JP7303532B2 - New fluorescent probe - Google Patents

Description

Application of Article 30, Paragraph 2 of the Patent Act Publication 1 ▲1 ▼ Date of issue: February 21, 2018 ▲2 ▼ Publication: Master's thesis presentation at the Graduate School of Pharmaceutical Sciences, the University of Tokyo (venue: National The University of Tokyo, Faculty of Pharmaceutical Sciences General Research Building, Date: March 6, 2018) ▲3▼ Publisher: Teru Ogasawara ▲4▼ Details of the disclosed invention: Teru Ogasawara, Graduate School of Pharmaceutical Sciences, the University of Tokyo Part of the research on novel fluorescent probes invented by Yasuteru Urano, Mako Kamiya, and Teru Ogasawara was published in the abstracts of the master's thesis presentation of the Graduate School of Science and Technology.

Application of Article 30, Paragraph 2 of the Patent Act Publication 2 ▲1 ▼Date: March 6, 2018 ▲2▼Meeting name, Venue: Graduate School of Pharmaceutical Sciences, University of Tokyo Master's thesis presentation, National The University of Tokyo Faculty of Pharmaceutical Sciences General Research Building ▲3▼ Publisher: Teru Ogasawara ▲4▼ Details of the disclosed invention: Teru Ogasawara, Yasuteru Urano and Mako Kamiya at the master's thesis presentation at the Graduate School of Pharmaceutical Sciences, the University of Tokyo , and part of the research on novel fluorescent probes invented by Teru Ogasawara.

Application of Article 30, Paragraph 2 of the Patent Law Publication 3 ▲1 ▼ Date of issue: May 10, 2018 ▲2 ▼ Publication: The 13th Annual General Meeting/Academic Meeting of the Japanese Society for Molecular Imaging (Place: National Ito International Research Center, The University of Tokyo, Date: May 31, 2018) ▲3▼ Publishers: Mako Kamiya and Yasuteru Urano ▲4▼ Details of the disclosed inventions: Mako Kamiya and Yasuteru Urano published a part of the research on novel fluorescent probes invented by Yasuteru Urano, Mako Kamiya, and Teru Ogasawara in the abstracts of the 13th Annual Meeting of the Molecular Imaging Society of Japan.

Application of Article 30, Paragraph 2 of the Patent Act Publication 4 ▲1 ▼ Date: May 31, 2018 ▲ 2 ▼ Meeting name, venue: The 13th Annual General Meeting/Academic Meeting of the Japanese Society for Molecular Imaging, National Ito International Research Center, The University of Tokyo ▲3▼Publisher: Mako Kamiya and Yasuteru Urano ▲4▼Details of the disclosed invention: Mako Kamiya and Yasuteru Urano at the 13th General Assembly and Scientific Meeting of the Molecular Imaging Society of Japan , Yasuteru Urano, Mako Kamiya, and Teru Ogasawara have published a part of their research on novel fluorescent probes.

The present invention relates to novel fluorescent probes for detecting puromycin-sensitive aminopeptidase (PSA), detection methods using the fluorescent probes, and detection kits containing the probes.

At present, the number of cancer patients and deaths is increasing year by year, and the development of treatment methods for cancer continues to be expected. At present, one of the most reliable cancer treatment methods is early detection and reliable surgical removal of cancer, but complete removal of minute cancer tissue that is difficult to see completely It is difficult to do so and causes recurrence.

Imaging technologies such as X-ray CT, magnetic resonance imaging (MRI), and positron emission tomography (PET) are extremely important in cancer diagnosis from the perspective of identifying minute cancer tissue. has become an important technology. Among them, fluorescence imaging is expected as a simple and safe technique with high sensitivity and excellent temporal and spatial resolution.

The inventors of the present application have so far developed a fluorescent probe for γ-glutamyl transpeptidase (GGT), which is enhanced in breast cancer cells, and this enables highly sensitive and specific detection of minute tumors in a short period of time. It has been demonstrated (for example, Non-Patent Documents 1 and 2). However, since there are false positive and false negative breast cancer specimens with this GGT fluorescent probe, development of a new probe having a criterion different from the GGT enzymatic activity has been desired. In particular, the GGT fluorescent probe of Non-Patent Document 1 uses a fluorescent response in the green region, so if a probe that exhibits a fluorescent response in a different region such as red can be developed, multiple fluorescent probes can be used together. It is expected that breast cancer can be detected with even higher sensitivity and specificity.

Y. Urano et al., Sci. Transl. Med., vol. 3, pp. 110ra119 (2011) H. Ueo et al., Sci. Rep. 5, 12080 (2015)

Therefore, the present invention provides a novel fluorescent probe that can detect with high sensitivity and high selectivity a biomarker enzyme activity that is highly specific to breast cancer, and that can detect it as a longer wavelength red fluorescence response. This is an issue.

As a result of intensive studies to solve the above problems, the present inventors found puromycin-sensitive aminopeptidase (PSA) as a new biomarker enzyme for detecting breast cancer cells, and have different characteristics from conventional green probes ( Breast cancer cells can be detected as a red fluorescence response by using a compound with a fluorophore having a wavelength, charge, lipid solubility) as a core and introducing an amino acid residue that is specifically cleaved by PSA in the side chain. The inventors have found that a novel fluorescent probe can be obtained, and have completed the present invention.

〔式中、
Ａは、アラニン残基であり、Ｂは、アミノ酸残基を表し、ここで、Ａは隣接するＮ原子とアミド結合を形成して連結し、ＢはＡとアミド結合を形成して連結しており、
Ｘは、Ｓｉ（Ｒ^ａ）（Ｒ^ｂ）、Ｇｅ（Ｒ^ａ）（Ｒ^ｂ）、Ｓｎ（Ｒ^ａ）（Ｒ^ｂ）、Ｃ（Ｒ^ａ）（Ｒ^ｂ）、Ｐ（＝Ｏ）（Ｒ^ａ）又はＯを表し（ここで、Ｒ^ａ及びＲ^ｂは、それぞれ独立に水素原子、アルキル基、又はアリール基を表す）；
Ｒ^１は、水素原子、又はそれぞれ置換されていてもよいアルキル基、カルボキシル基、エステル基、アルコキシ基、アミド基、及びアジド基よりなる群から独立に選択される１～４個の同一又は異なる置換基を表し；
Ｒ^２は、水素原子、ヒドロキシル基、シアノ基、それぞれ置換されていてもよいアルキル基、アルコキシ基、アリール、又はヘテロアリールを表し；
Ｒ^３及びＲ^４は、それぞれ独立に、水素原子、ヒドロキシル基、ハロゲン原子、それぞれ置換されていてもよいアルキル基、スルホ基、カルボキシル基、エステル基、アミド基及びアジド基よりなる群から独立に選択される１～３個の同一又は異なる置換基を表し；
Ｒ^５、Ｒ^６、及びＲ^７は、それぞれ独立に水素原子又はアルキル基を表し、
ここで、Ｒ^６又はＲ^７は、それぞれＲ^４と一緒になって、それらが結合する窒素原子を含む環構造を形成してもよい。〕；
＜２＞ Ｂが、アスパラギン残基又はリシン残基である、上記＜１＞に記載の蛍光プローブ；
＜３＞Ｒ^２が、置換されていてもよい炭素数１～６個のアルキル基、又は置換されていてもよい炭素数１～６個のアルコキシ基である、上記＜１＞又は＜２＞に記載の蛍光プローブ；
＜４＞Ｒ^２が、メチル基又はメトキシ基である、上記＜１＞～＜３＞のいずれか１に記載の蛍光プローブ；
＜５＞Ｒ^１、Ｒ^３、Ｒ^４、Ｒ^５、Ｒ^６、及びＲ^７が、いずれも水素原子である、上記＜１＞～＜４＞のいずれか１に記載の蛍光プローブ；
＜６＞上記＜１＞～＜５＞のいずれか１に記載の蛍光プローブを含む、がん細胞検出用組成物；
＜７＞前記がん細胞が、乳がん細胞である、上記＜６＞に記載の組成物；及び
＜８＞上記＜１＞～＜５＞のいずれか１項に記載の蛍光プローブを含む、ピューロマイシン感受性アミノペプチダーゼ（ＰＳＡ）検出用キット
を提供するものである。 That is, in one aspect of the present invention,
<1> A fluorescent probe for detecting puromycin-sensitive aminopeptidase (PSA), containing a compound represented by the following formula (I) or a salt thereof:

[In the formula,
A is an alanine residue and B represents an amino acid residue, wherein A is linked to an adjacent N atom by forming an amide bond, and B is linked to A by forming an amide bond. cage,
X is Si(R ^a )(R ^b ), Ge(R ^a )(R ^b ), Sn(R ^a )(R ^b ), C(R ^a )(R ^b ), P(=O)(R ^a ) or O (wherein R ^a and R ^b each independently represent a hydrogen atom, an alkyl group, or an aryl group);
R ¹ is a hydrogen atom or 1 to 4 identical or different groups independently selected from the group consisting of optionally substituted alkyl groups, carboxyl groups, ester groups, alkoxy groups, amido groups, and azido groups. represents a substituent;
R ² represents a hydrogen atom, a hydroxyl group, a cyano group, an optionally substituted alkyl group, an alkoxy group, an aryl, or a heteroaryl;
R ³ and R ⁴ are each independently selected from the group consisting of a hydrogen atom, a hydroxyl group, a halogen atom, an optionally substituted alkyl group, a sulfo group, a carboxyl group, an ester group, an amide group and an azide group. representing 1 to 3 identical or different substituents selected;
R ⁵ , R ⁶ and R ⁷ each independently represent a hydrogen atom or an alkyl group;
Here, R ⁶ or R ⁷ may each be taken together with R ⁴ to form a ring structure containing the nitrogen atom to which they are attached. ];
<2> The fluorescent probe according to <1> above, wherein B is an asparagine residue or a lysine residue;
<3> The above <1> or <2>, wherein R ² is an optionally substituted alkyl group having 1 to 6 carbon atoms or an optionally substituted alkoxy group having 1 to 6 carbon atoms. The fluorescent probe according to;
<4> The fluorescent probe according to any one of <1> to <3> above, wherein R ² is a methyl group or a methoxy group;
<5> The fluorescent probe according to any one of <1> to <4> above, wherein R ¹ , R ³ , R ⁴ , R ⁵ , R ⁶ , and R ⁷ are all hydrogen atoms;
<6> A composition for detecting cancer cells, comprising the fluorescent probe according to any one of <1> to <5>above;
<7> The cancer cell is a breast cancer cell, the composition according to <6>above; and <8> the fluorescent probe according to any one of <1> to <5> above. A kit for detection of mycin-sensitive aminopeptidase (PSA) is provided.

