U.S. patent application number 13/843546 was filed with the patent office on 2013-08-22 for probe for a hair cell, and labelling method for a hair cell using the probe for a hair cell.
This patent application is currently assigned to CANON KABUSHIKI KAISHA. The applicant listed for this patent is CANON KABUSHIKI KAISHA. Invention is credited to Takeshi Miyazaki, Norihiro Nishimura, Yuhei Nishimura, Tsuyoshi Nomoto, Yasuhito Shimada, Taichi Shintou, Kaoru Takahashi, Toshio Tanaka, Kohei Watanabe.
Application Number | 20130219529 13/843546 |
Document ID | / |
Family ID | 42027841 |
Filed Date | 2013-08-22 |
United States Patent
Application |
20130219529 |
Kind Code |
A1 |
Nomoto; Tsuyoshi ; et
al. |
August 22, 2013 |
PROBE FOR A HAIR CELL, AND LABELLING METHOD FOR A HAIR CELL USING
THE PROBE FOR A HAIR CELL
Abstract
Provided are a novel probe for a hair cell for clearly
identifying various conditions of a hair cell, and a labelling
method for a hair cell using the probe for a hair cell, more
particularly, a probe for a hair cell containing, as an active
agent, at least one kind selected from staining compounds
represented by one of the general formulae (I) and (II), and a
labelling method for a hair cell using the probe for a hair cell.
##STR00001##
Inventors: |
Nomoto; Tsuyoshi; (Tokyo,
JP) ; Miyazaki; Takeshi; (Yokohama-shi, JP) ;
Watanabe; Kohei; (Yokohama-shi, JP) ; Shintou;
Taichi; (Saitama-shi, JP) ; Takahashi; Kaoru;
(Saitama-shi, JP) ; Tanaka; Toshio; (Tsu-shi,
JP) ; Nishimura; Yuhei; (Tsu-shi, JP) ;
Shimada; Yasuhito; (Nagoya-shi, JP) ; Nishimura;
Norihiro; (Tsu-shi, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CANON KABUSHIKI KAISHA; |
|
|
US |
|
|
Assignee: |
CANON KABUSHIKI KAISHA
Tokyo
JP
|
Family ID: |
42027841 |
Appl. No.: |
13/843546 |
Filed: |
March 15, 2013 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12644472 |
Dec 22, 2009 |
8460639 |
|
|
13843546 |
|
|
|
|
Current U.S.
Class: |
800/3 ;
435/29 |
Current CPC
Class: |
A61P 27/16 20180101;
A61K 49/006 20130101; G01N 33/5005 20130101; G01N 33/5044 20130101;
G01N 2800/20 20130101; A61K 49/0032 20130101; G01N 33/6893
20130101; A61K 49/0041 20130101; C09B 23/105 20130101; C09B 23/0066
20130101; C09B 23/0075 20130101; C09B 23/06 20130101; C09B 23/0016
20130101; G01N 2800/14 20130101; C09B 23/107 20130101; C09B 23/0058
20130101; C09B 11/24 20130101 |
Class at
Publication: |
800/3 ;
435/29 |
International
Class: |
G01N 33/50 20060101
G01N033/50 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 25, 2008 |
JP |
2008-330979 |
Claims
1-17. (canceled)
18. A labelling method for a hair cell comprising administering to
a biological specimen a probe for a hair cell which contains as an
active agent, a staining compound represented by general formula
(I): ##STR00023## wherein: R.sub.1 represents a hydrogen atom, an
alkyl group, or an aryl group; R.sub.2 to R.sub.5 each
independently represent a hydrogen atom, an alkyl group, an aryl
group, an alkoxy group, a carboxylic acid group, a sulfonic acid
group, a heterocyclic group, an amino group, or a halogen atom, and
R.sub.2 and R.sub.3, R.sub.3 and R.sub.4, or R.sub.4 and R.sub.5
are optionally bonded to each other to form a ring; R.sub.6
represents a hydrogen atom or an alkyl group; R.sub.7 represents a
hydrogen atom, an alkyl group, a carboxylic acid group, or a cyano
group; X.sub.1.sup.- represents an anionic group; Y represents a
sulfur atom, an oxygen atom, --N(R.sub.8)--, or
--C(R.sub.9)(R.sub.10)--, wherein R.sub.8 to R.sub.10 each
independently represent a hydrogen atom, an alkyl group, or an aryl
group, and R.sub.9 and R.sub.10 are optionally bonded to each other
to form a ring; n represents an integer of 1 to 3; and A represents
the general formula (III): ##STR00024## wherein: R.sub.18 to
R.sub.21 each independently represent a hydrogen atom, an alkyl
group, an aryl group, an alkoxy group, a carboxylic acid group, a
sulfonic acid group, a heterocyclic group, an amino group, or a
halogen atom, and R.sub.18 and R.sub.19, R.sub.19 and R.sub.20, or
R.sub.20 and R.sub.21 are optionally bonded to each other to form a
ring; R.sub.22 represents a hydrogen atom, an alkyl group, or an
aryl group; and Z represents a sulfur atom, an oxygen atom, or
--C(R.sub.23)(R.sub.24)--, wherein R.sub.23 and R.sub.24 each
represent a hydrogen atom, an alkyl group, or an aryl group, and
R.sub.23 and R.sub.24 are optionally bonded to each other to form a
ring.
19. The labelling method for a hair cell according to claim 18,
wherein the compound represented by the general formula (I)
comprises at least one of a carboxylic acid group or a sulfonic
acid group.
20. An imaging method for a hair cell, comprising: administering to
a biological specimen a probe for a hair cell; and observing
fluorescence derived from the probe for a hair cell by irradiating
the biological specimen with excitation light; wherein the probe
for a hair cell contains as an active agent a staining compound
represented by the general formula (I) ##STR00025## wherein:
R.sub.1 represents a hydrogen atom, an alkyl group, or an aryl
group; R.sub.2 to R.sub.5 each independently represent a hydrogen
atom, an alkyl group, an aryl group, an alkoxy group, a carboxylic
acid group, a sulfonic acid group, a heterocyclic group, an amino
group, or a halogen atom, and R.sub.2 and R.sub.3, R.sub.3 and
R.sub.4, or R.sub.4 and R.sub.5 are optionally bonded to each other
to form a ring; R.sub.6 represents a hydrogen atom or an alkyl
group; R.sub.7 represents a hydrogen atom, an alkyl group, a
carboxylic acid group, or a cyano group; X.sub.1.sup.- represents
an anionic group; Y represents a sulfur atom, an oxygen atom,
--N(R.sub.8)--, or --C(R.sub.9)(R.sub.10)--, wherein R.sub.8 to
R.sub.10 each independently represent a hydrogen atom, an alkyl
group, or an aryl group, and R.sub.9 and R.sub.10 are optionally
bonded to each other to form a ring; n represents an integer of 1
to 3, and A represents the general formula (III): ##STR00026##
wherein: R.sub.18 to R.sub.21 each independently represent a
hydrogen atom, an alkyl group, an aryl group, an alkoxy group, a
carboxylic acid group, a sulfonic acid group, a heterocyclic group,
an amino group, or a halogen atom, and R.sub.18 and R.sub.19,
R.sub.19 and R.sub.20, or R.sub.20 and R.sub.21 are optionally
bonded to each other to form a ring; R.sub.22 represents a hydrogen
atom, an alkyl group, or an aryl group; and Z represents a sulfur
atom, an oxygen atom, or --C(R.sub.23)(R.sub.24)--, wherein
R.sub.23 and R.sub.24 each represent a hydrogen atom, an alkyl
group, or an aryl group, and R.sub.23 and R.sub.24 are optionally
bonded to each other to form a ring.
21. An evaluation method for auditory toxicity of a chemical
substance, comprising: administering the chemical substance to an
organism; administering to the organism a probe for a hair cell;
and observing fluorescence derived from the probe for a hair cell
by irradiating the organism with excitation light; wherein the
probe for a hair cell contains as an active agent a staining
compound represented by the general formula (I) ##STR00027##
wherein: R.sub.1 represents a hydrogen atom, an alkyl group, or an
aryl group; R.sub.2 to R.sub.5 each independently represent a
hydrogen atom, an alkyl group, an aryl group, an alkoxy group, a
carboxylic acid group, a sulfonic acid group, a heterocyclic group,
an amino group, or a halogen atom, and R.sub.2 and R.sub.3, R.sub.3
and R.sub.4, or R.sub.4 and R.sub.5 are optionally bonded to each
other to form a ring; R.sub.6 represents a hydrogen atom or an
alkyl group; R.sub.7 represents a hydrogen atom, an alkyl group, a
carboxylic acid group, or a cyano group; X.sub.1.sup.- represents
an anionic group; Y represents a sulfur atom, an oxygen atom,
--N(R.sub.8)--, or --C(R.sub.9)(R.sub.10)--, wherein R.sub.8 to
R.sub.10 each independently represent a hydrogen atom, an alkyl
group, or an aryl group, and R.sub.9 and R.sub.10 are optionally
bonded to each other to form a ring; n represents an integer of 1
to 3, and A represents the general formula (III): ##STR00028##
wherein: R.sub.18 to R.sub.21 each independently represent a
hydrogen atom, an alkyl group, an aryl group, an alkoxy group, a
carboxylic acid group, a sulfonic acid group, a heterocyclic group,
an amino group, or a halogen atom, and R.sub.18 and R.sub.19,
R.sub.19 and R.sub.20, or R.sub.20 and R.sub.21 are optionally
bonded to each other to form a ring; R.sub.22 represents a hydrogen
atom, an alkyl group, or an aryl group; and Z represents a sulfur
atom, an oxygen atom, or --C(R.sub.23)(R.sub.24)--, wherein
R.sub.23 and R.sub.24 each represent a hydrogen atom, an alkyl
group, or an aryl group, and R.sub.23 and R.sub.24 are optionally
bonded to each other to form a ring.
22. The evaluation method for auditory toxicity of a chemical
substance according to claim 21, wherein the organism comprises
Zebrafish.
23. A screening method for a therapeutic drug for hearing loss,
comprising: administering a test substance to a hearing loss model
animal; administering a diagnostic composition for an auditory
function to the hearing loss model animal; and examining a staining
condition of the diagnostic composition for an auditory function
for a hair cell of the hearing loss model animal; wherein the
diagnostic composition for an auditory function comprises a probe
for a hair cell, and wherein the probe for a hair cell contains as
an active agent a staining compound represented by the general
formula (I) ##STR00029## wherein: R.sub.1 represents a hydrogen
atom, an alkyl group, or an aryl group; R.sub.2 to R.sub.5 each
independently represent a hydrogen atom, an alkyl group, an aryl
group, an alkoxy group, a carboxylic acid group, a sulfonic acid
group, a heterocyclic group, an amino group, or a halogen atom, and
R.sub.2 and R.sub.3, R.sub.3 and R.sub.4, or R.sub.4 and R.sub.5
are optionally bonded to each other to form a ring; R.sub.6
represents a hydrogen atom or an alkyl group; R.sub.7 represents a
hydrogen atom, an alkyl group, a carboxylic acid group, or a cyano
group; X.sub.1.sup.- represents an anionic group; Y represents a
sulfur atom, an oxygen atom, --N(R.sub.8)--, or
--C(R.sub.9)(R.sub.10)--, wherein R.sub.8 to R.sub.10 each
independently represent a hydrogen atom, an alkyl group, or an aryl
group, and R.sub.9 and R.sub.10 are optionally bonded to each other
to form a ring; n represents an integer of 1 to 3, and A represents
the general formula (III): ##STR00030## wherein: R.sub.18 to
R.sub.21 each independently represent a hydrogen atom, an alkyl
group, an aryl group, an alkoxy group, a carboxylic acid group, a
sulfonic acid group, a heterocyclic group, an amino group, or a
halogen atom, and R.sub.18 and R.sub.19, R.sub.19 and R.sub.20, or
R.sub.20 and R.sub.21 are optionally bonded to each other to form a
ring; R.sub.22 represents a hydrogen atom, an alkyl group, or an
aryl group; and Z represents a sulfur atom, an oxygen atom, or
--C(R.sub.23)(R.sub.24)--, wherein R.sub.23 and R.sub.24 each
represent a hydrogen atom, an alkyl group, or an aryl group, and
R.sub.23 and R.sub.24 are optionally bonded to each other to form a
ring.
24. The screening method according to claim 23, wherein the hearing
loss model animal comprises Zebrafish.
25. An evaluation method for a therapeutic drug for hearing loss,
comprising: administering a test substance to a hearing loss model
animal; administering a diagnostic composition for an auditory
function to the hearing loss model animal; and examining a staining
condition of the diagnostic composition for an auditory function
for a hair cell of the hearing loss model animal; wherein the
diagnostic composition for an auditory function comprises a probe
for a hair cell, and wherein the probe for a hair cell contains as
an active agent a staining compound represented by the general
formula (I) ##STR00031## wherein: R.sub.1 represents a hydrogen
atom, an alkyl group, or an aryl group; R.sub.2 to R.sub.5 each
independently represent a hydrogen atom, an alkyl group, an aryl
group, an alkoxy group, a carboxylic acid group, a sulfonic acid
group, a heterocyclic group, an amino group, or a halogen atom, and
R.sub.2 and R.sub.3, R.sub.3 and R.sub.4, or R.sub.4 and R.sub.5
are optionally bonded to each other to form a ring; R.sub.6
represents a hydrogen atom or an alkyl group; R.sub.7 represents a
hydrogen atom, an alkyl group, a carboxylic acid group, or a cyano
group; X.sub.1.sup.- represents an anionic group; Y represents a
sulfur atom, an oxygen atom, --N(R.sub.8)--, or
--C(R.sub.9)(R.sub.10)--, wherein R.sub.8 to R.sub.10 each
independently represent a hydrogen atom, an alkyl group, or an aryl
group, and R.sub.9 and R.sub.10 are optionally bonded to each other
to form a ring; n represents an integer of 1 to 3, and A represents
the general formula (III): ##STR00032## wherein: R.sub.18 to
R.sub.21 each independently represent a hydrogen atom, an alkyl
group, an aryl group, an alkoxy group, a carboxylic acid group, a
sulfonic acid group, a heterocyclic group, an amino group, or a
halogen atom, and R.sub.18 and R.sub.19, R.sub.19 and R.sub.20, or
R.sub.20 and R.sub.21 are optionally bonded to each other to form a
ring; R.sub.22 represents a hydrogen atom, an alkyl group, or an
aryl group; and Z represents a sulfur atom, an oxygen atom, or
--C(R.sub.23)(R.sub.24)--, wherein R.sub.23 and R.sub.24 each
represent a hydrogen atom, an alkyl group, or an aryl group, and
R.sub.23 and R.sub.24 are optionally bonded to each other to form a
ring
26. The evaluation method according to claim 25, wherein the
hearing loss model animal comprises Zebrafish.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to a probe for a hair cell,
and a labelling method for a hair cell using the probe for a hair
cell.
[0003] 2. Description of the Related Art
[0004] In a human, a hair cell exists in the cochlea which is an
auditory receptor, and in the semicircular canals and vestibular
organs both of which are vestibular sensory receptors. The hair
cell is covered with special cilia. The cilia perceive the movement
of lymph generated depending on a sound, a motion, and a posture,
and then cause an electrical change.
[0005] The abnormality of the hair cell is said to relate to
factors of disorders such as peripheral sensorineural auditory
impairment (hearing loss), tinnitus, and vertigo. The hair cell
itself is active in metabolism, and in particular, is fragile to
and easily damaged by the expose to noise and chemicals. A list of
medicaments and chemical substances which may have hair cell
toxicity has been reported. The list includes, for example, an
antibiotic such as an aminoglycoside antibiotic, an
anti-inflammatory drug, a diuretic drug, an antimalarial drug, an
antitumor drug, and a topical agent (Drug Safety: an International
Journal of Medical Toxicology and Drug Experience, 14(3), pp.
