Chymotrypsin detection reagent and detection method

Abstract

The present invention provides a chymotrypsin detection reagent containing a compound represented by general formula (I) or a salt thereof. In the formula, R1 represents a substituent bonded to a benzene ring, p represents an integer of 0-4, R2, R3, R4, R5, R6, and R7 each independently represent a hydrogen atom or the like, R8 and R9 may each independently represent a hydrogen atom or the like, X represents a linear or branched C1-C3 alkylene group, R10 and R11 may each independently represent a hydrogen atom or the like, R12 may represent a hydrogen atom or the like, and L may represent a single bond or the like.

Description

Chymotrypsin detection reagent and detection method

[0001] The present invention relates to a chymotrypsin detection reagent, for example, a fluorescent probe that emits fluorescence upon reaction with chymotrypsin.

[0002] In recent years, fluorescent imaging has attracted attention as a method for detecting enzymes or enzyme activities (hereinafter, collectively referred to as "enzymes") in various measurement samples, such as biological samples, in real time, and development of fluorescent probes (detection reagents) capable of detecting various enzymes has been progressing. For example, Patent Document 1 discloses a fluorescent probe capable of detecting chymotrypsin.

[0003] Patent No. 6204147

[0004] The fluorescent probe disclosed in Patent Document 1 leaves room for improvement in terms of substrate specificity. Specifically, the fluorescent probe disclosed in Patent Document 1 (e.g., compound C1 described in the Examples below) is said to be usable for detecting chymotrypsin, but the present inventors have discovered a new problem: that the fluorescent probe (e.g., compound C1 described in the Examples below) also exhibits reactivity with trypsin, which is added to induce chymotrypsinogen to chymotrypsin. An object of the present invention is to provide a chymotrypsin detection reagent that has low reactivity with trypsin and is capable of detecting chymotrypsin with high specificity.

[0005] [1] According to one aspect of the present invention, there is provided a chymotrypsin detection reagent comprising a compound represented by the following general formula (I) or a salt thereof: [In the formula, R １ are the same or different and represent substituents attached to the benzene ring; p represents an integer of 0 to 4; R ２ , R ３ , R ４ , R ５ , R ６ , and R ７ each independently represents a hydrogen atom, a hydroxyl group, a halogen atom, or an optionally substituted linear, branched, or cyclic alkyl group; R ８ and R ９each independently represents a hydrogen atom or an optionally substituted linear, branched, or cyclic alkyl group, or R ８ and R ９ may be bonded to form a ring, or R ３ and R ８ may be bonded to form a ring, and / or R ４ and R ９ may be bonded to form a ring; X represents a linear or branched C1-C3 alkylene group; R １０ and R １１ each independently represents a hydrogen atom, a hydroxyl group, an alkoxy group, or an optionally substituted linear, branched, or cyclic alkyl group, or R １０ and R １１ may be bonded to form a ring; １２ represents a hydrogen atom, an optionally substituted linear, branched, or cyclic alkyl group, an optionally substituted linear or branched alkenyl group, an optionally substituted linear or branched alkynyl group, an optionally substituted aryl group, or an optionally substituted heterocyclic group, or R １０ or R １１ L may be bonded to form a ring; L is an amide bond, an ester bond, or -(linear or branched alkylene-O-) ｎ - (n is an integer of 1 to 10), or a single bond.] [2] The chymotrypsin detection reagent according to the above [1], wherein in the formula (I), -OL-R １２ The group represented by the formula (II) may be a group represented by the formula (II) below: [wherein n represents an integer of 0 to 10.] [3] In the chymotrypsin detection reagent according to the above [1], in the formula (I), -OL-R １２ The group represented by the formula (I) may be a hydroxyl group or a group selected from the following: [4] The chymotrypsin detection reagent according to the above [1], wherein in the formula (I), The partial structure represented by the following formula (wherein m is an integer of 1 to 3, and q is 0 or 1): [5] In the chymotrypsin detection reagent according to any one of [1] to [3] above, R １０ and R １１ may be an alkyl group or may be bonded to each other to form a cycloalkyl group. [6] In the chymotrypsin detection reagent according to the above item [1], the compound may be represented by the following formula (III): [7] According to another aspect of the present invention, there is provided a method for detecting chymotrypsin in a sample, comprising contacting the sample with the chymotrypsin detection reagent according to any one of [1] to [6] above and measuring fluorescence from the compound or a salt thereof after the contact. [8] In the method according to [7] above, the contacting of the sample with the chymotrypsin detection reagent may be carried out in the presence of trypsin.

