Luminescent substrate compounds

Abstract

Provided is a novel compound or the like useful as a luminescent molecule that serves as a substrate for a human-derived protein. The compound according to the present invention is a compound represented by formula [I], or a salt thereof, or a hydrate or a solvate thereof.

Description

[Technical Field]

[0001] The present invention relates to a compound that serves as a luminescent substrate and uses thereof. [Background technology]

[0002] Traditional protein analysis techniques have a wide variety of detection methods depending on the application, such as ultraviolet absorptiometry (using 280 nm ultraviolet light absorption) and the BCA method (detection by copper reduction). However, these methods are adversely affected by other biomolecules such as nucleic acids and phospholipids. Furthermore, while fluorescence protein analysis is relatively sensitive, the excitation light source used during observation induces a high background signal. Therefore, current protein analysis techniques require tedious sample preparation and are time-inefficient. In particular, fluorescence methods are unsuitable for continuous observation of protein dynamics in vivo because the excitation light source can cause protein denaturation and phototoxicity. Furthermore, developing fluorescence and absorption assay reagents that react specifically to proteins is challenging, and a simple and highly sensitive quantitative analysis method for proteins in solutions of various compositions has not yet been fully established. On the other hand, human serum albumin (HSA), a human-derived protein, is widely recognized as an important serum protein that maintains the osmotic pressure of blood and transports endogenous ligands such as bilirubin. It also functions as a catalytic enzyme in the Kemp elimination reaction (a chemical reaction in which a proton is removed from carbon), making it an important protein with multiple functions in the body (e.g., Non-Patent Documents 1 and 2). [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Japanese Patent Application Publication No. 2018-158896 [Patent Document 2] Japanese Patent Application Laid-Open No. 2018-165265 [Non-patent literature]

[0004] [Non-Patent Document 1] F. Hollfeider et al., J.Am.Chem,Soc., 2000, 122, 1022-1029. [Non-patent document 2] D. Rothlisberger et al., Nature, 2008, 453, 190-195. [Non-patent document 3] T. Hirano et al., Tetrahedron Letters, 1992, 33, 5771-5774. [Non-patent document 4] R. Nishihara et al., Theranostics, 2019, 9, 2646-2661. Summary of the Invention [Problem to be solved by the invention]

[0005] Under these circumstances, there was a need for the development of a protein analysis technology that would enable simple and highly sensitive quantitative analysis of desired proteins in solutions of various compositions; more specifically, the development of a new luminescent substrate compound that would produce light through an enzymatic reaction with human-derived proteins. [Means for solving the problem]

[0006] The present invention has been made in consideration of the above circumstances, and provides the compounds shown below. (1) Formula [I] below: [ka] [In formula [I], R1 is -CH2-A (where A is hydrogen or a group of the formula: [ka] (In the formula, R3 is hydrogen, a hydroxyl group, fluorine, an alkyl group having 1 to 5 carbon atoms, a methoxy group, or a trifluoromethyl group, and m is an integer of 0 to 5. or a group represented by The following formula: [ka] is a group represented by and R2 has the following formula: [ka] (Wherein R4 is (i) -O-(CH2) n -R6 (wherein R6 is a hydroxyl group, a methoxy group, a methyl group, a trifluoromethyl group, or an azide group, and n is an integer of 1 to 5), (ii) an alkyl group having 1 to 5 carbon atoms, or (iii) the following formula: [ka] Any one of the groups represented by ) or a group represented by The following formula: [ka] (Wherein R5 is (i) a hydrogen atom, a hydroxyl group, a methoxy group, a methyl group, a trifluoromethyl group, a dimethylamino group, a phenyl group, or an azide group; (ii) an alkyl group having 1 to 5 carbon atoms; (iii) -O-(CH2) p -R7 (wherein R7 is a hydroxyl group, a methoxy group, a methyl group, a trifluoromethyl group, a dimethylamino group, an azide group, or an alkyl group having 1 to 5 carbon atoms, and p is an integer of 1 to 5), or (iv) the following formula: [ka] Any one of the groups represented by where n is an integer from 0 to 5. is a group represented by the following formula: or a salt thereof, or a hydrate or solvate thereof.

[0007] (2) The compound represented by the formula [I] is a compound represented by the following formula [II]: [ka] [In formula [II], R3 is hydrogen, a hydroxyl group, fluorine, an alkyl group having 1 to 5 carbon atoms, a methoxy group, or a trifluoromethyl group; R4 is (i) -O-(CH2) n -R6 (wherein R6 is a hydroxyl group, a methoxy group, a methyl group, a trifluoromethyl group, or an azide group, and n is an integer of 1 to 5), (ii) an alkyl group having 1 to 5 carbon atoms, or (iii) the following formula: [ka] A group represented by any one of ] The compound according to (1) above, or a salt thereof, or a hydrate or solvate thereof, wherein the compound is a compound represented by the formula:

[0008] (3) A compound according to (1) or (2) above, or a salt thereof, or a hydrate or solvate thereof, wherein R3 and R4 in the formula [I] or formula [II] are each a combination of groups or atoms shown in the table below. [Table 1]

[0009] (4) The compound according to (3) above, wherein R3 is -H and R4 is -O-(CH2)3-OCH3, or a salt thereof, or a hydrate or solvate thereof. (5) The compound according to (3) above, wherein R3 is -OH and R4 is -O-(CH2)3-OCH3, or a salt thereof, or a hydrate or solvate thereof.

[0010] (6) The compound represented by the formula [I] is a compound represented by the following formula [III]: [ka] [In formula [III], R4 is (i) -O-(CH2) n -R6 (wherein R6 is a hydroxyl group, a methoxy group, a methyl group, a trifluoromethyl group, or an azide group, and n is an integer of 1 to 5), (ii) an alkyl group having 1 to 5 carbon atoms, or (iii) the following formula: [ka] A group represented by any one of ] The compound according to (1) above, or a salt thereof, or a hydrate or solvate thereof, wherein the compound is a compound represented by the formula:

[0011] (7) The compound according to (6) above, or a salt thereof, or a hydrate or solvate thereof, wherein R4 in the formula [III] above is a group shown in the table below. [Table 2]

[0012] (8) The compound according to (7) above, or a salt thereof, or a hydrate or solvate thereof, wherein R4 is -O-(CH2)3-OCH3.

