Serum albumin detection agent

Abstract

To provide a novel serum albumin detection agent.SOLUTION: The serum albumin detection agent contains a compound represented by general formula (1) or a salt thereof. [In the formula, R1 represents an unsaturated heterocyclic group or an aryl group optionally having a substituent; -R2- represents -S-, -CH2-, -CH=CH-, or -C≡C-; R3 represents an aryl group optionally having a substituent; R4 represents an aryl group having a substituent; l represents 0 or 1; and m represents 0 or 1, provided that if -R2- represents -CH2- then m represents 1.]SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a detection agent for serum albumin. [Background technology]

[0002] Albumin is the most abundant protein in blood, accounting for approximately 60% of the total protein. Albumin plays a role in transporting vitamins, enzymes, hormones, etc. throughout the body. Therefore, abnormalities in blood albumin levels are an indicator of life-threatening diseases. For example, hypoalbuminemia is a sign of diseases such as heart failure, liver disease, malnutrition, inflammatory bowel disease, infections, and kidney disease (Non-Patent Documents 1 and 2). Albumin analysis is particularly important in the field of forensic medicine. For example, many medico-legal investigations require the estimation of time since death. The albumin concentration in blood decreases at a constant rate up to 72 hours after death, so the time since death can be estimated by albumin assay (Non-Patent Document 3).

[0003] In addition, blood in the stool is an indicator of colon cancer and colitis, and is an important indicator of health. It is also known that even if it does not lead to colon cancer, it can cause complications such as hemolytic uremic syndrome and encephalopathy. Conventionally, colonoscopy and stool culture tests have been performed to confirm this. It is also an essential test item in regular checkups (Non-Patent Document 11). Albuminuria tests are also an indicator of kidney failure caused by diabetes. This test plays a role in early detection of diabetes (Non-Patent Document 16). However, the conventional methods described above cause great pain to patients and require a lot of testing costs and time. Therefore, if a simple and highly sensitive albumin test could be performed to diagnose related diseases early, it would be a great advantage in this diagnostic field.

[0004] Traditionally, albumin concentration has been measured by methods such as immunoassays, colorimetric assays, and luminescence assays. Typical immunoassay systems have relied on measurements using anti-human serum albumin (HSA) antibodies, fluorophore- or luminophore-labeled secondary antibodies, and their specific detectors (Non-Patent Document 4). Other measurement methods include colorimetric assays using transition metals (Non-Patent Documents 5 and 6), methods using gold nanoparticles (AuNPs) in fluidic microchannel devices (Non-Patent Document 7), and paper-based ratiometric luminescence analysis systems for point-of-care testing (Non-Patent Document 8). However, considering the inconvenience, cost, long measurement times, and patient pain of these methods, a new method for detecting serum albumin is still desired. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] WO2021 / 187531 Brochure [Patent Document 2] Patent Publication No. 2011-219392 [Non-patent literature]

[0006] [Non-Patent Document 1] Mendez, CM; McClain, CJ; Marsano, LS, Albumin therapy in clinical practice. Nutr Clin Pract 2005, 20(3), 314-320. [Non-Patent Document 2] Infusino, I.; Panteghini, M., Serum albumin: accuracy and clinical use. Clin Chim Acta 2013, 419, 15-18. [Non-Patent Document 3] Ankita K. Parmar, SKM, Estimation of postmortem interval through albumin in CSF by simple dye binding method. Science & Justice 2015, 55(6), 388-393.

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[0007] An object of the present invention is to provide a novel serum albumin detection agent that uses a compound that has not been known to be capable of detecting serum albumin. [Means for solving the problem]

[0008] Under such circumstances, the present inventors have conducted intensive research and found that a compound having a structure in which an aryl group is bound to imidazo[1,2-a]pyrazin-3(7H)-one via a carbon-carbon double bond, a carbon-carbon triple bond, or a sulfur atom, more specifically, a structure in which an aryl group is bound to the 8th position of imidazo[1,2-a]pyrazin-3(7H)-one via a carbon-carbon double bond, a carbon-carbon triple bond, or a sulfur atom, or a structure in which an aryl group is bound to the 6th position of imidazo[1,2-a]pyrazin-3(7H)-one via a carbon-carbon double bond, reacts with serum albumin to generate luminescence, thereby enabling specific detection of serum albumin derived from various animals. The present invention is based on the above-mentioned novel findings. Therefore, the present invention provides the following:

[0009] Item 1. A serum albumin detection agent comprising a compound represented by general formula (1) or a salt thereof:

[0010] [ka]

[0011] [In the formula, R 1 represents an aryl group or an unsaturated heterocyclic group which may have a substituent. 2 - is -S-, -CH 2 -, -CH=CH- or -C≡C-. 3 R represents an aryl group which may have a substituent. 4 represents an aryl group having a substituent; l represents 0 or 1; m represents 0 or 1. However, -R 2 -Ga-CH 2 -, then m is 1.]

[0012] Section 2.R 1 Item 2. The serum albumin detection agent according to Item 1, wherein, when the aryl group or unsaturated heterocyclic group represented by the formula (I) has a substituent, the substituent is at least one selected from the group consisting of a hydroxyl group, a thiol group, a hydroxyalkyl group, and a mercaptoalkyl group.

[0013] Section 3.R 3 Item 2. The serum albumin detection agent according to Item 1, wherein when the aryl group represented by the formula (I) has a substituent, the substituent is at least one selected from the group consisting of a halogen, a hydroxyl group, an alkyl group, an alkoxy group, an alkoxyalkyl group, a hydroxyalkyl group, and an alkylamino group.

[0014] Section 4.R 4 Item 2. The serum albumin detection agent according to Item 1, wherein the aryl group represented by the formula:

[0015] Item 5. The serum albumin detection agent according to item 1, wherein the compound represented by general formula (1) or a salt thereof is a compound represented by the following general formula (2) or a salt thereof:

[0016] [ka]

[0017] [In the formula, -R 2 - is -S-, -CH 2 -, -CH=CH- or -C≡C-. 5 R is a hydroxyl group, a thiol group, a hydroxyalkyl group, or a mercaptoalkyl group. 5 If there are multiple R 5 may be the same or different. 6 R is a halogen, a hydroxyl group, an alkyl group, an alkoxy group, an alkoxyalkyl group, a hydroxyalkyl group, or an alkylamino group. 6 If there are multiple R 6 may be the same or different. 7 R is a hydroxyl group, a hydroxyalkyl group, an alkoxy group, or an alkylamino group. 7 If there are multiple R 7 may be the same or different. l represents 0 or 1. m represents 0 or 1. n represents an integer of 0 to 2. p represents an integer of 0 to 2. q represents an integer of 1 or 2. However, -R 2-Ga-CH 2 -, then m is 1.]

[0018] Section 6. General formula (1') [ka] [In the formula, R 1 R represents an aryl group or an unsaturated heterocyclic group which may have a substituent. 3 R represents an aryl group which may have a substituent. 4 represents an aryl group having a substituent. l represents 0 or 1.] A compound represented by the formula:

[0019] Item 7. A method for detecting serum albumin in a sample, comprising the step of adding the serum albumin detection agent according to any one of items 1 to 5 or the compound or a salt thereof according to item 6 to a target sample.

[0020] Item 8. The method according to item 7 for measuring one or both of the content of albumin and the origin of albumin in a sample. Effect of the Invention

[0021] According to the present invention, it is possible to provide a novel serum albumin detection reagent that uses a compound that has not been known so far to be capable of detecting serum albumin. [Brief description of the drawings]

[0022] [Figure 1]Absolute luminescence intensity of the CTZ indicator in reaction with various serum proteins. Arrows indicate the luminescence intensity indicating the specificity of the CTZ indicator for the respective serum protein. The p-value (Student's t-test) is **** ≤ 0.0001. Inset a highlights the relative intensity of the CTZ indicator in response to serum albumin. Asterisks * and ** on the bars indicate HSA and BSA specificity for S6 and S6h, respectively. Inset b shows the corresponding luminescence image. Abbreviations: HSA, human serum albumin; BSA, bovine serum albumin; OVA, ovalbumin; HIgG, human immunoglobulin; BIGG, bovine immunoglobulin; HγG, human gamma globulin; BγG, bovine gamma globulin; HTF, human transferrin; HFG, human fibrinogen; STI, soybean trypsin inhibitor; HFN, human fibronectin; PBS, phosphate-buffered saline. [Diagram 2] Verification of the selectivity of CTZ indicators for serum proteins other than albumin. K6 specifically radiated light to soybean trypsin inhibitor (STI), and S5h selectively radiated light to human fibronectin (HFN). Insert a shows the breakdown of serum proteins used in this experiment. Insert b shows the chemical structures of K6 and S5h. [Diagram 3] Characterization of emission spectra of CTZ indicators with HSA or BSA and their mixtures. (A) Emission spectra of CTZ indicators with HSA (left) or BSA (right). Numbers indicate the peak wavelength of the spectrum. (B) Normalized emission spectra of S6h in response to different ratios of HSA and BSA mixtures. Numbers indicate the peak wavelength of the spectrum. Inset a shows the change in emission intensity of normalized spectra at 430 nm and 600 nm depending on the mixing ratio. Percentages indicate the maximum intensity variance of S6h by changing the mixing ratio of HSA and BSA. Inset b summarizes the maximum emission spectra of CTZ indicators with HSA and BSA. [Figure 4]Determination of kinetic constants of CTZ indicator with HSA and BSA. (A) Initial luminescence intensity of CTZ indicator in response to various concentrations of HSA. (B) Initial luminescence intensity of CTZ indicator in response to various concentrations of BSA. (C) Summary of kinetic constants showing the enzymatic properties of HSA and BSA in their reaction with CTZ indicator. [Diagram 5] A wide range of dose-response characteristics of selected CTZ indicators in various concentrations of serum albumin. (A) A wide range of dose-response curves of selected CTZ indicators in response to various concentrations of HSA, BSA, and OVA. The arrows highlight the gaps in intensity between the CTZ indicators. K6, S6, and S6h generally reacted with HSA in the applied concentration range of 0–1 mg / mL. In contrast, S6h showed relatively better luminescence intensity than other CTZ indicators when BSA was used. (B) Summarized dose-response curves of S6 and S6h in response to varying concentrations of serum albumin. (C) Corresponding optical images of S6 and S6h in reaction with various concentrations of serum albumin. The * and ** arrows indicate the detection limit and peak point, respectively. Inset a shows the chemical structures of S6 and S6h. The yellow shading highlights the characteristic functional groups of S6 that are different from S6h. [Figure 6] Inhibitors: ibuprofen and warfarin, and competition of the CTZ indicator for the drug binding site II of BSA. (A) Competition between the CTZ indicator and the inhibitor for the specific interaction site of BSA. The percentages indicate the ratio of the remaining final intensity of the CTZ indicator in the presence of 100 μM inhibitors compared to the absence of inhibitors ibuprofen and warfarin. Inset a shows the corresponding luminescence image. (B) Competition between the CTZ indicator and the inhibitor for the specific interaction site of HSA. The percentages indicate the intensity remaining after inhibition of 100 μM inhibitors compared to the intensity in the absence of inhibitors. Inset b shows the corresponding luminescence image. (C) Lineweaver-Burk inhibition plot showing the inhibitory effect of ibuprofen on the reaction of S6h with BSA. The term r2 means the coefficient of determination in linear regression. [Figure 7] Dose-response luminescence intensity of S6 and S6h in response to actual FBS. p-value (Student's t-test) is ** ≤ 0.01. Numbers on the graph indicate the fold-intensity of luminescence of S6h relative to S6. Inset a shows the corresponding luminescence image. Arrows highlight the approximate detection limit of the CTZ indicator. [Figure 8] Luminescence measurement of albumin (HSA or BSA) using the CTZ indicators nCTZ, K6, 11, 12, and 13. PBS stands for phosphate buffered saline and was used as a negative control. [Figure 9] Luminescence response spectra of CTZ indicators 11, 12, 13, and K6 to BSA and HSA. [Figure 10] Measurement results of specific luminescence reactivity of CTZ indicators (K6, S5, S5h, S6) with albumin [Figure 11] Measurement results of specific luminescence reactivity of CTZ indicators with albumin [Figure 12] Enlarged view of Fig. 11 [Figure 13] Measurement results of specific luminescence reactivity of CTZ indicators (P1, P2, TS1, TS2) with albumin [Figure 14] Measurement results of dose-response curves of the specific luminescence reactivity of CTZ indicators (P1, P2, TS1, TS2) with albumin [Figure 15] Emission spectra of CTZ indicators using HSA or MSA. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] Serum albumin detection agent In one embodiment, the present invention relates to a detection agent for serum albumin, comprising a compound represented by general formula (1) or a salt thereof:

[0024] [ka]

[0025] [In the formula, R 1 represents an aryl group or an unsaturated heterocyclic group which may have a substituent. 2 - is -S-, -CH 2 -, -CH=CH- or -C≡C-. 3 R represents an aryl group which may have a substituent. 4 represents an aryl group having a substituent; l represents 0 or 1; m represents 0 or 1. However, -R 2 -Ga-CH 2 -, then m is 1.] In this specification, the compound represented by general formula (1) may be simply referred to as compound (1). Similarly, the compound represented by general formula (2) may be simply referred to as compound (2). In addition, in the present invention, a coelenterazine (CTZ) derivative that emits luminescence when reacted with serum albumin may be referred to as a CTZ indicator. The compound (1) or a salt thereof, which is an active ingredient of the present invention, reacts with serum albumin to generate luminescence. The serum albumin in a sample can be detected by detecting the luminescence. Therefore, the serum albumin detection agent of the present invention can also be referred to as a luminescence detection agent for serum albumin.