[0006] According to an embodiment of the present invention, there is provided a chymotrypsin detection reagent capable of detecting chymotrypsin with high specificity. The chymotrypsin detection reagent according to an embodiment of the present invention has a significantly lower reaction responsiveness with trypsin than with chymotrypsin, and therefore can effectively detect chymotrypsin activity even in the presence of trypsin.

[0007] 1 is a graph showing the change in fluorescence intensity over time when the chymotrypsin detection reagents of Examples and Comparative Examples are reacted with chymotrypsin, and chymotrypsinogen is contacted with the chymotrypsin detection reagents of Examples and Comparative Examples in the presence or absence of trypsin.

[0008] Representative embodiments of the present invention will be described below, but the present invention is not limited to these embodiments. Each embodiment can be combined as appropriate unless it is clearly inappropriate.

[0009] A. Chymotrypsin Detection Reagent A chymotrypsin detection reagent according to an embodiment of the present invention comprises a compound represented by the above general formula (I) (hereinafter, may be referred to as "compound of formula (I)") or a salt thereof.

[0010] In formula (I), R １represents a substituent bonded to the benzene ring. p is an integer of 0 to 4, and may be 0 to 3, 0 to 2, 0 to 1, 1, or 0. R １ Examples of R include, but are not limited to, an optionally substituted linear or branched alkyl group; an optionally substituted linear or branched alkoxy group; a halogen atom; an optionally substituted amino group; a substituted silyl group; or an acyl group. １ may be the same or different, and "Me" represents a methyl group.

[0011] R ８ and R ９ When R ３ and R ８ When R ４ and R ９ When R １０ and R １１ When R and R are bonded to form a ring, the ring may contain a heteroatom (such as an oxygen atom, a nitrogen atom, or a sulfur atom), and may be, for example, a cycloalkyl or a cyclic ether. １２ and R １０ or R １１ When they combine to form a ring, the ring may be a cyclic ether, and these rings may be 3 to 14 members, 3 to 10 members, 3 to 8 members, or 3 to 6 members.

[0012] Throughout this specification, substituents on alkyl groups or alkyl moieties of other groups can include halogen atoms, hydroxyl groups, amino groups, alkylamino groups, dialkylamino groups, thiol groups, alkylthiol groups, sulfonyl groups, alkylsulfonyl groups, alkoxy groups, cyclic ethers, carboxyl groups, alkylcarbonyl groups, alkoxycarbonyl groups, alkoxycarbonylamino groups, alkylcarbonyloxy groups, alkylaminocarbonyl groups, alkylcarbonylamino groups, carbonylamino groups, hydrazinyl groups, etc. When an alkyl group is substituted, the number of substituents can be, for example, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 1, 2, or 3.

[0013] The substituents on the amino group may be linear or branched alkyl groups which may be substituted, and when the amino group is substituted, the number of the substituents may be 1 to 2, 1, or 2.

[0014] The substituents of the substituted silyl group may be an optionally substituted linear or branched alkyl group or an optionally substituted phenyl group. The number of substituents of the silyl group may be, for example, 1 to 4, 1 to 3, 1 to 2, 1, 2, 3, or 4.

[0015] As used herein, the term "alkyl group" or the "alkyl" moiety in other groups refers to a linear, branched, or cyclic saturated hydrocarbon group, preferably a saturated hydrocarbon group having 1 to 6 carbon atoms, such as a methyl group, ethyl group, n-propyl group, i-propyl group, n-butyl group, sec-butyl group, t-butyl group, isobutyl group, pentyl group, isopentyl group, 2,3-dimethylpropyl group, hexyl group, cyclopropyl group, cyclobutyl group, cyclopentyl group, and cyclohexyl group; more preferably a C1-5 alkyl group, such as a methyl group, ethyl group, n-propyl group, i-propyl group, n-butyl group, sec-butyl group, t-butyl group, isobutyl group, pentyl group, isopentyl group, or 2,3-dimethylpropyl group; even more preferably a C1-3 alkyl group, such as a methyl group, ethyl group, n-propyl group, or i-propyl group; and most preferably a methyl group or an ethyl group. Furthermore, the term "alkylene group" refers to a divalent group obtained by removing one hydrogen atom from the above alkyl group.

