Classifications

• • C—CHEMISTRY; METALLURGY
  • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
  • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
  • C09B11/00—Diaryl- or thriarylmethane dyes
  • C09B11/04—Diaryl- or thriarylmethane dyes derived from triarylmethanes, i.e. central C-atom is substituted by amino, cyano, alkyl
  • C09B11/10—Amino derivatives of triarylmethanes
  • C09B11/24—Phthaleins containing amino groups ; Phthalanes; Fluoranes; Phthalides; Rhodamine dyes; Phthaleins having heterocyclic aryl rings; Lactone or lactame forms of triarylmethane dyes
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
  • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
  • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
  • C07H19/06—Pyrimidine radicals
  • C07H19/10—Pyrimidine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
  • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
  • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
  • C07H19/16—Purine radicals
  • C07H19/20—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
  • C12Q1/44—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase

Definitions

• the present invention relates to a fluorescent probe for detecting pancreatic juice. More specifically, the present invention relates to a fluorescent probe that specifically labels pancreatic juice, a detection method using the fluorescent probe, and a detection kit including the probe.
• pancreatic cancer The number of deaths from pancreatic cancer is steadily increasing year by year, and it ranks among the top causes of cancer deaths worldwide.
• pancreatic cancer there are very few effective treatments for pancreatic cancer, and surgical removal of the cancer site is the only treatment that can hope for a complete cure.
• surgical removal of the cancer site is the only treatment that can hope for a complete cure.
• simultaneous removal of the pancreas is selected.
• pancreatic juice leakage in which pancreatic juice leaks from the cut surface of the pancreas after pancreatic resection.
• Pancreatic juice leakage not only leads to bacterial infection, but also poses the risk of blood vessels being digested by the autodigestion of pancreatic juice, causing massive bleeding and even death (Non-Patent Document 1). For this reason, pancreatic juice leakage is the most important issue in pancreatic resection.
• various pancreatic resection methods and postoperative management methods have been investigated to prevent the occurrence and aggravation of pancreatic juice leakage, pancreatic juice leakage still occurs at a frequency of 30-50%.
• pancreatic juice leakage is proposed by the International Study Group on Pancreatic Fistula (ISGPF) to measure the concentration of amylase, a glycolytic enzyme, in intraperitoneal drainage fluid (Non-Patent Documents 2 and 3).
• ISGPF International Study Group on Pancreatic Fistula
• Urano, and co-researchers reported a new fluorescence imaging technique using an activatable probe, which is initially non-fluorescent but emits a visible fluorescent signal immediately after hydrolysis by ⁇ -glutamyltranspeptidase overexpressed in cancer cells (Non-Patent Document 6).
• Non-Patent Document 6 By applying such fluorescent imaging technology, we have been working on developing a technology to visualize colorless and transparent pancreatic juice in real time during surgery, and a technology to rapidly measure the pancreatic juice enzyme activity of the patient's body fluids at the bedside after surgery, realizing safe and efficient postoperative management.
• the object of the present invention is to provide a novel fluorescent probe for detecting pancreatic juice.
• the inventors used an enzyme-activatable probe library (a variety of fluorescent probes for detecting proteolytic enzymes and fluorescent probes for detecting lipid metabolic enzymes) to comprehensively analyze and explore the enzyme activity of postoperative body fluids (digestive fluid and ascites) obtained in biliary and pancreatic surgery, and investigated their use as probes for detecting pancreatic juice. They found that fluorescent probes for detecting lipid metabolism that target enzymes with phosphate groups are useful. The inventors then discovered that among these, fluorescent probes with a specific structure are capable of labeling pancreatic juice, and completed the present invention.
• enzyme-activatable probe library a variety of fluorescent probes for detecting proteolytic enzymes and fluorescent probes for detecting lipid metabolic enzymes
• a fluorescent probe for detecting pancreatic juice comprising a compound represented by the following general formula (I) or a salt thereof:
• N is selected from any of the groups represented by the following formulas (1) to (6):
• the * in formulas (1) to (6) represents the site of bonding with the oxygen of the phosphate di- or monoester group.
• Y is an oxygen atom (O) or a nitrogen atom (N);
• L is selected from -NH-L 1 -, -OL 1 -, or a bond; where L1 represents a linker:
• S represents a fluorescent group represented by any one of the following formulas (II) to (IV).
