WO2021070906A1 - Fluorescent probe for detecting puromycin-sensitive aminopeptidase or bleomycin hydrolase - Google Patents

Fluorescent probe for detecting puromycin-sensitive aminopeptidase or bleomycin hydrolase Download PDF

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WO2021070906A1
WO2021070906A1 PCT/JP2020/038123 JP2020038123W WO2021070906A1 WO 2021070906 A1 WO2021070906 A1 WO 2021070906A1 JP 2020038123 W JP2020038123 W JP 2020038123W WO 2021070906 A1 WO2021070906 A1 WO 2021070906A1
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present
fluorescent probe
alkyl group
puromycin
hydrogen atom
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泰照 浦野
真子 神谷
克行 保科
大輔 赤木
優五 栗木
明彦 瀬尾
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国立大学法人 東京大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
    • C07D493/02—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains two hetero rings
    • C07D493/10—Spiro-condensed systems
    • 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
    • C09B11/28—Pyronines ; Xanthon, thioxanthon, selenoxanthan, telluroxanthon dyes
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances

Definitions

  • the present invention relates to a fluorescent probe capable of detecting puromycin-sensitive aminopeptidase and bleomycin hydrolase.
  • the present invention also provides a method for evaluating the vascular properties of an aortic aneurysm lesion or a non-dilated arterial wall using such a fluorescent probe, and a method for detecting a normal arterial wall in an aortic aneurysm lesion or a non-dilated arterial wall. Also involved.
  • An object of the present invention is to provide a fluorescent probe capable of objectively analyzing and evaluating vascular properties.
  • Non-Patent Document 1 One of the present inventors has developed a fluorescent probe gGlu-HMRG and succeeded in visualizing cancer (Non-Patent Document 1). It has also been reported that EP-HMRG, which reacts with DPP-4, can visualize esophageal cancer (Non-Patent Document 2).
  • the present inventors have focused on the HMRG fluorophore and aimed to clarify what kind of probe is visualized in the dilated aneurysm lesions and normal arteries of clinically obtained specimens.
  • a compound in which methionine was introduced into the HMRG skeleton can objectively analyze and evaluate the vascular properties, and completed the present invention.
  • the present inventors have found that the target enzymes of the compound in which methionine is introduced into the HMRG skeleton are puromycin-sensitive aminopeptidase and bleomycin hydrolase, and these aminopeptidases are highly expressed in the intima of the normal arterial wall. I found that. Based on such findings, the present inventors can evaluate the vascular properties of aortic aneurysm lesions or non-dilated arterial walls by using fluorescent probes containing HMRG fluorescent groups capable of detecting these aminopeptidases. The present invention was completed with the idea that.
  • R 1 if present, is the same or different monovalent substituent present on the benzene ring
  • R 2 and R 3 are each independently a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom
  • R 4 and R 5 are each independently a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom
  • m is an integer from 0 to 4.
  • a fluorescent probe used to detect puromycin-sensitive aminopeptidase or bleomycin hydrolase which contains the following compound or a salt thereof.
  • a method for evaluating the vascular properties of aortic aneurysm lesions or non-dilated arterial walls which comprises (a) a compound represented by the following formula (1) or a salt thereof, and puromycin-sensitive aminopeptidase or bleomycin. This includes applying a fluorescent probe capable of detecting a hydrolytic enzyme to a clinical sample of an aortic aneurysm lesion or a non-dilated arterial wall, and (b) measuring a fluorescent image of the clinical sample to which the fluorescent probe is applied. ,Method.
  • R 1 if present, is the same or different monovalent substituent present on the benzene ring;
  • R 2 and R 3 are each independently a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom;
  • R 4 and R 5 are each independently a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom;
  • R 6 and R 6 ' are each independently hydrogen atom or a C 1-5 alkyl group having a carbon;
  • m is an integer from 0 to 4;
  • P 1 and P 2 represent amino acid residues, and P 1 may or may not be present.
  • a method for detecting a normal arterial wall in an aortic aneurysm lesion or a non-dilated arterial wall which comprises (a) a compound represented by the following formula (1) or a salt thereof, and puromycin-sensitive aminopeptidase or The step of applying a fluorescent probe capable of detecting puromycin hydrolase to a clinical sample of an aortic aneurysm lesion or a non-dilated arterial wall, and (b) measuring the fluorescent image of the clinical sample to which the fluorescent probe is applied. Including, method.
  • R 1 if present, is the same or different monovalent substituent present on the benzene ring;
  • R 2 and R 3 are each independently a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom;
  • R 4 and R 5 are each independently a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom;
  • R 6 and R 6 ' are each independently hydrogen atom or a C 1-5 alkyl group having a carbon;
  • m is an integer from 0 to 4;
  • P 1 and P 2 represent amino acid residues, and P 1 may or may not be present.
  • Comparison of new probe Met-HMRG and DPPIV lobe by fluorescence intensity An example of fluorescence reaction of aneurysm wall, borderline lesion, and anastomotic artery wall using Met-HMRG immersion gel. Changes in fluorescence intensity over time and sensitivity / specificity in Met-HMRG Contrast of anastomotic artery wall and aneurysm wall according to the results of the DEG method. The results of an inhibition experiment in the fluorescence reaction between the anastomotic artery wall and the aneurysm wall using Puromycin and Bleomycin are shown. The time course of fluorescence intensity due to various purified enzymes and Met-HMRG is shown.
  • the alkyl moiety of an "alkyl group” or a substituent containing an alkyl moiety has, for example, 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms, unless otherwise specified. More preferably, it means an alkyl group composed of a straight chain, a branched chain, a cyclic chain, or a combination thereof having about 1 to 3 carbon atoms.
  • the alkyl groups include, for example, methyl group, ethyl group, n-propyl group, isopropyl group, cyclopropyl group, n-butyl group, sec-butyl group, isobutyl group, tert-butyl group and cyclopropyl.
  • Methyl group, n-pentyl group, n-hexyl group and the like can be mentioned.
  • halogen atom may be any of 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.
  • Fluorescent probe 1 of the present invention is a fluorescent probe used to detect a puromycin-sensitive aminopeptidase or bleomycin hydrolase containing a compound represented by the following formula (I) or a salt thereof. (Hereinafter, also referred to as "fluorescent probe 1 of the present invention").
  • R 1 if present, is the same or different monovalent substituent present on the benzene ring.
  • the monovalent substituent include halogen, an optionally substituted alkyl group and the like.
  • n is an integer from 0 to 4. In one preferred aspect of the invention, m is 0 and R 1 is absent and unsubstituted benzene ring.
  • R 2 and R 3 are each independently 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, a carboxy group, a sulfonyl group, a hydroxyl group, an amino group, an alkoxy group and the like.
  • the alkyl group indicated by R 2 or R 3 may be an alkyl halide group, a hydroxyalkyl group, a carboxyalkyl group or the like. It is preferable that both R 2 and R 3 are hydrogen atoms.
  • R 4 and R 5 are independently hydrogen atoms, alkyl groups having 1 to 6 carbon atoms, or halogen atoms.
  • the details of R 4 and R 5 are the same as those described for R 2 and R 3. It is preferable that both R 4 and R 5 are hydrogen atoms.
  • R 6 and R 6 ' are each independently ⁇ 1 hydrogen atom or a carbon 5 alkyl groups.
  • alkyl group of R 6 and R 6 ' a methyl group and an ethyl group are preferable.
  • R 6 and R 6 ' are both hydrogen atoms.
  • HMRG fluorescein Focusing on the HMRG fluorescein, the present inventors diligently investigated what kind of probe is useful for response visualization in dilated aneurysm lesions and normal arteries of clinically obtained specimens. It was found that the compound in which methionine (Met) was introduced into the skeleton significantly changed the fluorescence intensity in a test using a human abdominal aortic aneurysm surgical specimen, and also showed excellent results in an imaging experiment.
  • One preferred embodiment of the present invention is a fluorescent probe used to detect puromycin-sensitive aminopeptidase or bleomycin hydrolase, which contains the following compounds or salts thereof.
  • the compound having the above structure is also referred to as "Met-HMRG” below.
  • the compound of the formula (I) of the present invention and Met-HMRG can exist as an acid addition salt or a base addition salt.
  • the acid addition salt include mineral salts such as hydrochlorides, sulfates and nitrates, or organic acid salts such as methanesulfonates, p-toluenesulfonates, oxalates, citrates and tartrates.
  • the base addition salt include metal salts such as sodium salt, potassium salt, calcium salt and magnesium salt, ammonium salt, and organic amine salt such as triethylamine salt. In addition to these, it may form a salt with an amino acid such as glycine.
  • the compounds of the present invention or salts thereof may exist as hydrates or solvates, but these substances are also within the scope of the present invention.
  • the compound of the present invention may have one or two or more asymmetric carbons depending on the type of the substituent, but an optically active substance based on one or two or more asymmetric carbons or two or more asymmetric carbons.
  • stereoisomers such as diastereoisomers based on homocarbons, any mixture of stereoisomers, racemates and the like are all included in the scope of the present invention.
  • the Diced Electrophoresis Gel method (DEG method) was performed, and the target enzyme listed in the DEG method was examined by the Western blotting method.
  • DEG method Diced Electrophoresis Gel method
  • puromycin-sensitive aminopeptidase was examined.
  • bleomycin hydrolase were found to be expressed together.
  • puromycin-sensitive aminopeptidase was highly expressed in the endometrium.
  • one aspect of the present invention is a fluorescent probe used to detect puromycin-sensitive aminopeptidase, which contains the compound of the present invention or a salt thereof.
  • Another aspect of the present invention is a fluorescent probe used to detect a bleomycin hydrolase containing the compound of the present invention or a salt thereof.
  • Another aspect of the present invention is a fluorescent probe used to detect puromycin-sensitive aminopeptidase and bleomycin hydrolase, which contain the compound of the present invention or a salt thereof.
  • another embodiment of the present invention is a method for evaluating the vascular properties of an aortic aneurysm lesion or a non-dilated arterial wall, wherein (a) a compound represented by the following formula (1) or a salt thereof is used.
  • a method comprising measuring a fluorescent image of.
  • R 1 ⁇ R 6 ' and m are as defined in formula (I). That is, R 1 is the same or different monovalent substituent present on the benzene ring, if present; m is an integer of 0-4; R 2 and R 3 are independent of each other.
  • R 4 and R 5 are independently hydrogen atoms, an alkyl group having 1 to 6 carbon atoms, or a halogen atom;
  • R 6 and R 6 ' are independently a hydrogen atom or a C 1-5 alkyl group having a carbon.
  • P 1 and P 2 represent amino acid residues. Further, P 1 may or may not exist.
  • ⁇ -Amino acid residues glycine, alanine, leucine, isoleucine, valine, lysine, cysteine, threonine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, serine, histidine, phenylalanine, methionine, tryptophan, tyrosine, proline
  • Residues of derivatives N-methylleucine, 2,3-diaminopropanoic acid, 2,4-diaminobutyric acid, ornithine, ⁇ -hydroxyleucine, etc.
  • the amino acid residue and its derivative may be either an L-form amino acid or a D-form amino acid residue.
  • P 1 is represented by the following formula (a).
  • R 7 is a natural amino acid (glycine, alanine, leucine, isoleucine, valine, lysine, cysteine, threonine, arginine, asparagine, aspartic acid, glutamic acid, glutamic acid, serine, histidine, etc. , Phenylalanine, methionine, tryptophan, tyrosine, proline). Further, R 7 also includes a group other than the group constituting the side chain of the natural amino acid, for example, an alkyl group having various substituents and the like.
  • P 2 represents an N-terminal amino acid residue.
  • the amino acid residues of P 2 are the residue of glycine, alanine, leucine, isoleucine, valine, lysine, cysteine, threonine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, serine, histidine, phenylalanine, methionine, tryptophan, tyrosine and proline Selected from the group.
  • the amino acid residue of P 2 may be either an L-amino acid or a D-amino acid residue.
  • P 2 is represented by the following equation (b).
  • R 8 is hydrogen, such as a methyl group, a natural amino acid (glycine, alanine, leucine, isoleucine, valine, lysine, cysteine, threonine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, serine, histidine , Phenylalanine, methionine, tryptophan, tyrosine, proline).
  • a natural amino acid glycine, alanine, leucine, isoleucine, valine, lysine, cysteine, threonine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, serine, histidine , Phenylalanine, methionine, tryptophan, tyrosine, proline).
  • Met-HMRG is a fluorescent probe represented by the formula (1) and capable of detecting a puromycin-sensitive aminopeptidase or bleomycin hydrolase (hereinafter, also referred to as “fluorescent probe 2 of the present invention”).
  • P 1 does not exist and P 2 is a methionine residue
  • P 2 is a methionine residue
  • a method for evaluating the vascular properties of an aortic aneurysm lesion or a non-dilated arterial wall using a fluorescent probe represented by the formula (1) or the formula (1a) is within the scope of the present invention.
  • the fluorescent probe 2 of the present invention in the step (a) to a clinical specimen of an aortic aneurysm lesion or a non-dilated arterial wall, for example, dilute with a solution of the fluorescent probe 2 of the present invention (for example, 1xTBS buffer).
  • a solution of the fluorescent probe 2 of the present invention for example, 1xTBS buffer.
  • the concentration is about 50 ⁇ M) so that the entire sample is immersed.
  • clinical specimens include surgical specimens for aortic aneurysm lesions and non-dilated arterial walls.
  • the portion showing bright fluorescence has a high proportion of the normal arterial wall.
  • the proportion of the normal arterial wall is high because the fluorescence intensity itself is high, but preferably, the fluorescent probe 2 of the present invention is applied for a certain period of time (for example, 30 minutes, 60 minutes, etc.).
  • the rate of increase in fluorescence intensity after the lapse as an index it can be determined that the site with a high rate of increase also has a high rate of normal arterial wall.
  • one preferred aspect of the present invention is a method for evaluating the vascular properties of an aortic aneurysm lesion or a non-dilated arterial wall, in which a fluorescent probe containing Met-HMRG is used for clinical aortic aneurysm lesion or non-dilated arterial wall. It is a method including a step of applying to a sample and (b) measuring a fluorescent image of a clinical sample to which the fluorescent probe is applied.
  • the method of using the fluorescent probes 1 and 2 of the present invention is not particularly limited, and can be used in the same manner as the conventionally known fluorescent probes.
  • it is contained in fluorescent probes 1 and 2 in an aqueous medium such as physiological saline or a buffer solution, or a mixture of an aqueous medium and an aqueous solvent such as ethanol, acetone, ethylene glycol, dimethyl sulfoxide, or dimethyl formamide.
  • the compounds or salts thereof may be dissolved, the solution may be added to a suitable buffer containing cells or tissues, and the fluorescence spectrum may be measured.
  • the fluorescent probe of the present invention may be used in the form of a composition in combination with a suitable additive. For example, it can be combined with additives such as buffers, solubilizers and pH regulators.
  • Another aspect of the present invention is a method for detecting an aortic aneurysm lesion or a normal arterial wall in a non-dilated arterial wall, wherein (a) a compound represented by the following formula (1) or a salt thereof is used.
  • a method comprising measuring a fluorescent image of.
  • R 1 ⁇ R 6 ' and m are as defined above, P 1 and P 2 represents an amino acid residue. Further, P 1 may or may not exist.
  • the fluorescent probe 1 or 2 of the present invention will prevent complications such as anastomotic aneurysm and contribute to more accurate surgery.
  • A6 Synthesis A5 20% piperidine / N of, N'- dimethylformamide (v / v) 3 minutes shaken added 1200MyuL, the solution was removed by filtration. 1200 ⁇ L of 20% piperidine / N, N'-dimethylformamide solution (v / v) was added again and shaken for 12 minutes, and the solution was removed by filtration. 1350 ⁇ L of N, N'-dimethylformamide was added, and the mixture was shaken for 1 minute to remove the solution 6 times. The resin after the reaction was used as it was for the next reaction.
  • Example 1 Screening using human abdominal aortic aneurysm surgical specimens We compared the DPPIV fluorescent probes (EP-HMRG and GP-HMRG) that have been proven for esophageal cancer with the newly developed Met-HMRG. The experimental conditions are shown below. As a result, Met-HMRG showed a significant increase in fluorescence intensity compared to other probes (see FIG. 1).
  • the intima (including some media) of the iliac artery used as the anastomotic site was removed and converted to Lysate.
  • the protein concentration of Lysate was adjusted to 0.1 mg / ml so that the amount of protein was 2.25 ⁇ g.
  • Each probe was added dropwise to 1 ⁇ M, and the fluorescence intensity was measured at 1 min and 1 hr with Envision® at 37 ° C.
  • Example 2 Imaging Using Human Abdominal Aortic Aneurysm Surgery Specimens were collected from the aneurysm wall and anastomotic artery wall (normal artery wall) for open surgery cases performed on the abdominal aortic aneurysm, and gels immersed in Met-HMRG were used. After adaptation from the lumen side of the blood vessel, the fluorescence intensity was measured over time. As a result, an increase in fluorescence intensity was observed in the anastomotic artery wall as compared with the aneurysm wall (FIGS. 2 and 3). The experimental conditions are shown below.
  • Example 3 Identification of the target enzyme by the Diced Electrophoresis Gel method (DEG method) of Met-HMRG
  • DEG method Diced Electrophoresis Gel method
  • the DG method using the Lysate of the aneurysm wall and anastomotic artery of the case used in the screening to search for the target enzyme that reacts with Met-HMRG. was carried out.
  • the fluorescence intensity of the subdivided gel was measured using Envision (registered trademark), and a spot having a strong fluorescence reaction was recovered and mass spectrometry was performed.
  • Puromycin sensitive aminopeptidase puromycin-sensitive aminopeptidase
  • bleomycin hydrolase bleomycin hydrolase
  • Example 4 Inhibition experiment of Met-HMRG against target enzyme Puromycin and bleomycin hydrolase inhibitors, which are candidates for target enzyme by the DEG method, are used as puromycin and bleomycin. It was investigated whether the fluorescence reaction between wall Lysate and Met-HMRG was inhibited. The results showed that both of them inhibited the fluorescence reaction in a concentration-dependent manner, but in the Lysate of the anastomotic artery wall, Puromycin was more concentrated than bleomycin, and better concentration-dependent inhibition was observed (Fig. 5). The experimental conditions are shown below.
  • Example 5 Confirmation of Fluorescence Reaction Using Purified Enzyme with Met-HMRG (Puromycin sensitive aminopeptidase and Bleomycin hydrolase) From the inhibition experiment, it was confirmed whether the target enzymes Puromycin sensitive aminopeptidase and bleomycin hydrolase actually show a fluorescence reaction with Met-HMRG. As a result, it was shown that both were fluorescently reacted with Met-HMRG. The experimental conditions are shown below.
  • Example 6 Confirmation of target enzyme expression by Western blotting Western blotting was performed using Lysate of the arterial wall of the joint. As a result, the expression of Puromycin sensitive aminopeptidase and bleomycin hydrolase was observed in the Lysate of the anastomotic artery wall, and it was considered that the activity was also related to the expression of both. (Fig. 7). The experimental conditions are shown below.
  • the Lysate of the anastomotic artery used for screening was adjusted to a protein concentration of 0.5 mg / ml, and heat treatment was performed at 95 ° C. This protein was separated by SDS-PAGE. A PVDF membrane was used as the blotting membrane, and PSA antibody and BLMH antibody were used as primary antibodies at a concentration of 1/500. In addition, GAPDH antibody was used as a loading control at a concentration of 1/1000. The detection was performed by a color development method using HRP.
  • Example 7 Immunostaining of Puromycin sensitive aminopeptidase and Bleomycin hydrolase on the arterial wall of the anastomotic site Imaging with Met-HMRG was performed. As a result, it was confirmed that Puromycin sensitive aminopeptidase was more strongly expressed in the intima of the anastomotic artery than in the aneurysm wall (Fig. 9). Regarding bleomycin hydrolase, no difference was observed in the expression between the anastomotic artery wall and the aneurysm wall (Fig. 10). The experimental conditions are shown below.