[0016] The "alkoxy group" refers to a group bonded to the above-mentioned alkyl group via an oxygen atom ((alkyl)-O- group), and the alkyl group portion is as defined above. For example, the alkoxy group may have an alkyl group portion having 1 to 6 carbon atoms. Examples of the alkoxy group include a methoxy group, an ethoxy group, a 1-propyloxy group, a 2-propyloxy group, a 2-methyl-1-propyloxy group, a 2-methyl-2-propyloxy group, a 2,2-dimethyl-1-propyloxy group, a 1-butyloxy group, a 2-butyloxy group, a 2-methyl-1-butyloxy group, a 3-methyl-1-butyloxy group, a 2-methyl-2-butyloxy group, a 3-methyl-2-butyloxy group, a 1-pentyloxy group, a 2-pentyloxy group, a 3-pentyloxy group, a 2-methyl-1-pentyloxy group, a 3-methyl-1-pentyloxy group, a 2-methyl-2-pentyloxy group, a 3-methyl-2-pentyloxy group, a 1-hexyloxy group, a 2-hexyloxy group, and a 3-hexyloxy group. The C1-6 alkoxy group is preferably a C1-5 alkoxy group, and more preferably a methoxy group, an ethoxy group, an n-propyloxy group, an i-propyloxy group, an n-butyloxy group, a sec-butyloxy group, a t-butyloxy group, an isobutyloxy group, a pentyloxy group, an isopentyloxy group, or a 2,3-dimethylpropyloxy group.

[0017] An "alkenyl group" is a monovalent group obtained by removing one hydrogen atom from any carbon atom of a straight-chain, branched, or cyclic unsaturated hydrocarbon having one or more carbon-carbon double bonds, and may have, for example, 2 to 10, 2 to 6, or 2 to 4 carbon atoms. Examples of C2-10 alkenyl groups include vinyl, propenyl, isopropenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-methyl-1-butenyl, 1-methyl-2-butenyl, 1-methyl-3-butenyl, 1-methylidenebutyl, 2-methyl-1-butenyl, 2- Methyl-2-butenyl group, 2-methyl-3-butenyl group, 2-methylidenebutyl group, 3-methyl-1-butenyl group, 3-methyl-2-butenyl group, 3-methyl-3-butenyl group, 1-ethyl-1-propenyl group, 1-ethyl-2-propenyl group, 1-hexenyl group, 2-hexenyl group, 3-hexenyl group, 4-hexenyl group, 5-hexenyl group, 1-methyl-1-pentenyl group, 1- Methyl-2-pentenyl group, 1-methyl-3-pentenyl group, 1-methyl-4-pentenyl group, 1-methylidenepentyl group, 2-methyl-1-pentenyl group, 2-methyl-2-pentenyl group, 2-methyl-3-pentenyl group, 2-methyl-4-pentenyl group, 2-methylidenepentyl group, 3-methyl-1-pentenyl group, 3-methyl-2-pentenyl group, 3-methyl-3-pentenyl group Examples of such groups include 3-methyl-4-pentenyl, 3-methylidenepentyl, 4-methyl-1-pentenyl, 4-methyl-2-pentenyl, 4-methyl-3-pentenyl, 4-methyl-4-pentenyl, 1-heptenyl, 2-heptenyl, 3-heptenyl, 4-heptenyl, 5-heptenyl, 6-heptenyl, octenyl, nonenyl, and decenyl groups.

[0018] An "alkynyl group" is a monovalent group formed by removing one hydrogen atom from any carbon atom of a straight-chain or branched unsaturated hydrocarbon having one or more carbon-carbon triple bonds, and may have, for example, 2 to 6 or 2 to 4 carbon atoms. Examples of the alkynyl group include an ethynyl group, a 1-propynyl group, a 2-propynyl group, a 1-butynyl group, a 2-butynyl group, a 3-butynyl group, a pentynyl group, a hexynyl group, and a phenylethynyl group.

[0019] An "acyl group" is synonymous with an alkanoyl group and is a group represented by R-C(=O)-. The R portion in an acyl group represented by this structure includes a hydrogen atom, an optionally substituted linear or branched alkyl group, or an optionally substituted aryl group. For example, the acyl group may have 2 to 7 carbon atoms and includes an acetyl group, a propionyl group, a butyryl group, an isobutyryl group, a valeryl group, an isovaleryl group, a pivaloyl group, a hexanoyl group, and a heptanoyl group.