[0026] In the present invention, the alkyl group refers to a linear or branched monovalent hydrocarbon. The number of carbon atoms in the alkyl group is, for example, preferably 1 to 6, more preferably 1 to 3, and even more preferably 1. Examples of such an alkyl group include methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl, n-pentyl, and n-hexyl. In the present invention, the alkyl group may have (for example, 1 to 2) carbon-carbon double bonds (vinyl group, propenyl group, etc.), or may be a saturated hydrocarbon having no carbon-carbon double bond.

[0027] In the present invention, the hydroxyalkyl group may be the above-mentioned alkyl group having one or more hydroxyl groups. Therefore, the number of carbon atoms of the hydroxyalkyl group is, for example, preferably 1 to 6, more preferably 1 to 3, and even more preferably 1. The number of hydroxyl groups may be, for example, 1 to 3, preferably 1 to 2, and more preferably 1. Examples of such hydroxyalkyl groups include hydroxymethyl, 1-hydroxyethyl, 2-hydroxyethyl, 1-hydroxypropyl, 3-hydroxypropyl, 2,3-dihydroxypropyl, 1,2,3-trihydroxypropyl, 4-hydroxybutyl, 4-hydroxybutan-2-yl, 5-hydroxypentyl, and 6-hydroxyhexyl.

[0028] In the present invention, the mercaptoalkyl group may be the above-mentioned alkyl group having one or more thiol groups. Therefore, the number of carbon atoms of the mercaptoalkyl group is, for example, preferably 1 to 6, more preferably 1 to 3, and even more preferably 1. The number of thiol groups may be, for example, 1 to 3, preferably 1 to 2, and more preferably 1. Examples of such mercaptoalkyl groups include mercaptomethyl, 1-mercaptoethyl, 2-mercaptoethyl, 1-mercaptopropyl, 3-mercaptopropyl, 2,3-dimercaptopropyl, 1,2,3-trimercaptopropyl, 4-mercaptobutyl, 4-mercaptobutan-2-yl, 5-mercaptopentyl, and 6-mercaptohexyl.

[0029] In the present invention, the alkoxy group includes those in which the alkyl portion is the above-mentioned alkyl group. Therefore, the number of carbon atoms in the alkoxy group is, for example, preferably 1 to 6, more preferably 1 to 3, and further preferably 1. Examples of such alkoxy groups include methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, i-butoxy, t-butoxy, n-pentyloxy, n-hexyloxy, etc.

[0030] In the present invention, examples of the alkoxyalkyl group include an alkyl group having one alkoxy group 1 as a substituent, and the alkyl portion is the alkyl group described above. Therefore, the number of carbon atoms of the alkoxyalkyl group is, for example, preferably 2 to 12, more preferably 2 to 6, and even more preferably 2. Examples of such an alkoxy group include methoxymethyl, 2-methoxyethyl, 3-methoxypropyl, 2-methoxypropyl, ethoxymethyl, 2-n-propoxyethyl, i-propoxymethyl, n-butoxymethyl, i-butoxymethyl, t-butoxymethyl, n-pentyloxymethyl, and n-hexyloxymethyl.

[0031] In the present invention, examples of alkylamino include groups in which one or two of the two hydrogen atoms of an amino group are substituted with the above-mentioned alkyl group. Therefore, the number of carbon atoms of the alkylamino group is, for example, preferably 1 to 6, more preferably 1 to 3, and even more preferably 1. Examples of such alkyl groups include N-monoalkylamino such as N-methylamino, N-ethylamino, Nn-propylamino, Ni-propylamino, Nn-butylamino, Ni-butylamino, Nt-butylamino, Nn-pentylamino, and Nn-hexylamino; and N,N-dialkylamino such as N,N-dimethylamino, N,N-ethylamino, N-methyl-N-diethylamino, N,N-di-n-propylamino, N,N-di-i-propylamino, N,N-di-n-butylamino, N,N-di-n-pentylamino, and Nn-hexylamino.

[0032] In the present invention, the aryl group is not particularly limited, and for example, both a monocyclic aryl group (phenyl group) and a polycyclic aryl group can be used, but a monocyclic aryl group is preferred. The number of carbon atoms of such an aryl group is preferably 6 to 12, more preferably 6 to 11, and even more preferably 6 to 10. Specific examples of such an aryl group include a phenyl group, a naphthyl group, and a biphenyl group, and preferably a phenyl group.

[0033] In the present invention, examples of the unsaturated heterocyclic group include unsaturated heterocyclic groups having a 5-10 membered ring (preferably a 5-6 membered ring, more preferably a 5 membered ring) containing at least one heteroatom (preferably oxygen) selected from the group consisting of oxygen, nitrogen, and sulfur. The number of heteroatoms is not limited, but may be, for example, 1-4, preferably 1-2, and more preferably 1. The unsaturated heterocyclic group may be either monocyclic or polycyclic, but is preferably monocyclic. Specific examples of such unsaturated heterocyclic groups include furanyl, thienyl, thiazolyl, pyridinyl, imidazolyl, indolyl, pyrimidinyl, oxazolyl, indolizinyl, benzofuranyl, quinolinyl, and isoquinolinyl groups, with furanyl being preferred.

[0034] In the present invention, halogen includes bromine, chlorine, fluorine, iodine, etc., and preferably includes fluorine, etc., unless otherwise specified.

[0035] R 1 When the aryl group or unsaturated heterocyclic group represented by the formula (I) has a substituent, the substituent is preferably a hydroxyl group, a thiol group, a hydroxyalkyl, a mercaptoalkyl, etc., more preferably a hydroxyl group, a hydroxyalkyl, etc., and further preferably a hydroxyl group. 1 When the aryl group or unsaturated heterocyclic group represented by the formula (I) has a substituent, the number of the substituents is not limited, and may be, for example, 1 to 5, preferably 1 to 4, more preferably 1 to 3, even more preferably 1 to 2, and still more preferably 1. 1 When the aryl group or unsaturated heterocyclic group represented by R has multiple substituents, the multiple substituents may be the same or different. 1 When the aryl group represented by the formula (I) has a substituent, the position of the substituent is not limited and may be any of the ortho, meta, and para positions, but the para position is preferred. 1 When the aryl group represented by R has multiple substituents, it is preferable that at least one of the substituents is located at the para position. 1 When the unsaturated heterocyclic group represented by the formula (I) has a substituent, the position of the substituent is not limited. In a preferred embodiment of the present invention, the luminescence generated by the reaction of compound (1) or a salt thereof with human serum albumin and the luminescence generated by the reaction of compound (1) or a salt thereof with bovine serum albumin tend to occur in different wavelength ranges. Specifically, in a typical embodiment, the luminescence generated by compound (1) or a salt thereof in response to human serum albumin may have a peak at 490 to 500 nm, and the luminescence generated by compound (1) or a salt thereof in response to bovine serum albumin may have a peak at 480 to 535 nm. Therefore, this embodiment is preferable in that human serum albumin and bovine serum albumin can be measured simultaneously by using compound (1) or a salt thereof. Also, R 1 In a preferred embodiment of the present invention, compound (1) or a salt thereof can detect serum albumin, particularly human albumin, with high sensitivity when the aryl group or unsaturated heterocyclic group (preferably an aryl group) represented by the general formula (1) has a substituent. 1 The present invention provides a detection agent for human serum albumin, comprising a compound or a salt thereof in which an aryl group or an unsaturated heterocyclic group (preferably an aryl group) represented by the formula: 2 -Ga-CH 2 Even in the case of -R - or -S-), compound (1) or a salt thereof can detect serum albumin, particularly human albumin, with high sensitivity. Therefore, in a preferred embodiment of the present invention, compound (1) or a salt thereof is a compound represented by the general formula (1) in which m is 1 (preferably, m is 1 and -R 2 -Ga-CH 2 The present invention also provides a method for detecting human serum albumin, comprising the steps of: 1 In a preferred embodiment of the present invention, when the aryl group or unsaturated heterocyclic group (preferably an aryl group) represented by the general formula (1) has no substituent, compound (1) or a salt thereof can detect serum albumin, particularly bovine albumin, with high sensitivity. 1is an unsubstituted aryl group or an unsaturated heterocyclic group (preferably an aryl group), or a salt thereof. 2 In a preferred embodiment of the present invention, compound (1) or a salt thereof is a compound represented by the general formula (1) in which m is 1 (preferably, m is 1 and -R 2 The present invention provides a detection agent for mouse serum albumin, comprising a compound or a salt thereof, in which - is -S-. As can be seen from the fact that human and bovine albumin can be selectively visualized as described above, and that mouse albumin can also be detected, it is expected that within the scope of general formula (1), selective visualization of albumin derived not only from bovine, human, and mouse, but also from egg, rabbit, dog, monkey, chicken, cat, etc. is also possible.

[0036] R 3 In the case where the aryl group represented by the formula (I) has a substituent, the substituent is preferably a halogen, a hydroxyl group, an alkyl group, an alkoxy group, an alkoxyalkyl group, a hydroxyalkyl group, or an alkylamino group, and more preferably a halogen, etc. 3 When the aryl group represented by the formula (I) has a substituent, the number of the substituents is not limited, and may be, for example, 1 to 5, preferably 1 to 4, more preferably 1 to 3, even more preferably 1 to 2, and still more preferably 1. 3 When the aryl group represented by R has multiple substituents, the multiple substituents may be the same or different. 3 When the aryl group represented by the formula (I) has a substituent, the position of the substituent is not limited and may be any of the ortho, meta, and para positions, but the para and / or meta positions are preferred. 3 When the aryl group represented by the formula (I) has a plurality of substituents, it is preferable that at least one of the substituents is located at the para position and / or meta position. R 4When the aryl group represented by the formula (I) has a substituent, the substituent is preferably a hydroxyl group, a hydroxyalkyl, an alkoxy, an alkylamino, etc., more preferably a hydroxyl group, a hydroxyalkyl, etc., and even more preferably a hydroxyl group, etc. 4 When the aryl group represented by the formula (I) has a substituent, the number of the substituents is not limited, and may be, for example, 1 to 5, preferably 1 to 4, more preferably 1 to 3, even more preferably 1 to 2, and still more preferably 1. 4 When the aryl group represented by R has multiple substituents, the multiple substituents may be the same or different. 4 When the aryl group represented by the formula (I) has a substituent, the position of the substituent is not limited and may be any of the ortho, meta, and para positions, but the para position is preferred. 4 When the aryl group represented by R has multiple substituents, it is preferable that at least one of the substituents is located at the para position. 4 When the aryl group represented by the formula (I) has a substituent, the position of the substituent is not limited, but it is preferable that the substituent is not present at the meta position.

