Compounds or their salts, compositions, chemiluminescence methods, chemiluminescence signal measurement methods, reagents, reagent kits, and methods for measuring test substances.

Abstract

To provide a 1,2-dioxetane derivative that exhibits high luminous efficiency.SOLUTION: For example, a compound represented by the structural formula [I-P] in the figure or a salt thereof is presented.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to compounds or their salts, compositions, chemiluminescence methods, chemiluminescence signal measurement methods, reagents, reagent kits, and methods for measuring test substances. [Background technology]

[0002] 1,2-Dioxetane derivatives are compounds that can induce chemiluminescence triggered by reactions with chemical substances such as enzymes. For example, Patent Document 1 describes formula IVb: [ka] It is represented as follows, where A is a π* acceptor group such as -CN, -CH=CH-E, and the π* acceptor group is -YLR 4 Compounds are disclosed in which the group is bonded to the ortho or para position. [Prior art documents] [Patent Documents]

[0003] [Patent Document 1] U.S. Patent Application Publication No. 2019 / 290787 [Overview of the Initiative] [Problems that the invention aims to solve]

[0004] The present invention aims to provide compounds or salts thereof that exhibit high luminescence efficiency, compositions containing the same, reagents, and reagent kits. Furthermore, the present invention aims to provide a chemiluminescence method using the compound or a salt thereof, a chemiluminescence signal measurement method, and a test substance measurement method. [Means for solving the problem]

[0005] As a result of intensive studies to solve the above problems, it has been found that a compound having a benzene ring bonded to the 3-position of 1,2-dioxetane and having a specific substituent, that is, -C≡C-E (where E is -COOH, -H, -CN, -COO-alkyl, or an aryl group, a pyridinyl group, a pyridinium group, a 3H-indolyl group, or a 3H-indol-1-ium group which may be substituted with a substituent), or an aryl group substituted with an electron-withdrawing group exhibits high luminescence efficiency.

[0006] This disclosure includes the following embodiments: [Item 1] A compound represented by formula [I] or a salt thereof. [Chemical formula] [In the formula, R 1 is a C 1-8 alkyl group; R 2 and R 3 are each independently a C 3-18 alkyl group or a C 3-7 cycloalkyl group; R 1 and R 2 , R 1 and R 3 , or R 2 and R 3 may together with the carbon atom to which they are attached form a ring which may be substituted with a substituent; X is H or a caging group; L is absent or is a linker represented by formula L1, L2, L3, or L4: [Chemical formula] (In each formula, M is absent or is -O- or -NH-, the wavy line represents the bonding point with X, the asterisk represents the bonding point with Y) is a linker represented by; The benzene rings in formulas L1, L2, and L3 may be substituted with substituents selected from the group consisting of halo groups, alkyl groups, cycloalkyl groups, and alkoxy groups; This is a linker represented by; Y is either absent, -O-, or -NH-; R 4 is either H or an electron-withdrawing group bonded to the -YLX group at the ortho or para position; and A is -C≡CE (wherein E is -COOH, -H, -CN, -COO-alkyl, or an aryl group, pyridinyl group, pyridinium group, 3H-indolyl group, or 3H-indole-1-ium group which may be substituted with a substituent), or an aryl group which is substituted with an electron-withdrawing group. [Section 2] The compound or salt thereof according to item 1, wherein A is bonded to the -YLX group in the ortho or para position. [Section 3] The compound or salt thereof according to item 1 or 2, wherein A is -C≡CE bonded to the ortho position relative to the -YLX group. [Section 4] E is -COOH, -H, -CN, -COO-C 1-8 Alkyl, phenyl, -C6H4O-C 1-8 Alkyl, -C6H4COOH, -C6H4COO-C 1-8 A compound or salt thereof as described in any of items 1 to 3, which is alkyl, 4-pyridinyl, methylpyridinium-4-yl, 3,3-dimethyl-3H-indolyl, or 1,3,3-trimethyl-3H-indole-1-ium-2-yl. [Section 5] A is an aryl group bonded to the ortho position relative to the -YLX group. The aryl group in question is -COOR 8 , halo group, -OH, -NO2, -CN, -CO-R 9 , and -SO2-R 10 It is substituted with an electron-withdrawing group selected from the group consisting of the following: R 8 , R 9, and R 10 Each of them is independently H or C 1-18 It is an alkyl group. A compound or salt thereof as described in item 1 or 2. [Section 6] A is a phenyl group bonded to the ortho position relative to the -YLX group. The phenyl group in question is -COOR 8 , halo group, -OH, -NO2, -CN, -CO-R 9 , and -SO2-R 10 It is substituted with an electron-withdrawing group selected from the group consisting of the following: R 8 , R 9 , and R 10 Each of them is independently H or C 1-8 It is an alkyl group. A compound or salt thereof as described in item 1 or 2. [Section 7] R 4 The compound or salt thereof according to any one of items 1 to 6, wherein the compound is H, or a halo group or -CN bonded to the ortho or para position relative to the -YLX group. [Section 8] R 4 The compound or salt thereof according to any one of items 1 to 7, wherein the compound is H, or a halo group or -CN bonded to the ortho position relative to the -YLX group. [Section 9] X is H, a trialkylsilyl group, a (2,4-dinitrophenyl)sulfonyl group, a 3,4,6-trimethyl-2,5-dioxobenzyl group, a 2-(3-carboxy-4-nitrophenyl)-SS-ethyloxycarbonyl group, a 4-azidobenzyloxycarbonyl group, or formulas X1, X2, X3, X4, X5, X6, or X7: [ka] (In the formula, R 7 This is a group consisting of one amino acid residue or multiple amino acid residues linked together, with the C-terminus of these groups linked to NH. (The wavy line indicates the connection point with L.) A compound or salt thereof, which is a group represented by any of items 1 to 8. [Section 10] A compound or salt thereof described in any of items 1 to 9 that satisfies any of the following (1) to (4): (1) Y is -O- or -NH-, L is absent, and X is H; (2) Y is -O- or -NH-, L is absent, and X is given by equation X3: [ka] It is a base represented by; (3) Y is -O- or -NH-, L is a linker represented by formula L1, L2, L3 or L4, M is -O- or -NH-, and X is a casing group; or (4) Y is absent, L is absent, and X is expression X1 or X2: [ka] It is a base represented by . [Section 11] R 2 and R 3 A compound or salt thereof according to any one of items 1 to 10, wherein together with carbon atoms bonded to these, it forms a fused ring, spiro ring, or bridging ring which may be substituted with substituents. [Section 12] R 2 and R 3 A compound or salt thereof according to any one of items 1 to 11, wherein, together with the carbon atoms bonded to these, it forms an adamantane ring which may be substituted with a halo group. [Section 13] A composition comprising a compound or a salt thereof as described in any of items 1 to 12, and an aqueous solvent. [Section 14] The composition according to item 13, wherein X is a casing group. [Section 15] The composition according to item 13 or 14, further comprising a surfactant. [Section 16] A chemiluminescence method comprising the step of reacting a compound or a salt thereof described in any of items 1 to 12, wherein X is a casing group, with a substance that liberates the casing group. [Section 17] X is given by equation X3: [ka] The chemiluminescence method according to item 16, wherein the casing group is represented by , and the substance that liberates the casing group is alkaline phosphatase. [Section 18] A method for measuring a chemiluminescence signal, comprising the step of measuring a chemiluminescence signal generated by a chemiluminescence method described in item 16 or 17. [Section 19] A reagent for measuring a test substance in a sample, comprising a compound or salt thereof as described in any of items 1 to 12, wherein X is a casing group, or a composition as described in any of items 13 to 15. [Section 20] A first reagent comprising a compound or salt thereof according to any one of claims 1 to 12, wherein X is a casing group, or a composition according to any one of claims 13 to 15, A second reagent containing a substance that liberates the aforementioned casing group, A reagent kit for measuring test substances in a sample, including [specific reagents / reagents]. [Section 21] A method for measuring a test substance in a sample, A step of forming an immune complex on a solid phase, comprising the substance to be tested, a capture body that binds to the substance to be tested, and a detection body that contains a substance that binds to the substance to be tested and releases a saging group, A step of reacting a substance that releases the saging group in the immune complex with a compound or salt thereof according to any one of items 1 to 12, in which X is a saging group, to generate a chemiluminescent signal; The process of measuring the substance to be tested by measuring the chemiluminescence signal, Methods that include... [Effects of the Invention]

[0007] The present invention provides compounds exhibiting high luminescence efficiency or salts thereof, compositions containing the same, reagents, and reagent kits. Furthermore, the present invention provides a chemiluminescence method using the compound or a salt thereof, a chemiluminescence signal measurement method, and a test substance measurement method. [Brief explanation of the drawing]

[0008] [Figure 1A] Figure 1A is a graph comparing the luminous efficiency of Examples 1 to 3 with that of Comparative Examples 1 and 2. [Figure 1B] Figure 1B is a graph comparing the luminous efficiency of Example 4 with that of Comparative Examples 1 and 2. [Figure 2A] Figure 2A is a graph showing the relationship between the amount of alkaline phosphatase used and the luminescence signal in Example 1. [Figure 2B] Figure 2B is a graph showing the relationship between the amount of alkaline phosphatase used and the luminescence signal in Example 2. [Figure 2C] Figure 2C is a graph showing the relationship between the amount of alkaline phosphatase used and the luminescence signal in Example 3 (with Sapphire-II® Enhancer added). [Figure 2D] Figure 2D is a graph showing the relationship between the amount of alkaline phosphatase used and the luminescence signal in Example 3 (without the addition of Sapphire-II® Enhancer). [Figure 2E] Figure 2E is a graph showing the relationship between the amount of alkaline phosphatase used and the luminescence signal in Example 4. [Figure 3] Figure 3 is a graph showing the results of immunological measurement of hepatitis B virus surface antigen (HBsAg) using Example 3 (without the addition of Sapphire-II® Enhancer). [Figure 4] Figure 4 shows an example of the form in which the reagents of this disclosure are provided. [Figure 5] Figure 5 shows an example of how the reagent kit of this disclosure is provided. [Modes for carrying out the invention]

[0009] 1.Definition In this specification, a halo group refers to a monovalent group composed of a halogen. Examples of halo groups include a fluoro group (-F), a chloro group (-Cl), a bromo group (-Br), and an iodo group (-I).