[0020] An "aryl group" is a monovalent aromatic hydrocarbon group which may have, for example, 6 to 10 carbon atoms and includes phenyl and naphthyl groups.

[0021] A "heterocyclic group" is a monovalent group containing at least one heteroatom selected from a nitrogen atom, an oxygen atom, and a sulfur atom, and is preferably 5 to 14 members. The heterocyclic group may be a monocyclic heterocyclic group or a fused heterocyclic group. The number of heteroatoms contained in a 5 to 14-membered heterocyclic group may be, for example, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2, or 1. For example, various combinations exist, such as a heterocyclic group containing one nitrogen atom, a heterocyclic group containing two nitrogen atoms, a heterocyclic group containing three nitrogen atoms, a heterocyclic group containing one oxygen atom, a heterocyclic group containing two oxygen atoms, a heterocyclic group containing one oxygen atom and one nitrogen atom, and a heterocyclic group containing one sulfur atom. The 5 to 14-membered heterocyclic group may be aromatic or non-aromatic. The monocyclic heterocyclic group is preferably a 5- or 6-membered ring. The fused heterocyclic group is preferably an 8- to 10-membered ring.Examples of 5- to 14-membered heterocyclic groups include piperidyl, piperazyl, morpholyl, quinuclidyl, pyrrolidinyl, azetidyl, oxetyl, azetidin-2-one-yl, aziridinyl, tropanyl, furyl, tetrahydrofuryl, thienyl, pyrrolyl, pyrrolinyl, pyrrolidinyl, dioxolanyl, oxazolyl, oxazolinyl, isoxazolyl, thiazolyl, thiazolinyl, isothiazolyl, imidazolyl, imidazolinyl, and imidazo. Rizinyl, oxazolidinyl, thiazolidinyl, pyrazolyl, pyrazolinyl, pyrazolidinyl, oxadiazolyl, furazanyl, thiadiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, tetrazolyl, pyranyl, pyridyl, piperidinyl, pyridazinyl, pyrimidinyl, pyrazinyl, piperazinyl, dioxanyl, oxazinyl, morpholinyl, thiazinyl, triazinyl, benzofuranyl, isobenzofuranyl, dihydrido Isobenzofuranyl, dihydroisobenzofuranyl, benzothienyl, isobenzothienyl, dihydrobenzothienyl, dihydroisobenzothienyl, tetrahydrobenzothienyl, quinolyl, isoquinolyl, quinazolinyl, phthalazinyl, pteridinyl, coumaryl, chromonyl, 1,4-benzodiazepinyl, indolyl, isoindolyl, benzimidazoyl, benzofuryl, purinyl, acridinyl, phenoxazinyl, phenothiazinyl Examples of the alkyl group include benzoxazolyl, benzothiazolyl, indazolyl, benzimidazolyl, benzodioxolanyl, benzodioxanylchromenyl, chromanyl, isochromanyl, chromanonyl, cinnolinyl, quinoxalinyl, indolizinyl, quinolidinyl, imidazopyridyl, naphthyridinyl, dihydrobenzoxazinyl, dihydrobenzoxazolinonyl, dihydrobenzoxazinonyl, and benzothioxanyl.

[0022] Examples of the substituents on the aryl group and heterocyclic group include an alkoxy group, a halogen atom, an amino group, a mono- or di-substituted amino group, a substituted silyl group, and an acyl group. When the aryl group and the heterocyclic group have two or more substituents, they may be the same or different.

[0023] As used herein, the term "halogen atom" refers to a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom, and is preferably a fluorine atom, a chlorine atom, or a bromine atom, and more preferably a fluorine atom or a chlorine atom. As used herein, the term "carboxyl group" refers to a group represented by -C(=O)-OH.

[0024] R １ is preferably a linear or branched alkyl group. Also, p is preferably 0 or 1, more preferably 0.

[0025] R ２ is preferably a hydrogen atom.

[0026] R ３ is preferably a hydrogen atom.

[0027] R ４ is preferably a hydrogen atom.

[0028] R ５ is preferably a hydrogen atom.

[0029] R ６ is preferably a hydrogen atom.

[0030] R ７ is preferably a hydrogen atom.

