The present invention yet still further provides an
evaluating method of the condition of the retina, which comprises
using the above-described imaging method of the intraocular
tissue.
[0020] The present invention yet still further provides a screening
method comprising using a model animal labeled with the
above-described labeling composition for the intraocular
tissue.
[0021] The present invention yet still further provides a screening
method of a disease curing medicine or disease preventing medicine,
which comprises a step of administering a test substance to a model
animal, a step of administering the above-described labeling
composition for the intraocular tissue to the model animal, and a
step of checking the condition of the labeling of the intraocular
tissue of the model animal with the labeling composition for the
intraocular tissue.
EFFECTS OF THE INVENTION
[0022] The present invention provides a labeling composition for an
intraocular tissue, whereby noninvasive labeling of the intraocular
tissue, which has heretofore been impossible to conduct, becomes
feasible, and so it is possible to image the condition of a layer
structure of the intraocular tissue and the cell morphology thereof
in a simple and easy way and with high definition. It is thereby
possible to conduct evaluation and analysis of the intraocular
tissue with high precision. In addition, the labeling composition
for the intraocular tissue according to the present invention is
combined with an observing and analyzing apparatus for intraocular
tissues, whereby the condition of an intraocular tissue, or the
retina in particular, of a living individual, which has heretofore
been difficult to be observed, can be grasped with accuracy and
high sensitivity, and so a new tool effective for researches and
diagnoses in an ophthalmic field can be provided.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIG. 1 illustrates labeling of a retinal tissue observed in
Example 4.
[0024] FIG. 2 illustrates labeling of a retinal tissue observed in
Example 2.
[0025] FIG. 3 illustrates a fluorescent observation image of a
retinal tissue observed in Comparative Example 1.
[0026] FIG. 4 illustrates an observation image of a retinal tissue
in a living body observed in Example 118.
[0027] FIG. 5 illustrates a labeled image of a frozen section of a
Zebrafish eyeball observed in Example 120.
[0028] FIG. 6 illustrates a labeled image of a frozen section of a
man eyeball observed in Example 122.
[0029] FIG. 7 illustrates a labeled image of a frozen section of a
mouse eyeball observed in Example 123.
BEST MODE FOR CARRYING OUT THE INVENTION
[0030] The present invention will hereinafter be described in
detail.
<Labeling of Intraocular Tissue>
Definition of Labeling
[0031] In the present invention, the labeling of an intraocular
tissue means that a pigment composition, which is an active agent
in the above-described composition, is held in an intraocular
tissue, or on the surface thereof, or at the periphery thereof,
thereby becoming a state capable of detecting at least one of the
form, position and function of the intraocular tissue. Detections
include a method of acquiring a fluorescent image or labeled image
by an image acquiring unit, which will be described subsequently,
and a visually observing method.
[0032] A typical example of the labeling is staining, and the
labeling composition for the intraocular tissue, by which the
so-called cell staining or tissue staining becomes feasible, is
provided by the present invention.
Labeling Mechanism
[0033] A detailed mechanism by which the labeling composition for
the intraocular tissue according to the present invention labels a
specific intraocular tissue is not known. However, it is inferred
that the group of compounds provided by the present invention is
easy to permeate a cell membrane and migrate because they are
stable compounds with a low molecular weight and have a structure
easy to be held in a specific cell, thereby being held in the
specific tissue of the cell.
[0034] In addition, the labeling composition for the intraocular
tissue according to the present invention favorably labels the
intraocular tissue immediately after administration to a living
body. However, it may take a certain period of time to label the
intraocular tissue according to a method of administration. From
this point of view, the composition favorably contains a staining
compound capable of labeling within several minutes or several
hours after the administration.
[0035] In the case where one wished to observe a change with time,
it is favorable to select a staining compound remaining for a long
period of time after being labeled. However, it is favorable to
select a staining compound easy to be discharged by metabolism
after the administration for reducing an influence on the living
body.
<Living Sample>
[0036] No particular limitation is imposed on a living individual,
the intraocular tissue of which can be labeled with the labeling
composition for the intraocular tissue according to the present
invention. However, examples of vertebrate animals include bony
fishes such as T. rubripes, T. niphobles, T. nigrovirides, Japanese
killifish and Zebrafish; Amphibia such as X. laevis; birds such as
domestic fowl and quail; small animals such as rat, mouse and
hamster; large animals such as goat, pig, dog, cat, cattle and
horse; monkey; chimpanzee; and human. In particular, the
intraocular tissues of these living individuals can be labeled in a
living state. The human may be excluded from the living
samples.
[0037] In Zebrafish, main organs are formed in 6 and 7 days after
fertilization, and the histological form of an eyeball is also
almost completed. Therefore, the use of a 7-day-old embryo makes it
possible to conduct a test almost without having an influence of
individual differences. In Zebrafish, at least about 200 fertilized
ova are obtained in one spawning, and so there is a merit of
obtaining Zebrafishes having the same genetic background to be a
good convenience to screening.
<Intraocular Tissue>
[0038] No particular limitation is imposed on an intraocular
tissue, which can be labeled with the labeling composition for the
intraocular tissue according to the present invention, so far as it
is an intraocular tissue forming an intraocular structure being in
no direct contact with the outside in an eyeball and is a tissue
present in the eye, excluding tissues on the surface of the
eyeball. Examples thereof include a retinal tissue comprising
retinal pigment epithelium layer, photoreceptor cell layer, outer
limiting membrane, outer granular layer, outer reticular layer,
internal granular layer, internal reticular layer, ganglion cell
layer and internal limiting membrane, iris, ciliary body, choroid,
lens, vitreous body, lacrimal gland, optic nerve, optic disk, optic
tract, retinal blood vessel, diseased-condition tissues of these
tissues, and neogenetic tissues and cancer tissues caused by
diseases. When intraocular tissues different from the
above-described tissues are present according to the kind of a
living body or development stage or due to developmental anomaly or
a disease, their tissues may also be included.
[0039] The labeling composition for the intraocular tissue
according to the present invention is favorably used in staining of
a retinal tissue in particular. In the staining of the retinal
tissue, one or more tissues including the photoreceptor cell layer
among the above-described plural layers forming the retinal tissue
or cells thereof may be stained, so that availability becomes high
in uses such as visualization of a retinal disease.
[0040] No particular limitation is imposed on the cells included in
the above-described retinal tissue. However, examples thereof
include amacrine cells, horizontal cells, bipolar cells,
interplexiform cells, cone cells, rod cells, fibroblasts, Muller
glia cells, and tumor cells and undifferentiated cells (stem cells)
thereof. The labeling composition for the intraocular tissue
according to the present invention can favorably stain one or more
cells of the rod cells and plural kinds of cone cells different in
the sensitivity to a wavelength region.
[0041] In the present invention, to stain the cell morphology of
the retina means that at least one of cells making up the retinal
cells is stained to become a state that the cell morphology can be
clearly determined one by one.
<Administering Method>
[0042] In the present invention, to label the intraocular tissue
without damaging the ocular tissue or the nerve tissue linking to
the ocular tissue means labeling the intraocular tissue with the
labeling composition for the intraocular tissue without applying a
surgical damage such as incision of the ocular tissue or needling
into the ocular tissue or the nerve tissue linking to the ocular
tissue to the ocular tissue.
[0043] No particular limitation is imposed on the labeling method
without the surgical damage. However, examples thereof include a
method of exposing a part or the whole of a living individual to
the labeling composition for the intraocular tissue, a method by
oral contact, a method by pneumonic contact, a method by nasal
contact, a method by transgastrointestinal contact, a method by
transmucosal contact, a method by transhumoral contact, a method by
hypoglossal contact, a method by intravascular contact such as
intravenous or intra-arterial contact, a method by intra-abdominal
contact, an intra-abdominal, subcutaneous, intracutaneous,
intravesical or endotracheal (intrabronchial) injection method, and
a method by contact with the interior of a living body by a device
such as spraying or coating.
[0044] In the present invention, the nerve tissue linking to the
ocular tissue means a nerve tissue histologically linking to the
ocular tissue. Specifically, examples thereof include an optic
nerve tissue and a cranial nerve tissue though not particularly
limited thereto.
[0045] In the case of administration to an animal, the
administering form, administering route and dose thereof are
suitably selected according to the body weight and condition of the
subject animal.
<Compound>
[0046] The compound contained in the labeling composition for the
intraocular tissue according to the present invention includes a
compound capable of labeling at least a photoreceptor cell layer of
the retina. Favorably, the compound includes a compound selectively
labeling one or more cells selected from rod cells present in the
photoreceptor cell layer of the retina and plural kinds of cone
cells or a part of the cell bodies of these cells. "Selective
labeling" in the present invention means a state that at least a
specific cell or a part thereof is labeled, and unlabeled cells or
a part thereof exist in the retinal tissue.
[0047] The compound contained in the labeling composition for the
intraocular tissue according to the present invention is favorably
a low-molecular weight compound because the compound is caused to
migrate to the intraocular tissue as an object of labeling, in
particular, the retinal tissue, and so a compound having a
molecular weight of 2,000 or less is selected. The compound is
favorably a compound having a molecular weight of favorably 1,500
or less, particularly 1,000 or less.
[0048] The compound of the present invention is favorably a
fluorescent compound having fluorescence. Since the fluorescent
compound is high in sensitivity, labeling becomes feasible at a low
concentration, and so a necessary amount of the compound can be
relatively reduced. Compounds different in labeling sites and
fluorescent spectrum are combined and selected, whereby multiple
labeling of plural tissues becomes feasible, so that information on
a plurality of tissues can be obtained in one observation, and so
availability becomes high.
[0049] The compound used in the labeling composition for the
intraocular tissue according to the present invention is favorably
a staining compound having a structure represented by the following
general formula (I) or general formula (II) as a partial structure
thereof.
##STR00007##
[0050] In the general formula (I), R.sub.1 and R.sub.2 are,
independently of each other, a hydrogen atom, an alkyl group or an
aryl group, and R.sub.1 and R.sub.2 may bond to each other to form
a ring.
[0051] No particular limitation is imposed on the alkyl group in
R.sub.1 and R.sub.2. However, examples thereof include linear,
branched or cyclic alkyl groups having 1 to 20 carbon atoms, such
as methyl, ethyl, propyl, butyl, pentyl, hexyl, octyl, nonyl,
decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl,
hexadecyl, heptadecyl, octadecyl, nonadecyl, cyclopropyl,
cyclobutyl and cyclopentyl groups.
[0052] No particular limitation is imposed on the aryl group in
R.sub.1 and R.sub.2. However, examples thereof include 6- to
14-membered monocyclic or polycyclic aryl groups such as phenyl,
naphthyl, phenanthryl and anthracenyl groups.
[0053] R.sub.1 and R.sub.2 may additionally have substituent(s),
and no particular limitation is imposed on the substituent(s) so
far as the storage stability of the staining compound is not
markedly impaired. Examples thereof include alkyl groups such as
methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl
and tert-butyl groups; aryl groups such as phenyl and naphthyl
groups; alkoxy groups such as methoxy, ethoxy and butoxy groups;
aryloxy groups such as phenoxy and naphtyloxy groups; alkylsulfanyl
groups such as thiomethyl, thioethyl, thiopropyl, thiobutyl and
thiophenyl groups; mono-substituted amino groups such as
methylamino and butylamino groups; di-substituted amino groups such
as dimethylamino, N-ethyl-N-phenylamino and diphenylamino groups;
acyl groups such as acetyl, benzoyl, carboxyl, carboxylate and
carbamoyl groups; sulfonyl groups such as sulfonic, sulfonate and
sulfamoyl groups; heterocyclic groups such as pyridyl, triazinyl
and benzothiazolyl groups; a nitro group; halogen atoms such as
fluorine, chlorine, bromine and iodine atoms; a polyethylene glycol
groups; and salts such as quaternary ammonium salts, carboxylates
and sulfonates. The groups favorably have a substituent having such
a nature that the water solubility is improved, among these
substituents. Particularly favorable examples of such a substituent
include carboxyl, sulfonic, polyethylene glycol, carboxylate and
sulfonate groups though not limited thereto. Description of these
substituents exemplified above is hereinafter omitted as described
as "favorable examples of additional substituents" in the present
specification.
[0054] No particular limitation is imposed on the ring formed by
bonding R.sub.1 and R.sub.2 to each other. However, examples
thereof include heterocycles such as pyridine, piperazine,
morpholine, thiomorpholine and pyridinium rings.
Description of A in General Formula (I)
[0055] A is a structure represented by any one of the following
general formulae (III) to (IX) and (VIII').
##STR00008## ##STR00009##
[0056] Each structure will hereinafter be described in detail.
Description of General Formula (III)
[0057] In the general formula (III), R.sub.6 is an aryl group.
R.sub.7, R.sub.8, R.sub.10 and R.sub.11 are, independently of one
another, a hydrogen atom, an alkyl group, an aryl group or a
halogen atom. R.sub.9 is an ammonium salt group having a counter
anion.
[0058] No particular limitation is imposed on the aryl group in
R.sub.6. However, examples thereof include 6- to 14-membered
monocyclic or polycyclic aryl groups such as phenyl, naphthyl,
phenanthryl and anthracenyl groups. The ring may additionally have
substituent(s), and no particular limitation is imposed on the
substituent(s) so far as the storage stability of the staining
compound is not markedly impaired. Examples thereof include
substituents described as "favorable examples of additional
substituents" above.
[0059] The group favorably has a substituent having such a nature
that the water solubility is improved, among these substituents.
Particularly favorable examples of such a substituent include
carboxyl, sulfonic, polyethylene glycol, carboxylate and sulfonate
groups though not limited thereto.
[0060] R.sub.6 is favorably a phenyl or naphthyl group. In
particular, a phenyl group having a water-soluble substituent such
as a carboxyl, sulfonic, polyethylene glycol, carboxylate or
sulfonate group is favorable from the viewpoint of improving the
water solubility of the compound.