198-212, 1996).
[0006] However, thus far, there is no standardized screening method
for auditory toxicity in a drug development stage, and further,
many of already approved medicaments remain unknown for their
auditory toxicity.
[0007] For means for evaluating the auditory toxicity of a chemical
substance, for example, means for evaluating the life and death of
a hair cell of Zebrafish with a fluorescent dye such as
2-(4-(dimethylamino)styryl)-N-ethylpyridinium iodide (DASPEI) is
reported (Hearing Research, 208, pp. 79-88, 2005). Further, FM1-43
is known as a hair cell staining dyestuff (The Journal of
Histochemistry and Cytochemistry, 44(7), pp. 733-741, 1996).
[0008] For an expression mechanism of the auditory toxicity with a
chemical substance, various modes may be estimated. That is, there
is a diversity in the type of injuries on a hair cell function due
to a difference in a target biomolecule (such as a protein, an
enzyme, a nucleic acid, and a gene) of a chemical substance.
Accordingly, it is important to identify various conditions of a
hair cell (for example, loss of a specific cell function as well as
life and death of a cell) depending on the diversity in the type of
injuries. Therefore, there is a demand for hair cell staining
agents having various chemical structures.
[0009] However, known compounds for staining a hair cell are only
the two kinds (DASPEI and FM1-43) exemplified above. Those
compounds are close to each other in terms of the excitation
wavelength and fluorescence emission wavelength, and they do not
sufficiently contribute to enlargement of variations for selection
of staining technologies depending on the above mentioned diversity
in modes and on the purposes such as multiple staining.
SUMMARY OF THE INVENTION
[0010] It is an object of the present invention to provide a novel
probe for a hair cell for clearly identifying various conditions of
a hair cell. Further, it is another object of the present invention
to provide an evaluation method for an auditory function such as
evaluation of the auditory toxicity of a chemical substance and
imaging of the hair cell, by labeling a hair cell specifically
using the novel probe for a hair cell.
[0011] It is still another object of the present invention to
provide a diagnostic composition for an auditory function for
evaluating a condition of a hair cell in a living body.
[0012] It is yet still another object of the present invention to
provide a screening method for one of a therapeutic drug and a
preventive drug for hearing loss.
[0013] It is even yet still another object of the present invention
to provide an evaluation method for one of a therapeutic drug and a
preventive drug for hearing loss.
[0014] The probe for a hair cell according to the present invention
contains, as an active agent, at least one kind selected from
staining compounds represented by one of the general formulae (I)
and (II).
##STR00002##
[0015] where; R.sub.1 represents one of a hydrogen atom, an alkyl
group, and an aryl group, R.sub.2 to R.sub.5 each independently
represent one of a hydrogen atom, an alkyl group, an aryl group, an
alkoxy group, a carboxylic acid group, a sulfonic acid group, a
heterocyclic group, an amino group, and a halogen atom, one of
R.sub.2 and R.sub.3, R.sub.3 and R.sub.4, and R.sub.4 and R.sub.5
may be bonded to each other to form a ring, R.sub.6 represents one
of a hydrogen atom and an alkyl group, and R.sub.7 represents one
of a hydrogen atom, an alkyl group, a carboxylic acid group, and a
cyano group, X.sub.1.sup.- represents an anionic group, Y
represents one of a sulfur atom, an oxygen atom, --N(R.sub.8)--,
and --C(R.sub.9)(R.sub.10)--, R.sub.8 to R.sub.10 each
independently represent one of a hydrogen atom, an alkyl group, and
an aryl group, and R.sub.9 and R.sub.10 may be bonded to each other
to form a ring, A represents one of an aryl group and an alkenyl
group, and n represents an integer of 1 to 3, and when n represents
1, A and R.sub.6 may be condensed with each other to form a ring,
and
##STR00003##
where; R.sub.11 and R.sub.12 each independently represent one of a
carboxylic acid group, a carboxylic acid ester group, a carboxylic
acid amide group, a sulfonic acid group, a sulfonic acid ester
group, a sulfonic acid amide group, a carboxylic acid salt, and a
sulfonic acid salt, R.sub.13 and R.sub.14 each independently
represent one of a hydrogen atom, an alkyl group, and an aryl
group, and R.sub.15, R.sub.16, R.sub.17A, and R.sub.17B each
independently represent one of a hydrogen atom, an alkyl group, an
aryl group, and a heterocyclic group, and X.sub.2.sup.- represents
an anionic group.
[0016] Further, an imaging method for a hair cell according to the
present invention includes; bringing a biological specimen into
contact with the probe for a hair cell of the present invention,
and observing fluorescence derived from the probe for a hair cell
by irradiating the biological specimen with excitation light.
[0017] An evaluation method for auditory toxicity of a chemical
substance according to the present invention includes;
administering the chemical substance to an organism, bringing the
organism into contact with the probe for a hair cell of the present
invention, and observing fluorescence derived from the probe for a
hair cell by irradiating the organism with excitation light.
[0018] A diagnostic composition for an auditory function according
to the present invention includes, as an active agent, the probe
for a hair cell of the present invention.
[0019] A screening method for one of a therapeutic drug and a
preventive drug for hearing loss according to the present invention
includes; administering a test substance to a hearing loss model
animal, administering the diagnostic composition for an auditory
function of the present invention to the model animal, and
examining a staining condition of the diagnostic composition for an
auditory function for a hair cell of the model animal.
[0020] An evaluation method for one of a therapeutic drug and a
preventive drug for hearing loss according to the present invention
includes; administering a test substance to a hearing loss model
animal, administering the diagnostic composition for an auditory
function of the present invention to the model animal, and
examining a staining condition of the diagnostic composition for an
auditory function for a hair cell of the model animal.
[0021] According to the present invention, there is provided a
novel application of a compound different in wavelength property
from a conventional hair cell staining agent. Further, a compound
group used for the probe for a hair cell according to the present
invention is rich in diversity in combinations of the excitation
wavelength/fluorescence emission wavelength, and can clearly
visualize a neuromast present in lateral line organs.
[0022] Further features of the present invention will become
apparent from the following description of exemplary embodiments
with reference to the attached drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIG. 1 shows a fluorescence observation image of a Zebrafish
neuromast observed in Example 10.
[0024] FIG. 2 shows a fluorescence observation image of a Zebrafish
neuromast observed in Example 21.
[0025] FIG. 3 shows a fluorescence observation image of a Zebrafish
neuromast observed in Example 23.
[0026] FIG. 4 illustrates a comparison (intensity/msec) in
fluorescence intensity (relative fluorescence unit, RFU) measured
in Example 38.
DESCRIPTION OF THE EMBODIMENTS
[0027] Hereinafter, the present invention is described in more
detail.
[0028] The inventors of the present invention have found that a
probe for a hair cell containing, as an active agent, at least one
kind selected from staining compounds represented by one of the
general formulae (I) and (II) is a novel probe for a hair cell that
labels a hair cell with high sensitivity, and enables more precise
diagnosis and drug screening. Thus, the present invention has been
completed.
[0029] It should be noted that the phrase "labelling of a hair
cell" as used herein means that the above-mentioned active agent is
retained inside, on a surface of, or in the periphery of a hair
cell, resulting in such a condition that at least one of the shape,
location, and function of the hair cell can be detected. For
example, a method of capturing a fluorescence image and a staining
image using an image capturing unit as described below, and a
visual observation method are employed for the detection.
##STR00004##
[0030] In the general formula (I); R.sub.1 represents one of a
hydrogen atom, an alkyl group, and an aryl group, R.sub.2 to
R.sub.5 each independently represent one of a hydrogen atom, an
alkyl group, an aryl group, an alkoxy group, a carboxylic acid
group, a sulfonic acid group, a heterocyclic group, an amino group,
and a halogen atom, one of R.sub.2 and R.sub.3, R.sub.3 and
R.sub.4, and R.sub.4 and R.sub.5 may be bonded to each other to
form a ring, R.sub.6 represents one of a hydrogen atom and an alkyl
group, R.sub.7 represents one of a hydrogen atom, an alkyl group, a
carboxylic acid group, and a cyano group, X.sub.1.sup.- represents
an anionic group, Y represents one of a sulfur atom, an oxygen
atom, --N(R.sub.8)--, and --C(R.sub.9)(R.sub.10)--, R.sub.8 to
R.sub.10 each independently represent one of a hydrogen atom, an
alkyl group, and an aryl group, R.sub.9 and R.sub.10 may be bonded
to each other to form a ring, A represents one of an aryl group and
an alkenyl group; and n represents an integer of 1 to 3, and when n
represents 1, A and R.sub.6 may be condensed together to form a
ring.
[0031] In the general formula (II); R.sub.11 and R.sub.12 each
represent one of a carboxylic acid group, a carboxylic acid ester
group, a carboxylic acid amide group, a sulfonic acid group, a
sulfonic acid ester group, a sulfonic acid amide group, a
carboxylic acid salt, and a sulfonic acid salt, R.sub.13 and
R.sub.14 each independently represent one of a hydrogen atom, an
alkyl group, and an aryl group, R.sub.15, R.sub.16, R.sub.17A, and
R.sub.17B each independently represent one of a hydrogen atom, an
alkyl group, an aryl group, and a heterocyclic group, and
X.sub.2.sup.- represents an anionic group.
[0032] The alkyl group represented by R.sub.1 in the general
formula (I) is not particularly limited and examples thereof
include linear, branched, and cyclic alkyl groups having 1 to 20
carbon atoms such as a methyl group, an ethyl group, a propyl
group, a butyl group, a pentyl group, a hexyl group, an octyl
group, a nonyl group, a decyl group, an undecyl group, a dodecyl
group, a tridecyl group, a tetradecyl group, a pentadecyl group, a
hexadecyl group, a heptadecyl group, an octadecyl group, a
nonadecyl group, a cyclopropyl group, a cyclobutyl group, and a
cyclopentyl group.
[0033] R.sub.1 may further have a substituent and the substituent
is not particularly limited as long as the storage stability of the
staining compound is not significantly inhibited. Examples thereof
include; alkyl groups such as a methyl group, an ethyl group, an
n-propyl group, an isopropyl group, an n-butyl group, an isobutyl
group, a sec-butyl group, and a tert-butyl group, aryl groups such
as a phenyl group and a naphthyl group, alkoxy groups such as a
methoxy group, an ethoxy group, and a butoxy group, aryloxy groups
such as a phenoxy group and a naphthyloxy group, alkylsulfanyl
groups such as a thiomethyl group, a thioethyl group, a thiopropyl
group, a thiobutyl group, and a thiophenyl group, monosubstituted
amino groups such as a methylamino group and a butylamino group,
disubstituted amino groups such as a dimethylamino group, an
N-ethyl-N-phenylamino group, and a diphenylamino group, acyl groups
such as an acetyl group, a benzoyl group, a carboxylic acid group,
a carboxylic acid ester group, and a carbamoyl group, sulfonyl
groups such as a sulfonic acid group, a sulfonic acid ester group,
and a sulfamoyl group, heterocyclic groups such as a pyridyl group,
a triazinyl group, and a benzothiazolyl group, a nitro group;
halogen atoms such as a fluorine atom, a chlorine atom, a bromine
atom, and an iodine atom; a polyethylene glycol group, and salts
such as a quarternary ammonium salt, a carboxylic acid salt, and a
sulfonic acid salt. Of those substituents, it is preferred that
R.sub.1 have a substituent which has property of improving the
water solubility and, for example, a carboxylic acid group, a
sulfonic acid group, a polyethylene glycol group, a carboxylic acid
salt, and a sulfonic acid salt are particularly preferably used,
but are not limited thereto.
[0034] The aryl group represented by R.sub.1 is not particularly
limited and examples thereof include 6 to 14-membered monocyclic
and polycyclic aryl groups such as a phenyl group, a naphthyl
group, a phenanthryl group, and an anthracenyl group.
[0035] A preferred example of R.sub.1 includes an alkyl group, and
more preferred is an alkyl group having a substituent such as a
carboxylic acid group, a sulfonic acid group, a polyethylene glycol
group, a carboxylic acid salt, and a sulfonic acid salt because the
alkyl group increases the water solubility of the compound and also
increases the fluorescence intensity. The alkyl group represented
by each of R.sub.2 to R.sub.5 in the general formula (I) is not
particularly limited and examples thereof include linear, branched,
and cyclic alkyl groups having 1 to 20 carbon atoms such as a
methyl group, an ethyl group, a propyl group, a butyl group, a
pentyl group, a hexyl group, an octyl group, a nonyl group, a decyl
group, an undecyl group, a dodecyl group, a tridecyl group, a
tetradecyl group, a pentadecyl group, a hexadecyl group, a
heptadecyl group, an octadecyl group, a nonadecyl group, a
cyclopropyl group, a cyclobutyl group, and a cyclopentyl group.
[0036] The aryl group represented by each of R.sub.2 to R.sub.5 is
not particularly limited and examples thereof include 6 to
14-membered monocyclic and polycyclic aryl groups such as a phenyl
group, a naphthyl group, a phenanthryl group, and an anthracenyl
group.
[0037] The alkoxy group represented by each of R.sub.2 to R.sub.5
is not particularly limited and examples thereof include alkoxy
groups having 1 to 20 carbon atoms such as a methoxy group, an
ethoxy group, a propoxy group, a butoxy group, a pentyloxy group, a
decyloxy group, a hexyloxy group, a heptyloxy group, an octyloxy
group, a nonyloxy group, a decyloxy group, a dodecyloxy group, and
an octadecyloxy group.
[0038] The heterocyclic group represented by each of R.sub.2 to
R.sub.5 is not particularly limited and examples thereof include 4
to 10-membered monocyclic and bicyclic heterocyclic groups having 1
to 4 atoms selected from nitrogen, oxygen, and sulfur, such as a
pyridyl group, a pyrazinyl group, a pyrimidinyl group, a pyrrolyl
group, a thienyl group, a furyl group, a pyranyl group, an oxazolyl
group, a thiazolyl group, a triazolyl group, a tetrazolyl group, an
imidazolyl group, a pyrazolyl group, a morpholinyl group, a
thiomorpholinyl group, a piperidinyl group, a piperazinyl group, a
quinolyl group, an isoquinolyl group, an indolyl group, an
isoindolyl group, a benzofuryl group, and a benzothienyl group.
[0039] The amino group represented by each of R.sub.2 to R.sub.5 is
not particularly limited and examples thereof include; an
unsubstituted amino group, monosubstituted amino groups such as an
N-methylamino group, an N-butylamino group, an N-hexylamino group,
an N-tetradecylamino group, an N-phenylamino group, and an
N-naphthylamino group, disubstituted amino groups such as an
N,N-dimethylamino group, an N,N-diethylamino group, an
N,N-diphenylamino group, and an N,N-methylpropylamino group,
carbonylamino groups such as an acetylamino group, an
ethylcarbonylamino group, a tert-butylcarbonylamino group, a
benzoylamino group, a naphthoylamino group, and a
methoxycarbonylamino group, and sulfonylamino groups such as a
methylsulfonylamino group, an ethylsulfonylamino group, a
tert-butylsulfonylamino group, and an iso-propoxysulfonylamino
group.
[0040] Examples of the halogen atom represented by each of R.sub.2
to R.sub.5 include a fluorine atom, a chlorine atom, a bromine
atom, and an iodine atom.