In another aspect, the present invention also provides
<9> A step of contacting the fluorescent probe according to any one of <1> to <5> above with a sample to be measured, and puromycin-sensitive aminopeptidase (PSA) contained in the sample and the fluorescent probe A method for detecting PSA, comprising the step of observing a fluorescence response or absorbance change due to the reaction of
<10> The detection method according to <9> above, wherein the fluorescence response is visualized using a fluorescence imaging means;
<11> The method according to <9> or <10> above, wherein the sample to be measured is a cell expressing puromycin-sensitive aminopeptidase (PSA);
<12> The method according to <11> above, wherein the cells are cancer cells;
<13> The method according to <12> above, wherein the cancer cells are breast cancer cells.

According to the present invention, puromycin-sensitive aminopeptidase (PSA), which is expressed at high levels in breast cancer tissue, can be detected by fluorescence response, thereby accurately, rapidly and sensitively identifying the presence of breast cancer. and imaging.

In addition, the fluorescent probe of the present invention detects PSA activity as a fluorescence response as a new breast cancer biomarker enzyme different from the conventionally known γ-glutamyl transpeptidase (GGT). It is also possible to identify breast cancer specimens that could not be detected with detection fluorescent probes.

Furthermore, since the fluorescent probe of the present invention can detect PSA activity as a fluorescent response in the red region, it can be used in combination with a conventional green fluorescent probe to detect breast cancer cells with higher specificity than when each probe is used alone. Along with enabling detection, multi-color imaging using multiple fluorescence response regions is also possible. As a result, cancer cells such as breast cancer can be visualized and detected more precisely and with high sensitivity.

The fluorescent probe of the present invention is expected to be used not only for medical applications such as distinguishing between cancer cells such as breast cancer cells and normal cells and detecting or diagnosing them in real time, but also as a basic research tool for elucidating pathological conditions.

FIG. 1 shows fluorescence imaging images (a and b) of a tumor sample and a normal sample treated with the fluorescent probe 1, and changes in fluorescence intensity over time (c). FIG. 2 is a graph showing the time change of the T/N ratio associated with the addition of the fluorescent probe of the present invention to the tumor site. FIG. 3 is a graph showing changes in fluorescence intensity due to DEG assay (a) between fluorescent probe 1 and tumor site, and reaction with PSA. FIG. 4 shows absorption spectra (left) and fluorescence spectra (right) of fluorescent probes 1 and 2. FIG. 5 is a table showing fluorescence quantum yields of fluorescent probes 1 and 2. FIG. FIG. 6 is a graph showing changes in fluorescence intensity due to reaction of fluorescent probe 2 with PSA and GGT. FIG. 7 is a multicolor imaging image using fluorescent probe 2 and gGlu-HMRG.

Embodiments of the present invention will be described below. The scope of the present invention is not restricted by these explanations, and other than the following examples can be appropriately changed and implemented without impairing the gist of the present invention.

1. definition

As used herein, "amino acid" can be any compound having both an amino group and a carboxyl group, including natural and non-natural compounds. It may be a neutral amino acid, a basic amino acid, or an acidic amino acid. In addition to amino acids that themselves function as transmitters such as neurotransmitters, physiologically active peptides (dipeptides, tripeptides, tetrapeptides, oligopeptides) and polypeptide compounds such as proteins can be used, for example, α-amino acids, β-amino acids, γ-amino acids and the like. As the amino acid, it is preferable to use an optically active amino acid. For example, for α-amino acids, either D- or L-amino acids may be used, but it may be preferable to select optically active amino acids that function in vivo. As used herein, the term "amino acid residue" refers to a structure corresponding to a partial structure (acyl group) remaining after removing the hydroxyl group from the carboxyl group of an amino acid.

As used herein, "halogen atom" means a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom.

In the present specification, "alkyl" may be straight chain, branched chain, cyclic, or an aliphatic hydrocarbon group consisting of a combination thereof. _The number _of carbon atoms in the alkyl group _is not particularly limited. ) and 1 to 20 carbon atoms (C _1-20 ). When the number of carbon atoms is specified, it means "alkyl" having the number of carbon atoms within the specified range. For example, 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 are included. In this specification, an alkyl group may have one or more optional substituents. Examples of such substituents include, but are not limited to, alkoxy groups, halogen atoms, amino groups, mono- or di-substituted amino groups, substituted silyl groups, acyl, and the like. When an alkyl group has more than one substituent, they may be the same or different. The same applies to the alkyl moieties of other substituents containing alkyl moieties (eg, alkoxy groups, arylalkyl groups, etc.).

In this specification, when a certain functional group is defined as "optionally substituted", the type of substituent, substitution position, and number of substituents are not particularly limited, and two or more substitutions When having groups, they may be the same or different. Examples of 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 a substituent. Examples of such groups include, but are not limited to, halogenated alkyl groups, dialkylamino groups, and the like.

As used herein, "alkenyl" refers to straight or branched chain hydrocarbon groups having at least one carbon-carbon double bond. For example, non-limiting examples thereof include vinyl, allyl, 1-propenyl, isopropenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1,3-butandienyl, 1-pentenyl, 2-pentenyl, 3-pentenyl. , 4-pentenyl, 1,3-pentandienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl and 1,4-hexanedienyl). It may be in either the cis or trans conformation about the double bond.

As used herein, "alkynyl" refers to straight or branched chain hydrocarbon groups having at least one carbon-carbon triple bond. Examples include, but are not limited to, ethynyl, propynyl, 2-butynyl, 3-methylbutynyl, and the like.

As used herein, "cycloalkyl" refers to a monocyclic or polycyclic non-aromatic ring system consisting of alkyl as described above. The cycloalkyl can be unsubstituted or substituted with one or more substituents which may be the same or different, non-limiting examples of monocyclic cycloalkyls include cyclopropyl, cyclopentyl, cyclohexyl and cycloheptyl, and non-limiting examples of polycyclic cycloalkyls include 1-decalinyl, 2-decalinyl, norbornyl, adamantyl, and the like. The cycloalkyl may also be heterocycloalkyl containing one or more heteroatoms (eg, oxygen atom, nitrogen atom, sulfur atom, etc.) as ring-constituting atoms. Any —NH in the heterocycloalkyl ring may be protected as, for example, —N(Boc), —N(CBz) and —N(Tos) groups, nitrogen atoms in the ring or A sulfur atom can also be oxidized to the corresponding N-oxide, S-oxide or S,S-dioxide. For example, non-limiting examples of monocyclic heterocycloalkyls include diazapanyl, piperidinyl, pyrrolidinyl, piperazinyl, morpholinyl, thiomorpholinyl, thiazolidinyl, 1,4-dioxanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydrothiophenyl, lactams and lactones and the like.

As used herein, "cycloalkenyl" refers to a monocyclic or polycyclic non-aromatic ring system containing at least one carbon-carbon double bond. The cycloalkenyl may be unsubstituted or substituted with one or more substituents which may be the same or different, non-limiting examples of monocyclic cycloalkenyls include cyclopentenyl, cyclohexenyl and cyclohepta-1,3-dienyl, and non-limiting examples of polycyclic cycloalkenyls include norbornylenyl and the like. In addition, the cycloalkyl may be heterocycloalkenyl containing one or more heteroatoms (e.g., oxygen atom, nitrogen atom, sulfur atom, etc.) as a ring-constituting atom. Nitrogen atom or sulfur in heterocycloalkenyl ring Atoms may also be oxidized to the corresponding N-oxide, S-oxide or S,S-dioxide.

In the present specification, "aryl" may be either a monocyclic or condensed polycyclic aromatic hydrocarbon group, and a heteroatom (e.g., an oxygen atom, a nitrogen atom, or a sulfur atom) as a ring-constituting atom. etc.) may be aromatic heterocycles containing one or more. In this case, it is sometimes referred to as "heteroaryl" or "heteroaromatic." Whether the aryl is a single ring or a condensed ring, it may be attached at all possible positions. Non-limiting examples of monocyclic aryl include phenyl (Ph), thienyl (2- or 3-thienyl), pyridyl, furyl, thiazolyl, oxazolyl, pyrazolyl, 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-oxadiazole-3 -yl group and the like. Non-limiting examples of fused polycyclic aryl include 1-naphthyl, 2-naphthyl, 1-indenyl, 2-indenyl, 2,3-dihydroinden-1-yl, 2,3 -dihydroinden-2-yl group, 2-anthryl group, indazolyl group, quinolyl group, isoquinolyl group, 1,2-dihydroisoquinolyl group, 1,2,3,4-tetrahydroisoquinolyl group, indolyl group, isoindolyl group, phthalazinyl group, quinoxalinyl group, benzofuranyl group, 2,3-dihydrobenzofuran-1-yl group, 2,3-dihydrobenzofuran-2-yl group, 2,3-dihydrobenzothiophen-1-yl group, 2 ,3-dihydrobenzothiophen-2-yl group, benzothiazolyl group, benzimidazolyl group, fluorenyl group, thioxanthenyl group and the like. In this specification, an aryl group may have one or more optional substituents on its ring. Examples of such substituents include, but are not limited to, alkoxy groups, halogen atoms, amino groups, mono- or di-substituted amino groups, substituted silyl groups, acyl groups, and the like. When an aryl group has two or more substituents, they may be the same or different. The same applies to the aryl moieties of other substituents containing aryl moieties (such as aryloxy groups and arylalkyl groups).

As used herein, "arylalkyl" represents alkyl substituted with the above aryl. Arylalkyl may have one or more optional substituents. Examples of such substituents include, but are not limited to, alkoxy groups, halogen atoms, amino groups, mono- or di-substituted amino groups, substituted silyl groups, acyl groups, and the like. When an acyl group has two or more substituents, they may be the same or different. Non-limiting examples of arylalkyl include benzyl, 2-thienylmethyl, 3-thienylmethyl, 2-pyridylmethyl, 3-pyridylmethyl, 4-pyridylmethyl, 2-furylmethyl, 3-furylmethyl group, 2-thiazolylmethyl group, 4-thiazolylmethyl group, 5-thiazolylmethyl group, 2-oxazolylmethyl group, 4-oxazolylmethyl group, 5-oxazolylmethyl group, 1-pyrazolylmethyl group , 3-pyrazolylmethyl group, 4-pyrazolylmethyl group, 2-pyrazinylmethyl group, 2-pyrimidinylmethyl group, 4-pyrimidinylmethyl group, 5-pyrimidinylmethyl group, 1-pyrrolylmethyl group, 2-pyrrolylmethyl group, 3-pyrrolylmethyl group , 1-imidazolylmethyl group, 2-imidazolylmethyl group, 4-imidazolylmethyl group, 3-pyridazinylmethyl group, 4-pyridazinylmethyl group, 3-isothiazolylmethyl group, 3-isoxazoly methyl group, 1,2,4-oxadiazol-5-ylmethyl group or 1,2,4-oxadiazol-3-ylmethyl group.