[0013] (9) The compound represented by the formula [I] is a compound represented by the following formula [IV]: [ka] [In formula [IV], R3 is hydrogen, a hydroxyl group, fluorine, an alkyl group having 1 to 5 carbon atoms, a methoxy group, or a trifluoromethyl group; R5 is (i) a hydrogen atom, a hydroxyl group, a methoxy group, a methyl group, a trifluoromethyl group, a dimethylamino group, a phenyl group, or an azide group; (ii) an alkyl group having 1 to 5 carbon atoms; (iii) -O-(CH2) p -R7 (wherein R7 is a hydroxyl group, a methoxy group, a methyl group, a trifluoromethyl group, a dimethylamino group, an azide group, or an alkyl group having 1 to 5 carbon atoms, and p is an integer of 1 to 5), or (iv) the following formula: [ka] Any one of the groups represented by and n is an integer from 0 to 5. The compound according to (1) above, or a salt thereof, or a hydrate or solvate thereof, wherein the compound is a compound represented by the formula:

[0014] (10) The compound according to (9) above, or a salt thereof, or a hydrate or solvate thereof, wherein n, and the groups or atoms represented by R3 and R5 in the formula [IV] are the combinations shown in the table below. [Table 3]

[0015] (11) The compound according to (10) above, wherein n is 1, R3 is -H, and R5 is -OCH3, or a salt thereof, or a hydrate or solvate thereof. (12) The compound according to (10) above, wherein n is 1, R3 is -H, and R5 is -CF3, or a salt thereof, or a hydrate or solvate thereof. (13) The compound according to (10) above, wherein n is 1, R3 is -H, and R5 is -C6H5, or a salt thereof, or a hydrate or solvate thereof. (14) The compound according to (10) above, wherein n is 1, R3 is -H, and R5 is -H, or a salt thereof, or a hydrate or solvate thereof.

[0016] (15) A luminescent substrate for a protein or peptide, comprising the compound according to any one of (1) to (14) above, or a salt thereof, or a hydrate or solvate thereof. (16) A method for analyzing a protein or peptide, comprising administering in vivo or adding in vitro the compound according to any one of (1) to (14) above or a salt thereof, or a hydrate or solvate thereof, or the luminescent substrate according to (15) above, and detecting a desired protein or peptide. [Effects of the Invention]

[0017] According to the present invention, it is possible to provide novel compounds and the like as luminescent molecules that serve as substrates for human-derived proteins (luminescent substrates that produce light through an enzymatic reaction with human-derived proteins). The compounds of the present invention have higher luminescence intensity than known luminescent substrate compounds such as coelenterazine derivatives, and are therefore extremely useful and practical in the development of protein analysis techniques that enable simple and highly sensitive quantitative analysis of desired proteins in solutions of various compositions. [Brief explanation of the drawings]

[0018] [Figure 1] FIG. 1 shows the luminescence intensity of each compound of the examples and comparative examples of the present invention in combination with human or bovine serum albumin. [Figure 2] 1 shows the luminescence intensity of a compound (HuLumino12) according to an example of the present invention in combination with various human-derived proteins or bovine serum albumin, where "a" shows a schematic diagram. [Figure 3] FIG. 1 is a diagram showing the luminescence characteristics (signal to noise ratio, S / N ratio) of each compound in the examples and comparative examples of the present invention in human serum albumin (HSA). [Figure 4] FIG. 1 is a diagram showing the luminescence characteristics (luminescence duration) of an example compound (HuLumino12) of the present invention in human serum albumin (HSA). [Figure 5]FIG. 1 shows the luminescence intensity of a compound (HuLumino12) according to an example of the present invention, depending on the concentration of human serum albumin (HSA). [Figure 6] FIG. 1 shows the emission wavelength (results of emission spectrum measurement) of a compound (HuLumino12) according to an example of the present invention in human serum albumin (HSA). DETAILED DESCRIPTION OF THE INVENTION

[0019] The present invention will be described in detail below. The scope of the present invention is not limited to these descriptions, and other than the following examples, the present invention can be appropriately modified and implemented within the scope that does not depart from the spirit of the present invention. This specification encompasses the entirety of Japanese Patent Application No. 2020-046137 (filed March 17, 2020), from which priority is claimed. All publications cited in this specification, such as prior art documents, publications, patent publications, and other patent documents, are incorporated herein by reference.

[0020] 1. Overview of the Invention Luminescent molecules, composed of amino acids and found in bioluminescent organisms, are utilized as chemical probes for converting molecular recognition or enzymatic reactions into spectroscopic information. Bioanalysis utilizing these luminescent reactions enables microanalysis and bioimaging without the need for an excitation light source, and is widely used as a highly sensitive analytical technique in life sciences. This is due to the fact that luminescent molecules use chemical reactions as excitation energy. Luminescence systems widely used in bioanalysis include the firefly luminescence system and marine bioluminescent species such as the bioluminescent jellyfish Aequorea victoria. In particular, the Renilla luciferase (RLuc) luminescence system, found in one marine bioluminescent species and using coelenterazine (CTZ) as a luminescent substrate, is a simple luminescence system that requires no cofactors other than oxygen molecules (specifically, a simple luminescence mechanism in which light is emitted in response to proton transfer within the CTZ chemical structure). This allows for highly reproducible analysis not only within cells but also outside the cell in the absence of ATP. Accordingly, numerous CTZ derivatives with modified chemical structures have been reported, and their optical properties have been extensively investigated (see Patent Documents 1 and 2, and Non-Patent Documents 3 and 4, cited above). The compound of the present invention is a compound that serves as a luminescent molecule (a luminescent substrate that produces light through an enzymatic reaction with a human-derived protein) that serves as a substrate for a human-derived protein, and has been found to have higher luminescence intensity than the known luminescent substrate compounds described above, and to be a compound with excellent usefulness and practicality.