• R 1 if present, represents the same or different monovalent substituents present on the benzene ring; m is an integer from 0 to 4, and when m is 2 or more, each R 1 may be the same or different;
• R2 represents a hydrogen atom or a monovalent substituent;
• R 3 and R 4 each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom;
• R 5 and R 6 each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom;
• Z is an oxygen atom or NR a (wherein R a is a hydrogen atom or a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms);
• X is an oxygen atom, SiR b R c , CR b R c , or P( ⁇ O)R b ; wherein R b and R c are each independently an al
• R 1 to R 6 , m and X are as defined in formula (II);
• R 7 and R 8 each independently represent a hydrogen atom or a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms;
• R 7 and R 8 together with the nitrogen atom to which they are bonded may form a 5- to 7-membered heterocyclyl which may have a substituent and which contains one or more heteroatoms selected from the group consisting of oxygen atoms, nitrogen atoms, and sulfur atoms;
• R 7 or R 8 , or both R 7 and R 8 may be taken together with R 4 or R 6 , respectively, to form a 5-7 membered heterocyclyl or heteroaryl containing the nitrogen atom to which R 7 or R 8 is bonded, the heterocyclyl or heteroaryl ring optionally containing 1-3 additional heteroatoms selected from the group consisting of oxygen atoms, nitrogen atoms and sulfur atoms as members, and the heterocyclyl or heteroaryl
• R 1 if present, represents the same or different monovalent substituents present on the benzene ring; m' is an integer from 0 to 4, and when m' is 2 or more, each R 1 may be the same or different; R 2 and R 3 each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom; R 4 and R 5 each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom; R6 is selected from NR a R b , OH or OR c ; R a and R b each independently represent a hydrogen atom, an alkyl group having 1 to 5 carbon atoms, or a fluorinated alkyl group having 1 to 5 carbon atoms; R c represents an alkyl group having 1 to 5 carbon atoms; When R6 is NR a R b , (i) R a and R b together with the
• B is a phosphorus atom
• R 7 or R 8 is ⁇ O
• A represents an oxygen atom or NR d , where R d represents a hydrogen atom or an alkyl group having 1 to 5 carbon atoms
• B represents an oxygen atom, a silicon atom, a carbon atom, or a phosphorus atom
• n' is an integer from 1 to 3
• ** represents the position of binding to L in general formula (I).
• a method for detecting pancreatic juice comprising: (a) contacting a fluorescent probe according to any one of [1] to [3] with a body fluid sample; and (b) detecting the presence of pancreatic juice by observing a fluorescent response or a change in absorbance due to a reaction between the sample and the fluorescent probe.
• pancreatic juice detection method described in [6] wherein the body fluid sample contains phosphodiesterase.
• the present invention provides a novel fluorescent probe for detecting pancreatic juice.
• Patient characteristics for cases #1-8 are shown. This shows a library (40 types) of probes for lipid metabolism analysis that target enzymes having phosphate groups.
• the ranking of 40 types of probes for PJ (pancreatic juice) ⁇ ascites (Ascites) in cases #1-8 is shown.
• 1 shows the results of evaluating the fluorescence intensity of pancreatic juice using a probe library for lipid metabolism analysis.
• 1 shows the results of evaluating the fluorescence intensity of ascites using a probe library for lipid metabolism analysis.
• the results of evaluating the fluorescence intensity of 40 types of probes in pancreatic juice/ascites are shown.
• the measurement results (arbitrary units) of the fluorescence intensity of Probe.17 in pancreatic juice (PJ) and ascites (Ascites) are shown.
• the results of the inhibitor test are shown.
• halogen atom means a fluorine atom, chlorine atom, bromine atom, or iodine atom.
• alkyl may be any of linear, branched, cyclic, and aliphatic hydrocarbon groups consisting of a combination thereof.
• the number of carbon atoms of the alkyl group is not particularly limited, and may be, for example, 1 to 6 carbon atoms (C 1-6 ), 1 to 10 carbon atoms (C 1-10 ), 1 to 15 carbon atoms (C 1-15 ), or 1 to 20 carbon atoms (C 1-20 ).
• the number of carbon atoms is specified, it means “alkyl” having the number of carbon atoms in the specified range.
• C 1-8 alkyl includes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neo-pentyl, n-hexyl, isohexyl, n-heptyl, n-octyl, and the like.
• the alkyl group may have one or more optional substituents.
• substituents include, but are not limited to, alkoxy groups, halogen atoms, amino groups, mono- or di-substituted amino groups, substituted silyl groups, or acyl.
• alkyl group When an alkyl group has two or more substituents, they may be the same or different. The same applies to the alkyl moiety of other substituents containing an alkyl moiety (e.g., alkoxy groups, arylalkyl groups, etc.).
• substituents include, but are not limited to, alkyl groups, alkoxy groups, hydroxyl groups, carboxyl groups, halogen atoms, sulfo groups, amino groups, alkoxycarbonyl groups, and oxo groups. These substituents may further have substituents. Examples of such substituents include, but are not limited to, halogenated alkyl groups and dialkylamino groups.
• aryl may be either a monocyclic or condensed polycyclic aromatic hydrocarbon group, or an aromatic heterocycle containing one or more heteroatoms (e.g., oxygen, nitrogen, or sulfur atoms) as ring constituent atoms. In this case, it may be called “heteroaryl” or “heteroaromatic”. Whether the aryl is a monocyclic or condensed ring, it may be bonded at any possible position.
• Non-limiting examples of monocyclic aryl include phenyl group (Ph), thienyl group (2- or 3-thienyl group), pyridyl group, furyl group, thiazolyl group, oxazolyl group, pyrazolyl group, 2-pyrazinyl group, pyrimidinyl group, pyrrolyl group, imidazolyl group, pyridazinyl group, 3-isothiazolyl group, 3-isoxazolyl group, 1,2,4-oxadiazol-5-yl group, or 1,2,4-oxadiazol-3-yl group.
