Fluorescent labeling reagents

Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Having -c- – wherein x is chalcogen – bonded directly to...

Reexamination Certificate

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C548S181000, C424S009600

Reexamination Certificate

active

06403625

ABSTRACT:

TECHNICAL FIELD
The present invention relates to 3-indocyanine green-acyl-1,3-thiazolidine-2-thiones useful for fluorescent labeling agents and the like.
BACKGROUND ART
Polymethyne dyes absorb light in the near infrared region (600 to 1200 nm) and emit fluorescence (Patonay, G. et al., Anal. Chem., 1991, 63, 321A). They are characterized by high molecular absorbance and very high fluorescent emission. One of these dyes, indocyanine green (ICG) (U.S. Pat. No. 2,895,955; 1959), has an excellent feature of extremely low interference by absorption scattering in the near infrared region and fluorescence of a molecule in a living body, and has been clinically used for diagnosis of liver functions. Accordingly, derivatives of the aforementioned fluorescent compound are expected to be useful as fluorescent labeling agents.
It has been known that proteins such as bovine serum albumin (BSA) and immunoglobulin G (IgG) can be labeled with fluorescence by using the aforementioned fluorescent compound per se or its derivative (Southwick P. L. et al., Cytometry, 1990, 11, 418; Ernst, L. A. et al., Cytometry, 1989, 10, 3; Mujumdar, R. B. et al., Cytometry, 1989, 10, 11; Mujumdar, R. B. et al., Bioconjugate Chem., 1933, 4, 105; Ito, S. et al., Bioorg. Med. Chem. Let., 1995, 5, 2689). In these reports, it is explained that a protein labeled with fluorescence can be used for ultramicroanalysis by fluorescence measurement (Mujumdar, R. B. et al., Bioconjugate Chem., 1933, 4, 105), and that the protein can be a diagnostic reagent for very small stomach cancers (Ito, S. et al., Bioorg. Med. Chem. Let., 1995, 5, 2689).
Nagao et al. elucidated some features in aminolysis of various 3-acyl-1,3-thiazolidine-2-thione (ATT) derivatives by utilizing active amide structures, and reported that the ATT moiety selectively reacts with an amino group (Nagao, Y. et al., J. Chem. Soc., Perkin Trans., 1, 1984, 2439; Nagao, Y. et al., Heterocycles, 1982, 17, 537).
DISCLOSURE OF THE INVENTION
An object of the present invention is to provide novel compounds useful as fluorescent labeling agents. In particular, the object of the present invention is to provide compounds which have high fluorescence intensity in the near infrared region and can function to group-selectively and efficiently label proteins and oligopeptide compounds. The inventors of the present invention made intensive studies to achieve the aforementioned objects. As a result, they found that indocyanine green amide derivatives of 1,3-thiazolidine-2-thione have the aforementioned features and are extremely useful as fluorescent labeling agents. The present invention was achieved on the basis of the above findings.
The present invention thus provides compounds represented by the following formula (I):
wherein R represents a lower alkyl group which may be substituted; n represents an integer of from 1 to 10; and X represents an anion species. According to preferred embodiments of this invention, there are provided the aforementioned compounds wherein R is a C
1-6
alkyl group which may have one sulfonic acid group, and n is an integer of from 4 to 6; the aforementioned compounds wherein R is a C
1-6
alkyl group, and n is 5; and the aforementioned compound wherein R is ethyl group, and n is 5. As a preferred embodiment of this invention, there is provided 2-[7-(1,3-dihydro-1,1-dimethyl-3-ethylbenz[e]indolin-2-ylidene)-1,3,5-heptatrienyl]-1,1-dimethyl-3-[[6-[(1,3-thiazolidine-2-thion)-1-yl]-6-oxo]hexyl]-1H-benz[e]indolium chloride.
According to another aspect of the present invention, there are provided a fluorescent labeling agent which comprises the compound represented by the aforementioned formula (I); a medicament for diagnosis which comprises the compound represented by the aforementioned formula (I), and is preferably used for fluorescence imaging method; and a medicament which comprises the compound represented by the aforementioned formula (I) and is preferably used for cancer treatment by using a laser, more preferably for endoscopic cancer treatment by using a near infrared laser.
According to a further aspect of the present invention, there are provided a use of the compound represented by the aforementioned formula (I) for the manufacture of the aforementioned medicament; a method of immunochemical staining of a living tissue by using the compound represented by the aforementioned formula (I); a method for diagnosis by fluorescence imaging method with the compound represented by the aforementioned formula (I), preferably a method for diagnosis of a cancer; and a method for endoscopic treatment of a cancer with a laser, preferably a near infrared laser, by using the compound represented by the aforementioned formula (I).
BEST MODE FOR CARRYING OUT THE INVENTION
In the aforementioned formula (I), R represents a lower alkyl group which may be substituted; n represents an integer of from 1 to 10; and X represents an anion species. As the lower alkyl group, a straight chain or branched chain alkyl group having carbon atoms of from 1 to about 6 can be used. More specifically, methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, sec-butyl group, tert-butyl group or the like can be used. The symbol “n” is preferably an integer of from 4 to 6, more preferably 5. The kind of X is not particularly limited. For example, as an monovalent anion species, a halogen ion such as chlorine ion, bromine ion, or iodine ion can be used.
When the lower alkyl group represented by R has substituents, the number, the kind, and the position of the substituents are not particularly limited, and examples of the substituents include sulfonic acid group, hydroxyl group, carboxyl group, halogen atoms (may be any of fluorine atom, chlorine atom, bromine atom, or iodine atom), lower alkoxyl groups (e.g., methoxy group, ethoxy group and the like), amino group, mono- or di(lower alkyl)amino groups, keto group, lower alkanoyl groups and the like. The lower alkyl moiety in the aforementioned substituents may have one or more kinds of these substituents. R is preferably an unsubstituted lower alkyl group, or an alkyl group substituted with one sulfonic acid group, and more preferably, ethyl group can be used.
The compounds of the present invention may sometimes have one or more asymmetric carbon atoms depending on the kind of substituents, and isomers such as optical isomers and diastereoisomers based on one or more asymmetric carbon atoms may sometimes exist. Any of such isomers, any mixtures of the isomers, racemates and the like falls within the scope of the present invention. In addition, when the compound of the present invention has an alkyl group having an acidic substituent as group “R”, for example, sulfonic acid group and the like, the compound may sometimes form an intermolecular twitter ion without need of an anion species represented by X

. It should be understood that such compounds also fall within the scope of the present invention. A compound which forms a twitter ion in the molecule may sometimes have reduced ionicity and fails to have sufficient water solubility. Accordingly, when an alkyl group having a sulfonic acid group or the like is used as R, the sulfonic acid group is desirably in the form of a metal salt such as sodium sulfonate. Examples of the combination of a metal ion which forms the metal salt and X

preferably include, for example, NaI, KI, NaCl and the like.
A preferred compound among those of the present invention includes, for example, 2-[7-(1,3-dihydro-1,1-dimethyl-3-ethylbenz[e]indolin-2-ylidene)-1,3,5-heptatrienyl]-1,1-dimethyl-3-[[6-[(1,3-thiazolidine-2-thione)-1-yl]-6-oxo]hexyl]-1H-benz[e]indolium chloride (herein sometimes referred to as “ICG-ATT”). However, the compound of the present invention is not limited to the aforementioned specific compound.
Methods for preparation of the compounds of the present invention are not particularly limited, and those prepared by any methods of p

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