[0010] In this specification, an alkyl group refers to a monovalent group obtained by removing one hydrogen atom from a linear or branched saturated hydrocarbon. The number of carbon atoms in an alkyl group is not particularly limited, but is, for example, 1 to 20. Examples of alkyl groups include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group, tert-butyl group, n-pentyl group, isopentyl group, neopentyl group, tert-pentyl group, n-hexyl group, isohexyl group, n-heptyl group, isoheptyl group, n-octyl group, isooctyl group, 2-ethylhexyl group, n-nonyl group, isononyl group, and n-decyl group. Examples include isodecyl group, n-undecyl group, n-dodecyl group (lauryl group), isolauryl group, n-tridecyl group, isotridecyl group, n-tetradecyl group (myristyl group), isomiristyl group, n-pentadecyl group, isopentadecyl group, n-hexadecyl group (palmityl group), isopalmityl group, n-heptadecyl group, isoheptadecyl group, n-octadecyl group (stearyl group), and isostearyl group.

[0011] In this specification, C 1-8 An alkyl group is a linear or branched alkyl group with 1 to 8 carbon atoms. 1-8 Examples of alkyl groups include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group, tert-butyl group, n-pentyl group, isopentyl group, neopentyl group, tert-pentyl group, n-hexyl group, isohexyl group, n-heptyl group, isoheptyl group, n-octyl group, isooctyl group, and 2-ethylhexyl group.

[0012] In this specification, C3-18 An alkyl group refers to a linear or branched alkyl group with 3 to 18 carbon atoms. 3-18 Examples of alkyl groups include n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group, tert-butyl group, n-pentyl group, isopentyl group, neopentyl group, tert-pentyl group, n-hexyl group, isohexyl group, n-heptyl group, isoheptyl group, n-octyl group, isooctyl group, 2-ethylhexyl group, n-nonyl group, isononyl group, n-decyl group, isodecyl Examples include the syl group, n-undecyl group, n-dodecyl group (lauryl group), isolauryl group, n-tridecyl group, isotridecyl group, n-tetradecyl group (myristyl group), isomiristyl group, n-pentadecyl group, isopentadecyl group, n-hexadecyl group (palmityl group), isopalmityl group, n-heptadecyl group, isoheptadecyl group, n-octadecyl group (stearyl group), and isostearyl group.

[0013] In this specification, a cycloalkyl group refers to a monovalent group derived from a saturated aliphatic hydrocarbon ring. The number of carbon atoms in a cycloalkyl group is not particularly limited, but is for example 3 to 20. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, norbornyl, and adamantyl groups.

[0014] In this specification, C 3-7 A cycloalkyl group is a cycloalkyl group with 3 to 7 carbon atoms. Specifically, C 3-7 Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl groups.

[0015] In this specification, an aryl group refers to a monovalent group derived from an aromatic hydrocarbon ring. The number of carbon atoms in an aryl group is not particularly limited, but is for example 6 to 20. Examples of aryl groups include phenyl, indenyl, naphthyl, fluorenyl, phenantrenyl, and anthracenyl groups.

[0016] In this specification, an alkoxy group refers to a group represented by -O-alkyl. The alkyl in this group is synonymous with the "alkyl group" described above. Examples of alkoxy groups include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, n-pentoxy, isopentoxy, neopentoxy, tert-pentoxy, n-hexoxy, and isohexoxy groups.

[0017] In this specification, a caging group (also called a protecting group) refers to a group used to prevent the generation of unstable chemical species, such as 1,2-dioxetanyl phenolate. Caging groups can be uncaged (also called deprotected) by chemical processes such as hydrolysis, hydrolysis, and enzymatic reactions, or by physical processes such as electrolysis and photolysis. Chemical species generated by uncaging can, for example, enter an excited state and emit light when returning from the excited state to the ground state.

[0018] Examples of casing groups include trialkylsilyl groups, dialkylmonoarylsilyl groups, monoalkyldiarylsilyl groups, and triarylsilyl groups. Examples of trialkylsilyl groups include trimethylsilyl groups, triethylsilyl groups, triisopropylsilyl groups, and tert-butyldimethylsilyl groups. 1-4 Examples include alkylsilyl groups. Dialkylmonoarylsilyl groups include, for example, dimethylphenylsilyl groups and other diC groups. 1-4 Alkyl mono-C 6-10 Examples include arylsilyl groups. Monoalkyldiarylsilyl groups include monoC such as tert-butyldiphenylsilyl group.1-4 AlkyldiC 6-10 Examples include arylsilyl groups. Examples of triarylsilyl groups include triphenylsilyl groups and other tri-C groups. 6-10 Examples include arylsilyl groups.

[0019] Other examples of casing groups include the (2,4-dinitrophenyl)sulfonyl group, the 3,4,6-trimethyl-2,5-dioxobenzyl group, the 2-(3-carboxy-4-nitrophenyl)-SS-ethyloxycarbonyl group, and the 4-azidobenzyloxycarbonyl group.

[0020] Another example of a caging group is formula X1, X2, X3, X4, X5, X6, or X7: [ka] (In the formula, R 7 This is a group consisting of one amino acid residue or multiple amino acid residues linked together, with the C-terminus of these groups linked to NH. (The wavy line indicates the connection point with L.) Examples include the group represented by [the symbol].

[0021] In this specification, a substance that liberates (or removes) a saging group may be referred to as an uncaging substance. Examples of uncaging substances include glutathione when X is a (2,4-dinitrophenyl)sulfonyl group, hydrogen peroxide when X is a group represented by formula X2, alkaline phosphatase when X is a group represented by formula X3, and R 7However, if an amino acid sequence is cleavable by an enzyme (e.g., an amino acid sequence cleavable by cathepsin B, such as an amino acid sequence containing Phe-Lys, citrulline-Val, or Gly-Phe-Leu-Gly; an amino acid sequence cleavable by legmine, such as an amino acid sequence containing N-carbobenzoyl-Ala-Ala-Asn-ethylenediamine; an amino acid sequence cleavable by γ-glutamyl transpeptidase, such as an amino acid sequence containing γ-Glu-), then it is the enzyme; and if X is a group represented by formula X5 (a β-galactosyl group), then it is a β-galactosidase.

[0022] In this specification, an amino acid residue is typically defined as follows: [ka] (In the formula, R 71 and R 72 (Each of these is independently a side chain of an amino acid.) This refers to a group represented by . Examples of amino acid residues include glycine residues, alanine residues, valine residues, leucine residues, isoleucine residues, serine residues, threonine residues, cysteine ​​residues, methionine residues, aspartic acid residues, glutamic acid residues, lysine residues, arginine residues, asparagine residues, glutamine residues, phenylalanine residues, tyrosine residues, histidine residues, tryptophan residues, and proline residues.

[0023] In this specification, an electron-withdrawing group refers to a functional group whose Hammett substituent constant σp value is positive. For the definition of σp value and the σp values ​​of each functional group, refer to, for example, Hansch, C et al., Chem. Rev., 91, pp. 165-195 (1991). Examples of "electron-withdrawing groups" include halo groups, -OH, -NO2, -CN, -COOH, and -COO-C. 1-18 Alkyl, -COH, -CO-C 1-18 Alkyl, -SO2H, -SO2-C 1-18 Examples include alkyl groups.

[0024] In this specification, a specimen refers to a sample taken from a living organism that may contain a test substance. A specimen may be either a liquid or solid sample. Examples of liquid samples include blood samples, cerebrospinal fluid, sputum, bronchoalveolar lavage fluid, nasopharyngeal swabs, lymph, urine, feces, and saliva. Blood samples may be plasma, serum, or whole blood. A liquid sample may also be a solubilized solid sample. Examples of solid samples include excised tissue. Detection of the test substance includes in vitro detection of the test substance in a specimen and in vivo detection of the test substance in a specimen.

[0025] In this specification, "test substance" refers to a substance in a sample that is detected by a chemiluminescent probe, composition, reagent, reagent kit, or test substance measurement method described later. Examples of test substances include proteins, peptides, and nucleic acids. These test substances may be present in a free state in the sample, or they may be present on or inside cells, lipoproteins, vesicles, viruses, etc.