[0037] Next, preferred examples of the type, number, etc. of the substituent in general formula (1) are shown below. In general formula (1), l represents 0 or 1, and is preferably 1. m represents 0 or 1, and is preferably 1.

[0038] As described above, the aryl group in the general formula (1) is preferably a phenyl group. Therefore, in a preferred embodiment, the present invention provides a serum albumin detection agent comprising a compound represented by the general formula (2) or a salt thereof:

[0039] [ka]

[0040] [In the formula, -R 2 - is -S-, -CH 2 -, -CH=CH- or -C≡C-. 5 R is a hydroxyl group, a thiol group, a hydroxyalkyl group, or a mercaptoalkyl group.5 If there are multiple R 5 may be the same or different. 6 R is a halogen, a hydroxyl group, an alkyl group, an alkoxy group, an alkoxyalkyl group, a hydroxyalkyl group, or an alkylamino group. 6 If there are multiple R 6 may be the same or different. 7 R is a hydroxyl group, a hydroxyalkyl group, an alkoxy group, or an alkylamino group. 7 If there are multiple R 7 may be the same or different. l represents 0 or 1. m represents 0 or 1. n represents an integer of 0 to 2. p represents an integer of 0 to 2. q represents an integer of 1 or 2. However, -R 2 -Ga-CH 2 -, then m is 1.] R 5 , R 6 and R 7 Preferred embodiments (type, position, etc.) of the group represented by the formula (phenyl group substituent) are as described in the description of R 1 R is a substituent of an aryl group or an unsaturated heterocyclic group represented by the formula 3 The substituent of the aryl group represented by R 4 The substituents are the same as those of the aryl group represented by the following formula:

[0041] In formula (2), n represents an integer of 0 to 2, and preferably 0 to 1. p represents an integer of 0 to 2, and preferably 0 to 1. m represents 0 or 1, and preferably 1. q represents an integer of 1 to 2, and preferably 1. In the general formula (2), when p is 1 or 2, the group R 6 is preferably located at the para-position and / or meta-position of the phenyl group. 7 At least one of the groups R is preferably located at the para position of the phenyl group. 7 is preferably not present at the meta position of a phenyl group.

[0042] Compound (1), which is an active ingredient of the present invention, may be used in the form of a salt. The salt of compound (1) includes an acid addition salt and a base salt. Specific examples of the acid addition salt include inorganic acid salts such as hydrochloride, hydrobromide, hydroiodide, sulfate, perchlorate, and phosphate, organic acid salts such as oxalate, malonate, succinate, maleate, fumarate, lactate, malate, citrate, tartrate, benzoate, trifluoroacetate, acetate, methanesulfonate, p-toluenesulfonate, and trifluoromethanesulfonate, and acidic amino acid salts such as glutamate and aspartate. Specific examples of the salt with a base include alkali metal or alkaline earth metal salts such as lithium salt, sodium salt, potassium salt, or calcium salt, and salts with organic bases such as pyridine salt and triethylamine salt.

[0043] In the present invention, the compound (1) or a salt thereof, which is the active ingredient of the present invention, may be used as a serum albumin detector itself, or may be used as a detector composition in combination with various pharma- ceutically acceptable carriers (e.g., isotonicity agents, chelating agents, stabilizers, pH regulators, preservatives, antioxidants, solubilizing agents, thickening agents, etc.).

[0044] Examples of the isotonicity agent include sugars such as glucose, trehalose, lactose, fructose, mannitol, xylitol, sorbitol, etc., polyhydric alcohols such as glycerin, polyethylene glycol, propylene glycol, etc., inorganic salts such as sodium chloride, potassium chloride, calcium chloride, etc. These isotonicity agents can be used alone or in combination of two or more.

[0045] Examples of the chelating agent include edetate salts such as disodium edetate, calcium disodium edetate, trisodium edetate, tetrasodium edetate, calcium edetate, ethylenediaminetetraacetate, nitrilotriacetic acid or a salt thereof, sodium hexametaphosphate, citric acid, etc. These chelating agents may be used alone or in combination of two or more.

[0046] An example of the stabilizer is sodium hydrogen sulfite.

[0047] Examples of pH adjusters include acids such as hydrochloric acid, carbonic acid, acetic acid, citric acid, etc., and further include alkali metal hydroxides such as sodium hydroxide, potassium hydroxide, etc., alkali metal carbonates or hydrogen carbonates such as sodium carbonate, alkali metal acetates such as sodium acetate, alkali metal citrates such as sodium citrate, bases such as trometamol, etc. These pH adjusters can be used alone or in combination of two or more.

[0048] Examples of preservatives include sorbic acid, potassium sorbate, paraoxybenzoic acid esters such as methyl paraoxybenzoate, ethyl paraoxybenzoate, propyl paraoxybenzoate, and butyl paraoxybenzoate, chlorhexidine gluconate, quaternary ammonium salts such as benzalkonium chloride, benzethonium chloride, and cetylpyridinium chloride, alkylpolyaminoethylglycine, chlorobutanol, polyquad, polyhexamethylene biguanide, and chlorhexidine, etc. These preservatives may be used alone or in combination of two or more.

[0049] Examples of antioxidants include sodium hydrogen sulfite, dry sodium sulfite, sodium pyrosulfite, concentrated mixed tocopherols, etc. These antioxidants may be used alone or in combination of two or more.

[0050] Examples of solubilizing agents include sodium benzoate, glycerin, D-sorbitol, glucose, propylene glycol, hydroxypropylmethylcellulose, polyvinylpyrrolidone, macrogol, D-mannitol, etc. These solubilizing agents can be used alone or in combination of two or more.

[0051] Examples of thickening agents include polyethylene glycol, methylcellulose, ethylcellulose, carmellose sodium, xanthan gum, sodium chondroitin sulfate, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, polyvinyl alcohol, etc. These thickening agents can be used alone or in combination of two or more.

[0052] In an embodiment of the detection agent composition, the content of compound (1) or a salt thereof in the composition is not particularly limited and can be appropriately set based on conditions such as, for example, 99% by mass or more, 95% by mass or more, 90% by mass or more, 70% by mass or more, 50% by mass or more, 30% by mass or more, 10% by mass or more, 5% by mass or more, or 1% by mass or more.

[0053] According to the present invention, luminescence can be generated by reacting the active ingredient, compound (1) or a salt thereof, with serum albumin. It has not been reported that compound (1) or a salt thereof produces luminescence by reacting with serum albumin, and this is a novel finding. Here, Non-Patent Document 9 describes that HuLumino 1, which has the following structure, produces luminescence by reacting with human serum albumin:

[0054] [ka]

[0055] However, Non-Patent Document 9 describes that the luminescence generated by DeepBlueC or Blue2.3, which has a benzyl group at the 8th position of the imidazopyranodine skeleton, and human serum albumin is much weaker than that of HuLumino 1 (Non-Patent Document 9, Figure 1. (f)). In contrast, as shown in the Examples of the present application described later, compound (1) has a substituent at the C-8 position of the imidazopyranodine skeleton, but can react with serum albumin to emit luminescence. Therefore, the effects of the present invention are not predictable from the prior art. In addition, due to the structural feature that HuLumino 1 does not have a substituent at the 8th position of the imidazopyranodine skeleton, it becomes chemically unstable and self-luminesces (decomposes) (Non-Patent Document 10). In contrast, compound (1), which is the active ingredient of the present invention, has a substituent at the 8th position of the imidazopyranodine skeleton, and therefore has the advantage of excellent chemical stability. In the present invention, compound (1) has a substituent at the 8-position of the imidazopyranodine skeleton, and therefore has excellent chemical stability, while having sufficient reactivity and generating sufficient luminescence to detect serum albumin. The fact that such stability and luminescence can be achieved at the same time is something that could not be predicted from the prior art.

[0056] Furthermore, as described above, compound (1) or a salt thereof exhibits luminescence in response to bovine albumin, and in particular, in the general formula (1), m is 0 and R 1 When the aryl group or unsaturated heterocyclic group (preferably an aryl group) represented by the formula (1) has no substituent, compound (1) or a salt thereof can detect serum albumin, particularly bovine albumin, with high sensitivity. In contrast, Non-Patent Document 9 describes that addition of HuLumino 1 to bovine albumin did not result in luminescence (Non-Patent Document 9, page 2683, left column, lines 13-18). Therefore, the effect of the present invention using compound (1) or a salt thereof was unexpected from the above viewpoints, and furthermore, when R 1 The effect obtained by the embodiment using a compound or a salt thereof in which the aryl group or unsaturated heterocyclic group (preferably an aryl group) represented by the formula (I) has no substituent is particularly unexpected.

[0057] Compound or its salt Among the compounds (1) or salts thereof which are the active ingredients of the serum albumin detection agent of the present invention, the compound represented by the following general formula (1') or salts thereof is a novel compound. Therefore, in one embodiment, the present invention relates to a compound represented by the general formula (1') [ka] [In the formula, R 1 R represents an aryl group or an unsaturated heterocyclic group which may have a substituent. 3 R represents an aryl group which may have a substituent. 4 represents an aryl group having a substituent. l represents 0 or 1.] or a salt thereof. In this specification, the compound represented by general formula (1') may be simply referred to as compound (1'). Similarly, the compound represented by general formula (2') may be simply referred to as compound (2'). The preferred types and numbers of each substituent in the general formula (1') are the same as those described above. In the present invention, among the compound (1') or a salt thereof, the compound (2') or a salt thereof is preferred: [ka] [In the formula, R 5 R is a hydroxyl group, a thiol group, a hydroxyalkyl group, or a mercaptoalkyl group. 5 If there are multiple R 5 may be the same or different. 6 R is a halogen, a hydroxyl group, an alkyl group, an alkoxy group, an alkoxyalkyl group, a hydroxyalkyl group, or an alkylamino group. 6 If there are multiple R 6 may be the same or different. 7 R is a hydroxyl group, a hydroxyalkyl group, an alkoxy group, or an alkylamino group. 7 If there are multiple R 7 may be the same or different. m represents 0 or 1. n represents an integer of 0 to 2. p represents an integer of 0 to 2. q represents an integer of 1 or 2.] In the general formula (2'), R 5 R is preferably a hydroxyl group or a hydroxyalkyl group, more preferably a hydroxyl group. 6 R is preferably a hydroxyl group or a hydroxyalkyl group, more preferably a hydroxyl group. 6 is preferably a hydroxyl group or a hydroxyalkyl group, and more preferably a hydroxyl group. n is preferably 0 or 1. p is preferably 0 or 1, and more preferably 0. q is preferably 1.

[0058] Compound (1') can be produced by various methods, for example, the method shown in the following reaction scheme:

[0059] Reaction Scheme-1 [ka]

[0060] [In the formula, R 1 , R 3 , R 4 , and l are as described above. R 4 ' represents an aryl group which may have a substituent, and when the aryl group has a substituent, a part (e.g., 1 or 2) or all of the substituents are protected with a protecting group. X represents a halogen.] In the above reaction scheme, R 4 When the aryl group represented by ' has a substituent, the substituent includes a hydroxyl group, a hydroxyalkyl group, an alkoxy group, an alkylamino group, an acyloxy group, etc. Examples of the protecting group include an alkyl group (e.g., a methyl group), an acyl group (e.g., an acetyl group), etc. Examples of the halogen represented by X include bromine, chlorine, fluorine, iodine, etc., and preferably bromine, etc.

[0061] In reaction formula 1, first, compound 1' is reacted with compound 2' to obtain compound 3'. The ratio of compound 1' to compound 2' is not particularly limited, but for example, the latter can be used in an amount of about 1 mole for every 1 mole of the former. This reaction can be carried out using the Suzuki-Miyaura cross coupling (SMC) reaction, with a yield of about 80%. Next, R 4 When the aryl group represented by ' has a group protected by a protecting group, compound 4' can be obtained by deprotection. The reaction can be carried out in the presence of boron tribromide, hydrochloric acid, tetrabutylammonium fluoride, potassium carbonate, potassium hydroxide, or the like. Compound 4' can be reacted with compound 5' to obtain compound (1'). The ratio of compound 4' to compound 5' is not particularly limited, but for example, the latter can be used in an amount of about 1 mole per mole of the former. The reaction can be carried out in the presence of a catalyst such as hydrochloric acid.