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Abstract

[Problem] To provide a fluorescent probe that enables objective analysis and evaluation of blood vessel properties. [Solution] A fluorescent probe to be used for detecting puromycin-sensitive aminopeptidase or bleomycin hydrolase, the fluorescent probe comprising a compound represented by formula (I) or a salt thereof.

Description

ピューロマイシン感受性アミノペプチダーゼ又はブレオマイシン加水分解酵素検出用蛍光プローブ。Fluorescent probe for detecting puromycin-sensitive aminopeptidase or bleomycin hydrolase.
　本発明は、ピューロマイシン感受性アミノペプチダーゼ及びブレオマイシン加水分解酵素を検出することができる蛍光プローブに関わる。また、本発明は、かかる蛍光プローブを用いた、大動脈瘤病変又は非拡張部動脈壁の血管性状を評価する方法、及び、大動脈瘤病変又は非拡張部動脈壁における正常動脈壁を検知する方法にも関わる。 The present invention relates to a fluorescent probe capable of detecting puromycin-sensitive aminopeptidase and bleomycin hydrolase. The present invention also provides a method for evaluating the vascular properties of an aortic aneurysm lesion or a non-dilated arterial wall using such a fluorescent probe, and a method for detecting a normal arterial wall in an aortic aneurysm lesion or a non-dilated arterial wall. Also involved.
　血管外科手術に際し手術対象の血管壁の性状を適切に評価することは重要である。ヨード造影剤を用いたＣＴをはじめとする画像診断の進歩は著しいが、手術の際の血管壁の性状の評価は最終的には術者の経験に頼っているのが現状である。 It is important to properly evaluate the properties of the blood vessel wall to be operated on during vascular surgery. Although advances in diagnostic imaging such as CT using iodine contrast media have been remarkable, the current situation is that the evaluation of the properties of blood vessel walls during surgery ultimately depends on the experience of the surgeon.
　例えば、腹部大動脈瘤の開腹手術において、瘤切除後人工血管吻合部位をいかに選択するかは術者の主観的経験により決定されている。これは吻合部血管の性状に対して客観性を担保する物質や方法が存在しないためである。 For example, in open surgery for an abdominal aortic aneurysm, how to select an artificial blood vessel anastomosis site after aneurysm resection is determined by the subjective experience of the operator. This is because there is no substance or method that guarantees objectivity for the properties of the anastomotic vessel.
　また、時折、どこまで瘤（拡張病変）が続いているか判定し難い症例がある。　食道癌のように病変部位と反応するトルイジンブルーや正常部位と反応するヨードのような判定材料があるが、血管の正常部位を判定する材料や方法は見出されていない。 In addition, there are occasional cases where it is difficult to determine how long the aneurysm (dilated lesion) continues. There are judgment materials such as toluidine blue that reacts with the lesion site like esophageal cancer and iodine that reacts with the normal part, but no material or method has been found to judge the normal part of the blood vessel.
　従って、生体内における血管性状を客観的に分析評価することが可能となれば、より精度の高い手術を行える可能性があり、手術合併症軽減などに貢献する可能性がある。 Therefore, if it becomes possible to objectively analyze and evaluate the vascular properties in the living body, there is a possibility that more accurate surgery can be performed, which may contribute to reduction of surgical complications.
　本発明は、血管性状を客観的に分析評価することが可能な蛍光プローブを提供することを目的とする。 An object of the present invention is to provide a fluorescent probe capable of objectively analyzing and evaluating vascular properties.
　本発明者らの一人は、これまで蛍光プローブｇＧｌｕ－ＨＭＲＧを開発し、癌の可視化を成功させた（非特許文献１）。
また、ＤＰＰ－４と反応するＥＰ－ＨＭＲＧでは、食道癌の可視化が可能であることも報告されている（非特許文献２）。 One of the present inventors has developed a fluorescent probe gGlu-HMRG and succeeded in visualizing cancer (Non-Patent Document 1).
It has also been reported that EP-HMRG, which reacts with DPP-4, can visualize esophageal cancer (Non-Patent Document 2).
　そこで、本発明者らは、ＨＭＲＧ蛍光団に着目して、臨床から得られた検体の拡張性瘤化病変や正常動脈において、どのようなプローブに応答可視化されるかを明らかにすることを目標にして鋭意検討した結果、ＨＭＲＧ骨格にメチオニンを導入した化合物が血管性状を客観的に分析評価することが可能であることを見出し、本発明を完成した。 Therefore, the present inventors have focused on the HMRG fluorophore and aimed to clarify what kind of probe is visualized in the dilated aneurysm lesions and normal arteries of clinically obtained specimens. As a result of diligent studies, it was found that a compound in which methionine was introduced into the HMRG skeleton can objectively analyze and evaluate the vascular properties, and completed the present invention.
　また、本発明者らは、ＨＭＲＧ骨格にメチオニンを導入した化合物のターゲット酵素がピューロマイシン感受性アミノペプチダーゼ及びブレオマイシン加水分解酵素であることを見出し、これらアミノペプチダーゼが正常動脈壁の内膜において高発現していることを発見した。かかる知見に基づき、本発明者らは、これらアミノペプチダーゼを検出することができるＨＭＲＧ蛍光団を含む蛍光プローブを用いることにより、大動脈瘤病変又は非拡張部動脈壁の血管性状を評価することが可能であると着想して、本発明を完成した。 In addition, the present inventors have found that the target enzymes of the compound in which methionine is introduced into the HMRG skeleton are puromycin-sensitive aminopeptidase and bleomycin hydrolase, and these aminopeptidases are highly expressed in the intima of the normal arterial wall. I found that. Based on such findings, the present inventors can evaluate the vascular properties of aortic aneurysm lesions or non-dilated arterial walls by using fluorescent probes containing HMRG fluorescent groups capable of detecting these aminopeptidases. The present invention was completed with the idea that.
　即ち、本発明は、
［１］以下の式（Ｉ）で表される化合物又はその塩を含有する、ピューロマイシン感受性アミノペプチダーゼ又はブレオマイシン加水分解酵素を検出するのに用いられる蛍光プローブ。
Figure JPOXMLDOC01-appb-I000005


（式中、
Ｒ１は、存在する場合は、ベンゼン環上に存在する同一又は異なる一価の置換基であり；
Ｒ２及びＲ３は、それぞれ独立に、水素原子、炭素数１～６個のアルキル基又はハロゲン原子であり；
Ｒ４及びＲ５は、それぞれ独立に、水素原子、炭素数１～６個のアルキル基又はハロゲン原子であり；
Ｒ６及びＲ６’は、それぞれ独立に、水素原子又は炭素数１～５個のアルキル基であり；
ｍは、０～４の整数である。）
［２］以下の化合物又はその塩を含有する、ピューロマイシン感受性アミノペプチダーゼ又はブレオマイシン加水分解酵素を検出するのに用いられる蛍光プローブ。
Figure JPOXMLDOC01-appb-I000006


［３］大動脈瘤病変又は非拡張部動脈壁の血管性状を評価する方法であって、（ａ）以下の式（１）で表される化合物又はその塩を含み、ピューロマイシン感受性アミノペプチダーゼ又はブレオマイシン加水分解酵素を検出することができる蛍光プローブを大動脈瘤病変又は非拡張部動脈壁の臨床検体に適用する工程、及び（ｂ）前記蛍光プローブを適用した臨床検体の蛍光像を測定することを含む、方法。
Figure JPOXMLDOC01-appb-I000007