[0041] R.sub.2 to R.sub.5 each preferably represent one of a
hydrogen atom, a carboxylic acid group, a sulfonic acid group, an
amino group, and a halogen atom, and more preferably represent a
hydrogen atom and a sulfonic acid group, each of which improves the
water solubility of the compound.
[0042] The ring which is formed by one of R.sub.2 and R.sub.3,
R.sub.3 and R.sub.4, and R.sub.4 and R.sub.5 bonded to each other
is not particularly limited and examples thereof include; aromatic
rings having 3 to 10 carbon atoms such as a benzene ring and a
naphthalene ring, saturated rings such as a cyclooctane ring, a
cycloheptane ring, a cyclohexane ring, a cyclopentane ring, and a
cyclobutane ring, partially saturated rings such as a cyclopentene
ring and a cyclohexene ring, and heterocycles such as a pyridine
ring and a pyrimidine ring. Further, the ring may have a
substituent and the substituent is not particularly limited as long
as the storage stability of the staining compound is not
significantly inhibited. Examples thereof include alkyl groups such
as a methyl group, an ethyl group, an n-propyl group, an isopropyl
group, an n-butyl group, an isobutyl group, a sec-butyl group, and
a tert-butyl group; aryl groups such as a phenyl group and a
naphthyl group; alkoxy groups such as a methoxy group, an ethoxy
group, and a butoxy group; aryloxy groups such as a phenoxy group
and a naphthyloxy group; disubstituted amino groups such as a
dimethylamino group, an N-ethyl-N-phenylamino group, and a
diphenylamino group; acyl groups such as an acetyl group, a benzoyl
group, a carboxylic acid group, a carboxylic acid ester group, and
a carbamoyl group; sulfonyl groups such as a sulfonic acid group, a
sulfonic acid ester group, and a sulfamoyl group; heterocyclic
groups such as a pyridyl group, a triazinyl group, and a
benzothiazolyl group; a nitro group; halogen atoms such as a
fluorine atom, a chlorine atom, a bromine atom, and an iodine atom;
a polyethylene glycol group; and salts such as a quarternary
ammonium salt, a carboxylic acid salt, and a sulfonic acid
salt.
[0043] The ring which is formed by one of R.sub.2 and R.sub.3,
R.sub.3 and R.sub.4, and R.sub.4 and R.sub.5 bonded to each other
is preferably a benzene ring, because the storage stability of the
compound is improved.
[0044] The alkyl group represented by each of R.sub.6 and R.sub.7
in the general formula (I) is not particularly limited and examples
thereof include linear, branched, and cyclic alkyl groups having 1
to 20 carbon atoms such as a methyl group, an ethyl group, a propyl
group, a butyl group, a pentyl group, a hexyl group, an octyl
group, a nonyl group, a decyl group, an undecyl group, a dodecyl
group, a tridecyl group, a tetradecyl group, a pentadecyl group, a
hexadecyl group, a heptadecyl group, an octadecyl group, a
nonadecyl group, a cyclopropyl group, a cyclobutyl group, and a
cyclopentyl group.
[0045] R.sub.6 preferably represents one of a hydrogen atom, a
methyl group, an ethyl group, a propyl group, and a butyl group,
and more preferably represents, in terms of stability of the
compound, one of a hydrogen atom, a methyl group, and an ethyl
group.
[0046] R.sub.7 preferably represents one of a hydrogen atom and a
cyano group, and more preferably represents a hydrogen atom.
[0047] X.sub.1.sup.- in the general formula (I) represents an
anionic group. Here, the anionic group is not particularly limited
and examples thereof include: halogen ions such as a fluoride ion,
a chloride ion, a bromide ion, and an iodide ion; inorganic acid
ions such as a sulfuric acid ion, a phosphoric acid ion, a nitric
acid ion, a tetrafluoroboric acid ion, and a hexafluorophosphoric
acid ion; Lewis acid-containing ions such as a tetrachloroaluminum
ion; and organic acid ions such as an acetic acid ion, a lactic
acid ion, a methanesulfonic acid ion, a benzenesulfonic acid ion, a
p-toluenesulfonic acid ion, a trifluoroacetic acid ion, a
trifluoromethanesulfonic acid ion, and a tetraphenylboric acid
ion.
[0048] The anionic group represented by X.sub.1.sup.- is preferably
one of a chloride ion, a bromide ion, an iodide ion, a sulfuric
acid ion, a nitric acid ion, and a methanesulfonic acid ion, and
more preferably, in terms of ease of synthesis of the compound, one
of a bromide ion and an iodide ion.
[0049] Y in the general formula (I) represents one of a sulfur
atom, an oxygen atom, --N(R.sub.8)--, and
--C(R.sub.9)(R.sub.10)--.
[0050] In Y, the alkyl group represented by each of R.sub.8 to
R.sub.10 is not particularly limited and examples thereof include
linear, branched, and cyclic alkyl groups having 1 to 20 carbon
atoms such as a methyl group, an ethyl group, a propyl group, a
butyl group, a pentyl group, a hexyl group, an octyl group, a nonyl
group, a decyl group, an undecyl group, a dodecyl group, a tridecyl
group, a tetradecyl group, a pentadecyl group, a hexadecyl group, a
heptadecyl group, an octadecyl group, a nonadecyl group, a
cyclopropyl group, a cyclobutyl group, and a cyclopentyl group.
[0051] In Y, the aryl group represented by each of R.sub.8 to
R.sub.10 is not particularly limited and examples thereof include 6
to 14-membered monocyclic and polycyclic aryl groups such as a
phenyl group, a naphthyl group, a phenanthryl group, and an
anthracenyl group.
[0052] Y preferably represents, in terms of storage stability of
the compound, one of an oxygen atom, a sulfur atom, and
--C(CH.sub.3)(CH.sub.3)--.
[0053] The aryl group represented by A in the general formula (I)
is not particularly limited and examples thereof include 6 to
14-membered monocyclic and polycyclic aryl groups such as a phenyl
group, a naphthyl group, a phenanthryl group, an indolyl group, and
an anthracenyl group. Further, the ring may have a substituent and
the substituent is not particularly limited as long as the storage
stability of the staining compound is not significantly inhibited.
Examples thereof include: alkyl groups such as a methyl group, an
ethyl group, an n-propyl group, an isopropyl group, an n-butyl
group, an isobutyl group, a sec-butyl group, and a tert-butyl
group; aryl groups such as a phenyl group and a naphthyl group;
alkoxy groups such as a methoxy group, an ethoxy group, and a
butoxy group; aryloxy groups such as a phenoxy group and a
naphthyloxy group; alkylsulfanyl groups such as a thiomethyl group,
a thioethyl group, a thiopropyl group, a thiobutyl group, and a
thiophenyl group; monosubstituted amino groups such as a
methylamino group and a butylamino group; disubstituted amino
groups such as a dimethylamino group, an N-ethyl-N-phenylamino
group, and a diphenylamino group; acyl groups such as an acetyl
group, a benzoyl group, a carboxylic acid group, a carboxylic acid
ester group, and a carbamoyl group; sulfonyl groups such as a
sulfonic acid group, a sulfonic acid ester group, and a sulfamoyl
group; heterocyclic groups such as a pyridyl group, a triazinyl
group, and a benzothiazolyl group; a nitro group; halogen atoms
such as a fluorine atom, a chlorine atom, a bromine atom, and an
iodine atom; a polyethylene glycol group; and salts such as a
quarternary ammonium salt, a carboxylic acid salt, and a sulfonic
acid salt. Of those substituents, it is preferred that the ring
have a substituent which has property of improving the water
solubility and, for example, a carboxylic acid group, a sulfonic
acid group, a polyethylene glycol group, a carboxylic acid salt,
and a sulfonic acid salt are particularly preferably used, but are
not limited thereto.
[0054] The aryl group represented by A is preferably a compound
represented by the following general formula (IV).
##STR00005##
[0055] In the general formula (IV): R.sub.25 represents one of a
hydrogen atom, an alkyl group, an aralkyl group, an alkenyl group,
an aryl group, a heterocyclic group, and an acyl group; R.sub.26 to
R.sub.29 each independently represent one of a hydrogen atom, an
alkyl group, an aryl group, a carboxylic acid group, a carboxylic
acid ester group, and an acyl group, and R.sub.26 and R.sub.28 may
be bonded to each other to form a ring; and R.sub.30 represents one
of a hydrogen atom, an alkyl group, an alkoxy group, and a halogen
atom.
[0056] The alkyl group represented by R.sub.25 in the general
formula (IV) is not particularly limited and examples thereof
include linear, branched, and cyclic alkyl groups having 1 to 20
carbon atoms such as a methyl group, an ethyl group, a propyl
group, a butyl group, a pentyl group, a hexyl group, an octyl
group, a nonyl group, a decyl group, an undecyl group, a dodecyl
group, a tridecyl group, a tetradecyl group, a pentadecyl group, a
hexadecyl group, a heptadecyl group, an octadecyl group, a
nonadecyl group, a cyclopropyl group, a cyclobutyl group, and a
cyclopentyl group.
[0057] The aralkyl group represented by R.sub.25 is not
particularly limited and examples thereof include a benzyl group
and a phenethyl group.
[0058] The alkenyl group represented by R.sub.25 is not
particularly limited and examples thereof include alkenyl groups
having 2 to 20 carbon atoms such as a vinyl group, a
2,2-diphenylvinyl group, a 3-butenyl group, and a cyclohexenyl
group.
[0059] The aryl group represented by R.sub.25 is not particularly
limited and examples thereof include 6 to 14-membered monocyclic
and polycyclic aryl groups such as a phenyl group, a naphthyl
group, a phenanthryl group, and an anthracenyl group.
[0060] The heterocyclic group represented by R.sub.25 is not
particularly limited and examples thereof include 4 to 10-membered
monocyclic and bicyclic heterocyclic groups having 1 to 4 atoms
selected from nitrogen, oxygen, and sulfur, such as a pyridyl
group, a pyrazinyl group, a pyrimidinyl group, a pyrrolyl group, a
thienyl group, a furyl group, a pyranyl group, an oxazolyl group, a
thiazolyl group, a triazolyl group, a tetrazolyl group, an
imidazolyl group, a pyrazolyl group, a morpholinyl group, a
thiomorpholinyl group, a piperidinyl group, a piperazinyl group, a
quinolyl group, an isoquinolyl group, an indolyl group, an
isoindolyl group, a benzofuryl group, and a benzothienyl group.
[0061] R.sub.25 may further have a substituent and the substituent
is not particularly limited as long as the storage stability of the
staining compound is not significantly inhibited. Examples thereof
include: alkyl groups such as a methyl group, an ethyl group, an
n-propyl group, an isopropyl group, an n-butyl group, an isobutyl
group, a sec-butyl group, and a tert-butyl group; aryl groups such
as a phenyl group and a naphthyl group; alkoxy groups such as a
methoxy group, an ethoxy group, and a butoxy group; aryloxy groups
such as a phenoxy group and a naphthyloxy group; alkylsulfanyl
groups such as a thiomethyl group, a thioethyl group, a thiopropyl
group, a thiobutyl group, and a thiophenyl group; monosubstituted
amino groups such as a methylamino group and a butylamino group;
disubstituted amino groups such as a dimethylamino group, an
N-ethyl-N-phenylamino group, and a diphenylamino group; acyl groups
such as an acetyl group, a benzoyl group, a carboxylic acid group,
a carboxylic acid ester group, and a carbamoyl group; sulfonyl
groups such as a sulfonic acid group, a sulfonic acid ester group,
and a sulfamoyl group; heterocyclic groups such as a pyridyl group,
a triazinyl group, and a benzothiazolyl group; a nitro group;
halogen atoms such as a fluorine atom, a chlorine atom, a bromine
atom, and an iodine atom; a polyethylene glycol group; and salts
such as a quarternary ammonium salt, a carboxylic acid salt, and a
sulfonic acid salt. Of those substituents, it is preferred that
R.sub.25 have a substituent which has property of improving the
water solubility and, for example, a carboxylic acid group, a
sulfonic acid group, a polyethylene glycol group, a carboxylic acid
salt, and a sulfonic acid salt are particularly preferably used,
but are not limited thereto.
[0062] R.sub.25 may be independently and arbitrarily selected from
the substituents exemplified above, and because the fluorescence
intensity is large, preferred examples include an aralkyl group, an
alkenyl group, and an aryl group. Specifically, a phenyl group, a
bromophenyl group, a benzyl group, a bromobenzyl group, a
methylthiophenyl group, a methoxyphenyl group, a methoxynaphthyl
group, a benzylphenyl group, a 2,2-diphenylvinyl group, and a
2,2-diphenylvinylphenyl group are preferred. More preferred are a
phenyl group, a bromophenyl group, a benzyl group, a
methylthiophenyl group, a methoxyphenyl group, and a
methoxynaphthyl group. In particular, a methylthiophenyl group is
preferred because there is a tendency that a Stokes' shift becomes
remarkable large.
[0063] The alkyl group represented by each of R.sub.26 to R.sub.29
in the general formula (IV) is not particularly limited and
examples thereof include linear, branched, and cyclic alkyl groups
having 1 to 20 carbon atoms such as a methyl group, an ethyl group,
a propyl group, a butyl group, a pentyl group, a hexyl group, an
octyl group, a nonyl group, a decyl group, an undecyl group, a
dodecyl group, a tridecyl group, a tetradecyl group, a pentadecyl
group, a hexadecyl group, a heptadecyl group, an octadecyl group, a
nonadecyl group, a cyclopropyl group, a cyclobutyl group, and a
cyclopentyl group.
[0064] The aryl group represented by each of R.sub.26 to R.sub.29
is not particularly limited and examples thereof include 6 to
14-membered monocyclic and polycyclic aryl groups such as a phenyl
group, a naphthyl group, a phenanthryl group, and an anthracenyl
group.
[0065] The carboxylic acid ester group represented by each of
R.sub.26 to R.sub.29 is not particularly limited and examples
thereof include a carboxylic acid methyl ester group, a carboxylic
acid ethyl ester group, a carboxylic acid propyl ester group, and a
carboxylic acid butyl ester group.
[0066] The acyl group represented by each of R.sub.26 to R.sub.29
is not particularly limited and examples thereof include an acetyl
group, a propionyl group, a butyryl group, a pentanoyl group, a
benzoyl group, a 1-naphthoyl group, and a 2-naphthoyl group.
[0067] Each of R.sub.26 to R.sub.29 may further have a substituent
and the substituent is not particularly limited as long as the
storage stability of the staining compound is not significantly
inhibited. Examples thereof include: alkyl groups such as a methyl
group, an ethyl group, an n-propyl group, an isopropyl group, an
n-butyl group, an isobutyl group, a sec-butyl group, and a
tert-butyl group; aryl groups such as a phenyl group and a naphthyl
group; alkoxy groups such as a methoxy group, an ethoxy group, and
a butoxy group; aryloxy groups such as a phenoxy group and a
naphthyloxy group; alkylsulfanyl groups such as a thiomethyl group,
a thioethyl group, a thiopropyl group, a thiobutyl group, and a
thiophenyl group; monosubstituted amino groups such as a
methylamino group and a butylamino group; disubstituted amino
groups such as a dimethylamino group, an N-ethyl-N-phenylamino
group, and a diphenylamino group; acyl groups such as an acetyl
group, a benzoyl group, a carboxylic acid group, a carboxylic acid
ester group, and a carbamoyl group; sulfonyl groups such as a
sulfonic acid group, a sulfonic acid ester group, and a sulfamoyl
group; heterocyclic groups such as a pyridyl group, a triazinyl
group, and a benzothiazolyl group; a nitro group; halogen atoms
such as a fluorine atom, a chlorine atom, a bromine atom, and an
iodine atom; a polyethylene glycol group; and salts such as a
quarternary ammonium salt, a carboxylic acid salt, and a sulfonic
acid salt. Of those substituents, it is preferred that each of
R.sub.26 to R.sub.29 have a substituent which has property of
improving the water solubility and, for example, a carboxylic acid
group, a sulfonic acid group, a polyethylene glycol group, a
carboxylic acid salt, and a sulfonic acid salt are particularly
preferably used, but are not limited thereto. The ring which is
formed by R.sub.26 and R.sub.28 bonded to each other is not
particularly limited and examples thereof include: saturated
aliphatic rings such as a cyclooctane ring, a cycloheptane ring, a
cyclohexane ring, a cyclopentane ring, and a cyclobutane ring; and
partially saturated aliphatic rings such as a cyclopentene ring and
a cyclohexene ring. Further, the ring may have a substituent and
the substituent is not particularly limited as long as the storage
stability of the staining compound is not significantly inhibited.