Similarly, as used herein, "arylalkenyl" refers to alkenyl substituted with the above aryl.

As used herein, the term "alkoxy group" refers to a structure in which the aforementioned alkyl group is bonded to an oxygen atom, and examples thereof include saturated alkoxy groups that are linear, branched, cyclic, or a combination thereof. For example, methoxy, ethoxy, n-propoxy, isopropoxy, cyclopropoxy, n-butoxy, isobutoxy, s-butoxy, t-butoxy, cyclobutoxy, cyclopropylmethoxy, n- Pentyloxy group, cyclopentyloxy group, cyclopropylethyloxy group, cyclobutylmethyloxy group, n-hexyloxy group, cyclohexyloxy group, cyclopropylpropyloxy group, cyclobutylethyloxy group, cyclopentylmethyloxy group and the like are preferred. Examples include:

As used herein, the term "alkylene" refers to a linear or branched saturated hydrocarbon divalent group, such as 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, 1,2-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-dimethyltetramethylene, 2,2-dimethyltetramethylene, 2,2-di-n-propyltrimethylene and the like.

As used herein, the term “alkenylene” refers to a divalent group consisting of an unsaturated hydrocarbon having at least one straight or branched carbon-carbon double bond, such as ethenylene , 1-methylethenylene, 1-ethylethenylene, 1,2-dimethylethenylene, 1,2-diethylethenylene, 1-ethyl-2-methylethenylene, propenylene, 1-methyl-2-propenylene, 2-methyl -2-propenylene, 1,1-dimethyl-2-propenylene, 1,2-dimethyl-2-propenylene, 1-ethyl-2-propenylene, 2-ethyl-2-propenylene, 1,1-diethyl-2-propenylene , 1,2-diethyl-2-propenylene, 1-butenylene, 2-butenylene, 1-methyl-2-butenylene, 2-methyl-2-butenylene, 1,1-dimethyl-2-butenylene, 1,2-dimethyl -2-butenylene and the like.

As used herein, "arylene" and "arylalkylene" mean divalent groups based on the above "aryl" and "arylalkyl" respectively. Similarly, "oxyalkylene" and "aryleneoxy" refer to divalent radicals based on the above "alkoxy" and "aryloxy" respectively.

As used herein, "amido" includes both RNR'CO- (alkaminocarbonyl- when R=alkyl) and RCONR'- (alkylcarbonylamino- when R=alkyl).

As used herein, "ester" includes both ROCO- (alkoxycarbonyl- when R=alkyl) and RCOO- (alkylcarbonyloxy- when R=alkyl).

As used herein, the term "ring structure" means a heterocyclic or carbocyclic group when formed by the combination of two substituents, and such groups may be saturated, unsaturated, or aromatic. can be. Thus, we include cycloalkyl, cycloalkenyl, aryl, and heteroaryl as defined above. Examples include cycloalkyl, phenyl, naphthyl, morpholinyl, piperdinyl, imidazolyl, pyrrolidinyl, pyridyl, and the like. As used herein, substituents can form a ring structure with another substituent, and when such substituents are attached, the skilled artisan will appreciate the particular substitution, e.g. is formed. Thus, when certain substituents are described as forming a ring structure together, those skilled in the art will appreciate that the ring structure can be formed or readily produced by conventional chemical reactions. All such ring structures and processes for their formation are within the purview of those skilled in the art.

2. Fluorescent Probe for PSA Detection of the Present Invention In one aspect, the fluorescent probe of the present invention contains a compound having a structure represented by the following formula (I).

In the above formula (I), A and B are sites that have a structure in which amino acid residues are linked and can be selectively hydrolyzed by puromycin-sensitive aminopeptidase (PSA). More specifically, A is an alanine residue and B represents an amino acid residue. Here, A is linked to N(R ⁵ ) in the formula by forming an amide bond, that is, the N atom of N(R ⁵ ) with the carbonyl moiety (acyl group) of the alanine residue A is linked to the xanthene skeleton by forming an amide bond. Furthermore, A can be linked to amino acid residue B in the same manner as a normal peptide chain, so that B is linked to A by forming an amide bond. Therefore, B has the same structure as the so-called N-terminal residue, and the intermediate amino acid residue A can be linked to B in the same way as in a normal peptide chain.

Puromycin-sensitive aminopeptidase (PSA) is a cytoplasmic alanyl aminopeptidase that uses Ala-AMC as a standard substrate, and is reported to be highly expressed particularly in the central nervous system. In clinical medicine, it is used as a biomarker for estimating renal function, and its function is known to be inhibited by puromycin.

From the viewpoint of the purpose of the present invention, which is to detect puromycin-sensitive aminopeptidase (PSA), the above A and B are substrate peptides of PSA and are a combination of amino acid residues that are easily hydrolyzed selectively. preferable. More specifically, as described above, A is an alanine residue (“Ala” or “A”) and B is an asparagine residue (“Asn” or “N”) or a lysine residue (“ Lys" or "K"). More preferably, A is an alanine residue and B is an asparagine residue.

In the above formula (I), X is Si(R ^a )(R ^b ), Ge(R ^a )(R ^b ), Sn(R ^a )(R ^b ), C(R ^a )(R ^b ), represents P(=O) (R ^a ) or O; Here, Ra ^{and Rb} each independently represent a hydrogen atom, an alkyl group, or an aryl group. When R ^a and R ^b are alkyl groups, they may have one or more substituents such as alkyl groups, alkoxy groups, halogen atoms, hydroxyl groups, carboxyl groups, It may have one or more amino groups, sulfo groups and the like. R ^a and R ^b can each be an optionally substituted alkyl group having 1 to 6 carbon atoms, and both are preferably methyl groups. In some cases, R ^a and R ^b may combine with each other to form a ring structure. For example, when R ^a and R ^b are both alkyl groups, R ^a and R ^b can be joined together to form a spirocarbocycle. The ring formed is preferably, for example, a 5- to 8-membered ring. X is preferably Si(R ^a )(R ^b ), more preferably Si(CH ₃ ) ₂ .

R ¹ represents a hydrogen atom or 1 to 4 substituents bonded to a benzene ring. Preferably, R ¹ is a hydrogen atom or 1 to 4 groups independently selected from the group consisting of an alkyl group, a carboxyl group, an ester group, an alkoxy group, an amide group, and an azide group, each of which may be substituted. represent the same or different substituents. When R ¹ is other than a hydrogen atom, its position on the benzene ring is not particularly limited, but it is preferably meta to R ² . Moreover, when the benzene ring has two or more substituents, they may be the same or different. Preferably R ¹ is a hydrogen atom.

^R2 represents a hydrogen atom, a hydroxyl group, a cyano group, an optionally substituted alkyl group, an alkoxy group, an aryl, or a heteroaryl. Preferred are a hydroxyl group, a cyano group, an optionally substituted C ₁ -C ₄ alkyl group, a C ₁ -C ₄ alkoxy group, or a phenyl group. More preferably, it is a methyl group or a methoxy group.

R ³ and R ⁴ are each independently selected from the group consisting of a hydrogen atom, a hydroxyl group, a halogen atom, an optionally substituted alkyl group, a sulfo group, a carboxyl group, an ester group, an amide group and an azide group; represents 1 to 3 identical or different substituents. Preferably, both R ³ and R ⁴ are hydrogen atoms. Also, like R ¹ above, R ³ or R ⁴ can also have a fluorophore that serves as a fluorescence resonance energy transfer (FRET) donor.

R ⁵ , R ⁶ and R ⁷ each independently represent a hydrogen atom or an alkyl group. When R ⁵ , R ⁶ and R ⁷ all represent an alkyl group, they may be the same or different. For example, R ⁵ , R ⁶ and R ⁷ can each independently be a methyl group or an ethyl group. Preferably, R ⁵ , R ⁶ and R ⁷ are all hydrogen atoms.

Here, R ⁶ or R ⁷ may each be taken together with R ⁴ to form a ring structure containing the nitrogen atom to which they are attached. Preferably, the ring structure is a 5- to 8-membered heterocyclic ring structure. In addition, the ring structure can further contain heteroatoms other than the nitrogen atom to which ^R6 and ^R7 are attached.

Preferably, R ¹ , R ³ , R ⁴ , R ⁵ , R ⁶ and R ⁷ are all hydrogen atoms.

が挙げられる。ただし、これらに限定されるものではない。これらの化合物では、式（Ｉ）中の「Ａ－Ｂ」は、いずれもアラニン残基－アスパラギン残基（Ａｌａ－Ａｓｎ）となっている例である。 Specific examples of the compound of formula (I) (trifluoroacetate) particularly suitable as the PSA detection fluorescent probe of the present invention include:

is mentioned. However, it is not limited to these. In these compounds, "AB" in formula (I) is an example in which all are alanine residue-asparagine residue (Ala-Asn).

The compound represented by the above formula (I) usually exists as a salt because it has a monovalent positive charge at the N atom to which ^R6 and ^R7 are connected. Such salts include base addition salts, acid addition salts, amino acid salts and the like. Examples of base addition salts include metal salts such as sodium salts, potassium salts, calcium salts and magnesium salts, ammonium salts, or organic amine salts such as triethylamine salts, piperidine salts and morpholine salts. Examples include mineral salts such as hydrochlorides, sulfates, and nitrates; organic acid salts such as carboxylates, trifluoroacetates, methanesulfonates, paratoluenesulfonates, citrates, and oxalates; can be mentioned. Glycine salt etc. can be illustrated as an amino acid salt. However, it is not limited to these salts.

The compound represented by formula (I) may have one or more asymmetric carbon atoms depending on the type of substituent, and stereoisomers such as optical isomers or diastereoisomers exist. Sometimes. All stereoisomers in pure form, any mixtures of stereoisomers, racemates, etc. are included within the scope of the present invention.

Although the compound represented by formula (I) or a salt thereof may exist as a hydrate or solvate, both of these substances are included within 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 above fluorescent probe may be used as a composition by blending additives that are commonly used for preparation of reagents, if necessary. For example, as additives for use in a physiological environment, additives such as dissolution aids, pH adjusters, buffers, and tonicity agents can be used, and the blending amounts of these can be appropriately selected by those skilled in the art. be. These compositions can be provided as a composition in an appropriate form such as a mixture of powder forms, a freeze-dried product, granules, tablets, liquids, and the like.