[0021] 2. Luminescent substrate compounds The compound according to the present invention (hereinafter also referred to as the compound of the present invention) is a compound represented by the following formula [I]. [ka]

[0022] Here, in formula [I], R1 is —CH2—A. A is hydrogen or a group of the following formula: [ka] (In the formula, R3 is hydrogen, a hydroxyl group, fluorine, an alkyl group having 1 to 5 carbon atoms, a methoxy group, or a trifluoromethyl group, and m is an integer of 0 to 5. or The following formula: [ka] The former group is preferred.

[0023] In addition, in formula [I], R2 is the following formula: [ka] (Wherein R4 is (i) -O-(CH2) n -R6 (wherein R6 is a hydroxyl group, a methoxy group, a methyl group, a trifluoromethyl group, or an azide group, and n is an integer of 1 to 5), (ii) an alkyl group having 1 to 5 carbon atoms, or (iii) the following formula: [ka] Any one of the groups represented by ) or a group represented by The following formula: [ka] (Wherein R5 is (i) a hydrogen atom, a hydroxyl group, a methoxy group, a methyl group, a trifluoromethyl group, a dimethylamino group, a phenyl group, or an azide group; (ii) an alkyl group having 1 to 5 carbon atoms; (iii) -O-(CH2) p -R7 (wherein R7 is a hydroxyl group, a methoxy group, a methyl group, a trifluoromethyl group, a dimethylamino group, an azide group, or an alkyl group having 1 to 5 carbon atoms, and p is an integer of 1 to 5), or (iv) the following formula: [ka] Any one of the groups represented by where n is an integer from 0 to 5. It is a group represented by the following formula:

[0024] The compound represented by the formula [I] is not limited to, but preferred examples include a compound represented by the following formula [II], a compound represented by the following formula [III], and a compound represented by the following formula [IV].

[0025] [ka] [ka] [ka]

[0026] Here, the explanations regarding R3, R4, R5 and the value of n in formula [I] can be similarly applied to R3 and R4 in formula [II] above, R4 in formula [III] above, and R3, R5 and n in formula [IV] above.

[0027] In the above formula [II], the combination of R3 and R4 is not limited, but preferred examples include those shown in the table below.

[0028] [Table 4]

[0029] Furthermore, specific examples of the compound represented by the above formula [II] include the compounds represented by the following formulas.

[0030] [ka]

[0031] In the above formula [III], R4 is not limited, but preferred examples include those shown in the table below.

[0032] [Table 5]

[0033] In the above formula [IV], the value of n and the combination of R3 and R5 are not limited, but preferred examples include those shown in the table below.

[0034] [Table 6]

[0035] The compounds of the present invention described above may be used together with or instead of the compounds in the form of salts (preferably, pharmacologically acceptable salts, etc.). Preferred examples of such salts include, but are not limited to, hydrohalides (e.g., hydrochlorides, hydrobromides, and hydroiodides), inorganic acid salts (e.g., sulfates, nitrates, perchlorates, phosphates, carbonates, and bicarbonates), organic carboxylates (e.g., acetates, trifluoroacetates, maleates, tartrates, fumarates, and citrates), organic sulfonates (e.g., methanesulfonates, trifluoromethanesulfonates, ethanesulfonates, benzenesulfonates, toluenesulfonates, and camphorsulfonates), amino acid salts (e.g., aspartates and glutamates), quaternary amine salts, alkali metal salts (e.g., sodium salts and potassium salts), and alkaline earth metal salts (e.g., magnesium salts and calcium salts).

[0036] The compounds of the present invention also encompass all isomers (e.g., geometric isomers, optical isomers based on asymmetric carbons, rotational isomers, stereoisomers, tautomers, etc.) that may arise from the structure of the compounds, as well as mixtures of two or more of these isomers, and are not limited to the descriptions of convenient structural formulas, etc. Furthermore, the compounds of the present invention may be in any of the S-, R-, or RS-forms, and are not limited thereto. Furthermore, the compounds of the present invention may exist in the form of hydrates or solvates depending on the type thereof, and in the present invention, such hydrates and solvates are also included in the compounds of the present invention and can be used for the same purposes as the compounds of the present invention. Examples of such solvates include, but are not limited to, solvates with ethanol, for example.

[0037] The compound of the present invention (compound of formula [II]) can be prepared, for example, by reacting a ketoacetal compound (wherein R 3a can be produced by condensing a TBS (t-butyldimethylsilyl) protected hydroxyl group or hydrogen) with a coelenteramine derivative (wherein R4 has the same meaning as above, the same applies hereinafter).

[0038] [ka]

[0039] In the above reaction scheme 1, the coelenteramine derivative used as a starting material is a compound in which R4 in the above formula [II] is -O-(CH2) n The compound used to produce the compound having -R6 can be produced, for example, by the following Reaction Scheme 2. In all of the following reaction schemes, "boronic acid" refers to the boronic acid derivative shown in each reaction scheme.

[0040] [ka]

[0041] Furthermore, in the above Reaction Scheme 1, the coelenteramine derivative used as a starting material for producing the compound of the formula [II] in which R4 is an alkyl group having 1 to 5 carbon atoms can be produced, for example, by the following Reaction Scheme 3.

[0042] [ka]

[0043] Specific conditions for each step in each of the above schemes can be appropriately determined by a person skilled in the art, and are also specifically described individually in the synthesis examples below.