[0031] R ８ is preferably a hydrogen atom.

[0032] R ９ is preferably a hydrogen atom.

[0033] R １０ is preferably a hydrogen atom, an alkyl group or an alkoxy group, more preferably a hydrogen atom, a methyl group or a methoxy group.

[0034] R １１ is preferably a hydrogen atom, an alkyl group or an alkoxy group, more preferably a hydrogen atom, a methyl group or a methoxy group.

[0035] Or, R １０ and R １１are preferably linked together to form a cycloalkyl group, more preferably a C3-C5 cycloalkyl group.

[0036] R １２ is preferably a hydrogen atom or an optionally substituted linear, branched, or cyclic alkyl group, more preferably a hydrogen atom or a linear, branched, or cyclic alkyl group having 1 to 6 carbon atoms which may be substituted with one or more groups selected from a hydroxyl group, a carboxyl group, a cycloether, an alkoxy group (e.g., a methoxy group, an ethoxy group), a sulfonyl group, a halogen atom, an amino group, a methylamino group, and a dimethylamino group, and even more preferably a hydrogen atom or a linear or branched alkyl group having 1 to 4 carbon atoms which may be substituted with one or more groups selected from a hydroxyl group, a carboxyl group, a cycloether, an alkoxy group (e.g., a methoxy group, an ethoxy group), a sulfonyl group, a halogen atom, an amino group, a methylamino group, and a dimethylamino group (e.g., a methyl group, an ethyl group, a propyl group, an isopropyl group, a t-butyl group). Alternatively, R １２ is preferably R １０ or R １１ and combine with each other to form a 3- to 8-membered heterocyclic group containing 1 or 2 oxygen atoms (eg, dioxolanyl, dioxanyl).

[0037] L is preferably -(linear or branched alkylene-O-) ｎ - (n is an integer of 1 to 10) or a single bond. The straight-chain or branched alkylene may be, for example, an alkylene having 1 to 4 carbon atoms, preferably 2 or 3 carbon atoms, and more preferably 2 carbon atoms.

[0038] For example, in the above formula (I), -OL-R １２ The group represented by the formula (II) may be a group represented by the formula (II) below.

[0039] [In the formula, n represents an integer of 0 to 10.]

[0040] Furthermore, for example, in the above formula (I), -OL-R １２ The group represented by the formula (I) may be a hydroxyl group or a group selected from the following:

[0041]

[0042] Furthermore, for example, in the above formula (I),

[0043] The partial structure represented by the formula (wherein m is an integer of 1 to 3, and q is 0 or 1) may be a structure represented by the formula (wherein m is an integer of 1 to 3, and q is 0 or 1).

[0044]

[0045] A preferred example of the compound of formula (I) is a compound of formula (III).

[0046] Salts of compound (I) include base addition salts, acid addition salts, amino acid salts, etc. Examples of base addition salts include metal salts such as lithium salt, sodium salt, potassium salt, calcium salt, and magnesium salt; organic amine salts such as ammonium salt, methylamine salt, dimethylamine salt, dicyclohexylamine salt, tris(hydroxymethyl)aminomethane salt, N,N-bis(hydroxyethyl)piperazine salt, 2-amino-2-methyl-1-propanol salt, ethanolamine salt, N-methylglucamine salt, L-glucamine salt, triethylamine salt, piperidine salt, and morpholine salt; and salts with basic amino acids such as lysine, δ-hydroxylysine, and arginine. Examples of acid addition salts include mineral acid salts such as hydrochloride, hydrobromide, sulfate, nitrate, and phosphate, and organic acid salts such as methanesulfonate, benzenesulfonate, paratoluenesulfonate, citrate, oxalate, acetate, trifluoroacetate, propionate, tartrate, fumarate, maleate, malate, succinate, benzoate, mandelate, cinnamate, lactate, glycolate, glucuronate, ascorbate, nicotinate, and salicylate. Examples of amino acid salts include glycine salts.

