JP2002173478A - Naphthol compound - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a compound useful for dyeing a thin membrane, etc., containing a polypeptide such as gelatin, etc. SOLUTION: This naphthol compound is expressed by the following general formula (I) [wherein, X is -CO- or -SO2; L is H or a halogen atom; Y is -NHCOR2 or -NHSO2R2 (R2 is a substituted or non-substituted 1-6C alkyl, a substituted or non-substituted 1-6C alkoxy, or a substituted or non-substituted 6-12C aryl); and R1 is a substituted or non-substituted alkyl, a substituted or non-substituted alkylamino, a substituted or non-substituted anilino, or a substituted or non-substituted heterocyclic group] or its salt.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel naphthol compound or a salt thereof useful for staining a thin film containing a polypeptide such as gelatin.

[0002]

BACKGROUND ART In the invasion and metastasis of cancer cells, progression of periodontal disease such as periodontitis, progression of tissue destruction in rheumatoid arthritis, wound healing process, ontogenesis process, etc., matrix metalloproteinase, plasminogen It is known that various proteases such as an activator are involved, and as a method for detecting and quantifying those proteases, an immunoassay using an antibody, an immunoblotting method, an electrophoretic zymography method, and the like are known. . Also, as a method for measuring protease activity in tissues, see The FASEB Journal, Vol. 9, Juice.
The so-called in situ zymography method described in ly, pp. 974-980, 1995 or PCT / JP97 / 00588 is known.

In the method for measuring protease activity disclosed in WO 97/32035, dyes such as amide black, Coomassie blue or Ponceau can be used for staining thin films such as gelatin. Is known to have an undesirable effect on the human body in terms of mutagenicity and the like, and the development of a more safe staining method has been desired.

[0004]

SUMMARY OF THE INVENTION An object of the present invention is to provide a compound for dyeing a thin film containing a polypeptide such as gelatin. More specifically, when dyeing a thin film such as gelatin, it gives the same or higher digestibility discrimination as compared with a dyeing method using a dye such as amide black, Coomassie blue, or Ponceau, and performs safe staining. To provide compounds that can

[0005]

Means for Solving the Problems The inventors of the present invention have conducted intensive studies to solve the above-mentioned problems, and as a result, a compound represented by the following general formula (I) and a reducing compound (color developing agent) It has been found that by oxidatively coupling the compound and the compound, the thin film containing the polypeptide can be stained safely and efficiently, and the above-mentioned problem can be solved. The present invention has been completed based on the above findings.

That is, the present invention provides the following general formula (I):

Embedded image Wherein, X is -CO- or -SO ₂ - indicates; L represents a hydrogen atom or a halogen atom; Y is -NHCO-R ² or -NHSO ^₂ -R ₂ (R ₂ is a substituted or unsubstituted C _1-6
An alkyl group, a substituted or unsubstituted C _1-6 alkoxy group, or a substituted or unsubstituted C _6-12 aryl group); R ¹ represents a substituted or unsubstituted alkyl group;
A substituted or unsubstituted alkylamino group, a substituted or unsubstituted anilino group, or a substituted or unsubstituted heterocyclic group] or a salt thereof.

According to a preferred embodiment of the present invention, R ¹ is an alkylamino group having 2 to 6 carbon atoms, an anilino group having 6 to 8 carbon atoms, or a heterocyclic group having 4 to 6 carbon atoms, and Y is acetyl. Amino group, propionylamino group, methanesulfonylamino group, ethanesulfonylamino group, ethoxycarbonylamino group, propoxycarbonylamino group, butoxycarbonylamino group, benzenesulfonylamino group, 3-sulfobenzenesulfonylamino group, (3-methanesulfonyl Aminophenyl) sulfonylamino group,
Or the above compound which is a (3-carboxyphenyl) sulfonylamino group or a salt thereof;

X is --CO--, and R ¹ is a diethylamino group, a morpholino group, a piperidino group, a 3-carboxypiperidino group, a 4-carboxypiperidino group, a 3-sulfoanilino group, a 4-sulfoanilino group, or An oxazolidine-3-yl group, L is a hydrogen atom, Y is an acetylamino group, a trifluoroacetylamino group, a methanesulfonylamino group, a butoxycarbonylamino group, a benzenesulfonylamino group, a 3-sulfobenzenesulfonylamino group The above-mentioned compound or a salt thereof, which is a (3-methanesulfonylaminophenyl) sulfonylamino group or a (3-carboxyphenyl) sulfonylamino group;

X is --SO _2- , R ¹ is an ethylamino group, a propylamino group, a butylamino group, or a t-butylamino group; L is a hydrogen atom; Y is an acetylamino group; The above compound or a salt thereof, which is a fluoroacetylamino group, a benzenesulfonylamino group, a 3-sulfobenzenesulfonylamino group, a (3-methanesulfonylaminophenyl) sulfonylamino group, or a (3-carboxyphenyl) sulfonylamino group, is provided. Is done.

[0010] From another viewpoint, the above-mentioned compound or a salt thereof used for staining; the above-mentioned compound or a salt thereof used for staining a thin film containing a polypeptide; A salt thereof; a dyeing composition containing the above compound or a salt thereof; a dyeing composition containing a reducing compound which is a color developing agent; use of the above compound or a salt thereof for dyeing; A method of dyeing a thin film containing a polypeptide using the compound or a salt thereof; a method of oxidizing the reducing compound as a color developing agent with the compound or a salt thereof. A method as described above comprising the step of coupling; the above method performed in the presence of an oxidizing agent is provided by the present invention.

[0011]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The terms used in the present specification have the following meanings. The alkyl moiety in the alkyl group or a substituent containing an alkyl moiety (such as an alkoxy group) may be linear, branched, cyclic, or a combination thereof. For example, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, sec-butyl, isobutyl, cyclobutyl, cyclopropylmethyl, n-pentyl, neopentyl, n -Hexyl group, cyclohexyl group and the like. In the case of a halogen atom, any of a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom may be used. The alkylamino group may be either a monoalkylamino group or a dialkylamino group.