（式中、
Ｒ１は、存在する場合は、ベンゼン環上に存在する同一又は異なる一価の置換基であり；
Ｒ２及びＲ３は、それぞれ独立に、水素原子、炭素数１～６個のアルキル基又はハロゲン原子であり；
Ｒ４及びＲ５は、それぞれ独立に、水素原子、炭素数１～６個のアルキル基又はハロゲン原子であり；
Ｒ６及びＲ６’は、それぞれ独立に、水素原子又は炭素数１～５個のアルキル基であり；
ｍは、０～４の整数であり；
Ｐ１及びＰ２は、アミノ酸残基を表し、Ｐ１は、存在しても、存在しなくてもよい。）
［４］大動脈瘤病変又は非拡張部動脈壁における正常動脈壁を検知する方法であって、（ａ）以下の式（１）で表される化合物又はその塩を含み、ピューロマイシン感受性アミノペプチダーゼ又はブレオマイシン加水分解酵素を検出することができる蛍光プローブを大動脈瘤病変又は非拡張部動脈壁の臨床検体に適用する工程、及び（ｂ）前記蛍光プローブを適用した臨床検体の蛍光像を測定することを含む、方法。
Figure JPOXMLDOC01-appb-I000008


（式中、
Ｒ１は、存在する場合は、ベンゼン環上に存在する同一又は異なる一価の置換基であり；
Ｒ２及びＲ３は、それぞれ独立に、水素原子、炭素数１～６個のアルキル基又はハロゲン原子であり；
Ｒ４及びＲ５は、それぞれ独立に、水素原子、炭素数１～６個のアルキル基又はハロゲン原子であり；
Ｒ６及びＲ６’は、それぞれ独立に、水素原子又は炭素数１～５個のアルキル基であり；
ｍは、０～４の整数であり；
Ｐ１及びＰ２は、アミノ酸残基を表し、Ｐ１は、存在しても、存在しなくてもよい。） That is, the present invention
[1] A fluorescent probe used for detecting a puromycin-sensitive aminopeptidase or bleomycin hydrolase containing a compound represented by the following formula (I) or a salt thereof.
Figure JPOXMLDOC01-appb-I000005


(During the ceremony,
R 1 , if present, is the same or different monovalent substituent present on the benzene ring;
R 2 and R 3 are each independently a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom;
R 4 and R 5 are each independently a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom;
R 6 and R 6 'are each independently hydrogen atom or a C 1-5 alkyl group having a carbon;
m is an integer from 0 to 4. )
[2] A fluorescent probe used to detect puromycin-sensitive aminopeptidase or bleomycin hydrolase, which contains the following compound or a salt thereof.
Figure JPOXMLDOC01-appb-I000006


[3] A method for evaluating the vascular properties of aortic aneurysm lesions or non-dilated arterial walls, which comprises (a) a compound represented by the following formula (1) or a salt thereof, and puromycin-sensitive aminopeptidase or bleomycin. This includes applying a fluorescent probe capable of detecting a hydrolytic enzyme to a clinical sample of an aortic aneurysm lesion or a non-dilated arterial wall, and (b) measuring a fluorescent image of the clinical sample to which the fluorescent probe is applied. ,Method.
Figure JPOXMLDOC01-appb-I000007


(During the ceremony,
R 1 , if present, is the same or different monovalent substituent present on the benzene ring;
R 2 and R 3 are each independently a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom;
R 4 and R 5 are each independently a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom;
R 6 and R 6 'are each independently hydrogen atom or a C 1-5 alkyl group having a carbon;
m is an integer from 0 to 4;
P 1 and P 2 represent amino acid residues, and P 1 may or may not be present. )
[4] A method for detecting a normal arterial wall in an aortic aneurysm lesion or a non-dilated arterial wall, which comprises (a) a compound represented by the following formula (1) or a salt thereof, and puromycin-sensitive aminopeptidase or The step of applying a fluorescent probe capable of detecting puromycin hydrolase to a clinical sample of an aortic aneurysm lesion or a non-dilated arterial wall, and (b) measuring the fluorescent image of the clinical sample to which the fluorescent probe is applied. Including, method.
Figure JPOXMLDOC01-appb-I000008


(During the ceremony,
R 1 , if present, is the same or different monovalent substituent present on the benzene ring;
R 2 and R 3 are each independently a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom;
R 4 and R 5 are each independently a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom;
R 6 and R 6 'are each independently hydrogen atom or a C 1-5 alkyl group having a carbon;
m is an integer from 0 to 4;
P 1 and P 2 represent amino acid residues, and P 1 may or may not be present. )
　本発明により、血管性状を客観的に分析評価することが可能な蛍光プローブを提供することが可能であり、より精度の高い手術を行える可能性があり、手術合併症軽減などに貢献する可能性がある。 INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a fluorescent probe capable of objectively analyzing and evaluating vascular properties, and there is a possibility that more accurate surgery can be performed, which may contribute to reduction of surgical complications and the like. There is.
新規プローブＭｅｔ－ＨＭＲＧとＤＰＰＩＶローブの蛍光強度による比較。Comparison of new probe Met-HMRG and DPPIV lobe by fluorescence intensity. Ｍｅｔ－ＨＭＲＧ浸漬ゲルを用いた瘤壁、境界型病変、吻合部動脈壁の蛍光反応の一例。An example of fluorescence reaction of aneurysm wall, borderline lesion, and anastomotic artery wall using Met-HMRG immersion gel. Ｍｅｔ－ＨＭＲＧにおける蛍光強度の経時的変化と感度／特異度Changes in fluorescence intensity over time and sensitivity / specificity in Met-HMRG ＤＥＧ法の結果による吻合部動脈壁及び瘤壁の対比。Contrast of anastomotic artery wall and aneurysm wall according to the results of the DEG method. ＰｕｒｏｍｙｃｉｎとＢｌｅｏｍｙｃｉｎを使用した吻合部動脈壁と瘤壁の蛍光反応における阻害実験の結果を示す。The results of an inhibition experiment in the fluorescence reaction between the anastomotic artery wall and the aneurysm wall using Puromycin and Bleomycin are shown. 各種精製酵素とＭｅｔ－ＨＭＲＧによる蛍光強度の経時的変化を示す。The time course of fluorescence intensity due to various purified enzymes and Met-HMRG is shown. 吻合部動脈ＬｙｓａｔｅにおけるＰｕｒｏｍｙｃｉｎ　ｓｅｎｓｉｔｉｖｅ　ａｍｉｎｏｐｅｐｔｉｄａｓｅとＢｌｅｏｍｙｃｉｎ　ｈｙｄｒｏｌａｓｅの発現とＡｃｔｉｖｉｔｙ。Expression and activity of Puromycin sensitive aminopeptidase and bleomycin hydrolase in the anastomotic artery Lysate. Ｈｅｍａｔｏｘｏｘｙｌｉｎ－Ｅｏｓｉｎ染色。Hematoxylin-Eosin staining. ＤＡＢ法によるＰｕｒｏｍｙｃｉｎ　ｓｅｎｓｉｔｉｖｅ　ａｍｉｎｏｐｅｐｔｉｄａｓｅの発現。Expression of Puromycin sensitive aminopeptidase by the DAB method. ＤＡＢ法によるＢｌｅｏｍｙｃｉｎ　ｈｙｄｒｏｌａｓｅの発現。Expression of bleomycin hydrolase by the DAB method.
　本明細書において、「アルキル基」又はアルキル部分を含む置換基（例えばアルコキシ基など）のアルキル部分は、特に言及しない場合には例えば炭素数１～６個、好ましくは炭素数１～４個、更に好ましくは炭素数１～３個程度の直鎖、分枝鎖、環状、又はそれらの組み合わせからなるアルキル基を意味している。より具体的には、アルキル基として、例えば、メチル基、エチル基、ｎ－プロピル基、イソプロピル基、シクロプロピル基、ｎ－ブチル基、ｓｅｃ－ブチル基、イソブチル基、ｔｅｒｔ－ブチル基、シクロプロピルメチル基、ｎ－ペンチル基、ｎ－ヘキシル基などを挙げることができる。 In the present specification, the alkyl moiety of an "alkyl group" or a substituent containing an alkyl moiety (for example, an alkoxy group) has, for example, 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms, unless otherwise specified. More preferably, it means an alkyl group composed of a straight chain, a branched chain, a cyclic chain, or a combination thereof having about 1 to 3 carbon atoms. More specifically, the alkyl groups include, for example, methyl group, ethyl group, n-propyl group, isopropyl group, cyclopropyl group, n-butyl group, sec-butyl group, isobutyl group, tert-butyl group and cyclopropyl. Methyl group, n-pentyl group, n-hexyl group and the like can be mentioned.
　本明細書において「ハロゲン原子」という場合には、フッ素原子、塩素原子、臭素原子、又はヨウ素原子のいずれでもよく、好ましくはフッ素原子、塩素原子、又は臭素原子である。 In the present specification, the term "halogen atom" may be any of 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.
１．蛍光プローブ
　本発明の１つの実施態様は、以下の式（Ｉ）で表される化合物又はその塩を含有する、ピューロマイシン感受性アミノペプチダーゼ又はブレオマイシン加水分解酵素を検出するのに用いられる蛍光プローブである（以下「本発明の蛍光プローブ１」ともいう）。
Figure JPOXMLDOC01-appb-I000009

 1. 1. Fluorescent probe One embodiment of the present invention is a fluorescent probe used to detect a puromycin-sensitive aminopeptidase or bleomycin hydrolase containing a compound represented by the following formula (I) or a salt thereof. (Hereinafter, also referred to as "fluorescent probe 1 of the present invention").
Figure JPOXMLDOC01-appb-I000009
　式（Ｉ）において、Ｒ１は、存在する場合は、ベンゼン環上に存在する同一又は異なる一価の置換基である。一価の置換基としては、ハロゲン、置換されていてもよいアルキル基等が挙げられる。 In formula (I), R 1 , if present, is the same or different monovalent substituent present on the benzene ring. Examples of the monovalent substituent include halogen, an optionally substituted alkyl group and the like.
　ｍは、０～４の整数である。
　本発明の１つの好ましい側面においては、ｍが０であり、Ｒ１存在せずに無置換のベンゼン環である。 m is an integer from 0 to 4.
In one preferred aspect of the invention, m is 0 and R 1 is absent and unsubstituted benzene ring.
　式（Ｉ）において、Ｒ２及びＲ３は、それぞれ独立に、水素原子、炭素数１～６個のアルキル基又はハロゲン原子である。
　Ｒ２及びＲ３がアルキル基を示す場合には、該アルキル基にはハロゲン原子、カルボキシ基、スルホニル基、水酸基、アミノ基、アルコキシ基などが１個又は２個以上存在していてもよく、例えばＲ２又はＲ３が示すアルキル基はハロゲン化アルキル基、ヒドロキシアルキル基、カルボキシアルキル基などであってもよい。Ｒ２及びＲ３が共に水素原子であることが好ましい。 In formula (I), R 2 and R 3 are each independently a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom.
When R 2 and R 3 represent an alkyl group, the alkyl group may contain one or more halogen atoms, a carboxy group, a sulfonyl group, a hydroxyl group, an amino group, an alkoxy group and the like. For example, the alkyl group indicated by R 2 or R 3 may be an alkyl halide group, a hydroxyalkyl group, a carboxyalkyl group or the like. It is preferable that both R 2 and R 3 are hydrogen atoms.
　Ｒ４及びＲ５は、それぞれ独立に、水素原子、炭素数１～６個のアルキル基、又はハロゲン原子である。Ｒ４及びＲ５の詳細については、Ｒ２及びＲ３について説明したものと同様である。Ｒ４及びＲ５が共に水素原子であることが好ましい。 R 4 and R 5 are independently hydrogen atoms, alkyl groups having 1 to 6 carbon atoms, or halogen atoms. The details of R 4 and R 5 are the same as those described for R 2 and R 3. It is preferable that both R 4 and R 5 are hydrogen atoms.
　Ｒ６及びＲ６’は、それぞれ独立に、水素原子又は炭素数１～５個のアルキル基である。Ｒ６及びＲ６’のアルキル基としては、メチル基、エチル基が好ましい。
　本発明の１つの好ましい側面においては、Ｒ６及びＲ６’が共に水素原子である。 R 6 and R 6 'are each independently ~ 1 hydrogen atom or a carbon 5 alkyl groups. As the alkyl group of R 6 and R 6 ', a methyl group and an ethyl group are preferable.
In one preferred aspect of the present invention, R 6 and R 6 'are both hydrogen atoms.
　本発明者らは、ＨＭＲＧ蛍光団に着目して、臨床から得られた検体の拡張性瘤化病変や正常動脈において、どのようなプローブが応答可視化に有用であるかを鋭意検討したところ、ＨＭＲＧ骨格にメチオニン（Ｍｅｔ）を導入した化合物がヒト腹部大動脈瘤手術検体を用いた試験において蛍光強度が有意に変化し、またイメージング実験においても優れた結果を示すことを見出した。 Focusing on the HMRG fluorescein, the present inventors diligently investigated what kind of probe is useful for response visualization in dilated aneurysm lesions and normal arteries of clinically obtained specimens. It was found that the compound in which methionine (Met) was introduced into the skeleton significantly changed the fluorescence intensity in a test using a human abdominal aortic aneurysm surgical specimen, and also showed excellent results in an imaging experiment.
　本発明の１つの好ましい態様は、以下の化合物又はその塩を含有する、ピューロマイシン感受性アミノペプチダーゼ又はブレオマイシン加水分解酵素を検出するのに用いられる蛍光プローブである。
Figure JPOXMLDOC01-appb-I000010