[0026] 2. Compounds represented by formula [I] or their salts We found that compounds in formula [I] where A is -C≡CE (wherein E is -COOH, -H, -CN, -COO-alkyl, or an aryl group, pyridinyl group, pyridinium group, 3H-indolyl group, or 3H-indole-1-ium group which may be substituted with a substituent), or an aryl group substituted with an electron-withdrawing group, exhibit high luminescence efficiency.

[0027] A is preferably bonded to the -YLX group at the ortho or para position, and more preferably bonded to the -YLX group at the ortho position.

[0028] In one embodiment, A is preferably -C≡CE bonded to the -YLX group at the ortho or para position, and more preferably -C≡CE bonded to the -YLX group at the ortho position.

[0029] If E is a substituent-substituted aryl group, pyridinyl group, pyridinium group, 3H-indolyl group, or 3H-indole-1-ium group, the number of substituents may be one or more (for example, two, three, or four).

[0030] E is -COOH, -H, -CN, -COO-C 1-8 Alkyl, phenyl, -C6H4O-C 1-8 Alkyl, -C6H4COOH, -C6H4COO-C 1-8 Preferably, the elements are alkyl, 4-pyridinyl, methylpyridinium-4-yl, 3,3-dimethyl-3H-indolyl, or 1,3,3-trimethyl-3H-indole-1-ium-2-yl, and include -COOH, -H, -CN, and -COO-C. 1-4 Alkyl, -C6H4COOH, or -C6H4COO-C 1-4 It is more preferably alkyl, -COOH, -H, -COO-C 1-4 Alkyl, -C6H4COOH, or -C6H4COO-C 1-4 It is more preferably alkyl, and even more preferably -COOH, -H, -COOCH3, -C6H4COOH, or -C6H4COOCH3.

[0031] In one embodiment, A is preferably an aryl group substituted with an electron-withdrawing group bonded to the -YLX group at the ortho or para position, and more preferably an aryl group substituted with an electron-withdrawing group bonded to the -YLX group at the ortho position. The aryl group is a C such as a phenyl group. 6-10 It is preferable that it be an aryl group. The electron-withdrawing group is -COOR 8 , halo group, -OH, -NO2, -CN, -CO-R 9 , and -SO2-R 10 Preferably selected from the group consisting of -COOR 8 It is more preferable that it be -COOH. The number of electron-withdrawing groups may be one or more (for example, two, three, four, or five).

[0032] R 8 , R 9 , and R 10 Each of them is independently H or C 1-18 Preferably an alkyl group, H or C 1-8 It is more preferable that it be an alkyl group.

[0033] R 1 Preferably C 1-6 It is an alkyl group, more preferably a methyl group, an ethyl group, etc. 1-4 It is an alkyl group.

[0034] R 2 and R 3 Each of them is independently branched C 3-18 Alkyl or C 3-7 It is preferably a cycloalkyl group. Branched C 3-18 Examples of alkyl groups include isopropyl group, isobutyl group, sec-butyl group, tert-butyl group, isopentyl group, neopentyl group, tert-pentyl group, isohexyl group, isoheptyl group, isooctyl group, 2-ethylhexyl group, isononyl group, isodecyl group, isolauryl group, isotridecyl group, isomiristyl group, isopentadecyl group, isopalmityl group, isoheptadecyl group, and isostearyl group.

[0035] R 2 and R 3 These, for example, together with the carbon atoms to which they are bonded, form a ring which may be substituted with substituents, preferably a fused ring which may be substituted with substituents, a spiro ring, or a crosslinking ring which may be substituted with substituents, more preferably a crosslinking ring which may be substituted with substituents. The crosslinking ring is preferably a crosslinking ring having 5 to 10 carbon atoms, and examples of such crosslinking rings include norbornane rings, cubane rings, and adamantane rings. Of these, adamantane rings are particularly preferred. The substituents are preferably halo groups, more preferably chloro groups. When the ring is substituted with substituents, the number of substituents may be one or more (for example, two, three, four, or five).

[0036] R 4 Preferably, it is H, or a halo group, -OH, -NO2, -CN, -COOH, -COO-C bonded to the -YLX group at the ortho or para position. 1-18 Alkyl, -COH, -CO-C 1-18 Alkyl, -SO2H, or -SO2-C 1-18 The group is alkyl, more preferably H, or a halo group or -CN bonded to the ortho or para position relative to the -YLX group, even more preferably H, or a halo group or -CN bonded to the ortho position relative to the -YLX group.

[0037] X is H, a trialkylsilyl group, a (2,4-dinitrophenyl)sulfonyl group, a 3,4,6-trimethyl-2,5-dioxobenzyl group, a 2-(3-carboxy-4-nitrophenyl)-SS-ethyloxycarbonyl group, a 4-azidobenzyloxycarbonyl group, or formulas X1, X2, X3, X4, X5, X6, or X7: [ka] (In the formula, R 7 This is a group consisting of one amino acid residue or multiple amino acid residues linked together, with the C-terminus of these groups linked to NH. (The wavy line indicates the connection point with L.) It is preferable that the group is represented by .

[0038] Y, L, and X are preferably any combination of (1) to (4) below. (1) Y is -O- or -NH-, L is absent, and X is H; (2) Y is -O- or -NH-, L is absent, and X is a casing group, preferably a group represented by formula X3; (3) Y is -O- or -NH-, L is a linker represented by formula L1, L2, L3 or L4, M is -O- or -NH-, and X is a casing group; or (4) Y is absent, L is absent, and X is a caging group, preferably a group represented by formula X1 or X2.

[0039] The compound represented by formula [I] or a salt thereof may be in an ionic state in solution. For example, when the compound or a salt thereof has a carboxy group or a hydroxy group, in solution, it may be -COO - or -O - and may become.

[0040] In one embodiment, A is -C≡C-E bonded to the ortho position with respect to the -Y-L-X group, E is -COOH, -H, -CN, -COO-C 1-8 alkyl, phenyl, -C6H4O-C 1-8 alkyl, -C6H4COOH, -C6H4COO-C 1-8 alkyl, 4-pyridinyl, methylpyridinium-4-yl, 3,3-dimethyl-3H-indolyl, or 1,3,3-trimethyl-3H-indol-1-ium-2-yl, R 2 and R 3 are each independently a branched C3- 18 alkyl group or a C3-7 cycloalkyl group, or together with the carbon atom to which they are attached, form a condensed ring, a spiro ring, or a bridged ring which may be substituted with a substituent; R 4 is preferably H, or a halo group or -CN bonded to the ortho position or para position with respect to the -Y-L-X group.

[0041] In one embodiment, A is -C≡C-E bonded to the ortho position with respect to the -Y-L-X group, E is -COOH, -H, -CN, -COO-C 1-4 alkyl, -C6H4COOH, or -C6H4COO-C 1-4 alkyl, R 2 and R 3However, together with the carbon atoms bonded to them, they form an adamantane ring which may be substituted with a halo group; R 4 However, it is preferable that the group is H, or a halo group or -CN bonded to the ortho position relative to the -YLX group.

[0042] In one embodiment, A is -C≡CE bonded to the ortho position relative to the -YLX group, (i) E is -H, Y is -O-, L is absent, and X is H, a trialkylsilyl group, or a group represented by formula X3; or (ii) E is -COOH, Y is -O-, L is absent, and X is H, a trialkylsilyl group, or a group represented by formula X3; or (iii) E is -C6H4COOH, Y is -O-, L is absent, and X is H, a trialkylsilyl group, or a group represented by formula X3. R 2 and R 3 However, together with the carbon atoms bonded to them, they form an adamantane ring which may be substituted with a halo group. R 4 However, it is preferable that the group is H, or a halo group or -CN bonded to the ortho position relative to the -YLX group.

[0043] In one embodiment, A is an aryl group substituted with a -COOH bonded to the ortho position of the -YLX group. R 2 and R 3 However, together with the carbon atoms bonded to them, they form an adamantane ring which may be substituted with a halo group. R 4 However, it is preferable that the group is H, or a halo group or -CN bonded to the ortho position relative to the -YLX group.

[0044] The compound represented by formula [I] is preferably a compound represented by the following formulas: [IA], [IB], [IC], [ID], [IE], [IF], [IG], [IH], [II], [IJ], [IK], [IL], [IM], [IN], or [IO]:

[0045] [ka] (In the formula, Ar is an aryl group substituted with an electron-withdrawing group. X 1 is a base represented by formula X1 or X2, X 2 is H, a trialkylsilyl group, or a group represented by formula X3, X 3 is H, a trialkylsilyl group, or a group represented by formula X3, X5, X6, or X7. X 4 is a (2,4-dinitrophenyl)sulfonyl group, or represented by formula X4 and R 7 This is a group having an amino acid sequence in which multiple amino acid residues are linked together and can be cleaved by an enzyme. A, E, R 1 , R 2 , R 3 , R 4 (X, L, and Y are the same as above).

[0046] The compound represented by formula [I] is more preferably a compound represented by the following formulas [IP], [IQ], [IR], or [IS]: [ka]

[0047] The compound represented by formula [I] may exist, for example, in the form of an enantiomer, a diastereomer, or a racemic mixture.