[0044] The compounds of the present invention can be used as luminescent substrates for desired proteins or peptides, including, but not limited to, those derived from humans. Examples of the desired protein or peptide include, but are not limited to, luciferase-based enzymes (e.g., RLuc8 of the Renilla luciferase (RLuc) luminescence system (see Loening, AM et al., Protein Eng. Des. Sel., 2006, 19, 391-400; Loening, AM et al., J. Mol. Biol., 2007, 374, 1017-1028; Loening, AM et al., Protein Eng. Des. Sel., 2006, 19, 391-400), RLuc8.6 (see Loening, AM et al., Nat. Methods, 2007, 4, 641-643), and the CLuc system (see Mitani, Y. et al., Protein Expression and Purification, 2017, 133, 102-109), human serum albumin (HSA), and bovine serum albumin (BSA) are examples of such antibodies.

[0045] The compounds of the present invention can also be used in methods for analyzing proteins or peptides, including administering them in vivo or adding them in vitro and detecting the desired protein or peptide. Preferably, the protein or peptide is derived from a human, and the above-described explanations are applicable. The in vivo administration method and administration conditions, and the in vitro addition method and addition conditions, are not particularly limited and can be appropriately selected and set with reference to commonly known methods and conditions. The detection of the protein or peptide is also not particularly limited, as long as it can detect the luminescence derived from the compounds of the present invention as luminescent molecules. Well-known detection devices can be used and detection conditions can be set.

[0046] The present invention will be explained in more detail below by way of examples, but the present invention is not limited to these examples. [Example]

[0047] Synthesis method Reagents were purchased from Wako Pure Chemical Industries, Kanto Chemical, Tokyo Chemical, or Sigma-Aldrich and used as is without purification. For the synthesis of luminescent substrates, silica gel (Merck 1.07734.9025, silica gel 60 (0.063-0.200 mm), column chromatography grade (70-230 mesh ASTM)) was used for silica column chromatography. 1 H-NMR also 13 C-NMR was performed using a Bruker AvanceIII-500 with tetramethylsilane (TMS, 0 ppm) as the internal standard. Coupling constants (J) are shown in Hz. The abbreviations s, d, t, q, m, and br represent singlet, doublet, triplet, quartet, multiplet, and broad line, respectively.

[0048] [Example 1] Synthesis of 2-benzyl-6(4-(3-methoxypropoxy)phenyl)imidazo[1,2-a]pyrazine-3(7H)-one (HuLumino12)

[0049] [ka]

[0050] <Synthesis method> (1) Under a nitrogen atmosphere, 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (300.0 mg, 1.3 mmol, 1 eq.) and potassium carbonate (246 mg, 1.7 mmol, 1.3 eq.) were dissolved in acetone (20 ml) and stirred at room temperature. To this solution, 1-bromo-3-methoxypropane (416.2 mg, 2.7 mmol, 2 eq.) and potassium iodide (12 mg, 0.06 mmol, 0.05 eq.) dissolved in acetone (20 ml) were added and stirred at 70 °C overnight (19 hours). After cooling to room temperature, the mixture was concentrated under reduced pressure. The resulting residue was extracted with ethyl acetate, washed with distilled water and saturated brine, dried over sodium sulfate, and concentrated under reduced pressure again. The resulting residue was purified by column chromatography (eluent: hexane / ethyl acetate=9 / 1) to give 2-(4-(3-methoxypropoxy)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane as a white solid (287.3 mg, 73%).

[0051] (2) Under a nitrogen atmosphere, 5-bromopyrazin-2-amine (54.0 mg, 0.3 mmol, 1 eq.) and 2-(4-(3-methoxypropoxy)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (90.0 mg, 0.6 mmol, 1 eq.) obtained in (1) above were dissolved in ethanol (1 ml) and toluene (8 ml). 1 M aqueous sodium carbonate solution (3 ml) was added and stirred at room temperature. The reaction solution was degassed under vacuum, and a catalytic amount of tetrakistriphenylphosphinepalladium(0) (approximately one microspatula's worth) was added. The solution was degassed under vacuum again and stirred at 100 °C overnight (12 hours). After cooling to room temperature, the palladium catalyst was removed by filtration through Celite. The resulting residue was extracted with ethyl acetate, washed with distilled water and saturated brine, dried over sodium sulfate, and concentrated under reduced pressure. The resulting residue was purified by column chromatography (eluent: hexane / ethyl acetate=7 / 3→1 / 1) to give 5-(4-(3-methoxypropoxy)phenyl)pyrazin-2-amine as a yellow solid (80 mg, 100%).

[0052] 1 H-NMR (500 MHz, CDCl3): δ(ppm) = 7.73 (d, J = 8.5 Hz, 2H), 6.89 (d, J= 8.5 Hz, 2H), 4.05 (t, J = 5.1 Hz, 2H), 3.97 (t, J = 5.4 Hz, 2H), 1.33(m, 12H), 0.90 (s, 9H), 0.09 (s, 6H). 13 C-NMR (150 MHz, CDCl3): δ(ppm) = -5.05, 18.54, 24.99, 26.05, 62.07, 69.18, 83.66, 114.02, 136.61, 161.64.

[0053] (3) Under an argon atmosphere, 5-(4-(3-methoxypropoxy)phenyl)pyrazin-2-amine (30.0 mg, 0.08 mmol, 1 eq.) obtained in (2) above and 1,1-diethoxy-3-phenylpropan-2-one (38.0 mg, 0.17 mmol, 2 eq.) were dissolved in ethanol (2 ml) and milliQ (0.2 ml) and cooled to 0 °C. The reaction solution was degassed under vacuum, concentrated hydrochloric acid (0.1 ml) was added, and the mixture was stirred at 80 °C overnight (16 hours). After cooling to room temperature, the mixture was concentrated under reduced pressure. The residue was purified by silica column chromatography (methylene chloride / methanol = 20 / 1) to give 2-benzyl-6(4-(3-methoxypropoxy)phenyl)imidazo[1,2-a]pyrazin-3(7H)-one as a yellow solid (22.6 mg, 55%).