Examples thereof include alkyl groups such as a methyl group, an
ethyl group, an n-propyl group, an isopropyl group, an n-butyl
group, an isobutyl group, a sec-butyl group, and a tert-butyl
group; aryl groups such as a phenyl group and a naphthyl group;
alkoxy groups such as a methoxy group, an ethoxy group, and a
butoxy group; aryloxy groups such as a phenoxy group and a
naphthyloxy group; alkylsulfanyl groups such as a thiomethyl group,
a thioethyl group, a thiopropyl group, a thiobutyl group, and a
thiophenyl group; monosubstituted amino groups such as a
methylamino group and a butylamino group; disubstituted amino
groups such as a dimethylamino group, an N-ethyl-N-phenylamino
group, and a diphenylamino group; acyl groups such as an acetyl
group, a benzoyl group, a carboxylic acid group, a carboxylic acid
ester group, and a carbamoyl group; sulfonyl groups such as a
sulfonic acid group, a sulfonic acid ester group, and a sulfamoyl
group; heterocyclic groups such as a pyridyl group, a triazinyl
group, and a benzothiazolyl group; a nitro group; halogen atoms
such as a fluorine atom, a chlorine atom, a bromine atom, and an
iodine atom; a polyethylene glycol group; and salts such as a
quarternary ammonium salt, a carboxylic acid salt, and a sulfonic
acid salt. Of those substituents, it is preferred that the ring
have a substituent which has property of improving the water
solubility and, for example, a carboxylic acid group, a sulfonic
acid group, a polyethylene glycol group, a carboxylic acid salt,
and a sulfonic acid salt are particularly preferably used, but are
not limited thereto.
[0068] It is preferred that R.sub.26 to R.sub.29 each independently
represent one of a hydrogen atom, an alkyl group, and an aryl
group, and R.sub.26 and R.sub.28 be bonded to each other to form a
ring. It is more preferred that R.sub.26 and R.sub.28 be bonded to
each other to form a ring, which is a stable chemical structure.
Specific examples thereof include a cyclooctane ring, a
cycloheptane ring, a cyclohexane ring, a cyclopentane ring, and a
cyclobutane ring. In terms of storage stability, a cyclopentane
ring is more preferred.
[0069] The alkyl group represented by R.sub.30 in the general
formula (IV) is not particularly limited and examples thereof
include linear, branched, and cyclic alkyl groups having 1 to 20
carbon atoms such as a methyl group, an ethyl group, a propyl
group, a butyl group, a pentyl group, a hexyl group, an octyl
group, a nonyl group, a decyl group, an undecyl group, a dodecyl
group, a tridecyl group, a tetradecyl group, a pentadecyl group, a
hexadecyl group, a heptadecyl group, an octadecyl group, a
nonadecyl group, a cyclopropyl group, a cyclobutyl group, and a
cyclopentyl group.
[0070] The alkoxy group represented by R.sub.30 is not particularly
limited and examples thereof include alkoxy groups having 1 to 20
carbon atoms such as a methoxy group, an ethoxy group, a propoxy
group, a butoxy group, a pentyloxy group, a decyloxy group, a
hexyloxy group, a heptyloxy group, an octyloxy group, a nonyloxy
group, a decyloxy group, a dodecyloxy group, and an octadecyloxy
group.
[0071] Examples of the halogen atom represented by R.sub.30 include
a fluorine atom, a chlorine atom, a bromine atom, and an iodine
atom.
[0072] R.sub.30 preferably represents one of a hydrogen atom, a
halogen atom, and an alkoxy group, and more preferably represents
one of a hydrogen atom and a halogen atom.
[0073] The alkenyl group represented by A is preferably a compound
represented by the following general formula (III).
##STR00006##
[0074] In the general formula (III): R.sub.18 to R.sub.21 each
independently represent one of a hydrogen atom, an alkyl group, an
aryl group, an alkoxy group, a carboxylic acid group, a sulfonic
acid group, a heterocyclic group, an amino group, and a halogen
atom; one of R.sub.18 and R.sub.19, R.sub.19 and R.sub.20, and
R.sub.20 and R.sub.21 may be bonded to each other to form a ring;
R.sub.22 represents one of a hydrogen atom, an alkyl group, and an
aryl group; Z represents one of a sulfur atom, an oxygen atom, and
--C(R.sub.23)(R.sub.24)--; R.sub.23 and R.sub.24 each represent one
of a hydrogen atom, an alkyl group, and an aryl group; and R.sub.23
and R.sub.24 may be bonded to each other to form a ring.
[0075] The alkyl group represented by each of R.sub.18 to R.sub.21
in the general formula (III) is not particularly limited and
examples thereof include linear, branched, and cyclic alkyl groups
having 1 to 20 carbon atoms such as a methyl group, an ethyl group,
a propyl group, a butyl group, a pentyl group, a hexyl group, an
octyl group, a nonyl group, a decyl group, an undecyl group, a
dodecyl group, a tridecyl group, a tetradecyl group, a pentadecyl
group, a hexadecyl group, a heptadecyl group, an octadecyl group, a
nonadecyl group, a cyclopropyl group, a cyclobutyl group, and a
cyclopentyl group.
[0076] The aryl group represented by each of R.sub.18 to R.sub.21
is not particularly limited and examples thereof include 6 to
14-membered monocyclic and polycyclic aryl groups such as a phenyl
group, a naphthyl group, a phenanthryl group, and an anthracenyl
group.
[0077] The alkoxy group represented by each of R.sub.18 to R.sub.21
is not particularly limited and examples thereof include alkoxy
groups having 1 to 20 carbon atoms such as a methoxy group, an
ethoxy group, a propoxy group, a butoxy group, a pentyloxy group, a
decyloxy group, a hexyloxy group, a heptyloxy group, an octyloxy
group, a nonyloxy group, a decyloxy group, a dodecyloxy group, and
an octadecyloxy group.
[0078] The heterocyclic group represented by each of R.sub.18 to
R.sub.21 is not particularly limited and examples thereof include 4
to 10-membered monocyclic and bicyclic heterocyclic groups having 1
to 4 atoms selected from nitrogen, oxygen, and sulfur, such as a
pyridyl group, a pyrazinyl group, a pyrimidinyl group, a pyrrolyl
group, a thienyl group, a furyl group, a pyranyl group, an oxazolyl
group, a thiazolyl group, a triazolyl group, a tetrazolyl group, an
imidazolyl group, a pyrazolyl group, a morpholinyl group, a
thiomorpholinyl group, a piperidinyl group, a piperazinyl group, a
quinolyl group, an isoquinolyl group, an indolyl group, an
isoindolyl group, a benzofuryl group, and a benzothienyl group.
[0079] The amino group represented by each of R.sub.18 to R.sub.21
is not particularly limited and examples thereof include: an
unsubstituted amino group; monosubstituted amino groups such as an
N-methylamino group, an N-butylamino group, an N-hexylamino group,
an N-tetradecylamino group, an N-phenylamino group, and an
N-naphthylamino group; disubstituted amino groups such as an
N,N-dimethylamino group, an N,N-diethylamino group, an
N,N-diphenylamino group, and an N,N-methylpropylamino group;
carbonylamino groups such as an acetylamino group, an
ethylcarbonylamino group, a tert-butylcarbonylamino group, a
benzoylamino group, a naphthoylamino group, and a
methoxycarbonylamino group; and sulfonylamino groups such as a
methylsulfonylamino group, an ethylsulfonylamino group, a
tert-butylsulfonylamino group, and an iso-propoxysulfonylamino
group.
[0080] Examples of the halogen atom represented by each of R.sub.18
to R.sub.21 include a fluorine atom, a chlorine atom, a bromine
atom, and an iodine atom.
[0081] R.sub.18 to R.sub.21 each preferably represent one of a
hydrogen atom, a carboxylic acid group, a sulfonic acid group, an
amino group, and a halogen atom, and more preferably represent one
of a hydrogen atom and a sulfonic acid group because the water
solubility of the compound is improved.
[0082] The ring which is formed by one of R.sub.18 and R.sub.19,
R.sub.19 and R.sub.20, and R.sub.20 and R.sub.21 bonded to each
other is not particularly limited and examples thereof include:
aromatic rings having 3 to 10 carbon atoms such as a benzene ring
and a naphthalene ring; saturated rings such as a cyclooctane ring,
a cycloheptane ring, a cyclohexane ring, a cyclopentane ring, and a
cyclobutane ring; partially saturated rings such as a cyclopentene
ring and a cyclohexene ring; and heterocycles such as a pyridine
ring and a pyrimidine ring. Further, the ring may have a
substituent and the substituent is not particularly limited as long
as the storage stability of the staining compound is not
significantly inhibited. Examples thereof include: alkyl groups
such as a methyl group, an ethyl group, an n-propyl group, an
isopropyl group, an n-butyl group, an isobutyl group, a sec-butyl
group, and a tert-butyl group; aryl groups such as a phenyl group
and a naphthyl group; alkoxy groups such as a methoxy group, an
ethoxy group, and a butoxy group; aryloxy groups such as a phenoxy
group and a naphthyloxy group; alkylsulfanyl groups such as a
thiomethyl group, a thioethyl group, a thiopropyl group, a
thiobutyl group, and a thiophenyl group; monosubstituted amino
groups such as a methylamino group and a butylamino group;
disubstituted amino groups such as a dimethylamino group, an
N-ethyl-N-phenylamino group, and a diphenylamino group; acyl groups
such as an acetyl group, a benzoyl group, a carboxylic acid group,
a carboxylic acid ester group, and a carbamoyl group; sulfonyl
groups such as a sulfonic acid group, a sulfonic acid ester group,
and a sulfamoyl group; heterocyclic groups such as a pyridyl group,
a triazinyl group, and a benzothiazolyl group; a nitro group;
halogen atoms such as a fluorine atom, a chlorine atom, a bromine
atom, and an iodine atom; a polyethylene glycol group; and salts
such as a quarternary ammonium salt, a carboxylic acid salt, and a
sulfonic acid salt. Of those substituents, it is preferred that the
ring have a substituent which has property of improving the water
solubility and, for example, a carboxylic acid group, a sulfonic
acid group, a polyethylene glycol group, a carboxylic acid salt,
and a sulfonic acid salt are particularly preferably used, but are
not limited thereto.
[0083] The ring which is formed by one of R.sub.18 and R.sub.19,
R.sub.19 and R.sub.20, and R.sub.20 and R.sub.21 bonded to each
other is preferably a benzene ring, because the storage stability
of the compound is improved.
[0084] The alkyl group represented by R.sub.22 in the general
formula (III) is not particularly limited and examples thereof
include linear, branched, and cyclic alkyl groups having 1 to 20
carbon atoms such as a methyl group, an ethyl group, a propyl
group, a butyl group, a pentyl group, a hexyl group, an octyl
group, a nonyl group, a decyl group, an undecyl group, a dodecyl
group, a tridecyl group, a tetradecyl group, a pentadecyl group, a
hexadecyl group, a heptadecyl group, an octadecyl group, a
nonadecyl group, a cyclopropyl group, a cyclobutyl group, and a
cyclopentyl group.
[0085] R.sub.22 may further have a substituent and the substituent
is not particularly limited as long as the storage stability of the
staining compound is not significantly inhibited. Examples thereof
include: alkyl groups such as a methyl group, an ethyl group, an
n-propyl group, an isopropyl group, an n-butyl group, an isobutyl
group, a sec-butyl group, and a tert-butyl group; aryl groups such
as a phenyl group and a naphthyl group; alkoxy groups such as a
methoxy group, an ethoxy group, and a butoxy group; aryloxy groups
such as a phenoxy group and a naphthyloxy group; alkylsulfanyl
groups such as a thiomethyl group, a thioethyl group, a thiopropyl
group, a thiobutyl group, and a thiophenyl group; monosubstituted
amino groups such as a methylamino group and a butylamino group;
disubstituted amino groups such as a dimethylamino group, an
N-ethyl-N-phenylamino group, and a diphenylamino group; acyl groups
such as an acetyl group, a benzoyl group, a carboxylic acid group,
a carboxylic acid ester group, and a carbamoyl group; sulfonyl
groups such as a sulfonic acid group, a sulfonic acid ester group,
and a sulfamoyl group; heterocyclic groups such as a pyridyl group,
a triazinyl group, and a benzothiazolyl group; a nitro group;
halogen atoms such as a fluorine atom, a chlorine atom, a bromine
atom, and an iodine atom; a polyethylene glycol group; and salts
such as a quarternary ammonium salt, a carboxylic acid salt, and a
sulfonic acid salt. Of those substituents, it is preferred that
R.sub.22 have a substituent which has property of improving the
water solubility and, for example, a carboxylic acid group, a
sulfonic acid group, a polyethylene glycol group, a carboxylic acid
salt, and a sulfonic acid salt are particularly preferably used,
but are not limited thereto.
[0086] The aryl group represented by R.sub.22 is not particularly
limited and examples thereof include 6 to 14-membered monocyclic
and polycyclic aryl groups such as a phenyl group, a naphthyl
group, a phenanthryl group, and an anthracenyl group.
[0087] R.sub.22 preferably represents an alkyl group, and the alkyl
group further preferably has a substituent such as a carboxylic
acid group, a sulfonic acid group, a polyethylene glycol group, a
carboxylic acid salt, and a sulfonic acid salt, because those
substituents improve the water solubility of the compound and also
increase the fluorescence intensity. Specific examples thereof
include a methyl group, an ethyl group, a propyl group, a butyl
group, a pentyl group, a hexyl group, an octyl group, a nonyl
group, an acetic acid group, a propanoic acid group, and an
ethanesulfonic acid group.
[0088] The alkyl group represented by R.sub.23 and R.sub.24 in Z of
the general formula (III) is not particularly limited and examples
thereof include linear, branched, and cyclic alkyl groups having 1
to 20 carbon atoms such as a methyl group, an ethyl group, a propyl
group, a butyl group, a pentyl group, a hexyl group, an octyl
group, a nonyl group, a decyl group, an undecyl group, a dodecyl
group, a tridecyl group, a tetradecyl group, a pentadecyl group, a
hexadecyl group, a heptadecyl group, an octadecyl group, a
nonadecyl group, a cyclopropyl group, a cyclobutyl group, and a
cyclopentyl group.
[0089] In Z, the aryl group represented by R.sub.23 and R.sub.24 is
not particularly limited and examples thereof include 6 to
14-membered monocyclic and polycyclic aryl groups such as a phenyl
group, a naphthyl group, a phenanthryl group, and an anthracenyl
group.
[0090] In the general formula (III), Z particularly preferably
represents, in terms of storage stability of the compound, one of
an oxygen atom, a sulfur atom, and --C(CH.sub.3)(CH.sub.3)--.