[0048] The salt of the compound represented by formula [I] may be an inorganic salt or an organic salt. Examples of such salts include alkali metal salts (e.g., sodium salt, potassium salt, lithium salt), alkaline earth metal salts (e.g., calcium salt, magnesium salt), other metal salts (e.g., aluminum salt, iron salt, zinc salt, copper salt, nickel salt, cobalt salt), ammonium salt, tetramethylammonium salt, amine salt (e.g., t-octylamine salt, dibenzylamine salt, morpholine salt, glucosamine salt, phenylglycine alkyl ester salt, ethylenediamine salt, N-methylglucamine salt, guanidine salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N,N'-dibenzylethylenediamine salt, chloroprocaine salt, procaine salt) Examples include ethanolamine salts, diethanolamine salts, N-benzylphenethylamine salts, piperazine salts, tris(hydroxymethyl)aminomethane salts, inorganic salts (e.g., hydrofluoric acid salts, hydrochloride salts, hydrobromide salts, hydroiodide salts, nitrates, perchlorates, sulfates, phosphates), organic salts (e.g., methanesulfonate salts, trifluoromethanesulfonate salts, ethanesulfonate salts, benzenesulfonate salts, p-toluenesulfonate salts, acetate salts, malate salts, fumarate salts, succinate salts, citrate salts, tartrate salts, oxalate salts, maleate salts), and amino acid salts (e.g., glycine salts, lysine salts, arginine salts, ornithine salts, glutamate salts, aspartate salts).

[0049] The compound represented by formula [I] or a salt thereof may combine with a fluorophore to form a conjugate. Examples of fluorophores include fluorescein dyes (e.g., FAM), rhodamine dyes (e.g., TAMRA), coumarin dyes, cyanine dyes, pyrene dyes, and borondipyrrometene dyes.

[0050] The compound represented by formula [I] or its salts can be prepared, for example, according to the following reaction equation. [ka] (In the formula, Halo is a halo group, R 5 This is a base represented by H, or by formula X1 or X2 (where the dashed line indicates the bond point with A), Q 1 and Q 2 Each of these is an alkyl group which may be independently substituted with one or more halo groups, A, R 1 , R 2 , R 3 , R 4 (X, L, and Y are the same as above).

[0051] Step (i) is the Horner-Wadsworth-Emmons reaction. Typically, the compound represented by formula [VII] is dissolved in a solvent such as an alcohol, tetrahydrofuran, 1,2-dimethoxyethane, or dimethyl sulfoxide, and a base such as sodium hydride, sodium methoxide, potassium carbonate, or n-butyllithium is added to generate an anion, after which the compound represented by formula [VI] is added to carry out the reaction. This reaction can be carried out, for example, at temperatures from -78°C to reflux.

[0052] Step (ii) is a halogenation reaction. Typically, the reaction is carried out by dissolving the compound represented by formula [V] in a solvent such as toluene and adding a halogenating agent such as N-iodosuccinimide. This reaction can be carried out, for example, at 0 to 30°C.

[0053] Step (iii) is a coupling reaction [e.g., Sonogashira coupling reaction, Suzuki coupling reaction]. In the case of Sonogashira coupling, typically a mixture of the compound represented by formula [IV], a palladium catalyst such as tetrakis(triphenylphosphine)palladium (Pd(PPh3)4), a copper salt such as copper(I) iodide, and a base (which can also be a solvent) such as isopropylamine or triethylamine is mixed with the compound represented by formula [III] and R 5The reaction is carried out by adding a compound in which is H. Ligands such as trialkylphosphine and triarylphosphine may be added to the mixture. The reaction can be carried out, for example, at 5 to 30°C. In the case of Suzuki coupling, typically, in the presence of a palladium catalyst such as tris(dibenzylideneacetone)dipalladium (Pd2(dba)3), a ligand such as 2-dicyclohexylphosphino-2',6'-dimethoxybiphenyl (Sphos), and a base such as potassium phosphate, the compound represented by formula [IV] is reacted with the compound represented by formula [III] and R 5 The compound is reacted with a group represented by formula X1 or X2. The reaction can be carried out, for example, in a mixed solvent of water and an organic solvent such as tetrahydrofuran, toluene, or dioxane. The reaction can also be carried out, for example, at 5 to 100°C.

[0054] Step (iv) is a step of reacting the compound represented by formula [II] with singlet oxygen. This reaction can be carried out, for example, by irradiating the compound represented by formula [II] with light in an oxygen atmosphere in the presence of a solvent (e.g., halogenated hydrocarbons such as dichloromethane, dichloroethane, carbon tetrachloride and / or alcohols such as methanol and ethanol) and a photosensitizer (e.g., methylene blue, rose bengal, tetraphenylporfin). This reaction can be carried out, for example, at 5 to 30°C.

[0055] If the compounds used in the reaction of each step have amino groups, carboxyl groups, hydroxyl groups, etc., these groups may be protected with a general-purpose protecting group as needed, and the protecting group may be deprotected after the reaction. The reaction products of each step may be purified as needed by washing, distillation, filtration, chromatography, recrystallization, etc.

[0056] 3. Chemiluminescent probes The compound represented by formula [I] or a salt thereof may be used as a probe (hereinafter also referred to as a "chemiluminescent probe") for the detection of a test substance in vitro or in vivo. Detection of the test substance in a sample in vitro can be performed, for example, by enzyme immunoassay (EIA). Detection of the test substance in vivo is called in vivo imaging and can be performed, for example, by the method described in U.S. Patent Application Publication No. 2019 / 290787.

[0057] 4. Composition In one embodiment, the composition comprises a compound represented by formula [I] or a salt thereof, and a solvent. The solvent may be an organic solvent or an aqueous solvent. Examples of aqueous solvents include water, physiological saline, and buffer solutions. Examples of buffer solutions include Good's buffers such as HEPES, TAPS, MOPS, BES, and TES, Tris-HCl buffer, Orenveronal buffer, and imidazole-HCl buffer. The buffer solution may contain one or more buffering agents. Glycine may be added to the buffer solution as needed.

[0058] In the above composition, the amount of aqueous solvent is, for example, 10 parts by mass of the compound represented by formula [I] or its salt. 4 ~10 10 Parts by mass, preferably 10 5 ~10 9 Mass portion, more preferably 10 6 ~10 8 This is the mass part.

[0059] The composition may further contain additives. Examples of additives include surfactants and salts. Additives can be used individually or in combination of two or more.

[0060] The above composition exhibits high luminescence efficiency even without a surfactant, but can exhibit even higher luminescence efficiency by including a surfactant. The surfactant is not particularly limited as long as it has surface activity. The surfactant can be a nonionic surfactant, anionic surfactant, cationic surfactant, or amphoteric surfactant. Of these, nonionic surfactants are preferred. Examples of nonionic surfactants include sorbitan fatty acid esters such as sorbitan monolaurate; polyoxyethylene sorbitan fatty acid esters such as Tween(trademark)-20, Tween(trademark)-40, Tween(trademark)-60, and Tween(trademark)-80; glycerin fatty acid esters such as glycerin monostearate; polyoxyethylene glycerin fatty acid esters such as polyoxyethylene glyceryl monostearate; polyoxyethylene polyoxypropylene glycol such as poloxamer 188; and polyoxyethylene alkylphenyl ethers such as Triton(trademark) X-100.

[0061] The amount of surfactant is, for example, 10 to 10 parts by mass of the compound represented by formula [I] or its salt. 6 Parts by mass, preferably 100 to 10 5 This is the mass part.

[0062] Examples of salts include chlorides such as magnesium chloride and acetates such as sodium acetate. The amount of salt is, for example, 1 to 10 parts by mass of the compound represented by formula [I] or its salt. 4 The amount is parts by mass, preferably 10 to 1000 parts by mass.

[0063] The composition may be used for the detection of a test substance in vitro or in vivo. In vitro detection of a test substance in a sample can be performed, for example, by EIA. In vivo detection of a test substance is called in vivo imaging and can be performed, for example, by the method described in U.S. Patent Application Publication No. 2019 / 290787.

[0064] 5. Chemiluminescence Method In one embodiment, the chemiluminescence method includes the step of reacting a compound or a salt thereof, in which X in formula [I] is a saging group, with an uncaging substance. The uncaging substance can be the same as those described in "1. Definitions" above. The uncaging substance is preferably an enzyme. For example, if X in formula [I] is a saging group represented by formula X3, the uncaging substance may be an alkaline phosphatase.

[0065] In the above reaction, for 100 parts by mass of the compound or salt thereof in which X is a casing group in formula [I], the uncasing material is, for example, 10 ―10 ~10 10 Parts by mass, preferably 10 ―7 ~10 7 It can be used for mass parts.

[0066] The reaction temperature and reaction time of the above reaction are not particularly limited as long as the reaction proceeds. The reaction temperature can be the optimal temperature of the enzyme, for example, 25 to 45°C. The reaction time is, for example, 30 seconds to 3 hours, preferably 50 seconds to 2 hours, and more preferably 160 seconds to 2 hours.

[0067] 6. Method for measuring chemiluminescence signals In one embodiment, the method for measuring a chemiluminescence signal includes the step of measuring a chemiluminescence signal generated by the chemiluminescence method described in "5. Chemiluminescence Method" above. In this step, the measurement of the chemiluminescence signal includes detecting the presence or absence of a signal and quantifying the intensity of the signal. The measurement of the chemiluminescence signal can be carried out by known methods. Specifically, commercially available luminometers, chemiluminescence immunoassay (CLIA) devices, etc., can be used.