The aryl moiety of the aryl group or the other substituent having an aryl moiety may be a single ring or a condensed ring, and preferably an aryl group having 6 to 20 carbon atoms can be used. For example, a phenyl group, a 1-naphthyl group, a 2-naphthyl group and the like can be mentioned. As the heterocyclic group, an aromatic or non-aromatic heterocyclic group containing at least one heteroatom as a ring constituent atom can be used, and the heterocyclic group may be a single ring or a condensed ring. As the hetero atom, for example, one or more hetero atoms selected from an oxygen atom, a nitrogen atom, a sulfur atom, and the like can be used. When the heterocyclic group has two or more hetero atoms as ring constituent atoms, May be the same or different. As the aromatic heterocyclic group,
For example, a 5- to 8-membered aromatic heterocyclic group having 1 to 10 carbon atoms (e.g., 4-pyridyl, 2-furyl, 2-pyrimidyl, 1-pyridyl, 2-benzothiazolyl, 1-imidazolyl group, 1-pyrazolyl group) and the like, and as the non-aromatic heterocyclic group, 1-
Pyrrolidinyl group, 1-piperazinyl group, morpholino group,
A piperidino group or the like can be used.

In the present specification, the term "substituted or unsubstituted" with respect to a certain functional group is not particularly limited as to the type of the substituent, the number of the substituents, and the position of the substituents. In that case they may be the same or different. Examples of the substituent include a halogen atom, an alkyl group, an aryl group, a hydroxyl group, a carboxyl group, an amino group, an oxo group, and an alkoxy group (preferably having 1 carbon atom).
To 6 alkoxy groups, for example, a methoxy group,
Ethoxy group, propyloxy group, isopropyloxy group, 1-butoxy group, etc.), alkoxycarbonyl group and the like, but are not limited thereto.

R ¹ is an alkylamino group having 2 to 6 carbon atoms, a heterocyclic group having 4 to 6 carbon atoms, or a 6 to 8 carbon atoms.
Is preferred, and Y is an acetylamino group, a propionylamino group, a methanesulfonylamino group, an ethanesulfonylamino group, an ethoxycarbonylamino group, a propoxycarbonylamino group, a butoxycarbonylamino group, a benzenesulfonylamino group, or a 3-sulfonyl group. Benzenesulfonylamino group, (3-methanesulfonylaminophenyl) sulfonylamino group, or (3-
A (carboxyphenyl) sulfonylamino group is preferred.

When X represents --CO--, R ¹ represents a diethylamino group, a morpholino group, a piperidino group, a 3-carboxypiperidino group, a 4-carboxypiperidino group, a 3-sulfoanilino group, or a 4-sulfoanilino group. It is preferred that In this case, L is preferably a hydrogen atom, and Y is an acetylamino group, trifluoroacetylamino group, methanesulfonylamino group, butoxycarbonylamino group, benzenesulfonylamino group, 3-sulfobenzenesulfonylamino group, (3 -A methanesulfonylaminophenyl) sulfonylamino group or a (3-carboxyphenyl) sulfonylamino group is preferable.

When X represents —SO ₂ —, R ¹ represents an ethylamino group, a propylamino group, a butylamino group, or a t-
It is preferably a butylamino group. In this case, L is preferably a hydrogen atom, Y is an acetylamino group, a trifluoroacetylamino group, a benzenesulfonylamino group, a 3-sulfobenzenesulfonylamino group,
It is preferably a (3-methanesulfonylaminophenyl) sulfonylamino group or a (3-carboxyphenyl) sulfonylamino group.

Preferred compounds include the following compounds, but the scope of the present invention is not limited to these compounds.

[0018]

Embedded image

[0019]

Embedded image

The compounds of the present invention may exist as salts. As the salt, a metal salt such as a sodium salt, a potassium salt, or a calcium salt, an organic amine salt such as a triethylamine salt, or a base addition salt such as an ammonium salt, a hydrochloride, a sulfate, or a mineral acid salt such as a nitrate, or Oxalates, p-toluenesulfonate, or organic acid salts such as citrate, and the like,
It is not limited to these. It may form salts with amino acids such as glycine. Further, the compound of the present invention or a salt thereof may exist as a hydrate or a solvate in some cases. All of these substances are included in the scope of the present invention.

Further, the compound of the present invention may have one or more asymmetric carbons depending on the type of the substituent, and may exist as an optically active substance or a stereoisomer such as a diastereomer. There is. Pure stereoisomers as well as any mixtures, racemates and the like of the stereoisomers are included in the scope of the present invention.

The compound represented by the above formula (I) can be synthesized by a combination of general synthesis methods. For example,
The compound can be synthesized with reference to the synthesis methods described in JP-A-10-55055 and JP-A-10-55047 or the literature cited therein. In the examples of the present specification, production methods for typical compounds of the present invention are specifically shown. Therefore, those skilled in the art can appropriately select reaction raw materials, reagents, reaction conditions, and the like while referring to the descriptions of the above-mentioned publications and the examples of the present specification, and can appropriately apply these methods as necessary. By adding the modification or modification of
It is possible to produce any of the compounds encompassed by the above formula (I).

The use of the compound represented by the general formula (I) or a salt thereof of the present invention is not particularly limited. For example, it can be used for staining a thin film formed of a polypeptide such as gelatin. A dye can be formed by oxidatively coupling the compound represented by the general formula (I) of the present invention or a salt thereof with a compound having a reducing action, which is a color developing agent. Can be easily and safely stained. This reaction can be performed in the presence of an oxidizing agent, if necessary.