• Non-limiting examples of fused polycyclic aryls include 1-naphthyl, 2-naphthyl, 1-indenyl, 2-indenyl, 2,3-dihydroinden-1-yl, 2,3-dihydroinden-2-yl, 2-anthryl, indazolyl, quinolyl, isoquinolyl, 1,2-dihydroisoquinolyl, 1,2,3,4-tetrahydroisoquinolyl, indolyl, isoindolyl, phthalazinyl, quinoxalinyl, benzofuranyl, 2,3-dihydrobenzofuran-1-yl, 2,3-dihydrobenzofuran-2-yl, 2,3-dihydrobenzothiophen-1-yl, 2,3-dihydrobenzothiophen-2-yl, benzothiazolyl, benzimidazolyl, fluorenyl, or thioxanthenyl.
• an aryl group may have one or more optional substituents on its ring.
• substituents include, but are not limited to, an alkoxy group, a halogen atom, an amino group, a mono- or di-substituted amino group, a substituted silyl group, or an acyl.
• an aryl group has two or more substituents, they may be the same or different. The same applies to the aryl moiety of other substituents containing an aryl moiety (e.g., an aryloxy group or an arylalkyl group).
• alkoxy group refers to a structure in which the alkyl group is bonded to an oxygen atom, and examples of such alkoxy groups include saturated alkoxy groups that are linear, branched, cyclic, or a combination thereof.
• Suitable examples include methoxy, ethoxy, n-propoxy, isopropoxy, cyclopropoxy, n-butoxy, isobutoxy, s-butoxy, t-butoxy, cyclobutoxy, cyclopropylmethoxy, n-pentyloxy, cyclopentyloxy, cyclopropylethyloxy, cyclobutylmethyloxy, n-hexyloxy, cyclohexyloxy, cyclopropylpropyloxy, cyclobutylethyloxy, and cyclopentylmethyloxy.
• alkylene refers to a divalent group consisting of a linear or branched saturated hydrocarbon, and examples of such groups include methylene, 1-methylmethylene, 1,1-dimethylmethylene, ethylene, 1-methylethylene, 1-ethylethylene, 1,1-dimethylethylene, 1,2-dimethylethylene, 1,1-diethylethylene, 1,2-diethylethylene, 1-ethyl-2-methylethylene, trimethylene, 1-methyltrimethylene, 2-methyltrimethylene, 1,1-dimethyltrimethylene, 1, Examples include 2-dimethyltrimethylene, 2,2-dimethyltrimethylene, 1-ethyltrimethylene, 2-ethyltrimethylene, 1,1-diethyltrimethylene, 1,2-diethyltrimethylene, 2,2-diethyltrimethylene, 2-ethyl-2-methyltrimethylene, tetramethylene, 1-methyltetramethylene, 2-methyltetramethylene, 1,1-dimethyltetramethylene, 1,2-d
• Fluorescent probe for detecting pancreatic juice comprising a compound represented by general formula (I) or a salt thereof
• One embodiment of the present invention is a fluorescent probe for detecting pancreatic juice comprising a compound represented by the following general formula (I) or a salt thereof (hereinafter also referred to as the "fluorescent probe of the present invention").
• N is selected from any of the groups represented by the following formulas (1) to (6).
• the inventors have investigated the possibility of using various fluorescent probes for detecting protease and lipid metabolic enzymes as probes for detecting pancreatic juice, and have found that fluorescent probes for detecting lipid metabolism that target enzymes having a phosphate group are useful. Furthermore, as a result of carrying out detailed studies using a library of phosphate ester type probes and phosphate diester type probes as such probes, they have found that a compound in which a group selected from any of formulas (1) to (6) is bound to a fluorophore via a phosphate diester or phosphate monoester group can be used as a probe for detecting pancreatic juice.
• the fluorescent probe of the present invention having any of the groups represented by formulae (1) to (6) can effectively detect pancreatic juice, and among these, those having the groups represented by formulae (4) and (5) are preferred, and those having the group represented by formula (4) are more preferred due to their high specificity for pancreatic juice.
• the level of specificity to pancreatic juice can be determined from the ratio of the fluorescence intensity of the probe in the pancreatic juice to the fluorescence intensity of the probe in the ascites.
• Y is an oxygen atom (O) or a nitrogen atom (N).
• L is selected from -NH-L 1 -, -OL 1 -, or a bond.
• bond means that there is no -NH-L 1 - or -OL 1 - group as L, and an oxygen atom (O) or nitrogen atom (N) represented by Y is directly bonded to S.
• L1 represents a linker. Any group can be used as the linker, but preferably, for example, —CO—, —CH 2 —, and a combination of any two or more thereof.
• S represents a fluorescent group.
• the fluorophore is preferably one represented by any one of the following formulae (II) to (IV).
• R 1 represents the same or different monovalent substituents present on the benzene ring.
• the type of monovalent substituent represented by R 1 is not particularly limited, but is preferably selected from the group consisting of, for example, an alkyl group having 1 to 6 carbon atoms, an alkenyl group having 1 to 6 carbon atoms, an alkynyl group having 1 to 6 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, a hydroxyl group, a carboxy group, a sulfonyl group, an alkoxycarbonyl group, a halogen atom, and an amino group.
• These monovalent substituents may further have one or more optional substituents.
• the alkyl group represented by R 1 may have one or more halogen atoms, carboxy groups, sulfonyl groups, hydroxyl groups, amino groups, alkoxy groups, etc., and for example, the alkyl group represented by R 1 may be a halogenated alkyl group, a hydroxyalkyl group, a carboxyalkyl group, or an aminoalkyl group.