[0068] 7. Reagents Compounds in which X is a casing group in formula [I], salts thereof, chemiluminescent probes comprising such compounds or salts thereof, or compositions containing such compounds or salts thereof may be provided as reagents. Such reagents are suitably used for in vitro detection of test substances. In vitro detection of test substances in a sample can be performed, for example, by EIA. The chemiluminescent probes and compositions described in "3. Chemiluminescent Probes" and "4. Compositions" above may be used, respectively.

[0069] In the reagent described above, the concentration of the compound or salt in which X of formula [I] is a casing group can be appropriately set according to the type of substance to be tested and the measurement conditions, for example, 0.01 μM to 10 mM, preferably 0.1 μM to 1 mM.

[0070] The reagent typically contains a solvent. Examples of solvents include those described in "4. Composition" above. The reagent may also contain other optional components, such as additives like preservatives, antioxidants, and stabilizers.

[0071] 8. Reagent kit The reagents described above are usually provided to the user in containers. These containers may be packaged in a box and provided to the user as a reagent kit. The box may also include an instruction manual describing how to use the reagent kit. Figure 4 shows an example of how the reagent kit is provided. In the reagent kit 11, 12 represents the packaging box, 13 represents the container containing the reagents, and 14 represents the instruction manual.

[0072] Another embodiment of the reagent kit comprises a first reagent comprising a compound or a salt thereof in which X is a casing group in formula [I], a chemiluminescent probe comprising the compound or a salt thereof, or a composition containing the compound or a salt thereof, and a second reagent comprising the uncaging substance. The first reagent may be one of those described in "7. Reagents" above.

[0073] In the second reagent, the uncaging substance is the same as that described in "1. Definition" above. The concentration of the uncaging substance can be set appropriately depending on the type of uncaging substance and the measurement conditions, for example, 10 ―18 M to 1M, preferably 10 ―15 It is M to 1M.

[0074] The second reagent typically contains a solvent. Examples of solvents include those described in "4. Composition" above. The reagent may also contain other optional components, such as preservatives, antioxidants, stabilizers, and other additives.

[0075] The first and second reagents may be contained in separate containers, and these containers may be packaged in a box and provided to the user. The box may also include an instruction manual describing how to use the reagent kit. Figure 5 shows an example of how the reagent kit is provided. In the reagent kit 21, 22 indicates box packaging, 23a indicates the container containing the first reagent, 23b indicates the container containing the second reagent, and 24 indicates the instruction manual.

[0076] 9. Method for measuring the test substance In one embodiment, a method for measuring a test substance in a sample is: A step of forming an immune complex on a solid phase, comprising the substance to be tested, a capture body that binds to the substance to be tested, and a detection body that binds to the substance to be tested and contains an uncaging substance, The steps include: reacting the uncaging substance in the immune complex with a compound or a salt thereof in which X is a caging group in formula [I] to generate a chemiluminescent signal; The process of measuring the substance to be tested by measuring the chemiluminescence signal, Includes.

[0077] The method for measuring the test substance in the sample may be any known immunoassay method, preferably an enzyme-linked immunosorbent assay (ELISA) method, and more preferably a sandwich ELISA method. Alternatively, the immunocomplex transfer method described in U.S. Patent No. 5,236,849 may be used.

[0078] In the process of forming an immune complex, the solid phase is not particularly limited, but examples include latex, rubber, polyethylene, polypropylene, polystyrene, styrene-butadiene copolymer, polyvinyl chloride, polyvinyl acetate, polyacrylamide, polymethacrylate, styrene-methacrylate copolymer, polyglycidyl methacrylate, acrolein-ethylene glycol dimethacrylate copolymer, polyvinylidene difluoride (PVDF), silicone, agarose, gelatin, red blood cells, silica gel, glass, inert alumina, and magnetic materials. These may be used individually or in combination of two or more. Examples of solid phase shapes include particles, microplates, microtubes, membranes, and test tubes, with particles being particularly preferred. In one embodiment, the solid phase is preferably magnetic particles. Examples of such magnetic particles include particles containing Fe2O3 and / or Fe3O4, cobalt, nickel, filite, magnetite, etc., as a base material.

[0079] The capture agent that binds to the test substance is not particularly limited as long as it can bind to the solid phase and capture the test substance, but may be an antigen, antibody, etc. The detector may contain an uncaging substance that binds to the test substance. Examples of such uncaging substances are the same as those described in "1. Definitions" above, and react with a compound or a salt thereof in which X is a caging group in formula [I] to generate a chemiluminescent signal. Examples of the detector include antibodies labeled with an uncaging substance.

[0080] If the test substance is antibody A, the antigen functions as a capture agent to capture antibody A. If the sample contains antibody A, an immune complex can be formed containing the sample, the antigen, and the detection antibody B. The immune complex can be formed on a solid phase by contacting a solution containing the immune complex with a solid phase on which the antigen can be immobilized. Alternatively, the immune complex can be formed on a solid phase by contacting a solid phase on which the antigen has been pre-immobilized with the sample and the detection antibody.

[0081] The manner in which the capture material is immobilized on the solid phase is not particularly limited. For example, the capture material and the solid phase may be directly bound together, or they may be indirectly bound together via another substance. Examples of direct binding include physical adsorption and covalent bonding by a crosslinking agent. Examples of indirect binding include combinations of biotins (including biotin and biotin analogs such as desthiobiotin and oxybiotin) and avidins (including avidin and avidin analogs such as streptavidin and tamavidin®), and combinations of haptens and anti-hapten antibodies (e.g., compounds having a 2,4-dinitrophenyl group (DNP group) and anti-DNP antibodies). In one embodiment, by using a capture material that has been pre-modified with biotins and a solid phase to which avidins have been pre-bound, the capture material can be immobilized on the solid phase via binding between biotins and avidins.

[0082] Between the immunocomplex formation step and the chemiluminescent signal generation step, bound / free (B / F) separation may be performed to remove unreacted free components that have not formed a complex. Unreacted free components refer to components that do not constitute an immunocomplex. Examples include captures and detects that did not bind to the test substance. The means of B / F separation are not particularly limited, but if the solid phase is particles, B / F separation can be performed by centrifugation. If the solid phase is a container such as a microplate or microtube, B / F separation can be performed by removing the liquid containing the unreacted free components. Furthermore, if the solid phase is magnetic particles, B / F separation can be performed by magnetically confining the magnetic particles with a magnet and then aspirating and removing the liquid containing the unreacted free components with a nozzle, which is preferable from the viewpoint of automation. After removing the unreacted free components, the solid phase containing the captured immunocomplexes may be washed with a suitable aqueous medium such as PBS.

[0083] In the process of generating a chemiluminescent signal, the amount of uncaking material is, for example, 10 parts by mass of a compound or salt thereof in which X is a causing group in formula [I]. ―10 ~10 10 Parts by mass, preferably 10 ―7 ~10 7 It is preferable to react the two so that one component is formed by mass.

[0084] The process of measuring the chemiluminescence signal can be carried out, for example, by the method described in "6. Method for Measuring Chemiluminescence Signal" above. [Examples]

[0085] The present invention will be described in more detail below based on examples, but the present invention is not limited to these examples.

[0086] <Example of synthesis> (Reference example 1)

[0087] [ka]

[0088] To a methanol (MeOH) solution (500 mL) of 3-hydroxybenzaldehyde (50.0 g, 0.41 mol), trimethyl orthoformate (130 g, 1.22 mol) and tetrabutylammonium tribromide (Bu4NBr3) (15.0 g, 0.03 mol) were added and the mixture was stirred overnight at room temperature. The reaction solution was added to water and extracted twice with ethyl acetate. The organic layer was washed with saturated brine, dried over sodium sulfate, and the solvent was removed under reduced pressure to obtain 3-(dimethoxymethyl)phenol as a yellow liquid. (75.0 g, quant.) 1 HNMR (CDCl3, 400MHz): δ7.21-7.25(m, 1H), 6.99-7.01(m, 2H), 6.85-6.87(m, 1H), 5.36(s, 1H), 3.35(s, 6H).

[0089] (Reference example 2)

[0090] [ka]

[0091] To a 750 mL solution (DCM) of the compound from Reference Example 1 (75.0 g) and imidazole (41.8 g, 0.61 mol) of tert-butyldimethylchlorosilane (TBSCl) (74.1 g, 0.49 mol) was added and the mixture was stirred at room temperature for 1 hour. The reaction solution was added to water and extracted twice with dichloromethane. The organic layer was washed with saturated brine, dried over sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by silica gel chromatography (petroleum ether / ethyl acetate = 50:1~20:1) to obtain tert-butyl(3-(dimethoxymethyl)phenoxy)dimethylsilane as a colorless liquid (80.0 g, 69% (2 steps)). 1HNMR (CDCl3, 400MHz): δ7.23(t, J=8.0Hz, 1H), 7.04d, J=7.6Hz, 1H), 6.96(s, 1H) 6.80(dd, J=2.0, 8.0Hz, 1H), 5.39(s, 1H), 3.33(s, 9H), 1.00(s, 6H), 0.21(s, 6H).