The examples herein specifically illustrate methods of preparation for representative compounds encompassed by the compounds of the present invention represented by formula (I), and therefore, a person skilled in the art can refer to the disclosures of the present specification. Any compound encompassed by formula (I) can be easily produced by referring to and optionally selecting starting materials, reagents, reaction conditions, and the like.

3. Method for Detecting PSA Using the Fluorescent Probe of the Present Invention The fluorescent probe of the present invention detects PSA enzymatic activity by fluorescence control using photoinduced electron transfer (PET). More specifically, as shown in the scheme below, in a state in which an amino acid residue (peptide chain) of "AB" is linked to the side chain at the 3-position of the xanthene ring, irradiation with excitation light Since the xanthene ring site is highly electron-accepting, it is in a state of being quenched by electron transfer from the benzene ring site. On the other hand, when the fluorescence probe molecule reacts with PSA, the "AB" peptide chain of the side chain is cleaved by hydrolysis and the 3-position is changed to an amino group, the xanthene ring site electron-accepting , and as a result, fluorescence emission will be observed.

That is, in the compound represented by formula (I), the fluorescence is quenched by PET from the benzene ring site before the reaction with PSA, but when the peptide chain is cleaved by PSA, the electron density of the xanthene ring site becomes is changed, and fluorescence quenching by PET is suppressed, so that the activity of PSA can be detected as a fluorescence response. As a result, when the cells incorporating the fluorescent probe represented by formula (I) do not express a peptidase (PSA) capable of cleaving by hydrolyzing "AB", the fluorescent substance is However, if such a peptidase is present, cleavage of "A-B" will result in a strong fluorescence emission, thereby indicating the presence of the targeted peptidase. It is possible to detect the presence of cancer cells expressing peptidases such as PSA by observing on/off changes in intensity.

Further, in the compound represented by formula (I), X which is the 10th element of the xanthene ring
By adjusting the type of and the type of substituents (R ¹ and R ² ) on the benzene ring linked to the xanthene skeleton, fluorescence emission due to “AB” cleavage is changed to fluorescence in the red region with a fluorescence wavelength of 600 nm or more It has the feature that it can be This makes it possible to detect the presence of cancer cells or the like as a red fluorescence response.

When detection is performed using the fluorescent probe of the present invention, visible light of about 400 to 600 nm may be applied as excitation light. The fluorescence wavelength to be observed is usually in the range of about 580-700 nm.

According to the above-described luminescence mechanism, the PSA detection method using the fluorescent probe of the present invention specifically detects or visualizes the presence of PSA by observing the fluorescence response or absorbance change due to the reaction between PSA and the fluorescent probe. can be done. As used herein, the term "detection" should be interpreted in the broadest sense, including measurement for various purposes such as quantitative and qualitative.

in particular,
A) the step of contacting the fluorescent probe of the present invention with a sample to be measured; and
B) By including the step of observing the fluorescence response or absorbance change due to the reaction between the puromycin-sensitive aminopeptidase (PSA) contained in the sample and the fluorescent probe, only target cells expressing PSA are specifically fluorescent It can be detected or visualized as a response.

Also, the method of the invention can further comprise observing the fluorescence response using a fluorescence imaging means. As means for observing the fluorescence response, a fluorometer having a wide measurement wavelength can be used, but the fluorescence response can also be visualized using fluorescence imaging means capable of displaying a two-dimensional image. By using the means of fluorescence imaging, the fluorescence response can be visualized in two dimensions, thus making it possible to visualize PSA instantaneously. A device known in the art can be used as the fluorescence imaging device. In some cases, it is also possible to detect the reaction between the sample to be measured and the fluorescent probe by means of a change in the ultraviolet-visible absorption spectrum (for example, a change in absorbance at a specific absorption wavelength).

In the above step A), the means for bringing the sample to be measured and the fluorescent probe into contact typically includes adding, coating, or spraying a solution containing the fluorescent probe to the sample. It is possible to appropriately select according to the measurement environment and the like. Such contacting preferably takes place in an ex vivo environment. In addition, when the fluorescent probe of the present invention is applied to diagnose or assist diagnosis in individual animals, or to detect specific cells or tissues, the means for contacting the fluorescent probe with target cells or tissues is particularly Without limitation, administration means common in the art can be used, for example, intravenous administration.

The application concentration of the fluorescent probe of the present invention is not particularly limited, but for example, a solution with a concentration of about 0.1 to 10 μM can be applied.

In addition, the target cells can be irradiated with light directly or through a waveguide (optical fiber or the like). As the light source, any light source can be used as long as it can irradiate light containing the absorption wavelength of the fluorescent probe of the present invention after enzymatic cleavage. It can be selected as appropriate.

As the fluorescent probe of the present invention, the compound represented by the above formula (I) or a salt thereof may be used as it is. may be used. For example, as additives for using the reagent in a physiological environment, additives such as dissolution aids, pH adjusters, buffers, and tonicity agents can be used, and the amount of these additives can be appropriately selected by those skilled in the art. It is possible. These compositions are generally provided as compositions in suitable forms such as powdered mixtures, lyophilized products, granules, tablets, and liquids. It is possible to apply by dissolving in

The sample to be measured in the above step A) can be cells expressing puromycin-sensitive aminopeptidase (PSA), but when such cells are cancer cells or cancer tissues expressing PSA Furthermore, cancer cells and cancer tissues can be detected or visualized by the detection method of the present invention. That is, the fluorescent probe of the present invention, the composition containing the fluorescent probe, and the detection method of the present invention can also be used for cancer diagnosis.

As used herein, the term "cancer tissue" means any tissue containing cancer cells. The term "tissue" should be interpreted in the broadest sense, including a part or the whole of an organ, and should not be interpreted restrictively in any way. The fluorescent probe of the present invention or the composition for detecting cancer cells containing the fluorescent probe has the effect of detecting PSA that is specifically and strongly expressed in cancer tissues. is preferred. In addition, the term "diagnosis" as used herein should be interpreted in the broadest sense, including macroscopically or microscopically confirming the presence of cancerous tissue in any living body site. The sample to be measured in step A) above is preferably cancer cells, more preferably breast cancer cells.

Moreover, as one aspect of the detection method of the present invention, a fluorescent probe for γ-glutamyltranspeptidase (GGT) can be used in combination with the fluorescent probe of the present invention. As a result, both GGT and PSA expressed in breast cancer cells can be detected as biomarker enzymes, so breast cancer cells can be detected with higher specificity than using individual probes alone. Become. In particular, when the fluorescent probe of the present invention has fluorescence emission in the red region, it can be used in combination with a conventional green fluorescent probe to enable multi-color imaging using a plurality of fluorescence response regions.

4. PSA Detection Kit Using the Fluorescent Probe of the Present Invention In the detection method of the present invention, it is preferable to use a PSA detection kit containing the above fluorescent probe. In the kit, the fluorescent probe of the present invention is usually prepared as a solution. It can also be applied by dissolving in distilled water for injection or an appropriate buffer solution.

In addition, the kit may contain other reagents and the like as necessary. For example, additives such as dissolution aids, pH adjusters, buffers, tonicity agents, and the like can be used, and the amount of these additives can be appropriately selected by those skilled in the art. In particular, as described above, in addition to the fluorescent probes of the present invention, a green fluorescent probe for GGT detection may be included.

EXAMPLES The present invention will be described in more detail below with reference to Examples, but the present invention is not limited to these.

1. Synthesis of Fluorescent Probes Fluorescent probe compounds were synthesized by introducing various combinations of dipeptides into the 3-position of the xanthene ring according to the reaction scheme shown below. Here, the reaction scheme (General Procedure) is described first, followed by the synthesis of individual compounds having specific dipeptides.

(a) Fmoc-A.A., HATU, DIEA, DMF, r.t. y. # Fmoc-A.A. = Gly 76.4 %, Glu(OtBu) 52.0 %, Lys(Boc) 46.6 %, Tyr(tBu) 42.6 %, Leu 52.4 %, Pro 49.6 %, Ile 63.2 %, Ala 42.2 %, Val 71.6 %, Trp(Boc) 63.5 %, Thr(tBu) 76.0 %, His(Trt) 53.8 %, Ser(tBu) 64.4 %, Phe 67.5 %, Cys(Trt) 66.6 %, Met 55.3 %, Asn(Trt) 64.4 %, Gln(Trt) 71.3 %, Asp(OtBu) 66.3 %, Arg(Pbf) 69.3 %, (b) 2-Chlorotrityl resin, DIEA, DMF/CH2Cl2, r.t. (c’) DDQ, DMF, r.t. (d) (i) piperidine, DMF/NMP, r.t. (ii) Fmoc-A.A., HATU, DIEA, DMF/NMP, r.t. (iii) piperidine, DMF/NMP, r.t. (e) TFA, Anisole, H2O, r.t. [Reaction Scheme (General Procedure)]

(a) Fmoc-AA, HATU, DIEA, DMF, rty# Fmoc-AA = Gly 76.4%, Glu(OtBu) 52.0%, Lys(Boc) 46.6%, Tyr(tBu) 42.6%, Leu 52.4%, Pro 49.6 %, Ile 63.2 %, Ala 42.2 %, Val 71.6 %, Trp(Boc) 63.5 %, Thr(tBu) 76.0 %, His(Trt) 53.8 %, Ser(tBu) 64.4 %, Phe 67.5 %, Cys(Trt) 66.6%, Met 55.3%, Asn(Trt) 64.4%, Gln(Trt) 71.3%, Asp(OtBu) 66.3%, Arg(Pbf) 69.3%, (b) 2-Chlorotrityl resin, DIEA, DMF/CH2Cl2, rt (c') DDQ, DMF, rt (d) (i) piperidine, DMF/NMP, rt (ii) Fmoc-AA, HATU, DIEA, DMF/NMP, rt (iii) piperidine, DMF/NMP, rt (e ) TFA, Anisole, H2O, rt

文献（Kushida, Y.ら、 Bioorganic & medicinal chemistry letters 2012, 22 (12), 3908-3911.）に従い、出発物質である化合物１を合成した。 [Synthesis of Compound 1 (Leuco-2-Me SiR600)]

The starting material compound 1 was synthesized according to the literature (Kushida, Y. et al., Bioorganic & medicinal chemistry letters 2012, 22 (12), 3908-3911.).