[0047] Compound (I) may have one or more asymmetric carbon atoms depending on the type of substituent, and may exist as stereoisomers such as optical isomers or diastereoisomers. Compound (I) may be in any form, such as a pure stereoisomer, any mixture of stereoisomers, or a racemate. Compound (I) or a salt thereof may also exist as a hydrate or solvate, and all of these substances are encompassed within the scope of the present invention. The type of solvent that forms the solvate is not particularly limited, and examples include solvents such as ethanol, acetone, and isopropanol. In this specification, reference to "Compound (I)" includes any mixture of isomers or a specific stereoisomer of Compound (I), pharmacologically acceptable salts, hydrates, and solvates of Compound (I), as well as hydrates or solvates of pharmacologically acceptable salts of Compound (I), even if not explicitly stated, unless clearly incompatible.

[0048] Compound (I) can be obtained by any suitable method. For example, compound (I) can be obtained by the synthesis method described in WO2022 / 270607A1.

[0049] The chymotrypsin detection reagent according to an embodiment of the present invention may contain any appropriate additive component depending on the intended use, dosage form, etc. Dosage forms include powders, lyophilized preparations, liquids, oils, etc. Additives include excipients, pH adjusters, buffers, isotonicity agents, solubilizers, solvents, etc. The blending ratio of compound (I) and each additive in the detection reagent is not limited as long as the effects of the present invention can be obtained, and can be appropriately set depending on the intended use, dosage form, etc. The detection reagent may be diluted or dissolved in an aqueous medium such as water, physiological saline, or a buffer solution before use.

[0050] Chymotrypsin detected by the chymotrypsin detection reagent according to the embodiment of the present invention is, for example, mammalian chymotrypsin, preferably human chymotrypsin (e.g., α-chymotrypsin). Chymotrypsin is produced by cleaving an inactive precursor, chymotrypsinogen, with trypsin or pre-existing chymotrypsin, resulting in three polypeptide chains that are disulfide-bridged. In this specification, the phrase "detecting chymotrypsin" may also refer to detecting chymotrypsin activity.

[0051] Compound (I), in the form of a closed ring isomer in which the upper part of the xanthene skeleton represented by general formula (I) is closed, is substantially non-fluorescent in the neutral region (e.g., pH 5 to 9). On the other hand, the amide bond between the xanthene skeleton and the methionine residue is cleaved, rapidly opening the ring to form an open ring isomer, which is a fluorescent substance. Therefore, chymotrypsin detection using the above-mentioned chymotrypsin detection reagent can be performed by measuring the fluorescence emitted by Compound (I) in the open ring state. Detection of fluorescence in the fluorescence measurement can indicate the presence of chymotrypsin or chymotrypsin activity in the sample to be measured. Furthermore, the detected fluorescence can indicate the concentration of chymotrypsin in the sample or the degree of chymotrypsin activity.

[0052] Fluorescence may be measured directly using a spectrofluorometer or the like, or may be measured by analyzing imaging data obtained with a fluorescence imaging device. The excitation wavelength of compound (I) is, for example, 440 nm to 500 nm, preferably 445 nm to 490 nm, and more preferably 450 nm to 480 nm. The fluorescence detection wavelength of compound (I) is, for example, 510 nm to 800 nm, preferably 516 nm to 556 nm.

[0053] B. Detection Method An example of a method for detecting chymotrypsin according to an embodiment of the present invention includes contacting a sample with the chymotrypsin detection reagent described in Section A, and measuring the fluorescence from Compound (I) after the contact. This detection method allows for the detection of chymotrypsin present in the sample. This detection method can be performed in vivo, in vitro, or ex vivo, and is preferably performed in vivo or ex vivo. The use of this in vivo or ex vivo detection method is useful, for example, in in vivo or in vitro diagnostic applications.

[0054] Examples of the sample that can be used include biological tissues, cells, and fluids (blood, saliva, urine, tears, exudates, lymph, serous fluid, spinal fluid, cerebrospinal fluid, synovial fluid, digestive fluid (e.g., pancreatic juice), drainage fluid, etc.), or processed products thereof; cell cultures, cell culture supernatants, cell lysates, cell lysates, or processed products thereof; and isolated and purified enzymes.

[0055] The organism from which the sample is derived is typically a mammal such as a human, cow, horse, dog, cat, pig, sheep, rabbit, or rat, and is preferably a human.

[0056] The sample can be contacted with the detection reagent by any suitable method. Specific examples of contacting methods include adding, applying, immersing, spraying, and injecting. The sample after contacting with the detection reagent or a mixture of the detection reagent and the sample can be subjected to fluorescence measurement. The fluorescence measurement can be performed after a predetermined time has elapsed since contacting, or over time.