[0062] Each reaction in the above reaction formula is usually carried out in a conventional solvent that does not adversely affect the reaction, such as water, methanol, ethanol, isopropanol, n-butanol, trifluoroethanol, ethylene glycol, dioxane, tetrahydrofuran, triethylamine, acetone, methyl ethyl ketone, diethyl ether diglyme, acetonitrile, N,N-dimethylformamide, dimethyl sulfoxide, acetic acid, a mixed solvent thereof, etc. The reaction temperature of each reaction is not particularly limited, and the reaction is usually carried out at room temperature, under cooling or heating. Preferably, the reaction temperature is -78 to 150°C, etc. The reaction time of each reaction is also not particularly limited, and the reaction can be carried out for, for example, 1 to 30 hours.

[0063] In addition, compounds in the form of solvates (e.g., hydrates, ethanolates, etc.) added to the raw materials and target compounds shown in each reaction scheme are also included in each general formula. Preferred solvates include hydrates.

[0064] Each target compound obtained in each of the above reaction schemes can be isolated and purified from the reaction mixture by, for example, cooling the reaction mixture, separating the crude reaction product by an isolation procedure such as filtration, concentration, extraction, etc., and then subjecting it to a conventional purification procedure such as column chromatography, recrystallization, etc. Among the compounds (1), those having a structure other than the above compound (1') are either known compounds or can be produced by the above method or a similar known method.

[0065] Detection Method In one embodiment, the present invention provides a method for detecting serum albumin in a sample, comprising the step of adding a compound represented by general formula (1) or a salt thereof to a sample of interest:

[0066] [ka]

[0067] [In the formula, R 1 represents an aryl group or an unsaturated heterocyclic group which may have a substituent. 2 - is -S-, -CH 2 -, -CH=CH- or -C≡C-. 3 R represents an aryl group which may have a substituent. 4 represents an aryl group having a substituent; l represents 0 or 1; m represents 0 or 1. However, -R 2 -Ga-CH 2 -, then m is 1.] In general formula (1), R 1 , R 3 , R 4 Preferred embodiments of l and m (types of aryl groups, unsaturated heterocyclic groups, etc., types and positions of substituents, etc.) are described in Serum albumin detection agent In the general formula (1), -R 2 -Preferred aspects etc. of Serum albumin detection agent " This is similar to what was explained in the "

[0068] Examples of target samples include blood (whole blood, plasma, serum, etc.), urine, sweat, feces, etc. Examples of samples include those derived from vertebrates, such as those derived from mammals (humans, cows, mice, rats, monkeys, dogs, cats, guinea pigs, rabbits, horses, humans, etc., with humans, cows, mice, etc. being preferred, and humans, cows, etc. being more preferred) and non-mammals (for example, fish, birds, amphibians, reptiles, etc.). These target samples may be pretreated by dilution, centrifugation, filtration, dialysis, pH adjustment, etc.

[0069] The amount of compound (1) or a salt thereof added to the sample may be, for example, 2 to 500 μM, preferably 2 to 100 μM, more preferably 20 to 50 μM, calculated as the final concentration of free compound (1). The temperature in the above step is not particularly limited, and the step is usually carried out at room temperature, or under cooling or heating. A preferred temperature is 20 to 37° C., etc.

[0070] In this embodiment, the method of the present invention may further include a step of detecting luminescence. In this step, a luminescence detector known per se can be appropriately used. In this embodiment, the method of the present invention may include a step of leaving a mixture of the sample and the compound (1) or a salt thereof after adding the compound (1) or a salt thereof to the sample before the luminescence measurement, but it is preferable to keep this step to a minimum. In this embodiment, the time for leaving the sample is not particularly limited, and can be appropriately set within the range of, for example, immediate (0 minutes) to 1 minute. As described above, according to the present invention, the origin of albumin (human origin, bovine origin, etc.) can also be measured by utilizing the difference in the peak wavelength of luminescence. In addition, according to the present invention, the content of albumin in a sample can also be quantitatively measured. Since albumin precipitates in a biological sample over time, it is possible to estimate how much time has passed since the albumin was produced by measuring the albumin concentration.

[0071] Specific embodiments of the present invention will be described in more detail below using examples, but the present invention is not limited to the following examples. EXAMPLES

[0072] Example 1 1. Reagents and samples The following serum proteins were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan): human serum albumin (HSA); bovine serum albumin (BSA); human immunoglobulin (HIgG); human gamma-globulin (HγG); human transferrin (HTF); human fibrinogen (HFG); soybean trypsin inhibitor (STI). The following serum proteins were purchased from Jackson Immuno Research, Inc. (West Grove, PA, USA): bovine immunoglobulin (BIgG) and bovine gamma-globulin (BγG); whereas ovalbumin (OVA) and human fibronectin (HFN) were obtained from MP Biomedicals, Inc. (Santa Ana, CA, USA) and CORNING, Inc. (Corning, NY, USA), respectively. Native coelenterazine (nCTZ) was purchased from NanoLights, Inc. (Pine Top, AZ, USA). The CTZ analogs K6, S5, and S6 were synthesized based on the descriptions in non-patent documents 12 and 13.

[0073] [ka]

[0074] 2. Synthesis of new CTZ indicators S5h and S6h for selective imaging of serum albumin The CTZ indicators, S5h and S6h, were synthesized according to the following scheme, with reference to non-patent documents 12 and 13.

[0075] [ka]

[0076] Compound 2 was synthesized by Suzuki-Miyaura cross-coupling reaction of commercially available 2-amino-5-bromoaminopyrazine 1 with commercially available phenylboronic acid and tetrakis(triphenylphosphine)palladium(0). Compound 3 was further synthesized by bromination using compound 2 and commercially available N-bromosuccinimide. Compounds 4 and 5 were synthesized from compound 3 using 4-fluorobenzenethiol substitution reaction with benzenethiol or sodium hydride. Compounds 4 and 5 were then demethylated with boron tribromide to give compounds 6 and 7. Finally, compounds 6 and 7 were condensed and cyclized with ketoacetal derivative 12 under hydrochloric acid conditions. The synthesized CTZ indicators were named S5h and S6h. The reaction conditions for each step were as follows: a: Tetrakis(triphenylphosphine)palladium(0), 1,4-dioxane, 2 M Na 2 CO 3 a: aqueous solution, 110 °C. b: N-bromosuccinimide, chloroform, room temperature c: sodium hydride, N,N-dimethylformamide, 0 to 110 °C. d: boron tribromide, chloroform, -80 °C to room temperature e: 12 M aqueous HCl, eta, 60 °C. f: thionyl chloride, dichloromethane, 0 °C. g: 1) Mg, 1,2-dibromoethane, ethyl diethoxyacetate, tetrahydrofuran, room temperature. Reflux from 0 to -80 °C. 2) palladium on activated carbon, methanol, hydrogen, room temperature h: ethyl diethoxyacetate, tetrahydrofuran, -80 °C.

[0077] 3. Specific luminescence visualization of serum proteins using the CTZ indicator Each serum protein solution was previously prepared by diluting the original stock with phosphate-buffered saline (PBS) to 1 mg / mL. Then, 20 μL of each serum protein solution was dispensed into each well of a 96-well black frame microplate (Thermo Fisher Scientific, Waltham, MA, USA). A total of four wells were used per protein solution (n = 4).

[0078] Separately, a series of CTZ indicator solutions were prepared by first dissolving nCTZ and its analogs (CTZ indicators) in methanol and then diluting them in PBS to 100 μM. Using a multichannel micropipette (Gilson, Middleton, WI, USA), 40 μL of CTZ indicator solution was simultaneously injected into each well of the microplate. The luminescence intensity from the microplate was immediately measured (Figure 1) with an IVIS Spectrum imaging system (PerkinElmer, MA, USA) and analyzed with Living Image version 4.7 software. 4. Some CTZ indicators show specific luminescence intensity for certain albumins. The CTZ indicator was used to measure the luminescence reactivity with major serum proteins (Figure 1). First, to clarify the protein specificity of the CTZ indicator, the following serum proteins were selected as test candidates based on their abundance in serum: (i) human, bovine, and ovalbumin (HAS, BSA, and OVA) as the most abundant proteins in serum (35,000-52,000 mg / L); (ii) HIgG and BIgG as the second most abundant proteins (7,000-16,000 mg / L); and (iii) HTF, the third most abundant protein in serum (2,000-3,600 mg / mL). Furthermore, (iv) HγG and BγG were selected in this test because gamma globulins, classified as IgA, IgM, and IgY, are the fourth most abundant serum proteins (e.g., 700–4,000 mg / mL for IgA); (v) HFG was also selected because it is the sixth most abundant protein in serum. On the other hand, (vi) STI and HFN were selected because, although they are the 19th and 26th most abundant proteins in serum, they are important indicators of growth retardation, gastrointestinal disorders, metabolic disorders, and infectious diseases in humans, respectively.

[0079] According to the results in Figure 1, when tested with nCTZ, it showed no reactivity with any serum proteins. However, the other CTZ indicators, namely K6, S5, S5h, S6, and S6h, were interestingly found to emit luminescence of considerable intensity with both HSA and BSA. Among them, S6h reacted specifically with BSA, showing luminescence intensity 8.3 times stronger with BSA than with HSA (about 12%). The absolute intensity of this "S6h-BSA combination" was at least 8 times better than the other combinations. On the other hand, S6 reacted selectively with HSA, showing luminescence intensity 5.9 times stronger with HSA than with BSA (about 17%). Similarly, S5h was more reactive with BSA than with HSA, whereas S5 selectively emitted luminescence with HSA over BSA. On the other hand, all the tested CTZ indicators did not show strong reactivity with OVA and other serum proteins. When examining the specificity profiles of each of the CTZ indicators mentioned above, it was found that there was a significant difference in selectivity between "S5 and S6" and "S5h and S6h". Considering that the only difference between these two groups is the "hydroxy (OH) group", this indicates that the hydroxy group (OH) at the C-2 position of the CTZ indicator is the determining factor for specificity to serum albumin. In addition, when comparing the structures of S5h and S6h, it is found that the only difference between S5h and S6h is the presence or absence of a fluoro (F) residue at the C-8 position. This indicates that the fluoro (F) group at the C-8 position enhances the specificity.

[0080] 5. CTZ indicators K6 and S5h show specific luminescence for serum proteins STI and HFN The selectivity of the CTZ indicator for serum proteins other than albumin was examined (Fig. 2).

[0081] For this experiment, serum proteins and CTZ indicators were diluted and prepared in the same manner as shown in Fig. 1. In addition, serum proteins and CTZ indicators were reacted in the same manner as shown in Fig. 1, and the resulting luminescence images were immediately measured with an IVIS Spectrum imaging system (PerkinElmer, MA, USA) and analyzed with Living Image version 4.7 software.

[0082] As a result, nCTZ did not show any luminescence reaction to any of the proteins tested. On the other hand, K6 luminesced specifically to soybean trypsin inhibitor (STI). The absolute brightness was 5.1 x 10 4 p / s / cm 2 / sr. K6 showed background level luminescence intensity against other serum proteins. Measurements were then performed using S5 and S5h. S5 did not react with any serum proteins, but S5h selectively luminesced with human fibronectin (HFN). The absolute luminescence intensity was 4.0 x 10 4 p / s / cm 2 It was / sr.

[0083] These results indicate that the K6 and S5h indicators can be used as specific luminescent reagents for STI and HFN, respectively. The proportion of STI and HFN as serum proteins is 19th and 26th, respectively, and they are important indicators of growth retardation, digestive disorders, metabolic disorders, and human infectious diseases, respectively. Therefore, the ability to assay these serum proteins means that this method can be used for the diagnosis of the aforementioned diseases.