　上記の構造を有する化合物を、以下で「Ｍｅｔ－ＨＭＲＧ」ともいう。 One preferred embodiment of the present invention is a fluorescent probe used to detect puromycin-sensitive aminopeptidase or bleomycin hydrolase, which contains the following compounds or salts thereof.
Figure JPOXMLDOC01-appb-I000010


The compound having the above structure is also referred to as "Met-HMRG" below.
　本発明の式（Ｉ）の化合物及びＭｅｔ－ＨＭＲＧ（以下「本発明の化合物」ともいう）は、酸付加塩又は塩基付加塩として存在することができる。酸付加塩としては、例えば、塩酸塩、硫酸塩、硝酸塩などの鉱酸塩、又はメタンスルホン酸塩、ｐ－トルエンスルホン酸塩、シュウ酸塩、クエン酸塩、酒石酸塩などの有機酸塩などを挙げることができ、塩基付加塩としては、ナトリウム塩、カリウム塩、カルシウム塩、マグネシウム塩などの金属塩、アンモニウム塩、又はトリエチルアミン塩などの有機アミン塩などを挙げることができる。これらのほか、グリシンなどのアミノ酸との塩を形成する場合もある。本発明の化合物又はその塩は、水和物又は溶媒和物として存在する場合もあるが、これらの物質も本発明の範囲内である。 The compound of the formula (I) of the present invention and Met-HMRG (hereinafter, also referred to as “compound of the present invention”) can exist as an acid addition salt or a base addition salt. Examples of the acid addition salt include mineral salts such as hydrochlorides, sulfates and nitrates, or organic acid salts such as methanesulfonates, p-toluenesulfonates, oxalates, citrates and tartrates. Examples of the base addition salt include metal salts such as sodium salt, potassium salt, calcium salt and magnesium salt, ammonium salt, and organic amine salt such as triethylamine salt. In addition to these, it may form a salt with an amino acid such as glycine. The compounds of the present invention or salts thereof may exist as hydrates or solvates, but these substances are also within the scope of the present invention.
　本発明の化合物は、置換基の種類により、１個又は２個以上の不斉炭素を有する場合があるが、１個又は２個以上の不斉炭素に基づく光学活性体や２個以上の不斉炭素に基づくジアステレオ異性体などの立体異性体のほか、立体異性体の任意の混合物、ラセミ体などは、いずれも本発明の範囲に包含される。 The compound of the present invention may have one or two or more asymmetric carbons depending on the type of the substituent, but an optically active substance based on one or two or more asymmetric carbons or two or more asymmetric carbons. In addition to stereoisomers such as diastereoisomers based on homocarbons, any mixture of stereoisomers, racemates and the like are all included in the scope of the present invention.
　本発明の化合物の製造方法を本明細書の実施例に具体的に示した。従って、当業者は、これらの説明をもとにして、反応原料、反応条件、反応試薬などを適宜選択して、必要に応じてこれらの方法に修飾や改変を加えることにより、本発明の化合物を製造することができる。 The method for producing the compound of the present invention is specifically shown in the examples of the present specification. Therefore, those skilled in the art can appropriately select reaction raw materials, reaction conditions, reaction reagents, etc. based on these explanations, and modify or modify these methods as necessary to modify the compounds of the present invention. Can be manufactured.
　また、本発明の蛍光プローブ１のターゲット酵素を調べたところ、Ｄｉｃｅｄ　Ｅｌｅｃｔｒｏｐｈｏｒｅｓｉｓ　Ｇｅｌ法（ＤＥＧ法）を施行し、ＤＥＧ法で挙げられたターゲット酵素をウエスタンブロッティング法で調べたところ、ピューロマイシン感受性アミノペプチダーゼ及びブレオマイシン加水分解酵素がともに発現していることが見出された。
　また、ＤＥＧ法で挙げられたターゲット酵素を免疫染色法で標本内に発現しているか確認したところ、ピューロマイシン感受性アミノペプチダーゼが内膜に高発現していることが確認された。 Further, when the target enzyme of the fluorescent probe 1 of the present invention was examined, the Diced Electrophoresis Gel method (DEG method) was performed, and the target enzyme listed in the DEG method was examined by the Western blotting method. As a result, puromycin-sensitive aminopeptidase was examined. And bleomycin hydrolase were found to be expressed together.
In addition, when it was confirmed by immunostaining whether the target enzyme listed in the DEG method was expressed in the specimen, it was confirmed that puromycin-sensitive aminopeptidase was highly expressed in the endometrium.
　即ち、本発明の１つの側面は、本発明の化合物又はその塩を含有する、ピューロマイシン感受性アミノペプチダーゼを検出するのに用いられる蛍光プローブである。 That is, one aspect of the present invention is a fluorescent probe used to detect puromycin-sensitive aminopeptidase, which contains the compound of the present invention or a salt thereof.
　また、本発明のもう１つの側面は、本発明の化合物又はその塩を含有する、ブレオマイシン加水分解酵素を検出するのに用いられる蛍光プローブである。 Another aspect of the present invention is a fluorescent probe used to detect a bleomycin hydrolase containing the compound of the present invention or a salt thereof.
　また、本発明のもう１つの側面は、本発明の化合物又はその塩を含有する、ピューロマイシン感受性アミノペプチダーゼ及びブレオマイシン加水分解酵素を検出するのに用いられる蛍光プローブである。 Another aspect of the present invention is a fluorescent probe used to detect puromycin-sensitive aminopeptidase and bleomycin hydrolase, which contain the compound of the present invention or a salt thereof.
２．ピューロマイシン感受性アミノペプチダーゼ又はブレオマイシン加水分解酵素の検出方法
　上記の通り、正常動脈壁の内膜においてピューロマイシン感受性アミノペプチダーゼ又はブレオマイシン加水分解酵素が高発現している事が判明した。従って、これら２つのアミノペプチターゼと蛍光反応を示すＭｅｔ－ＨＭＲＧを含むＨＭＲＧ蛍光団を用いることにより正常動脈壁を高感度に評価する事が可能となる。 2. Method for detecting puromycin-sensitive aminopeptidase or bleomycin hydrolase As described above, it was found that puromycin-sensitive aminopeptidase or bleomycin hydrolase was highly expressed in the intima of the normal arterial wall. Therefore, it is possible to evaluate the normal arterial wall with high sensitivity by using an HMRG fluorescent group containing Met-HMRG that exhibits a fluorescent reaction with these two aminopeptidases.
　従って、本発明のもう１つの実施態様は、大動脈瘤病変又は非拡張部動脈壁の血管性状を評価する方法であって、（ａ）以下の式（１）で表される化合物又はその塩を含み、ピューロマイシン感受性アミノペプチダーゼ又はブレオマイシン加水分解酵素を検出することができる蛍光プローブを大動脈瘤病変又は非拡張部動脈壁の臨床検体に適用する工程、及び（ｂ）前記蛍光プローブを適用した臨床検体の蛍光像を測定することを含む、方法である。 Therefore, another embodiment of the present invention is a method for evaluating the vascular properties of an aortic aneurysm lesion or a non-dilated arterial wall, wherein (a) a compound represented by the following formula (1) or a salt thereof is used. A step of applying a fluorescent probe containing a puromycin-sensitive aminopeptidase or a bleomycin hydrolyzing enzyme to a clinical specimen of an aortic aneurysm lesion or a non-dilated arterial wall, and (b) a clinical specimen to which the fluorescent probe is applied. A method comprising measuring a fluorescent image of.
Figure JPOXMLDOC01-appb-I000011
Figure JPOXMLDOC01-appb-I000011
　式（１）において、Ｒ１～Ｒ６’及びｍは、式（Ｉ）で定義した通りである。
　即ち、Ｒ１は、存在する場合は、ベンゼン環上に存在する同一又は異なる一価の置換基であり；ｍは、０～４の整数であり；Ｒ２及びＲ３は、それぞれ独立に、水素原子、炭素数１～６個のアルキル基又はハロゲン原子であり；Ｒ４及びＲ５は、それぞれ独立に、水素原子、炭素数１～６個のアルキル基、又はハロゲン原子であり；Ｒ６及びＲ６’は、それぞれ独立に、水素原子又は炭素数１～５個のアルキル基である。Ｒ１～Ｒ６’及びｍの詳細については、式（Ｉ）について上述した通りである。 In the formula (1), R 1 ~ R 6 ' and m are as defined in formula (I).
That is, R 1 is the same or different monovalent substituent present on the benzene ring, if present; m is an integer of 0-4; R 2 and R 3 are independent of each other. A hydrogen atom, an alkyl group or halogen atom having 1 to 6 carbon atoms; R 4 and R 5 are independently hydrogen atoms, an alkyl group having 1 to 6 carbon atoms, or a halogen atom; R 6 and R 6 'are independently a hydrogen atom or a C 1-5 alkyl group having a carbon. For more information about the R 1 ~ R 6 'and m, are as described above for formula (I).
　式（１）において、Ｐ１及びＰ２は、アミノ酸残基を表す。また、Ｐ１は、存在しても、存在しなくてもよい。 In formula (1), P 1 and P 2 represent amino acid residues. Further, P 1 may or may not exist.
　式（１）において、Ｐ１は－ＮＨ－ＣＲＲ’－Ｃ（＝Ｏ）－＊構造（＊は、ベンゼン環に結合したＮＨ基と結合する側を示す）を有する部位であり、一般的なα－アミノ酸残基（グリシン、アラニン、ロイシン、イソロイシン、バリン、リジン、システイン、トレオニン、アルギニン、アスパラギン、アスパラギン酸、グルタミン、グルタミン酸、セリン、ヒスチジン、フェニルアラニン、メチオニン、トリプトファン、チロシン、プロリン）や、その誘導体（Ｎ－メチルロイシン、２，３－ジアミノプロパン酸、２，４－ジアミノ酪酸、オルニチン、α－ヒドロキシロイシン等）の残基が挙げられる。アミノ酸残基、その誘導体とも、Ｌ体アミノ酸及びＤ体アミノ酸の残基のいずれであってもよい。 In the formula (1), P 1 is a site having a -NH-CRR'-C (= O)-* structure (* indicates the side bonded to the NH group bonded to the benzene ring), which is general. α-Amino acid residues (glycine, alanine, leucine, isoleucine, valine, lysine, cysteine, threonine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, serine, histidine, phenylalanine, methionine, tryptophan, tyrosine, proline) and their Residues of derivatives (N-methylleucine, 2,3-diaminopropanoic acid, 2,4-diaminobutyric acid, ornithine, α-hydroxyleucine, etc.) can be mentioned. The amino acid residue and its derivative may be either an L-form amino acid or a D-form amino acid residue.
　本発明の１つの側面において、Ｐ１は以下の式（ａ）で表される。
Figure JPOXMLDOC01-appb-I000012

In one aspect of the present invention, P 1 is represented by the following formula (a).
Figure JPOXMLDOC01-appb-I000012
　式（ａ）において、Ｒ７は、水素、メチル基等の、天然アミノ酸（グリシン、アラニン、ロイシン、イソロイシン、バリン、リジン、システイン、トレオニン、アルギニン、アスパラギン、アスパラギン酸、グルタミン、グルタミン酸、セリン、ヒスチジン、フェニルアラニン、メチオニン、トリプトファン、チロシン、プロリン）の側鎖を構成する基を表す。また、Ｒ７には、天然アミノ酸の側鎖を構成する基以外の基、例えば、種々の置換基を有するアルキル基なども含まれる。 In formula (a), R 7 is a natural amino acid (glycine, alanine, leucine, isoleucine, valine, lysine, cysteine, threonine, arginine, asparagine, aspartic acid, glutamic acid, glutamic acid, serine, histidine, etc. , Phenylalanine, methionine, tryptophan, tyrosine, proline). Further, R 7 also includes a group other than the group constituting the side chain of the natural amino acid, for example, an alkyl group having various substituents and the like.
　式（１）において、Ｐ２は、Ｎ末端アミノ酸残基を表す。Ｐ２のアミノ酸残基は、グリシン、アラニン、ロイシン、イソロイシン、バリン、リジン、システイン、トレオニン、アルギニン、アスパラギン、アスパラギン酸、グルタミン、グルタミン酸、セリン、ヒスチジン、フェニルアラニン、メチオニン、トリプトファン、チロシン、プロリンの残基から選択される。
　Ｐ２のアミノ酸残基としては、Ｌ体アミノ酸、Ｄ体アミノ酸の残基のいずれであってもよい。 In formula (1), P 2 represents an N-terminal amino acid residue. The amino acid residues of P 2 are the residue of glycine, alanine, leucine, isoleucine, valine, lysine, cysteine, threonine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, serine, histidine, phenylalanine, methionine, tryptophan, tyrosine and proline Selected from the group.
The amino acid residue of P 2 may be either an L-amino acid or a D-amino acid residue.
　本発明の１つの側面において、Ｐ２は、以下の式（ｂ）で表される、
Figure JPOXMLDOC01-appb-I000013

In one aspect of the present invention, P 2 is represented by the following equation (b).
Figure JPOXMLDOC01-appb-I000013
　式（ｂ）において、Ｒ８は、水素、メチル基等の、天然アミノ酸（グリシン、アラニン、ロイシン、イソロイシン、バリン、リジン、システイン、トレオニン、アルギニン、アスパラギン、アスパラギン酸、グルタミン、グルタミン酸、セリン、ヒスチジン、フェニルアラニン、メチオニン、トリプトファン、チロシン、プロリン）の側鎖を構成する基を表す。 In formula (b), R 8 is hydrogen, such as a methyl group, a natural amino acid (glycine, alanine, leucine, isoleucine, valine, lysine, cysteine, threonine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, serine, histidine , Phenylalanine, methionine, tryptophan, tyrosine, proline).
　式（１）で表される化合物の１つの側面は、以下の式で表される化合物である。
Figure JPOXMLDOC01-appb-I000014