The compound represented by the general formula (I) or a salt thereof can form a dye by coupling with an oxidized form of a color developing agent. For this purpose, the compound represented by the general formula (I) It is preferable that the represented compound or a salt thereof has appropriate water solubility. Further, it is preferable that the dye has a high dye-forming efficiency over a wide range from weakly acidic to strongly basic, and that the polypeptide thin film can be efficiently dyed. Those described as preferable compounds in the above description of the compounds have such characteristics remarkably.

The reducing compound used in dye formation using the compound represented by the general formula (I) or a salt thereof is as follows:
"Color developing agents" in the field of silver halide photographic materials
(Hereinafter, referred to as “color developing agent” in the present specification). For example, a paraphenylenediamine derivative, a sulfonylhydrazine derivative, a semicarbazide derivative, an acylhydrazide derivative, a parasulfonamidophenol derivative and the like can be suitably used. Among them, paraphenylenediamine derivatives, sulfonylhydrazine derivatives, and parasulfonamidophenol derivatives can be more preferably used.

Preferable examples of these color developing agents and synthesis methods are described in West German Patent No. 1,158,836, British Patent No. 2,018,453, and US Pat. No. 3,734,735. Specification, U.S. Pat.
9,906, JP-A-47-37539,
JP-A-52-42725, JP-A-52-5782
7, JP-A-54-31737, U.S. Pat. No. 4,126,461, JP-B-63-366.4
No. 87, JP-A-58-33,238, JP-A-59-53,831, JP-B-2-4,890, JP-B-3-60,418, JP-B-4- 20,
177, JP-B 5-48,901, JP-B 5-48,902, JP-B 4-69,776, JP-A-60-181,742, JP-A-60-181.
191 and 251; JP-A-62-227141; JP-A-62-288835;
15,052, JP-A-3-33,743,
JP-A-3-125145, JP-A-5-224
No. 381, JP-A-49-46,427, JP-B-2-15,855, JP-B-2-15,856, JP-A-59-111,148, U.S. Pat. No. 430,420, JP-B-46-7,782
JP, JP-B-45-9,031, Belgian Patent No. 602,250, U.S. Pat. No. 3,357,2
No. 03, JP-B-48-39,165, JP-B-3-74,817, JP-A-62-131,25.
No. 3, JP-B 5-33,781, JP-B 5-33,781
33,782, JP-A-63-8,645,
JP-A-63-123043, JP-A-63-12
3,044, JP-A-63-123,045, JP-A-63-123,046, JP-A-08-163
227,131, JP-A-06-194,793, JP-A-05-241,282, JP-A-09.
-152,703, JP-A-08-122,994
And JP-A-08-110,608 and JP-A-09-146,248. The disclosures of these publications are incorporated herein by reference.

As the oxidizing agent for oxidizing the color developing agent, any oxidizing agent may be used.
Silver salts such as silver oxide and silver acetate, persulfates such as ammonium persulfate and potassium persulfate, and quinones such as benzoquinone and dichlorodicyanobenzoquinone can be preferably used. Among these, an oxidizing agent having high water solubility can be used particularly effectively. When an oxidized form of a sulfonylhydrazine derivative or a parasulfonamidophenol derivative is used as the color developing agent, it is not necessary to add an oxidizing agent.

As a solvent for dyeing, a solvent which dissolves the compound of the present invention or a salt thereof and a color developing agent is preferable.
In order to stain a thin film containing a polypeptide, a solvent containing water or methanol can be particularly preferably used. A solvent having a water content of 50 to 100% can be particularly preferably used. As a solvent used by mixing with water and methanol, preferably propanol, butanol, t-butanol, isopropyl alcohol, dimethylformamide, dimethylsulfoxide, dimethylacetamide, sulfolane, acetonitrile, N-methylpyrrolidone, acetic acid, ethyl acetate, methyl acetate , Pyridine,
Examples thereof include triethylamine, tetrahydrofuran, and acetone. These may be used in combination of two or more.

In the dyeing step, the method of adding the compound of the present invention represented by the above formula (I) or a salt thereof and a color developing agent is not particularly limited. For example, after dissolving both in the above organic solvent, A method of mixing with water or a method of directly dissolving both in water is preferably employed. The order of addition of the coupler, the color developing agent, and, if necessary, the oxidizing agent is not particularly limited, and may be added in any order, but a mixed solution containing the compound of the present invention or a salt thereof and the color developing agent Most preferred is a method of adding an oxidizing agent.

In dyeing a thin film containing a polypeptide, the compound represented by the above formula (I) or a salt thereof may be added to the thin film, but a form in which the compound is supplied as an aqueous solution to the thin film is preferably used. The color developing agent may be present in the thin film or in the solution, but a method in which the color developing agent is present in the solution is preferred. The oxidizing agent is preferably supplied to the thin film as a solution.

When measuring protease in a biological sample using a thin film containing a polypeptide such as gelatin,
By staining the thin film on which the biological sample has been acted with the compound of the present invention represented by the above formula (I) or a salt thereof,
Protease activity contained in a biological sample can be easily and safely measured. The membrane may be cross-linked,
A hardener may be included. An example of a preferred thin film for use in measuring protease activity is a cross-linked and / or substantially water-insoluble thin film formed on a support and containing at least one dye and a protease substrate. Can be.

The term measurement method as used herein should be interpreted in the broadest sense, including qualitative and quantitative. The principle of the above protease measurement method is
A method for measuring protease by digesting a protease substrate with a protease contained in a sample and forming a digestion mark in a thin film (so-called in situ zymogr).
aphy method: disclosed, for example, in PCT / JP97 / 0588), but for efficient measurements, crosslinked and / or substantially water-containing protease substrates are used. It is preferable to use an insoluble thin film. By washing the thin film with water, the digested part of the substrate is washed away,
Digestion marks are detected under a microscope as low optical density parts,
The presence of protease activity in the sample can be detected. In this method, the presence of protease activity can be detected with high detection sensitivity by staining the thin film with the compound represented by the formula (I) of the present invention or a salt thereof and a color developing agent. Further, by blending the thin film with a dye having a color tone different from the color at the time of staining, it is possible to achieve higher detection sensitivity.