[0084] 6. Variation of the emission spectrum of the CTZ indicator depending on serum albumin The emission spectrum of the CTZ indicator was measured in the presence of serum albumin (Figure 3). HSA and BSA solutions were first prepared by dissolving them in PBS to a concentration of 10 mg / mL. Then, 50 μL of the solution was dispensed into a 200 μL PCR tube. Separately, each CTZ indicator was first dissolved in methanol and then further diluted with PBS to a concentration of 100 μM.

[0085] For the measurements, 50 μL of CTZ indicator solution was injected into each PCR tube, and the emission spectrum was immediately measured with a precision spectrophotometer (AB-1850, ATTO, Tokyo, Japan) that simultaneously measures all wavelengths in the visible and near-infrared regions. The integration time was 5 min.

[0086] In Figure 3(A), S6h showed two different colors depending on the type of serum albumin (HSA or BSA), so the spectra were measured while changing the mixture ratio of HSA and BSA (Figure 3(B)).

[0087] HSA and BSA were diluted with PBS to 5 mg / mL and 0.2 mg / mL, respectively. These initial HSA and BSA solutions were mixed in the following ratios: 100:0, 80:20, 60:40, 40:60, 20:80, and 0:100. 50 μL of each albumin mixture was placed in a 200 μL PCR tube. Separately, S6h indicator was first dissolved in methanol and then further diluted to 100 μM in PBS. 50 μL of this indicator solution was dispensed into PCR tubes, and the emission spectrum of each was measured using a precision spectrophotometer. The integration time was 5 min.

[0088] 7. The CTZ indicator showed different emission color spectra depending on whether it was BSA or HSA. The emission spectra of the CTZ indicators differed in absolute brightness and peak height depending on whether they were BSA or HSA (Figure 2). The highest peak height was obtained with the "S6h-HSA combination," followed by the "S6h-BSA combination" (Figure 3(A)). The color of S6h was blue with a peak at 480 nm for HSA and yellow-green with a peak at 535 nm for BSA. The gap between the peaks was approximately 55 nm.

[0089] On the other hand, the S6 indicator did not show any significant color change when reacting with HSA and BSA. The color of HSA and BSA was normal green, with peaks at approximately 490 and 510 nm, respectively. There was also a 30 nm gap between the HSA and BSA spectral peaks in the presence of the S5h indicator, which was the second largest gap measured. All S-series CTZ indicators generally showed short and long wavelength peaks with HSA and BSA, respectively. However, this feature was consistent with K6, which showed a longer wavelength emission spectrum (535 nm) with HSA and a shorter wavelength peak (515 nm) with BSA.

[0090] Inspired by the dramatic color change of S6h due to HSA and BSA, we investigated whether the spectral peak of S6h changed according to the mixing ratio of HSA and BSA solutions (Figure 3(B)).

[0091] The results confirmed that the S6h peak at 480 nm (100% HAS) gradually shifted to longer wavelengths (maximum shift position: 535 nm) by increasing the ratio of BSA. The reddest peak was obtained with 100% BSA solution, and the proportion of the red part longer than 600 nm in the spectrum was 21% of the total area. When slices were taken at 430 nm and 600 nm, the slice height (brightness) at the 430 nm slice position gradually decreased by increasing the ratio of BSA in the mixed solution, eventually resulting in a loss of 42% of the initial intensity. On the other hand, the intensity at the 600 nm slice position increased by a maximum of 26% by increasing the BSA portion in the mixed solution (Figure 3(B) inset a).

[0092] This blue-to-red peak shift can be explained by the difference in energy level of the S6h intermediate during the reaction with BSA. It is known that during the luminescence reaction, the CTZ substrate generates four intermediates with different energy levels, namely, a neutral species, an amide anion, a phenolate anion, and a pyrazine anion, and shows a wide emission peak from blue to green (400 to 535 nm) (Non-Patent Document 14). In the case of S6h, compared with nCTZ, it has a longer π-conjugation due to the presence of a sulfur (S) elbow at the C-8 position. This feature should have fundamentally contributed to the red-shifted emission of the intermediate. Furthermore, the blue-to-red shift of the emission spectrum of S6h depending on the HSA-BSA ratio means that the major intermediates generated by HSA and BSA are fundamentally different. That is, they are amide anion and phenolate anion, respectively, or phenolate anion and pyrazine anion, respectively. Due to the difference in the enzyme properties within the active site of HSA and BSA, S6h is analyzed to generate two different major intermediates.

[0093] 8. Determination of the rate constant of the CTZ indicator in response to serum albumin To determine representative rate constants for the aforementioned albumin-specific CTZ indicators, luminescence experiments were performed in the presence of HSA or BSA (Figure 4).

[0094] First, CTZ substrates (i.e., nCTZ, K6, S5, S5h, S6, and S6h) were first dissolved in methanol and further diluted with PBS to 0, 2, 5, 10, 20, 30, 50, and 100 μM. 40 μL of substrate solution was placed into each well of a 96-well black frame microplate (Thermo Fisher Scientific). The microplate was then loaded into a microplate reader (TriStar2 S LB 942, Berthold, Germany).

[0095] Separately, HSA and BSA were diluted with PBS to 1.0 mg / mL each. This albumin solution was primed into the injector of the microplate reader. During measurement, 40 μL of the albumin solution was automatically injected into each well of the microplate by the program, and the corresponding luminescence intensity was immediately measured with 0.1 s integration and 20 points acquisition mode. The rate constants were calculated using Prism version 9 (GraphPad, Boston, MA, USA).

[0096] 9. Kinetic experiments explained why S6 is specific for HSA and why BSA is the most immunoreactive for S6h.

[0097] The enzymatic performance of the CTZ indicator was examined in terms of the rate constant (Figure 4). The lowest Michaelis-Menten constant (Km) was 22.1 μM for the S6-HSA combination, followed by 23.7 μM for the S5h-HSA combination. These values ​​indicate that the S6 and S5h substrates have the strongest binding affinity with HSA compared to the other substrates. The turnover rate (K cat ) and maximum speed (V max ) was the highest for the "K6 and HSA combination," with 7.64 s -1 and 2.8 x 10 15 s -1 In contrast, the K in the "K6 and HSA combination" m The catalytic efficiency of the K6 / HAS combination was 1.2 x 10 -2 μM -1 s -1 The catalytic efficiency of the combination of S6 and HAS is 3.5 x 10 -2 μM -1 s -1 When comparing the "combination of S6 and HSA" with the "combination of S6 and BSA," the catalytic efficiency of the "combination of S6-HSA" (3.5 x 10 -2 μM -1 s -1 ) for the "S6-BSA combination" (2.3 x 10 -3 μM -1 s -1) is more than 15 times larger than that of the catalysis. This large difference in catalytic efficiency is mainly due to the cat The difference in values, i.e. 0.78 s -1 vs. 0.11 s -1 This comparison confirms that S6 is more specific to HAS than BSA. As shown in Figure 1, the combination of S6h-BSA showed the highest brightness. The reason is that the maximum K cat Value 1.66 xs -1 This explains the high metabolic turnover rate (K cat ), implying that BSA consumes S6h substrates more rapidly than the other substrates. Interestingly, HSA consumes less S6h per second than BSA, at 0.65 s -1 This comparison illustrates that when sufficient amounts of S6h are provided, the "S6h-BSA pair" is brighter than the other pairs.

[0098] 10. Determination of a wide range calibration curve for serum albumin in promising CTZ indicators Calibration curves of promising CTZ indicators over a wide range of serum albumin concentrations were determined (Figure 5).

[0099] HSA, BSA, and OVA solutions were initially dissolved in PBS to a concentration of 10 mg / mL. The stock solutions were further serially diluted in PBS to the following concentrations: 0, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, and 1 mg / mL. 40 μL of each dilution was sequentially placed in each well of a 9-well black frame microplate (Thermo Fisher Scientific). Separately, in addition to nCTZ, other CTZ indicators K6, S6, and S6h were each dissolved in methanol and further diluted in PBS to a concentration of 20 μM. 40 μL of CTZ indicator solution per well was simultaneously injected into each well of the microplate using a 12-channel micropipette. The luminescence intensity from the microplate was immediately measured using an IVIS Spectrum imaging system and analyzed using Living Image version 4.7 software. 11. The broad-range calibration curve described above showed the albumin specificity and luminescence intensity of the CTZ indicator depending on the albumin level. To obtain a wide range of calibration curves for the selected CTZ indicators, luminescence measurements were performed with varying concentrations of serum albumin (Figure 5). nCTZ did not show a significant increase in luminescence intensity with increasing HSA concentration (Figure 5(A)). However, K6, S6, and S6h rapidly increased their luminescence intensity in an albumin concentration-dependent manner. K6, S6, and S6h generally increased their luminescence intensity with increasing HSA levels, but S6h showed the most rapid increase in luminescence, especially in the low HSA concentration range (0-0.2 mg / mL). By increasing the BSA level, S6h emitted the strongest luminescence intensity. For example, the brightness of this S6h was 21 times brighter at the 0.2 mg / mL BSA point than the second brightest combination, "K6-BSA." The luminescence intensity decreased with BSA concentrations in the range of 0.5-1.0 mg / mL. These decreases in luminescence intensity can be explained by the rapid turnover rate of BSA in the presence of S6h, as shown in Figure 4(C). catConsidering the values, the decrease in luminescence intensity at the high concentration range is analyzed as being due to BSA consuming all of the S6h indicator (substrate) and the light was already dark at the time of measurement. When the OVA concentration was increased, no luminescence intensity was observed with any of the CTZ indicators in the tested concentration range (0-1.0 mg / mL).

[0100] The serum albumin specificity of S6 and S6h is summarized in Figure 5(B). The "S6h-BSA pair" showed 5.3 times stronger luminescence intensity than the "S6h-HSA pair" at a serum albumin concentration of 0.2 mg / mL. These albumin specificities of the CTZ indicators were also confirmed by visual images in Figure 4(C).

[0101] 12. Study on the competitive reaction between CTZ indicator and inhibitor for the enzyme active sites of HSA and BSA To identify the enzyme active sites in HSA and BSA, we investigated the competitive reaction between the CTZ indicator and specific inhibitors (Figure 6).

[0102] First, fatty acid-free HSA and BSA solutions were prepared by diluting them to 1.0 mg / mL in PBS. Then, 40 μL of HSA and BSA solutions were premixed with 0, 1, 5, 10, 20, 40, 80, or 100 μM warfarin to block DBS I of albumin, or with 20 μL of ibuprofen of the same concentration scale to block DBS II of albumin. Next, the premixes were pre-mixed sequentially into each well of a 96-well black frame microplate (Thermo Fisher Scientific), and then 40 μL of 50 μM S6 or S6h solutions were simultaneously injected into each well using a 12-channel micropipette, and the resulting luminescence images were measured using an IVIS Spectrum imaging system and analyzed using Living Image version 4.7 software.

[0103] Furthermore, to investigate the competitive inhibition of ibuprofen by the substrate S6h, the details of the luminescence reaction were examined by Lineweaver-Burk analysis (Figure 6(C)). The S6h indicator was first dissolved in methanol and diluted with PBS to 20, 40, 50, 80, and 100 μM. Then, 40 μL of each S6h solution was premixed with 20 μL of 0, 10, or 50 μM ibuprofen solution. The mixtures were sequentially placed in a 96-well black frame microplate. Separately, BSA was dissolved in PBS to 100 μg / mL. Then, 40 μL of BSA solution per well was simultaneously injected into each well of the microplate using a 12-channel micropipette. Luminescence images were captured using an IVIS Spectrum imaging system and analyzed using Living Image version 4.7 software. 13. CTZ indicators and inhibitors show competitive reactions with HSA and BSA Serum albumin is known to have multiple drug-binding sites. In this study, we investigated the competitive reaction of CTZ indicators with inhibitors for these binding sites (Figure 6).

[0104] The inhibitors warfarin and ibuprofen are known to block DBS I and II of serum albumin, respectively, so we pretreated a fixed amount of HSA or BSA with various amounts of the inhibitors ranging from 0 to 100 μM. We then simultaneously injected the CTZ indicator into this mixture and measured the luminescence intensity emitted from it.