[0061] No particular limitation is imposed on the alkyl group in
R.sub.7, R.sub.8, R.sub.10 and R.sub.11. However, examples thereof
include linear, branched or cyclic alkyl groups having 1 to 20
carbon atoms, such as methyl, ethyl, propyl, butyl, pentyl, hexyl,
octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl,
pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl,
cyclopropyl, cyclobutyl and cyclopentyl groups.
[0062] No particular limitation is imposed on the aryl group in
R.sub.7, R.sub.8, R.sub.10 and R.sub.11. However, examples thereof
include 6- to 14-membered monocyclic or polycyclic aryl groups such
as phenyl, naphthyl, phenanthryl and anthracenyl groups.
[0063] Examples of the halogen atom in R.sub.7, R.sub.8, R.sub.10
and R.sub.11 include fluorine, chlorine, bromine and iodine
atoms.
[0064] R.sub.7, R.sub.8, R.sub.10 and R.sub.11 are each favorably a
hydrogen atom, an alkyl group or a halogen atom, and the hydrogen
atom is particularly favorable from the viewpoint of stability of
the compound.
[0065] No particular limitation is imposed on the ammonium salt
group having a counter anion in R.sub.9. However, examples thereof
include amino groups obtained by converting an amino group to a
quaternary salt. No particular limitation is imposed on the counter
anion. However, examples thereof include halide ions such as
fluoride, chloride, bromide and iodide ions; inorganic acid ions
such as sulfate, phosphate, nitrate, tetrafluoroborate and
hexafluorophosphate ions; Lewis-acid-containing ions such as
tetrachloroaluminate ion; and organic acid ions such as acetate,
lactate, methanesulfonate, benzenesulfonate, p-toluenesulfonate,
trifluoroacetate, trifluoromethanesulfonate and tetraphenylborate
ions.
[0066] When R.sub.6 in the molecular structure represented by the
general formula (III) is an aromatic ring having a sulfonic or
carboxyl group at an ortho position, a tautomer of the following
general formula (III') or (III'') exists. The structure represented
by the general formula (III) that the staining compound according
to the present invention has also includes a structure represented
by the following general formula (III') or (III'').
##STR00010##
wherein R.sub.7 to R.sub.11 in the general formulae (III') and
(III'') have the same meanings as R.sub.7 to R.sub.11 in the
general formulae (III).
Description of General Formula (IV)
[0067] In the general formula (IV), R.sub.12 and R.sub.13 are,
independently of each other, a hydrogen atom, an alkyl group, an
aryl group or a heterocyclic group. R.sub.14 to R.sub.16 are,
independently of one another, a hydrogen atom, an alkyl group, an
aryl group, a heterocyclic group, a halogen atom or an amino
group.
[0068] No particular limitation is imposed on the alkyl group in
R.sub.12, R.sub.13, and R.sub.14 to R.sub.16. However, examples
thereof include linear, branched or cyclic alkyl groups having 1 to
20 carbon atoms, such as methyl, ethyl, propyl, butyl, pentyl,
hexyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl,
pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl,
cyclopropyl, cyclobutyl and cyclopentyl groups.
[0069] No particular limitation is imposed on the aryl group in
R.sub.12, R.sub.13, and R.sub.14 to R.sub.16. However, examples
thereof include 6- to 14-membered monocyclic or polycyclic aryl
groups such as phenyl, naphthyl, phenanthryl and anthracenyl
groups.
[0070] No particular limitation is imposed on the heterocyclic
group in R.sub.12, R.sub.13, and R.sub.14 to R.sub.16. However,
examples thereof include 4- to 10-membered monocyclic or bicyclic
heterocyclic groups containing 1 to 4 atoms selected from nitrogen,
oxygen and sulfur. Examples of these groups include pyridyl,
pyrazinyl, pyrimidinyl, pyrrolyl, thienyl, furyl, pyranyl,
oxazolyl, thiazolyl, triazolyl, tetrazolyl, imidazolyl, pyrazolyl,
morpholinyl, thiomorpholinyl, piperidinyl, piperazinyl, quinolyl,
isoquinolyl, indolyl, isoindolyl, benzofuryl and benzothienyl
groups.
[0071] R.sub.12 and R.sub.13 are each favorably an alkyl group,
with a methyl, ethyl, propyl or butyl group being particularly
favorable.
[0072] Examples of the halogen atom in R.sub.14 to R.sub.16 include
fluorine, chlorine, bromine and iodine atoms.
[0073] No particular limitation is imposed on the amino group in
R.sub.14 to R.sub.16. However, examples thereof include an
unsubstituted amino group; mono-substituted amino groups such as
N-methylamino, N-butylamino, N-hexylamino, N-tetradecylamino,
N-phenylamino and N-naphthylamino groups; di-substituted amino
groups such as N,N-dimethylamino, N,N-diethylamino,
N,N-diphenylamino and N,N-methylpropylamino groups; carbonylamino
groups such as acetylamino, ethylcarbonylamino,
tert-butylcarbonylamino, benzoylamino, naphthoylamino and
methoxycarbonylamino groups; and sulfonylamino groups such
methylsulfonylamino, ethylsulfonylamino, tert-butylsulfonylamino
and isopropoxysulfonylamino groups.
[0074] R.sub.14 to R.sub.16 are each favorably a hydrogen atom, an
alkyl group, a halogen atom or an amino group, with the hydrogen
atom being particularly favorable.
Description of General Formula (V)
[0075] In the general formula (V), R.sub.17 and R.sub.18 are,
independently of each other, a hydrogen atom, an alkyl group or an
alkoxy group. R.sub.19 and R.sub.20 are, independently of each
other, a hydrogen atom, an alkyl group, an alkoxy group, a hydroxyl
group or a cyano group. R.sub.21 is a heterocyclic group or
--CH.dbd.C(R.sub.22)(R.sub.23). R.sub.22 and R.sub.23 are,
independently of each other, a hydrogen atom, a cyano group, a
heterocyclic group, a carboxyl group or a carboxylate group.
R.sub.1 and R.sub.17, R.sub.2 and R.sub.18, and R.sub.22 and
R.sub.23 may, independently of one another, bond to each other to
form a ring.
[0076] No particular limitation is imposed on the alkyl group in
R.sub.17 to R.sub.20. However, examples thereof include linear,
branched or cyclic alkyl groups having 1 to 20 carbon atoms, such
as methyl, ethyl, propyl, butyl, pentyl, hexyl, octyl, nonyl,
decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl,
hexadecyl, heptadecyl, octadecyl, nonadecyl, cyclopropyl,
cyclobutyl and cyclopentyl groups.
[0077] No particular limitation is imposed on the alkoxy group in
R.sub.17 to R.sub.20. However, examples thereof include alkoxy
groups having 1 to 20 carbon atoms, such as methoxy, ethoxy,
propoxy, butoxy, pentyloxy, hexyloxy, heptyloxy, octyloxy,
nonyloxy, decyloxy, dodecyloxy and octadecyloxy groups.
[0078] R.sub.17 to R.sub.20 are each favorably a hydrogen atom or
an alkoxy group, with the hydrogen atom being particularly
favorable from the viewpoint of stability of the compound.
[0079] No particular limitation is imposed on the heterocyclic
group in R.sub.21 to R.sub.23. However, examples thereof include 4-
to 10-membered monocyclic or bicyclic heterocyclic groups
containing 1 to 4 atoms selected from nitrogen, oxygen and sulfur.
Examples of these groups include pyridyl, pyrazinyl, pyrimidinyl,
pyrrolyl, thienyl, furyl, pyranyl, oxazolyl, thiazolyl, triazolyl,
tetrazolyl, imidazolyl, pyrazolyl, morpholinyl, thiomorpholinyl,
piperidinyl, piperazinyl, quinolyl, isoquinolyl, indolyl,
isoindolyl, benzofuryl and benzothienyl groups.
[0080] The heterocyclic group in R.sub.21 is favorably an oxazolyl,
thiazolyl or imidazolyl group, with the oxazolyl group being
particularly favorable from the viewpoint of staining ability.
[0081] No particular limitation is imposed on the carboxylate group
in R.sub.21 to R.sub.23. However, examples thereof include methyl
carboxylate, ethyl carboxylate, propyl carboxylate and butyl
carboxylate groups.
[0082] R.sub.21 to R.sub.23 are each favorably a pyridyl,
pyrazinyl, pyrimidinyl, oxazolyl, thiazolyl, triazolyl, tetrazolyl,
imidazolyl or pyrazolyl group, with the oxazolyl, thiazolyl or
imidazolyl group being particularly favorable from the viewpoint of
stability of the compound.
[0083] No particular limitation is imposed on the ring formed by
bonding, independently of one another, R.sub.1 and R.sub.17,
R.sub.2 and R.sub.18, and R.sub.22 and R.sub.23 to each other.
However, examples thereof include aromatic rings having 3 to 10
carbon atoms, such as benzene and naphthalene rings; saturated
rings such as cyclooctane, cycloheptane, cyclohexane, cyclopentane
and cyclobutane rings; partially saturated rings such as
cyclopentene and cyclohexene rings; and heterocycles such as
pyridine, tetrahydropyridine and pyrimidine rings. These rings may
additionally have substituent(s), and no particular limitation is
imposed on the substituent(s) so far as the storage stability of
the staining compound is not markedly impaired. Examples thereof
include substituents described as "favorable examples of additional
substituents" above.
[0084] The ring favorably has a substituent having such a nature
that the water solubility is improved, among these substituents.
Particularly favorable examples of such a substituent include
carboxyl, sulfonic, polyethylene glycol, carboxylate and sulfonate
groups though not limited thereto.
[0085] The ring formed by bonding, independently of one another,
R.sub.1 and R.sub.17, R.sub.2 and R.sub.18, and R.sub.22 and
R.sub.23 to each other is favorably a heterocycle, with a
tetrahydropyridine ring being particularly favorable.
Description of General Formula (VI)
[0086] In the general formula (VI), R.sub.24 is a hydrogen atom, an
alkyl group, an aryl group or a heterocyclic group.
[0087] No particular limitation is imposed on the alkyl group in
R.sub.24. However, examples thereof include linear, branched or
cyclic alkyl groups having 1 to 20 carbon atoms, such as methyl,
ethyl, propyl, butyl, pentyl, hexyl, octyl, nonyl, decyl, undecyl,
dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl,
octadecyl, nonadecyl, cyclopropyl, cyclobutyl and cyclopentyl
groups. The group may additionally have substituent(s), and no
particular limitation is imposed on the substituent(s) so far as
the storage stability of the staining compound is not markedly
impaired. Examples thereof include substituents described as
"favorable examples of additional substituents" above.
[0088] The group favorably has a substituent having such a nature
that the water solubility is improved, among these substituents.
Particularly favorable examples of such a substituent include
carboxyl, sulfonic, polyethylene glycol, carboxylate and sulfonate
groups though not limited thereto.
[0089] No particular limitation is imposed on the aryl group in
R.sub.24. However, examples thereof include 6- to 14-membered
monocyclic or polycyclic aryl groups such as phenyl, naphthyl,
phenanthryl and anthracenyl groups.
[0090] No particular limitation is imposed on the heterocyclic
group in R.sub.24. However, examples thereof include 4- to
10-membered monocyclic or bicyclic heterocyclic groups containing 1
to 4 atoms selected from nitrogen, oxygen and sulfur. Examples of
these groups include pyridyl, pyrazinyl, pyrimidinyl, pyrrolyl,
thienyl, furyl, pyranyl, oxazolyl, thiazolyl, triazolyl,
tetrazolyl, imidazolyl, pyrazolyl, morpholinyl, thiomorpholinyl,
piperidinyl, piperazinyl, quinolyl, isoquinolyl, indolyl,
isoindolyl, benzofuryl and benzothienyl groups.
[0091] R.sub.24 is favorably an alkyl group, with a methyl, ethyl,
propyl, butyl, pentyl or hexyl group being particularly favorable
from the viewpoint of stability of the compound.
Description of General Formula (VII)
[0092] In the general formula (VII), R.sub.25 is an oxygen atom, a
sulfur atom or N(R.sub.27). R.sub.26 is a hydrogen atom, an alkyl
group, an alkoxy group or a sulfonic group. R.sub.27 is a hydrogen
atom, an alkyl group or an aryl group.
[0093] R.sub.25 is favorably an oxygen atom or N(R.sub.27), with NH
being particularly favorable from the viewpoint of staining
ability.
[0094] No particular limitation is imposed on the alkyl group in
R.sub.26 and R.sub.27. However, examples thereof include linear,
branched or cyclic alkyl groups having 1 to 20 carbon atoms, such
as methyl, ethyl, propyl, butyl, pentyl, hexyl, octyl, nonyl,
decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl,
hexadecyl, heptadecyl, octadecyl, nonadecyl, cyclopropyl,
cyclobutyl and cyclopentyl groups.
[0095] No particular limitation is imposed on the alkoxy group in
R.sub.26. However, examples thereof include alkoxy groups having 1
to 20 carbon atoms, such as methoxy, ethoxy, propoxy, butoxy,
pentyloxy, hexyloxy, heptyloxy, octyloxy, nonyloxy, decyloxy,
dodecyloxy and octadecyloxy groups.
[0096] R.sub.26 is favorably a hydrogen atom or a sulfonic group,
with the hydrogen atom being particularly favorable.
[0097] No particular limitation is imposed on the aryl group in
R.sub.27. However, examples thereof include 6- to 14-membered
monocyclic or polycyclic aryl groups such as phenyl, naphthyl,
phenanthryl and anthracenyl groups.
Description of General Formula (VIII)
[0098] In the general formula (VIII), R.sub.28 is a hydrogen atom,
an alkyl group or an aryl group.
[0099] No particular limitation is imposed on the alkyl group in
R.sub.28. However, examples thereof include linear, branched or
cyclic alkyl groups having 1 to 20 carbon atoms, such as methyl,
ethyl, propyl, butyl, pentyl, hexyl, octyl, nonyl, decyl, undecyl,
dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl,
octadecyl, nonadecyl, cyclopropyl, cyclobutyl and cyclopentyl
groups. The group may additionally have substituent(s), and no
particular limitation is imposed on the substituent(s) so far as
the storage stability of the staining compound is not markedly
impaired. Examples thereof include substituents described as
"favorable examples of additional substituents" above.