[0057] In one embodiment, the sample may be contacted with the chymotrypsin detection reagent in the presence of trypsin. As described above, chymotrypsinogen, an inactive precursor, is cleaved by trypsin to produce chymotrypsin. According to this embodiment, chymotrypsin produced by cleavage of chymotrypsinogen present in a sample with trypsin can also be detected. Therefore, for example, detection of fluorescence from compound (I) in a sample contacted with a detection reagent in the presence of trypsin suggests the presence of chymotrypsin and / or chymotrypsinogen in the sample. Furthermore, for example, chymotrypsinogen present in a sample can be detected by using a sample not treated with trypsin as a control. Here, compound (I) has the characteristic of exhibiting a significantly lower fluorescence response with trypsin than with chymotrypsin, allowing chymotrypsin to be detected with a high S / N ratio even in the presence of trypsin. Therefore, compound (I) can detect chymotrypsin activity in a sample with high accuracy even in the presence of trypsin.

[0058] The trypsin may be derived from an organism of the same or a different species as the sample, as long as it is capable of cleaving chymotrypsinogen that may be contained in the sample. Trypsin may also be purified from an organism or produced by genetic engineering techniques. Trypsin may be added to the reaction system before, after, or simultaneously with contact of the sample with the detection reagent.

[0059] The same explanation as in Section A can be applied to the method of measuring fluorescence. For example, fluorescence can be measured by measuring the fluorescence intensity of a sample (e.g., tissue, cells, or liquid derived from a living body) after contact with a chymotrypsin detection reagent, or by performing fluorescence imaging and analyzing the resulting image.

[0060] Chymotrypsin detection can be a quantitative or qualitative assessment of chymotrypsin expression or activity, for example, by using the measured fluorescence intensity as an absolute value, by obtaining a change in the measured fluorescence intensity over time, and / or by comparing the measured fluorescence intensity with that of a control, or by identifying a site where fluorescence of a given intensity is observed.

[0061] The present invention will be specifically explained below with reference to examples, but the present invention is not limited to these examples in any way.

[0062] Experimental Example 1 Synthesis of Compound 1 Compound 1 represented by the following formula (III) was synthesized in the same manner as described in paragraphs

[0126] to

[0128] of WO2022 / 270607A1.

[0063] Experimental Example 2: Synthesis of Compound C1 The following compound C1 was synthesized by the same method as described in Japanese Patent No. 6204147, paragraphs

[0067] to

[0073] .

[0064] Experimental Example 3: Chymotrypsin Assay 1 Solutions were prepared as follows. 1. 2 mg of chymotrypsin (Nacalai Tesque, product number "09041-71", 35 U / mg) was dissolved in 0.7 mL of PBS to prepare a 100 U / mL chymotrypsin solution. 45 μL of the 100 U / mL chymotrypsin solution was added to 1,455 μL of PBS to prepare a 3 U / mL chymotrypsin solution. 2. 10 μL of a 0.5 mM DMSO solution of Compound C1 was dissolved in 990 μL of PBS to prepare probe solution C1, which had a Compound C1 concentration of 5 μM. 3. 10 μL of a 0.5 mM DMSO solution of Compound 1 was dissolved in 990 μL of PBS to prepare probe solution 1, which had a Compound 1 concentration of 5 μM.

[0065] The probe solution, PBS, and chymotrypsin were added in this order, and the enzyme reaction was initiated at 37°C. The final concentration of Compound 1 or C1 in the reaction solution was 2.5 μM, and the final concentration of chymotrypsin was 1.5 U / mL. Fluorescence intensity was measured every 5 minutes for 35 minutes after the start of the reaction. Fluorescence intensity was measured using a plate reader (Ex. 480 nm, Em. 530 nm). The results are shown in Figure 1.

[0066] As shown in FIG. 1, both Compound 1 and Compound C1 showed a fluorescent response upon reaction with chymotrypsin.

[0067] Experimental Example 4: Chymotrypsin Assay 2 Solutions were prepared as follows: 1. 5 μL of chymotrypsinogen (hereinafter "CTG", Merck, product number "C4879", 56 U / mg, 10 mg / mL) was dissolved in 275 μL of PBS to prepare a 10 U / mL CTG solution. 2. 10 μL of trypsin (Roche, product number "06369880103", 235 U / mL, 73 mg / mL) was diluted in 355 μL of PBS to prepare a 2 mg / mL trypsin solution. 3. 10 μL of a 10 mM DMSO solution of compound C1 was dissolved in 990 μL of PBS to prepare probe solution C2 with a compound C1 concentration of 100 μM. 4. 10.4 mg of a 3.65 wt % polysorbate 20 solution of Compound 1 was dissolved in 6 mL of PBS to prepare probe solution 2 having a Compound 1 concentration of 100 μM.