[0092] (Reference example 3)

[0093] [ka]

[0094] A dichloromethane solution (800 mL) of the compound from Reference Example 2 (80.0 g, 0.28 mol) and trimethyl phosphite (52.7 g, 0.42 mol) was cooled to 0°C, and titanium(IV) chloride (80.5 g, 0.42 mol) was added dropwise. The reaction solution was stirred at 0°C for 1 hour. The reaction solution was added to water and extracted twice with dichloromethane. The organic layer was washed with saturated brine, dried over sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by silica gel chromatography (petroleum ether / ethyl acetate = 30 / 1 to 10 / 1) to obtain dimethyl((3-((tert-butyldimethylsilyl)oxy)phenyl)(methoxy)methyl)phosphonate as a colorless liquid (57.0 g, 56%). 1 HNMR (CDCl3, 400MHz): δ7.24(t, J=8.0Hz, 1H), 7.03(d, J=7.2Hz, 1H), 6.96(d, J=2.0Hz, 1H) 6.83(d , J=8.4Hz, 1H), 4.50(d, J=15.6Hz, 1H), 3.66-3.73(m, 6H), 3.39(s, 3H), 0.99(s, 9H), 0.21(s, 6H).

[0095] (Reference example 4)

[0096] [ka]

[0097] A 570 mL solution of the compound from Reference Example 3 (57.0 g, 0.16 mol) in anhydrous tetrahydrofuran (THF) was cooled to -78°C, and n-butyllithium (n-BuLi) (2.5 M, 110 mL, 0.28 mol) was added under a nitrogen atmosphere. The reaction solution was stirred under a nitrogen atmosphere at -78°C for 1 hour. A 150 mL solution of 5-chloro-2-adamantanone (35.0 g, 0.19 mol) in anhydrous tetrahydrofuran was added dropwise at -78°C. The reaction solution was stirred under a nitrogen atmosphere at -78°C for 30 minutes. The reaction solution was added to a saturated aqueous solution of ammonium chloride and extracted twice with ethyl acetate. The organic layer was washed with saturated brine, dried over sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by silica gel chromatography (petroleum ether / ethyl acetate = 30 / 1 to 10 / 1) to obtain tert-butyl(3-((Z)-((1R,3S,5S,7S)-5-chloroadamantan-2-ylidene)(methoxy)methyl)phenoxy)dimethylsilane as a colorless liquid (30.0 g, 45%). 1 HNMR (CDCl3, 400MHz): δ7.23(t, J=8.0Hz, 1H), 6.90(d, J=7.6Hz, 1H), 6.78-6.83(m, 2H) 3.46(s, 1H), 3.30(s, 3H), 2.79(s, 1H), 2.16-2.28(m, 7H), 1.71-1.85(m, 4H).1.00(s, 9H), 0.22(s, 6H).

[0098] (Reference example 5)

[0099] [ka]

[0100] A tetrahydrofuran solution (300 mL) of the compound from Reference Example 4 (30.0 g, 71.6 mmol) was cooled to 0°C, and a tetrahydrofuran solution (1 M, 80 mL, 80 mmol) of tetrabutylammonium fluoride (TBAF) was added. The reaction solution was stirred at 0°C for 30 minutes. The reaction solution was added to water and extracted twice with ethyl acetate. The organic layer was washed with saturated brine, dried over sodium sulfate, and the solvent was removed under reduced pressure to obtain 3-((Z)-((1R,3S,5S,7S)-5-chloroadamantane-2-ylidene)(methoxy)methyl)phenol as a colorless liquid (24 g, quant.). 1 HNMR (CDCl3, 400MHz): δ7.21-7.25(m, 1H), 6.81-6.86(m, 3H), 3.45(s, 1H), 3.3 2(s, 3H), 2.81(s, 1H), 2.15-2.30(m, 7H), 1.85-1.86(m, 1H), 1.71-1.79(m, 3H).

[0101] (Reference example 6)

[0102] [ka]

[0103] A toluene solution (500 mL) of the compound (24 g) from Reference Example 5 was cooled to 0°C, and N-iodosuccinimide (NIS) (17.7 g, 78.6 mmol) was added. The reaction solution was stirred at room temperature for 1 hour. The reaction solution was added to water and extracted twice with ethyl acetate. The organic layer was washed with saturated brine, dried over sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by silica gel chromatography (petroleum ether / ethyl acetate = 20 / 1 to 3 / 1) to obtain 5-((Z)-((1R,3S,5S,7S)-5-chloroadamantane-2-ylidene)(methoxy)methyl)-2-iodophenol as a white solid (9.3 g, 30% (2 steps)). 1HNMR (CDCl3, 400MHz): δ7.66(d, J=8.0Hz, 1H), 6.94(d, J=1.6Hz, 1H), 6.64(dd, J=1.6, 8.0Hz1H), 5.41(s, 1H), 3.45(s, 1H), 3.31(s, 3H), 2.80(s, 1H), 2.15-2.27(m, 7H), 1.85-1.86(m, 1H), 1.71-1.78(m, 3H).Mass Calcd.:430;MS Found:431[M+H] + .

[0104] (Reference example 7)

[0105] [ka]

[0106] The compound from Reference Example 6 (430.7 mg, 1.0 mmol, 1 eq), tetrakis(triphenylphosphine)palladium(0)(Pd(PPh3)4) (115.6 mg, 0.1 mmol, 0.1 eq), copper(I) iodide (38.1 mg, 0.2 mmol, 0.2 eq), triphenylphosphine (PPh3) (52.5 mg, 0.2 mmol, 0.2 eq), diisopropylamine (iPr2NH) (2 mL), and triethylamine (TEA) (4 mL) were mixed and stirred at room temperature under an argon atmosphere for 30 minutes. Methyl 4-ethynylbenzoate (320.4 mg, 2.0 mmol, 2 eq) was added and the mixture was stirred at 80°C for 1 hour. After cooling to room temperature, the reaction solution was diluted with dichloromethane and filtered by Celite. The organic layer was washed with 1N hydrochloric acid, water, and saturated brine, dried over sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by silica gel chromatography (n-hexane / ethyl acetate = 4 / 1) to obtain the compound represented by formula [II-A] as a yellow solid (222.4 mg, 48%). ESI-MS m / z: 461 [MH] - .

[0107] (Reference example 8)

[0108] [ka]

[0109] A 2 mL solution of anhydrous dichloromethane containing triallyl phosphite (P(OAllyl)3) (71.6 μL, 0.51 mmol, 6 eq) was cooled to 0°C, and iodine (108.5 mg, 0.43 mmol, 5 eq) was added. The mixture was stirred at 0°C for 5 minutes and then returned to room temperature. The compound from Reference Example 7 (39.6 mg, 0.09 mmol, 1 eq) and DMAP (62.7 mg, 0.51 mmol, 6 eq) were added dropwise at room temperature to a 2 mL solution of anhydrous dichloromethane. After stirring at room temperature for 40 minutes, the mixture was diluted with dichloromethane, and the organic layer was washed with saturated ammonium chloride aqueous solution, saturated sodium bicarbonate aqueous solution, and saturated brine. After drying over sodium sulfate, the solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by silica gel chromatography (n-hexane / ethyl acetate = 3 / 1) to obtain the compound represented by formula [II-B] as a red liquid (28.7 mg, 55%). ESI-MS m / z:624[M+H] + .

[0110] (Reference example 9)

[0111] [ka]

[0112] To a tetrahydrofuran solution (3 mL) of the compound from Reference Example 8 (127.1 mg, 0.20 mmol, 1 eq), formic acid (38.5 μL, 1.02 mmol, 5 eq), triethylamine (34.1 μL, 0.24 mmol, 1.2 eq), and tetrakis(triphenylphosphine)palladium (0) (23.6 mg, 0.02 mmol, 0.1 eq) were added, and the mixture was stirred at 50°C for 1 hour. After cooling to room temperature, the solvent was removed under reduced pressure. The residue was dissolved in tetrahydrofuran (4 mL) and 1 mL of 1N sodium hydroxide aqueous solution, and the mixture was stirred at room temperature for 1 hour. The solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography (dichloromethane / methanol = 10 / 1 to 3 / 1) to obtain the compound represented by formula [II-C] as a yellow solid (160.4 mg, quant.). ESI-MS m / z: 527 [MH] - .

[0113] (Example 1)

[0114] [ka]

[0115] The compound from Reference Example 9 (60.8 mg, 0.12 mmol, 1 eq) and methylene blue (2 mg) were dissolved in dichloromethane (2 mL) / methanol (0.5 mL) and stirred for 45 minutes under an oxygen atmosphere while irradiating with light (LED, 25 W, 5000 K). The reaction solution was purified by silica gel chromatography (ethyl acetate / methanol = 1 / 1) to obtain the crude product. The crude product was purified by HPLC (eluent A: water, 5 mM ammonium carbonate aqueous solution, eluent B: acetonitrile, A / B = 90 / 10 - 10 / 90 (20 minutes)) to obtain the compound represented by formula [IP] as a white solid (8.6 mg, 13%). ESI-MS m / z: 559 [MH] - .