[0100] The group favorably has a substituent having such a nature
that the water solubility is improved, among these substituents.
Particularly favorable examples of such a substituent include
carboxyl, sulfonic, polyethylene glycol, carboxylate and sulfonate
groups though not limited thereto.
[0101] No particular limitation is imposed on the aryl group in
R.sub.28. However, examples thereof include 6- to 14-membered
monocyclic or polycyclic aryl groups such as phenyl, naphthyl,
phenanthryl and anthracenyl groups.
Description of General Formula (VIII')
[0102] In the general formula (VIII'), R.sub.28' is a halogen atom,
an alkoxy group or an aryloxy group.
[0103] The halogen atom in R.sub.28' is a chlorine, bromine or
iodine atom.
[0104] No particular limitation is imposed on the alkoxy group in
R.sub.28'. However, examples thereof include alkoxy groups having 1
to 20 carbon atoms, such as methoxy, ethoxy, propoxy, butoxy,
pentyloxy, decyloxy, hexyloxy, heptyloxy, octyloxy, nonyloxy,
decyloxy, dodecyloxy and octadecyloxy groups. The group may
additionally have substituent(s), and no particular limitation is
imposed on the substituent(s) so far as the storage stability of
the staining compound is not markedly impaired. Examples thereof
include substituents described as "favorable examples of additional
substituents" above.
[0105] The group favorably has a substituent having such a nature
that the water solubility is improved, among these substituents.
Particularly favorable examples of such a substituent include
carboxyl, sulfonic, polyethylene glycol, carboxylate and sulfonate
groups though not limited thereto.
[0106] No particular limitation is imposed on the aryl group in
R.sub.28'. However, examples thereof include 6- to 14-membered
monocyclic or polycyclic aryl groups such as phenyl, naphthyl,
phenanthryl and anthracenyl groups.
Description of General Formula (IX)
[0107] In the general formula (IX), R.sub.29 is a hydrogen atom, an
alkyl group or an aryl group.
[0108] No particular limitation is imposed on the alkyl group in
R.sub.29. However, examples thereof include linear, branched or
cyclic alkyl groups having 1 to 20 carbon atoms, such as methyl,
ethyl, propyl, butyl, pentyl, hexyl, octyl, nonyl, decyl, undecyl,
dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl,
octadecyl, nonadecyl, cyclopropyl, cyclobutyl and cyclopentyl
groups. The group may additionally have substituent(s), and no
particular limitation is imposed on the substituent(s) so far as
the storage stability of the staining compound is not markedly
impaired. Examples thereof include substituents described as
"favorable examples of additional substituents" above.
[0109] The group favorably has a substituent having such a nature
that the water solubility is improved, among these substituents.
Particularly favorable examples of such a substituent include
carboxyl, sulfonic, polyethylene glycol, carboxylate and sulfonate
groups though not limited thereto.
[0110] No particular limitation is imposed on the aryl group in
R.sub.29. However, examples thereof include 6- to 14-membered
monocyclic or polycyclic aryl groups such as phenyl, naphthyl,
phenanthryl and anthracenyl groups.
Another Mode of A in the General Formula (I)
[0111] When A is a structure represented by the following general
formula (X), R.sub.1 and R.sub.2 in the general formula (I) bond to
each other to form one substituent group: .dbd.N--R.sub.30.
R.sub.30 is an aryl group or a heterocyclic group.
##STR00011##
[0112] In the general formula (X), R.sub.31 and R.sub.32 are
individually a sulfonic group or a salt thereof. R.sub.33 and
R.sub.34 are individually a hydrogen atom, an alkyl group, an
alkoxy group or a halogen atom.
[0113] No particular limitation is imposed on the alkyl group in
R.sub.33 and R.sub.34. However, examples thereof include linear,
branched or cyclic alkyl groups having 1 to 20 carbon atoms, such
as methyl, ethyl, propyl, butyl, pentyl, hexyl, octyl, nonyl,
decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl,
hexadecyl, heptadecyl, octadecyl, nonadecyl, cyclopropyl,
cyclobutyl and cyclopentyl groups.
[0114] No particular limitation is imposed on the alkoxy group in
R.sub.33 to R.sub.34. However, examples thereof include alkoxy
groups having 1 to 20 carbon atoms, such as methoxy, ethoxy,
propoxy, butoxy, pentyloxy, decyloxy, hexyloxy, heptyloxy,
octyloxy, nonyloxy, decyloxy, dodecyloxy and octadecyloxy
groups.
[0115] Examples of the halogen atom in R.sub.33 and R.sub.34
include fluorine, chlorine, bromine and iodine atoms.
[0116] R.sub.33 to R.sub.34 are each favorably a hydrogen atom, an
alkoxy group or a halogen atom, with the hydrogen atom being
particularly favorable.
[0117] No particular limitation is imposed on the aryl group in
R.sub.30. However, examples thereof include 6- to 14-membered
monocyclic or polycyclic aryl groups such as phenyl, naphthyl,
phenanthryl and anthracenyl groups.
[0118] R.sub.30 is favorably an aryl group, with a naphthyl group
being particularly favorable.
Description of General Formula (II)
[0119] In addition, in the general formula (II), R.sub.3 and
R.sub.4 are, independently of each other, a hydrogen atom, an
alkenyl group, a cyano group, a carboxyl group, a carboxylate
group, a sulfonic group, an acyl group or a heterocyclic group.
R.sub.3 and R.sub.4 may bond to each other to form a ring. R.sub.5
is a hydrogen atom, an alkyl group, an aryl group or a heterocyclic
group.
[0120] No particular limitation is imposed on the alkenyl group in
R.sub.3 and R.sub.4 of the general formula (II). However, examples
thereof include alkenyl groups having 2 to 20 carbon atoms, such as
vinyl, 2,2-diphenylvinyl, 3-butenyl and cyclohexenyl groups.
[0121] No particular limitation is imposed on the carboxylate group
in R.sub.3 and R.sub.4. However, examples thereof include methyl
carboxylate, ethyl carboxylate, propyl carboxylate and butyl
carboxylate groups.
[0122] No particular limitation is imposed on the acyl group in
R.sub.3 and R.sub.4. However, examples thereof include acetyl,
propionyl, butyryl, pentanoyl, benzoyl, 1-naphthoyl and 2-naphthoyl
groups.
[0123] No particular limitation is imposed on the heterocyclic
group in R.sub.3 and R.sub.4. However, examples thereof include 4-
to 10-membered monocyclic or bicyclic heterocyclic groups
containing 1 to 4 atoms selected from nitrogen, oxygen and sulfur.
Examples of these groups include pyridyl, pyrazinyl, pyrimidinyl,
pyrrolyl, thienyl, furyl, pyranyl, oxazolyl, thiazolyl, triazolyl,
tetrazolyl, imidazolyl, pyrazolyl, morpholinyl, thiomorpholinyl,
piperidinyl, piperazinyl, quinolyl, isoquinolyl, indolyl,
isoindolyl, benzofuryl and benzothienyl groups.
[0124] Either one of R.sub.3 and R.sub.4 is favorably a cyano,
carboxyl or heterocyclic group from the viewpoint of ease of
synthesis of the compound, and either one of R.sub.3 and R.sub.4 is
particularly favorably a cyano group.
Description of the Case where One of R.sub.3 and R.sub.4 in General
Formula (II) is Hydrogen Atom, and the Other is Heterocyclic Group
of General Formula (XI)
[0125] R.sub.3 and R.sub.4 are favorably such that one of them is a
hydrogen atom and the other is a heterocyclic group of the general
formula (XI).
##STR00012##
wherein in the general formula (XI), R.sub.35 is an alkyl group or
an aryl group, R.sub.36 to R.sub.39 are, independently of one
another, a hydrogen atom, an alkyl group, an aryl group, an alkoxy
group, a carboxyl group, a sulfonic group, a heterocyclic group, an
amino group or a halogen atom, R.sub.36 and R.sub.37, R.sub.37 and
R.sub.38, or R.sub.38 and R.sub.39 may bond to each other to form a
ring, X.sup.- is an anionic group, and Q.sub.1 is a sulfur atom, an
oxygen atom, --C(R.sub.40)(R.sub.41)--, --CH.dbd.CH-- or
--N(R.sub.42)--, in which R.sub.40 to R.sub.42 are individually a
hydrogen atom, an alkyl group or an aryl group, and R.sub.40 and
R.sub.41 may bond to each other to form a ring.
[0126] No particular limitation is imposed on the alkyl group in
R.sub.35, and R.sub.36 to R.sub.39. However, examples thereof
include linear, branched or cyclic alkyl groups having 1 to 20
carbon atoms, such as methyl, ethyl, propyl, butyl, pentyl, hexyl,
octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl,
pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl,
cyclopropyl, cyclobutyl and cyclopentyl groups.
[0127] No particular limitation is imposed on the aryl group in
R.sub.35, and R.sub.36 to R.sub.39. However, examples thereof
include 6- to 14-membered monocyclic or polycyclic aryl groups such
as phenyl, naphthyl, phenanthryl and anthracenyl groups.
[0128] R.sub.35 may additionally have substituent(s), and no
particular limitation is imposed on the substituent(s) so far as
the storage stability of the staining compound is not markedly
impaired. Examples thereof include substituents described as
"favorable examples of additional substituents" above.
[0129] The group favorably has a substituent having such a nature
that the water solubility is improved, among these substituents.
Particularly favorable examples of such a substituent include
carboxyl, sulfonic, polyethylene glycol, carboxylate and sulfonate
groups though not limited thereto.
[0130] R.sub.35 is favorably an alkyl group, and it is favorable
that the alkyl group additionally has a substituent such as a
carboxyl, sulfonic, polyethylene glycol, carboxylate or sulfonate
group, since the water solubility of the resulting compound is
increased, and the fluorescence intensity thereof is also
increased.
[0131] No particular limitation is imposed on the alkoxy group in
R.sub.36 to R.sub.39. However, examples thereof include alkoxy
groups having 1 to 20 carbon atoms, such as methoxy, ethoxy,
propoxy, butoxy, pentyloxy, decyloxy, hexyloxy, heptyloxy,
octyloxy, nonyloxy, decyloxy, dodecyloxy and octadecyloxy
groups.
[0132] No particular limitation is imposed on the heterocyclic
group in R.sub.36 to R.sub.39. However, examples thereof include 4-
to 10-membered monocyclic or bicyclic heterocyclic groups
containing 1 to 4 atoms selected from nitrogen, oxygen and sulfur.
Examples of these groups include pyridyl, pyrazinyl, pyrimidinyl,
pyrrolyl, thienyl, furyl, pyranyl, oxazolyl, thiazolyl, triazolyl,
tetrazolyl, imidazolyl, pyrazolyl, morpholinyl, thiomorpholinyl,
piperidinyl, piperazinyl, quinolyl, isoquinolyl, indolyl,
isoindolyl, benzofuryl and benzothienyl groups.
[0133] No particular limitation is imposed on the amino group in
R.sub.36 to R.sub.39. However, examples thereof include an
unsubstituted amino group; mono-substituted amino groups such as
N-methylamino, N-butylamino, N-hexylamino, N-tetradecylamino,
N-phenylamino and N-naphthylamino groups; di-substituted amino
groups such as N,N-dimethylamino, N,N-diethylamino,
N,N-diphenylamino and N,N-methylpropylamino groups; carbonylamino
groups such as acetylamino, ethylcarbonylamino,
tert-butylcarbonylamino, benzoylamino, naphthoylamino and
methoxycarbonylamino groups; and sulfonylamino groups such
methylsulfonylamino, ethylsulfonylamino, tert-butylsulfonylamino
and isopropoxysulfonylamino groups.
[0134] Examples of the halogen atom in R.sub.36 to R.sub.39 include
fluorine, chlorine, bromine and iodine atoms.
[0135] R.sub.36 to R.sub.39 are each favorably a hydrogen atom, a
carboxyl group, a sulfonic group, an amino group or a halogen atom,
and a hydrogen atom or a sulfonic group is particularly favorable
because the water solubility of the resulting compound is
improved.
[0136] No particular limitation is imposed on the ring formed by
bonding R.sub.36 and R.sub.37, R.sub.37 and R.sub.38, or R.sub.38
and R.sub.39 to each other. However, examples thereof include
aromatic rings having 3 to 10 carbon atoms, such as benzene and
naphthalene rings; saturated rings such as cyclooctane,
cycloheptane, cyclohexane, cyclopentane and cyclobutane rings;
partially saturated rings such as cyclopentene and cyclohexene
rings; and heterocycles such as pyridine and pyrimidine rings. The
ring may additionally have substituent(s), and no particular
limitation is imposed on the substituent(s) so far as the storage
stability of the staining compound is not markedly impaired.
Examples thereof include substituents described as "favorable
examples of additional substituents" above.
[0137] The ring favorably has a substituent having such a nature
that the water solubility is improved, among these substituents.
Particularly favorable examples of such a substituent include
carboxyl, sulfonic, polyethylene glycol, carboxylate and sulfonate
groups though not limited thereto.
[0138] The ring formed by bonding R.sub.36 and R.sub.37, R.sub.37
and R.sub.38, or R.sub.38 and R.sub.39 to each other is favorably a
benzene ring because the storage stability of the resulting
compound is improved.
Description of X.sup.- in General Formula (XI)
[0139] In the general formula (XI), X.sup.- is an anionic group. No
particular limitation is imposed on the anionic group. However,
examples thereof include halide ions such as fluoride, chloride,
bromide and iodide ions; inorganic acid ions such as sulfate,
phosphate, nitrate, tetrafluoroborate and hexafluorophosphate ions;
Lewis-acid-containing ions such as tetrachloroaluminate ion; and
organic acid ions such as acetate, lactate, methanesulfonate,
benzenesulfonate, p-toluenesulfonate, trifluoroacetate,
trifluoromethanesulfonate and tetraphenylborate ions.
[0140] The anionic group of X.sup.- is favorably a chloride,
bromide, iodide, sulfate, nitrate or methanesulfonate ion and more
favorably a bromide or iodide ion from the viewpoint of ease of
synthesis of the compound.