DMF中の化合物１（２当量）の溶液に、Ｆｍｏｃ－アミノ酸（１当量）、ＨＡＴＵ（１当量）およびＤＩＥＡ（５当量）を加え、室温で１．５時間撹拌した。反応混合物を溶媒留去し、Ｈ_２Ｏを加え、次いで水層をＣＨ_２Ｃｌ_２で抽出した。有機層を塩水で洗浄し、Ｎａ_２ＳＯ_４で乾燥し、蒸発させた。残渣をカラムクロマトグラフィーまたはＭＰＬＣで精製した。不純物が含まれている場合は、溶離液としてクロロホルムを用いたＧＰＣリサイクルカラムによる追加精製を行い、純粋な化合物を得た。 [Synthesis of Compound 2]

To a solution of compound 1 (2 eq) in DMF was added Fmoc-amino acid (1 eq), HATU (1 eq) and DIEA (5 eq) and stirred at room temperature for 1.5 hours. The reaction mixture was evaporated and H ₂ O was added, then the aqueous layer was extracted with CH ₂ Cl ₂ . _The organic layer was washed with brine, dried over _Na2SO4 and evaporated. The residue was purified by column chromatography or MPLC. If impurities were present, additional purification by GPC recycle column using chloroform as eluent was performed to obtain pure compounds.

ＤＭＦ ０．４ｍＬおよびＣＨ_２Ｃｌ_２ １．６ｍＬ中の化合物２（１－１．１当量）の溶液に、塩化２－クロロトリチル樹脂（１当量）およびＤＩＥＡ ２００μＬを加えた。反応混合物を室温で撹拌した。Ａｒ雰囲気下で一晩放置した。 反応溶液を濾過により除去し、樹脂をＣＨ_２Ｃｌ_２で複数回洗浄した。 この樹脂を分割し、ペプチド合成機で次の反応に使用した。 [Synthesis of Compound 3]

To a solution of compound 2 (1-1.1 eq) in 0.4 mL DMF and 1.6 mL CH ₂ Cl ₂ was added 2-chlorotrityl chloride resin (1 eq) and 200 μL DIEA. The reaction mixture was stirred at room temperature. It was left overnight under an Ar atmosphere. The reaction solution was removed by filtration and the resin was washed multiple times with CH ₂ Cl ₂ . The resin was split and used for the next reaction on the peptide synthesizer.

上記で得られた樹脂（０．０２４５ｍｍｏｌ）にＤＭＦ １．１ｍＬを加え、１時間振とうして樹脂を膨潤させた。ＤＭＦを濾過により除去し、ＤＭＦ中のＤＤＱ（４当量）を加え、１時間振とうした。反応液をろ別した。樹脂に、ＤＭＦ中の４０％ピペリジンを加え、８００μＬを添加し、３分間振とうし、反応溶液を濾過により除去した。ＤＭＦ中の４０％ピペリジン４００μＬを添加し、１２分間振とうし、反応溶液を濾過により除去した。樹脂にＤＭＦ９００μＬを加え、１分間振とうし、ＤＭＦを濾過により除去した。この洗浄操作をさらに５回繰り返した。 [Synthesis of compound 4]

1.1 mL of DMF was added to the resin (0.0245 mmol) obtained above and shaken for 1 hour to swell the resin. DMF was removed by filtration and DDQ (4 eq) in DMF was added and shaken for 1 hour. The reaction liquid was filtered off. To the resin was added 40% piperidine in DMF, 800 μL was added, shaken for 3 minutes and the reaction solution was filtered off. 400 μL of 40% piperidine in DMF was added, shaken for 12 minutes and the reaction solution was filtered off. 900 μL of DMF was added to the resin, shaken for 1 minute, and the DMF was removed by filtration. This washing operation was repeated 5 more times.

上記で得られた樹脂（０．０２４５ｍｍｏｌ）にＤＭＦ中のＦｍｏｃ－アミノ酸（４当量）、ＤＭＦ中のＨＡＴＵ（４当量）およびＮＭＰ中の２Ｍ ＤＩＥＡ ２００μＬを加え、２時間振とうした。反応液をろ別した。樹脂にＤＭＦ ９００μＬを加え、１分間振とうし、ＤＭＦを濾過により除去した。この洗浄操作をさらに２回繰り返した。この凝縮操作をもう一度繰り返した。ＤＭＦ中のＦｍｏｃ－アミノ酸（４当量）、ＤＭＦ中のＨＡＴＵ（４当量）およびＮＭＰ中の２Ｍ ＤＩＥＡ ２００μＬを樹脂（０．０２４５ｍｍｏｌ）に添加し、１時間振とうした。反応液をろ別した。樹脂にＤＭＦ ９００μＬを加え、１分間振とうし、ＤＭＦを濾過により除去した。この洗浄操作をさらに２回繰り返した。樹脂に、ＤＭＦ中の４０％ピペリジン８００μＬを加え、３分間振とうし、反応溶液を濾過により除去した。ＤＭＦ中の４０％ピペリジン４００μＬを添加し、１２分間振とうし、反応溶液を濾過により除去した。樹脂にＤＭＦ９００μＬを加え、1分間振とうし、ＤＭＦを濾過により除去した。この洗浄操作をさらに５回繰り返した。 [Synthesis of compound 5]

To the resin obtained above (0.0245 mmol) was added Fmoc-amino acid (4 eq.) in DMF, HATU (4 eq.) in DMF and 200 μL of 2M DIEA in NMP and shaken for 2 hours. The reaction liquid was filtered off. 900 μL of DMF was added to the resin, shaken for 1 minute, and the DMF was removed by filtration. This washing operation was repeated two more times. This condensation operation was repeated once more. Fmoc-amino acid (4 eq.) in DMF, HATU (4 eq.) in DMF and 200 μL of 2M DIEA in NMP were added to the resin (0.0245 mmol) and shaken for 1 hour. The reaction liquid was filtered off. 900 μL of DMF was added to the resin, shaken for 1 minute, and the DMF was removed by filtration. This washing operation was repeated two more times. 800 μL of 40% piperidine in DMF was added to the resin, shaken for 3 minutes, and the reaction solution was filtered off. 400 μL of 40% piperidine in DMF was added, shaken for 12 minutes and the reaction solution was filtered off. 900 μL of DMF was added to the resin, shaken for 1 minute, and the DMF was removed by filtration. This washing operation was repeated 5 more times.

ペプチド合成機との反応後の樹脂（０．０２４ｍｍｏｌ）に、ＴＦＡ ２ｍＬ、アニソール５０μＬ、水５０μＬを加え、２時間攪拌した。反応混合物を蒸発乾固させた。残渣を４℃で５０ｍＬのチューブ中の４０ｍＬのＥｔ_２Ｏ中に注ぎ、沈殿させ、遠心分離（３，０００ｒｐｍ、４℃で５分間）を行った。 上清を除去し、残渣を風乾して粗生成物（化合物６）を得た。粗生成物をＤＭＳＯに溶解し、ＬＣ－ＭＳで分析した。２５４ｎｍまたは４９０ｎｍのいずれかでの粗生成物の純度が８０％未満である場合、４０分間でＡ / Ｂ = ７０ / ３０～０ / １００（溶出液Ａ ：０．１％ＴＦＡを含有するＨ_２Ｏ、溶出液Ｂ：８０％アセトニトリルおよび２０％Ｈ_２Ｏ中の０．１％ＴＦＡ）で精製して十分な純度の化合物６を得た。 [Synthesis of compound 6]

2 mL of TFA, 50 μL of anisole, and 50 μL of water were added to the resin (0.024 mmol) after the reaction with the peptide synthesizer, and the mixture was stirred for 2 hours. The reaction mixture was evaporated to dryness. The residue was poured into 40 mL Et ₂ O in a 50 mL tube at 4° C., pelleted, and centrifuged (3,000 rpm, 4° C. for 5 minutes). The supernatant was removed and the residue was air dried to give the crude product (compound 6). The crude product was dissolved in DMSO and analyzed by LC-MS. A/B = 70/30 to 0/100 in 40 min (Eluent A: _H2 containing 0.1% TFA O, eluent B: 0.1% TFA in 80% acetonitrile and 20% _H2O ) to give compound 6 of sufficient purity.

[Synthesis of fluorescent probe 1 (NA-2-Me SiR600) of the present invention]
According to the reaction scheme described above, the fluorescent probe compound (NA-2-Me SiR600) of the present invention having Asn-Ala (NA) in the side chain was synthesized as follows.

上記化合物２に相当する化合物７を化合物１から合成した。上記反応スキームに従い、化合物１（417mg、1.21mmol）、Ｆｍｏｃ－Ａｌａ－ＯＨ（380mg、1.22mmol）、ＨＡＴＵ（232mg、0.611mmol）およびＤＩＥＡ（515μL、3.03mmol）から、化合物７を合成した。ＡｃＯＥｔ / ｎ－ヘキサン（１／１）を溶出液としてとするＭＰＬＣ、及びクロロホルムを溶出液とするＧＰＣリサイクルカラムを用いて精製した後、化合物７を得た（163mg、42.2％）。
¹H-NMR (300 MHz, CD2Cl2) δ: 0.40 (d, 3H, J = 2.9 Hz), 0.56 (d, 3H, J = 5.9 Hz), 1.46 (d, 3H, J = 6.6 Hz), 2.36 (s, 3H), 3.58 (br, 2H), 4.20 (t, 1H, J = 6.6 Hz), 4.42 (d, 2H, J = 7.3 Hz), 4.43-4.46 (m, 1H), 5.66 (s, 1H), 5.95 (d, 1H, J = 7.3 Hz), 6.60 (dd, 1H, J = 8.4, 2.6 Hz), 6.88 (d, 1H, J = 8.1 Hz), 6.92 (d, 1H, J = 2.2 Hz), 7.00-7.10 (m, 4H), 7.17-7.18 (m, 1H), 7.26-7.28 (m, 2H), 7.31-7.34 (m, 1H), 7.38 (t, 2H, J = 7.0 Hz), 7.55-7.61 (m, 2H), 7.76-7.81 (m, 2H), 7.89 (s, 1H), 8.58 (s, 1H).
¹³C-NMR (75 MHz, CD2Cl2, isomer mixture) δ: -0.57, -0.53, -0.33, 18.72, 20.77, 47.46, 50.35, 51.71, 67.43, 117.29, 119.16, 120.31, 121.63, 124.50, 125.35, 125.41, 126.27, 126.56, 127.44, 128.08, 130.12, 130.43, 130.95, 131.58, 133.93, 134.55, 135.24, 135.28, 135.66, 138.65, 141.61, 144.01, 144.22, 144.46, 145.50, 146.39, 156.73, 171.21.
HRMS (ESI+): calcd for [M+H]+, 638.28389 ; found, 638.28361 (-0.28 mmu) [Synthesis of compound 7]