• R 1 is an alkoxy group having 1 to 6 carbon atoms, more preferably an alkoxy group having 1 to 3 carbon atoms, still more preferably an ethoxy group or a methoxy group, and particularly preferably a methoxy group.
• R 1 is introduced as R 1 , it is possible to control the fluorescence by increasing the electron density, which is preferable.
• R1 is at least one selected from the group consisting of an alkyl group having 1 to 6 carbon atoms and a hydroxyl group. Introduction of these groups is also preferred because it is possible to increase the electron density.
• n is an integer of 0 to 4, and when m is 2 or more, each R 1 may be the same or different.
• R 1 can be introduced at any position depending on the type of the substituent.
• R2 represents a hydrogen atom or a monovalent substituent.
• the type of the monovalent substituent represented by R2 is not particularly limited, but like R1 , examples thereof include an alkyl group having 1 to 6 carbon atoms, an alkenyl group having 1 to 6 carbon atoms, an alkynyl group having 1 to 6 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, a hydroxyl group, a carboxy group, a sulfonyl group, an alkoxycarbonyl group, a halogen atom, and an amino group.
• R2 is an alkyl group having 1 to 6 carbon atoms (preferably a methyl group), a carboxyl group, a methoxy group or a hydroxymethyl group.
• R 3 and R 4 each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom.
• the alkyl group may contain one or more halogen atoms, carboxy groups, sulfonyl groups, hydroxyl groups, amino groups, alkoxy groups, etc., and for example, the alkyl group represented by R3 or R4 may be a halogenated alkyl group, a hydroxyalkyl group, a carboxyalkyl group, etc.
• R3 and R4 each independently represent a hydrogen atom or a halogen atom, and it is more preferable that R3 and R4 both represent a hydrogen atom, or that R3 and R4 both represent a fluorine atom or a chlorine atom.
• R 5 and R 6 each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom.
• the alkyl group may contain one or more halogen atoms, carboxy groups, sulfonyl groups, hydroxyl groups, amino groups, alkoxy groups, etc., and for example, the alkyl group represented by R5 or R6 may be a halogenated alkyl group, a hydroxyalkyl group, a carboxyalkyl group, etc. It is preferable that R 5 and R 6 both represent a hydrogen atom, both represent a chlorine atom, or both represent a fluorine atom.
• Z is an oxygen atom or NR a .
• R a is a hydrogen atom or a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms.
• R a is preferably an ethyl group.
• X is an oxygen atom.
• R b and R c are each independently an alkyl group or an aryl group having 1 to 6 carbon atoms.
• R b and R c are each preferably independently an alkyl group having 1 to 3 carbon atoms, and more preferably both R b and R c are a methyl group.
• the alkyl group represented by R b and R c may have one or more halogen atoms, carboxy groups, sulfonyl groups, hydroxyl groups, amino groups, alkoxy groups, etc., and for example, the alkyl group represented by R b or R c may be a halogenated alkyl group, a hydroxyalkyl group, a carboxyalkyl group, etc.
• R b or R c represents an aryl group
• the aryl group may be either a monocyclic aromatic group or a condensed aromatic group, and the aryl ring may contain one or more ring-constituting heteroatoms (e.g., nitrogen atoms, oxygen atoms, sulfur atoms, etc.).
• the aryl group is preferably a phenyl group.
• One or more substituents may be present on the aryl ring.
• one or more halogen atoms, carboxy groups, sulfonyl groups, hydroxyl groups, amino groups, alkoxy groups, etc. may be present as the substituents.
• ** represents the point of bonding with L in general formula (I).
• R7 and R8 each independently represent a hydrogen atom or a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, preferably an ethyl group.
• R 7 and R 8 may be taken together with the nitrogen atom to which they are bonded to form a 5- to 7-membered heterocyclyl which may have a substituent and which contains one or more heteroatoms selected from the group consisting of oxygen atoms, nitrogen atoms, and sulfur atoms.
• R 7 or R 8 may be taken together with R 4 or R 6 to form a 5-7 membered heterocyclyl or heteroaryl containing the nitrogen atom to which R 7 or R 8 is bonded
• the heterocyclyl or heteroaryl ring may contain 1-3 additional heteroatoms selected from the group consisting of oxygen atoms, nitrogen atoms and sulfur atoms as constituent members
• the heterocyclyl or heteroaryl may be substituted with at least one group selected from the group consisting of alkyl having 1-6 carbon atoms, alkenyl having 2-6 carbon atoms, alkynyl having 2-6 carbon atoms, aralkyl group having 6-10 carbon atoms and alkyl-substituted alkenyl group having 6-10 carbon atoms.
• the heterocyclyl or heteroaryl may have one or more substituents.
• R 9 represents a hydrogen atom or a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms.
• the alkyl group is preferably an ethyl group.
• T is --CO-- or --CH 2 --.
• R 1 represents the same or different monovalent substituents present on the benzene ring.
• the type of monovalent substituent represented by R 1 is not particularly limited, but is preferably selected from the group consisting of, for example, an alkyl group having 1 to 6 carbon atoms, an alkenyl group having 1 to 6 carbon atoms, an alkynyl group having 1 to 6 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, a hydroxyl group, a carboxy group, a sulfonyl group, an alkoxycarbonyl group, a halogen atom, and an amino group.