[0105] In the competitive reaction in Figure 6(A), it was found that the luminescence intensity emitted by the "S6h-BSA pair" was inhibited by ibuprofen by up to 71%, whereas the luminescence intensity of the other pairs was not significantly inhibited by ibuprofen or warfarin. These results are interpreted as DBS II of BSA being the luminescence reaction site of S6h. Meanwhile, the "S6h-HSA pair" was partially inhibited by ibuprofen by up to 28%, whereas the same pair was not affected by warfarin (Figure 6(B)). This result implies that DBS II of HSA is a partial luminescence reaction site of S6h, but DBS I is not involved in the binding of S6h. Meanwhile, the "S6-HSA pair" was inhibited by both ibuprofen and warfarin by up to 31% and up to 47%, respectively. These results indicate that both DBS I and II of HSA interact with S6, but DBS I binds to S6 at a higher rate. Focusing on the effective inhibitory effect of ibuprofen on the interaction between S6h and BSA, the inhibitory effect of ibuprofen on the interaction between S6h and BSA was further confirmed by Lineweaver-Burk analysis (Figure 6(C)).

[0106] As a result, it was found that the slope of the plot of 1 / [S6h] increased with increasing ibuprofen concentration. The correlation coefficients were found to be in the range of approximately 0.912 to 0.975. In the Lineweaver-Burk plot, three fitting lines were found at different negative values ​​on the X-axis (-1 / K m Next, by increasing the amount of ibuprofen, the Y-section point (1 / V maxThe results support the "mixed inhibition" mode of competition between S6h and ibuprofen for the BSA site. This indicates that the binding site of BSA can interact with both S6h and ibuprofen simultaneously. It suggests that BSA bound to the first ligand is also reactive to the second ligand. This result on the competition between S6h and ibuprofen is slightly different from the conclusion of a recent study by Nishihara et al. that "the CTZ indicator HuLumino1 "competitively inhibits" DBS II of HSA with ibuprofen" (Non-Patent Document 9).

[0107] Combining these results with the references, we conclude that (i) the ligand-binding reaction of BSA is more complicated than expected, which should not be interpreted simply as "competitive inhibition", but may be in "noncompetitive" or "semi-mixed inhibition" mode. It should also be noted that (ii) the first ligand binding of BSA may have some effect on the second ligand binding of BSA. This suggests that the order of pretreatment of the ligand and BSA may also produce different results.

[0108] 14. Quantitative measurement of fetal bovine serum using the S6h indicator Taking advantage of the BSA specificity of the S6h indicator, we applied it to quantitatively measure real fetal bovine serum (FBS) (Figure 7). First, FBS stock solution was diluted in PBS to the following percentages: 0, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, and 10%. Then, 40 μL of the aforementioned diluted solution was pre-dispensed per well into a 96-well black frame microplate (Thermo Fisher Scientific). Separately, S6 and S6h solutions were diluted to 100 μM in PBS. Then, 40 μL of substrate solution per well was injected simultaneously into the microplate using a 12-channel micropipette, and the resulting luminescence images were acquired with an IVIS Spectrum imaging system and analyzed with Living Image version 4.7 software.

[0109] 15. Albumin in real fetal bovine serum can be measured using the CTZ indicator. Finally, to verify the usefulness of the CTZ indicator, quantitative imaging of actual FBS was performed (Figure 7). As a result, S6h significantly increased the luminescence intensity from the 0.1% point of FBS, while S6 started to increase significantly from about 1% point of FBS. Next, S6h rapidly increased the luminescence intensity in the range of 0.5-10% FBS, and its maximum luminescence intensity was observed near 5% FBS. The luminescence intensity of S6h at the 5% point of FBS was 16 times greater than that of S6. This large S / B ratio proves that S6h is a practical and efficient luminescence indicator for identifying BSA. Considering that the main protein in serum is albumin, these results suggest that this indicator can be used to measure the origin and concentration of albumin in blood in the forensic field. That is, it can be applied to estimate the postmortem time, which is a very important issue in many forensic investigations. The reason why this postmortem time estimation is possible is because the albumin concentration in the blood of a person after death decreases linearly from 2 to 72 hours (Non-Patent Document 3). Albumin assays are important because the concentration of albumin in the blood is an indicator of many albumin-related diseases, such as liver disease, heart failure, malnutrition, vitamin deficiency, inflammatory bowel disease, and kidney disease.

[0110] 16. Quantitative visualization of BSA and HSA using novel CTZ indicators 11, 12, and 13 Furthermore, luminescence measurements of HSA and BSA were carried out using the following compounds 11, 12, and 13 (FIG. 8).

[0111] [ka]

[0112] First, to prepare BSA and HSA solutions, the stock solutions were diluted to 1 mg / mL with PBS. Then, 20 μL of each serum protein solution was dispensed into each well of a 96-well black frame microplate. A total of four wells were used for each protein solution (n=4).

[0113] Separately, a series of CTZ indicator solutions was prepared by first dissolving the CTZ indicators nCTZ, K6, 11, 12, and 13 in methanol and then diluting them in PBS to 100 μM. Using a multichannel micropipette (Gilson, Middleton, WI, USA), 40 μL of CTZ indicator solution was simultaneously injected into each well of the microplate. The luminescence intensity from the microplate was immediately measured (Figure 1) with an IVIS Spectrum imaging system (PerkinElmer, MA, USA) and analyzed with Living Image version 4.7 software.

[0114] As a result, none of the CTZ indicators showed any luminescence intensity for the negative control PBS. On the other hand, CTZ indicators 11, 12, and 13 selectively luminesced for HSA, and CTZ indicator 13 in particular showed the highest signal-to-background ratio (S / B ratio) and brightness. As an example of the S / B ratio, it showed a 41-fold S / B ratio for 1 mg / mL HSA compared to the negative control PBS. Similarly, CTZ indicator 12 showed a 255-fold S / B ratio under the same conditions. In terms of selectivity between albumins, CTZ indicators 11, 12, and 13 also showed excellent HSA selectivity. For example, CTZ indicators 11, 12, and 13 showed S / B ratios of 3, 6, and 9, respectively, for the same concentration of HSA compared to 1 mg / mL BSA. On the other hand, the conventional CTZ indicators nCTZ and K6, which are the control groups, showed S / B ratios of only 0.3 and 1.8, respectively.

[0115] These experimental results demonstrated that the CTZ indicators 11, 12, and 13 selectively reacted to HSA and had sufficiently strong luminescence intensity.

[0116] 16. Emission spectra of new CTZ indicators, 11, 12, and 13 The emission spectra of CTZ indicators 11, 12, 13, and K6 reacting with BSA and HSA were measured (Figure 9). For this experiment, HSA and BSA solutions were first prepared by dissolving them in PBS to a concentration of 10 mg / mL. Then, 50 μL of the solution was dispensed into a 200 μL PCR tube. Separately, each of the CTZ indicators 11, 12, 13, and K6 was dissolved in methanol and further diluted with PBS to a concentration of 100 μM.

[0117] During the measurement, 50 μL of the CTZ indicator solution was placed into the PCR tube mentioned above, and the emission spectrum was immediately measured using a precision spectrophotometer (AB-1850, ATTO, Tokyo, Japan) that simultaneously measures all wavelengths. The integration time was 4 min.

[0118] As a result, CTZ indicator 12 showed the shortest wavelength emission color (blue, peak at 455 nm). On the other hand, CTZ indicator 13 showed the longest wavelength shifted emission color (yellow, peak at 560 nm). However, interestingly, we discovered that CTZ indicator 11 simultaneously emitted the two emission colors mentioned above (blue and yellow). For reference, CTZ indicator K6 emitted yellow-green light at around 540 nm. All of these emission spectra were specific to HSA. No emission spectrum was observed when reacting with BSA, which was measured simultaneously. This result supports the fact that CTZ indicators 11, 12, and 13 are selective for HSA.

[0119] Furthermore, the specific luminescence reactivity of the CTZ indicators developed in the present invention (K6, S5, S5h, S6) with albumin was further measured (FIG. 10). For this example, the reactivity of the CTZ indicators developed in the present invention (K6, S5, S5h, S6) to four types of albumin, including mouse serum albumin (MSA) in addition to the albumins used in the previous example (HSA, BSA, OVA), was examined. To carry out this experiment, each albumin was first dissolved in pure water to prepare a 10 mg / mL solution. This albumin solution was diluted 10-fold with phosphate buffered saline (PBS) to prepare albumin solutions with final concentrations of 1 mg / mL. Meanwhile, the CTZ indicators K6, S5, S5h, and S6 of the present invention were first dissolved in methanol and then further diluted with PBS to a final concentration of 100 μM. Next, 30 μL of the albumin solution prepared in advance was dispensed into each well of a 96-well black frame microplate, while the CTZ indicator solution prepared in advance was prepared in each well of a separate microplate. During luminescence measurement, 30 μL of CTZ indicator solution was simultaneously injected into each well of the 96-well black frame microplate containing the albumin described above using a 12-channel micropipette. The luminescence intensity from the microplate was then immediately measured using an IVIS Spectrum imaging system (PerkinElmer, MA, USA) (Figure 1) and analyzed using Living Image version 4.7 software.

[0120] As a result, the order of luminescence intensity for HSA was K6>S6>S5>S5h. On the other hand, for BSA, K6 and S6h emitted strong light (Figure 10(A)). For OVA, S6 emitted a slight amount of light. Finally, for MSA, K6 emitted the brightest light, followed by S6. However, S5h emitted a slight amount of light for MSA. These results are shown as quantitative values ​​in Figure 10(B). These results reaffirm that the CTZ indicators examined here each have selectivity for albumin, and that they also emit light with unique selectivity for mouse serum albumin such as MSA, and for human and bovine albumin.

[0121] Example 1 Summary Recent scientific research has highlighted the importance of serum albumin measurement in the prognosis evaluation of many life-threatening diseases (Non-Patent Documents 1, 2). The present invention relates to the "development and demonstration of a synthetic CTZ indicator" as a means of quantitatively measuring the luminescence signal caused by HSA and BSA with high specificity and colorimetric diversity. To accomplish the invention, the functional group of the CTZ indicator was chemically modified to emit light specifically with HSA or BSA. It was demonstrated that the CTZ indicator instantly emits a "switch-on" type luminescence signal with HSA and BSA, respectively. Depending on the origin of serum albumin, the luminescence color changed dramatically from blue to yellow-green. The height of the luminescence peak was quantitative and proportional to the mixture ratio of HSA-BSA. The reaction rate experiments and inhibition experiments of HSA and BSA confirmed that serum albumin exerts the role of luciferase itself in catalyzing the CTZ indicator. Finally, the usefulness of the CTZ indicator was demonstrated by actual quantitative imaging of FBS. Furthermore, as is clear from the absence of background luminescence in the luminescence photographs of Examples such as Figures 1, 5 and 7, compound (1) or a salt thereof also has chemical stability.