Description of Q.sub.1 in General Formula (XI)
[0141] In the general formula (XI), Q.sub.1 is a sulfur atom,
oxygen atom, --C(R.sub.40)(R.sub.41)--, --CH.dbd.CH-- or
--N(R.sub.42)--.
[0142] No particular limitation is imposed on the alkyl group in
R.sub.40 to R.sub.42. However, examples thereof include linear,
branched or cyclic alkyl groups having 1 to 20 carbon atoms, such
as methyl, ethyl, propyl, butyl, pentyl, hexyl, octyl, nonyl,
decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl,
hexadecyl, heptadecyl, octadecyl, nonadecyl, cyclopropyl,
cyclobutyl and cyclopentyl groups.
[0143] No particular limitation is imposed on the aryl group in
R.sub.40 to R.sub.42. However, examples thereof include 6- to
14-membered monocyclic or polycyclic aryl groups such as phenyl,
naphthyl, phenanthryl and anthracenyl groups.
[0144] R.sub.40 and R.sub.41 in Q.sub.1 may bond to each other to
form a ring, and examples thereof include cyclohexane, piperidine
and piperidinium rings.
[0145] Q.sub.1 is particularly favorably an oxygen atom, sulfur
atom or --C(CH.sub.3)(CH.sub.3)-- because the storage stability of
the compound is improved.
[0146] It is also favorable that Q.sub.1 is --C(CH.sub.2).sub.5--,
since the staining ability of the compound becomes excellent.
Description of Ring Formed by Bonding R.sub.3 and R.sub.4 in
General Formula (II) to Each Other
[0147] The ring formed by bonding R.sub.3 and R.sub.4 in the
general formula (II) to each other is represented by any one of the
following general formulae (XII), (XIII) and (XV).
##STR00013##
[0148] In the general formula (XII), R.sub.43 is a hydrogen atom,
an alkyl group, an aryl group or a heterocyclic group, and R.sub.44
is an alkyl group, an aryl group, a carboxyl group, a carboxylate
group, a hydroxyl group or an amino group.
[0149] No particular limitation is imposed on the alkyl group in
R.sub.43 and R.sub.44. However, examples thereof include linear,
branched or cyclic alkyl groups having 1 to 20 carbon atoms, such
as methyl, ethyl, propyl, butyl, pentyl, hexyl, octyl, nonyl,
decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl,
hexadecyl, heptadecyl, octadecyl, nonadecyl, cyclopropyl,
cyclobutyl and cyclopentyl groups.
[0150] No particular limitation is imposed on the aryl group in
R.sub.43 and R.sub.44. However, examples thereof include 6- to
14-membered monocyclic or polycyclic aryl groups such as phenyl,
naphthyl, phenanthryl and anthracenyl groups.
[0151] The ring may additionally have substituent(s), and no
particular limitation is imposed on the substituent(s) so far as
the storage stability of the staining compound is not markedly
impaired. Examples thereof include substituents described as
"favorable examples of additional substituents" above.
[0152] The ring favorably has a substituent having such a nature
that the water solubility is improved, among these substituents.
Particularly favorable examples of such a substituent include
carboxyl, sulfonic, polyethylene glycol, carboxylate and sulfonate
groups though not limited thereto.
[0153] No particular limitation is imposed on the heterocyclic
group in R.sub.43. However, examples thereof include 4- to
10-membered monocyclic or bicyclic heterocyclic groups containing 1
to 4 atoms selected from nitrogen, oxygen and sulfur. Examples of
these groups include pyridyl, pyrazinyl, pyrimidinyl, pyrrolyl,
thienyl, furyl, pyranyl, oxazolyl, thiazolyl, triazolyl,
tetrazolyl, imidazolyl, pyrazolyl, morpholinyl, thiomorpholinyl,
piperidinyl, piperazinyl, quinolyl, isoquinolyl, indolyl,
isoindolyl, benzofuryl and benzothienyl groups.
[0154] R.sub.43 is favorably an aryl group from the viewpoint of
stability of the compound. It is also favorable that the aryl group
has a water-soluble substituent such as a carboxyl group, since the
water solubility of the compound is improved.
[0155] No particular limitation is imposed on the carboxylate group
in R.sub.44. However, examples thereof include methyl carboxylate,
ethyl carboxylate, propyl carboxylate and butyl carboxylate
groups.
[0156] No particular limitation is imposed on the amino group in
R.sub.44. However, examples thereof include an unsubstituted amino
group; mono-substituted amino groups such as N-methylamino,
N-butylamino, N-hexylamino, N-tetradecylamino, N-phenylamino and
N-naphthylamino groups; di-substituted amino groups such as
N,N-dimethylamino, N,N-diethylamino, N,N-diphenylamino and
N,N-methylpropylamino groups; carbonylamino groups such as
acetylamino, ethylcarbonylamino, tert-butylcarbonylamino,
benzoylamino, naphthoylamino and methoxycarbonylamino groups; and
sulfonylamino groups such methylsulfonylamino, ethylsulfonylamino,
tert-butylsulfonylamino and isopropoxysulfonylamino groups.
[0157] R.sub.44 is favorably an alkyl, aryl, carboxyl or amino
group from the viewpoint of easy of synthesis of the compound, with
the alkyl or carboxyl group being particularly favorable.
Description of General Formula (XIII)
[0158] In the general formula (XIII), Q.sub.2 is an oxygen atom, a
sulfur atom or --N(R.sub.52)--. R.sub.45 is a hydrogen atom, an
alkyl group, an aryl group or a heterocyclic group, R.sub.46 is a
sulfur atom, an oxygen atom, .dbd.NR.sub.53, a heterocyclic group
or a dicyanomethylene group, and R.sub.52 and R.sub.53 are
individually a hydrogen atom, an alkyl group, an aryl group or a
heterocyclic group.
[0159] No particular limitation is imposed on the alkyl group in
R.sub.45, R.sub.52 and R.sub.53. However, examples thereof include
linear, branched or cyclic alkyl groups having 1 to 20 carbon
atoms, such as methyl, ethyl, propyl, butyl, pentyl, hexyl, octyl,
nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl,
hexadecyl, heptadecyl, octadecyl, nonadecyl, cyclopropyl,
cyclobutyl and cyclopentyl groups. The group may additionally have
substituent(s), and no particular limitation is imposed on the
substituent(s) so far as the storage stability of the staining
compound is not markedly impaired. Examples thereof include
substituents described as "favorable examples of additional
substituents" above.
[0160] The group favorably has a substituent having such a nature
that the water solubility is improved, among these substituents.
Particularly favorable examples of such a substituent include
carboxyl, sulfonic, polyethylene glycol, carboxylate and sulfonate
groups though not limited thereto.
[0161] No particular limitation is imposed on the aryl group in
R.sub.45, R.sub.52 and R.sub.53. However, examples thereof include
6- to 14-membered monocyclic or polycyclic aryl groups such as
phenyl, naphthyl, phenanthryl and anthracenyl groups.
[0162] No particular limitation is imposed on the heterocyclic
group in R.sub.45, R.sub.52 and R.sub.53. However, examples thereof
include 4- to 10-membered monocyclic or bicyclic heterocyclic
groups containing 1 to 4 atoms selected from nitrogen, oxygen and
sulfur. Examples of these groups include pyridyl, pyrazinyl,
pyrimidinyl, pyrrolyl, thienyl, furyl, pyranyl, oxazolyl,
thiazolyl, triazolyl, tetrazolyl, imidazolyl, pyrazolyl,
morpholinyl, thiomorpholinyl, piperidinyl, piperazinyl, quinolyl,
isoquinolyl, indolyl, isoindolyl, benzofuryl and benzothienyl
groups.
[0163] R.sub.45 is favorably an alkyl group, and it is favorable
that the alkyl group additionally has a substituent such as a
carboxyl, sulfonic, polyethylene glycol, carboxylate or sulfonate
group, since the water solubility of the resulting compound is
increased, and the fluorescence intensity thereof is also
increased.
[0164] R.sub.46 is favorably a sulfur atom, oxygen atom or
heterocycle.
[0165] It is favorable that R.sub.46 is a sulfur atom, since the
compound tends to improve the staining ability thereof, and that
R.sub.46 is 2-thioxothiazolidin-4-one having a substituent at the
3-position, since detection of a maximum fluorescence wavelength is
often conducted in such a larger wavelength region as a
near-infrared wavelength region, so that the compound can be used
in near-infrared applications.
Description of General Formula (XV)
[0166] In the general formula (XV), R.sub.49 and R.sub.50 are,
independently of each other, a hydrogen atom, an alkyl group or an
aryl group, and R.sub.51 is an oxygen atom or a sulfur atom.
[0167] No particular limitation is imposed on the alkyl group in
R.sub.49 and R.sub.50. However, examples thereof include linear,
branched or cyclic alkyl groups having 1 to 20 carbon atoms, such
as methyl, ethyl, propyl, butyl, pentyl, hexyl, octyl, nonyl,
decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl,
hexadecyl, heptadecyl, octadecyl, nonadecyl, cyclopropyl,
cyclobutyl and cyclopentyl groups.
[0168] No particular limitation is imposed on the aryl group in
R.sub.49 and R.sub.50. However, examples thereof include 6- to
14-membered monocyclic or polycyclic aryl groups such as phenyl,
naphthyl, phenanthryl and anthracenyl groups.
(Description of B in General Formula (II))
[0169] B is a structure represented by any one of the following
general formulae (XVI) to (XVIV) and (XVII')
##STR00014##
[0170] Each structure will hereinafter be described in detail.
Description of General Formula (XVI)
[0171] In the general formula (XVI), R.sub.54 is a hydrogen atom,
an alkyl group, an aralkyl group, an alkenyl group, an aryl group,
a heterocyclic group or an acyl group. R.sub.55 to R.sub.58 are,
independently of one another, a hydrogen atom, an alkyl group, an
aryl group, a carboxyl group, a carboxylate group or an acyl group,
and R.sub.55 and R.sub.57 may bond to each other to form a ring.
R.sub.59 is a hydrogen atom, an alkyl group, an alkoxy group or a
halogen atom.
[0172] No particular limitation is imposed on the alkyl group in
R.sub.54, R.sub.55 to R.sub.58, and R.sub.59. However, examples
thereof include linear, branched or cyclic alkyl groups having 1 to
20 carbon atoms, such as methyl, ethyl, propyl, butyl, pentyl,
hexyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl,
pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl,
cyclopropyl, cyclobutyl and cyclopentyl groups.
[0173] No particular limitation is imposed on the aralkyl group in
R.sub.54. However, examples thereof include benzyl and phenethyl
groups.
[0174] No particular limitation is imposed on the alkenyl groups in
R.sub.54. However, examples thereof include alkenyl groups having 2
to 20 carbon atoms, such as vinyl, 2,2-diphenylvinyl, 3-butenyl and
cyclohexenyl groups.
[0175] No particular limitation is imposed on the aryl group in
R.sub.54, and R.sub.55 to R.sub.58. However, examples thereof
include 6- to 14-membered monocyclic or polycyclic aryl groups such
as phenyl, naphthyl, phenanthryl and anthracenyl groups.
[0176] No particular limitation is imposed on the heterocyclic
group in R.sub.54. However, examples thereof include 4- to
10-membered monocyclic or bicyclic heterocyclic groups containing 1
to 4 atoms selected from nitrogen, oxygen and sulfur. Examples of
these groups include pyridyl, pyrazinyl, pyrimidinyl, pyrrolyl,
thienyl, furyl, pyranyl, oxazolyl, thiazolyl, triazolyl,
tetrazolyl, imidazolyl, pyrazolyl, morpholinyl, thiomorpholinyl,
piperidinyl, piperazinyl, quinolyl, isoquinolyl, indolyl,
isoindolyl, benzofuryl and benzothienyl groups.
[0177] No particular limitation is imposed on the acyl group in
R.sub.54, and R.sub.55 to R.sub.58. However, examples thereof
include acetyl, propionyl, butyryl, pentanoyl, benzoyl, 1-naphthoyl
and 2-naphthoyl groups.
[0178] R.sub.54, and R.sub.55 to R.sub.58 may additionally have
substituent(s), and no particular limitation is imposed on the
substituent(s) so far as the storage stability of the staining
compound is not markedly impaired. Examples thereof include
substituents described as "favorable examples of additional
substituents" above.
[0179] The groups favorably have a substituent having such a nature
that the water solubility is improved, among these substituents.
Particularly favorable examples of such a substituent include
carboxyl, sulfonic, polyethylene glycol, carboxylate and sulfonate
groups though not limited thereto.
[0180] R.sub.54, and R.sub.55 to R.sub.58 may be selected
independently of one another and arbitrarily from the substituents
mentioned above. However, as a favorable mode, the aralkyl, alkenyl
or aryl group is favorable because the fluorescence intensity of
the resulting compound becomes strong. Favorable specific examples
thereof include phenyl, bromophenyl, benzyl, bromobenzyl,
methylthiophenyl, methoxyphenyl, methoxynaphthyl, benzylphenyl,
2,2-diphenylvinyl and 2,2-diphenylvinylphenyl groups. More
favorable examples thereof include phenyl, bromophenyl, benzyl,
methylthiophenyl, methoxyphenyl and methoxynaphthyl groups, with
the methylthiophenyl group being particularly favorable because a
tendency for a Stokes shift (a difference between maximum
excitation wavelength and maximum fluorescence wavelength) to
remarkably become large is recognized.
[0181] No particular limitation is imposed on the carboxylate group
in R.sub.55 to R.sub.58. However, examples thereof include methyl
carboxylate, ethyl carboxylate, propyl carboxylate and butyl
carboxylate groups.
[0182] No particular limitation is imposed on the ring formed by
bonding R.sub.55 and R.sub.57 to each other. However, examples
thereof include saturated aliphatic rings such as cyclooctane,
cycloheptane, cyclohexane, cyclopentane and cyclobutane rings; and
partially saturated aliphatic rings such as cyclopentene and
cyclohexene rings. These rings may additionally have
substituent(s), and no particular limitation is imposed on the
substituent(s) so far as the storage stability of the staining
compound is not markedly impaired. Examples thereof include
substituents described as "favorable examples of additional
substituents" above.