[0054] 1 H-NMR (500 MHz, CD3OD): δ(ppm) = 7.88 (s, 1H), 7.63 (s, 1H), 7.55 (d, J = 8.5 Hz, 2H), 7.28-7.11 (m, 5H), 7.00 (d, J = 8.6 Hz, 2H), 4.09 (s, 2H), 4.06 (t, J = 6.2 Hz, 2H), 3.53 (t, J = 6.1 Hz, 2H), 3.26 (q, J = 3.2 Hz, 3H). 13 C-NMR (125 MHz, CD3OD): δ(ppm) = 161.75, 139.52, 129.94, 129.53, 128.74, 127.48, 116.23, 108.44, 70.17, 66.10, 58.91, 34.43, 30.52.

[0055] [Example 2] Synthesis of 6(4-(3-methoxypropoxy)phenyl)-2-methylimidazo[1,2-a]pyrazine-3(7H)-one (HuLumino22)

[0056] [ka]

[0057] <Synthesis method> Under an argon atmosphere, 5-(4-(3-methoxypropoxy)phenyl)pyrazin-2-amine (30.0 mg, 0.11 mmol, 1 eq.) and diacetyl (19.9 mg, 0.23 mmol, 2 eq.) were dissolved in ethanol (2 ml) and milliQ (0.2 ml) and cooled to 0 °C. The reaction solution was degassed under vacuum, concentrated hydrochloric acid (0.1 ml) was added, and the mixture was stirred at 80 °C overnight (16 h). After cooling to room temperature, the mixture was concentrated under reduced pressure. The residue was purified by silica column chromatography (methylene chloride / methanol = 20 / 1) to give 6(4-(3-methoxypropoxy)phenyl)-2-methylimidazo[1,2-a]pyrazin-3(7H)-one as a yellow solid (22.6 mg, 10%).

[0058] 1 H-NMR (500 MHz, CD3OD): δ(ppm) = 7.88 (s, 1H), 7.60 (d, J = 8.7 Hz, 2H), 7.06 (d, J = 8.7 Hz, 2H), 4.12 (t, J = 6.2 Hz, 2H), 3.58 (t, J = 6.2 Hz, 2H), 3.35 (s, 3H), 2.05 (t, J = 12.4 Hz, 2H)..

[0059] [Example 3] Synthesis of (E)-2-benzyl-6-(4-(trifluoromethyl)styryl)imidazo[1,2-a]pyrazine-3(7H)-one (HuLumino32)

[0060] [ka]

[0061] <Synthesis method> (1) Under a nitrogen atmosphere, 5-bromopyrazin-2-amine (100.0 mg, 0.57 mmol, 1 eq.) and (E)-(4-(trifluoromethyl)styryl)boronic acid (196 mg, 0.91 mmol, 1.6 eq.) were dissolved in ethanol (1.6 ml) and toluene (10 ml). 1 M aqueous sodium carbonate solution (4 ml) was added and stirred at room temperature. The reaction solution was degassed under vacuum, and a catalytic amount of tetrakistriphenylphosphinepalladium(0) (approximately one microspatula's worth) was added. The solution was degassed under vacuum again and stirred overnight (12 hours) at 100°C. After cooling to room temperature, the palladium catalyst was removed by filtration through Celite. The resulting residue was extracted with ethyl acetate, washed with distilled water and saturated brine, dried over sodium sulfate, and concentrated under reduced pressure. The resulting residue was purified by column chromatography (eluent: hexane / ethyl acetate=1 / 1) to give (E)-5-(4-(trifluoromethyl)styryl)pyrazin-2-amine as a yellow solid (144.4 mg, 95%).

[0062] 1 H-NMR (500 MHz, CDCl3): δ(ppm) = 8.07 (d, J = 1.1 Hz, 2H), 8.02 (d, J= 1.2 Hz, 2H), 7.60 (q, J = 1.8 Hz, 4H), 7.46 (d, J = 16.0 Hz, 1H), 7.12 (d, J = 16.0 Hz, 1H),4.73 (s, 2H). 13 C-NMR (125 MHz, CDCl3): δ(ppm) = 153.43, 141.61, 140.78, 140.58, 132.42, 129.66, 129.40, 128.10, 126.88, 125.79, 125.76.

[0063] (2) Under an argon atmosphere, (E)-5-(4-(trifluoromethyl)styryl)pyrazin-2-amine (30.0 mg, 0.05 mmol, 1 eq.) obtained in (1) above and 1,1-diethoxy-3-phenylpropan-2-one (30.1 mg, 0.13 mmol, 2 eq.) were dissolved in ethanol (2 ml) and milliQ (0.2 ml) and cooled to 0° C. The reaction solution was degassed under vacuum, concentrated hydrochloric acid (0.1 ml) was added, and the mixture was stirred at 80° C. overnight (16 hours). After cooling to room temperature, the mixture was concentrated under reduced pressure, and the residue was purified by silica column chromatography (methylene chloride / methanol = 20 / 1) to give (E)-2-benzyl-6-(4-(trifluoromethyl)styryl)imidazo[1,2-a]pyrazine-3(7H)-one as a yellow solid (22.6 mg, 55%).

[0064] 1 H-NMR (500 MHz, CDCl3): δ(ppm) = 7.76 (s, 1H), 7.70-7.61 (m, 6H), 7.34-7.29 (m, 3H), 7.21-7.16 (m, 2H), 7.06 (d, J = 16.5 Hz, 1H),4.40 (s, 2H).

[0065] [Comparative Examples 1 to 4 and Comparative Examples 5 to 7] NCTZ (Native CTZ; Fujifilm Wako Pure Chemical Industries, Ltd.), DeepBlueC, shown by the following structural formula: TM (NanoLight), MCLA TM (Cayman Chemical Co.), and BBlue2.3 (see Non-Patent Document 4: R. Nishihara et al., Theranostics, 2019, 9, 2646-2661.) were designated as the compounds according to Comparative Examples 1 to 4, respectively.