[0091] In the general formula (I), n represents an integer of 1 to
3, and in terms of stability of the compound, n preferably
represents 1. Further, when n represents 1, A and R.sub.6 may be
condensed together to form a ring.
[0092] Examples of the ring which is formed by A and R.sub.6
condensed together include a squarylium ring and a
thiazolidin-4-one ring.
[0093] Further, in the case of using light in a near-infrared area
as excitation light, in the general formula (I), when A represents
the general formula (III), n preferably represents one of 2 and
3.
[0094] Further, the compound represented by the general formula (I)
preferably has at least one of a carboxylic acid group and a
sulfonic acid group.
[0095] In the general formula (I), the ring formed by A and R.sub.6
bonded to each other is not particularly limited and examples
thereof include a 2,3-dihydroindene ring, an indene-1,3-dione ring,
a 4-cyclopentene-1,3-dione ring, a fluorene ring, a cyclohexene
ring, a hydroxycyclobutenone ring, a cyclohexanone ring, and a
5,5-dimethyl-1-cyclohexene ring.
[0096] In the general formula (II), the carboxylic acid ester group
represented by each of R.sub.11 and R.sub.12 is not particularly
limited and examples thereof include a carboxylic acid methyl ester
group, a carboxylic acid ethyl ester group, a carboxylic acid
propyl ester group, and a carboxylic acid butyl ester group.
[0097] The carboxylic acid amide group represented by each of
R.sub.11 and R.sub.12 is not particularly limited and examples
thereof include a carboxylic acid monomethyl amide group, a
carboxylic acid monobutyl amide group, a carboxylic acid diethyl
amide group, and a carboxylic acid 2-ethylhexyl group.
[0098] The sulfonic acid ester group represented by each of
R.sub.11 and R.sub.12 is not particularly limited and examples
thereof include a sulfonic acid methyl ester group, a sulfonic acid
ethyl ester group, a sulfonic acid propionic acid ester group, and
a sulfonic acid butyl ester group.
[0099] The sulfonic acid amide group represented by each of
R.sub.11 and R.sub.12 is not particularly limited and examples
thereof include a sulfonic acid monomethyl amide group, a sulfonic
acid monobutyl amide group, a sulfonic acid diethyl amide group,
and a sulfonic acid 2-ethylhexyl amide group.
[0100] The sulfonic acid salt and carboxylic acid salt represented
by each of R.sub.11 and R.sub.12 are not particularly limited and
examples thereof include: alkali metal salts such as a lithium
salt, a sodium salt, and a potassium salt; and ammonium salts such
as an ammonium salt, a methylammonium salt, a dimethylammonium
salt, a trimethylammonium salt, a tetramethylammonium salt, an
ethylammonium salt, a diethylammonium salt, a triethylammonium
salt, and a tetraethylammonium salt, an n-propylammonium salt, an
isopropylammonium salt, a diisopropylammonium salt, an
n-butylammonium salt, a tetra-n-butylammonium salt, an
isobutylammonium salt, a monoethanolammonium salt, a
diethanolammonium salt, and a triethanolammonium salt.
[0101] R.sub.11 and R.sub.12 each preferably represent one of a
sulfonic acid group, a carboxylic acid group, a sulfonic acid
methyl ester group, a sulfonic acid ethyl ester group, a carboxylic
acid methyl ester group, a carboxylic acid ethyl ester group, a
carboxylic acid monobutyl amide group, a sulfonic acid monobutyl
amide group, a sulfonic acid sodium salt, a sulfonic acid potassium
salt, a sulfonic acid ammonium salt, a carboxylic acid sodium salt,
a carboxylic acid potassium salt, and a carboxylic acid ammonium
salt, and more preferably represent, in terms of improving the
water solubility, one of a sulfonic acid group, a carboxylic acid
group, a sulfonic acid sodium salt, and a carboxylic acid sodium
salt.
[0102] The alkyl group represented by each of R.sub.13 and R.sub.14
in the general formula (II) is not particularly limited and
examples thereof include linear, branched, and cyclic alkyl groups
having 1 to 20 carbon atoms such as a methyl group, an ethyl group,
a propyl group, a butyl group, a pentyl group, a hexyl group, an
octyl group, a nonyl group, a decyl group, an undecyl group, a
dodecyl group, a tridecyl group, a tetradecyl group, a pentadecyl
group, a hexadecyl group, a heptadecyl group, an octadecyl group, a
nonadecyl group, a cyclopropyl group, a cyclobutyl group, and a
cyclopentyl group.
[0103] The aryl group represented by each of R.sub.13 and R.sub.14
is not particularly limited and examples thereof include 6 to
14-membered monocyclic and polycyclic aryl groups such as a phenyl
group, a naphthyl group, a phenanthryl group, and an anthracenyl
group.
[0104] R.sub.13 and R.sub.14 each preferably represent one of an
alkyl group and a phenyl group, and more preferably one of a methyl
group, an ethyl group, a propyl group, and a butyl group.
[0105] The alkyl group represented by each of R.sub.15, R.sub.16,
R.sub.17A, and R.sub.17B in the general formula (II) is not
particularly limited and examples thereof include linear, branched,
and cyclic alkyl groups having 1 to 20 carbon atoms such as a
methyl group, an ethyl group, a propyl group, a butyl group, a
pentyl group, a hexyl group, an octyl group, a nonyl group, a decyl
group, an undecyl group, a dodecyl group, a tridecyl group, a
tetradecyl group, a pentadecyl group, a hexadecyl group, a
heptadecyl group, an octadecyl group, a nonadecyl group, a
cyclopropyl group, a cyclobutyl group, and a cyclopentyl group.
[0106] The aryl group represented by each of R.sub.15, R.sub.16,
R.sub.17A, and R.sub.17B is not particularly limited and examples
thereof include 6 to 14-membered monocyclic and polycyclic aryl
groups such as a phenyl group, a naphthyl group, a phenanthryl
group, and an anthracenyl group. Further, the ring may have a
substituent and the substituent is not particularly limited as long
as the storage stability of the staining compound is not
significantly inhibited. Examples thereof include: alkyl groups
such as a methyl group, an ethyl group, an n-propyl group, an
isopropyl group, an n-butyl group, an isobutyl group, a sec-butyl
group, and a tert-butyl group; aryl groups such as a phenyl group
and a naphthyl group; alkoxy groups such as a methoxy group, an
ethoxy group, and a butoxy group; aryloxy groups such as a phenoxy
group and a naphthyloxy group; disubstituted amino groups such as a
dimethylamino group, an N-ethyl-N-phenylamino group, and a
diphenylamino group; acyl groups such as an acetyl group, a benzoyl
group, a carboxylic acid group, a carboxylic acid ester group, and
a carbamoyl group; sulfonyl groups such as a sulfonic acid group, a
sulfonic acid ester group, and a sulfamoyl group; heterocyclic
groups such as a pyridyl group, a triazinyl group, and a
benzothiazolyl group; a nitro group; halogen atoms such as a
fluorine atom, a chlorine atom, a bromine atom, and an iodine atom;
a polyethylene glycol group; and salts such as a quarternary
ammonium salt, a carboxylic acid salt, and a sulfonic acid
salt.
[0107] The heterocyclic group represented by each of R.sub.15,
R.sub.16, R.sub.17A, and R.sub.17B is not particularly limited and
examples thereof include 4 to 10-membered monocyclic and bicyclic
heterocyclic groups having 1 to 4 atoms selected from nitrogen,
oxygen, and sulfur, such as a pyridyl group, a pyrazinyl group, a
pyrimidinyl group, a pyrrolyl group, a thienyl group, a furyl
group, a pyranyl group, an oxazolyl group, a thiazolyl group, a
triazolyl group, a tetrazolyl group, an imidazolyl group, a
pyrazolyl group, a morpholinyl group, a thiomorpholinyl group, a
piperidinyl group, a piperazinyl group, a quinolyl group, an
isoquinolyl group, an indolyl group, an isoindolyl group, a
benzofuryl group, and a benzothienyl group.
[0108] R.sub.15, R.sub.16, R.sub.17A, and R.sub.17B each preferably
represent one of an alkyl group and an aryl group, and more
preferably represent one of a methyl group, an ethyl group, a
propyl group, a butyl group, and an unsubstituted and substituted
phenyl group.
[0109] X.sub.2.sup.- in the general formula (II) represents an
anionic group. Here, the anionic group is not particularly limited
and examples thereof include: halogen ions such as a fluoride ion,
a chloride ion, a bromide ion, and an iodide ion; inorganic acid
ions such as a sulfuric acid ion, a phosphoric acid ion, a nitric
acid ion, a tetrafluoroboric acid ion, and a hexafluorophosphoric
acid ion; Lewis acid-containing ions such as a tetrachloroaluminum
ion; and organic acid ions such as an acetic acid ion, a lactic
acid ion, a methanesulfonic acid ion, a benzenesulfonic acid ion, a
p-toluenesulfonic acid ion, a trifluoroacetic acid ion, a
trifluoromethanesulfonic acid ion, and a tetraphenylboric acid
ion.
[0110] The anionic group represented by X.sub.2.sup.- is preferably
one of a chloride ion, a bromide ion, an iodide ion, a sulfuric
acid ion, a nitric acid ion, and a methanesulfonic acid ion, and
more preferably, in terms of ease of synthesis of the compound, one
of a bromide ion and an iodide ion.
[0111] When R.sub.11 represents one of a sulfonic acid group and a
carboxylic acid group in a molecular structure represented by the
general formula (II), there exists a tautomer represented by one of
the following general formulae (II') and (II''). The structure
represented by the general formula (II) of the staining compound of
the present invention also encompasses a structure represented by,
for example, one of the following general formulae (II') and
(II'').
##STR00007##
R.sub.12 to R.sub.16, R.sub.17A and R.sub.17B in the staining
compound represented by one of the general formulae (II') and
(II'') have the same meanings as those of R.sub.12 to R.sub.16,
R.sub.17A, and R.sub.17B in the general formula (II).
[0112] The staining compound according to the present invention can
be utilized for labelling of a hair cell as a staining agent that
is retained by itself in the hair cell and stains the hair cell
based on a structure of the compound itself that has a coloring
property. Further, the staining compound according to the present
invention can be used as a probe in such a form that a compound
capable of producing an optical signal is further added to the
compound, by utilizing a feature of the compound of being retained
in a hair cell. The compound to be added may bonded directly or via
a linker molecule. For the compound to be added, such a low
molecular weight compound that can penetrate a cell membrane and
permeate into a hair cell can be suitably used.
[0113] Next, a production method for the staining compound having a
structure represented by the general formula (I) of the present
invention is described below. The staining compound represented by
the general formula (I) according to the present invention may be
synthesized by a known method. An exemplary synthesis scheme is
described below.
##STR00008##
[0114] R.sub.1 to R.sub.5, R.sub.25 to R.sub.30, and X.sub.1.sup.-
in the above general formulae (I), (A), and (B) have the same
meanings as those of R.sub.1 to R.sub.5, R.sub.25 to R.sub.30, and
X.sub.1.sup.- in the general formulae (I) to (IV).
[0115] The staining step can be easily performed by a known method
(for example, Yakugaku Zasshi, 69, pp. 237-239, 1949, Indian
Journal of Chemistry, Vol. 6, pp. 136-139, 1968, and Synthesis, pp.
37-38, 1976). A specific coupling method is not particularly
limited, and a method described below is exemplified as one
aspect.
[0116] That is, the coupling of an aldehyde derivative (A) and a
compound (B) yields a staining compound (I).
[0117] The amount of the compound (B) to be used is 0.1 to 10-fold
mol, preferably 0.5 to 3-fold mol, and more preferably 0.8 to
2-fold mol with respect to 1 mol of the aldehyde derivative
(A).
[0118] This step may also be performed without using any solvent
but is preferably performed in the presence of a solvent. The
solvent is not particularly limited as long as it is not involved
in the reaction, and examples thereof include: an ester-based
solvent such as methyl acetate, ethyl acetate, isopropyl acetate,
and butyl acetate; a nitrile-based solvent such as acetonitrile,
propionitrile, and benzonitrile; an aromatic solvent such as
benzene, toluene, xylene, ethylbenzene, chlorobenzene, and
mesitylene; an ether-based solvent such as diisopropyl ether,
methyl tert-butyl ether, and tetrahydrofuran; an alcohol-based
solvent such as methanol, ethanol, n-propyl alcohol, iso-propyl
alcohol, butyl alcohol, and diethylene glycol; a ketone-based
solvent such as acetone and methyl ethyl ketone; dimethylformamide
(DMF); dimethylsulfoxide (DMSO); water; and acetic acid. Preferred
examples include an alcohol-based solvent such as methanol,
ethanol, n-propyl alcohol, iso-propyl alcohol, butyl alcohol, and
diethylene glycol, water, and acetic acid, and more preferred
examples include ethanol, iso-propyl alcohol, diethylene glycol,
and acetic acid. Further, two or more kinds of solvents may be used
in mixture, and the mixing ratio may be arbitrarily set during use
in mixture.
[0119] The amount of a reaction solvent to be used in this step is
in the range of 0.1 to 1,000-fold weight, preferably 0.5 to
500-fold weight, and more preferably 1.0 to 150-fold weight with
respect to the aldehyde derivative (A).
[0120] The reaction temperature at which this step is performed is
in the range of -80.degree. C. to 250.degree. C., preferably
-20.degree. C. to 200.degree. C., and more preferably -5.degree. C.
to 150.degree. C. In general, the reaction is completed within 24
hours.
[0121] In this step, the reaction rapidly proceeds by the addition
of an acid or a base as necessary. The acid to be used is not
limited as long as it is not directly involved in the reaction, and
examples thereof include: an inorganic acid such as hydrochloric
acid, sulfuric acid, and phosphoric acid; an organic acid such as
p-toluenesulfonic acid, formic acid, acetic acid, propionic acid,
and trifluoroacetic acid; a strongly acidic ion-exchange resin such
as Amberlite (Rohm and Haas Company) and Amberlyst (Rohm and Haas
Company); and an inorganic acid salt such as ammonium formate and
ammonium acetate. More preferred is an inorganic acid salt such as
ammonium formate and ammonium acetate, and still more preferred is
ammonium acetate. The amount of the acid to be used is 0.001 to
50-fold mol, preferably 0.01 to 10-fold mol, and more preferably
0.1 to 5-fold mol with respect to 1 mol of the aldehyde derivative
(A).
[0122] Specific examples of the base to be used in this step
include: a metal alkoxide such as potassium tert-butoxide, sodium
tert-butoxide, sodium methoxide, and sodium ethoxide; an organic
base such as piperidine, pyridine, 2-methylpyridine,
dimethylaminopyridine, diethylamine, triethylamine,
isopropylethylamine, sodium acetate, potassium acetate,
1,8-diazabicyclo[5.4.0]undeca-7-ene (hereinafter, abbreviated as
DBU), and ammonium acetate; an organic base such as n-butyl lithium
and tert-magnesium chloride; and an inorganic base such as sodium
borohydride, metallic sodium, sodium hydride, and sodium carbonate.
Preferred examples include potassium tert-butoxide, sodium
methoxide, sodium ethoxide, piperidine, dimethylaminopyridine,
sodium acetate, and ammonium acetate, and more preferred examples
include sodium methoxide, piperidine, sodium acetate, and ammonium
acetate. The amount of the above-mentioned base to be used is 0.1
to 20-fold mol, preferably 0.5 to 8-fold mol, and more preferably
1.0 to 4-fold mol with respect to 1 mol of the aldehyde derivative
(A).