As used herein, the term “crosslinked and / or substantially water-insoluble thin film” refers to a thin film formed on the surface of a support when substantially immersed in water at 30 ° C. for 30 minutes. This means that the protease substrate does not elute in water. This thin film can generally be produced by crosslinking a protease substrate using a hardener, but in some cases, a hardener is not necessarily required. For example, cross-linking by baking a collagen thin film is described in US Pat. No. 4,931,386, and it is also possible to cross-link a protein with an aminotransferase.

For the production of a thin film, generally, an organic or inorganic hardener can be used. Such a hardening agent may be appropriately selected from hardening agents available for accelerating hardening, such as gelatin, but it is necessary to select a hardening agent which does not affect the activity of the protease to be measured. is there. For example, active halogen compounds (such as 2,4-dichloro-6-hydroxy-1,3,5-triazine and its sodium salt) and active vinyl compounds (1,3-bisvinylsulfonyl-2-propanol, 1,2- Bis (vinylsulfonylacetamide) ethane, bis (vinylsulfonylmethyl) ether, and a vinyl-based polymer having a vinylsulfonyl group in its side chain can be used, and 1,2-bis (vinylsulfonylacetamido) ethane is used. Is preferred.

The proteases to be measured include:
For example, mention may be made of matrix metalloproteases and serine proteases, which are described in detail in Takaru Tsuruo, “Molecular Mechanisms of Cancer Metastasis”, pp. 92-107, published by Medical View, 1993. ing.
Particularly suitable proteases are, for example, matrix metalloproteases such as interstitial collagenase (MMP-1), gelatinase A (MMP-2), and gelatinase B (MMP-9); and plasminogen activator (PA )
And the like, but the measurement target is not limited to the above specific protease.

The polypeptide used as a protease substrate is not particularly limited as long as it is a polypeptide that is degraded as a protease substrate. For example, collagen, gelatin, proteoglycan, fibronectin, laminin, elastin, casein, or the like can be used. Preferably, collagen, gelatin, fibronectin, or casein can be used, and more preferably, gelatin or casein can be used. When gelatin is used, the kind of gelatin is not particularly limited.Examples include, for example, beef bone alkali-treated gelatin, pork skin alkali-treated gelatin, bovine bone-treated gelatin, bovine bone phthalated gelatin, and pig skin acid-treated gelatin. Can be used. As the protease substrate, one kind may be used, or two or more kinds may be used in combination.

By using two or more different protease substrates in combination, the type of protease contained in a biological sample may be accurately specified in some cases. For example, one of the substantially continuous sections in a biological sample may be contacted with a thin film containing a certain protease substrate to detect digestion marks, and the other section may be contacted with a thin film containing a different protease substrate. Digestion marks on the thin film can be detected and the results can be compared. For comparison, two or more thin films containing different protease substrates may be used. As the thin film containing the protease substrate, it is preferable to use a thin film having a thickness of 1 to 10 μm, preferably 4 to 7 μm after drying.

The above thin film may be formed on the surface of a support. The material and shape of the support are not particularly limited, but when the surface change on the thin film is observed under a microscope, or when the surface change is detected by spectroscopic means such as absorbance measurement or fluorescence measurement, for example, The thin film is preferably formed on a flat transparent or translucent support. As such a transparent or translucent polymer support, for example,
Polyethylene terephthalate, polyethylene naphthalate, atactic polystyrene, syndiotactic polystyrene, polycarbonate, triacetyl cellulose, polymethyl methacrylate, polysulfone,
A transparent or translucent plastic film made of polyarylate or the like can be used. Particularly preferred are polyethylene terephthalate, syndiotactic polystyrene and polyarylate, and it is most preferred to use polyethylene terephthalate. The support used may be colored.

The thickness of the support is not particularly limited.
It is preferably from μm to 300 μm, more preferably from 100 μm to 200 μm. Particularly preferably 17
Those having a size of about 5 μm can be used. The thin film on the support can be formed in a single layer or a multilayer, but the thin film should be prepared so as to give as uniform a surface as possible. For example, it is preferable to adjust the film thickness after drying to 1 to 10 μm, preferably about 4 to 7 μm.

For the preparation of a thin film, for example, a predetermined amount of a hardener and a dye solution or a dye dispersion are added to an aqueous solution of a protease substrate and mixed uniformly, and the resulting solution or dispersion is applied to the surface of a support. What is necessary is just to apply and dry. As a coating method, for example, a dip coating method, a roller coating method, a curtain coating method, an extrusion coating method, or the like can be adopted. However, the method of preparing the thin film is not limited to these, and for example, a thin film forming method widely used in the technical field of photographic films and the like can be appropriately adopted.

In forming the thin film on the support, an undercoat layer may be provided between the thin film and the surface of the support to improve the adhesion between the thin film and the support. For example, polymers or copolymers obtained by polymerizing one or more monomers selected from vinyl chloride, vinylidene chloride, butadiene, styrene, methacrylic acid, acrylic acid, itaconic acid, maleic anhydride, etc., polyethylene Polymers such as imines, epoxy resins, grafted gelatin, or nitrocellulose can be formed as a subbing layer. When a polyester-based support is used, instead of the undercoat layer, the surface of the support may be subjected to corona treatment, ultraviolet treatment, or glow treatment to improve the adhesive strength between the support and the thin film. . Corona treatment, UV treatment,
Alternatively, a method of applying the undercoat layer after performing the glow treatment can also improve the adhesive strength between the support and the thin film.

As used herein, the term "thin film formed on a support surface" or a synonym thereof excludes such treatment of one or more subbing layers and / or the support surface. Should not be interpreted as
However, the means for improving the adhesion between the thin film and the support is not limited to the above, and for example, means commonly used in the technical field of photographic films and the like can be appropriately employed. In the case where the thin film is formed by stacking a plurality of layers, an intermediate layer may be further provided between the two layers to be stacked, and the interpretation is limited to the case where the two layers are in direct contact. must not. Means for appropriately arranging such an intermediate layer is widely used, for example, in the technical field of photographic films. It is also preferable to provide a protective layer on the surface of the film formed on the support surface,
It is widely used in the technical field of photographic films.