• These monovalent substituents may further have one or more optional substituents.
• the alkyl group represented by R 1 may have one or more halogen atoms, carboxy groups, sulfonyl groups, hydroxyl groups, amino groups, alkoxy groups, etc., and for example, the alkyl group represented by R 1 may be a halogenated alkyl group, a hydroxyalkyl group, a carboxyalkyl group, or an aminoalkyl group.
• R 1 is absent (ie, m is 0).
• n' is an integer of 0 to 4, and when m' is 2 or more, each R 1 may be the same or different.
• R 1 can be introduced at any position depending on the type of the substituent.
• R 2 and R 3 each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom.
• the alkyl group may contain one or more halogen atoms, carboxy groups, sulfonyl groups, hydroxyl groups, amino groups, alkoxy groups, etc., and for example, the alkyl group represented by R2 or R3 may be a halogenated alkyl group, a hydroxyalkyl group, a carboxyalkyl group, etc.
• R2 and R3 each independently represent a hydrogen atom or a halogen atom, and it is more preferable that R2 and R3 both represent a hydrogen atom, or that R2 and R3 both represent a fluorine atom or a chlorine atom.
• R 4 and R 5 each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom.
• the alkyl group may contain one or more halogen atoms, carboxy groups, sulfonyl groups, hydroxyl groups, amino groups, alkoxy groups, etc., and for example, the alkyl group represented by R4 or R5 may be a halogenated alkyl group, a hydroxyalkyl group, a carboxyalkyl group, etc. It is preferable that R 4 and R 5 both represent a hydrogen atom, both represent a chlorine atom, or both represent a fluorine atom.
• R6 is selected from NRaRb , OH or ORc .
• R a and R b each independently represent a hydrogen atom, a substituted or unsubstituted alkyl group having 1 to 5 carbon atoms, or a fluorinated alkyl group having 1 to 5 carbon atoms.
• R c represents an alkyl group having 1 to 5 carbon atoms.
• R 6 when R 6 is NR a R b , R a and R b may be taken together with the nitrogen atom to which they are bonded to form a 5- to 7-membered heterocyclyl which may have a substituent and which contains one or more heteroatoms selected from the group consisting of oxygen atoms, nitrogen atoms, and sulfur atoms.
• R6 when R6 is NR a R b , R a or R b , or both R a and R b , may be taken together with R 2 or R 4 , respectively, to form a 5-7 membered heterocyclyl or heteroaryl containing the nitrogen atom to which R a or R b is bonded.
• the heterocyclyl or heteroaryl ring may contain 1 to 3 additional heteroatoms selected from the group consisting of oxygen atoms, nitrogen atoms, and sulfur atoms as constituent members. Furthermore, the heterocyclyl or heteroaryl may be substituted with at least one group selected from the group consisting of alkyl groups having 1 to 6 carbon atoms, alkenyl groups having 2 to 6 carbon atoms, alkynyl groups having 2 to 6 carbon atoms, aralkyl groups having 6 to 10 carbon atoms, and alkyl-substituted alkenyl groups having 6 to 10 carbon atoms.
• R 7 and R 8 when present, each independently represent an alkyl group having 1 to 6 carbon atoms or an aryl group. However, when B is an oxygen atom, R 7 and R 8 do not exist. When B is a phosphorus atom, either R 7 or R 8 is ⁇ O.
• R 7 and R 8 are each preferably independently an alkyl group having 1 to 3 carbon atoms, and more preferably both R 7 and R 8 are methyl groups.
• the alkyl group represented by R 7 and R 8 may have one or more halogen atoms, carboxy groups, sulfonyl groups, hydroxyl groups, amino groups, alkoxy groups, etc., and for example, the alkyl group represented by R 7 or R 8 may be a halogenated alkyl group, a hydroxyalkyl group, a carboxyalkyl group, etc.
• the aryl group may be either a monocyclic aromatic group or a condensed aromatic group, and the aryl ring may contain one or more ring-constituting heteroatoms (e.g., nitrogen atoms, oxygen atoms, sulfur atoms, etc.).
• the aryl group is preferably a phenyl group.
• One or more substituents may be present on the aryl ring. For example, one or more halogen atoms, carboxy groups, sulfonyl groups, hydroxyl groups, amino groups, alkoxy groups, etc. may be present as the substituents.
• A represents an oxygen atom or NR d , where R d represents a hydrogen atom or an alkyl group having 1 to 5 carbon atoms.
• B represents an oxygen atom, a silicon atom, a carbon atom, or a phosphorus atom, and is preferably an oxygen atom or a silicon atom.
• n' is an integer from 1 to 3, preferably 1 or 2, and more preferably 1.
• R 1 if present, represents the same or different monovalent substituents present on the benzene ring. Details of R 1 in formula (IVa) are the same as those described above for R 1 in formula (IV).
• m' is an integer of 0 to 4.
• each R 1 may be the same or different.
• R 2 and R 3 each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom. Details of R2 and R3 in formula (IVa) are the same as those described above for R2 and R3 in formula (IV).
• R 4 and R 5 each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom. Details of R 4 and R 5 in formula (IVa) are the same as those described above for R 4 and R 5 in formula (IV).
• R a and R b each independently represent a hydrogen atom, an alkyl group having 1 to 5 carbon atoms, or a fluorinated alkyl group having 1 to 5 carbon atoms.