　Ｐ１及びＰ２は、式（１）で説明したのと同様である。 One aspect of the compound represented by the formula (1) is the compound represented by the following formula.
Figure JPOXMLDOC01-appb-I000014


P 1 and P 2 are the same as those described in the equation (1).
　式（１）で表され、かつ、ピューロマイシン感受性アミノペプチダーゼ又はブレオマイシン加水分解酵素を検出することができる蛍光プローブ（以下、「本発明の蛍光プローブ２」ともいう）としては、上記したＭｅｔ－ＨＭＲＧ（Ｐ１は存在せず、Ｐ２はメチオニン残基である）が代表的な例であるが、これに限定されるものではない。
　即ち、正常動脈壁の内膜においてピューロマイシン感受性アミノペプチダーゼ又はブレオマイシン加水分解酵素が高発現していることは、本発明者らにより初めて見出されたことであり、これらアミノペプチダーゼを検出することができる式（１）又は式（１ａ）で表される蛍光プローブを用いる大動脈瘤病変又は非拡張部動脈壁の血管性状を評価する方法は、本発明の範囲内である。 The above-mentioned Met-HMRG is a fluorescent probe represented by the formula (1) and capable of detecting a puromycin-sensitive aminopeptidase or bleomycin hydrolase (hereinafter, also referred to as “fluorescent probe 2 of the present invention”). (P 1 does not exist and P 2 is a methionine residue) is a typical example, but is not limited to this.
That is, it was first discovered by the present inventors that puromycin-sensitive aminopeptidase or bleomycin hydrolase is highly expressed in the intima of the normal arterial wall, and these aminopeptidases can be detected. A method for evaluating the vascular properties of an aortic aneurysm lesion or a non-dilated arterial wall using a fluorescent probe represented by the formula (1) or the formula (1a) is within the scope of the present invention.
　（ａ）の工程の本発明の蛍光プローブ２を大動脈瘤病変又は非拡張部動脈壁の臨床検体に適用するには、例えば、本発明の蛍光プローブ２の溶液（例えば、１ｘＴＢＳバッファーで希釈して、濃度が５０μＭ程度）を検体全体が浸るように滴下することが挙げられる。また、本発明の蛍光プローブ２適用する前に、臨床検体を１ｘＴＢＳバッファー等で洗浄することが好ましい。 To apply the fluorescent probe 2 of the present invention in the step (a) to a clinical specimen of an aortic aneurysm lesion or a non-dilated arterial wall, for example, dilute with a solution of the fluorescent probe 2 of the present invention (for example, 1xTBS buffer). , The concentration is about 50 μM) so that the entire sample is immersed. Further, it is preferable to wash the clinical sample with a 1xTBS buffer or the like before applying the fluorescent probe 2 of the present invention.
　臨床検体としては、大動脈瘤病変又は非拡張部動脈壁の手術検体等が挙げられる。 Examples of clinical specimens include surgical specimens for aortic aneurysm lesions and non-dilated arterial walls.
　本発明の蛍光プローブ２を適用した大動脈瘤病変又は非拡張部動脈壁の臨床検体の蛍光像を測定して、明るい蛍光を示す部位が正常動脈壁の割合が高いと判断することができる。この場合、蛍光強度自体が高いことで正常動脈壁の割合が高いと判断してもよいが、好ましくは、本発明の蛍光プローブ２を適用して一定時間（例えば、３０分、６０分等）経過後の蛍光強度の増加率を指標として、増加率が高い部位が正常動脈壁も割合が高いと判断することができる。 By measuring the fluorescence image of the clinical specimen of the aortic aneurysm lesion or the non-dilated arterial wall to which the fluorescent probe 2 of the present invention is applied, it can be determined that the portion showing bright fluorescence has a high proportion of the normal arterial wall. In this case, it may be determined that the proportion of the normal arterial wall is high because the fluorescence intensity itself is high, but preferably, the fluorescent probe 2 of the present invention is applied for a certain period of time (for example, 30 minutes, 60 minutes, etc.). Using the rate of increase in fluorescence intensity after the lapse as an index, it can be determined that the site with a high rate of increase also has a high rate of normal arterial wall.
　本発明の前大動脈瘤病変又は非拡張部動脈壁の血管性状を評価する方法においては、本発明の蛍光プローブ２の中でも、Ｐ１は存在せず、Ｐ２はメチオニン残基であるＭｅｔ－ＨＭＲＧを用いると、血管性状をより高精度に評価することができ好ましい。
　即ち、本発明の１つの好ましい側面は、大動脈瘤病変又は非拡張部動脈壁の血管性状を評価する方法であって、Ｍｅｔ－ＨＭＲＧを含む蛍光プローブを大動脈瘤病変又は非拡張部動脈壁の臨床検体に適用する工程、及び（ｂ）前記蛍光プローブを適用した臨床検体の蛍光像を測定することを含む、方法である。 In the method for evaluating the vascular properties of the anterior aortic aneurysm lesion or the non-dilated arterial wall of the present invention, P 1 is absent among the fluorescent probes 2 of the present invention, and P 2 is a methionine residue Met-HMRG. Is preferable because the vascular properties can be evaluated with higher accuracy.
That is, one preferred aspect of the present invention is a method for evaluating the vascular properties of an aortic aneurysm lesion or a non-dilated arterial wall, in which a fluorescent probe containing Met-HMRG is used for clinical aortic aneurysm lesion or non-dilated arterial wall. It is a method including a step of applying to a sample and (b) measuring a fluorescent image of a clinical sample to which the fluorescent probe is applied.
　本発明の蛍光プローブ１及び２の使用方法は特に限定されず、従来公知の蛍光プローブと同様に用いることが可能である。通常は、生理食塩水や緩衝液などの水性媒体、又はエタノール、アセトン、エチレングリコール、ジメチルスルホキシド、ジメチルホルムアミドなどの水混合性の有機溶媒と水性媒体との混合物などに蛍光プローブ１及び２に含まれる化合物又はそれらの塩を溶解し、細胞や組織を含む適切な緩衝液中にこの溶液を添加して、蛍光スペクトルを測定すればよい。本発明の蛍光プローブを適切な添加物と組み合わせて組成物の形態で用いてもよい。例えば、緩衝剤、溶解補助剤、ｐＨ調節剤などの添加物と組み合わせることができる。 The method of using the fluorescent probes 1 and 2 of the present invention is not particularly limited, and can be used in the same manner as the conventionally known fluorescent probes. Usually, it is contained in fluorescent probes 1 and 2 in an aqueous medium such as physiological saline or a buffer solution, or a mixture of an aqueous medium and an aqueous solvent such as ethanol, acetone, ethylene glycol, dimethyl sulfoxide, or dimethyl formamide. The compounds or salts thereof may be dissolved, the solution may be added to a suitable buffer containing cells or tissues, and the fluorescence spectrum may be measured. The fluorescent probe of the present invention may be used in the form of a composition in combination with a suitable additive. For example, it can be combined with additives such as buffers, solubilizers and pH regulators.
　また、本発明のもう１つの態様は、大動脈瘤病変又は非拡張部動脈壁における正常動脈壁を検知する方法であって、（ａ）以下の式（１）で表される化合物又はその塩を含み、ピューロマイシン感受性アミノペプチダーゼ又はブレオマイシン加水分解酵素を検出することができる蛍光プローブを大動脈瘤病変又は非拡張部動脈壁の臨床検体に適用する工程、及び（ｂ）前記蛍光プローブを適用した臨床検体の蛍光像を測定することを含む、方法である。 Another aspect of the present invention is a method for detecting an aortic aneurysm lesion or a normal arterial wall in a non-dilated arterial wall, wherein (a) a compound represented by the following formula (1) or a salt thereof is used. A step of applying a fluorescent probe containing a puromycin-sensitive aminopeptidase or a bleomycin hydrolyzing enzyme to a clinical specimen of an aortic aneurysm lesion or a non-dilated arterial wall, and (b) a clinical specimen to which the fluorescent probe is applied. A method comprising measuring a fluorescent image of.
Figure JPOXMLDOC01-appb-I000015
Figure JPOXMLDOC01-appb-I000015
　式（１）において、Ｒ１～Ｒ６’及びｍは、上記した通りであり、Ｐ１及びＰ２は、アミノ酸残基を表す。また、Ｐ１は、存在しても、存在しなくてもよい。 In the formula (1), R 1 ~ R 6 ' and m are as defined above, P 1 and P 2 represents an amino acid residue. Further, P 1 may or may not exist.
　腹部大動脈瘤の開腹手術において、本発明の蛍光プローブ１又は２を用いれば吻合部瘤などの合併症を未然に防ぎ、より精度の高い手術に寄与するものと期待される。 In open surgery for abdominal aortic aneurysm, it is expected that the fluorescent probe 1 or 2 of the present invention will prevent complications such as anastomotic aneurysm and contribute to more accurate surgery.
　以下、本発明を実施例によって具体的に説明するが、本発明は、これらの実施例によって限定されるものではない。 Hereinafter, the present invention will be specifically described with reference to Examples, but the present invention is not limited to these Examples.
１．Ｍ－ＨＭＲＧの合成
　Ｍ－ＨＭＲＧ（１）は以下のスキーム１に従って合成した。
Figure JPOXMLDOC01-appb-I000016
 1. 1. Synthesis of M-HMRG M-HMRG ( 1 ) was synthesized according to Scheme 1 below.
Figure JPOXMLDOC01-appb-I000016
２．合成法および機器データ2. Synthesis method and equipment data
Ａ１の合成
　Rhodamine 110 Chloride １１２７ｍｇ（３．１２ｍｍｏｌ）をメタノール１８０ｍＬに溶解し、硫酸９ｍＬを加えた後にアルゴン雰囲気下で８０℃で一晩撹拌した。室温まで冷却した後、反応溶媒を減圧除去し、残渣を飽和炭酸水素ナトリウム水溶液を加えて中和後、濾過した。濾過して得られた個体をメタノールに溶解させ回収し、メタノールを減圧除去した。残渣をテトラヒドロフラン　２００ｍＬに溶解させ、氷浴下で撹拌させながら水素化リチウムアルミニウム　１５４５ｍｇ（４０．７ｍｍｏｌ）を加えアルゴン雰囲気下、アルミホイルで遮光して一晩室温で撹拌した。反応溶液を氷浴下で撹拌しながらメタノール３０ｍＬを加えた後、反応溶媒を減圧除去した。残渣に飽和ロッシェル塩水溶液　２００ｍＬと酢酸エチル１００ｍＬを加えてアルゴン雰囲気下、アルミホイルで遮光して一晩撹拌した。反応溶液から分液操作で酢酸エチル層を抽出した後に酢酸エチルを減圧除去し、残渣をシリカゲルクロマトグラフィーで精製して（ジクロロメタン／メタノール＝９０／１０）目的化合物（５５７ｍｇ、５６％）を得た。
1H NMR (300 MHz, CD3OD): δ 4.63 (s, 2H), 5.38 (s, 1H), 6.30 (dd, 2H, J = 2.1, 9.0 Hz), 6.41 (d, 2H, J = 2.1 Hz), 6.64 (d, 2H, J = 9.0 Hz), 7.04-7.08 (m, 1H), 7.14-7.16 (m, 2H), 7.39-7.42 (m, 1H)
13C NMR (100 MHz, CD3OD): δ 40.2, 63.0, 103.4, 112.2, 115.4, 127.3, 128.7, 128.9, 131.2, 131.9, 145.7, 148.5, 152.8
HRMS (ESI+): calcd for [M+H]+, 319.14018 ; found, 319.14465 (-4.48 mmu) Synthesis of A1 1127 mg (3.12 mmol) of Rhodamine 110 Chloride was dissolved in 180 mL of methanol, 9 mL of sulfuric acid was added, and the mixture was stirred overnight at 80 ° C. under an argon atmosphere. After cooling to room temperature, the reaction solvent was removed under reduced pressure, and the residue was neutralized by adding a saturated aqueous sodium hydrogen carbonate solution and then filtered. The solid obtained by filtration was dissolved in methanol and recovered, and methanol was removed under reduced pressure. The residue was dissolved in 200 mL of tetrahydrofuran, 1545 mg (40.7 mmol) of lithium aluminum hydride was added while stirring under an ice bath, and the mixture was stirred at room temperature overnight under an argon atmosphere with light shielding from aluminum foil. After adding 30 mL of methanol while stirring the reaction solution under an ice bath, the reaction solvent was removed under reduced pressure. 200 mL of a saturated aqueous solution of Rochelle salt and 100 mL of ethyl acetate were added to the residue, and the mixture was stirred under an argon atmosphere, shielded from light with aluminum foil, and stirred overnight. After extracting the ethyl acetate layer from the reaction solution by a liquid separation operation, ethyl acetate was removed under reduced pressure, and the residue was purified by silica gel chromatography (dichloromethane / methanol = 90/10) to obtain the target compound (557 mg, 56%). ..