In dyeing a thin film using the compound represented by the formula (I) or a salt thereof according to the present invention and a color developing agent, the dye formed on the thin film is not particularly limited as long as it has absorption in the visible region. No restrictions. One type of dye may be formed, or two or more types of dyes may be formed using a plurality of compounds of the formula (I) or salts thereof or a plurality of color developing agents. For example, when a multilayered thin film is used, a different color developing agent can be added to each layer to form a different color dye in each layer. It is preferable to form the dye in an amount of about 0.001 to 10 mmol / m ² , preferably about 0.01 to 1 mmol / m ² as the total amount of the dye with respect to the area of the thin film.

A dye may be previously blended into the thin film for the purpose of adjusting the color. Examples of the dye in this case include an azo dye, an azomethine dye, an indoaniline dye,
Benzoquinone dye, naphthoquinone dye, anthraquinone dye, diphenylmethane dye, triphenylmethane dye, xanthene dye, acridine dye, azine dye, oxazine dye, thiazine dye, oxonol dye, merocyanine dye, cyanine dye, arylidene dye, styryl dye, phthalocyanine dye, Perinone dyes, indigo dyes, thioindigo dyes, quinoline dyes, nitro dyes, nitroso dyes and the like can be mentioned. For specific compounds, refer to "New Edition Dye Handbook" (edited by The Society of Synthetic Organic Chemistry, Maruzen, 1970), "Color Index" (The S
Society of Dyers and Colorists), "Color Material Engineering Handbook" (edited by the Color Material Association; Asakura Shoten, 1989) and the like. A water-soluble dye can be used for the production of the thin film, but an oil-soluble dye is preferred because it does not adversely affect the enzymatic reaction.

Further, a pigment may be used as a coloring matter. The type of pigment is not particularly limited, and any organic or inorganic pigment may be used. As the pigment, commercially available pigments, known pigments described in various documents, and novel compounds can be used. Specifically, as the organic pigment, for example, azo pigments (azo lake pigments, insoluble monoazo pigments,
Insoluble disazo pigments, condensed azo pigments, metal complex azo pigments, chelate azo pigments), polycyclic pigments (phthalocyanine pigments, anthraquinone pigments, perylene and perinone pigments, indigo pigments, thioindigo pigments, quinacridone pigments, dioxazine pigments Pigments, isoindolinone pigments, quinophthalone pigments, diketopyrrolopyrrole pigments, dye lake pigments (lake pigments of acidic or basic dyes), azine pigments, and other pigments (nitroso pigments, alizarin lake pigments, Alkali blue), and inorganic pigments such as ultramarine blue and cobalt blue.

In the thin film, various additives may be added in addition to the protease substrate polypeptide and, if necessary, a dye and / or a hardener. Examples of the additives include a surfactant for facilitating coating of a thin film, an oil or an emulsifier for dispersing a dye, a preservative, a fungicide, an acid or base for adjusting pH, and an enzyme activity. in order to
Examples thereof include inorganic ions such as Ca ^++, but are not limited thereto. The thin film may be provided with antistatic means. For example, those having a surface electrical resistance of 10 ¹² Ω or less on the protease substrate layer side or on the opposite side can be preferably used. As a means for reducing the surface electric resistance of the film, for example, a technique used for a photographic film can be adopted.

For example, in the production of a thin film, the following additives can be used as needed. Hardener (RD17643: page 26; RD18716: page 651, left column; RD307105: 874
875 pages), binder (RD17643: page 26; RD18716: 651)
RD307105: pages 873 to 874, left column, plasticizer or lubricant (RD17643: page 27; RD18716: page 650, right column; RD307105: 876)
Page), coating aids or surfactants (RD17643: pages 26 to 27; R
D18716: 650 right column; RD307105: 875-876), antistatic agent (RD17643: 27 pages; RD18716: 650 right column; RD30710)
5: 876-877), matting agent (RD307105: 878-879)
page). All of these additives are widely used in the technical field of photographic films, and can be similarly used for producing thin films.

As the biological sample, a biological sample separated and collected from mammals including humans can be used.
For example, a biological sample separated and collected from an affected mammal, a mammal suspected of having a disease, or an experimental animal can be used. The form of the biological sample is not particularly limited, but a solid sample such as a tissue section or a non-solid sample such as a body fluid can be used. As the non-solid sample, for example, a sample containing cells or a piece of tissue collected by suction from a tissue, or a bodily fluid such as blood, lymph, or saliva can be used. For example, lung cancer, stomach cancer, esophageal cancer, colon cancer, breast cancer,
Cancer tissue, lymph node, periodontal disease tissue, rheumatism isolated and collected from cancer tissues such as uterine cancer, ovarian cancer, thyroid cancer, liver cancer, oral cancer, prostate cancer, kidney cancer, and bladder cancer by surgery or histological examination Tissue isolated / collected by surgery or histological examination from tissues such as synovium and bone tissue of osteoarthritis, gingival crevicular fluid, fluid contained in destructive lesion tissue (for example, joint fluid of rheumatic lesion or alveolar purulent tissue) Extract), pleural effusion, ascites,
Cerebrospinal fluid, abnormal mammary gland secretion, ovarian cyst fluid, kidney cyst fluid,
Sputum, blood or blood cells can be used.