[0123] After the completion of the reaction, the dilution with
water, the acid deposition with hydrochloric acid, or the like may
be performed to obtain a staining compound (I).
[0124] The obtained staining compound (I) may be subjected to a
conventional method for isolation and purification of an organic
compound. For example, after acid deposition has been performed by
acidifying a reaction solution with hydrochloric acid and the like,
a solid is separated by filtration, followed by neutralization with
sodium hydroxide and the like and concentration. Thus, a crude
product is obtained. In addition, the crude product is
recrystallized from acetone, methanol, and the like, and purified
by, for example, silica gel column chromatography. The purification
may be performed by using one of those methods alone or by using
two or more kinds thereof in combination to afford a product with
high purity.
[0125] Next, a production method for a staining compound having a
structure represented by the general formula (II) of the present
invention is described below.
[0126] The staining compound represented by the general formula
(II) according to the present invention can be synthesized
according to a known method. An exemplary synthesis scheme is
described below.
##STR00009##
[0127] R.sub.11 to R.sub.16, R.sub.17A, and R.sub.17B in the above
general formulae (II) and (V) to (VIII) have the same meanings as
those of R.sub.11 to R.sub.16, R.sub.17A, and R.sub.17B in the
general formula (II).
[0128] The staining step can be easily performed by a known method
(for example, Japanese Patent Application Laid-Open No.
2008-94897). That is, the staining compound (II) of the present
invention can be synthesized by a condensation (1) step and a
condensation (2) step.
[0129] First, in the condensation (1) step, a compound (V) and a
compound (VI) are subjected to heat condensation in an organic
solvent (or in the absence of a solvent) in the presence of a
condensation agent (or in the absence of a condensation agent) to
afford a compound (VII). Next, the compound (VII) is subjected to
second heat condensation with such a compound (VIII) as described
above. Thus, a staining compound (II) according to the present
invention is obtained.
[0130] A preferred organic solvent that can be used in the
condensation (1) step of the condensation reaction of such a
synthesis scheme as exemplified above is not particularly limited
as long as the solvent is not involved in the reaction, and for
example, methanol, ethanol, n-propanol, isopropanol, and n-butanol
may be used alone or in mixture. A preferred organic solvent that
can be used in the condensation (2) step is not particularly
limited as long as the solvent is not involved in the reaction, and
examples thereof include ethylene glycol, N-methylpyrrolidone,
N,N-dimethylacetamide, N,N-dimethylformamide, dimethylsulfoxide,
sulfolane, chlorobenzene, dichlorobenzene, trichlorobenzene, and
nitrobenzene.
[0131] The reaction temperature at which the condensation (1) step
is performed is in the range of 0.degree. C. to 200.degree. C.,
preferably 10.degree. C. to 150.degree. C., and more preferably
20.degree. C. to 100.degree. C. Further, the reaction temperature
at which the condensation (2) step is performed is in the range of
50.degree. C. to 250.degree. C., preferably 100.degree. C. to
230.degree. C., and more preferably 150.degree. C. to 220.degree.
C.
[0132] In the case of a compound where R.sub.14 and R.sub.15 in the
general formula (II) are the same substituent to each other, and
where R.sub.16 and R.sub.17 are the same substituent to each other,
because the compounds (VI) and (VIII) in the scheme are identical
to each other, the staining compound of the general formula (II)
can be obtained in one condensation step from the compound (V). In
this case, the reaction temperature conforms to that in the
condensation (2) step.
[0133] The condensation agent to be used in the condensation
step(s) is not particularly limited as long as the agent is not
involved in the reaction, and may be selected from magnesium oxide,
zinc chloride, and aluminum chloride, for example. The obtained
staining compound (II) may be subjected to a conventional method
for isolation and purification of an organic compound. For example,
after acid deposition has been performed by acidifying a reaction
solution with hydrochloric acid and the like, a solid is separated
by filtration, followed by neutralization with sodium hydroxide and
the like and concentration. Thus, a crude product is obtained. In
addition, the crude product is purified by, for example,
recrystallization from acetone, methanol, and the like, and silica
gel column chromatography. The purification may be performed by
using one kind of those methods alone or by using two or more kinds
thereof in combination to afford a product with high purity.
[0134] Hereinafter, specific examples (1) to (70) of the present
invention are described. However, the present invention is not
limited to the following examples.
##STR00010## ##STR00011## ##STR00012## ##STR00013## ##STR00014##
##STR00015## ##STR00016## ##STR00017## ##STR00018## ##STR00019##
##STR00020## ##STR00021## ##STR00022##
[0135] A hair cell staining (labelling) solution of the present
invention is prepared by dissolving the probe for a hair cell of
the present invention in an appropriate solvent. The solvent is not
particularly limited as long as it has no influence on a living
body, but an aqueous liquid with high biocompatibility is
preferred. Examples of the solvent include: water; physiological
saline; a buffer such as a phosphoric buffer saline (PBS) and Tris;
an alcohol-based solvent such as ethanol, ethylene glycol, and
glycerin; an organic solvent such as N,N-dimethylsulfoxide
(hereinafter, abbreviated as DMSO); a cell culture medium such as
D-MEM and HBSS; and an infusion solution such as a lactated
Ringer's solution, and particularly preferred is a solvent
containing 50% or more of water. Further, two or more kinds of
those solvents may be used in mixture.
[0136] To the hair cell staining (labelling) solution, preferably
added is at least one of a humectant, a surface tension adjusting
agent, and a thickener, for example. If a salt concentration and a
pH suitable for a living body must be controlled, one of salts such
as sodium chloride, various pH adjusting agents, preservatives,
antibacterial agents, sweeteners, and flavors may be appropriately
added, for example.
[0137] The pH adjusting agent is not particularly limited, but
preferably adjusts a pH to 5 to 9. Examples thereof include
hydrochloric acid, acetic acid, phosphoric acid, citric acid, malic
acid, sodium hydroxide, and sodium bicarbonate.
[0138] An organism to be stained with the probe for a hair cell of
the present invention is not particularly limited as long as the
organism has a hair cell, and examples thereof include: small bony
fishes such as Japanese putter fish, Japanese rice fish, and
Zebrafish; a small animal such as a rat and a mouse; a large animal
such as a primate, a pig, and a dog; and a human. In addition to an
individual organism, there are exemplified a tissue and a tissue
section derived from the organism, and a culture cell derived from
the organism.
[0139] The form of the probe for a hair cell of the present
invention is not particularly limited, and the probe for a hair
cell can be used in a form such as a liquid, a granule, a tablet, a
capsule, and a patch.
[0140] The administration route of the probe for a hair cell for
staining a hair cell present in the auditory organs is not
particularly limited, and the probe for a hair cell can be orally
or parenterally administered. Systemic administration is also
possible, but local administration is preferred in order to enhance
an effect of staining properties.
[0141] In the case of local administration, the probe for a hair
cell can be directly administered to a tissue with a hair cell
(i.e. inner ear), or may be administered through the external
auditory canal and the middle ear. Further, the probe for a hair
cell may be administered via the endolymphatic sac, the
endolymphatic duct, and the auditory capsule. Local administration
is more preferably performed by exposure of the probe for a hair
cell to a living body (a liquid etc.), and administration to the
living body including intravascular administration such as
intravenous administration and intraarterial administration, oral
administration, sublingual administration, intrarectal
administration, intraperitoneal administration, transdermal
administration, subcutaneous administration, intradermal
administration, intravesical administration, endotracheal
(intrabronchial) administration, intraocular administration,
transnasal administration, and intraaural administration, which
utilize means such as injection, catheter infusion, nebulization,
application, and the like. In addition, such an apparatus as
described in, for example, U.S. Pat. Nos. 5,476,446 and 5,350,580
may be used.
[0142] The dosage of the probe for a hair cell is not particularly
limited as long as a target site can be finally detected, and can
be appropriately increased or decreased depending on the kind of
the target site and the probe to be used. In particular, when the
probe for a hair cell is administered to an individual organism,
the smallest possible amount is preferred. Further, when being
exposed to a tissue and a cell, the probe for a hair cell may be
used in an amount that has selectivity for tissue staining
(labelling) and is easy to be distinguished.
[0143] The concentration of the probe for a hair cell to be used is
in the range of generally 0.001 nM to 1,000 .mu.M and preferably
0.01 nM to 100 .mu.M.
[0144] The administration form, administration route, and dosage
for an animal are appropriately selected depending on the body
weight and condition of an animal of interest.
[0145] An evaluation method for an auditory function of the present
invention involves staining a hair cell of an organism with the
probe for a hair cell of the present invention, and then observing,
detecting, or measuring a staining condition of the hair cell. The
observation, measurement, and detection in the evaluation method
for an auditory function of the present invention are performed by
a method known to those skilled in the art.
[0146] An observation method for a staining condition to be used in
the present invention is not particularly limited as long as the
method has no influence on both the organism and the probe for a
hair cell, and is a method of capturing a condition and a change of
the organism as an image. Examples of the observation method
include infra red imaging, which involves irradiating an organism
with one of visible light, near-infrared light, and infrared light,
and observing with a camera, CCD, and the like, laser microscopy;
fluorescent imaging, fluorescent microscopy, fluorescent
endomicroscopy, confocal endomicroscopy, multiphoton-excited
fluorescence microscopy, narrow band imaging, and optical coherence
tomography (OCT), each of which involves observing fluorescence
derived from an organism with a fluorescence endoscope etc. by
irradiating a biological specimen with excitation light from an
excitation light source; and further, soft X-ray microscopy.
[0147] The probe for a hair cell of the present invention may be
labelled with a radionuclide.
[0148] The probe for a hair cell labelled with a radionuclide may
be imaged with one of autoradiography, positron emission tomography
(PET), and single photon emission computed tomography (SPECT).
Further, the probe for a hair cell may be detected by magnetic
resonance imaging (MRI) utilizing an MR signal derived from a
fluorine nucleus and .sup.13C. In addition, the probe for a hair
cell can be imaged by using a compton camera (GREI) capable of
simultaneously imaging multiple molecules as a next-generation
molecular imaging apparatus. A distribution condition of a hair
cell can be time-dependently measured and imaged in a noninvasive
manner by those methods. Further, a probe for a biological specimen
can be quantitatively determined by using, for example, a liquid
scintillation counter, an X-ray film, and an imaging plate.
[0149] Further, the measurement of the blood (or urinary or fecal)
concentration of the probe for a hair cell labelled with a
radioisotope such as .sup.14C using, for example, accelerator mass
spectrometry (AMS) can provide pharmacokinetic information (such as
area under the blood concentration-time curve (AUC), blood
concentration half life (T.sub.1/2), maximum blood concentration
(C.sub.max), time-to-maximum blood concentration (T.sub.max),
distribution volume, first-pass effect, bioavailability, and
urinary and fecal excretion rate) on an unchanged product and a
metabolite of a labelled substance.
[0150] The radionuclide is not particularly limited and may be
appropriately selected depending on the usage mode.
[0151] Specifically, in the case of measurement with PET, a
positron emitting nuclide such as .sup.11C, .sup.14C, .sup.13N,
.sup.15O, .sup.18F, .sup.19F, .sup.62CU, .sup.68Ga, and .sup.78Br
may be used, for example.
[0152] Preferred examples include .sup.11C, .sup.13N, .sup.15O, and
.sup.18F, and particularly preferred examples include .sup.11C and
.sup.18F. Further, in the case of measurement with SPECT, a
.gamma.-ray emitting nuclide such as .sup.99mTC, .sup.111IN,
.sup.67Ga, .sup.201Tl, .sup.123I, and .sup.33Xe may be used, for
example. Preferred examples include .sup.99mTc and .sup.123I. In
addition, in the case of measurement of animals other than a human,
a radionuclide having a longer half life such as .sup.125I may be
used, for example. In the case of measurement with GREI, one of
.sup.131I, .sup.85Sr, and .sup.65Zn may be used, for example.
[0153] The radionuclide may be contained in or bonded to the
compound represented by one of the general formula (I) and (II). A
labelling method with the radionuclide is not particularly limited,
and at least one part of elements of which the compound represented
by one of the general formula (I) and (II) is formed may be
replaced by or bonded to the radionuclide.
[0154] When the compound represented by one of the general formula
(I) and (II) is labelled with the radionuclide, the compound
preferably has radioactivity of about 1 to 100 .mu.Ci per mM.
[0155] In this case, the dosage of the probe for a hair cell to be
used is not particularly limited as long as it has no influence,
and is appropriately selected depending on the kind of the compound
and the kind of the radionuclide used as a label.
[0156] For example, for an adult human, the amount of the probe for
a hair cell to be used is 0.0001 .mu.g to 1,000 .mu.g and
preferably 0.01 .mu.g to 10 .mu.g per day.
[0157] The wavelength of excitation light to be used in the present
invention is not particularly limited as long as it has no
influence on both the organism and the probe for a hair cell,
varies depending on the kind of the probe to be used, and is not
particularly limited as long as the probe of the present invention
efficiently fluoresces. The wavelength is generally 200 to 1,010
nm, preferably 400 to 900 nm, and more preferably 480 to 800 nm.
The wavelength in the case of using light in a near-infrared area
is generally 600 to 1,000 nm and preferably 680 to 800 nm in which
wavelength biological permeability is excellent.
[0158] A fluorescence excitation light source to be used in the
present invention is not particularly limited as long as the light
source has no influence on both the organism and the probe for a
hair cell, and various laser light sources may be used. Examples
thereof include a dye laser, a semiconductor laser, an ion laser, a
near-infrared pulse laser, a fiber laser, a halogen lamp, a xenon
lamp, and a tungsten lamp. Further, the use of various optical
filters allows the acquisition of preferred excitation wavelengths
and the detection of only fluorescence.
[0159] As mentioned above, if an image of an individual organism is
captured in such a condition that the organism has been irradiated
with excitation light to cause light emission inside the individual
organism, a light emitting site can be easily detected. Further, a
bright field image obtained by irradiation with visible light and a
fluorescence image obtained by irradiation with excitation light
can be combined by using an image processing unit to observe the
individual organism in more detail.
[0160] Many of the probes for a hair cell of the present invention
each have large Stokes' shift, and hence can identify a hair cell
clearly. The term "Stokes' shift" as used herein represents a
difference between the maximum excitation wavelength and the
maximum fluorescence emission wavelength. In general, a small
Stokes' shift tends to generate a measurement error due to
excitation light and its scattering light.
[0161] Next, an evaluation method for the auditory toxicity of a
chemical substance by using the probe for a hair cell of the
present invention is described in detail below. That is, the
evaluation method involves the following steps: administering a
chemical substance to an organism; bringing the organism into
contact with a probe for a hair cell; and observing fluorescence
derived from the probe for a hair cell by irradiating the organism
with excitation light. Thus, the auditory toxicity of a chemical
substance can be evaluated (screening method).
[0162] First, a step of administering a chemical substance to an
organism is described.
[0163] The organism to be used in this step is not particularly
limited, and the use of small bony fishes such as Zebrafish allows
the high-throughput evaluation for the auditory toxicity of each of
various chemical substances, for example.
[0164] In recent years, in U.S. and U.K., Zebrafish has been
already recognized as a third model animal following a mouse and a
rat. Zebrafish is easily optically observed because Zebrafish has a
feature that a process in which the respective parts (organs such
as heart, liver, kidney, and gastrointestinal tract) are
differentiated and formed from a fertilized embryo can be observed
through its transparent body. Further, it is being clarified that,
in a comparison to human, Zebrafish has an 80% homology in terms of
the full genome sequence, is almost the same in terms of the number
of genes, and is very similar also in terms of the development and
structure of principal organs and tissues. Therefore, the auditory
toxicity of a chemical substance screened by using Zebrafish as a
model animal is highly likely to be applicable to a human. In
addition, a sensory hair cell lies deep within a temporal bone
portion in a vertebrate such as a mouse. Thus, the sensory hair
cell is anatomically difficult to handle, and is hardly observed
and subjected to an additional manipulation in an experiment. In
contrast, Zebrafish has a sensory hair cell of the inner ear, which
can be clearly observed even in a developmental stage of embryo
after spawning. Zebrafish also has a sensory hair cell in organs
present on the skin surface called lateral line organs. Thus, it is
possible to easily observe and manipulate the hair cell. Each of
the lateral line organs serves as a sensor of the movement of
water, and also shares a common origin with the inner ear.