[0116] (Reference example 10)

[0117] [ka]

[0118] The compound from Reference Example 6 (1.29 g, 3.0 mmol, 1 eq), tetrakis(triphenylphosphine)palladium(0) (173.3 mg, 0.15 mmol, 0.05 eq), copper(I) iodide (57.1 mg, 0.3 mmol, 0.1 eq), triphenylphosphine (78.7 mg, 0.3 mmol, 0.1 eq), diisopropylamine (5 mL), and triethylamine (10 mL) were mixed and stirred at room temperature under an argon atmosphere for 30 minutes. Trimethylsilylacetylene (1.24 mL, 9.0 mmol, 3 eq) was added and the mixture was stirred at 90°C for 2 hours. After cooling to room temperature, the reaction solution was diluted with dichloromethane and filtered by Celite. The organic layer was washed with saturated ammonium chloride aqueous solution and saturated brine, dried over sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude product. The crude product was dissolved in tetrahydrofuran (15 mL), and tetrabutylammonium fluoride solution (1 M, tetrahydrofuran) was added. The mixture was stirred at room temperature for 20 minutes, and the solvent was removed under reduced pressure. The residue was purified by silica gel chromatography (n-hexane / ethyl acetate = 4 / 1) to obtain the compound represented by formula [II-D] as a brown solid (820.2 mg, 83%). ESI-MS m / z:327[MH] - .

[0119] (Reference example 11)

[0120] [ka]

[0121] A 5 mL solution of the compound from Reference Example 10 (32.9 mg, 0.1 mmol, 1 eq) and anhydrous pyridine (169.2 μL, 2.10 mmol, 21 eq) in anhydrous dichloromethane was cooled to 0°C, and phosphoryl chloride (93.2 μL, 1.0 mmol, 10 eq) was added dropwise. The mixture was stirred under an argon atmosphere at 0°C for 3.5 hours, and the solvent was removed under reduced pressure. The process of adding toluene and removing the solvent under reduced pressure was repeated twice to obtain the crude product. The crude product was purified by HPLC (eluent A: water, 5 mM ammonium carbonate aqueous solution, eluent B: acetonitrile, A / B = 90 / 10 - 10 / 90 (20 minutes)) to obtain the compound represented by formula [II-E] as a white solid (17.1 mg, 42%). ESI-MS m / z: 407 [MH] - .

[0122] (Example 2)

[0123] [ka]

[0124] The compound from Reference Example 11 (14 mg, 0.027 mmol, 1 eq) and Rose Bengal B polystyrene-bound form (20 mg) were added to dichloromethane (2 mL) / methanol (0.5 mL) and stirred for 1 hour under an oxygen atmosphere while irradiating with light (LED, 25 W, 5000 K). The reaction solution was filtered through a cotton plug, and the solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by HPLC (eluent A: water, 5 mM ammonium carbonate aqueous solution, eluent B: acetonitrile, A / B = 90 / 10 - 10 / 90 (20 minutes)) to obtain the compound represented by formula [IQ] as a white solid (4.5 mg, 30%). ESI-MS m / z:439[MH] - .

[0125] (Reference example 12)

[0126] [ka]

[0127] To a 19 mL dichloromethane solution (611.7 mg, 1.86 mmol, 1 eq) of the compound from Reference Example 10 and imidazole (253.3 mg, 3.72 mmol, 2 eqs), tert-butyldimethylchlorosilane (420.5 mg, 2.8 mmol, 1.5 eq) was added. The reaction solution was stirred under an argon atmosphere at room temperature for 30 minutes. Imidazole (126 mg, 1.86 mmol, 1 eq) and tert-butyldimethylchlorosilane (210 mg, 1.4 mmol, 0.7 eq) were added to the reaction solution and stirred at room temperature for 4 hours. The reaction solution was filtered, and the filtrate was removed under reduced pressure to obtain the residue. The residue was purified by silica gel chromatography (n-hexane / ethyl acetate = 20 / 1) to obtain the compound represented by formula [II-F] as a colorless liquid (720.6 mg, 87%). ESI-MS m / z:443[M+H] + .

[0128] (Reference example 13)

[0129] [ka]

[0130] An anhydrous tetrahydrofuran solution (8 mL) of the compound from Reference Example 12 (680.5 mg, 1.54 mmol, 1 eq) was cooled to -78°C, and an n-hexane solution of n-butyllithium (2.6 M, 1.77 mL, 4.61 mol, 3 eq) was added dropwise under a nitrogen atmosphere. The reaction solution was stirred under a nitrogen atmosphere at -78°C for 30 minutes. Methyl chloroformate (589.9 μL, 7.68 mmol, 5 eq) was added, and the mixture was stirred at room temperature for 1 hour. Slowly, an aqueous solution of saturated ammonium chloride was added, and the mixture was extracted twice with acetic acid. The mixture was then washed with water and saturated brine. After drying over sodium sulfate, the solvent was removed under reduced pressure to obtain the residue. The residue was purified by silica gel chromatography (n-hexane / ethyl acetate = 20 / 1) to obtain the compound represented by formula [II-G] as a pale yellow liquid (774.1 mg, quant.). ESI-MS m / z:501[M+H] +

[0131] (Reference example 14)

[0132]

Chem.

[0133] To a solution of the compound of Reference Example 13 (751.6 mg, 1.50 mmol, 1 mmol) in tetrahydrofuran (7.5 mL) was added a solution of tetrabutylammonium fluoride in tetrahydrofuran (1 M, 1.8 mL, 1.8 mmol, 1.2 eq), and the mixture was stirred at room temperature for 20 minutes. Saturated aqueous sodium hydrogen carbonate solution was added to the reaction solution, and the mixture was extracted twice with ethyl acetate. The organic layer was washed with saturated brine, dried over sodium sulfate, and the solvent was removed under reduced pressure to obtain a residue. The residue was purified by silica gel chromatography (n-hexane / ethyl acetate = 3 / 1) to obtain the compound represented by Formula [II-H] as a white solid (456.3 mg, 79%). ESI-MS m / z: 385 [M+H] - 。

[0134] (Reference Example 15)

[0135]

Chem.

[0136] A dehydrated dichloromethane solution (2.6 mL) of the compound of Reference Example 14 (50.3 mg, 0.13 mmol, 1 eq) and dehydrated pyridine (219.9 μL, 2.73 mmol, 21 eq) was cooled to 0 °C, and phosphoryl chloride (121.2 μL, 1.3 mmol, 10 eq) was added dropwise. The mixture was stirred at 0 °C for 40 minutes under an argon atmosphere, and the solvent was removed under reduced pressure. The operation of adding toluene and removing the solvent under reduced pressure was repeated twice to obtain a residue. The residue was suspended in water (2 mL), potassium hydroxide (218.8 mg, 3.9 mmol, 30 eq) was added, and the mixture was stirred at room temperature for 5 minutes. The reaction solution was purified by HPLC (eluent A: water, 5 mM aqueous ammonium carbonate solution, eluent B: acetonitrile, A / B = 90 / 10 - 10 / 90 (for 20 minutes)) to obtain the compound represented by Formula [II-J] as a white solid (36.6 mg, 62%). ESI-MS m / z: 451 [M-H] - 。

[0137] (Example 3)

[0138]

Chem.

[0139] The compound of Reference Example 15 (36.6 mg, 0.081 mmol, 1 eq) and rose bengal B polystyrene-bound (30 mg) were added to dichloromethane (5 mL) / methanol (2 mL), and stirred for 2 hours while irradiating with light (LED, 25 W, 5000 K) under an oxygen atmosphere. The reaction solution was filtered through a cotton plug, and the solvent was removed under reduced pressure to obtain a crude product. The crude product was purified by HPLC (eluent A: water, 5 mM aqueous ammonium carbonate solution, eluent B: acetonitrile, A / B = 90 / 10 - 10 / 90 (20 minutes)) to obtain the compound represented by formula [I-R] as a white solid (18.4 mg, 47%). ESI-MS m / z: 483 [M-H] - 。

[0140] (Reference Example 16)

[0141]

Chem.

[0142] The compound from Reference Example 6 (215.4 mg, 0.50 mmol, 1 eq.), methyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate (196.6 mg, 0.75 mmol, 1.5 eq.), tris(dibenzylideneacetone)dipalladium(0)(Pd2(dba)3) (22.9 mg, 0.03 mmol, 0.05 eq.), SPhos (20.5 mg, 0.05 mmol, 0.1 eq.), potassium phosphate (212.3 mg, 1.00 mmol, 2 eq.), tetrahydrofuran (2 mL), and water (0.5 mL) were mixed and stirred at room temperature under an argon atmosphere for 28 hours. The reaction solution was diluted with ethyl acetate, the organic layer was washed with saturated ammonium chloride aqueous solution and saturated brine, dried over sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by silica gel chromatography (n-hexane / ethyl acetate = 3 / 1) to obtain the compound represented by formula [II-K] as a white solid (163.9 mg, 75%). ESI-MS m / z:439[MH] + .

[0143] (Reference example 17)

[0144] [ka]

[0145] A solution of triallyl phosphite (96.1 μL, 0.69 mmol, 2.5 eq) in anhydrous dichloromethane (2 mL) was cooled to 0°C, and iodine (139.9 mg, 0.55 mmol, 2 eq) was added. The mixture was stirred at 0°C for 5 minutes, and after returning to room temperature, the compound from Reference Example 7 (121.9 mg, 0.28 mmol, 1 eq) and DMAP (101.0 mg, 0.83 mmol, 3 eq) in anhydrous dichloromethane (3 mL) were added dropwise at room temperature. After stirring at room temperature for 2 hours, the mixture was diluted with dichloromethane, and the organic layer was washed with 1N hydrochloric acid, saturated sodium bicarbonate aqueous solution, and saturated brine. After drying over sodium sulfate, the solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by silica gel chromatography (n-hexane / ethyl acetate = 3 / 1) to obtain the compound represented by formula [II-L] (114.1 mg, 69%). ESI-MS m / z: 599 [M+H] + 。

[0146] (Reference Example 18)

Chem.