[0068] The probe solution, PBS, and CTG were added in this order, followed by the addition of a trypsin solution to initiate the enzyme reaction at 37°C (System 1). The final concentration of Compound 1 or C1 in the reaction solution was 50 µM, the final concentration of CTG was 1 U / mL, and the final concentration of trypsin was 0.4 mg / mL. Fluorescence intensity was measured every 5 minutes for 30 minutes after the start of the reaction. Fluorescence intensity was measured using a plate reader (Ex. 480 nm, Em. 530 nm). Furthermore, as controls, fluorescence intensity was similarly measured in a system without CTG (System 2) and a system without CTG or trypsin. The results are shown in Figure 2.

[0069] The S / N ratio in detecting chymotrypsin activity in the presence of trypsin was calculated using the formula: S / N ratio = (fluorescence intensity of system 1) / (fluorescence intensity of system 2). The results are shown in Table 1.

[0070] As shown in Table 1 and Figure 2, Compound 1 showed a smaller increase in fluorescence upon treatment with trypsin alone than Compound C1, and functioned as a fluorescent probe for chymotrypsin (chymotrypsinogen) with a good S / N ratio even in the presence of trypsin. Therefore, it can be said that Compound 1 can detect chymotrypsin activity in a sample with high accuracy even in the presence of trypsin.

[0071] The detection reagent according to the embodiment of the present invention can be suitably used in the detection of chymotrypsin, etc.

Claims

1. A chymotrypsin detection reagent comprising a compound represented by the following general formula (I) or a salt thereof: [In the formula, R １ are the same or different and represent substituents attached to the benzene ring; p represents an integer of 0 to 4; R ２ , R ３ , R ４ , R ５ , R ６ , and R ７ each independently represents a hydrogen atom, a hydroxyl group, a halogen atom, or an optionally substituted linear, branched, or cyclic alkyl group; R ８ and R ９ each independently represents a hydrogen atom or an optionally substituted linear, branched, or cyclic alkyl group; ８ and R ９ may be bonded to form a ring, or R ３ and R ８ and may be bonded to form a ring, and / or R ４ and R ９ may be bonded to form a ring; X represents a linear or branched C1-C3 alkylene group; R １０ and R １１ each independently represents a hydrogen atom, a hydroxyl group, an alkoxy group, or an optionally substituted linear, branched, or cyclic alkyl group; １０ and R １１ may be bonded to form a ring; １２ represents a hydrogen atom, an optionally substituted linear, branched or cyclic alkyl group, an optionally substituted linear or branched alkenyl group, an optionally substituted linear or branched alkynyl group, an optionally substituted aryl group, or an optionally substituted heterocyclic group, or R １０ Or R １１ and L may be bonded to form a ring; L is an amide bond, an ester bond, or -(linear or branched alkylene-O-) ｎ - (n is an integer of 1 to 10), or a single bond.

2. In the above formula (I), -OL-R １２ The chymotrypsin detection reagent according to claim 1 , wherein the group represented by the formula: [In the formula, n represents an integer of 0 to 10.] 3. In the above formula (I), -OL-R １２ The chymotrypsin detection reagent according to claim 1 , wherein the group represented by the formula:

4. In the above formula (I), The partial structure represented by the following formula (wherein m is an integer of 1 to 3, and q is 0 or 1): The chymotrypsin detection reagent according to claim 1 , wherein the structure is represented by:

5. R １０ and R １１ The chymotrypsin detection reagent according to claim 1 , wherein:

6. The chymotrypsin detection reagent according to claim 1, wherein the compound is represented by the following formula (III):

7. A method for detecting chymotrypsin in a sample, comprising: contacting the sample with the chymotrypsin detection reagent described in claim 1; and measuring the fluorescence from the compound or a salt thereof after said contact.

8. The method of claim 7, wherein contacting the sample with the chymotrypsin detection reagent is performed in the presence of trypsin.

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