1 H NMR (300 MHz, CD 3 OD): δ 4.63 (s, 2H), 5.38 (s, 1H), 6.30 (dd, 2H, J = 2.1, 9.0 Hz), 6.41 (d, 2H, J = 2.1) Hz), 6.64 (d, 2H, J = 9.0 Hz), 7.04-7.08 (m, 1H), 7.14-7.16 (m, 2H), 7.39-7.42 (m, 1H)
13 C NMR (100 MHz, CD 3 OD): δ 40.2, 63.0, 103.4, 112.2, 115.4, 127.3, 128.7, 128.9, 131.2, 131.9, 145.7, 148.5, 152.8
HRMS (ESI + ): calcd for [M + H] + , 319.14018; found, 319.14465 (-4.48 mmu)
Ａ２の合成
　化合物Ａ１ ５５７ｍｇ（１．７５ｍｍｏｌ）、ｔ－ブチルジメチルシリルクロライド４０２ｍｇ（２．６６ｍｍｏｌ）、イミダゾール２４２ｍｇ（３．５５ｍｍｏｌ）をＮ，Ｎ－ジメチルホルムアミド２５ｍＬに溶解させ、アルゴン雰囲気下、アルミホイルで遮光して４時間撹拌した。水を１００ｍＬ加えた後、酢酸エチルを加え分液操作で酢酸エチル層を抽出した。酢酸エチルを減圧除去し、残渣をシリカゲルカラムクロマトグラフィーで精製して（酢酸エチル／ヘキサン＝２／３）目的化合物（７１３ｍｇ、９４％）を得た。
1H NMR (400 MHz, aceton-d6 ): δ 0.11 (s, 6H), 0.98 (s,9H), 4.72 (s, 4H), 4.79 (s, 2H), 5.44 (s, 1H), 6.35 (dd, 2H, J = 2.9, 10.7 Hz), 6.46 (d, 2H, J = 2.9 Hz), 6.71 (d, 2H, J = 10.7 Hz), 7.22-7.29 (m, 3H), 7.53-7.57 (m, 1H)
13C NMR (75 MHz, CDCl3): δ-5.47, 18.3, 25.9, 39.1, 62.9, 102.0, 110.6, 114.1, 126.4, 127.2, 127.4, 130.4, 130.7, 138.5, 143.9, 146.0, 151.3
HRMS (ESI+): calcd for [M+H]+, 433.23113 ; found, 433.23114 (0.01 mmu) Synthesis of A2 Compound A1 557 mg (1.75 mmol), t-butyldimethylsilyl chloride 402 mg (2.66 mmol), and imidazole 242 mg (3.55 mmol) were dissolved in 25 mL of N, N-dimethylformamide, and aluminum foil was used under an argon atmosphere. The mixture was shaded with light and stirred for 4 hours. After adding 100 mL of water, ethyl acetate was added and the ethyl acetate layer was extracted by a liquid separation operation. Ethyl acetate was removed under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate / hexane = 2/3) to give the target compound (713 mg, 94%).
1 H NMR (400 MHz, aceton-d 6 ): δ 0.11 (s, 6H), 0.98 (s, 9H), 4.72 (s, 4H), 4.79 (s, 2H), 5.44 (s, 1H), 6.35 (dd, 2H, J = 2.9, 10.7 Hz), 6.46 (d, 2H, J = 2.9 Hz), 6.71 (d, 2H, J = 10.7 Hz), 7.22-7.29 (m, 3H), 7.53-7.57 ( m, 1H)
13 C NMR (75 MHz, CDCl 3 ): δ-5.47, 18.3, 25.9, 39.1, 62.9, 102.0, 110.6, 114.1, 126.4, 127.2, 127.4, 130.4, 130.7, 138.5, 143.9, 146.0, 151.3
HRMS (ESI + ): calcd for [M + H] + , 433.23113; found, 433.23114 (0.01 mmu)
Ａ３の合成
　化合物Ａ２ ８４ｍｇ（０．１９４ｍｍｏｌ）、Ｆｍｏｃ－メチオニン４８ｍｇ（０．１２９ｍｍｏｌ）、ＨＡＴＵ７４ｍｇ（０．１９５ｍｍｏｌ）をＮ，Ｎ’－ジメチルホルムアミド５ｍＬに溶解させた後、Ｎ，Ｎ－ジイソプロピルエチルアミン１００μＬ（０．５８１ｍｍｏｌ）を加えてアルゴン雰囲気下にて５０℃で９０分間撹拌した。反応溶媒を減圧除去した後、残渣を酢酸エチルに溶解し、飽和食塩水を加えて分液操作を行った。酢酸エチル層を抽出し、減圧除去によって得られた残渣をシリカゲルカラムクロマトグラフィー（酢酸エチル／ヘキサン＝２／１→１／１）で精製し、目的化合物（８０ｍｇ、７９％）を得た。
1H NMR (400 MHz, CD2Cl2): δ 0.04 (s, 6H), 0.89 (s, 9H), 1.99-2.06 (m, 1H), 2.09 (s, 3H), 2.12-2.19 (m, 1H), 2.53-2.61 (m, 2H), 4.23 (t, J = 6.6 Hz, 1H), 4.44-4.46 (m, 3H), 4.70 (d, J = 12.8 Hz, 1H), 4.76 (d, J = 12.8 Hz, 1H), 5.48 (s, 1H), 5.60 (s, 1H), 6.29 (dd, J = 2.3, 8.2 Hz, 1H), 6.41 (d, J = 1.8 Hz, 1H), 6.69 (d, J = 8.2 Hz, 1H), 6.85-6.95 (m, 2H), 7.05 (d, J = 6.9 Hz, 1H), 7.15-7.21 (m, 2H), 7.29-7.32 (m, 2H), 7.36-7.45 (m, 4H), 7.58-7.61 (m, 2H), 7.77 (d, J = 7.3 Hz, 2H), 8.18 (s, 1H)
13C NMR (75 MHz, CDCl3): δ-5.40, 15.2, 18.4, 25.9, 30.1, 31.3, 38.9, 47.1, 54.4, 63.2, 67.2, 102.1, 107.6, 107.7, 110.9, 113.8, 114.5 (containing 2 peaks), 120.0, 120.7, 124.9, 126.5, 127.1, 127.5, 127.7, 130.1, 130.2, 130.4, 130.8, 136.7, 138.3, 141.2, 143.5, 143.6, 143.8, 146.2, 150.9, 151.1, 156.4, 169.3
HRMS (ESI+): calcd for [M+H]+, 786.33969 ; found, 786.33663 (-3.06 mmu) Synthesis of A3 84 mg (0.194 mmol) of compound A2 , 48 mg (0.129 mmol) of Fmoc-methionine, and 74 mg (0.195 mmol) of HATU were dissolved in 5 mL of N, N'-dimethylformamide, and then 100 μL of N, N-diisopropylethylamine. (0.581 mmol) was added, and the mixture was stirred at 50 ° C. for 90 minutes under an argon atmosphere. After removing the reaction solvent under reduced pressure, the residue was dissolved in ethyl acetate, saturated brine was added, and a liquid separation operation was performed. The ethyl acetate layer was extracted, and the residue obtained by removal under reduced pressure was purified by silica gel column chromatography (ethyl acetate / hexane = 2/1 → 1/1) to obtain the target compound (80 mg, 79%).
1 H NMR (400 MHz, CD 2 Cl 2 ): δ 0.04 (s, 6H), 0.89 (s, 9H), 1.99-2.06 (m, 1H), 2.09 (s, 3H), 2.12-2.19 (m, 1H), 2.53-2.61 (m, 2H), 4.23 (t, J = 6.6 Hz, 1H), 4.44-4.46 (m, 3H), 4.70 (d, J = 12.8 Hz, 1H), 4.76 (d, J = 12.8 Hz, 1H), 5.48 (s, 1H), 5.60 (s, 1H), 6.29 (dd, J = 2.3, 8.2 Hz, 1H), 6.41 (d, J = 1.8 Hz, 1H), 6.69 (d , J = 8.2 Hz, 1H), 6.85-6.95 (m, 2H), 7.05 (d, J = 6.9 Hz, 1H), 7.15-7.21 (m, 2H), 7.29-7.32 (m, 2H), 7.36- 7.45 (m, 4H), 7.58-7.61 (m, 2H), 7.77 (d, J = 7.3 Hz, 2H), 8.18 (s, 1H)
13 C NMR (75 MHz, CDCl 3 ): δ-5.40, 15.2, 18.4, 25.9, 30.1, 31.3, 38.9, 47.1, 54.4, 63.2, 67.2, 102.1, 107.6, 107.7, 110.9, 113.8, 114.5 (containing 2 peaks) ), 120.0, 120.7, 124.9, 126.5, 127.1, 127.5, 127.7, 130.1, 130.2, 130.4, 130.8, 136.7, 138.3, 141.2, 143.5, 143.6, 143.8, 146.2, 150.9, 151.1, 156.4, 169.3
HRMS (ESI + ): calcd for [M + H] + , 786.33969; found, 786.33663 (-3.06 mmu)
Ａ４の合成
　化合物Ａ３ ２６４ｍｇ（０．３４４ｍｍｏｌ）をジクロロメタン３．２ｍＬ、Ｎ，Ｎ’－ジメチルホルムアミド０．８ｍＬに溶解させた後、２－クロロトリチルクロライドレジン６３３ｍｇ（１．０３１ｍｍｏｌ）、Ｎ，Ｎ－ジエチルイソプロピルアミン４００μＬ（２．２６ｍｍｏｌ）を加え、アルゴン雰囲気下、アルミホイルで遮光して２４時間撹拌した。濾過により反応溶媒を取り除いた後、レジンをジクロロメタン／メタノール／Ｎ，Ｎ－ジイソプロピルエチルアミン＝１７／２／１混合液およびジクロロメタンで洗浄した。得られたレジンを１４等分し、１４分の１量を次の合成に使用した。 Synthesis of A4 264 mg (0.344 mmol) of compound A3 was dissolved in 3.2 mL of dichloromethane and 0.8 mL of N, N'-dimethylformamide, and then 633 mg (1.031 mmol) of 2-chlorotrityl chloride resin, N, N- 400 μL (2.26 mmol) of diethyl isopropylamine was added, and the mixture was stirred with aluminum foil under an argon atmosphere for 24 hours. After removing the reaction solvent by filtration, the resin was washed with dichloromethane / methanol / N, N-diisopropylethylamine = 17/2/1 mixture and dichloromethane. The obtained resin was divided into 14 equal parts, and 1/14 amount was used for the next synthesis.
Ａ５の合成
　Ａ４に、Ｎ，Ｎ’－ジメチルホルムアミド１．６５ｍＬを加え、１時間振とう後濾過により溶液を除き、テトラクロロ－ｐ－ベンゾキノン１２０ｍＭ　Ｎ，Ｎ’－ジメチルホルムアミド溶液　２．４ｍＬを加え３０分振とうした。溶液を濾過によって除き、Ｎ，Ｎ’－ジメチルホルムアミド１．６５ｍＬを加え１分間振とうし、溶液を濾過によって除いた。反応後のレジンをそのまま次の反応に使用した。 Synthesis of A5 To A4 , 1.65 mL of N, N'-dimethylformamide was added, the solution was removed by filtration after shaking for 1 hour, and 2.4 mL of tetrachloro-p-benzoquinone 120 mM N, N'-dimethylformamide solution was added. Shake for 30 minutes. The solution was removed by filtration, 1.65 mL of N, N'-dimethylformamide was added and shaken for 1 minute, and the solution was removed by filtration. The resin after the reaction was used as it was for the next reaction.
Ａ６の合成
　Ａ５に２０％ピペリジン／Ｎ，Ｎ’－ジメチルホルムアミド溶液（ｖ／ｖ）　１２００μＬを加えて３分間振とうし、溶液を濾過によって除いた。再び２０％ピペリジン／Ｎ，Ｎ’－ジメチルホルムアミド溶液（ｖ／ｖ）１２００μＬを加えて１２分間振とうし、溶液を濾過によって除いた。Ｎ，Ｎ’－ジメチルホルムアミド　１３５０μＬを加え１分間振とうし、溶液を除く作業を６度行った。反応後のレジンをそのまま次の反応に使用した。 A6 Synthesis A5 20% piperidine / N of, N'- dimethylformamide (v / v) 3 minutes shaken added 1200MyuL, the solution was removed by filtration. 1200 μL of 20% piperidine / N, N'-dimethylformamide solution (v / v) was added again and shaken for 12 minutes, and the solution was removed by filtration. 1350 μL of N, N'-dimethylformamide was added, and the mixture was shaken for 1 minute to remove the solution 6 times. The resin after the reaction was used as it was for the next reaction.
Ｍ－ＨＭＲＧの合成
　得られたレジンＡ６を３０ｍＬバイアル瓶に移し、トリフルオロ酢酸２ｍＬ、水２００μＬ、トリエチルシラン２００μＬを加え２時間撹拌した。濾過によってレジンを除き、アセトニトリルで洗いこんだ後、濾液を減圧蒸留し残渣をジエチルエーテル２０ｍＬに加えて３，０００ｒｐｍで１０分間遠心した。上清のジエチルエーテルを除き、一晩風乾させた後、残渣をジメチルスルホキシドに溶解させて回収し、ＨＰＬＣ（ｅｌｕｅｎｔ　Ａ（Ｈ２Ｏ　０．１％　ＴＦＡ）ａｎｄ　ｅｌｕｅｎｔ　Ｂ（ＣＨ３ＣＮ　８０％、　Ｈ２Ｏ　２０％、　０．１％　ＴＦＡ）（Ａ／Ｂ＝８０／２０　ｔｏ　２５／７５　ｉｎ　４５ｍｉｎ））で精製し目的化合物を得た。
HRMS(ESI+): calcd for [M]+, 448.16949; found, 448.16828 (-1.21 mmu) Synthesis of M-HMRG The obtained resin A6 was transferred to a 30 mL vial, 2 mL of trifluoroacetic acid, 200 μL of water and 200 μL of triethylsilane were added, and the mixture was stirred for 2 hours. The resin was removed by filtration, washed with acetonitrile, the filtrate was distilled under reduced pressure, the residue was added to 20 mL of diethyl ether, and the mixture was centrifuged at 3,000 rpm for 10 minutes. Except diethyl ether supernatant, after air-dry overnight, the residue was collected and dissolved in dimethyl sulfoxide, HPLC (eluent A (H 2 O 0.1% TFA) and eluent B (CH 3 CN 80%, H 2 O 20%, to give the desired compound, which was purified by 0.1% TFA) (a / B = 80/20 to 25/75 in 45min)).
HRMS (ESI + ): calcd for [M] + , 448.16949; found, 448.16828 (-1.21 mmu)
酸性条件分析
Figure JPOXMLDOC01-appb-I000017