[0066] [ka]

[0067] Furthermore, comparative compounds 1 to 3 (see Non-Patent Document 3: T. Hirano et al., Tetrahedron Letters, 1992, 33, 5771-5774) shown in the following structural formulas were designated as compounds according to comparative examples 5 to 7, respectively.

[0068] [ka]

[0069] [Example 4] Synthesis of (E)-2-benzyl-6-(4-methoxystyryl)imidazo[1,2-a]pyrazine-3(7H)-one (HuLumino30)

[0070] [ka]

[0071] <Synthesis method> (1) Under an argon atmosphere, 5-bromopyrazin-2-amine (500.0 mg, 2.87 mmol, 1 eq.) and (E)-(4-methoxystyryl)boronic acid (818 mg, 4.34 mmol, 1.6 eq.) were dissolved in ethanol (4.8 ml) and toluene (30 ml). 1M aqueous sodium carbonate solution (12 ml) was added and stirred at room temperature. The reaction solution was degassed under vacuum, and a catalytic amount of tetrakistriphenylphosphinepalladium(0) (approximately one microspatula's worth) was added. The mixture was degassed under vacuum again and stirred at 100 °C overnight. After cooling to room temperature, the palladium catalyst was removed by filtration through Celite. The resulting residue was extracted with ethyl acetate, washed with distilled water and saturated brine, dried over sodium sulfate, and concentrated under reduced pressure. The resulting residue was purified by column chromatography (eluent: hexane / ethyl acetate=1 / 1) to give (E)-5-(4-methoxystyryl)pyrazin-2-amine as a yellow solid (423.0 mg, 64%).

[0072] 1H-NMR (500 MHz, CDCl3): δ(ppm) = 8.04 (s, 1H),7.98 (s, 1H), 7.47 (d, J = 8.6 Hz, 2H), 7.39 (d, J = 16.0 Hz, 1H), 6.93 (d, 1H), 6.89 (d, J = 8.8 Hz, 2H), 4.61 (s, 2H), 3.82 (s, 3H). 13 C-NMR (125 MHz, CDCl3): δ(ppm) = 159.53, 152.71, 141.89, 140.61, 131.98, 129.77, 129.34, 128.00, 122.19, 114.17, 55.33.

[0073] (2) Under an argon atmosphere, (E)-4-(2-(5-aminopyrazin-2-yl)vinyl)phenol (30.0 mg, 0.14 mmol, 1 eq.) and 1,1-diethoxy-3-phenylpropan-2-one (37.3 mg, 0.16 mmol, 1.2 eq.) were dissolved in ethanol (2 ml) and milliQ (0.2 ml) and cooled to 0 °C. The reaction solution was degassed under vacuum, concentrated hydrochloric acid (0.1 ml) was added, and the mixture was stirred at 80 °C overnight. After cooling to room temperature, the mixture was concentrated under reduced pressure. The residue was purified by silica column chromatography (methylene chloride / methanol = 10 / 1) to give (E)-2-benzyl-6-(4-methoxystyryl)imidazo[1,2-a]pyrazin-3(7H)-one as a yellow solid (10.3 mg, 21%).

[0074] 1 H-NMR (500 MHz, CD3OD,CDCl3): δ(ppm) = 7.70 (s, 1H), 7.65 (s, 1H),7.50-7.17 (m, 7H), 7.07 (d, J = 16.5 Hz, 1H), 6.93 (d, J = 8.7 Hz, 2H), 6.76 (d, J = 16.4 Hz, 1H), 4.15 (s, 2H), 3.84 (s, 3H).

[0075] [Example 5] Synthesis of (E)-6-(2-([1,1'-biphenyl]-4-yl)vinyl)-2-benzylimidazo[1,2-a]pyrazine-3(7H)-one (HuLumino44)

[0076] [ka]

[0077] <Synthesis method> (1) Under an argon atmosphere, 5-bromopyrazin-2-amine (150.0 mg, 0.86 mmol, 1 eq.) and (E)-(2-([1,1'-biphenyl]-4-yl)vinyl)boronic acid (309 mg, 1.37 mmol, 1.6 eq.) were dissolved in ethanol (4 ml) and toluene (20 ml). 1 M aqueous sodium carbonate solution (8 ml) was added and stirred at room temperature. The reaction solution was degassed under vacuum, and a catalytic amount of tetrakistriphenylphosphinepalladium(0) (approximately one microspatula's worth) was added. The solution was degassed under vacuum again and stirred at 100°C overnight. After cooling to room temperature, the palladium catalyst was removed by filtration through Celite. The resulting residue was extracted with ethyl acetate, washed with distilled water and saturated brine, dried over sodium sulfate, and concentrated under reduced pressure. The resulting residue was purified by column chromatography (eluent: hexane / ethyl acetate=1 / 2) to give (E)-5-(2-([1,1′-biphenyl]-4-yl)vinyl)pyrazin-2-amine as a yellow solid (114.0 mg, 48%).

[0078] 1 H-NMR (500 MHz, CDCl3): δ(ppm) = 8.08 (s, 1H), 8.02 (s, 1H),7.62-7.33 (m, 11H),7.10 (d, J = 16.0 Hz, 1H), 4.63 (s, 2H). 13C-NMR (125 MHz, CDCl3): δ(ppm) = 152.92, 141.53, 141.03, 140.65, 140.60, 136.03, 132.12, 129.24, 128.81, 127.38, 127.17, 126.93, 124.29.