[0122] Example 2

[0123] 1. Synthesis of a new luminescent substrate (CTZ derivative) based on Example 1 Based on the results of Example 1 above, it was noted that the luminescent substrates S5, S5h, S6, S6h, etc. exhibited special performance in visualizing albumin, and a series of novel luminescent substrates were synthesized based on these luminescent substrates. [ka] These new luminescent substrates were named 1-6, P1-P6, M1-M6, and O1-O6 due to the similarity of their chemical structures. P, M, and O here stand for para, meta, and ortho, respectively. In addition, 1 and 2 refer to S5 and S5h, respectively, in the above examples, but the compound names will be changed to 1 and 2 for convenience in the examples below. Similarly, P1 and P2 refer to S6 and S6h, respectively, in the above examples, but the compound names will be changed to P1 and P2 for convenience in the examples below. Meanwhile, the above T1 and T2 are known compounds named 6piOH-CTZ and 6piOH-2H-CTZ, respectively, in the previous paper by the present inventors ("Luciferase-Specific Coelenterazine Analogues for Optical Contamination-Free Bioassays" Scientific Reports volume 7, Article number: 908 (2017)). However, here, they will be renamed T1 and T2, respectively, for convenience. Furthermore, TS1 and TS2 are new luminescent substrates developed in this invention. Specifically, TS1 and TS2 were synthesized by the following method: These CTZ analogs, "1-6, P1-P6, M1-M6, O1-O6," were synthesized based on the descriptions in Non-Patent Documents 12 and 13. In addition, T1, T2, TS1, and TS2 were synthesized based on the following procedure. [ka] 4a; (E)-2-(4-methoxystyryl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane Under argon atmosphere and 0°C reaction conditions, 2M lithium diisopropylamide tetrahydrofuran / hexane / ethylbenzene solution (3.0 mmol, 1.5 mL) was mixed with bis([pinacolato]boryl)methane (3.0 mmol, 803 mg) dissolved in tetrahydrofuran (15 mL) and stirred for 5 minutes. The reaction solution was then cooled to -78°C, and p-anisaldehyde (2.0 mmol, 243 μL) dissolved in tetrahydrofuran (4 mL) was slowly added and stirred at -78°C for 30 minutes. After the reaction was completed, 30 mL of pure water was added, and the reaction product was extracted with ethyl acetate (3 × 100 mL). The organic layer was evaporated under anhydrous sodium sulfate conditions. The resulting product was separated by flash chromatography (hexane / ethyl acetate = 10 / 1) to give 4a (494 mg, 1.89 mmol, 94%) as a colorless oil. 1 H-NMR (500 MHz, Acetone-d 6 ) δ 7.51 (td, J = 6.0, 3.4 Hz, 2H), 7.30 (d, J = 18.9 Hz, 1H), 6.94 (td, J = 5.9, 3.6 Hz, 2H), 5.98 (d, J = 18.3 Hz, 1H), 3.82 (s, 3H), 1.27 (s, 12H) 13 C-NMR (126 MHz, Acetone-d 6 ) δ 161.4, 149.7, 131.1, 129.2, 114.8, 83.7, 55.6, 25.1 HR-MALDI-MS m / z: [M+H] + calcd for C 15 H 22 BO 3 , 261.16357; found, 261.16565. [ka] 7a; (E)-3-benzyl-5-(4-methoxystyryl)pyrazin-2-amine Compound 5a (100 mg, 0.38 mmol) and compound 4a (148 mg, 0.57 mmol) were dissolved in 1,4-dioxane (4 mL) at room temperature. Tetrakis(triphenylphosphine)palladium(0) (21 mg, 0.02 mmol) and 2M Na 2 CO 3 aq (4 mL) was added and the reaction was carried out under argon atmosphere at 100°C for 2 hours. After the reaction was completed, 10 mL of pure water was added, and the synthesis result was extracted with ethyl acetate (3 × 30 mL). The organic layer was evaporated with anhydrous sodium sulfate. The residue was separated by column chromatography (hexane / ethyl acetate = 3 / 1) to obtain 7a (100 mg, 0.32 mmol, 84%) in the form of yellow crystals. 1 H-NMR (500 MHz, Acetone-d 6 ) δ 7.96 (s, 1H), 7.52 (dd, J = 11.5, 2.9 Hz, 2H), 7.43 (d, J = 16.0 Hz, 1H), 7.35 (d, J = 7.4 Hz, 2H), 7.30 (t, J = 7.4 Hz, 2H), 7.22 (t, J = 7.2 Hz, 1H), 7.04 (d, J = 16.0 Hz, 1H), 6.94 (dd, J = 12.0, 2.9 Hz, 2H), 5.53 (s, 2H), 4.15 (s, 2H), 3.82 (s, 3H) 13 C-NMR (126 MHz, Acetone-d 6) δ 160.4, 153.3, 141.5, 141.1, 140.2, 138.8, 131.1, 129.7, 129.3, 128.7, 128.6, 127.3, 123.9, 115.0, 55.6, 40.6 HR-MALDI-MS m / z: [M+H] + calcd for C 20 H 20 N 3 O, 318.15964; found, 318.16009. [ka] 8a; (E)-5-(4-methoxystyryl)-3-(phenylthio)pyrazin-2-amine Compound 6a (1.4 g, 4.98 mmol) and compound 4a (1.9 g, 7.47 mmol) were dissolved in 1,4-dioxane (40 mL) at room temperature. Tetrakis(triphenylphosphine)palladium(0) (287 mg, 0.25 mmol) and 2M Na 2 CO 3 aq (40 mL) was added and the reaction was carried out under argon atmosphere at 100 °C for 2 hours. After the reaction was completed, 60 mL of pure water was added and the reaction product was extracted with ethyl acetate (3 × 200 mL). The organic solvent layer was evaporated with anhydrous sodium sulfate. The residue was separated by column chromatography (hexane / ethyl acetate = 2 / 1 to 1 / 1) to obtain compound 8a (1.34 g, 3.99 mmol, 80%) in the form of yellow crystals. 1 H-NMR (500 MHz, Acetone-d 6) δ 7.89 (s, 1H), 7.56 (dt, J = 6.5, 1.7 Hz, 2H), 7.49-7.44 (m, 3H), 7.42 (dd, J = 6.9, 1.7 Hz, 2H), 7.14 (d, J = 16.0 Hz, 1H), 6.94 (d, J = 16.0 Hz, 1H), 6.91 (dd, J = 6.6, 2.0 Hz, 2H), 5.88 (s, 1H), 3.81 (s, 3H) 13 C-NMR (126 MHz, Acetone-d 6 ) δ 160.5, 152.9, 141.8, 139.7, 138.8, 134.2, 131.6, 130.8, 130.0, 129.5, 129.1, 128.7, 122.7, 115.0, 55.6 HR-MALDI-MS m / z: [M+H] + calcd for C 19 H 18 N 3 OS, 336.11611; found, 336.11651. [ka] 9a; (E)-4-(2-(5-amino-6-benzylpyrazin-2-yl)vinyl)phenol Compound 7a (95 mg, 0.29 mmol) was dissolved in dehydrated dichloromethane (7 mL) and stirred under argon atmosphere at -80 °C. Boron tribromide (17% in dichloromethane, 1 mol / L) (2.0 mL, 2.03 mmol) was added to the solution at -80 °C. The solution was stirred overnight under argon atmosphere at room temperature. After the reaction was completed, 10 mL of NaHCO 3Aq was added at 0 °C, and the compound was extracted with chloroform (3 × 20 mL) and ethyl acetate (3 × 20 mL). The organic layer was evaporated with anhydrous sodium sulfate. The residue was separated by column chromatography (chloroform / methanol = 10 / 1) to give yellow solid compound 9a (85 mg, 0.28 mmol, 96%). 1 H-NMR (500 MHz, Acetone-d 6 ) δ 7.95 (s, 1H), 7.47-7.43 (m, 2H), 7.41 (d, J = 16.6 Hz, 1H), 7.35 (d, J = 6.9 Hz, 2H), 7.32-7.28 (m, 2H), 7.22 (t, J = 7.2 Hz, 1H), 6.99 (d, J = 15.5 Hz, 1H), 6.85 (d, J = 8.6 Hz, 2H), 5.50 (s, 2H), 4.14 (s, 2H) 13 C-NMR (126 MHz, Acetone-d 6 ) δ 158.1, 153.2, 141.4, 141.2, 140.1, 138.8, 130.1, 129.7, 129.3, 129.0, 128.8, 127.3, 123.1, 116.4, 40.6 HR-MALDI-MS m / z: [M+H] + calcd for C 19 H 18 N 3 O, 304.14427; found, 304.14444. [ka] 10a; (E)-4-(2-(5-amino-6-(phenylthio)pyrazin-2-yl)vinyl)phenol Compound 8a (500 mg, 1.49 mmol) was dissolved in dehydrated dichloromethane (35 mL) under argon atmosphere at -80 °C with stirring. Boron tribromide (17% in dichloromethane, 1 mol / L) (3.7 mL, 3.73 mmol) at -80 °C was added to the solution. The solution was stirred overnight under argon atmosphere at room temperature. After the reaction was completed, the solution was diluted with 30 mL of NaHCO at 0 °C. 3 Aq was added, and the resulting mixture was extracted with ethyl acetate (3 × 20 mL). The organic layer was evaporated with anhydrous sodium sulfate. The residue was separated by column chromatography (chloroform / methanol= 1 / 1) to give 10a (325 mg, 0.28 mmol, 68%) as a yellow solid. 1 H-NMR (500 MHz, Acetone-d 6 ) δ 7.88 (s, 1H), 7.56-7.54 (m, 2H), 7.49-7.44 (m, 3H), 7.34 (d, J = 8.4 Hz, 2H), 7.12 (d, J = 15.9 Hz, 1H), 6.89 (d, J = 15.9 Hz, 1H), 6.82 (d, J = 8.6 Hz, 2H), 5.86 (s, 2H) 13 C-NMR (126 MHz, Acetone-d 6 ) δ 206.3, 206.2, 206.0, 158.3, 152.7, 141.9, 139.5, 138.6, 134.2, 131.6, 130.0, 129.8, 129.8, 129.1, 128.8, 122.0, 116.4 HR-MALDI-MS m / z: [M+H] + calcd for C 18 H 16 N 3 OS, 322.10054; found, 322.10086. [ka] T1; (E)-8-benzyl-2-(4-hydroxybenzyl)-6-(4-hydroxystyryl)imidazo[1,2-a]pyrazin-3(7H)-one Compound 9a (30 mg, 0.098 mmol) and compound 2a (1,1-diethoxy-3-(4-hydroxyphenyl)propan-2-one, 35 mg, 0.148 mmol) were dissolved in ethanol (2 mL) at room temperature. 12 M HCl aq. (100 μL) was added to the solution and reacted at 60 °C for 12 h under an argon atmosphere. The solution was evaporated. The residue was separated by automated flash chromatography (Smart Flash EPCLC AI-580S, Universal Columns, chloroform / methanol = 99 / 1 to 15 / 85) to obtain the CTZ analog T1 (11.1 mg, 0.048 mmol, 25 %, reddish-brown solid). 1 H-NMR (500 MHz, Methanol-d 4 ) δ 7.79 (s, 1H), 7.41 (d, J = 8.6 Hz, 2H), 7.37 (d, J = 6.9 Hz, 2H), 7.32-7.29 (m, 3H), 7.26-7.23 (m, 1H), 7.13 (d, J = 8.0 Hz, 2H), 6.86 (d, J = 16.6 Hz, 1H), 6.79 (d, J = 8.6 Hz, 3H), 6.71 (d, J = 8.6 Hz, 2H), 4.45 (s, 2H), 4.09 (s, 2H) HR-MALDI-MS m / z: [M+H] + calcd for C 28 H 24 N 3 O 2 , 450.18028; found, 450.18122. [ka] T2; (E)-2,8-dibenzyl-6-(4-hydroxystyryl)imidazo[1,2-a]pyrazin-3(7H)-one Compound 9a (30 mg, 0.098 mmol) and compound 3a (1,1-diethoxy-3-phenylpropan-2-one 33 mg, 0.148 mmol) were dissolved in ethanol (2 mL) at room temperature. 12M HCl aq (100 μL) was added to the solution under argon atmosphere and 60 °C, and the mixture was stirred and reacted for 12 hours. The solution was evaporated. The residue was separated by automated flash chromatography (Smart Flash EPCLC AI-580S, Universal Columns, chloroform / methanol = 99 / 1 to 15 / 85) to obtain compound T2 (15.7 mg, mmol, 37 %, reddish-brown solid). 1H-NMR (500 MHz, Methanol-d 4 ) δ 7.55 (s, 1H), 7.39-7.16 (m, 13H), 6.77 (t, J = 8.9 Hz, 3H), 4.40 (s, 2H), 4.15 (s, 2H) HR-MALDI-MS m / z: [M+H] + calcd for C 28 H 24 N 3 O 2 , 434.18574; found, 434.18630. [ka] TS1; (E)-2-(4-hydroxybenzyl)-6-(4-hydroxystyryl)-8-(phenylthio)imidazo[1,2-a]pyrazin-3(7H)-one Compound 10a (30 mg, 0.093 mmol) and compound 2a (33 mg, 0.14 mmol) were dissolved in ethanol (2 mL) at room temperature. 12M HCl aq. (100 μL) was added to this solution and reacted for 12 hours under argon atmosphere at 60°C. The residue obtained by evaporating this solution was separated by automated flash chromatography (Smart Flash EPCLC AI-580S, Universal Columns, chloroform / methanol = 99 / 1 to 15 / 85) to obtain TS1 (17.3 mg, 0.048 mmol, 39 %, reddish-brown solid). 1 H-NMR (500 MHz, Methanol-d 4 ) δ 7.77 (s, 1H), 7.68 (d, J = 6.9 Hz, 3H), 7.54 (d, J = 6.9 Hz, 4H), 7.21 (d, J = 8.6 Hz, 2H), 7.10 (d, J = 8.0 Hz, 2H), 6.99 (d, J = 15.5 Hz, 1H), 6.81 (d, J = 15.5 Hz, 1H), 6.73 (d, J = 8.6 Hz, 2H), 6.71 (d, J = 8.0 Hz, 2H), 4.02 (s, 2H) HR-MALDI-MS m / z: [M+H] + calcd for C 27 H 22 N 3 O 3 S, 468.13811; found, 468.13764. [ka] TS2; (E)-2-benzyl-6-(4-hydroxystyryl)-8-(phenylthio)imidazo[1,2-a]pyrazin-3(7H)-one Compound 10a (30 mg, 0.093 mmol) and compound 3a (31 mg, 0.14 mmol) were dissolved in ethanol (2 mL) at room temperature. 12M HCl aq (100 μL) was added to this solution and reacted for 12 hours under argon atmosphere at 60°C. The solution was evaporated, and the residue was separated by automated flash chromatography (Smart Flash EPCLC AI-580S, Universal Columns, chloroform / methanol = 99 / 1 to 15 / 85) to obtain TS2 (10.9 mg, 0.048 mmol, 26 %, reddish-brown solid). 1 H-NMR (500 MHz, Methanol-d 4 ) δ 7.72 (s, 1H), 7.68 (dd, J = 7.7, 2.0 Hz, 2H), 7.54-7.50 (m, 3H), 7.30-7.24 (m, 4H), 7.21 (d, J = 8.6 Hz, 2H), 7.15 (t, J = 7.2 Hz, 1H), 6.98 (d, J = 15.5 Hz, 1H), 6.79 (d, J = 15.5 Hz, 1H), 6.73 (d, J = 8.6 Hz, 2H), 4.11 (s, 2H) HR-MALDI-MS m / z: [M+H] + calcd for C 27 H 22 N 3 O 3 S, 452.14127; found, 452.14272.