Compound 7 corresponding to compound 2 above was synthesized from compound 1. Compound 7 was synthesized from compound 1 (417 mg, 1.21 mmol), Fmoc-Ala-OH (380 mg, 1.22 mmol), HATU (232 mg, 0.611 mmol) and DIEA (515 μL, 3.03 mmol) according to the above reaction scheme. After purification using MPLC with AcOEt/n-hexane (1/1) as eluent and GPC recycle column with chloroform as eluent, compound 7 was obtained (163 mg, 42.2%).
¹ H-NMR (300 MHz, CD2Cl2) δ: 0.40 (d, 3H, J = 2.9 Hz), 0.56 (d, 3H, J = 5.9 Hz), 1.46 (d, 3H, J = 6.6 Hz), 2.36 ( s, 3H), 3.58 (br, 2H), 4.20 (t, 1H, J = 6.6 Hz), 4.42 (d, 2H, J = 7.3 Hz), 4.43-4.46 (m, 1H), 5.66 (s, 1H ), 5.95 (d, 1H, J = 7.3 Hz), 6.60 (dd, 1H, J = 8.4, 2.6 Hz), 6.88 (d, 1H, J = 8.1 Hz), 6.92 (d, 1H, J = 2.2 Hz ), 7.00-7.10 (m, 4H), 7.17-7.18 (m, 1H), 7.26-7.28 (m, 2H), 7.31-7.34 (m, 1H), 7.38 (t, 2H, J = 7.0 Hz), 7.55-7.61 (m, 2H), 7.76-7.81 (m, 2H), 7.89 (s, 1H), 8.58 (s, 1H).
¹³ C-NMR (75 MHz, CD2Cl2, isomer mixture) δ: -0.57, -0.53, -0.33, 18.72, 20.77, 47.46, 50.35, 51.71, 67.43, 117.29, 119.16, 120.31, 121.63, 124.50 , 125.35, 125.41, 126.27, 126.56, 127.44, 128.08, 130.12, 130.43, 130.95, 131.58, 133.93, 134.55, 135.24, 135.28, 135.66, 138.65, 141.61, 1 44.01, 144.22, 144.46, 145.50, 146.39, 156.73, 171.21.
HRMS (ESI+): calcd for [M+H]+, 638.28389 ; found, 638.28361 (-0.28 mmu)

上記反応スキームに従い、化合物６に相当する化合物８を、上記反応スキームに従い化合物７（19.0mg、0.0298mmol）から合成した。粗生成物を以下の条件下でＨＰＬＣで精製し、化合物８を得た（10.2mg、53.3％）。４０分でＡ / Ｂ = ７０ / ３０～０ / １００（溶出液Ａ：０．１％ＴＦＡを含有するＨ_２Ｏ、溶離液Ｂ：８０％アセトニトリルおよび２０％Ｈ_２Ｏ中の０．１％ＴＦＡ）。
¹H-NMR (400 MHz, CD3OD) δ: 0.58 (s, 3H), 0.60 (s, 3H), 1.49 (d, 3H, J = 7.3 Hz), 2.04 (s, 3H), 2.78-2.87 (m, 1H), 2.94-3.02 (m, 1H), 4.22 (dd, 1H, J = 8.5, 4.8 Hz), 4.54 (q, 1H, J = 7.2 Hz), 6.77 (dd, 1H, J = 9.8, 2.5 Hz), 7.09 (d, 1H, J = 9.1 Hz), 7.17 (d, 1H, J = 7.3 Hz), 7.31 (d, 1H, J = 10.1 Hz), 7.38-7.46 (m, 3H), 7.51 (t, 1H, J = 7.3 Hz), 7.60-7.66 (m, 1H), 8.24-8.29 (m, 1H)
¹³C-NMR (100 MHz, CD3OD) δ: -1.96, -1.68, 17.80, 19.56, 36.23, 51.15, 51.57, 120.29, 122.07, 126.75, 127.01, 128.38, 130.20, 130.51, 131.16, 131.58, 136.06, 137.00, 138.71, 139.28, 144.01, 144.51, 147.36, 156.11, 162.41, 169.62, 169.76, 173.25, 173.65.
HRMS (ESI+): calcd for [M+H]+, 528.24309 ; found, 528.24304 (-0.05 mmu) [Synthesis of fluorescent probe 1 (compound 8: NA-2-Me SiR600)]

According to the above reaction scheme, compound 8 corresponding to compound 6 was synthesized from compound 7 (19.0 mg, 0.0298 mmol) according to the above reaction scheme. The crude product was purified by HPLC under the following conditions to give compound 8 (10.2 mg, 53.3%). A/B = 70/30 to 0/100 at 40 min (eluent A: _H2O containing 0.1% TFA, eluent B: 0.1% in 80% acetonitrile and 20% _H2O ). TFA).
¹ H-NMR (400 MHz, CD3OD) δ: 0.58 (s, 3H), 0.60 (s, 3H), 1.49 (d, 3H, J = 7.3 Hz), 2.04 (s, 3H), 2.78-2.87 (m , 1H), 2.94-3.02 (m, 1H), 4.22 (dd, 1H, J = 8.5, 4.8 Hz), 4.54 (q, 1H, J = 7.2 Hz), 6.77 (dd, 1H, J = 9.8, 2.5 Hz), 7.09 (d, 1H, J = 9.1 Hz), 7.17 (d, 1H, J = 7.3 Hz), 7.31 (d, 1H, J = 10.1 Hz), 7.38-7.46 (m, 3H), 7.51 ( t, 1H, J = 7.3Hz), 7.60-7.66 (m, 1H), 8.24-8.29 (m, 1H)
¹³ C-NMR (100 MHz, CD3OD) δ: -1.96, -1.68, 17.80, 19.56, 36.23, 51.15, 51.57, 120.29, 122.07, 126.75, 127.01, 128.38, 130.20, 130.51, 131.16, 131.58, 136.06, 137.00, 138.71, 139.28, 144.01, 144.51, 147.36, 156.11, 162.41, 169.62, 169.76, 173.25, 173.65.
HRMS (ESI+): calcd for [M+H]+, 528.24309 ; found, 528.24304 (-0.05 mmu)

(a) Fmoc-Ala-OH, HATU, DIEA, DMF, r.t. (b) 2-Chlorotrityl resin, DIEA, DMF/CH2Cl2, r.t. (c) DDQ, DMF, r.t. (d) (i) piperidine, DMF/NMP, r.t. (ii) Fmoc-Asn(Trt)-OH, HATU, DIEA, DMF/NMP, r.t. (iii) piperidine, DMF/NMP, r.t. (e) TFA, Anisole, H2O, r.t. [Synthesis of fluorescent probe 2 (NA-2-OMe SiR600) of the present invention]
According to the reaction scheme described above, the fluorescent probe 2 (NA-2-OMe SiR600) of the present invention having Asn-Ala (NA) on the side chain was synthesized as follows. Fluorescent probe 2 differs from fluorescent probe 1 (NA-2-Me SiR600) in that the substituent on the benzene ring corresponding to R ² in formula (I) is methoxy.

(a) Fmoc-Ala-OH, HATU, DIEA, DMF, rt (b) 2-Chlorotrityl resin, DIEA, DMF/CH2Cl2, rt (c) DDQ, DMF, rt (d) (i) Piperidine, DMF/NMP , rt (ii) Fmoc-Asn(Trt)-OH, HATU, DIEA, DMF/NMP, rt (iii) piperidine, DMF/NMP, rt (e) TFA, Anisole, HO, rt

N,N,N',N'-Tetraallyldiamino-Si-xanthoneを、文献（Kushida, Y.ら、 Bioorganic & medicinal chemistry letters 2012, 22 (12), 3908-3911.）に従い合成した。 [Synthesis of N,N,N',N'-Tetraallyldiamino-Si-xanthone]

N,N,N',N'-Tetraallyldiamino-Si-xanthone was synthesized according to the literature (Kushida, Y. et al., Bioorganic & medicinal chemistry letters 2012, 22 (12), 3908-3911.).

アルゴンでフラッシュした乾燥フラスコに、２－ブロモアニソール（5.57g、29.8mmol）および無水ＴＨＦ（30mL）を添加した。溶液を－７８℃に冷却し、１Ｍ ｓ－ＢｕＬｉ（26.7mmol）を加え、混合物を２０分間撹拌した。同じ温度で、無水ＴＨＦ（10mL）に溶解したN,N,N',N'-Tetraallyldiamino-Si-xanthone（1.28g、2.97mmol）をゆっくり加え、混合物を室温に温めた。次いで、１時間撹拌した。２Ｎ ＨＣｌの添加により反応を停止させ、混合物を室温で５分間撹拌した。飽和ＮａＨＣＯ_３を加え、全体をＣＨ_２Ｃｌ_２で抽出した。有機層を塩水で洗浄し、Ｎａ_２ＳＯ_４で乾燥させ、溶媒留去した。残渣をＭｅＯＨに溶解し、０℃で撹拌し、ＮａＢＨ_４（340mg、8.99mmol）を加えた。反応混合物を０℃で１０分間撹拌した。混合物を蒸発させ、Ｈ_２Ｏを加え、次いで水層をＣＨ_２Ｃｌ_２で抽出した。有機層を塩水で洗浄し、Ｎａ_２ＳＯ_４で乾燥させ、溶媒留去した。残渣をＭＰＬＣ（シリカゲル、ヘキサン/ ＡｃＯＥｔ = １００：０～８７：１３）で精製し、粗生成物を得た（2.58g）。粗生成物のＣＨ_２Ｃｌ_２（50mL）溶液に１，３-ジメチルバルビツール酸（3.28g、21.0mmol）とＰｄ（ＰＰｈ_３）_４（0.94g、0.81mmol）を加え、３５℃でＡｒ雰囲気中で２２時間加熱した。ＮａＨＣＯ_３水溶液を加えて反応を停止させ、水層をＣＨ_２Ｃｌ_２で抽出した。有機層を塩水で洗浄し、Ｎａ_２ＳＯ_４で乾燥させ、溶媒留去した。残渣をＭＰＬＣ（シリカゲル、ヘキサン/ ＡｃＯＥｔ = ７１：２９～５０：５０）で精製し、化合物９を得た（708mg、66.2％）。
¹H-NMR (300 MHz, CD2Cl2) δ: 0.50 (s, 3H), 0.57 (s, 3H), 3.65 (s, 4H), 3.98 (s, 3H), 6.04 (s, 1H), 6.64 (dd, 2H, J = 8.1, 2.2 Hz), 6.73-6.84 (m, 2H), 6.90-6.94 (m, 1H), 6.94 (d, 2H, J = 2.2 Hz), 7.06-7.12 (m, 1H), 7.20 (d, 2H, J = 8.1 Hz).
¹³C-NMR (75 MHz, CD2Cl2) δ: -0.69, 0.71, 45.91, 55.88, 111.55, 117.22, 119.35, 120.91, 126.87, 129.99, 130.82, 134.79, 137.68, 139.98, 144.43, 156.16
HRMS (ESI+): calcd for [M+H]+, 361.17361 ; found, 361.17301 (-0.60 mmu) [Synthesis of compound 9]
Compound 9 (Leuco-2-OMe SiR600) in which the substituent on the benzene ring of compound 1 was changed from methyl to methoxy was synthesized as follows.