Acid condition analysis
Figure JPOXMLDOC01-appb-I000017
［実施例１］
ヒト腹部大動脈瘤手術検体を用いたスクリーニング
　これまでに食道癌に実績のあるＤＰＰＩＶ蛍光プローブ（Ｅ－Ｐ－ＨＭＲＧ及びＧ－Ｐ－ＨＭＲＧ）と新規に開発したＭｅｔ－ＨＭＲＧを比較した。実験条件を以下に示す。結果としてＭｅｔ－ＨＭＲＧが他のプローブよりも有意に蛍光強度の上昇を認めた（図１参照）。 [Example 1]
Screening using human abdominal aortic aneurysm surgical specimens We compared the DPPIV fluorescent probes (EP-HMRG and GP-HMRG) that have been proven for esophageal cancer with the newly developed Met-HMRG. The experimental conditions are shown below. As a result, Met-HMRG showed a significant increase in fluorescence intensity compared to other probes (see FIG. 1).
（実験条件）
　吻合部として使用した腸骨動脈の内膜（一部中膜を含む）を摘出し、Ｌｙｓａｔｅ化。Ｌｙｓａｔｅの蛋白濃度は０．１ｍｇ／ｍｌに調整して蛋白量としては２．２５μｇになるように調整した。其々のプローブが１μＭになるように滴加し、ｉｎｃｕｂａｔｅは３７℃としてＥｎｖｉｓｉｏｎ（登録商標）で１ｍｉｎ，１ｈｒの蛍光強度を測定した。 (Experimental conditions)
The intima (including some media) of the iliac artery used as the anastomotic site was removed and converted to Lysate. The protein concentration of Lysate was adjusted to 0.1 mg / ml so that the amount of protein was 2.25 μg. Each probe was added dropwise to 1 μM, and the fluorescence intensity was measured at 1 min and 1 hr with Envision® at 37 ° C.
［実施例２］
ヒト腹部大動脈瘤手術検体を用いたイメージング
　腹部大動脈瘤に施行した開腹手術症例に対して、瘤壁と吻合部動脈壁（正常動脈壁）から各々試料を採取し、Ｍｅｔ－ＨＭＲＧに浸漬したゲルを血管内腔側から適応後、経時的に蛍光強度を測定した。その結果、瘤壁と比較して吻合部動脈壁で蛍光強度の上昇を認めた（図２及び３）。実験条件を以下に示す。 [Example 2]
Imaging Using Human Abdominal Aortic Aneurysm Surgery Specimens were collected from the aneurysm wall and anastomotic artery wall (normal artery wall) for open surgery cases performed on the abdominal aortic aneurysm, and gels immersed in Met-HMRG were used. After adaptation from the lumen side of the blood vessel, the fluorescence intensity was measured over time. As a result, an increase in fluorescence intensity was observed in the anastomotic artery wall as compared with the aneurysm wall (FIGS. 2 and 3). The experimental conditions are shown below.
（実験条件）
　蛍光プローブのＤＭＳＯ溶液（１０μＭ）のうち０．５μＬを１００μＬのＰＢＳ（－）溶液に溶かし（最終プローブ濃度５０μＭ）、ゲルを３０分間浸漬させる。各々の検体にプローブ浸漬ゲルを適応した後、Ｍａｅｓｔｒｏ　Ｉｎ　Ｖｉｖｏ　Ｉｍａｇｉｎｇ　Ｓｙｓｔｅｍ　Ｅｘを用いて蛍光強度を経時的に６０分間測定した。
・Ｆｉｌｔｅｒ　ｓｅｔ：Ｂｌｕｅ（Ｅｘ．４３５－４８０ｎｍ／Ｅｍ．４９０ｎｍ　ＬＰ），　
・Ａｃｑｕｉｓｉｔｉｏｎ　Ｓｅｔｔｉｎｇｓ：５００　ｔｏ　７２０ｎｍ　ｉｎ　１０ｎｍ　ｓｔｅｐｓ (Experimental conditions)
0.5 μL of the fluorescent probe DMSO solution (10 μM) is dissolved in 100 μL PBS (−) solution (final probe concentration 50 μM) and the gel is immersed for 30 minutes. After applying the probe immersion gel to each sample, the fluorescence intensity was measured over time for 60 minutes using Maestro In Vivo Imaging System Ex.
・ Filter set: Blue (Ex.435-480nm / Em.490nm LP),
-Acquisition Settings: 500 to 720nm in 10nm steps
［実施例３］
Ｍｅｔ－ＨＭＲＧのＤｉｃｅｄ　Ｅｌｅｃｔｒｏｐｈｏｒｅｓｉｓ　Ｇｅｌ法（ＤＥＧ法）によるターゲット酵素の同定
　Ｍｅｔ－ＨＭＲＧと反応するターゲット酵素を検索すべく、スクリーニングで使用した症例の瘤壁及び吻合部動脈のＬｙｓａｔｅを使用してＤＥＧ法を実施した。細分化されたゲルの蛍光強度をＥｎｖｉｓｉｏｎ（登録商標）を用いて蛍光強度を測定し、蛍光反応が強いＳｐｏｔを回収して質量分析を行った。結果は、腸骨動脈壁内膜側でＭｅｔ－ＨＭＲＧを分解するターゲット酵素として、Ｐｕｒｏｍｙｃｉｎ　ｓｅｎｓｉｔｉｖｅ　ａｍｉｎｏｐｅｐｔｉｄａｓｅ（ピューロマイシン感受性アミノペプチダーゼ）とＢｌｅｏｍｙｃｉｎ　ｈｙｄｒｏｌａｓｅ（ブレオマイシン加水分解酵素）が候補として挙げられた（図４）。実験条件を以下に示す。 [Example 3]
Identification of the target enzyme by the Diced Electrophoresis Gel method (DEG method) of Met-HMRG The DG method using the Lysate of the aneurysm wall and anastomotic artery of the case used in the screening to search for the target enzyme that reacts with Met-HMRG. Was carried out. The fluorescence intensity of the subdivided gel was measured using Envision (registered trademark), and a spot having a strong fluorescence reaction was recovered and mass spectrometry was performed. As a result, Puromycin sensitive aminopeptidase (puromycin-sensitive aminopeptidase) and bleomycin hydrolase (bleomycin hydrolase) were listed as candidates as target enzymes for degrading Met-HMRG on the intima side of the iliac artery wall (Fig. 4). .. The experimental conditions are shown below.
（実験条件）
　瘤壁及び吻合物動脈のＬｙｓａｔｅを蛋白量が２２．５μｇになるようにＬｏａｄｉｎｇし、非変性の２次元電気泳動を行った。２次元で泳動されているゲルと採取して、３８４プレートに細分化されるように適応してから、Ｍｅｔ－ＨＭＲＧの最終濃度が１μＭになるように３８４個の各ウェルに滴加した。その後、Ｅｎｖｉｓｉｏｎ（登録商標）を用いてＭｅｔ－ＨＭＲＧの滴加直後の蛍光強度を測定し、３８４プレートを３７℃で一晩ｉｎｃｕｂａｔｅした。Ｏｖｅｒｎｉｇｈｔした３８４プレートの蛍光強度を再度測定し、蛍光強度の上昇値が高いウェルを同定。同定したウェルを採取して質量分析にかけた。 (Experimental conditions)
Lysate of the aneurysm wall and anastomotic artery was loaded so that the amount of protein was 22.5 μg, and non-denatured two-dimensional electrophoresis was performed. The gel was collected in two dimensions and adapted to be subdivided into 384 plates and then added dropwise to each of the 384 wells to a final concentration of Met-HMRG of 1 μM. Then, using Envision®, the fluorescence intensity immediately after the addition of Met-HMRG was measured, and the 384 plate was incubated at 37 ° C. overnight. The fluorescence intensity of the Overnight 384 plate was measured again to identify wells with a high increase in fluorescence intensity. The identified wells were collected and subjected to mass spectrometry.
［実施例４］
Ｍｅｔ－ＨＭＲＧのターゲット酵素に対する阻害実験
　ＤＥＧ法によってターゲット酵素の候補として挙げられたＰｕｒｏｍｙｃｉｎ　ｓｅｎｓｉｔｉｖｅ　ａｍｉｎｏｐｅｐｔｉｄａｓｅとＢｌｅｏｍｙｃｉｎ　ｈｙｄｒｏｌａｓｅの阻害剤であるＰｕｒｏｍｙｃｉｎ（ピューロマイシン）とＢｌｅｏｍｙｃｉｎ（ブレオマイシン）を用いて、吻合部動脈及び瘤壁のＬｙｓａｔｅとＭｅｔ－ＨＭＲＧの蛍光反応が阻害されるか調べた。結果は両者ともに濃度依存性に蛍光反応を阻害したが、吻合部動脈壁のＬｙｓａｔｅにおいてＢｌｅｏｍｙｃｉｎよりもＰｕｒｏｍｙｃｉｎで、より良好な濃度依存性による阻害を認めた（図５）。実験条件を以下に示す。 [Example 4]
Inhibition experiment of Met-HMRG against target enzyme Puromycin and bleomycin hydrolase inhibitors, which are candidates for target enzyme by the DEG method, are used as puromycin and bleomycin. It was investigated whether the fluorescence reaction between wall Lysate and Met-HMRG was inhibited. The results showed that both of them inhibited the fluorescence reaction in a concentration-dependent manner, but in the Lysate of the anastomotic artery wall, Puromycin was more concentrated than bleomycin, and better concentration-dependent inhibition was observed (Fig. 5). The experimental conditions are shown below.
（実験条件）
　Ｍｅｔ－ＨＭＲＧ最終濃度を１μＭになるように調整し、インヒビター最終濃度は、１ｍＭ～１ｎＭの１０倍刻みと０Ｍ（ＤＭＳＯ）としてプローブと混和させた。３８４プレートを使用して各濃度の阻害剤入りプローブを１５μｌ滴加し、０．１ｍｇ／ｍｌになるように調整した吻合部動脈壁及び瘤壁のＬｙｓａｔｅ５μｌを各ウェルに適応した後にＥｎｖｉｓｉｏｎ（登録商標）を用いて０～１２０分まで１分毎に蛍光強度を測定した。 (Experimental conditions)
The final Met-HMRG concentration was adjusted to 1 μM and the final inhibitor concentration was mixed with the probe in 10-fold increments of 1 mM to 1 nM and 0 M (DMSO). Using a 384 plate, 15 μl of a probe containing an inhibitor of each concentration was added, and 5 μl of Lysate of the anastomotic artery wall and aneurysm wall adjusted to 0.1 mg / ml was applied to each well, and then Envision (registered trademark). ) Was used to measure the fluorescence intensity every minute from 0 to 120 minutes.
［実施例５］
Ｍｅｔ－ＨＭＲＧとの精製酵素を用いた蛍光反応の確認（Ｐｕｒｏｍｙｃｉｎ　ｓｅｎｓｉｔｉｖｅ　ａｍｉｎｏｐｅｐｔｉｄａｓｅ及びＢｌｅｏｍｙｃｉｎ　ｈｙｄｒｏｌａｓｅ）
　阻害実験よりターゲット酵素と考えられるＰｕｒｏｍｙｃｉｎ　ｓｅｎｓｉｔｉｖｅ　ａｍｉｎｏｐｅｐｔｉｄａｓｅ及びＢｌｅｏｍｙｃｉｎ　ｈｙｄｒｏｌａｓｅが実際にＭｅｔ－ＨＭＲＧと蛍光反応を示すか確認した。結果として両者とＭｅｔ－ＨＭＲＧと蛍光反応する事が示された。実験条件を以下に示す。 [Example 5]
Confirmation of Fluorescence Reaction Using Purified Enzyme with Met-HMRG (Puromycin sensitive aminopeptidase and Bleomycin hydrolase)
From the inhibition experiment, it was confirmed whether the target enzymes Puromycin sensitive aminopeptidase and bleomycin hydrolase actually show a fluorescence reaction with Met-HMRG. As a result, it was shown that both were fluorescently reacted with Met-HMRG. The experimental conditions are shown below.
（実験条件）
　Ｐｕｒｏｍｙｃｉｎ　ｓｅｎｓｉｔｉｖｅ　ａｍｉｎｏｐｅｐｔｉｄａｓｅ　蛋白量１０ｎｇとＢｌｅｏｍｙｃｉｎ　ｈｙｄｒｏｌａｓｅ　蛋白量２５０ｎｇと最終濃度が０．１μＭになるように調整したＭｅｔ－ＨＭＲＧを其々に滴加して反応させ、Ｅｎｖｉｓｉｏｎ（登録商標）により０～１２０分まで１分毎に蛍光強度を測定した。 (Experimental conditions)
Puromycin sensitive aminopeptidase protein amount of 10 ng, bleomycin hydrolase protein amount of 250 ng, and Met-HMRG adjusted to a final concentration of 0.1 μM were added dropwise to each of them to react, and from 0 to 120 minutes by Envision (registered trademark) Fluorescence intensity was measured every minute.
［実施例６］
Ｗｅｓｔｅｒｎ　Ｂｌｏｔｔｉｎｇ法によるターゲット酵素発現の確認
　合部動脈壁のＬｙｓａｔｅ用いてＷｅｓｔｅｒｎ　ｂｌｏｔｔｉｎｇを行った。この結果、吻合部動脈壁のＬｙｓａｔｅではＰｕｒｏｍｙｃｉｎ　ｓｅｎｓｉｔｉｖｅ　ａｍｉｎｏｐｅｐｔｉｄａｓｅ及びＢｌｅｏｍｙｃｉｎ　ｈｙｄｒｏｌａｓｅの発現を認め、Ａｃｔｉｖｉｔｙに関しても両者の発現と関係していると考えられた。（図７）。以下に実験条件を示す。 [Example 6]
Confirmation of target enzyme expression by Western blotting Western blotting was performed using Lysate of the arterial wall of the joint. As a result, the expression of Puromycin sensitive aminopeptidase and bleomycin hydrolase was observed in the Lysate of the anastomotic artery wall, and it was considered that the activity was also related to the expression of both. (Fig. 7). The experimental conditions are shown below.
（実験条件）
　スクリーニングにしようした吻合部動脈のＬｙｓａｔｅを蛋白濃度０．５ｍｇ／ｍｌに調整し、９５度分で熱処理を行った。この蛋白をＳＤＳ－ＰＡＧＥで分離した。ブロッティング膜はＰＶＤＦ膜を用い、１次抗体としてＰＳＡ抗体とＢＬＭＨ抗体を濃度１／５００で使用した。またｌｏａｄｉｎｇ　ｃｏｎｔｒｏｌとしてＧＡＰＤＨ抗体を濃度１／１０００で使用した。検出はＨＲＰを用いた発色法で行った。
　最終濃度１ｍＭに調整したＭｅｔ－ＨＭＲＧを１５μｌと吻合部動脈Ｌｙｓａｔｅ　５μｌを３８４プレートに其々滴加して反応させ、Ｅｎｖｉｓｉｏｎ（登録商標）により０～１２０分の蛍光強度を測定し、Ａｃｔｉｖｉｔｙを計測した。 (Experimental conditions)
The Lysate of the anastomotic artery used for screening was adjusted to a protein concentration of 0.5 mg / ml, and heat treatment was performed at 95 ° C. This protein was separated by SDS-PAGE. A PVDF membrane was used as the blotting membrane, and PSA antibody and BLMH antibody were used as primary antibodies at a concentration of 1/500. In addition, GAPDH antibody was used as a loading control at a concentration of 1/1000. The detection was performed by a color development method using HRP.
15 μl of Met-HMRG adjusted to a final concentration of 1 mM and 5 μl of anastomotic artery Lysate were added dropwise to a 384 plate to react, and the fluorescence intensity for 0 to 120 minutes was measured by Envision (registered trademark) to measure the activity. did.
［実施例７］
吻合部動脈壁におけるＰｕｒｏｍｙｃｉｎ　ｓｅｎｓｉｔｉｖｅ　ａｍｉｎｏｐｅｐｔｉｄａｓｅとＢｌｅｏｍｙｃｉｎ　ｈｙｄｒｏｌａｓｅの免疫染色
　Ｍｅｔ－ＨＭＲＧによるイメージングを行い、蛍光反応を確認した検体に関して、実際のＰｕｒｏｍｙｃｉｎ　ｓｅｎｓｉｔｉｖｅ　ａｍｉｎｏｐｅｐｔｉｄａｓｅとＢｌｅｏｍｙｃｉｎ　ｈｙｄｒｏｌａｓｅ発現を確認するために免疫染色を行った。結果として、Ｐｕｒｏｍｙｃｉｎ　ｓｅｎｓｉｔｉｖｅ　ａｍｉｎｏｐｅｐｔｉｄａｓｅは瘤壁よりも吻合物動脈内膜に強く発現している事が確認された（図９）。Ｂｌｅｏｍｙｃｉｎ　ｈｙｄｒｏｌａｓｅに関しては吻合部動脈壁と瘤壁で発現に差異は認められなかった（図１０）。以下に実験条件を示す。 [Example 7]
Immunostaining of Puromycin sensitive aminopeptidase and Bleomycin hydrolase on the arterial wall of the anastomotic site Imaging with Met-HMRG was performed. As a result, it was confirmed that Puromycin sensitive aminopeptidase was more strongly expressed in the intima of the anastomotic artery than in the aneurysm wall (Fig. 9). Regarding bleomycin hydrolase, no difference was observed in the expression between the anastomotic artery wall and the aneurysm wall (Fig. 10). The experimental conditions are shown below.
（実験条件）
　パラフィン固定の組織からスライド切片を作成し、１次抗体としてＰＳＡ抗体を濃度１／１０、ＢＬＭＨ抗体１／５０で使用した。発色はＤＡＢ法による。 (Experimental conditions)
Slide sections were prepared from paraffin-fixed tissue and PSA antibody was used as the primary antibody at a concentration of 1/10 and BLMH antibody 1/50. Color development is by the DAB method.
　以上の実験結果から、Ｍｅｔ－ＨＭＲＧのターゲット酵素はＰｕｒｏｍｙｃｉｎ　ｓｅｎｓｉｔｉｖｅ　ａｍｉｎｏｐｅｐｔｉｄａｓｅが最も強く関与しており、Ｂｌｅｏｍｙｃｉｎ　ｈｙｄｒｏｌａｓｅの関与も否定出来ない。この明らかとなった生物学的性質を基盤として、このプローブの蛍光強度の差を利用することで、血管性状を客観的に評価出来る可能性が示された。 From the above experimental results, Puromycin sensitive aminopeptidase is most strongly involved in the target enzyme of Met-HMRG, and the involvement of bleomycin hydrolase cannot be denied. Based on this clarified biological property, it was shown that it is possible to objectively evaluate the vascular properties by using the difference in fluorescence intensity of this probe.