Therefore, Zebrafish is suitable for screening as a model
animal.
[0165] A chemical substance to be evaluated by an evaluation method
for the auditory toxicity of a chemical substance of the present
invention means a collective name of substances, each of which may
have a biological action. The chemical substance is not
particularly limited and examples thereof include a pharmaceutical,
an organic compound, a therapeutic agent, an investigational drug,
an agricultural chemical, a cosmetic, an environmental pollutant,
and an endocrine disruptor.
[0166] A method of administering the chemical substance to
Zebrafish is not particularly limited. When the chemical substance
is water-soluble, there is given a method of allowing the chemical
substance to coexist in feeding water, and when the chemical
substance is non-water-soluble, there is given one of a method of
allowing the chemical substance alone to disperse and coexist in
feeding water, a method of allowing the chemical substance and a
trace amount of a surfactant or DMSO to coexist, and a method of
orally administering the chemical substance mixed in a feed for
Zebrafish, and a method of parenterally administrating the chemical
substance with injection and the like. Preferably, there is given a
method of allowing the chemical substance to coexist in feeding
water, which can be easily performed.
[0167] Next, there is described a step of bringing an organism into
contact with a probe for a hair cell.
[0168] After the administration of a chemical substance to
Zebrafish, if necessary, the chemical substance exposed is removed,
and then at least one kind of the probe for a hair cell of the
present invention is utilized for staining a hair cell of
Zebrafish. In this step, a method of administering the probe for a
hair cell to Zebrafish is not particularly limited. When the probe
for a hair cell is water-soluble, there is given a method of
allowing the probe for a hair cell to coexist in feeding water, and
when the probe for a hair cell is non-water-soluble, there is given
one of a method of allowing the probe for a hair cell alone to
disperse and coexist in feeding water, a method of allowing the
probe for a hair cell and a trace amount of a surfactant or DMSO to
coexist, a method of orally administering the probe for a hair cell
mixed in a feed for Zebrafish, and a method of parenterally
administering the probe for a hair cell with injection and the
like. Preferably, there is given a method of allowing the probe for
a hair cell to coexist in feeding water, which can be easily
performed.
[0169] The dosage of the probe for a hair cell is not particularly
limited as long as a target site can be finally detected, and may
be appropriately increased or decreased depending on the kind of
the target site and the probe to be used. In particular, in the
case of administration to an individual organism, the smallest
possible amount is preferred. Further, in the case of exposure to a
tissue and a cell, an amount that has selectivity for tissue
staining and is easy to be distinguished may be used.
[0170] Next, there is described a step of observing fluorescence
derived from the probe by irradiating the organism with excitation
light.
[0171] The wavelength of excitation light to be used in this step
is not particularly limited as long as it has no influence on both
the organism and the probe for a hair cell, varies depending on the
kind of the probe to be used, and is not particularly limited as
long as the probe of the present invention efficiently fluoresces.
The wavelength is generally 200 to 1,010 nm, preferably 400 to 900
nm, and more preferably 480 to 800 nm. The wavelength in the case
of using light in a near-infrared area is generally 600 to 1,000 nm
and preferably 680 to 800 nm in which biological permeability is
excellent.
[0172] A fluorescence excitation light source to be used in this
step is not particularly limited as long as the light source has no
influence on both the organism and the probe for a hair cell, and
various laser light sources may be used. Examples thereof include a
dye laser, a semiconductor laser, an ion laser, a fiber laser, a
halogen lamp, a xenon lamp, and a tungsten lamp. Further, the use
of various optical filters allows the acquisition of preferred
excitation wavelengths and the detection of only fluorescence.
[0173] As described above, the auditory function of Zebrafish is
evaluated by observing, detecting, or measuring a staining
condition in such a condition that light has emitted in a hair cell
of Zebrafish by irradiating Zebrafish with excitation light. The
auditory toxicity of the chemical substance subjected to a test can
be screened based on the evaluation.
[0174] Many special mechanoreceptors called lateral line organs
exist on the body surface of Zebrafish and form a lateral line
system as a whole. An individual end organ belonging to the system
serves as a neuromast and is innervated by a branch of a specific
cranial nerve. Embryologically, the neuromast and a nerve
distributed in the neuromast are derived from placodes resulting
from the thickening of an ectoderm of an embryo. The placodes are
formed of approximately 120 cells, and are differentiated from
primordia moving from the head toward the tail in a period of 20
hours to 40 hours after fertilization. The primordia leave 7 to 9
proneuromasts and interneuromast cells connecting the proneuromasts
with each other along the movement route. After that, second
smaller primordia move along the same route and leave 2 or 3
proneuromasts on the movement route. A posterior lateral line
neuromast gradually matures from the head toward the tail, and by
Day 5 of development, a functional hair cell is included in 9 to 11
neuromasts. After that, the system becomes complicated through the
additional development of the neuromast and the growth and
elongation of existing clusters.
[0175] The use of the probe for a hair cell of the present
invention allows the staining, staining with various patterns, and
identification of not only a Zebrafish neuromast (big size), which
has been conventionally reported, but also a microorgan (neuromast
of small size) on the body surface, which has not been visualized
thus far.
[0176] The staining of the neuromast with different colors can be
utilized for the evaluation for the toxicity of a chemical
substance with respect to a difference in the type of hair cell
injuries in a developmental process, and the evaluation for the
auditory toxicity of a chemical substance with respect to a hair
cell precursor, for example. An evaluation method (screening
method) for the auditory toxicity of a chemical substance of the
present invention is highly excellent in terms of a speed and a
cost compared with a method using a mouse, a rat, and the like
because Zebrafish is easy in its feeding and propagation and low in
its market price, and has principal organs and tissues, the basic
structures of which are formed in 48 to 72 hours after
fertilization.
[0177] Zebrafish is not limited to wild-type Zebrafish and various
disease-based models of Zebrafish may be used depending on the
purpose of screening. In the case of an auditory disease-based
model, the model can be applied for screening for finding out an
effect and toxicity of a new drug candidate compound by observing a
hair cell by using the probe for a hair cell according to the
present invention as an index. Examples of the auditory
disease-based model include, but not particularly limited to, a
model of Zebrafish such as a drug-induced hearing loss model and an
inherited hearing loss model.
[0178] In a vertebrate such as a mouse, a sensory hair cell lies
deep within a temporal bone portion. Thus, the sensory hair cell is
anatomically difficult to handle, and is relatively hardly observed
and subjected to an additional manipulation in an experiment. In
order to visualize the sensory hair cell of a vertebrate including
a human, out of the probe for a hair cell of the present invention,
particularly preferred are an agent that fluoresces in a
near-infrared area and an agent that has been labelled with a
radionuclide. This is because, in case of using the agent that
fluoresces in a near-infrared area, near-infrared light has no
absorption with water and hemoglobin in the area, and easily
transmits a body tissue, and as a result, test diagnosis can be
performed in the range of a thickness of 10 to 20 cm. Further, the
agent that has been labelled with a radionuclide can image a
condition of a hair cell in a noninvasive manner by using PET,
PET-CT, SPECT, and MRI, for example.
[0179] The probe for a hair cell of the present invention can be
used as a diagnosis drug for an auditory function disease. Further,
there can be provided a diagnostic composition for an auditory
function containing, as an active agent, the probe for a hair cell
of the present invention. The hair cell that has suffered an injury
is difficult to retrieve. It is also said that, in a human, once
the number of the hair cell is reduced, the number is extremely
difficult to increase. Also from such a viewpoint, there is a need
for finding out diseases through early diagnosis.
[0180] Examples of the auditory function disease include, but not
particularly limited to, peripheral sensorineural auditory
impairment (hearing reduction and hearing loss), tinnitus, vertigo,
feeling of fullness in the ear, and equilibrium loss. Examples of
hearing loss include, but not particularly limited to, age-related
hearing loss, sensorineural hearing loss, conductive hearing loss,
sudden hearing loss, noise-induced hearing loss, and acoustic
trauma hearing loss, and acute hearing loss such as Meniere's
disease, delayed endolymphatic hydrops, perilymph leak,
drug-induced hearing loss, viral labyrinthitis, acoustic neuroma,
and functional hearing loss. For high precision diagnosis and
treatment method for those auditory function diseases, the
application development such as a diagnosis with the staining
properties of the hair cell is expected. Further, when the
regeneration of the hair cell is attempted by using, for example, a
gene treatment, and an ES cell and an iPS cell, the application
development such as a following diagnosis of the hair cell is
expected by utilizing the staining properties of the hair cell.
[0181] The probe for a hair cell of the present invention can be
used for screening by the staining properties of a tissue and a
cell taken out from a biological specimen (in vitro). In addition,
the application development such as development of high precision
diagnosis and treatment method is expected by utilizing staining
properties of the probe for a hair cell. For example, it is
expected that the probe for a hair cell can be used for cytology
involving sampling one part of tissues and cells as targets in a
trace amount by aspiration with a puncturing cytodiagnostic device
and the like, staining the sample with the probe for a hair cell of
the present invention, and assessing the form, kind, and benignancy
and malignancy, and the like of the cell.
[0182] Further, for example, the introduction of an optical fiber
into the tympanum through the tympanic membrane dissected from the
external auditory canal or through the Eustachian tube from the
epipharynx, the irradiation of one of a vestibular window portion,
a cochlea portion, and a semicircular canal portion with excitation
light via the optical fiber, and the acquisition of fluorescence
allows in vivo acquisition of a hair cell labelling signal and a
diagnosis for an auditory function disease based on the signal. The
human inner ear is anatomically located in a pit of the skull, and
hence, there is no need to worry about light shielding by the bone
during light irradiation from the tympanum. Further, because the
tympanum is generally filled with a gas, the absorption of
near-infrared light by a liquid can be as minimum as absorption by
lymph with which one of the vestibular window portion, the cochlea
portion, and the semicircular canal portion is filled.
Near-infrared light with generally used intensity has a
permeability of 10 to 20 cm with respect to a living body, and
hence, the probe for a hair cell having absorption or fluorescence
in a near-infrared area can be suitably used as a component of a
diagnostic drug for an auditory function of the present invention.
Meanwhile, conventionally known probes for a hair cell such as
DASPEI and FM1-43 have no absorption and fluorescence in a
near-infrared area.
[0183] An image diagnosis of an auditory function using the probe
for a hair cell of the present invention may complement
conventional physiological function tests. As a physiological
function test of hearing, there are exemplified: a threshold for
air conduction and bone conduction of a pure sound, a speech
reception threshold, and a speech discrimination ability;
tympanometry; and a stapedial reflex test including impedance
audiometry. Those tests are being used for assessment of hearing
loss. The combination of an image using the diagnostic composition
for an auditory function of the present invention allows the
utilization of objective diagnosis information which may complement
the physiological function tests which tend to be dependent on
subjectivity of each of patients. Further, there is provided an
auditory function diagnosis system formed of a combination of the
probe for a hair cell of the present invention and a light signal
detecting unit such as an optical fiber. The auditory function
diagnosis system may further include units for imaging and
analyzing the obtained light signal.
[0184] The probe for a hair cell of the present invention includes
an agent that stains not only a somatic portion but also an axon
portion of the hair cell. The demyelination of the axon portion of
an auditory neuron is known as one of causes of hearing loss. The
type of hearing loss can be assessed by evaluating both the
staining properties of the somatic portion and the staining
properties of the axon portion of the hair cell based on an imaging
method for a hair cell of the present invention.
[0185] For the purpose of improving otorrhea and hearing loss due
to chronic otitis media, a treatment called tympanoplasty may be
performed. Further, for the purpose of extirpating a tumor and
treating an infection, a treatment called labyrinthoplasty may be
performed. Those operation techniques involve incising the skin at
the postauricular root, trimming the bone to remove an inflammation
in the bone, and restoring the tympanic membrane and the bone. As
risks of those operation techniques, there are exemplified inner
ear symptoms such as vertigo, tinnitus, and hearing loss. However,
in the operation, if a retention degree of an auditory function is
monitored by using the diagnostic composition for an auditory
function of the present invention, an image diagnosis can be
performed in an operation, leading to a more safe operation.
[0186] In particular, in the case of infants and senior adults with
dementia who can hardly communicate with each other, animals, and
the like, the diagnostic composition for an auditory function is
expected to be used as a test reagent for an auditory function
based on ex vivo staining properties using a hair cell extirpated
and cultured as one means for auditory test.
[0187] The diagnostic composition for an auditory function of the
present invention contains, as an active agent, at least one of the
probe for a hair cell of the present invention. The composition is
not particularly limited. The use of a substance for diagnosis
labelled with the probe for a hair cell of the present invention is
possible, and the use and application as a medicament for diagnosis
containing the substance for diagnosis are also possible. Further,
the use of the medicament for diagnosis containing the probe for a
hair cell labelled with a radionuclide allows the hair cell to be
easily imaged with one of PET, SPECT, and MRI, for example.
[0188] A screening method for one of a therapeutic drug and a
preventive drug for hearing loss of the present invention involves
the following steps: administering a test substance to a hearing
loss model animal; administering a diagnostic composition for an
auditory function to the model animal; and examining a staining
condition of the diagnostic composition for an auditory function
for the hair cell of the model animal. Examples of the hearing loss
model animal include an animal auditory impaired with a medicament
having auditory toxicity, an animal whose auditory related genes
have been knockdown by administration of an antisense nucleic acid,
and an animal whose auditory related genes have been knockout with
a genetic engineering procedure. As hearing loss genes involved in
inherited hearing loss, for example, a gene related to ion
transport such as connexin, a gene related to the tectorial
membrane construction and the extracellular matrix such as tectorin
and a type-XI collagen .alpha.2 domain, a gene related to a hair
cell stability such as type-VII myosin and type-XV myosin, a gene
related to cell differentiation and migration such as factor IV and
factor III of POU domain class III, a mitochondrial gene such as
A1555G mutation of 12S rRNA, and a COCH gene have been reported for
a human, and an animal including a mutation introduced into the
genes homologous to the above-mentioned genes is exemplified. In
particular, Zebrafish has been already mapped for its genome, and
further, has the above-mentioned superiority. Therefore,
gene-modified Zebrafish may be suitably used as a hearing loss
model animal.
EXAMPLES
[0189] Hereinafter, the present invention is described in more
detail by way of examples. Those examples are described for the
purpose of deeper appreciation of the present invention, and the
present invention is not in any way limited to those specific
examples. It should be noted that, unless otherwise indicated, "%"
means "mass %".