[0183] The ring favorably has a substituent having such a nature
that the water solubility is improved, among these substituents.
Particularly favorable examples of such a substituent include
carboxyl, sulfonic, polyethylene glycol, carboxylate and sulfonate
groups though not limited thereto.
[0184] It is favorable that R.sub.55 to R.sub.58 are, independently
of each other, a hydrogen atom, alkyl group or aryl group, and
R.sub.55 and R.sub.57 bond to each other to form a ring. It is more
favorable that R.sub.55 and R.sub.57 bond to each other to form a
ring, since the resulting compound becomes stable in chemical
structure. Specific examples of the ring include cyclooctane,
cycloheptane, cyclohexane, cyclopentane and cyclobutane rings, and
the cyclopentane ring is more favorable from the viewpoint of
storage stability.
[0185] No particular limitation is imposed on the alkoxy group in
R.sub.59. However, examples thereof include alkoxy groups having 1
to 20 carbon atoms, such as methoxy, ethoxy, propoxy, butoxy,
pentyloxy, hexyloxy, heptyloxy, octyloxy, nonyloxy, decyloxy,
dodecyloxy and octadecyloxy groups.
[0186] Examples of the halogen atom in R.sub.59 include fluorine,
chlorine, bromine and iodine atoms.
[0187] R.sub.59 is favorably a hydrogen atom, a halogen atom or an
alkoxy group, with the hydrogen or halogen atom being more
favorable.
Description of General Formula (XVII)
[0188] In the general formula (XVII), R.sub.60 is a hydrogen atom,
an alkyl group or an aryl group. R.sub.61 to R.sub.64 are,
independently of one another, a hydrogen atom, an alkyl group, an
aryl group, an alkoxy group, a carboxyl group, a sulfonic group, a
heterocyclic group, an amino group or a halogen atom. R.sub.61 and
R.sub.62, R.sub.62 and R.sub.63, or R.sub.63 and R.sub.64 may bond
to each other to form a ring.
[0189] No particular limitation is imposed on the alkyl group in
R.sub.60, and R.sub.61 to R.sub.64. However, examples thereof
include linear, branched or cyclic alkyl groups having 1 to 20
carbon atoms, such as methyl, ethyl, propyl, butyl, pentyl, hexyl,
octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl,
pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl,
cyclopropyl, cyclobutyl and cyclopentyl groups.
[0190] No particular limitation is imposed on the aryl group in
R.sub.60, and R.sub.61 to R.sub.64. However, examples thereof
include 6- to 14-membered monocyclic or polycyclic aryl groups such
as phenyl, naphthyl, phenanthryl and anthracenyl groups.
[0191] R.sub.60 may additionally have substituent(s), and no
particular limitation is imposed on the substituent(s) so far as
the storage stability of the staining compound is not markedly
impaired. Examples thereof include substituents described as
"favorable examples of additional substituents" above.
[0192] The group favorably has a substituent having such a nature
that the water solubility is improved, among these substituents.
Particularly favorable examples of such a substituent include
carboxyl, sulfonic, polyethylene glycol, carboxylate and sulfonate
groups though not limited thereto.
[0193] R.sub.60 is favorably an alkyl group, and it is favorable
that the alkyl group additionally has a substituent such as a
carboxyl, sulfonic, polyethylene glycol, carboxylate or sulfonate
group, since the water solubility of the resulting compound is
increased, and the fluorescence intensity thereof is also
increased.
[0194] No particular limitation is imposed on the alkoxy group in
R.sub.61 to R.sub.64. However, examples thereof include alkoxy
groups having 1 to 20 carbon atoms, such as methoxy, ethoxy,
propoxy, butoxy, pentyloxy, hexyloxy, heptyloxy, octyloxy,
nonyloxy, decyloxy, dodecyloxy and octadecyloxy groups.
[0195] No particular limitation is imposed on the heterocyclic
group in R.sub.61 to R.sub.64. However, examples thereof include 4-
to 10-membered monocyclic or bicyclic heterocyclic groups
containing 1 to 4 atoms selected from nitrogen, oxygen and sulfur.
Examples of these groups include pyridyl, pyrazinyl, pyrimidinyl,
pyrrolyl, thienyl, furyl, pyranyl, oxazolyl, thiazolyl, triazolyl,
tetrazolyl, imidazolyl, pyrazolyl, morpholinyl, thiomorpholinyl,
piperidinyl, piperazinyl, quinolyl, isoquinolyl, indolyl,
isoindolyl, benzofuryl and benzothienyl groups.
[0196] No particular limitation is imposed on the amino group in
R.sub.61 to R.sub.64. However, examples thereof include an
unsubstituted amino group; mono-substituted amino groups such as
N-methylamino, N-butylamino, N-hexylamino, N-tetradecylamino,
N-phenylamino and N-naphthylamino groups; di-substituted amino
groups such as N,N-dimethylamino, N,N-diethylamino,
N,N-diphenylamino and N,N-methylpropylamino groups; carbonylamino
groups such as acetylamino, ethylcarbonylamino,
tert-butylcarbonylamino, benzoylamino, naphthoylamino and
methoxycarbonylamino groups; and sulfonylamino groups such
methylsulfonylamino, ethylsulfonylamino, tert-butylsulfonylamino
and isopropoxysulfonylamino groups.
[0197] Examples of the halogen atom in R.sub.61 to R.sub.64 include
fluorine, chlorine, bromine and iodine atoms.
[0198] R.sub.61 to R.sub.64 are each favorably a hydrogen atom, a
carboxyl group, a sulfonic group, an amino group or a halogen atom,
and a hydrogen atom or a sulfonic group is particularly favorable
because the water solubility of the resulting compound is
improved.
[0199] No particular limitation is imposed on the ring formed by
bonding R.sub.61 and R.sub.62, R.sub.62 and R.sub.63, or R.sub.63
and R.sub.64 to each other. However, examples thereof include
aromatic rings having 3 to 10 carbon atoms, such as benzene and
naphthalene rings; saturated rings such as cyclooctane,
cycloheptane, cyclohexane, cyclopentane and cyclobutane rings;
partially saturated rings such as cyclopentene and cyclohexene
rings; and heterocycles such as pyridine and pyrimidine rings. The
ring may additionally have substituent(s), and no particular
limitation is imposed on the substituent(s) so far as the storage
stability of the staining compound is not markedly impaired.
Examples thereof include substituents described as "favorable
examples of additional substituents" above.
[0200] The ring favorably has a substituent having such a nature
that the water solubility is improved, among these substituents.
Particularly favorable examples of such a substituent include
carboxyl, sulfonic, polyethylene glycol, carboxylate and sulfonate
groups though not limited thereto.
[0201] The ring formed by bonding R.sub.61 and R.sub.62, R.sub.62
and R.sub.63, or R.sub.63 and R.sub.64 to each other is favorably a
benzene ring because the storage stability of the resulting
compound is improved.
Description of Q.sub.3 in General Formula (XVII)
[0202] In the general formula (XVII), Q.sub.3 is a sulfur atom, an
oxygen atom, --C(R.sub.65)(R.sub.66)-- or --CH.dbd.CH--, in which
R.sub.65 and R.sub.66 bond to each other to form a ring.
[0203] Examples of the ring formed by bonding R.sub.65 and R.sub.66
in Q.sub.3 to each other include cyclohexane, piperidine and
piperidinium rings.
[0204] Q.sub.3 is favorably a sulfur atom, oxygen atom or
--C(R.sub.65)(R.sub.66)--, and particularly favorably an oxygen
atom or sulfur atom.
[0205] It is also favorable that Q.sub.3 is --C(CH.sub.2).sub.5--,
since the staining ability of the compound becomes excellent.
Description of General Formula (XVII')
##STR00015##
[0207] In the general formula (XVII'), R.sub.60' is a hydrogen
atom, an alkyl group or an aryl group, Q.sub.3' is a sulfur atom or
an oxygen atom. Dotted lines in the general formula (XVII')
represent the case where a benzene ring is present or absent.
[0208] No particular limitation is imposed on the alkyl group in
R.sub.60'. However, examples thereof include linear, branched or
cyclic alkyl groups having 1 to 20 carbon atoms, such as methyl,
ethyl, propyl, butyl, pentyl, hexyl, octyl, nonyl, decyl, undecyl,
dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl,
octadecyl, nonadecyl, cyclopropyl, cyclobutyl and cyclopentyl
groups.
[0209] No particular limitation is imposed on the aryl group in
R.sub.60'. However, examples thereof include 6- to 14-membered
monocyclic or polycyclic aryl groups such as phenyl, naphthyl,
phenanthryl and anthracenyl groups.
[0210] R.sub.60' may additionally have substituent(s), and no
particular limitation is imposed on the substituent(s) so far as
the storage stability of the staining compound is not markedly
impaired. Examples thereof include substituents described as
"favorable examples of additional substituents" above.
[0211] The group favorably has a substituent having such a nature
that the water solubility is improved, among these substituents.
Particularly favorable examples of such a substituent include
carboxyl, sulfonic, polyethylene glycol, carboxylate and sulfonate
groups though not limited thereto.
[0212] R.sub.60' is favorably an alkyl group, and it is favorable
that the alkyl group additionally has a substituent such as a
carboxyl, sulfonic, polyethylene glycol, carboxylate or sulfonate
group, since the water solubility of the resulting compound is
increased, and the fluorescence intensity thereof is also
increased.
Description of General Formula (XVIII)
[0213] In the general formula (XVIII), R.sub.68 and R.sub.69 are,
independently of each other, a hydrogen atom, an alkyl group or an
aryl group. R.sub.70 is an oxygen atom or a sulfur atom.
[0214] No particular limitation is imposed on the alkyl group in
R.sub.68 and R.sub.69. However, examples thereof include linear,
branched or cyclic alkyl groups having 1 to 20 carbon atoms, such
as methyl, ethyl, propyl, butyl, pentyl, hexyl, octyl, nonyl,
decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl,
hexadecyl, heptadecyl, octadecyl, nonadecyl, cyclopropyl,
cyclobutyl and cyclopentyl groups.
[0215] No particular limitation is imposed on the aryl group in
R.sub.68 and R.sub.69. However, examples thereof include 6- to
14-membered monocyclic or polycyclic aryl groups such as phenyl,
naphthyl, phenanthryl and anthracenyl groups.
[0216] R.sub.68 and R.sub.69 may additionally have substituent(s),
and no particular limitation is imposed on the substituent(s) so
far as the storage stability of the staining compound is not
markedly impaired. Examples thereof include substituents described
as "favorable examples of additional substituents" above.
[0217] The groups favorably have a substituent having such a nature
that the water solubility is improved, among these substituents.
Particularly favorable examples of such a substituent include
carboxyl, sulfonic, polyethylene glycol, carboxylate and sulfonate
groups though not limited thereto.
Description of General Formula (XVIV)
[0218] In the general formula (XVIV), R.sub.71 is an alkyl
group.
[0219] No particular limitation is imposed on the alkyl group in
R.sub.71. However, examples thereof include linear, branched or
cyclic alkyl groups having 1 to 20 carbon atoms, such as methyl,
ethyl, propyl, butyl, pentyl, hexyl, octyl, nonyl, decyl, undecyl,
dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl,
octadecyl, nonadecyl, cyclopropyl, cyclobutyl and cyclopentyl
groups.
Description of General Formulae (I) to (XVIV), (VIII'), and
(XVII')
[0220] It is favorable that a compound of the general formulae (I)
to (XVIV), (VIII'), and (XVII') has at least one of carboxyl,
sulfonic and polyethylene glycol groups, since the water solubility
of the compound is improved. A salt of the carboxyl or sulfonic
group is also usable in the present invention. No particular
limitation is imposed on the salt of the carboxyl or sulfonic
group. Specific examples thereof include alkali metal salts such as
sodium and potassium salts; alkaline earth salts such as magnesium
and calcium salts; amine salts such as ammonium, pyridinium,
piperidinium and triethylammonium salts; and amino acid salts such
as tryptophan, lysine, leucine, phenylalanine, valine and arginine
salts. Favorable examples thereof include sodium, potassium,
ammonium, pyridinium and piperidinium salts.
SYNTHETIC EXAMPLES
[0221] The staining compound s according to the present invention,
which are represented by any one of the general formulae (I) to
(XVIV), (VIII'), and (XVII'), may be commercially available and can
be obtained with ease. The compounds can also be easily synthesized
according to publicly known respective processes. For example, a
staining compound in which A in the general formula (I) is
represented by the general formula (III) can be easily synthesized
by referring to, for example, Japanese Patent Application Laid-Open
No. 2008-94897. A staining compound in which A in the general
formula (I) is represented by the general formula (V) or (VII) can
be synthesized according to the process described in, for example,
Japanese Patent Application Laid-Open 2001-315437. On the other
hand, a staining compound in which B in the general formula (II) is
represented by the general formula (XVI) can be easily synthesized
by referring to, for example, Chem. Comm., Vol. 24, pp. 3036-3037
(2003).
[0222] An aspect about a production process of the staining
compound in which A in the general formula (I) is represented by
the general formula (III) will hereinafter be described, but the
synthetic process is not limited thereto. An exemplary synthetic
scheme will hereinafter be shown.
##STR00016##
R.sub.1, R.sub.2, and R.sub.6 to R.sub.11 in the above scheme have
the same meanings as described above.
[0223] First, in Condensation (1) step, a compound (A) and an amine
derivative are heated and condensed in the presence of a condensing
agent (or in the absence of the condensing agent) in an organic
solvent (or in the absence of a solvent) to obtain a compound (B).
The compound (B) is then heated and condensed again with the
above-described amine derivative. As a result, a staining compound
(III) according to the present invention is obtained.