• R a and R b may be taken together with the nitrogen atom to which they are bonded to form a 5- to 7-membered heterocyclyl which may have a substituent and which contains one or more heteroatoms selected from the group consisting of oxygen atoms, nitrogen atoms, and sulfur atoms.
• R a or R b may be taken together with R 2 or R 4 , respectively, to form a 5-7 membered heterocyclyl or heteroaryl containing the nitrogen atom to which R a or R b is bonded, and the heterocyclyl or heteroaryl ring may contain 1 to 3 additional heteroatoms selected from the group consisting of oxygen atoms, nitrogen atoms and sulfur atoms as constituent members, and the heterocyclyl or heteroaryl may be substituted with at least one group selected from the group consisting of alkyl having 1 to 6 carbon atoms, alkenyl having 2 to 6 carbon atoms, alkynyl having 2 to 6 carbon atoms, aralkyl groups having 6 to 10 carbon atoms and alkyl-substituted alkenyl groups having 6 to 10 carbon atoms.
• Y is an oxygen atom (O) and L is a bond, which is a compound represented by the following general formula (Ia):
• the compounds represented by the general formulas (I) and (Ia) or their salts can exist as acid addition salts or base addition salts.
• acid addition salts include mineral acid salts such as hydrochloride, sulfate, and nitrate, and organic acid salts such as methanesulfonate, p-toluenesulfonate, oxalate, citrate, tartrate, and trifluoroacetate.
• Examples of base addition salts include metal salts such as sodium salt, potassium salt, calcium salt, and magnesium salt, ammonium salt, and organic amine salts such as triethylamine salt.
• salts may be formed with amino acids such as glycine.
• the compounds represented by the general formulas (I) and (Ia) or their salts may exist as hydrates or solvates, and these substances can also be used in the present invention.
• the compounds represented by general formulas (I) and (Ia) or salts thereof may have one or more asymmetric carbons depending on the type of substituent, but in the present invention, stereoisomers such as optically active substances based on one or more asymmetric carbons and diastereoisomers based on two or more asymmetric carbons, as well as any mixture of stereoisomers and racemates can be used.
• the compounds represented by general formulas (I) and (Ia) or salts thereof may exist as hydrates or solvates, and all of these substances are included in the scope of the present invention.
• the type of solvent that forms the solvate is not particularly limited, but examples include solvents such as ethanol, acetone, and isopropanol.
• the method for producing the compounds represented by general formulas (I) and (Ia) is not particularly limited, but a person skilled in the art can produce a compound encompassed by general formula (I) by appropriately modifying or altering the starting materials, reaction reagents, reaction conditions, etc. as necessary, while referring to the description in this specification and the previous research of the present inventors (e.g., Kawaguchi et al., "Synthesis of Fluorescent probes targeting tumor-suppressor protein (FHIT) and identification of apoptosis-inducing FHIT inhibitors” (J. Med. Chem. 2021, 64, 9567-9576), etc.).
• FHIT tumor-suppressor protein
• Non-limiting examples of the compounds represented by general formulas (I) and (Ia) that can be used as the fluorescent probe of the present invention are shown below, but the compounds of the fluorescent probe of the present invention are not limited to these.
• the fluorescent probe of the present invention itself is substantially non-fluorescent in the neutral region (for example, in the range of pH 5 to 9).
• the fluorescent probe of the present invention which contains a compound represented by general formula (I) or (Ia) or a salt thereof as an active ingredient, is believed to be hydrolyzed by phosphodiesterase believed to be present in pancreatic juice, preferably ENPP1 (ectonucleotide pyrophosphatase/phosphodiesterase 1) or ENPP3, and has the property of giving a compound that emits strong fluorescence.
• ENPP1 ectonucleotide pyrophosphatase/phosphodiesterase 1
• ENPP3 ectonucleotide pyrophosphatase/phosphodiesterase 1
• the method of using the fluorescent probe of the present invention is not particularly limited, and it can be used in the same manner as the conventionally known fluorescent probe.
• the compound of formula (I) or its salt is dissolved in an aqueous medium such as physiological saline or a buffer solution, or a mixture of a water-miscible organic solvent such as ethanol, acetone, ethylene glycol, dimethyl sulfoxide, or dimethylformamide with an aqueous medium, and the fluorescence spectrum is measured by adding this solution to an appropriate buffer solution containing pancreatic juice.
• the fluorescent probe of the present invention may be used in the form of a composition in combination with an appropriate additive.
• the fluorescent probe of the present invention can be combined with additives such as a buffer, a solubilizing agent, or a pH adjusting agent.
• additives such as a buffer, a solubilizing agent, or a pH adjusting agent.
• concentrations of the compounds of general formulae (I) and (Ia) or salts thereof in the fluorescent probe of the present invention can be appropriately determined depending on the type of pancreatic juice sample to be measured, the measurement conditions, and the like.
• the fluorescent probe of the present invention can be used as a contrast agent to visualize pancreatic juice in the patient's body cavity during surgery, and can be observed with the naked eye through an imaging device or filter that can irradiate appropriate excitation light during pancreatic resection or digestive surgery where pancreatic juice leakage is a concern. If leakage of pancreatic juice from the pancreatic stump, anastomosis, or injury is identified, additional procedures such as additional sutures or strict placement of a peritoneal drain can be performed.