[0079] (2) Under an argon atmosphere, (E)-5-(2-([1,1'-biphenyl]-4-yl)vinyl)pyrazin-2-amine (31.7 mg, 0.11 mmol, 1 eq.) and 1,1-diethoxy-3-phenylpropan-2-one (30.6 mg, 0.13 mmol, 1.2 eq.) were dissolved in ethanol (2 ml) and milliQ (0.2 ml) and cooled to 0°C. The reaction solution was degassed under vacuum, concentrated hydrochloric acid (0.1 ml) was added, and the mixture was stirred at 80°C for 6 hours. After cooling to room temperature, the mixture was concentrated under reduced pressure, and the residue was purified by silica column chromatography (methylene chloride / methanol = 10 / 1) to give (E)-6-(2-([1,1'-biphenyl]-4-yl)vinyl)-2-benzylimidazo[1,2-a]pyrazine-3(7H)-one as a yellow solid (6.4 mg, 13%).

[0080] 1 H-NMR (500 MHz, CDCl3, CD3OD): δ(ppm) = 7.73-7.00 (m, 18H), 4.37 (s, 2H).

[0081] [Example 6] Synthesis of (E)-2-benzyl-6-styrylimidazo[1,2-a]pyrazine-3(7H)-one (HuLumino45)

[0082] [ka]

[0083] <Synthesis method> (1) Under an argon atmosphere, 5-bromopyrazin-2-amine (150.0 mg, 0.86 mmol, 1 eq.) and (E)-cystyrylboronic acid (204 mg, 1.37 mmol, 1.6 eq.) were dissolved in ethanol (4 ml) and toluene (20 ml). 1M aqueous sodium carbonate solution (8 ml) was added and stirred at room temperature. The reaction solution was degassed under vacuum, and a catalytic amount of tetrakistriphenylphosphinepalladium(0) (approximately one microspatula's worth) was added. The mixture was degassed under vacuum again and stirred at 100 °C overnight. After cooling to room temperature, the palladium catalyst was removed by filtration through Celite. The resulting residue was extracted with ethyl acetate, washed with distilled water and saturated brine, dried over sodium sulfate, and concentrated under reduced pressure. The resulting residue was purified by column chromatography (eluent: hexane / ethyl acetate=1 / 2) to give (E)-5-styrylpyrazin-2-amine as a yellow solid (153.6 mg, 90%).

[0084] 1 H-NMR (500 MHz, CDCl3): δ(ppm) = 8.06 (s, 1H), 8.00 (s, 1H), 7.54-7.25 (m, 6H), 7.05 (d, J= 16.0 Hz, 1H), 4.67 (s, 2H). 13 C-NMR (125 MHz, CDCl3): δ(ppm) = 153.09, 141.57, 141.08, 137.07, 132.21, 129.82, 128.82, 128.00, 126.84, 124.39.

[0085] (2) Under an argon atmosphere, (E)-5-styrylpyrazin-2-amine (30.0 mg, 0.15 mmol, 1 eq.) and 1,1-diethoxy-3-phenylpropan-2-one (40.6 mg, 0.18 mmol, 1.2 eq.) were dissolved in ethanol (2 ml) and milliQ (0.2 ml) and cooled to 0 °C. The reaction solution was degassed under vacuum, concentrated hydrochloric acid (0.1 ml) was added, and the mixture was stirred at 80 °C overnight. After cooling to room temperature, the mixture was concentrated under reduced pressure. The residue was purified by silica column chromatography (methylene chloride / methanol = 10 / 1) to give (E)-2-benzyl-6-styrylimidazo[1,2-a]pyrazin-3(7H)-one as a yellow solid (6.0 mg, 12%).

[0086] 1 H-NMR (500 MHz, CDCl3, CD3OD): δ(ppm) = 7.76-6.92 (m, 14H), 4.44 (s, 2H).

[0087] Luminescence activity measurement method and luminescence intensity comparison results Human serum albumin (human serum-derived lyophilized powder, fatty acid-free, globulin-free, Sigma-Aldrich), other human protein species, or bovine serum albumin (bovine serum-derived lyophilized powder, Sigma-Aldrich) were dissolved in PB Buffer (10 mM, pH 7.4) and used directly for luminescence measurement. The luminescent substrates used were NCTZ and DeepBlueC. TM , MCLA TM Comparative compounds 1 to 3, HuLumino12, and HuLumino22 were used. The luminescent substrate was dissolved in methanol to make a 2 mM solution. This was diluted to 10 μM with 10 mM PB buffer (pH 7.4) to make the substrate solution. 10 μL of 10 mM PB buffer (pH 7.4) containing human serum albumin (HSA) or bovine serum albumin (BSA) was added to 90 μL of this solution to start the luminescence reaction. The luminescence intensity was measured using a Promega GloMax (R)The measurements were taken for 60 seconds at 20 / 20 and the total luminescence (total luminescence / min) was shown and compared (Figure 1). The luminescence intensity of natural coelenterazine in HSA was normalized to 1.0, and the comparison results are shown in Table A below.

[0088] [Table 7]

[0089] As shown in Figure 1 and Table A, the novel compounds of the present invention are recognized by human serum albumin (HSA) and exhibit significant luminescence. In particular, the HuLumino12 / HSA combination was found to have a luminescence intensity 912 times higher than that of NCTZ / HSA. Furthermore, the Michaelis-Menten constants for HuLumino12 / HSA and NCTZ / HSA were 4.2 μM and 25.3 μM, respectively, demonstrating that the luminescent molecules of the present invention have a higher affinity for HSA than conventional molecules. Furthermore, HuLumino32, which has a relatively high fluorescence quantum yield, exhibited the highest luminescence intensity with both BSA and HSA.

[0090] From FIG. 2, it was revealed that the novel compound of the present invention, HuLumino12, is recognized by human serum albumin (HSA) among various human protein species and exhibits a specific luminescent reaction. As shown in Figure 3, the S / N ratio of the luminescence reaction between the novel compound HuLumino12 of the present invention and HSA was 37, and the luminescence duration reached 3 hours, as shown in Figure 4. Furthermore, as shown in Figure 3, the S / N ratio of the luminescence reaction between the novel compound HuLumino44 of the present invention and HSA was 150, which was about four times that of HuLumino12, demonstrating excellent optical properties. From FIG. 5, it was found that the detection limit of HSA using the novel compound HuLumino12 of the present invention was 7.4 μg / mL.