When the sample is a tissue, for example, the thickness of the sample is rapidly frozen with liquid nitrogen using a cryosectioning apparatus.
By preparing a section of 1 to 10 μm, preferably 4 to 6 μm, and attaching the section to the thin film, the sample can be brought into contact with the thin film. A non-solid sample containing cells or tissue pieces collected by fine needle aspiration can also be mixed with a molding material such as a compound, rapidly frozen with liquid nitrogen, and similarly used to prepare a slice. When a non-solid sample containing cells or tissue pieces collected from a tissue by puncture suction is used as it is, the sucked sample may be discharged onto a thin film, and the cells may be adhered to the thin film in a dispersed state. Furthermore, when the biological sample is a piece of tissue, the sample and the thin film can be brought into contact by gently wiping the water of the collected tissue and then allowing the sample to stand on the thin film containing the protease substrate for about 1 to 30 minutes. it can.

When a non-solid sample such as synovial fluid collected from a patient with rheumatoid arthritis is used, after diluting the sample to an appropriate concentration and / or performing a necessary pretreatment, 1 to 50 μL, preferably about 1 to 20 μL, may be dropped on the thin film. When using gingival crevicular fluid for periodontal disease as a sample, a method of inserting a filter paper into the gingival sulcus, collecting about 5 to 10 μL of gingival crevicular fluid, and applying the filter paper to a thin film is adopted. be able to. After collecting the gingival crevicular fluid, if necessary, use distilled water or an appropriate buffer (eg, 50 mM Tris
-HCl, pH 7.5, 10 mM CaCl ₂ , 0.2 M NaCl, etc.) to extract the gingival crevicular fluid from the filter paper, and the extract may be dropped on the thin film. In the case of a bodily fluid sample (such as cyst fluid) that can be collected in a larger amount, a method in which a part of the thin film is immersed in a container in which the sample is placed can obtain reproducible results.

After the tissue section containing the protease substrate is attached to the thin film, or the sample containing the protease is brought into contact with the thin film by such means as dropping a liquid sample,
The thin film is incubated at a temperature suitable for the expression of the protease activity, for example, at a saturated humidity of 37 ° C. for a time necessary for digestion of the substrate. The time required varies depending on the type of sample or thin film, but is preferably 1 to 4 at 37 ° C for a tissue section or a non-solid sample containing cells or tissue pieces obtained by suction.
Incubate for 8 hours, more preferably for 6 to 30 hours, for liquid samples such as exudates, for 0.5 to 24 hours, preferably for 1 to 6 hours, and allow proteases in the sample to form digestion marks in the thin film. Thereafter, the thin film is washed with water to remove the digested substrate.

One of two or more substantially continuous sections of the biological sample is attached to a thin film containing no protease inhibitor, and one of the other sections is attached to a thin film containing a protease inhibitor. By comparing the digestion marks of both thin films, it becomes possible to specify the type of protease. Although the type of the protease inhibitor is not particularly limited, for example, a chelating agent, a matrix metalloprotease inhibitor, a serine protease inhibitor, or the like can be suitably used.

By dyeing the thin film using the compound represented by the formula (I) or a salt thereof of the present invention and a color developing agent, the thin film is generally digested by the protease in the sample. The optical density of the upper digestion mark is reduced. When a thin film containing a protease substrate is applied in multiple layers, and a different color dye is added to each layer, the hue of the thin film changes with the optical density as the thin film is digested by the protease in the sample. By dyeing such a thin film using the compound represented by the formula (I) of the present invention or a salt thereof and a color developing agent, the digestion strength can be visually more accurately determined. It will be easier.

By measuring the protease activity contained in a biological sample, it is possible to examine the state of the living body from which the sample was derived, for example, the correspondence to the metastasis of cancer or the progress of rheumatism. To determine the digestive strength of digestion marks, a method of visual judgment under an optical microscope, a method of measuring the optical density of digestive marks by a spectroscope, and a method of analyzing the image obtained by an optical microscope into a computer and analyzing the image And any of the methods for digitizing various types of digestion marks by using the method.
When performing image analysis, various data processing methods can be used, and the type thereof is not particularly limited. The degree of digestion is quantified using the area of digestion marks or the integration of the concentration and area of digestion marks. Is preferred.

[0055]

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to Examples, but the scope of the present invention is not limited to these Examples.

Example 1 (Synthesis of Compound (9)) 1-1: Synthesis of (3-methanesulfonylamino) benzenesulfonyl chloride 3-aminobenzenesulfonic acid (methanilic acid) (21
To 6.5 g), pyridine (253 mL) and acetonitrile (253 mL) were added and suspended, followed by stirring under water cooling.
To this was added methanesulfonyl chloride (166 mL) dropwise over 45 minutes. Then, the mixture was cooled with ice water and phosphorus oxychloride (138 mL) was added dropwise over 5 minutes. The temperature of the reaction solution is 3
The temperature rose to 5 ° C. After reacting for 4 hours, ice water 3K
g, and the precipitated crystals were filtered under reduced pressure, washed sufficiently, and dried by blowing at 30 ° C. Yield 344 g, yield 10
0%

1-2: Synthesis of 5- (3-methanesulfonylaminobenzenesulfonamido) -1-naphthol-2-carboxylic acid 5-amino-1-naphthol-2-carboxylic acid (5-amino-1-naphthol) Kolbe-Schmidt (Kolb)
e-Schmitt) (reaction was carried out) and 150 g was dissolved in dimethylacetamide by heating and then cooled with ice water. Under stirring, (3-methanesulfonylamino) benzenesulfonyl chloride (438 g) synthesized above was added over 5 minutes. Next, 191 mL of pyridine was added dropwise while keeping the temperature at 30 ° C. or lower. Stir for 8 hours at room temperature,
Slowly poured into 9 liters of water. The precipitated crystals were filtered under reduced pressure, washed sufficiently with water, and then drawn with methanol. 5
Blow drying was performed at 0 ° C. or lower to obtain the desired product. Yield 28
6.6 g, yield 89.0%