• the fluorescent probe of the present invention is also expected to be clinically applicable as an in vitro diagnostic agent for diagnosing pancreatic leakage after gastrointestinal surgery.
• the fluorescent probe of the present invention is used to rapidly measure the fluorescence activity of ascites and pancreatic juice collected from a drain after surgery at the bedside. If the presence or absence of pancreatic leakage and the risk of it becoming severe can be determined quickly and accurately during treatment of the patient, the doctor in charge can decide on the spot whether or not to remove the drain, which will lead to safe and efficient management of patients after gastrointestinal surgery.
• the fluorescent probe is brought into contact with a body fluid sample, and a fluorescent response due to a reaction between phosphodiesterase contained in the sample and the fluorescent probe is observed, thereby making it possible to detect the presence of pancreatic juice.
• the phosphodiesterase is preferably ENPP1 or ENPP3, and more preferably ENPP1.
• another embodiment of the present invention is a method for detecting pancreatic juice, comprising: (a) a step of contacting a fluorescent probe of the present invention with a body fluid sample; and (b) a step of detecting the presence of pancreatic juice by observing a fluorescent response or a change in absorbance due to a reaction between the sample and the fluorescent probe (hereinafter also referred to as the "pancreatic juice detection method of the present invention").
• the body fluid sample preferably contains phosphodiesterase.
• the phosphodiesterase is preferably ENPP1 or ENPP3, and more preferably ENPP1.
• detection should be interpreted in the broadest sense to include measurements for various purposes, such as quantitative and qualitative measurements.
• the form of the body fluid sample that is the subject of fluorescence detection is not particularly limited, but examples include liquid collected from the abdominal cavity of a patient during or after surgery, a resected pancreas attached to any absorbent material such as a piece of paper such as filter paper or a towel, or liquid attached to or flowing out of the surface of the pancreas itself that remains in the body after removal of a tumor.
• Typical means for contacting the body fluid sample (subject) with the fluorescent probe include adding, applying, or spraying a solution containing the fluorescent probe to the sample, but the means can be appropriately selected depending on the form of the body fluid sample, the measurement environment, etc.
• the means for observing the fluorescence response can be a fluorometer with a wide measurement wavelength range, but it is also possible to use a fluorescence imaging device that can display the fluorescent emission site as a two-dimensional image.
• a fluorescence imaging means By using a fluorescence imaging means, the fluorescence response can be visualized in two dimensions, making it possible to instantly visually identify the position of the pancreatic duct or the location where pancreatic juice leakage is occurring.
• a small, portable fluorescence detector and fluorescence imaging device are preferable for measuring on-site body fluid samples collected from subjects during surgery, etc.
• the pancreatic juice detection method of the present invention can be performed, for example, during surgery, examination, or after surgery.
• the term "surgery” includes any surgery applied to treat pancreatic diseases such as pancreatic cancer and bile duct cancer, including endoscopic surgery using an endoscope or laparoscope.
• the term “examination” includes examinations using an endoscope and procedures such as tissue resection and collection associated with the examinations, as well as examinations performed on tissues separated or collected from a living body. These terms must be interpreted in the broadest sense and must not be interpreted in any restrictive way.
• the concentration of the fluorescent probe of the present invention is not particularly limited, but a solution with a concentration of, for example, about 1 to 1,000 ⁇ M can be used.
• the compound represented by the above general formula (I) or (Ia) or a salt thereof may be used as it is, but if necessary, additives normally used in the preparation of reagents may be added to the compound to be used as a composition.
• additives such as solubilizing agents, pH regulators, buffers, and isotonicity agents can be used as additives for using the reagent in a physiological environment, and the amounts of these additives can be appropriately selected by those skilled in the art.
• These compositions are generally provided as compositions in an appropriate form such as a powder mixture, lyophilized product, granules, tablets, or liquid, but may be applied by dissolving in distilled water for injection or an appropriate buffer solution when used.
• pancreatic juice detection kit In the pancreatic juice detection method of the present invention, it is preferable to use a pancreatic juice detection kit containing the above-mentioned fluorescent probe.
• the fluorescent probe of the present invention is usually prepared as a solution, but it can also be provided as a composition in an appropriate form, such as a powder mixture, a lyophilized product, granules, tablets, or liquid, and can be applied by dissolving it in distilled water for injection or an appropriate buffer solution at the time of use.
• the kit may also contain other reagents as necessary.
• additives such as solubilizing agents, pH adjusting agents, buffering agents, and isotonicity agents can be used, and the amounts of these additives can be appropriately selected by those skilled in the art.
• Sample collection 84 samples of waste fluid from pancreatic tubes and peritoneal drains were collected on the 3rd and 7th postoperative days from 8 pancreatic resection surgeries performed at the Department of Hepato-Biliary-Pancreatic Surgery and Artificial Organ Transplant Surgery at the University of Tokyo Hospital between June 2011 and August 2022. They were immediately frozen at -80°C and stored frozen until immediately before analysis. The samples were collected in the inpatient ward of the Department of Hepato-Biliary-Pancreatic Surgery and Artificial Organ Transplant Surgery at the University of Tokyo Hospital, and the waste fluid samples were analyzed in the laboratory of the Department of Drug Metabolism Chemistry, graduate School of Pharmaceutical Sciences, The University of Tokyo.