[0091] Emission spectrum measurement method Emission spectrum measurements were performed in the same manner as the emission intensity measurement method described above, and the maximum emission wavelength was found to be 427 nm (Figure 6). The emission spectrum was measured using a spectrometer. The maximum emission wavelength was determined by normalizing the maximum emission intensity to 1.0.

[0092] The CTZ derivatives designed in this study can also be applied to other bioluminescence systems. The giant salamander shrimp (Oplophorus gracilirostris) uses CTZ as a luminescent substrate. Recently, a luciferase derived from Oplophorus gracilirostris, NanoLuc (trade name) (Promega) (reference: Hall PM et al., ACS. Chem. Biol., 2012, 7, 1848-1857), has been developed. When combined with the substrate furimazine, it exhibits luminescence intensity approximately 100 times greater than that of the firefly luciferase luminescence system. Its application to various bioassay systems, including reporter assays and protein-protein interaction analysis using the bioluminescence resonance energy transfer (BRET) mechanism, has been demonstrated (reference: England GC et al., Bioconjugate Chem., 2016, 27, 1175-1187).

[0093] In substrate synthesis studies for NanoLuc (trade name) (Promega), derivatives at the 6th and 2nd positions of CTZ have been reported (references: Hall PM et al., ACS. Chem. Biol., 2012, 7, 1848-1857; Shakhmin A. et al., Chem. Eur. J., 2016, 22, 10369-10375). However, in all studies, the effect of the 2nd position substituent on enzyme activity was primarily investigated. The present invention demonstrates that modifying the 6th and 8th positions of CTZ significantly affects enzyme activity, and further enhancement of the luminescence intensity of NanoLuc (trade name) (Promega) may be achieved by developing novel substrates with modifications at the 6th and 8th positions of CTZ. [Industrial Applicability]

[0094] The compounds of the present invention can be used not only to detect human-derived proteins but also as reagents for luminescent detection of structural degradation and aggregation of antibody proteins. This can be used for quality control of antibody drugs and immunochromatographic in vitro diagnostic reagents. Furthermore, the binding affinity between serum proteins and drugs can be rapidly determined by conducting competitive inhibition tests with the compounds of the present invention, which are luminescent molecules. Therefore, the compounds of the present invention can contribute to accelerating drug discovery research as one of the methods for pharmacokinetic evaluation.

Claims

1. The following formula [II]: 【Chemistry 1】 [In formula [II], R 3 is hydrogen, a hydroxyl group, a fluorine atom, an alkyl group having 1 to 5 carbon atoms, a methoxy group, or a trifluoromethyl group, R 4 teeth, (i) -O-(CH 2 ) n -R 6 (where R 6 is a hydroxyl group, a methoxy group, a methyl group, a trifluoromethyl group, or an azido group, and n is an integer of 1 to 5. (ii) an alkyl group having 1 to 5 carbon atoms, or (iii) a compound of the formula: 【Chemistry 2】 A group represented by any one of ] or a salt thereof, or a hydrate or solvate thereof.

2. R in the formula [II] 3 and R 4 The compound according to claim 1, or a salt thereof, or a hydrate or solvate thereof, wherein each of the groups or atom combinations shown in the table below is selected from the group consisting of methyl, ... Table 1

3. R 3 is -H and R 4 -O-(CH 2 ) 3 -OCH 3 3. The compound according to claim 2, or a salt thereof, or a hydrate or solvate thereof:

4. R 3 is -OH and R 4 -O-(CH 2 ) 3 -OCH 3 3. The compound according to claim 2, or a salt thereof, or a hydrate or solvate thereof:

5. The following formula [IV]: 【Transformation 3】 [In formula [IV], R 3 is hydrogen, a hydroxyl group, a fluorine atom, an alkyl group having 1 to 5 carbon atoms, a methoxy group, or a trifluoromethyl group, R 5 teeth, (i) a hydrogen atom, a hydroxyl group, a methoxy group, a methyl group, a trifluoromethyl group, a dimethylamino group, a phenyl group, or an azide group; (ii) an alkyl group having 1 to 5 carbon atoms; (iii) -O-(CH 2 ) p -R 7 (where R 7 is a hydroxyl group, a methoxy group, a methyl group, a trifluoromethyl group, a dimethylamino group, an azide group, or an alkyl group having 1 to 5 carbon atoms, and p is an integer of 1 to 5; or (iv) a compound of the formula: 【Chemistry 4】 any one of the groups represented by and n is an integer from 0 to 5. A compound represented by (However, the following compounds: 【Chemistry 5-1】 【Chemistry 5-2】 【Chemistry 5-3】 【Chemistry 5-4】 ), or a salt thereof, or a hydrate or solvate thereof.

6. n and R in the formula [IV] 3 and R 5 The compound according to claim 5, or a salt thereof, or a hydrate or solvate thereof, wherein the groups or atoms shown in the following formula (I) are in the combinations shown in the table below. Table 2

7. n is 1 and R 3 is -H and R 5 Ga-OCH 3 7. The compound according to claim 6, or a salt thereof, or a hydrate or solvate thereof:

8. n is 1 and R 3 is -H and R 5 Ga-CF 3 7. The compound according to claim 6, or a salt thereof, or a hydrate or solvate thereof:

9. n is 1 and R 3 is -H and R 5 Ga-C 6 H 5 7. The compound according to claim 6, or a salt thereof, or a hydrate or solvate thereof:

10. n is 1 and R 3 is -H and R 5 The compound according to claim 6, or a salt thereof, or a hydrate or solvate thereof, wherein is -H.

11. A luminescent substrate for a protein or peptide, comprising the compound according to any one of claims 1 to 10, or a salt thereof, or a hydrate or solvate thereof.

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