To an argon-flushed dry flask was added 2-bromoanisole (5.57 g, 29.8 mmol) and anhydrous THF (30 mL). The solution was cooled to −78° C., 1M s-BuLi (26.7 mmol) was added and the mixture was stirred for 20 minutes. At the same temperature, N,N,N',N'-Tetraallyldiamino-Si-xanthone (1.28 g, 2.97 mmol) dissolved in anhydrous THF (10 mL) was slowly added and the mixture was allowed to warm to room temperature. It was then stirred for 1 hour. The reaction was quenched by the addition of 2N HCl and the mixture was stirred at room temperature for 5 minutes. Saturated NaHCO ₃ was added and the whole was extracted with CH ₂ Cl ₂ . The organic layer was washed with _brine , dried over _Na2SO4 and evaporated. The residue was dissolved in MeOH, stirred at 0° C. and NaBH ₄ (340 mg, 8.99 mmol) was added. The reaction mixture was stirred at 0° C. for 10 minutes. The mixture was evaporated, _{H2O was} added, then the aqueous layer was extracted with _CH2Cl2 . The organic layer was washed with _brine , dried over _Na2SO4 and evaporated. The residue was purified by MPLC (silica gel, hexane/AcOEt = 100:0 to 87:13) to give crude product (2.58g). A solution of the crude product in CH ₂ Cl ₂ (50 mL) was added with 1,3-dimethylbarbituric acid (3.28 g, 21.0 mmol) and Pd(PPh ₃ ) ₄ (0.94 g, 0.81 mmol) and stirred at 35° C. under Ar atmosphere. Heated in for 22 hours. Aqueous NaHCO ₃ solution was added to quench the reaction and the aqueous layer was extracted with CH ₂ Cl ₂ . The organic layer was washed with _brine , dried over _Na2SO4 and evaporated. The residue was purified by MPLC (silica gel, hexane/AcOEt = 71:29 to 50:50) to give compound 9 (708 mg, 66.2%).
¹ H-NMR (300 MHz, CD2Cl2) δ: 0.50 (s, 3H), 0.57 (s, 3H), 3.65 (s, 4H), 3.98 (s, 3H), 6.04 (s, 1H), 6.64 (dd , 2H, J = 8.1, 2.2 Hz), 6.73-6.84 (m, 2H), 6.90-6.94 (m, 1H), 6.94 (d, 2H, J = 2.2 Hz), 7.06-7.12 (m, 1H), 7.20 (d, 2H, J = 8.1Hz).
¹³ C-NMR (75 MHz, CD2Cl2) δ: -0.69, 0.71, 45.91, 55.88, 111.55, 117.22, 119.35, 120.91, 126.87, 129.99, 130.82, 134.79, 137.68, 139. 98, 144.43, 156.16
HRMS (ESI+): calcd for [M+H]+, 361.17361 ; found, 361.17301 (-0.60 mmu)

ＤＭＦ（10mL）中の化合物９（127mg、0.353mmol）に、Ｆｍｏｃ－Ａｌａ－ＯＨ（55mg、0.177mmol）、ＨＡＴＵ（67mg、0.176mmol）およびＤＩＥＡ（114μL、0.670mmol）を加え、Ａｒ雰囲気中で１．５時間室温で撹拌した。反応混合物を溶媒留去し、Ｈ_２Ｏを加え、次いで水層をＣＨ_２Ｃｌ_２で抽出した。有機層を塩水で洗浄し、Ｎａ_２ＳＯ_４で乾燥し、溶媒留去した。溶離液としてＡｃＯＥｔ / ｎ－ヘキサン（1/1）を用いたＭＰＬＣで精製した後、クロロホルムを溶離液として用いるＧＰＣリサイクルカラムで精製し、化合物１０を得た（81mg、70.3％）。
¹H-NMR (400 MHz, CD2Cl2) δ: 0.46 (d, 3H, J = 1.8 Hz), 0.53 (d, 3H, J = 3.2 Hz), 1.45 (d, 3H, J = 6.9 Hz), 3.67 (br, 2H), 3.95 (s, 3H), 4.23 (t, 1H, J = 6.9 Hz), 4.44 (d, 3H, J = 6.9 Hz), 5.71 (s, 1H), 6.08 (s, 1H), 6.64 (d, 1H, J = 7.3 Hz), 6.71-6.79 (m, 2H), 6.91 (d, 2H, J = 7.8 Hz), 7.06-7.10 (m, 1H), 7.19 (d, 1H, J = 8.2 Hz), 7.28 (d, 2H, J = 6.4 Hz), 7.34-7.46 (m, 4H), 7.57-7.61 (m, 2H), 7.74-7.79 (m, 3H), 8.32 (s, 1H)
¹³C-NMR (100 MHz, CD2Cl2, isomer mixture) δ: -0.74, 0.63, 18.58, 46.39, 47.54, 51.75, 55.87, 67.44, 111.58, 117.38, 119.35, 120.33, 120.95, 121.73, 124.65, 125.38, 125.42, 127.15, 127.47, 128.10, 130.04, 130.56, 130.85, 134.50, 135.04, 135.67, 136.89, 139.37, 141.66, 144.17, 144.25, 144.55, 146.23, 156.15, 156.71, 170.96.
HRMS (ESI+): calcd for [M+Na]+, 676.26075 ; found, 676.26088 (0.13 mmu) [Synthesis of compound 10]

To compound 9 (127 mg, 0.353 mmol) in DMF (10 mL) was added Fmoc-Ala-OH (55 mg, 0.177 mmol), HATU (67 mg, 0.176 mmol) and DIEA (114 μL, 0.670 mmol) in an Ar atmosphere. Stir at room temperature for 1.5 hours. The reaction mixture was evaporated and H ₂ O was added, then the aqueous layer was extracted with CH ₂ Cl ₂ . The organic layer was washed with _brine , dried over _Na2SO4 and evaporated. Purification by MPLC using AcOEt/n-hexane (1/1) as eluent followed by purification by GPC recycle column using chloroform as eluent gave compound 10 (81 mg, 70.3%).
¹ H-NMR (400 MHz, CD2Cl2) δ: 0.46 (d, 3H, J = 1.8 Hz), 0.53 (d, 3H, J = 3.2 Hz), 1.45 (d, 3H, J = 6.9 Hz), 3.67 ( br, 2H), 3.95 (s, 3H), 4.23 (t, 1H, J = 6.9 Hz), 4.44 (d, 3H, J = 6.9 Hz), 5.71 (s, 1H), 6.08 (s, 1H), 6.64 (d, 1H, J = 7.3Hz), 6.71-6.79 (m, 2H), 6.91 (d, 2H, J = 7.8Hz), 7.06-7.10 (m, 1H), 7.19 (d, 1H, J = 8.2 Hz), 7.28 (d, 2H, J = 6.4 Hz), 7.34-7.46 (m, 4H), 7.57-7.61 (m, 2H), 7.74-7.79 (m, 3H), 8.32 (s, 1H)
¹³ C-NMR (100 MHz, CD2Cl2, isomer mixture) δ: -0.74, 0.63, 18.58, 46.39, 47.54, 51.75, 55.87, 67.44, 111.58, 117.38, 119.35, 120.33, 120.95, 121 .73, 124.65, 125.38, 125.42, 127.15, 127.47, 128.10, 130.04, 130.56, 130.85, 134.50, 135.04, 135.67, 136.89, 139.37, 141.66, 144.17, 144.25, 144.55, 1 46.23, 156.15, 156.71, 170.96.
HRMS (ESI+): calcd for [M+Na]+, 676.26075 ; found, 676.26088 (0.13 mmu)

上記反応スキーム（Ｇｅｎｅｒａｌ Ｐｒｏｃｅｄｕｒｅ）に従い、化合物１０（18.0mg、0.0276mmol）から、化合物１１を合成した。粗生成物を以下の条件下でＨＰＬＣで精製し、化合物１１を得た（10.9mg、62.3％）。４０分でＡ / Ｂ = ７０ / ３０～０ / １００（溶出液Ａ：０．１％ＴＦAを含有するＨ_２Ｏ、溶離液Ｂ：８０％アセトニトリルおよび２０％Ｈ_２Ｏ中の０．１％ＴＦＡ）。
¹H-NMR (400 MHz, CD3OD, isomer mixture) δ: 0.47 (s, 0.6H), 0.57 (s, 2.4H), 0.60 (d, 3H, J = 3.2 Hz), 1.49 (d, 2.7H, J = 7.3 Hz), 1.62 (d, 0.3H, J = 6.8 Hz), 2.66 (br, 0.2H), 2.77-2.87 (m, 0.9H), 2.96-3.02 (m, 0.9H), 3.09 (s, 0.6H), 3.73 (s, 2.4H), 4.06-4.25 (m, 1H), 4.49-4.57 (m, 1H), 6.69 (d, 0.2H, J = 4.0 Hz), 6.76 (d, 0.8H, J = 4.8Hz), 7.09-7.26 (m, 4H), 7.32 (d, 0.2H, J = 4.2 Hz), 7.38-7.42 (m, 1.8H), 7.53 (s, 0.2H), 7.59-7.68 (m, 1.6 H), 7.87-7.91 (m, 0.2H), 8.14 (s, 0.1H), 8.23 (br, 1H)
¹³C-NMR (100 MHz, CD3OD) δ: -1.91, -1.66, 17.83, 36.23, 51.17, 51.57, 56.25, 112.51, 119.88, 121.64, 121.90, 126.52, 128.09, 128.26, 131.46, 131.67, 132.27, 136.56, 138.76, 143.68, 144.28, 147.93, 156.11, 158.10, 162.39, 168.37, 169.59, 173.27, 173.60
HRMS (ESI+): calcd for [M+H]+, 544.23800 ; found, 544.23644 (-1.57 mmu) [Synthesis of fluorescent probe 2 (compound 11: NA-2-OMe SiR600)]