• Figure 1 shows the characteristics of patients in cases #1-8.
• pancreatic juice and ascites fluids selected in the order of (1) Winslow (2) upper border of the pancreas, which are less susceptible to the anastomosis
• pancreatic juice/ascites fluid was compared.
• Forty types of probes were ranked, and among the probes that showed strong fluorescent activity in pancreatic juice, probes 14, 15, 16, and 17, which react with phosphodiesterase that hydrolyzes nucleotides with pyrimidine bases, showed high values in 4/8 cases ( Figures 3 and 4).
• Fig. 4 shows the results of fluorescence intensity evaluation of pancreatic juice using the lipid metabolism analysis probe library
• Fig. 5 shows the results of fluorescence intensity evaluation of ascites using the lipid metabolism analysis probe library.
• 1, 1 to 4 are phosphatase probes, 5 and 8 to 17 are phosphodiesterase probes, 18 to 26 and 29 to 34 are phosphodiesterase probes (FHIT/Ap 3 Aase), and 35 to 42 are Rhodol-based phosphodiesterase probes.
• Figure 3 shows the ranking of 40 types of probes for PJ (pancreatic juice)/ascites in cases #1-8
• Figure 6 shows the results of the fluorescence intensity evaluation of pancreatic juice/ascites for the 40 types of probes.
• Figures 3 and 6 confirm that Probe.14 (TG-CMP), Probe.15 (TG-dCMP), Probe.16 (TG-UMP) and Probe.17 (TG-TMP) showed a large increase in fluorescence intensity in pancreatic juice.
• FIG. 7 shows the measurement results (arbitrary units) of the fluorescence intensity of Probe. 17 in pancreatic juice (PJ) and ascites (Ascites).
• PJ pancreatic juice
• Ascites ascites
• Example 2 The time course of the fluorescence intensity was evaluated using inhibitors of ENPP1 and ENPP3, which are known to be related to the inhibitor test Probe.17.
• the fluorescence intensity was measured using an inhibitor with relatively high specificity for ENPP1 (compound C) and a compound that inhibits both ENPP1 and ENPP3 (compound 6-53) developed by Kawaguchi et al. (Synthesis of Fluorescent probes targeting tumor-suppressor protein (FHIT) and identification of apoptosis-inducing FHIT inhibitors (J. Med. Chem. 2021, 64, 9567-9576)).
• FHIT tumor-suppressor protein
• HMRG Derivative Probe A fluorescent probe of the HMRG derivative type, which is the fluorescent probe of the present invention, was synthesized.
• HMRG (19 mg, 0.050 mmol) was dissolved in t-BuOH/H 2 O (5 mL/1.7 mL), and guanosine 5'-monophosphate disodium salt hydrate (GMP-2Na, 61 mg, 0.15 mmol), 4-dimethylaminopyridine (DMAP, 10 mg, 0.082 mmol) and triethylamine (3 drops) were added.
• DMAP 4-dimethylaminopyridine
• DCC N,N'-dicyclohexylcarbodiimide
• HMRG (19 mg, 0.050 mmol) was dissolved in t-BuOH/H 2 O (3 mL/1 mL), and cytidine 5'-monophosphate (CMP, 49 mg, 0.15 mmol), 4-dimethylaminopyridine (DMAP, 6.1 mg, 0.050 mmol) and triethylamine (3 drops) were added. Then, N,N'-dicyclohexylcarbodiimide (DCC, 31 mg, 0.15 mmol) was added and the mixture was heated to reflux at 90°C for 30 minutes.
• CMP cytidine 5'-monophosphate
• DMAP 4-dimethylaminopyridine
• DMAP 4-dimethylaminopyridine
• triethylamine 3 drops
• HMRG 15 mg, 0.040 mmol was dissolved in t-BuOH/H 2 O (3 mL/1 mL), and uridine 5'-monophosphate disodium (UMP-2Na, 44 mg, 0.12 mmol), 4-dimethylaminopyridine (DMAP, 4.9 mg, 0.040 mmol) and triethylamine (3 drops) were added. N,N'-dicyclohexylcarbodiimide (DCC, 27 mg, 0.13 mmol) was then added and the mixture was heated to reflux at 90°C for 30 minutes.
• DCC N,N'-dicyclohexylcarbodiimide
• UMP-2Na 45 mg
• DCC 27 mg, 0.13 mmol
• DCC 7 mg, 0.13 mmol
• the residue was purified by preparative HPLC to obtain HMRG-UMP (0.9 mg, 0.0014 mmol, yield 3.6%) as a red solid.
• HMRG 15 mg, 0.040 mmol was dissolved in t-BuOH/H 2 O (3 mL/1 mL), and thymidine 5'-monophosphate disodium (TMP-2Na, 47 mg, 0.13 mmol), 4-dimethylaminopyridine (DMAP, 4.9 mg, 0.040 mmol) and triethylamine (3 drops) were added. Then, N,N'-dicyclohexylcarbodiimide (DCC, 27 mg, 0.13 mmol) was added and the mixture was heated to reflux at 90°C for 30 minutes.
• TMP-2Na thymidine 5'-monophosphate disodium
• DMAP 4-dimethylaminopyridine
• DCC N,N'-dicyclohexylcarbodiimide

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