Example 1
Staining with Probe for Hair Cell
[0190] A staining compound (1) was dissolved in DMSO to prepare a
stock solution at a concentration of 1 mg/mL. The stock solution
was diluted with distilled water to prepare a hair cell staining
solution 1 having a dye concentration of 1 .mu.g/mL. Further, Egg
Water was prepared by dissolving artificial seawater SEALIFE
(manufactured by Marinetech Co., Ltd.) in distilled water at a
concentration of 60 mg/L. Five juveniles (embryos on Day 7 after
fertilization) of Zebrafish were placed into any one of wells of a
24-well multiwell plate together with feeding water. The feeding
water was discharged and 1 mL of the hair cell staining solution 1
was added. After the whole had been left to stand for 1 hour, the
hair cell staining solution 1 in the well was discharged and
replaced by 1 mL of fresh Egg Water. In addition, such an operation
that Egg Water was discharged and replaced by 1 mL of fresh Egg
Water was repeated twice. One of the juveniles was taken out from
the well onto a dish, the motion of the juvenile was restricted by
addition of methylcellulose, and a fluorescence image was captured
with a fluorescence stereomicroscope. MZ16FA manufactured by Leica
Microsystems K.K. was used as a stereoscopic microscope.
Examples 2 to 33
[0191] The same operation as that in Example 1 was performed except
that the staining compound (1) used in Example 1 was changed to
each of the staining compounds described in Table 1. An image
pickup unit (a digital camera DP72 for a microscope manufactured by
Olympus Corporation) of a fluorescence stereomicroscope was
remodeled by removing an IR cut filter so as to capture a
fluorescence image in a near-infrared wavelength region. In the
case of using a staining compound having excitation and
fluorescence emission wavelengths in a near-infrared area, the same
operation as that in Example 1 was performed with a fluorescence
stereomicroscope equipped with the image pickup unit.
Comparative Example 1
[0192] The same operation as that in Example 1 was performed except
that the staining compound (1) used in Example 1 was changed to
2-(4-(dimethylamino)styryl)-N-ethylpyridinium iodide (DASPEI) and
the dye concentration was adjusted to 250 .mu.g/mL.
Comparative Example 2
[0193] The same operation as that in Example 1 was performed except
that the staining compound (1) used in Example 1 was changed to
FM1-43.
Comparative Example 3
[0194] The same operation as that in Example 1 was performed except
that the staining compound (1) used in Example 1 was changed to
indocyanine green (ICG) and the dye concentration was adjusted to
250 .mu.g/mL.
[0195] <Evaluation for Fluorescence Intensity>
[0196] Based on each of fluorescence observation images in Examples
1 to 33 and Comparative Examples 1 to 3, the fluorescence intensity
of a hair cell was visually evaluated (+++: strongly observed, ++:
moderately observed, +: weakly observed, and no stained). It should
be noted that the excitation wavelength and the fluorescence
emission wavelength of the staining compound were determined by
measuring an aqueous solution, which had been obtained by diluting
500-fold a 10 mg/mL solution in DMSO with purified water, with a
fluorescence spectrophotometer FL 4500 manufactured by Hitachi
High-Technologies Corporation.
[0197] <Evaluation for Staining Pattern>
[0198] By using a fluorescence observation image of each of
Examples 1 to 33 and Comparative Examples 1 to 3, the staining
properties of the hair cell was visually evaluated (B: a neuromast
of big size is mainly stained, S: a neuromast of small size is
mainly stained, and BS: both a neuromast of big size and a
neuromast of small size are stained).
TABLE-US-00001 TABLE 1 Exci- tation Fluorescence wave- emission
Fluores- Staining length wavelength cence Staining compound
.lamda.ex .lamda.em intensity pattern Example 01 1 570 657 +++ S
Example 02 4 401 516 +++ B Example 03 6 545 636 +++ B Example 04 8
560 677 +++ B Example 05 9 520 555 +++ S Example 06 15 566 611 +++
B Example 07 16 459 564 +++ S Example 08 20 650 770 +++ B Example
09 22 496 569 +++ B Example 10 25 470 558 +++ S Example 11 28 429
503 +++ BS Example 12 30 561 616 +++ BS Example 13 32 545 576 +++
BS Example 14 33 478 564 +++ BS Example 15 36 516 602 +++ BS
Example 16 39 563 569 +++ BS Example 17 40 344 381 +++ S Example 18
43 473 564 +++ B Example 19 45 560 628 +++ B Example 20 47 354 469
+++ BS Example 21 50 571 620 +++ B Example 22 51 611 720 +++ S
Example 23 53 614 669 +++ BS Example 24 54 528 550 +++ B Example 25
57 570 589 +++ B Example 26 58 480 509 +++ B Example 27 60 509 578
+++ B Example 28 63 490 560 +++ BS Example 29 66 770 800 +++ S
Example 30 67 750 780 +++ BS Example 31 68 730 760 +++ S Example 32
69 670 700 +++ BS Example 33 70 660 690 +++ BS Comparative DASPEI
461 589 + B Example 1 Comparative FM1-43 503 627 +++ B Example 2
Comparative ICG 784 811 No stained No stained Example 3
[0199] As clear from Table 1, the probe for a hair cell of the
present invention is rich in the diversity in the excitation
wavelength/fluorescence emission wavelength, and clearly stains a
neuromast present in lateral line organs with various patterns and
with high fluorescence intensity. Specifically, in a comparison of
FIGS. 1 to 3, i.e., an image stained with the probe for a hair cell
containing the staining compound (25) in Example 10 (FIG. 1), an
image stained with the probe for a hair cell containing the
staining compound (50) in Example 21 (FIG. 2), and an image stained
with the probe for a hair cell containing the staining compound
(53) in Example 23 (FIG. 3), a neuromast containing a hair cell of
Zebrafish is stained in all cases, but a large difference is
confirmed in patterns of the neuromast to be stained. That is, when
the staining compound (25) is used, a portion with a smaller
neuromast is selectively stained. Further, when the staining
compound (50) is used, a portion with a bigger neuromast is
selectively stained. Meanwhile, when the staining compound (53) is
used, both a portion with a bigger neuromast and a portion with a
smaller neuromast are simultaneously stained.
Example 34
[0200] It has been pointed out that an aminoglycoside antibiotic
such as gentamicin and an anticancer agent such as cisplatin each
have auditory toxicity to a human. Such a chemical substance
suspected of having auditory toxicity is exposed to a human model
organism, and a change in staining properties of a hair cell is
detected by using the probe for a hair cell of the present
invention, which enables the auditory toxicity of the chemical
substance to be evaluated.
[0201] In this example, Zebrafish was selected as the human model
organism, and the auditory toxicity of gentamicin was evaluated by
using the probe for a hair cell of the present invention as
follows. A gentamicin solution having a concentration of 5 .mu.M
was exposed to Zebrafish on Day 7 after fertilization for 24 hours.
Zebrafish after exposure was washed with distilled water. The same
operation as that in Example 1 was performed except that the
staining compound (1) used in Example 1 was changed to a staining
compound (54).
Example 35
[0202] The same operation as that in Example 34 was performed
except that the gentamicin solution used in Example 34 was changed
to a 5-.mu.M cisplatin solution.
Example 36
[0203] The same operation as that in Example 34 was performed
except that the gentamicin solution used in Example 34 was changed
to a 5-.mu.M taurine solution.
[0204] The results of Examples 34 to 36 are shown in Table 2.
TABLE-US-00002 TABLE 2 Chemical substance Staining Change in
Auditory to be evaluated compound staining intensity toxicity
Example 34 Gentamicin 54 Weakened Present Example 35 Cisplatin 54
Weakened Present Example 36 Taurine 54 No change Absent
[0205] As a result, changes in the staining intensity and staining
pattern confirmed that auditory neuron injury actions of gentamicin
and cisplatin can be evaluated, and further, the auditory toxicity
of gentamicin and cisplatin is extrapolated from Zebrafish to a
human. As clear also from the fact, the probe for a hair cell of
the present invention may be used to evaluate the auditory toxicity
of a chemical substance.
Example 37
[0206] The use of changes in cell staining conditions (staining
intensity and fluorescence properties) caused by the probe for a
hair cell of the present invention as an index allows the detection
and the evaluation of changes in cell conditions (for example,
transcriptome, proteome, and metabolome) and functions (for
example, viability and membrane potential).
[0207] For example, the administration of a specific chemical
substance, the depletion of a specific nutrient component, and the
administration of an antisense nucleic acid may change the cell
conditions and functions. The evaluation of such a change in the
staining condition due to artificial intervention to cell
conditions and functions enables an effect caused by artificial
intervention to be verified.
[0208] Meanwhile, an artificial intervention manipulation to cell
conditions and functions may also be regarded as the induction of a
cell toward a certain kind of disease condition. Therefore, the
screening of a substance that restores disease conditions to normal
by using a change in cell staining condition with the probe for a
hair cell of the present invention as an index allows the selection
of a drug candidate substance.
[0209] In order to confirm the fact, an experiment for examining
the artificial intervention to cell conditions and functions
involving the knockdown of a specific gene, and the change in
staining properties due to the artificial intervention was
performed as follows.
[0210] In order to knockdown a specific gene, a mixed solution
containing a morpholino antisense oligonucleotide (MO) was
prepared. MO was synthesized by using GeneTools, LLC (Philomath,
Oreg.). A morpholino antisense oligonucleotide (atgMO) designed so
that the translation from a start codon to a protein would be
inhibited was dissolved in distilled water to prepare a MO mixed
solution having the following composition.
TABLE-US-00003 atgMO 10 .mu.g/.mu.L Phenol red 0.005% EGFP 50
ng/.mu.L
[0211] A fertilized embryo within 1 hour after fertilization
(before a second cleavage period) of Zebrafish was microinjected
with the MO mixed solution. A capillary made of glass (inner
diameter: 0.6 mm) having a sharp extremity obtained by extending
and cutting a glass tube with a PC-10 puller (manufactured by
Narishige Co., Ltd.) and grinding the cross section with a EG-400
grinder (manufactured by Narishige Co., Ltd.) was used for the
microinjection. Feeding water and the fertilized embryo were placed
into a 10-cm dish in which 1% agarose had been bedded, and the
microinjection was performed under a stereoscopic microscope.
During injection, one of a manual injector IM-9A (manufactured by
Narishige Co., Ltd.) and an electrical microinjector IM-30
(manufactured by Narishige Co., Ltd.) was connected to the
capillary made of glass, to thereby perform infusion with pressure.
The infusion amount of MO was set to 1 to 10 ng per fertilized
embryo.
[0212] Because the MO mixed solution is supplemented with a dye
(0.005% phenol red), the success or failure of the infusion of the
MO mixed solution may be confirmed with the naked eye. Further,
because the MO mixed solution is supplemented with a fluorescent
protein expression vector (EGFP), on Day 3 after infusion, the
success or failure of the microinjection can be confirmed after the
fertilized embryo has hatched (a sample in which a signal derived
from the fluorescent protein was observed also retains MO in
Zebrafish).
[0213] Juveniles (embryos on Day 7 after fertilization) of
Zebrafish were stained in the same manner as that in Example 1, and
a change in staining condition of the hair cell was measured.
[0214] The compound (54) was selected as the probe for a hair cell
and compared with FM1-43. Further, SLC25A12 and TRPC2 were selected
as target genes. The staining intensity of a juvenile neuromast
having unknockdown genes was used as a control, and compared with
the staining intensity of a juvenile neuromast in which each of the
target genes has been knockdown by representing a fluorescence
image obtained with a stereoscopic microscope in numerical
values.
[0215] Table 3 shows the change in staining intensity of the
neuromast due to the knockdown (KD) of each of the target
genes.
TABLE-US-00004 TABLE 3 Ratio of Number of Number of staining Target
Com- sample sample intensity gene pound (N_KD) (N_control)
(KD/control) p value SLC25A12 (54) 27 26 2.06 0.009 SLC25A12 FM1-43
16 18 1.36 0.027 TRPC2 (54) 20 21 2.18 0.0003 TRPC2 FM1-43 11 12
1.35 0.024
[0216] The staining properties with the compound (54) were
significantly increased about 2-fold by the knockdown of the
SLC25A12 and TRPC2 genes, respectively. Meanwhile, the staining
properties with FM1-43 were also significantly increased by the
knockdown each of the SLC25A12 and TRPC2 genes, but the ratio was
about 30%, which was smaller than the compound (54).
[0217] The fact indicates that the probe for a hair cell of the
present invention may detect more perceptively a change in staining
properties due to artificial intervention to cell functions.
[0218] Further, because a chemical structure of compound (54) is
different from a chemical structure of FM1-43, the compound (54)
and FM1-43 may be different in the staining mechanism of the hair
cell.
Example 38
[0219] The probe for a hair cell of the present invention includes
an agent whose staining intensity is higher compared with that with
a conventionally known staining agent such as FM1-43. In order to
confirm the fact, an experiment for examining a change in staining
properties by changing the concentration of the staining agent was
performed as follows.
[0220] The compounds (43), (54), and (65) were selected as probes
for a hair cell, and compared with FM1-43. Three kinds of staining
solutions containing those staining agents were prepared at
concentrations of 10 ng/mL, 30 ng/mL, and 100 ng/mL. Juveniles of
(embryos on Day 7 after fertilization) of Zebrafish were stained in
the same manner as that in Example 1. When a fluorescence image is
acquired with a fluorescence stereomicroscope, the fluorescence
intensity per unit intake time (intensity/msec) was calculated by
changing an intake time (exposure time) and dividing the
fluorescence intensity (Relative Fluorescence Unit, RFU) of a
neuromast of the obtained fluorescence image by the intake
time.
[0221] FIG. 4 shows the results. The results confirmed that the
staining intensity of the hair cell with the compounds (43), (54),
and (65) were higher compared with the staining intensity with
FM1-43.
[0222] The probe for a hair cell having high staining intensity of
the present invention may be used at lower concentrations. Also, a
clear staining image with small background noise may be obtained.
In addition, the wide dynamic range of a staining signal allows the
more detailed analysis of the hair cell for its physiological
conditions.
INDUSTRIAL APPLICABILITY
[0223] Sensorineural hearing loss resulting from the induction of
cell death of an inner ear hair cell is hearing loss which widely
develops in the range of newborns to senior adults owing to
congenital and acquired causes, and there are many unclear points
about detailed pathogenesis. Therefore, there is a strong demand
for the establishment of an effective treatment method.
[0224] The probe for a hair cell of the present invention has a
variety of chemical structures, is rich in the diversity in the
excitation wavelength/fluorescence emission wavelength, and can
clearly stain a neuromast present in lateral line organs of
Zebrafish with various patterns and with high fluorescence
intensity. The difference in the staining pattern probably reflects
that each of the probes for a hair cell stains the hair cell by a
different mechanism, which allows the identification of various
conditions of the hair cell.
[0225] In the probe for a hair cell of the present invention, the
staining condition may greatly change depending on cell conditions
and functions, and hence, an auditory function can be evaluated
from various angles.
[0226] Based on the evaluation for an auditory function using the
probe for a hair cell of the present invention, an effective
treatment method, therapeutic drug, and preventive drug for
sensorineural hearing loss may be developed.
[0227] Further, the screening of one of a therapeutic drug and a
preventive drug for hearing loss is accelerated by use of
Zebrafish, which leads to a cost reduction. Still further, one of a
therapeutic drug and a preventive drug for hearing loss can be
easily evaluated.
[0228] Further, the probe for a hair cell may be used as an index
during screening for evaluating the auditory toxicity of a chemical
substance.
[0229] In addition, the analysis of a change in staining condition
due to artificial intervention to cell conditions and functions
allows the verification of an effect of the artificial
intervention. Thus, the probe for a hair cell may become an
effective tool for a bioscience research.
[0230] While the present invention has been described with
reference to exemplary embodiments, it is to be understood that the
invention is not particularly limited to the disclosed exemplary
embodiments. The scope of the following claims is to be accorded
the broadest interpretation so as to encompass all such
modifications and equivalent structures and functions.
[0231] This application claims the benefit of Japanese Patent
Application No. 2008-330979, filed on Dec. 25, 2008, which is
hereby incorporated by reference herein in its entirety.
* * * * *