[0224] No particular limitation is imposed on a favorable organic
solvent usable in the condensation reaction of the
above-illustrated synthetic scheme so far as the solvent does not
participate in the reaction in Condensation (1) step. However, for
example, methanol, ethanol, n-propanol, isopropanol and n-butanol
may be used either singly or in any combination thereof. No
particular limitation is imposed on a favorable organic solvent
usable in Condensation (2) step so far as the solvent does not
participate in the reaction. However, examples thereof include
ethylene glycol, N-methylpyrrolidone, N,N-dimethyl-acetamide,
N,N-dimethylformamide, dimethyl sulfoxide, sulfolane,
chlorobenzene, dichlorobenzene, trichlorobenzene and
nitrobenzene.
[0225] The reaction in Condensation (1) step is conducted in a
temperature range of from 0 to 200.degree. C., favorably from 10 to
150.degree. C., more favorably from 20 to 100.degree. C. The
reaction in Condensation (2) step is conducted in a temperature
range of from 50 to 250.degree. C., favorably from 100 to
230.degree. C., more favorably from 150 to 220.degree. C.
[0226] In the case of compounds in which R.sub.1 and R.sub.2, and
R.sub.1' and R.sub.2' in the substituents of the amine derivatives
to be reacted are the same substituents, the same amine derivatives
may be used, so that the staining compound of the general formula
(III) can be obtained through a one-stage condensation step. In
this case, the reaction temperature follows the condition of
Condensation (2) step.
[0227] No particular limitation is imposed on the condensing agent
used in the condensation step so far as it does not participate in
the reaction. However, the condensing agent may be chosen for use
from, for example, magnesium oxide, zinc chloride and aluminum
chloride.
[0228] The resultant staining compound (III) may be subjected to an
ordinary isolation/purification process for organic compounds. For
example, a reaction mixture is acidified with hydrochloric acid and
subjected to aciding-out, thereby separating solids by filtration,
and then subjected to neutralization with sodium hydroxide or the
like and concentration, thereby obtaining a crude product. The
crude product is further purified by recrystallization with acetone
or methanol, column purification using silica gel, or the like.
These methods may be used singly or in combination of two or more
methods thereof to conduct purification, thereby obtaining the
compound with higher purity.
[0229] Specific Exemplified Compounds (1) to (122) according to the
present invention will be shown below. However, compounds according
to the present invention are not limited thereto.
##STR00017## ##STR00018## ##STR00019## ##STR00020## ##STR00021##
##STR00022## ##STR00023## ##STR00024## ##STR00025## ##STR00026##
##STR00027## ##STR00028## ##STR00029## ##STR00030## ##STR00031##
##STR00032## ##STR00033## ##STR00034## ##STR00035## ##STR00036##
##STR00037## ##STR00038## ##STR00039##
<Radiation Labeling>
[0230] The labeling composition for the intraocular tissue
according to the present invention may also be used as a probe
labeled with a radioactive nuclide.
[0231] No particular limitation is imposed on the kind of the
radioactive nuclide used in labeling, and it may be suitably
selected according to the mode of use.
[0232] The labeling composition for the intraocular tissue labeled
with the radioactive nuclide may be used in imaging by, for
example, autoradiography, positron emission tomography (PET) using
a positron emission nuclide, or single photon emission computed
tomography (SPECT) using various kinds of gamma-ray emission
nuclides. Detection may also be made by magnetic resonance imaging
(MRI) making good use of an MR signal derived from a fluorine
atomic nucleus, or .sup.13C. In addition, imaging may also be
conducted by a Compton camera (GREI) capable of coincidentally
imaging a plurality of molecules as a next-generation molecular
imaging apparatus. It may also be possible to quantify a probe for
a retinal tissue using, for example, a liquid scintillation
counter, X-ray film or imaging plate.
[0233] A labeling composition for the intraocular tissue labeled
with a radioactive isotope such as .sup.14C permits measuring a
concentration in blood (or urine or feces) by an accelerator mass
spectrometry (AMS) or the like to obtain pharmacokinetic
information (area under the blood concentration-time curve (AUC),
blood concentration half-life period (T.sub.1/2), maximum drug
concentration (C.sub.max), maximum drug concentration time
(T.sub.max), distribution volume, first-pass effect, biological
utilization factor, excretion rates in feces and urine, etc.) of
the parent compound and metabolite of the substance labeled.
[0234] No particular limitation is imposed on the radioactive
nuclide, and it may be suitably selected according to the mode of
use.
[0235] Specifically, in the case of measurement by PET, a .sup.11C,
.sup.14C, .sup.13N, .sup.15O, .sup.18F, .sup.19F, .sup.62Cu,
.sup.68Ga or .sup.78Br may be used. .sup.11C, .sup.13N, .sup.15O or
.sup.18F positron emission nuclide of, for example, is favorable,
with .sup.11C or .sup.18F being particularly favorable.
[0236] In the case of measurement by SPECT, a .gamma.-ray emission
nuclide of, for example, .sup.99mTc, .sup.111In, .sup.67Ga,
.sup.201Tl, .sup.123I or .sup.133Xe may be used. .sup.99mTc or
.sup.123I is favorable.
[0237] In the case of measuring another animal than human, a
radioactive nuclide longer in half-life period, such as, for
example, .sup.125I may be used.
[0238] In the case of measurement by GREI, for example, .sup.131I,
.sup.85Sr or .sup.65Zn may be used.
[0239] The radioactive nuclide may be either contained in or bonded
to the compound represented by any one of the general formulae (I)
to (XVIV), (VIII'), and (XVII').
[0240] No particular limitation is imposed on the labeling method
with the radioactive nuclide, and a generally used method may be
used. At least part of the elements forming the compound
represented by any one of the general formulae (I) to (XVIV),
(VIII'), and (XVII') may be substituted by the radioactive nuclide
or bonded thereto.
[0241] When the compound represented by any one of the general
formulae (I) to (XVIV), (VIII'), and (XVII') is labeled with the
radioactive nuclide, the compound favorably has a radioactivity of
about 1 to 100 .mu.Ci per mM.
[0242] At this time, no particular limitation is imposed on the
dose of the labeling composition for the intraocular tissue used,
and it may be suitably selected according to the kind of the
compound and the kind of the radioactive nuclide used in
labeling.
<Preparation of Labeling Composition for Intraocular
Tissue>
[0243] No particular limitation is imposed on the concentration of
the compound contained in the labeling composition for the
intraocular tissue according to the present invention so far as the
intraocular tissue can be detected. However, the concentration may
be suitably adjusted according to the target site and the compound
used. The compound is used at a concentration of generally 0.001
ng/mL or more and 100 .mu.g/mL or less, favorably 0.001 ng/mL or
more and 10 .mu.g/mL or less, more favorably 0.001 ng/mL or more
and 5 .mu.la/mL or less.
[0244] The labeling composition for the intraocular tissue
according to the present invention is used by dissolving at least
one of the staining compound s represented by any one of the
general formulae (I) to (XVIV), (VIII'), and (XVII') in a proper
solvent. No particular limitation is imposed on the solvent so far
as it does not affect a living body. However, an aqueous liquid
high in affinity for the living body is favorable. Examples thereof
include water; physiological saline; buffer solutions such as
phosphate buffer solution (PBS) and Tris; alcoholic solvents such
as methanol, ethanol, isopropanol, butanol, ethylene glycol and
glycerol; organic solvents such as N,N-dimethyl sulfoxide
(hereinafter abbreviated as DMSO) and N,N-dimethylformamide
(hereinafter abbreviated as DMF); cell culture media such as D-MEM
and HBSS; and infusions such as lactate Ringer solution. It is
particularly favorable to contain at least 50% of water. These
solvents may also be used in combination of two or more
thereof.
[0245] No particular limitation is imposed on a preparation method
of the labeling composition for the intraocular tissue according to
the present invention. For example, the composition may be prepared
by diluting a thick solution of the compound dissolved in such a
solvent as described above. In the case where the water solubility
of the compound is low, the compound may be dissolved in a proper
solvent and then diluted with purified water to use it. The proper
solvent is particularly favorably methanol, ethanol or DMSO.
[0246] If it is necessary to control the salt concentration or pH
to be suitable for the living body, additives may be added to the
labeling composition for the intraocular tissue according to the
present invention either singly or in combination of two or more
thereof.
[0247] No particular limitation is imposed on the additives used in
the present invention so far as they do not affect the labeling
composition for the intraocular tissue. However, examples thereof
include humectants, surface tension adjustors, thickeners, salts
such as sodium chloride, various kinds of pH adjustors, pH buffers,
preservatives, antibacterial agents, sweetening agents, and perfume
bases.
[0248] The pH adjustors favorably adjust the pH to be 5 to 9, and
no particular limitation is imposed on the pH adjustors. However,
examples thereof include hydrochloric acid, acetic acid, phosphoric
acid, citric acid, malic acid, sodium hydroxide and sodium
hydrogencarbonate.
<Testing Method>
[0249] The compound contained in the labeling composition for the
intraocular tissue in the present invention is favorably a compound
with a maximum luminance ratio (La/Lb) of 3 or more when the
following testing method is performed. Details of the testing
method for selecting the compound used in the present invention are
described below.
[0250] An aqueous solution of a test compound is prepared to give a
concentration of 1 .mu.g/mL, and three Zebrafish 7-day-old embryos
are exposed to the aqueous solution for one hour. Thereafter, the
embryos are washed twice with purified water and immobilized with a
4% paraformaldehyde solution to provide immobilized sections a1 to
a3 of one eyeball of the respective Zebrafishes. On the other hand,
three untreated Zebrafish 7-day-old embryos are immobilized with a
4% paraformaldehyde solution to provide immobilized sections b1 to
b3 of one eyeballs of the respective Zebrafishes. In this testing
method, an immobilized section containing the entire layer
structures of at least vitreous body and retina is used.
[0251] The immobilized sections a1 to a3 are used to adjust the
magnification of an observation apparatus to a magnification
capable of observing a retinal section of each eyeball, thereby
setting the excitation wavelength and the fluorescence wavelength
to those at which the fluorescence intensity in the retinal tissue
becomes maximum. The magnification capable of observing the retinal
section is not required to be strictly adjusted so far as the layer
structures of the vitreous body and retina can be identified.
However, the magnification is favorably such that the whole tissue
section of the eye is included. Specifically, the structure of the
retinal tissue can be visualized by setting the magnification to
from 10 times to 400 times, favorably from 40 times to 200 times.
On the other hand, the average value of maximum luminance values in
arbitrarily-sized retinal regions between the retinal pigment
epithelium and vitreous body of the immobilized sections b1 to b3
is regarded as Lb to set the intensity of excitation light and
photographic sensitivity such that the value Lb falls within a
range of from 2 to 6.
[0252] Under the thus-set observation conditions, the average value
of maximum luminance values in arbitrarily-sized retinal regions
between the retinal pigment epithelium and vitreous body of the
immobilized sections a1 to a3 is regarded as La to calculate the
value La/Lb.
[0253] No particular limitation is imposed on the strain of
Zebrafishes used in this testing method. However, for example, a
wild type having no genetic variation, or a strain having no
genetic variation relating to eyes in particular is favorably used.
Specifically, RIKEN WT (RW), AB strain or TU strain is favorably
used.
[0254] The exposure to the solution of the test compound can be
conducted by putting a Zebrafish 7-day-old embryo in an arbitrary
well in, for example, a 24-well plate together with breeding water,
and taking the breeding water out of the well to be replaced by the
solution of the test compound, thereby bringing the solution into
contact with the embryo. A method of adding a proper amount of a
thick solution of the test compound to a Zebrafish embryo put in a
well together with distilled water in such a manner that the final
concentration is 1 .mu.g/mL may also be used.
[0255] In general, the compound migrating to the intraocular tissue
without damaging the ocular tissue or the nerve tissue linking to
the ocular tissue can label the intraocular tissue by exposing the
Zebrafish 7-day-old embryo to the solution of the test compound for
1 hour. No particular limitation is imposed on the observation of
the intraocular tissue. However, for example, observation can be
made by observing an immobilized section of an ocular tissue of
Zebrafish, which has been exposed to the solution of the test
compound.
[0256] No particular limitation is imposed on preparation of the
immobilized section of an eyeball of Zebrafish. However, for
example, the section can be prepared by embedding Zebrafish in a
low-temperature fused agarose gel and slicing it by a commercially
available microtome.
[0257] No particular limitation is imposed on the commercially
available microtome so far as a section can be prepared. However,
examples thereof include Vibratome and Linear Slicer. In
particular, Linear Slicer is favorably used because a section is
prepared more satisfactorily. The immobilized section prepared by
such a microtome is placed on a slide glass to observe it.
[0258] It may also be possible to embed Zebrafish in a compound for
a frozen tissue section, rapidly freeze it with liquid nitrogen,
and then prepare a section by a cryostat microtome. No particular
limitation is imposed on the cryostat microtome. However, a
commercially available Cryostat may be favorably used. The frozen
section prepared in such a manner is placed on a slide glass and
then dried to observe it.
[0259] No particular limitation is imposed on the thickness of a
section upon preparation of the section so far as the thickness is
fixed upon testing. However, for example, a thickness of 5 .mu.m or
more is favorable because its morphology is easily kept upon its
transfer to a slide glass.
[0260] The control to the excitation wavelength and fluorescence
wavelength at which the fluorescence intensity in the retinal
tissue becomes maximum can be made by changing the set of
fluorescent light filters in such a manner that when a section
sample exposed to an aqueous solution containing a test compound is
observed, the sample is irradiated with excitation light of an
arbitrary wavelength, and fluorescence of a wavelength longer by at
least 10 nm or more, favorably 20 nm or more than the excitation
wavelength is detected, the excitation light is scarcely visible
and the fluorescence intensity becomes highest.
[0261] For the measurement of the maximum luminance value in the
region including the retinal tissue, it is necessary to set the
excitation wavelength and fluorescence wavelength of the
observation apparatus according to the test compound used.
[0262] No particular limitation is imposed on the observation
apparatus used so far as it permits fluorescence observation.
However, examples thereof include a stereoscopic fluorescence
microscope, a fluorescence microscope and a confocal laser-scanning
fluorescence microscope.
[0263] In this testing method, an image containing the retinal
region of the immobilized section b is obtained, and a maximum
luminance value in an arbitrary region of the image thus obtained
is measured.