Claims (4)

  1. 　以下の式（Ｉ）で表される化合物又はその塩を含有する、ピューロマイシン感受性アミノペプチダーゼ又はブレオマイシン加水分解酵素を検出するのに用いられる蛍光プローブ。
    Figure JPOXMLDOC01-appb-I000001


    （式中、
    Ｒ１は、存在する場合は、ベンゼン環上に存在する同一又は異なる一価の置換基であり；
    Ｒ２及びＲ３は、それぞれ独立に、水素原子、炭素数１～６個のアルキル基又はハロゲン原子であり；
    Ｒ４及びＲ５は、それぞれ独立に、水素原子、炭素数１～６個のアルキル基又はハロゲン原子であり；
    Ｒ６及びＲ６’は、それぞれ独立に、水素原子又は炭素数１～５個のアルキル基であり；
    ｍは、０～４の整数である。）     A fluorescent probe used to detect a puromycin-sensitive aminopeptidase or bleomycin hydrolase containing a compound represented by the following formula (I) or a salt thereof.
    Figure JPOXMLDOC01-appb-I000001


    (During the ceremony,
    R 1 , if present, is the same or different monovalent substituent present on the benzene ring;
    R 2 and R 3 are each independently a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom;
    R 4 and R 5 are each independently a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom;
    R 6 and R 6 'are each independently hydrogen atom or a C 1-5 alkyl group having a carbon;
    m is an integer from 0 to 4. )
  2. 　以下の化合物又はその塩を含有する、ピューロマイシン感受性アミノペプチダーゼ又はブレオマイシン加水分解酵素を検出するのに用いられる蛍光プローブ。
    Figure JPOXMLDOC01-appb-I000002

    A fluorescent probe used to detect puromycin-sensitive aminopeptidase or bleomycin hydrolase, which contains the following compounds or salts thereof.
    Figure JPOXMLDOC01-appb-I000002
  3. 　大動脈瘤病変又は非拡張部動脈壁の血管性状を評価する方法であって、（ａ）以下の式（１）で表される化合物又はその塩を含み、ピューロマイシン感受性アミノペプチダーゼ又はブレオマイシン加水分解酵素を検出することができる蛍光プローブを大動脈瘤病変又は非拡張部動脈壁の臨床検体に適用する工程、及び（ｂ）前記蛍光プローブを適用した臨床検体の蛍光像を測定することを含む、方法。
    Figure JPOXMLDOC01-appb-I000003



    （式中、
    Ｒ１は、存在する場合は、ベンゼン環上に存在する同一又は異なる一価の置換基であり；
    Ｒ２及びＲ３は、それぞれ独立に、水素原子、炭素数１～６個のアルキル基又はハロゲン原子であり；
    Ｒ４及びＲ５は、それぞれ独立に、水素原子、炭素数１～６個のアルキル基又はハロゲン原子であり；
    Ｒ６及びＲ６’は、それぞれ独立に、水素原子又は炭素数１～５個のアルキル基であり；
    ｍは、０～４の整数であり；
    Ｐ１及びＰ２は、アミノ酸残基を表し、Ｐ１は、存在しても、存在しなくてもよい。）     A method for evaluating the vascular properties of aortic aneurysm lesions or non-dilated arterial walls, which comprises (a) a compound represented by the following formula (1) or a salt thereof, and is a puromycin-sensitive aminopeptidase or bleomycin hydrolyzing enzyme. A method comprising applying a fluorescent probe capable of detecting aneurysm lesion or a non-dilated arterial wall to a clinical sample, and (b) measuring a fluorescent image of the clinical sample to which the fluorescent probe is applied.
    Figure JPOXMLDOC01-appb-I000003



    (During the ceremony,
    R 1 , if present, is the same or different monovalent substituent present on the benzene ring;
    R 2 and R 3 are each independently a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom;
    R 4 and R 5 are each independently a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom;
    R 6 and R 6 'are each independently hydrogen atom or a C 1-5 alkyl group having a carbon;
    m is an integer from 0 to 4;
    P 1 and P 2 represent amino acid residues, and P 1 may or may not be present. )
  4. 　大動脈瘤病変又は非拡張部動脈壁における正常動脈壁を検知する方法であって、（ａ）以下の式（１）で表される化合物又はその塩を含み、ピューロマイシン感受性アミノペプチダーゼ又はブレオマイシン加水分解酵素を検出することができる蛍光プローブを大動脈瘤病変又は非拡張部動脈壁の臨床検体に適用する工程、及び（ｂ）前記蛍光プローブを適用した臨床検体の蛍光像を測定することを含む、方法。
    Figure JPOXMLDOC01-appb-I000004


    （式中、
    Ｒ１は、存在する場合は、ベンゼン環上に存在する同一又は異なる一価の置換基であり；
    Ｒ２及びＲ３は、それぞれ独立に、水素原子、炭素数１～６個のアルキル基又はハロゲン原子であり；
    Ｒ４及びＲ５は、それぞれ独立に、水素原子、炭素数１～６個のアルキル基又はハロゲン原子であり；
    Ｒ６及びＲ６’は、それぞれ独立に、水素原子又は炭素数１～５個のアルキル基であり；
    ｍは、０～４の整数であり；
    Ｐ１及びＰ２は、アミノ酸残基を表し、Ｐ１は、存在しても、存在しなくてもよい。）     A method for detecting a normal arterial wall in an aortic aneurysm lesion or a non-dilated arterial wall, which comprises (a) a compound represented by the following formula (1) or a salt thereof, and puromycin-sensitive aminopeptidase or bleomycin hydrolysis. A method comprising applying a fluorescent probe capable of detecting an enzyme to a clinical sample of an aortic aneurysm lesion or a non-dilated arterial wall, and (b) measuring a fluorescent image of the clinical sample to which the fluorescent probe is applied. ..
    Figure JPOXMLDOC01-appb-I000004


    (During the ceremony,
    R 1 , if present, is the same or different monovalent substituent present on the benzene ring;
    R 2 and R 3 are each independently a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom;
    R 4 and R 5 are each independently a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom;
    R 6 and R 6 'are each independently hydrogen atom or a C 1-5 alkyl group having a carbon;
    m is an integer from 0 to 4;
    P 1 and P 2 represent amino acid residues, and P 1 may or may not be present. )
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012111818A1 (en) * 2011-02-18 2012-08-23 国立大学法人 東京大学 Fluorescent probe
WO2013180181A1 (en) * 2012-05-30 2013-12-05 国立大学法人 東京大学 Fluorescent probe for high-sensitivity pancreatic fluid detection, and method for detecting pancreatic fluid
WO2016137004A1 (en) * 2015-02-27 2016-09-01 国立大学法人 東京大学 Fluorescent probe for detecting calpain activity
WO2019168199A1 (en) * 2018-03-02 2019-09-06 国立大学法人 東京大学 Fluorescent probe for detecting carboxypeptidase activity
WO2020095946A1 (en) * 2018-11-07 2020-05-14 国立大学法人 東京大学 Novel fluorescent probe

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012111818A1 (en) * 2011-02-18 2012-08-23 国立大学法人 東京大学 Fluorescent probe
WO2013180181A1 (en) * 2012-05-30 2013-12-05 国立大学法人 東京大学 Fluorescent probe for high-sensitivity pancreatic fluid detection, and method for detecting pancreatic fluid
WO2016137004A1 (en) * 2015-02-27 2016-09-01 国立大学法人 東京大学 Fluorescent probe for detecting calpain activity
WO2019168199A1 (en) * 2018-03-02 2019-09-06 国立大学法人 東京大学 Fluorescent probe for detecting carboxypeptidase activity
WO2020095946A1 (en) * 2018-11-07 2020-05-14 国立大学法人 東京大学 Novel fluorescent probe

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
KOMATSU, TORU ET AL.: "Diced Electrophoresis Gel Assay for Screening Enzymes with Specified Activities", J.AM.CHEM.SOC., vol. 135, no. 16, 12 April 2013 (2013-04-12), pages 6002 - 6005, XP055817448 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023228790A1 (en) * 2022-05-27 2023-11-30 五稜化薬株式会社 Fluorescent-probe solution

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