[0147] To a solution of the compound of Reference Example 17 (60.4 mg, 0.10 mmol, 1 eq) in tetrahydrofuran (2 mL) were added formic acid (19.0 μL, 0.50 mmol, 5 eq), triethylamine (16.9 μL, 0.12 mmol, 1.2 eq), and tetrakis(triphenylphosphine)palladium(0) (11.7 mg, 0.01 mmol, 0.1 eq), and the mixture was stirred at 60 °C for 10 minutes. After cooling to room temperature, the solvent was removed under reduced pressure. The residue was dissolved in tetrahydrofuran (3 mL) and 1N aqueous sodium hydroxide solution (0.5 mL), and the mixture was stirred at room temperature for 1 hour. The solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography (ethyl acetate / methanol = 4 / 1 to 1 / 1) to obtain the compound represented by Formula [II-N] as a white solid (44.1 mg, 87%). ESI-MS m / z: 503 [M-H] - 。

[0148] (Example 4)

[0149]

Chem.

[0150] Reference Example 18 (35.9 mg, 0.07 mmol, 1 eq) and methylene blue (2 mg) were dissolved in dichloromethane (2 mL) / methanol (0.5 mL) and stirred for 30 minutes under an oxygen atmosphere while irradiating with light (LED, 25 W, 5000 K). The reaction solution was purified by silica gel chromatography (ethyl acetate / methanol = 1 / 10) to obtain the crude product. The crude product was purified by HPLC (eluent A: water, 5 mM ammonium carbonate aqueous solution, eluent B: acetonitrile, A / B = 90 / 10 - 10 / 90 (20 minutes)) to obtain the compound represented by formula [IS] as a white solid (2.9 mg, 8%). ESI-MS m / z: 535 [MH] - .

[0151] <Test Example 1> Dimethyl sulfoxide solutions (1 mM, 5 μL) of 1,2-dioxetane derivatives (Examples 1-4, Comparative Examples 1-2) were added to 155 μL of the reaction solution and incubated at 42°C for 1 hour. 20 μL of a solution containing 1 pmol of alkaline phosphatase was added, and the luminescence signal was measured at 42°C for 10 hours, and the integrated value was calculated. The reaction solution used was a 2:1 mixture of buffer (buffer containing magnesium chloride and Sapphire-II® Enhancer (Thermo Fischer Scientific) (pH 9.6)) and HISCL R4 reagent (Sysmex Corporation).

[0152] The results of Test Example 1 are shown in Figures 1A and 1B. As shown in Figures 1A and 1B, Examples 1 to 4 had higher chemiluminescence efficiencies compared to Comparative Example 1 (CDP-Star®) and Comparative Example 2 (AquaSpark®).

[0153] <Test Example 2> Dimethyl sulfoxide solutions of 1,2-dioxetane derivatives (Examples 1-4) were added to 155 μL of the reaction solution and incubated at 42°C for 1 hour. 20 μL of a solution containing alkaline phosphatase was added, incubated at 42°C for 5 minutes, and then the chemiluminescence intensity was measured. The reaction solution used was a 2:1 mixture of buffer (buffer containing magnesium chloride and Sapphire-II® Enhancer (Thermo Fischer Scientific) (pH 9.6)) and HISCL R4 reagent (Sysmex Corporation). Example 3 used two types: one with Sapphire-II® Enhancer added (S (+)) and one without Sapphire-II® Enhancer added (S (-)).

[0154] The detection limits (LODs) for Examples 1-4 are shown in Table 1. Furthermore, as shown in Figures 2A-2E, the measured count values ​​were found to depend on the alkaline phosphatase concentration. [Table 1]

[0155] <Test Example 3> After several hours under the conditions of Test Example 1, the maximum emission wavelength was measured using a spectrofluorometer F-7000 (Hitachi High-Tech Science Corporation).

[0156] The maximum emission wavelengths for Examples 1-4 are shown in Table 2.

[0157] [Table 2]

[0158] <Test Example 4> The HISCL™ HBsAg calibrator (Sysmex Corporation) was measured using the HI-1000 fully automated high-sensitivity immunoassay analyzer (Sysmex Corporation) and the HISCL™ HBsAg reagent (Sysmex Corporation) for hepatitis B virus surface antigen. For the measurement in Example 3, instead of the HISCL R5 reagent, a dimethyl sulfoxide solution of the 1,2-dioxetane derivative (Example 3) was diluted in a buffer (buffer containing magnesium chloride (pH 9.6)) (final concentration in Example 3: 93 μM), incubated at 42°C for 1 hour under light-shielding conditions, and then returned to room temperature. Note that this buffer did not contain Sapphire-II™ Enhancer (Thermo Fischer Scientific).

[0159] As shown in Figure 3, the HISCL count value was found to be dependent on the concentration of HBsAg.

Claims

1. A compound represented by formula [I] or a salt thereof. 【Chemistry 1】 [In the formula, R １ is a methyl group; R ２ and R ３ These, together with the carbon atoms to which they are bonded, form an adamantane ring which may be substituted with a halo group; X is given by equation X3: 【Chemistry 2】 It is a casing group represented by; L is nonexistent; Y is either -O- or -NH-; R ４ H is; and A is a phenyl group substituted with -C≡C-E (wherein E is -COOH, -H, or -C6H4COOH), or -COOH.

2. The compound or salt thereof according to claim 1, wherein A is bonded to the -Y-L-X group at the ortho or para position.

3. The compound or salt thereof according to claim 1 or 2, wherein A is -C≡C-E bonded to the ortho position of the -Y-L-X group.

4. The compound or salt thereof according to claim 1 or 2, wherein A is a phenyl group substituted with -COOH bonded to the ortho position of the -Y-L-X group.

5. A composition comprising a compound or a salt thereof according to any one of claims 1 to 4 and an aqueous solvent.

6. The composition according to claim 5, further comprising a surfactant.

7. A chemiluminescence method comprising the step of reacting a compound represented by formula [I] or a salt thereof with a substance that liberates a casing group. 【Transformation 3】 [In the formula, R １ is a methyl group; R ２ and R ３ These, together with the carbon atoms to which they are bonded, form an adamantane ring which may be substituted with a halo group; X is given by equation X3: 【Chemistry 4】 It is a casing group represented by; L is nonexistent; Y is either -O- or -NH-; R ４ H is; and A is a phenyl group substituted with -C≡C-E (wherein E is -COOH, -H, or -C6H4COOH), or -COOH.

8. The chemiluminescence method according to claim 7, wherein the substance that liberates the casing group is alkaline phosphatase.

9. A method for measuring a chemiluminescence signal, comprising the step of measuring a chemiluminescence signal generated by the chemiluminescence method described in claim 7 or 8.

10. A reagent for measuring a test substance in a sample, comprising a compound represented by formula [I] or a salt thereof. 【Transformation 5】 [In the formula, R １ is a methyl group; R ２ and R ３ These, together with the carbon atoms to which they are bonded, form an adamantane ring which may be substituted with a halo group; X is given by equation X3: 【Transformation 6】 It is a casing group represented by; L is nonexistent; Y is either -O- or -NH-; R ４ H is; and A is a phenyl group substituted with -C≡C-E (wherein E is -COOH, -H, or -C6H4COOH), or -COOH.

11. A first reagent comprising a compound represented by formula [I] or a salt thereof, A second reagent containing a substance that liberates the caging group, A reagent kit for measuring test substances in a sample, including [specific reagents / reagents]. 【Transformation 7】 [In the formula, R １ is a methyl group; R ２ and R ３ These, together with the carbon atoms to which they are bonded, form an adamantane ring which may be substituted with a halo group; X is given by equation X3: 【Transformation 8】 It is a casing group represented by; L is nonexistent; Y is either -O- or -NH-; R ４ H is; and A is a phenyl group substituted with -C≡C-E (wherein E is -COOH, -H, or -C6H4COOH), or -COOH.

12. A method for measuring a test substance in a sample, A step of forming an immune complex on a solid phase, comprising the substance to be tested, a capture body that binds to the substance to be tested, and a detection body that contains a substance that binds to the substance to be tested and releases a saging group, A step of reacting a substance that releases the casing group in the immune complex with a compound represented by formula [I] or a salt thereof to generate a chemiluminescent signal, The process of measuring the substance to be tested by measuring the chemiluminescence signal, Methods that include... 【Chemistry 9】 [In the formula, R １ is a methyl group; R ２ and R ３ These, together with the carbon atoms to which they are bonded, form an adamantane ring which may be substituted with a halo group; X is given by equation X3: 【Chemistry 10】 It is a casing group represented by; L is nonexistent; Y is either -O- or -NH-; R ４ H is; and A is a phenyl group substituted with -C≡C-E (wherein E is -COOH, -H, or -C6H4COOH), or -COOH.

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