1-3: Synthesis of 5- (3-methanesulfonylaminobenzenesulfonamide) -1-naphthol-2-carboxylic acid phenyl ester 5- (3-methanesulfonylaminobenzenesulfonamide) -1-naphthol-2 -Carboxylic acid (39.2
g), phenol (25.4 g), acetonitrile (2
00 mL) and dimethylacetamide (5 mL) were mixed and heated to reflux. Under these conditions, phosphorus oxychloride 25.
1 mL was added dropwise. The reaction was performed for 10 hours and cooled to room temperature. The precipitated crystals were filtered under reduced pressure and washed with acetonitrile. Further, the obtained crystals were suspended in a saturated saline solution, vigorously stirred, filtered under reduced pressure, washed with a saturated aqueous solution of sodium hydrogen carbonate, water and acetonitrile, and air-dried. Yield 41.6 g, 90.4% yield

1-4: Synthesis of Compound (9) 5- (3-Methanesulfonylaminobenzenesulfonamide) -1-naphthol-2-carboxylic acid phenyl ester (15.0 g) was added to 1,3-dimethylimidazoline-
30 mL of 2-one was added and heated to 70 ° C. After adding 75 mL of acetonitrile, 15.2 of diethylamine was added.
mL was added. The reaction was carried out for 12 hours under reflux conditions and cooled to room temperature. 200 mL of ethyl acetate in the reaction solution
Was added, and the organic phase was washed twice with dilute hydrochloric acid and twice with water, and then dried over magnesium sulfate. After distilling off the solvent under reduced pressure, isopropyl alcohol / hexane = 100 mL
Crystallized from / 10 mL. The crystals were filtered under reduced pressure and air-dried. Yield 7.8 g, yield 54.2%, melting point 153-1
55 ° C

Example 2 (Synthesis of Compound (5)) 2-1: Synthesis of 4- (5-amino-1-naphthol-2-carbonyl) morpholine hydrochloride 5-amino-1-naphthol-2-carboxylic acid ( 707
g) was suspended in 2 liters of acetonitrile, triphenyl phosphite (1400 mL) was added, and the mixture was stirred at room temperature. To this, morpholine (606 mL) was added dropwise while maintaining the temperature at 60 ° C. or lower. Then, after reacting at 70 ° C for 2 hours,
Add 2 liters of acetonitrile, activate carbon (60 g),
Celite (60 g) was added and stirred for 30 minutes. Perform hot filtration (the washing solution for filtration is acetonitrile).
Heated to 8 ° C. When 270 mL of concentrated hydrochloric acid was slowly added dropwise, crystals precipitated. After cooling to room temperature, the crystals were filtered under reduced pressure. Washed with acetonitrile and blow dried. Yield 7
51 g, 69.9% yield

2-2: Synthesis of compound (5) 4- (5-amino-1-naphthol-2-carbonyl)
Morpholine hydrochloride (10 g) was added to dimethylacetamide 5
Dissolved in 0 mL and cooled with water. While stirring, 3.0 mL of methanesulfonyl chloride was added, and then 3.2 mL of pyridine was slowly added dropwise. After the reaction was carried out at room temperature for 2 hours, 100 mL of water was slowly added dropwise to the reaction solution to precipitate crystals. The crystals were filtered under reduced pressure, washed with water, methanol and acetonitrile in that order, and the crystals were air-dried. Yield 9.5 g, yield 83.7%, melting point 209-21
0

Example 3 (Synthesis of Compound (15)) 3-1: Synthesis of 5- (N, N-bis (methanesulfonyl) amino) -1-methanesulfonyloxy-2-naphthalenesulfonic acid sodium salt 5-amino 12 liters of water was added to -1-naphthol-2-sulfonic acid sodium salt (3 kg) and stirred.
A 30% aqueous sodium hydroxide solution (1.8 kg) was added thereto, and the mixture was further stirred. Next, methanesulfonyl chloride (11.7 kg) and a 30% aqueous sodium hydroxide solution (6.4 kg) were simultaneously added dropwise while maintaining the pH at 6.5 to 7.0. The reaction was carried out at a temperature of 40 ± 5 ° C., and the dripping time was 4 hours. After the completion of the dropwise addition, the reaction was carried out at 40 ± 5 ° C. for 1 hour, then cooled to 17 ° C., and the crystals were filtered. After washing with 4.5 liters of water, the crystals were taken out. The crystals are added to 31.5 liters of water, and stirred for 90 minutes.
Heated to ° C. After cooling to 17 ° C., the crystals were filtered, washed with 11 l of methanol and dried. Yield 4.
0 kg, yield 70.3%

3-2: 5- (N, N-bis (methanesulfonyl) amino) -1-methanesulfonyloxy-chloride
Synthesis of 2-naphthalenesulfonyl 5- (N, N-bis (methanesulfonyl) amino) -1
-Methanesulfonyloxy-2-naphthalenesulfonic acid sodium salt (4.0 Kg), acetonitrile (16 L), and N-methylpyrrolidone (4.04 Kg) were mixed and stirred. The temperature was raised to 35 ° C. and 8.36 kg of phosphorus oxychloride was added dropwise over 4 hours. Reaction temperature is 3
Performed at 5 ± 5 ° C. After the completion of dropping, the temperature is set to 40 ° C.
A time reaction was performed. When the reaction solution was poured into a container containing water (36 kg) and ice (36 kg) while stirring, crystals were precipitated. The precipitated crystals were filtered, washed with water, washed with 0.8 liter of acetonitrile, and dried to obtain the desired product. 3.3 Kg yield, 83.1% yield

3-3: Synthesis of Compound (15) 22.3 g of 5- (N, N-bis (methanesulfonyl) amino) -1-methanesulfonyloxy-2-naphthalenesulfonyl chloride and 45 mL of water were mixed. And stirred.
To this, propylamine (32.8 mL) was slowly added dropwise while maintaining the temperature at 40 ° C. or lower, and then heating was performed.
A time reaction was performed. After cooling with water, the temperature of the reaction solution was adjusted to 23 ° C., and 150 g of a 23% aqueous potassium hydroxide solution was added dropwise.
After setting the reaction temperature at 45 ° C. and performing the reaction for 30 minutes,
It was cooled to 30 ° C. with water. When 100 mL of concentrated hydrochloric acid was added dropwise to make it acidic, crystals were precipitated. The precipitated crystals are collected by filtration,
Washing was performed in the order of water and acetonitrile, followed by drying. 13.2 g, yield 81.2%, melting point 157-159 ° C.