[0264] The maximum luminance value is measured by means of a
software or the like. It is favorable to measure the value by
converting the image to a gray scale. No particular limitation is
imposed on the software used in the measurement of the maximum
luminance value so far as the maximum luminance value in the
arbitrary region is determined. However, examples thereof may
include NIH Image, Scion Image and Image J.
[0265] Such software is used to control the intensity of excitation
light and photographic sensitivity in such a manner that the
maximum luminance values of the immobilized sections b1 to b3 fall
within a range of from 2 to 6, thereby obtaining the average value
Lb of the maximum luminance values of the immobilized sections b1
to b3.
[0266] An image containing the retinal region of each of the
immobilized sections a1 to a3 is then obtained under the conditions
set above, whereby the average value La of the maximum luminance
values of the immobilized sections a1 to a3 can be obtained in the
same manner as described above.
[0267] The compound contained in the labeling composition for
intraocular tissue according to the present invention is favorably
such that the ratio (La/Lb) of La to Lb obtained in the
above-described testing method is 3 or more. The compound that the
ratio La/Lb is 3 or more can clearly label the intraocular tissue
without damaging the ocular tissue or the nerve tissue linking to
the ocular tissue. The ratio La/Lb is favorably 5 or more, more
favorably 10 or more, because the intraocular tissue is stained in
a clearly distinguishable state even when the concentration of the
compound is low.
<Labeling Method>
[0268] The labeling method for the intraocular tissue according to
the present invention can be conducted by using the labeling
composition for the intraocular tissue containing at least one of
the staining compound s represented by any one of the general
formulae (I) to (XVIV), (VIII'), and (XVII'). The method for
labeling the intraocular tissue of a living individual is to label
with the labeling composition without causing a surgical damage
such as incision of an ocular tissue or needling into the ocular
tissue or a nerve tissue linking to the ocular tissue.
[0269] No particular limitation is imposed on the labeling method
without the surgical damage. However, examples thereof include a
method of exposing a part or the whole of a living individual to
the labeling composition for the intraocular tissue, a method by
oral contact, a method by pneumonic contact, a method by nasal
contact, a method by transgastrointestinal contact, a method by
transmucosal contact, a method by transhumoral contact, a method by
hypoglossal contact, a method by intravascular contact such as
intravenous or intra-arterial contact, a method by intra-abdominal
contact, an intra-abdominal, subcutaneous, intracutaneous,
intravesical or endotracheal (intrabronchial) injection method, and
a method by contact with the interior of a living body by a device
such as spraying or coating.
[0270] The labeling composition for the intraocular tissue
according to the present invention may also be injected directly
into an eye to label the intraocular tissue. No particular
limitation is imposed on the method of injecting directly into the
eye. However, for example, the method is performed by making a hole
by sclerotomy, filling a posterior portion of a vitreous body with
air and then injecting several drops, generally 1 mL or less, of
the labeling composition for the intraocular tissue according to
the present invention into the eye.
<Observing Method>
[0271] The observing method of the present invention uses the
labeling composition for the intraocular tissue according to the
present invention. The measuring and detecting methods thereof are
performed by methods publicly known by a person with ordinary skill
in the art.
[0272] No particular limitation is imposed on the observing method
used in the present invention so far as the intraocular tissue is
not affected. The method is a method for capturing the condition
and change of a living sample as images. Examples thereof include
visible light observation, near-infrared light observation and
infrared light observation performed by irradiating an ocular
tissue with visible light, near-infrared light and infrared light,
respectively, and observing the ocular tissue by a camera or CCD;
laser microscope observation; fluorescence observation,
fluorescence microscope observation, fluorescence endoscope
observation, confocal microscope observation, multiple
photon-excited fluorescence microscope observation, and narrow-band
light observation performed by irradiating a living sample with
excitation light from an excitation light source like a
fluorescence endoscope to observe fluorescence of the living sample
causing emission; optical coherence tomographic (OCT) observation;
and observation by a soft X-ray microscope.
[0273] No particular limitation is imposed on the wavelength for
excitation used in the present invention. However, it varies
according to the staining compound represented by any one of the
general formulae (I) to (XVIV), (VIII'), and (XVII'), and no
particular limitation is imposed thereon so far as the staining
compound represented by any one of the general formulae (I) to
(XVIV), (VIII'), and (XVII') efficiently emits fluorescence. The
wavelength is generally from 200 to 1,010 nm, favorably from 400 to
900 nm, more favorably from 480 to 800 nm. In the case of using
light in a near-infrared region, a wavelength of generally from 600
to 1,000 nm, favorably from 680 to 900 nm, is used.
[0274] No particular limitation is imposed on the light source for
fluorescence excitation used in the present invention. However,
various kinds of laser beam sources may be used. Examples thereof
include dye lasers, semiconductor lasers, ion lasers, fiber lasers,
halogen lamps, xenon lamps and tungsten lamps. Various kinds of
optical filters may be used to obtain a favorable wavelength for
excitation or to detect fluorescence alone.
[0275] When an intraocular tissue is imaged in a state of causing
emission in the interior of an ocular tissue by irradiating a
living individual with excitation light in the above-described
manner, an emitting site can be easily detected. The intraocular
tissue can be observed in more detail by combining a bright field
image obtained by irradiation of visible light with a fluorescent
image obtained by irradiation of excitation light by an image
processing unit.
<Screening Method>
[0276] According to the labeling composition for the intraocular
tissue according to the present invention, general characteristics
with respect to staining of an intraocular tissue can be screened
in a living state, i.e., in vivo, by using a living individual, for
example, Zebrafish that is a small bony fish. In addition, since
Zebrafish that is a living individual is used, the safety of the
labeling composition for the intraocular tissue may also be
screened at the same time.
[0277] Zebrafish has been acknowledged as the third model animal
following mouse and rat in the United States and Britain in recent
years, and it has been found that compared with human the whole
genome sequence thereof has 80% homology, the number of genes is
also almost the same, and the geneses and structures of the main
organs and tissues are also well similar. The Zebrafish is
characterized in that the processes of differentiating from a
fertilized ovum to form respective parts (viscera and organs such
as heart, liver, kidney and alimentary canal) can be observed
through a transparent body, so that it is particularly favorable to
use Zebrafish in screening as a model animal.
[0278] As a specific example, is mentioned such a screening method
that the labeling composition for the intraocular tissue is brought
into contact with Zebrafish to observe an influence of the labeling
composition for the intraocular tissue on Zebrafish.
[0279] No particular limitation is imposed on the method for
bringing Zebrafish into contact. However, examples thereof include
a method of administering the labeling composition for the
intraocular tissue into breeding water in the case where the
labeling composition for the intraocular tissue is water-soluble,
and include, in the case where the labeling composition for the
intraocular tissue is water-insoluble, a method of dispersing the
labeling composition for the intraocular tissue in breeding water
by itself, a method of administering the composition together with
a trace quantity of a surfactant or DMSO, a method of mixing the
composition with a feed for Zebrafish to orally administering it,
and a method of parenterally administering the composition by
injection or the like. The method of administering the labeling
composition for the intraocular tissue into breeding water is
favorable because of its easiness. Thus, the staining compound
represented by any one of the general formulae (I) to (XVIV),
(VIII'), and (XVII') is desirably water-soluble, and a carboxyl
group or sulfonic is desirably contained in the compound.
[0280] One or more labeling compositions for the intraocular tissue
according to the present invention can be utilized as active agents
to conduct in vivo screening for influence on a living body, such
as effects, side effects or safety of a chemical substance in an
intraocular tissue, using a living individual, for example,
Zebrafish. The labeling compositions for the intraocular tissue can
be selected as needed according to target sites, objects,
inspection methods and/or the like. In addition, applied
developments such as high-precision diagnoses of diseases and
development of curing methods are expected from the staining
ability of the labeling composition for the intraocular tissue, and
so this composition may be used as a diagnostic composition.
[0281] The chemical substance means a generic name of a compound
having chemical action and is not particularly limited. However,
examples thereof include pharmaceuticals, organic compounds, curing
medicines, investigational drugs, agricultural chemicals,
cosmetics, environmental pollutants and endocrine disruptors.
[0282] Zebrafish is not limited to wild type Zebrafish, and
Zebrafishes of various disease type models may be used according to
the object of screening. In the case of using a disease type model,
the effects of a proposed new medicine compound can be found by
observation to apply them to conduct screening of a disease curing
or preventing medicine.
[0283] The screening method according to the present invention can
use small bony fishes. No particular limitation is imposed on the
small bony fishes used in the screening method according to the
present invention. However, examples thereof include Zebrafish,
globefish, goldfish, killifish and giant rerio. The small bony
fishes are favorably used because they are excellent in speed and
cost compared with mouse and rat. In particular, Zebrafish is
favorable because decoding of genome has been almost completed, its
breeding and propagation are easy, circulation cost is also cheap,
and the basic structures of main organs and tissues are formed in
48 to 72 hours after fertilization.
[0284] The labeling composition for the intraocular tissue
according to the present invention can be used in measures for
specifically and selectively labeling, for example, a diseased
cellular tissue at ophthalmic surgery or a site of a test substance
suspected to be a tumor, thereby ascertaining a difference from
normal cells, or in observation of a change of a tissue by a
disease.
[0285] The labeling composition for the intraocular tissue
according to the present invention can specifically label an
intraocular tissue of a living individual without need of an
invasive operation such as exposure of an ocular tissue or
injection of a staining agent into the ocular tissue or a nerve
tissue linking to the ocular tissue. Accordingly, the composition
can be applied to a diagnostic agent making good use of these
identification abilities.
[0286] No particular limitation is imposed on the diagnostic agent,
and the composition can be used as, for example, a diagnostic agent
for inspecting the functions of the eyes and a diagnostic agent of
ophthalmic diseases.
<Extrapolability to Human>
[0287] The labeling composition for the intraocular tissue
according to the present invention may also be applied to human.
The extrapolability to human is confirmed by overall approximation
in recognition of analogy and points of difference of intraocular
tissues between human and a laboratory animal. Examples are given
below though not limited thereto.
[0288] 1. Intraocular tissues of human and another living sample
than human are labeled to confirm analogy therebetween. Examples of
other living samples than human include mammals such as mouse,
hamster, rat, guinea pig, rabbit, dog, pig, cat and monkey; and
bony fishes such as Zebrafish.
[0289] 2. An immobilized tissue section of said another living
sample than human is used to confirm staining characteristics of
intraocular tissues.
[0290] 3. An immobilized tissue section of human is used to confirm
staining characteristics of intraocular tissues.
[0291] By confirming the above 3 points, it can be confirmed that
the labeling composition for the intraocular tissue according to
the present invention may also be applied to human.
[0292] As another method for confirming the extrapolability to
human, it can be confirmed by radiolabeling the labeling
composition for the intraocular tissue according to the present
invention, administering a trace quantity of the labeled
composition into a body of human and confirming localization of the
compound in an intraocular tissue. This method is called a
microdosing test.
[0293] As another method, there is a method of identifying and
confirming the following 4 points.
[0294] 1. A target biomolecule or labeling mechanism of the
labeling composition for the intraocular tissue according to the
present invention is identified in an intraocular tissue of another
living sample than human.
[0295] 2. A human biomolecule or labeling mechanism homologous to
said target biomolecule or labeling mechanism is identified.
[0296] 3. Said human biomolecule or staining mechanism is
introduced into another laboratory animal than human by a genetic
modification.
[0297] 4. The thus-obtained laboratory animal is used to confirm
the fact that staining is made through the biomolecule or staining
mechanism introduced.
[0298] As another living sample than human, Zebrafish may be
particularly favorably used. Eyes are very well preserved
anatomically, histologically and biochemically among many
vertebrate animals, and the same applies to the genesis. The retina
of Zebrafish is made up of 7 main cell types like many presently
existing vertebrate animals (Progress in Retinal and Eye Research,
27, pp. 89-110 (2008)), and comparison of retinal tissue section
images between human and Zebrafish shows very high commonality
though different in scale (Current Opinion in Pharmacology, 4,
504-512 (2004)). The use of Zebrafish has high advantage of low
breeding cost and small amount of the compound used compared with
mouse. In addition, commonality of not only the morphology, but
also the base sequence of rhodopsin that is a visual pigment is
somewhat higher than mouse, thus indicating high homology to human
at molecular levels. Rodent is nocturnal, while Zebrafish is
diurnal and has various cone cells in addition to rod cells at the
retina, so that human and Zebrafish have high homology both
histologically and functionally. From the above, it is favorable
that Zebrafish is used to confirm the extrapolability of the
labeling composition for the intraocular tissue according to the
present invention to human.
EXAMPLES
[0299] The present invention will hereinafter be described in more
detail by the following Examples. These Examples are specific
examples shown for understanding more deeply the present invention,
and the present invention is by no means limited to these specific
examples. Incidentally, "%" is based on mass unless expressly
noted.
Staining of Intraocular Tissue by Labeling Composition for
Intraocular Tissue
Example 1
[0300] Distilled water was added to a 1 mg/mL DMSO solution of
Staining compound 1 to obtain Staining Liquid 1 containing Staining
compound 1 at a concentration of 1 .mu.g/mL. In an arbitrary well
of a 24-well multiplate (product of IWAKI), were put 1 mL of
Staining Liquid 1 and a fry of Zebrafish 7-day-old embryo (7 dpf),
and the plate was left to stand for 1 hour. A process of removing
Staining Liquid 1 in the well and replacing it by 1 mL of distilled
water was then performed 3 times. Further, 1 mL of a phosphate
buffer solution containing 4% paraformaldehyde was added, and the
plate was left to stand for 1 hour. The fry was then taken out of
the well and embedded in a 5% low-temperature fused agarose gel to
prepare a section of an ocular tissue using Linear Slicer PRO 7
(manufactured by Dosaka EM Co., Ltd.). The thus-prepared ocular
tissue section was placed on a slide glass to observe the ocular
tissue through a confocal microscope (Pascal Exciter, manufactured
by Zeiss Co.).
Examples 2 to 106
[0301] Sections were prepared by the same process as in Example 1
except that Staining compound 1 in Example 1 was changed to
staining compound s described in Table 1, and observed.