Compound 11 was synthesized from compound 10 (18.0 mg, 0.0276 mmol) according to the above reaction scheme (General Procedure). The crude product was purified by HPLC under the following conditions to give compound 11 (10.9 mg, 62.3%). A/B = 70/30 to 0/100 at 40 min (eluent A: _H2O containing 0.1% TFA, eluent B: 0.1% in 80% acetonitrile and 20% _H2O ). TFA).
¹ H-NMR (400 MHz, CD3OD, isomer mixture) δ: 0.47 (s, 0.6H), 0.57 (s, 2.4H), 0.60 (d, 3H, J = 3.2 Hz), 1.49 (d, 2.7H, J = 7.3 Hz), 1.62 (d, 0.3H, J = 6.8 Hz), 2.66 (br, 0.2H), 2.77-2.87 (m, 0.9H), 2.96-3.02 (m, 0.9H), 3.09 (s , 0.6H), 3.73 (s, 2.4H), 4.06-4.25 (m, 1H), 4.49-4.57 (m, 1H), 6.69 (d, 0.2H, J = 4.0 Hz), 6.76 (d, 0.8H , J = 4.8Hz), 7.09-7.26 (m, 4H), 7.32 (d, 0.2H, J = 4.2 Hz), 7.38-7.42 (m, 1.8H), 7.53 (s, 0.2H), 7.59-7.68 (m, 1.6H), 7.87-7.91 (m, 0.2H), 8.14 (s, 0.1H), 8.23 (br, 1H)
¹³ C-NMR (100 MHz, CD3OD) δ: -1.91, -1.66, 17.83, 36.23, 51.17, 51.57, 56.25, 112.51, 119.88, 121.64, 121.90, 126.52, 128.09, 128.26, 131.46, 131.67, 132.27, 136.56, 138.76, 143.68, 144.28, 147.93, 156.11, 158.10, 162.39, 168.37, 169.59, 173.27, 173.60
HRMS (ESI+): calcd for [M+H]+, 544.23800 ; found, 544.23644 (-1.57 mmu)

In addition to these fluorescent probes 1 and 2, according to the reaction scheme described above, 20 types of natural amino acids × 20 types, a total of 400 types of dipeptides were introduced as the AB sites of formula (I), 400 types of fluorescent probes. Built a library.

Screening Using Human Breast Cancer Surgical Specimens Using the fluorescent probe library synthesized in Example 1, human breast cancer surgical specimens were screened. First, lysates were prepared by dividing the surgical sample into tumor and normal sites, and each probe with a different peptide chain in the AB region was reacted with each lysate to measure changes in fluorescence intensity. Thirty-six fluorescent probes that showed a sufficient increase in fluorescence and a high T/N ratio (Tumor to Normal ratio) were selected as candidate probes.

Subsequently, for the candidate probe group, a probe solution was dropped onto the excised specimen, fluorescence imaging was performed, and changes in fluorescence intensity were measured. Specifically, a large number of 3 to 5 mm square pieces were cut out from a human breast cancer sample with a scalpel, placed in each well of an 8-well chamber slide (Thermo Fisher 155411), and each fluorescent probe solution (50 μM) was added thereto. was added dropwise, and changes in fluorescence intensity were measured using the Maestro In-Vivo Imaging System.

As a result, while the fluorescent probe 1 (compound 8: NA-2-Me SiR600) in which the AB site in formula (I) is "Asn-Ala" (NA) exhibits a rapid fluorescence increase at the tumor site, , it was found that there was almost no increase in normal sites. In addition, fluorescent probes with AB sites of "Lys-Ala" (KA) and "Met-Ala" (MA) also showed high sensitivity.

Furthermore, when similar imaging was performed with fluorescent probe 1 on breast cancer specimens from other 13 cases, all showed a significant increase in fluorescence at the tumor site, and fluorescence could be detected at the tumor site at a high T/N ratio. was suggested. FIG. 1 shows fluorescence imaging images (a and b) of a tumor sample and a normal sample treated with the fluorescent probe 1, and changes in fluorescence intensity over time (c).

FIG. 2 shows the change in the T/N ratio with time upon addition to the tumor site for fluorescent probe 1 (NA) and compound (KA) whose AB site is "Lys-Ala". Fluorescent probe 1 (NA) showed a higher T/N ratio at each time except 1 minute later.

For the specific fluorescent probe 1 of the biomarker enzyme , a DEG (diced electrophoresis gel) assay was performed using a lysate prepared from the tumor site of the surgical specimen, and a single spot with high activity was observed (Fig. 3 ( a)). Focusing on this spot, PMF (Peptide Mass Fingerprinting) analysis was performed. As a result, puromycin-sensitive aminopeptidase (PSA) was identified as a metabolic enzyme for fluorescent probe 1. When the identified PSA was reacted with the fluorescent probe 1 using the purified enzyme, it showed a rapid fluorescence increase (Fig. 3(b)). From these results, it was found that fluorescent probe 1 is effective as a breast cancer detection fluorescent probe using PSA as a biomarker enzyme.

Fluorescent assay for PSA For fluorescent probe 1 (compound 8: NA-2-Me SiR600) and fluorescent probe 2 (compound 11: NA-2-OMe SiR600) synthesized in Example 1, absorption and fluorescence spectra were measured, and , the quantum yield was calculated. In both cases, the fluorescent probe concentration was 1 μM, and a 0.1 M Na phosphate buffer (pH 7.4, 0.1% DMSO) solution was used.

The obtained absorption and fluorescence spectra are shown in FIG. 4, and the calculated quantum yield is shown in FIG. Although there is no significant difference in the absorption spectra, on the other hand, when focusing on the fluorescence spectra, it was found that the fluorescence probe 2 is greatly quenched compared to the fluorescence probe 1. It was found that the fluorescent quantum yield of fluorescent probe 2 was also reduced to almost zero. This is because by using a methoxy group as the substituent of R ² in formula (I), fluorescence quenching due to photoinduced electron transfer is increased in the state before the AB site is cleaved, resulting in non-fluorescence. It is considered that Therefore, fluorescent probe 2 can be expected to exhibit a fluorescent response at a high T/N ratio, and was found to be a fluorescent probe suitable for in vivo imaging.

Next, in order to confirm the reactivity between the fluorescent probe 2 and PSA, the fluorescence intensity change was measured using the PSA purified enzyme. PSA (0.55 μg/well) solution containing 0.0015% DMSO and 0.1% CHAPS was added as an enzyme solution to a 1 μM fluorescent probe 2 solution in phosphate buffered saline (PBS). FIG. 6 shows the fluorescence intensity change at the time. As a comparative example, changes in fluorescence intensity when using a γ-glutamyl transpeptidase (GGT) solution (150 μg/well) are also shown. The excitation wavelength is 544 nm and the emission wavelength is 615 nm.

From the results of FIG. 6, fluorescent probe 2 showed a rapid increase in fluorescence with the addition of PSA. This is presumably because PET was suppressed and the fluorescence emission of the xanthene ring was recovered by cleavage of "Asn-Ala" at the AB site in formula (I) by reaction with PSA. . On the other hand, when GGT was added, no increase in fluorescence was observed, indicating that the AB site of fluorescent probe 2 was not cleaved by GGT. This result demonstrates that fluorescent probe 2 can specifically detect the presence of PSA.

Fluorescent probe 2, which has been shown to function as a red fluorescent probe for multicolor imaging PSA for breast cancer cells , and a breast cancer detection green fluorescent probe (gGlu-HMRG), which is known to exhibit a fluorescent response to GGT, are used in combination. We attempted multi-color imaging of breast cancer. gGlu-HMRG is a fluorescent probe having the following structure. Urano et al., Sci. Transl. Med., vol. 3, pp. 110ra119, 2011 (Non-Patent Document 1).

A tumor site and a normal site of about 5 mm square were separately cut out from a breast cancer sample, and a mixed solution of fluorescent probe 2 and gGlu-HMRG was dropped on each of them. Green and red fluorescence images were acquired 1, 3, 5, 10, 15, 20 and 30 minutes after dripping, and merged images were created by superimposing them. A total of 14 cases were imaged in the same manner. An image of one of these cases is shown in FIG. 7 as a representative example. In FIG. 7, for both fluorescent probe 2 and gGlu-HMRG, an increase in fluorescence was observed only in the tumor site, and no increase in fluorescence was observed in the normal site. The tumor site was detectable in 13 out of 14 cases (93%). This demonstrates that the combination of fluorescent probe 2 for PSA and gGlu-HMRG for GGT can detect breast cancer tumor sites with higher specificity.

The above results demonstrate that the fluorescent probe of the present invention can selectively detect PSA and is a highly practical fluorescent probe for breast cancer.

Claims (11)

A fluorescent probe for detecting puromycin-sensitive aminopeptidase (PSA), comprising a compound represented by the following formula (I) or a salt thereof:

[In the formula,
A is an alanine residue, B is an asparagine residue or a lysine residue, wherein A links adjacent N atoms by forming an amide bond, and B forms an amide bond with A. are connected by
X is Si(R ^a )(R ^b ), Ge(R ^a )(R ^b ), Sn(R ^a )(R ^b ), C(R ^a )(R ^b ), P(=O)(R ^a ) or O (wherein R ^a and R ^b each independently represent a hydrogen atom, an alkyl group, or an aryl group);
R ¹ is a hydrogen atom or 1 to 4 identical or different groups independently selected from the group consisting of optionally substituted alkyl groups, carboxyl groups, ester groups, alkoxy groups, amido groups, and azido groups. represents a substituent;
R ² represents an optionally substituted C ₁ -C ₄ alkoxy group;
R ³ and R ⁴ are each independently selected from the group consisting of a hydrogen atom, a hydroxyl group, a halogen atom, an optionally substituted alkyl group, a sulfo group, a carboxyl group, an ester group, an amide group and an azide group. representing 1 to 3 identical or different substituents selected;
R ⁵ , R ⁶ and R ⁷ each independently represent a hydrogen atom or an alkyl group;
Here, R ⁶ or R ⁷ may each be taken together with R ⁴ to form a ring structure containing the nitrogen atom to which they are attached. ]. ^2. The fluorescent probe of claim 1, wherein R2 is a methoxy group. 3. The fluorescent probe according to claim 1 or 2, wherein ^R1 , ^R3 , ^R4 , ^R5 , ^R6 , and ^R7 are all hydrogen atoms. A composition for detecting cancer cells, comprising the fluorescent probe according to any one of claims 1 to 3. 5. The composition of claim 4 , wherein said cancer cells are breast cancer cells. A kit for detecting puromycin-sensitive aminopeptidase (PSA), comprising the fluorescent probe according to any one of claims 1 to 3. A step of contacting the fluorescent probe according to any one of claims 1 to 3 with a sample to be measured, and a fluorescence response by reaction between puromycin-sensitive aminopeptidase (PSA) contained in the sample and the fluorescent probe, or A method for detecting PSA, comprising the step of observing changes in absorbance. 8. A method of detection according to claim 7, characterized in that the fluorescence response is visualized using fluorescence imaging means. The method according to claim 7 or 8, wherein the sample to be measured is a cell expressing puromycin-sensitive aminopeptidase (PSA). 10. The method of claim 9, wherein said cells are cancer cells. 11. The method of claim 10, wherein said cancer cells are breast cancer cells.

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