Example 4 (Synthesis of Compound (1)) 4-1: Synthesis of Compound (1) 4- (5-amino-1-naphthol-2) synthesized in Example 2
-Carbonyl) morpholine hydrochloride (10.0 g) in 50
Dissolve in 0 mL of dimethylacetamide, cool with ice water and add 1
The mixture was stirred at 0 ° C or lower. Trifluoroacetic anhydride (5.4m
L) was added, followed by slow dropwise addition of pyridine (3.2 mL). After reacting at room temperature for 2 hours, 100 mL of water was added dropwise to precipitate crystals. The precipitated crystals were collected by filtration and washed with water and acetonitrile in this order to obtain the desired product. Yield 10.2 g, yield 85.5%, melting point 189-1
90 ° C

Example 5 (Measurement of Protease Activity of Breast Cancer Tissue) A breast cancer specimen which was removed by surgery and frozen was sliced to a thickness of 4 μm at −25 ° C. using a cryosection preparation apparatus, and then PCT / JP97 / 0
It was adhered to a gelatin film disclosed in No. 0588. The gelatin having a thickness of 6 μm was used. This gelatin film is
The mixture was incubated at 100 ° C. and a relative humidity of 100% for 16 to 30 hours and air-dried. Staining was performed as follows. 10 mL of a 5% dimethyl sulfoxide solution of the compound of the present invention and a color developing agent in the combinations shown in Table 1 were added to 100 mL of a pH 8 buffer solution. Then, a gelatin film to which the prepared specimen was adhered was immersed in this solution, and a 3% aqueous solution of ammonium persulfate was slowly dropped into the immersion solution with gentle stirring. The immersion time was determined while visually checking the degree of staining of the gelatin film.

For comparison, a solution prepared by dissolving Ponceau 3R in a 6% aqueous solution of trichloroacetic acid so as to have a concentration of 0.8% was immersed at room temperature for 60 seconds for dyeing. Each membrane was washed with water for 5 minutes, immersed in Mayer's hematoxylin solution for 2 minutes to stain the nucleus, washed with water for 10 minutes, immersed in ethanol for 20 seconds, and air-dried. After drying, a cover aid film (Sakura Seiki) to cover the tissue section
Was adhered using xylene, and a breast cancer section was enclosed. When this film was held on a plastic mount and observed using an optical microscope, gelatin digestion was observed in breast cancer tissue sections at sites where cancer cells are thought to be present due to the morphology of the nucleus, revealing that it has protease activity. became. In addition, since the compound of the present invention was negative in both the mutagenicity test and the chromosome aberration test, it was proved that it could be used for a highly safe staining method.

[0068]

[Table 1]

[0069]

Embedded image

[0070]

The compound of the present invention or a salt thereof is useful for staining a thin film containing a polypeptide such as gelatin, and has high staining properties and safety.

 ────────────────────────────────────────────────── ─── Continued on the front page (72) Inventor ▲ Taka ▼ Bridge Kazunashi 210 Nakanaka, Minamiashigara-shi, Kanagawa Prefecture Fuji Photo Film F-term (reference) 4H006 AA01 AB20

Claims (4)

[Claims] 1. The following general formula (I): Wherein, X is -CO- or -SO ₂ - indicates; L represents a hydrogen atom or a halogen atom; Y is -NHCO-R ² or -NHSO ^₂ -R ₂ (R ₂ is a substituted or unsubstituted C _1-6
An alkyl group, a substituted or unsubstituted C _1-6 alkoxy group, or a substituted or unsubstituted C _6-12 aryl group); R ¹ represents a substituted or unsubstituted alkyl group;
A substituted or unsubstituted alkylamino group, a substituted or unsubstituted anilino group, or a substituted or unsubstituted heterocyclic group] or a salt thereof. 2. R ¹ is an alkylamino group having 2 to 6 carbon atoms, an anilino group having 6 to 8 carbon atoms, or a heterocyclic group having 4 to 6 carbon atoms, and Y is an acetylamino group, a propionylamino group, Methanesulfonylamino group, ethanesulfonylamino group, ethoxycarbonylamino group, propoxycarbonylamino group, butoxycarbonylamino group, benzenesulfonylamino group, 3-sulfobenzenesulfonylamino group, (3-methanesulfonylaminophenyl) sulfonylamino group, Or the compound or a salt thereof according to claim 1, which is a (3-carboxyphenyl) sulfonylamino group. 3. X is —CO—, and R ¹ is a diethylamino group, a morpholino group, a piperidino group, a 3-carboxypiperidino group, a 4-carboxypiperidino group, a 3-sulfoanilino group, a 4-sulfoanilino group. Or an oxazolidin-3-yl group, L is a hydrogen atom, Y is an acetylamino group, a trifluoroacetylamino group, a methanesulfonylamino group, a butoxycarbonylamino group,
The compound according to claim 1, which is a benzenesulfonylamino group, a 3-sulfobenzenesulfonylamino group, a (3-methanesulfonylaminophenyl) sulfonylamino group, or a (3-carboxyphenyl) sulfonylamino group, or a salt thereof. 4. X is —SO ₂ —, R ¹ is an ethylamino group, propylamino group, butylamino group or t-butylamino group, L is a hydrogen atom, and Y is an acetylamino group. The trifluoroacetylamino group, a benzenesulfonylamino group, a 3-sulfobenzenesulfonylamino group, a (3-methanesulfonylaminophenyl) sulfonylamino group, or a (3-carboxyphenyl) sulfonylamino group according to claim 1. A compound or a salt thereof.