Comparative Examples 1 to 3
[0302] Sections were prepared by the same process as in Example 1
except that Staining compound 1 in Example 1 was not used or
changed to indocyanine green (ICG) or fluorescein (FLU) as
described in Table 1, and observed.
Evaluation of Intraocular Tissue-Staining Ability
[0303] Regarding Examples 1 to 106, and Comparative Examples 1 to
3, staining ability (+++: intraocular tissue including
photoreceptor cell layer being strongly stained; ++: photoreceptor
cell layer being stained; -: not stained), and stained sites (1:
layer structure including photoreceptor cell layer in retinal
tissue being visualized by 1 to 5 layers thereof; 2: retinal tissue
being wholly visualized; 3: L: vitreous body being stained) were
evaluated.
[0304] The results thereof are shown in Table 1. Incidentally, the
excitation wavelengths and fluorescence wavelengths of the staining
compound s in Examples 1 to 106 were determined by subjecting an
aqueous solution obtained by diluting a 10 mg/mL DMSO solution to
1/500 with purified water to measurement using a fluorescence
spectrometer FL4500 (manufactured by Hitachi High-Technologies
Corporation). Regarding the staining compound s in Examples 95 to
106, values in a chloroform solution are shown.
TABLE-US-00001 TABLE 1 Staining Stained Compound No. .lamda.ex
.lamda.em ability site Ex. No. 1 1 599 619 ++ 2 2 2 486 553 +++ 2 3
3 480 556 ++ 2 4 4 473 560 ++ 1 5 5 556 577 +++ 2, L 6 6 555 576 ++
2, L 7 7 469 555 ++ 2, L 8 8 477 592 ++ 2 9 9 464 514 ++ 1 10 10
459 520 ++ 1 11 11 422 476 +++ 2, L 12 12 463 509 ++ 2, L 13 13 472
504 +++ 2 14 14 588 617 ++ 2, L 15 15 496 569 +++ 2, L 16 17 459
564 ++ 1 17 19 545 576 ++ 1 18 20 555 567 ++ 1 19 21 334 377 ++ 2,
L 20 22 571 620 ++ 1 21 24 609 692 ++ 1 22 25 635 671 ++ 1 23 26
679 715 ++ 2 24 27 485 575 ++ 1 25 29 474 509 ++ 1, L 26 30 353 481
++ 1 27 31 495 664 ++ 2 28 32 558 640 ++ 2, L 29 33 512 628 ++ 1, L
30 34 571 648 ++ 2, L 31 35 535 655 ++ 2 32 36 566 661 +++ 1 33 37
531 633 +++ 1 34 38 482 609 ++ 1 35 39 576 657 ++ 1 36 40 559 671
++ 1 37 41 582 679 ++ 1 38 44 564 651 ++ 1 39 45 481 586 ++ 1 40 47
522 611 ++ 1 41 48 541 619 ++ 2 42 49 569 649 ++ 1 43 50 482 580 ++
1 44 51 580 638 ++ 2 45 52 580 638 ++ 1 46 53 568 712 ++ 1 47 54
597 647 ++ 2 48 55 502 644 ++ 1 49 56 486 606 ++ 2 50 57 590 671 ++
2 51 58 546 676 ++ 2 52 59 581 625 ++ 1 53 60 560 677 ++ 2 54 61
545 636 ++ 2, L 55 62 610 675 ++ 2 56 63 566 611 ++ 1 57 64 401 516
++ 2 58 65 570 657 ++ 1 59 66 527 636 +++ 2 60 67 570 699 +++ 2 61
68 450 527 ++ 2 62 69 653 609 ++ 1 63 70 556 659 ++ 1 64 71 592 671
++ 2, L 65 72 611 720 ++ 1 66 73 548 649 ++ 1 67 74 614 669 ++ 1 68
75 619 683 ++ 1 69 76 674 724 ++ 1 70 77 583 668 ++ 1 71 78 598 726
++ 1 72 79 567 702 ++ 1 73 80 551 673 ++ 1 74 81 576 604 ++ 2, L 75
82 591 721 +++ 2 76 83 605 682 ++ 2 77 84 599 718 ++ 2 78 85 353
503 ++ 2 79 86 623 671 ++ 1 80 87 466 551 ++ 2, L 81 88 450 503 ++
1 82 90 528 550 ++ 2, L 83 95 509 578 ++ 2, L 84 95 528 590 ++ 1 85
97 530 680 .largecircle. 1 86 98 510 690 .largecircle. 1 87 100 430
600 .largecircle. 1 88 101 560 760 .largecircle. 1 89 102 590 700
.largecircle. 1 90 103 520 640 .largecircle. 1 91 106 510 650
.largecircle. 1 92 107 380 490 .largecircle. 1 93 108 530 680
.largecircle. 1 94 109 540 630 .largecircle. 1 95 110 538 614
.largecircle. 1 96 111 527 589 .largecircle. 1 97 112 513 568
.largecircle. 1 98 113 514 575 .largecircle. 1 99 114 513 568
.largecircle. 1 100 115 540 614 .largecircle. 1 101 116 537 584
.largecircle. 1 102 117 538 581 .largecircle. 1 103 118 540 634
.largecircle. 1 104 120 537 609 .largecircle. 1 105 121 537 609
.largecircle. 1 106 122 534 582 .largecircle. 1 Comp. Ex. No. 1 Not
used -- -- 2 ICG 784 811 -- -- 3 Fluorescein 494 521 -- --
[0305] As apparent from Table 1, the labeling compositions for the
intraocular tissue according to the present invention are excellent
in fluorescence sensitivity and able to stain living intraocular
tissues, so that the staining ability thereof is excellent. The
stained sites varied according to the staining compound s used.
Some of the compositions strongly stained the photoreceptor cell
layer, while some of them exhibited staining ability to the whole
retinal tissue. In addition, screening can be cheaply performed by
using Zebrafish as a model animal.
In Vivo Intraocular Observation
Example 107
[0306] Distilled water was added to a 1 mg/mL DMSO solution of
Staining compound 5 so as to give a concentration of 1 .mu.g/mL to
obtain Staining Liquid 2. In an arbitrary well of a 24-well
multiplate, were put 1 mL of Staining Liquid 2 and a fry of
Zebrafish, and the plate was left to stand for 1 hour. A process of
removing Staining Liquid 2 in the well and replacing it by 1 mL of
distilled water was then performed 3 times.
[0307] The fry was then put in a 0.7% agarose solution and placed
on a slide glass to observe the fundus of the fry through a
confocal microscope.
Examples 108 to 119
[0308] Fundus of fry were observed according to the same process as
in Example 107 except that the compound used in Example 107 was
changed to compounds shown in Table 2.
Comparative Examples 4 to 6
[0309] Fundus of fry were observed according to the same process as
in Example 107 except that the compound used in Example 107 was
changed to compounds shown in Table 2.
Evaluation of In Vivo Intraocular Observation
[0310] Regarding Examples 107 to 119 and Comparative Examples 4 to
6, staining ability (+++: both photoreceptor cell layer and further
inner layer structure of retina comprised of plural layers being
stained; ++: only photoreceptor cell layer of retina comprised of
plural layers being mainly stained; -: not observed) was
evaluated.
[0311] The results thereof are shown in Table 2.
TABLE-US-00002 TABLE 2 Ex. No. Compound No. Result of observation
107 5 +++ 108 6 ++ 109 7 +++ 110 8 +++ 111 9 ++ 112 10 ++ 113 11
+++ 114 12 ++ 115 13 +++ 116 36 ++ 117 48 ++ 118 66 ++ 119 81
+++
TABLE-US-00003 Comp. Ex. No. Compound No. Result of observation 4
Not used -- 5 ICG -- 6 Fluorescein --
[0312] In Examples 107 to 119, fluorescence was observed at the
fundus of Zebrafish. Specifically, as illustrated in, for example,
FIG. 4, it is understood that the labeling compositions for the
intraocular tissue according to the present invention noninvasively
migrate to the intraocular tissue to clearly image the intraocular
tissue.
Example 120
[0313] After an eyeball of a fry of Zebrafish 7-day-old embryo (7
dpf) was immobilized with a PBS solution of 4% paraformaldehyde
(PFA), the solution was replaced by a PBS solution of 30% sucrose,
and the eyeball was embedded in an OCT compound (product of
Tissue-Tek Co.) and frozen with liquid nitrogen. The frozen sample
was sliced into a 7-.mu.m section by Cryostat (Tissue.cndot.Tek
Cryo 3, manufactured by Sakura Finetek Japan Co., Ltd.) and
extended on a MAS-coated slide glass (product of MATSUNAMI GLASS
IND., LTD) to obtain a tissue section slide of the Zebrafish
eyeball. The slide glass was treated with a blocking solution
(Blocking One, product of Nakarai Co.) for 30 minutes at room
temperature, and an anti-Zpr-1 antibody (which stains red and green
cone cells) diluted with PBS was mounted as a primary antibody on
the slide glass and treated for 12 hours at 4.degree. C. The slide
glass was then washed 3 times with PBST for 10 minutes, and a
secondary antibody (Alexa 488-anti-mouse IgG antibody) and 1
.mu.g/mL of Compound 5 were diluted with PBS and mounted on the
slide glass for 1 hour at room temperature. The slide glass was
washed 3 times with PBST for 10 minutes, Fluoromount G (product of
Southern Biotechnology Co.) was mounted thereon, and a cover glass
was placed thereon. The thus-obtained stained slide of the tissue
section of the Zebrafish eye ball was observed through a confocal
microscope.
Example 121
[0314] A stained slide of a tissue section of a Zebrafish eye ball
was prepared according to the same process as in Example 120 except
that the primary antibody used in Example 120 was changed to an
anti-Zpr-3 antibody (which stains rod cells), and observed through
a confocal microscope.
Example 122
[0315] A stained slide of a tissue section of a human normal retina
was prepared according to the same process as in Example 120 except
that the tissue section slide of the Zebrafish eyeball was changed
to a tissue section slide (product of US Biomax Co.) of the human
normal retina, and the primary antibody and secondary antibody were
changed to anti-mGluR5 antibody (which stains cone cells) and Alexa
488-anti-rabbit IgG antibody, respectively, and observed through a
confocal microscope.
Evaluation of Stained Slide
[0316] In Example 120, the stained images with Compound 5 and
anti-Zpr-1 antibody in the photoreceptor cell layer did not overlap
(FIG. 5), but in Example 121, the stained images with Compound 5
and anti-Zpr-3 antibody in the photoreceptor cell layer partially
overlapped. This fact indicates that Compound 5 has staining
ability to the photoreceptor cell layer but does not stain the red
and green cone cells in the photoreceptor cell layer, or
non-stained portions exist in cell bodies existing in the
photoreceptor cell layer. On the other hand, the result of Example
121 reveals that Compound 5 has stained the rod cells and at least
one kind of cell type of blue or ultraviolet cone cells. From this
fact, it was confirmed that Compound 5 selectively stains a part of
the cell types in the photoreceptor cell layer or of the cell
bodies existing in the photoreceptor cell layer.
[0317] In Example 122, it was confirmed that the photoreceptor cell
layer common to the retina of Zebrafish in the frozen section of
the human normal retina is stained. It was also confirmed that the
stained site by the anti-mGluR5 antibody is different, and so a
part of the cell types in the photoreceptor cell layer or of the
cell bodies existing in the photoreceptor cell layer is selectively
stained even in human. From the above results, it was confirmed
that the retinas of human and Zebrafish are approximate in staining
characteristics, and the labeling composition for the intraocular
tissue according to the present invention is high in
extrapolability to human.
Staining of Mouse Intraocular Tissue
Example 123
[0318] Staining compound 81 was added to an equimolar amount of a
NaOH solution so as to give a concentration of 10 mg/mL, and the
resultant mixture was centrifuged for 5 minutes at 14 krpm to
obtain a supernatant. To an abdominal cavity of a 3-month-old B10
mouse, was administered 0.2 mL of this supernatant at one time.
After 1 hour, the thus-treated animal was sacrificed under
anesthesia by diethyl ether, and an eyeball thereof was taken out.
The eyeball taken was embedded in an OCT compound and frozen in
isopentane chilled with liquid nitrogen. This eyeball was sliced
into a thickness of about 5 .mu.m in a cryostat chilled to
-20.degree. C., and this slice was placed on a slide glass and
dried to prepare a section of an ocular tissue. The thus-prepared
ocular tissue section was observed through a confocal microscope
(Pascal Exciter, manufactured by Zeiss Co.). As a result, it was
confirmed that Compound 81 has staining ability to the retina by
administration to the abdominal cavity of the mouse.
Staining of Intraocular Tissue and Hair Cell
[0319] It was confirmed that the staining compound s used in
Examples 15 to 20, 24, 53 to 58, 65, 67, 82, and 83 stain hair
cells in vivo in addition to the effect of staining the intraocular
tissues. In other words, it was found that the compound represented
by Compound Nos. 15, 17, 19 to 22, 27, 60 to 65, 72, 74, 90, and 94
have an effect of coincidentally staining both intraocular tissues
and hair cells.
INDUSTRIAL APPLICABILITY
[0320] The present invention provide labeling compositions for
intraocular tissues, which can stain intraocular tissues of living
samples and can image cell morphologies of the intraocular tissues
with high sensitivity, so that the compositions become materials
essential to researches in the ophthalmic field and ophthalmologic
imaging techniques. In addition, evaluation with time becomes
feasible in drug discovery relating to ophthalmic diseases,
high-throughput and high-precision screening can be conducted at
low cost, and development of new diagnosing methods and curing
methods for diseases as well as ophthalmologic researches are
significantly advanced. Thus, the present invention becomes an
extremely effective platform technology from industrial and
practical points of view.
[0321] The present invention is not limited to the above
embodiments and various changes and modifications can be made
within the spirit and scope of the present invention. Therefore to
apprise the public of the scope of the present invention, the
following claims are made.
[0322] This application claims the benefit of Japanese Patent
Application No. 2008-330988, filed Dec. 25, 2008, which is hereby
incorporated by reference herein in its entirety.
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