[0124] 2. Measurement of albumin visualization ability of new luminescent substrates The albumin visualization ability of the luminescent substrates described above and conventional luminescent substrates was measured (Figure 11). The albumins used in this measurement were human serum albumin (HSA), murine serum albumin (MSA), bovine serum albumin (BSA), and ovine serum albumin (OVA). The absolute value results are shown in Figure 11. An enlarged view of Figure 11 is shown in Figure 12. The results can be considered as follows. First, each group, 1-6, P1-P6, M1-M6, and O1-O6, shows periodic selectivity characteristics for each albumin. The luminescent substrates 1, P1, M1, and O1 all have a common feature of being selective for HSA, and as mentioned above, they also have similar chemical structures. Similarly, 3, P3, M3, and O3 all selectively emitted light for HAS. Furthermore, 5, P5, M5, and O5 also showed the characteristic of selectively emitting light for HSA. On the other hand, luminescent substrates 2, P2, M2, and O2 showed a common luminescence response to HSA and BSA. Similarly, 4, P4, M4, and O4 showed a common luminescence response to HSA and BSA, although the brightness was weak. Similarly, 6, P6, M6, and O6 showed a common luminescence response to HSA and BSA. In summary, odd-numbered luminescent substrates (substrates with names ending in 1, 3, and 5) are selective for HSA only, whereas even-numbered luminescent substrates (substrates with names ending in 2, 4, and 6) show the characteristic of luminescence with both HSA and BSA. These luminescent substrates (group 1, group P, group M, and group O) showed more or less luminescence activity with HSA and BSA, but did not show significant luminescence intensity with MSA or OVA. Within the same group, luminescent substrates with names ending in 2 (i.e., 2, P2, M2, etc.) showed relatively high luminescence intensity. The order of increasing intensity was M2 > P2 > 2. Compared to the other groups, the T group had the characteristic of showing a certain level of luminescence brightness with HSA, BSA, and MSA. For example, T1 and T2 show similar selectivity to the even- and odd-numbered luminescent substrates mentioned above, respectively, but are also characterized by their fairly strong luminescence brightness with MSA. In addition, TS1 and TS2 showed the highest luminescence brightness of all the luminescent substrates tested this time. In particular, they showed 8x10 6The TS2 showed a strong luminescence response per p / s / cm2 / sr. The TS2 also showed a relatively strong luminescence brightness in MSA, proving that it can be used sufficiently for the luminescence measurement of mouse serum albumin. However, none of the conventional luminescent substrates (nCTZ, CTZh) measured in parallel showed a luminescent response to the serum albumins tested.

[0125] 3. Measurement of albumin visualization ability of selected novel luminescent substrates Since the serum albumin visualization ability of each luminescent substrate was confirmed from the above-mentioned Examples, nCTZ, P1, P2, TS1, and TS2 were selected to highlight their respective serum albumin visualization abilities (FIG. 13). As a result, nCTZ did not show any significant luminescence response to any serum albumin, while P1 and TS1 luminesced specifically to HSA. On the other hand, P2 luminesced specifically to BSA, with a luminescence intensity of about 3x10 6 The luminescence brightness was about p / s / cm2 / sr. In this highlighting experiment, TS2 showed a unique serum albumin selectivity that is different from other luminescent substrates. TS2 was most brightly luminescent with HSA, but was also more strongly luminescent with MSA than other luminescent substrates, at about 1x10 6 The luminescence intensity was about p / s / cm2 / sr, which is sufficient for various bioassays and luminescence imaging in individual animals.

[0126] 4. Measurement of dose-response curves between selected novel luminescent substrates and serum albumin From the above-mentioned Examples, it was confirmed that P1, P2, TS1, and TS2 responded specifically to each serum albumin by luminescence, and furthermore, their dose-response curves were measured (FIG. 14). The concentrations of serum albumin for this experiment were adjusted to 0, 0.01, 0.025, 0.05, 0.075, 0.1, 0.15, and 0.2 mg / mL. Meanwhile, each luminescent substrate was adjusted to 100 μM. Then, 40 μL of each prepared albumin solution and luminescent substrate were dispensed and reacted in each well of a 96-well microplate, and luminescence was measured using the IVIS Spectrum system. As a result, it was found that P1, TS1, and TS2 emitted light in response to only 0.01 mg / mL of HSA or BSA. The overall graph also showed that the detection limit was approximately 0.01 mg / mL of HSA or BSA. On the other hand, P2 does not seem to reach the relatively low concentration range of serum albumin detection limit, but the result graph shows that it still emitted light in response to 0.025 mg / mL of BSA. From the viewpoint of selectivity, P1, TS1, and TS2 showed strong selectivity for HSA, and P2 showed strong selectivity for BSA, and all of them showed almost linear response graphs in the entire concentration range measured. When comparing P1 and P2, it was confirmed that P1 was selective for HSA and P2 was selective for BSA regardless of the serum albumin concentration in the entire measurement range. In the case of TS1, it showed a strong luminescence response to HSA, but did not show a strong luminescence response to other serum albumins. On the other hand, it was found that TS2 also showed a strong luminescence response to HSA, and further showed a considerable luminescence response to MSA and BSA. These results showed that P1, P2, TS1, TS2, etc. selected in this example are all luminescence indicators that have both a very low detection limit and unique serum albumin selectivity.

[0127] 5. Measurement of the emission spectrum of the selected luminescent substrate From the results of the above-mentioned examples, it was found that the selected luminescent substrates exhibited strong selectivity and low detection limits for each serum albumin. Therefore, in this example, the luminescence spectra were measured for each combination of luminescent substrate and serum albumin (Figure 15). To carry out this example, each luminescent substrate was first prepared to a concentration of 100 μM. Meanwhile, serum albumin was prepared to a concentration of 0.5-10 mg / mL by diluting with PBS. Each solution was dispensed into PCR tubes at 100 μM, and each spectrum was measured using a precision luminescence spectrophotometer (AB-1850, ATTO, Tokyo, Japan) with 1 minute of integration. As a result, when HSA reacted with TS1 or TS2, a spectrum showing an emission peak (blue color) at around 499-502 nm was measured. On the other hand, when BSA reacted with M2 or P2, a spectrum with an emission peak at around 547-558 nm (yellow-green color) was observed. Furthermore, when MSA reacted with TS1 or TS2, an emission spectrum with a peak (green color) at around 500-495 nm was measured. These results confirmed that the combination of BSA with M2 or P2 showed the emission spectrum shifted to the longest wavelength, and therefore had good tissue penetration of the emission light when applied to animal imaging models, making this the most advantageous combination for animal imaging.

[0128] This invention contributes to expanding the toolbox of luminescent indicators for efficient analysis of serum proteins and ovalbumin in various biological fluid samples with high specificity and luminescent color. Considering that CTZ indicators emit instantaneous luminescent signals with high specificity and quantitative luminescent signals, this luminescent indicator may be a solution to overcome the traditional technical hurdles in point-of-care assay of serum albumin.

Claims

1. A serum albumin detection agent comprising a compound represented by general formula (1) or a salt thereof: 【Chemistry 1】 [In the formula, R １ represents an aryl group or an unsaturated heterocyclic group which may have a substituent. ２ - is -S-, -CH ２ -, -CH=CH- or -C≡C-. ３ R represents an aryl group which may have a substituent. ４ represents an aryl group having a substituent; l represents 0 or 1; m represents 0 or 1. ２ - is -CH ２ -, m represents 1.

2. R １ 2. The serum albumin detection agent according to claim 1, wherein, when the aryl group or unsaturated heterocyclic group represented by the formula (I) has a substituent, the substituent is at least one selected from the group consisting of a hydroxyl group, a thiol group, a hydroxyalkyl group and a mercaptoalkyl group.

3. R ３ The serum albumin detection agent according to claim 1, wherein, when the aryl group represented by the formula (I) has a substituent, the substituent is at least one selected from the group consisting of a halogen, a hydroxyl group, an alkyl group, an alkoxy group, an alkoxyalkyl group, a hydroxyalkyl group and an alkylamino group.

4. R ４ 2. The serum albumin detection agent according to claim 1, wherein the aryl group represented by the formula: is substituted with at least one group selected from the group consisting of a hydroxyl group, a hydroxyalkyl group, an alkoxy group and an alkylamino group.

5. The serum albumin detection agent according to claim 1, wherein the compound represented by the general formula (1) or a salt thereof is a compound represented by the following general formula (2) or a salt thereof: 【Chemistry 2】 [In the formula, -R ２ - is -S-, -CH ２ -, -CH=CH- or -C≡C-. ５ R is a hydroxyl group, a thiol group, a hydroxyalkyl group, or a mercaptoalkyl group. ５ If there are multiple R ５ may be the same or different. ６ R is a halogen, a hydroxyl group, an alkyl group, an alkoxy group, an alkoxyalkyl group, a hydroxyalkyl group, or an alkylamino group. ６ If there are multiple R ６ may be the same or different. ７ R is a hydroxyl group, a hydroxyalkyl group, an alkoxy group, or an alkylamino group. ７ If there are multiple R ７ may be the same or different. l represents 0 or 1. m represents 0 or 1. n represents an integer of 0 to 2. p represents an integer of 0 to 2. q represents an integer of 1 or 2. However, -R ２ - is -CH ２ -, m represents 1.

6. General formula (1') 【Chemistry 3】 [In the formula, R １ R represents an aryl group or an unsaturated heterocyclic group which may have a substituent. ３ R represents an aryl group which may have a substituent. ４ represents an aryl group having a substituent; and l represents 0 or 1. A compound represented by the formula:

7. A method for detecting serum albumin in a sample, comprising the step of adding the serum albumin detection agent according to any one of claims 1 to 5 or the compound or a salt thereof according to claim 6 to a target sample.

8. 8. The method of claim 7 for determining the content of albumin and / or the origin of albumin in a sample.

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