Functional compound

Abstract

Provided is a novel compound useful for, e.g., comprehensive analysis of cell surface proteins. Disclosed is a functional compound or a salt thereof in which a tyrosine residue reaction site R is linked to a protein purification tag site Tag via a spacer S. The tyrosine residue reaction site R is a group represented by formula (1). (In the formula, * denotes a bond with a spacer S, and R1 and R2 are each independently selected from the group consisting of H, alkyl which optionally be substituted with one or more substituents, aryl which optionally be substituted with one or more substituents, and heteroaryl which optionally be substituted with one or more substituents.)

Description

functional compound

[0001] The present disclosure encompasses functional compounds.

[0002] In recent years, in the research field of chemical biology, molecular biology and organic chemistry techniques have been utilized to detect and quantify trace amounts of biological molecules, such as proteins or physiologically active substances, and to elucidate the functions or reactions of these biological molecules. As part of these efforts, chemical probes, or compounds that specifically interact with biological molecules, have been developed to elucidate the functions or reactions of biological molecules.

[0003] In this research field, chemoproteomics is a method for elucidating the structure and function of proteins using organic chemical techniques.

[0004] Chemoproteomics is a method that enables unbiased and comprehensive direct identification of target proteins from living cells or tissues that reflect the phenotype, and is thought to make a significant contribution to drug development, such as identifying unknown target proteins of drug candidate compounds discovered through phenotypic screening and identifying antigens of unknown antibodies.

[0005] Conventional methods for comprehensively analyzing cell surface proteins, which are important in drug discovery research, involve comprehensive labeling using amine-labeling reagents, but these methods are dependent on reaction temperature and time and are therefore unsuitable for capturing transient phenomena in living cells. Furthermore, methods using biotin tyramide, which can capture transient phenomena (e.g., peroxidase-mediated cell surface labeling (PECSL) described in Non-Patent Document 1), have problems such as low reaction efficiency and difficulty in identifying the labeling site. Even proximity-dependent labeling methods capable of detecting specific membrane proteins (e.g., selective proteomic proximity labeling assay using tyramide (SPPLAT) described in Non-Patent Document 2) have problems with site-specific labeling and difficulty in identifying the labeling site. Other proximity labeling methods, such as the μMAP method (Non-Patent Document 3), require a light irradiation device and expensive reagents.

[0006] In addition, as reactive groups that selectively react with specific amino acids in protein molecules, NHS (N-hydroxysuccinimide), which reacts with lysine, and tyramide, which reacts with tyrosine, as well as luminol derivatives have been reported (Non-Patent Document 4).

[0007] Analytical Chemistry 2021, 93, 4542-4551Current Protocols in Protein Science 19.27.1-18Science 367, 1091-1097 (2020)ChemBioChem 2017, 18, 475-478

[0008] Objects of the present disclosure include providing novel compounds that are useful for the comprehensive analysis of cell surface proteins, etc.

[0009] In order to achieve the above-mentioned object, the present inventors have conducted extensive research and found that a functional compound or a salt thereof in which a tyrosine residue reactive site R is linked to a protein purification tag site Tag via a spacer S is useful for the comprehensive analysis of cell surface proteins.

[0010] The present invention was completed based on these findings and further investigations, and includes the following aspects: [Item 1] A functional compound or a salt thereof, in which a tyrosine residue reactive site R is linked to a protein purification tag site Tag via a spacer S, wherein the tyrosine residue reactive site R is a group represented by the following formula (1): (wherein * means a bond to the spacer S, R １ and R ２ are each independently H, alkyl optionally substituted with one or more substituents (preferably C １－６ alkyl), aryl (preferably C) optionally substituted with one or more substituents ６－１０ aryl), and heteroaryl (preferably 5- to 15-membered heteroaryl) optionally substituted with one or more substituents. The spacer S is selected from the group consisting of -S1-, -S2-, and -S3-E-S4-, and S1 is selected from the group consisting of one or more substituents R ３alkylene (preferably C １－３０ alkylene), and R ３ is -N(R ３１ ) ２ and heteroaryl (preferably 5- to 15-membered heteroaryl) optionally substituted with one or more substituents, ３１ are each independently H, alkyl optionally substituted with one or more substituents (preferably C １－６ alkyl), substituted carbonyl, and -C(=NH)NH ２ S2 is an alkylene having 2 or more carbon atoms (preferably an alkylene having 2 or more and 30 or less carbon atoms), and has at least one terminal and / or one or more adjacent —CH ２ Between -O-, -CO-, -NR ａ -, -CO-NR ａ -, -NR ａ -CO-, a group represented by formula (L-1), or a group represented by formula (L-2) is bonded to the group, and the group is one or more substituents R ３ R ａ is H or alkyl optionally substituted with one or more substituents (preferably C １－６ alkyl), and the formulas (L-1) and (L-2) are the following formulas: (wherein m is 0 or 1, and n is 0 or 1.) S3 and S4 are each independently selected from the group consisting of S1 and S2, and E is a cleavable site. [Item 2] The functional compound or salt thereof according to Item 1, wherein S is -S3-E-S4-. [Item 3] The functional compound or salt thereof according to Item 1 or 2, wherein E is selected from the group consisting of the following formulae (E-1), (E-2), (E-3), (E-4), (E-5), (E-6), (E-7), (E-8), and (E-9). (wherein, one wavy line represents a bond with S3, the other wavy line represents a bond with S4, and Q １ , Q ２ , Q ３ , Q ４ , Q ５ , and Q ６are each independently H or alkyl (preferably C １－６ alkyl), and A １ , A ２ , A ３ , and A ４ are each independently an arylene (preferably C ６－１８ Pep is ENLYFQG (SEQ ID NO: 1), ENLYFQS (SEQ ID NO: 2), or X １ X ２ X ３ X ４ X ５ X ６ X ７ (X １ , X ２ , X ３ , X ５ , X ６ , and X ７ are each independently an amino acid residue other than K or R, and X ４ is K or R.) [Item 4] S3 and S4 are each independently selected from S2, preferably S3 is an alkylene having 2 or more carbon atoms (preferably an alkylene having 2 to 30 carbon atoms) in which -O- is bonded to the R-terminal and -CH ２ Between -O-, -CO-, -NR ａ -, -CO-NR ａ -, -NR ａ -CO-, a group represented by formula (L-1), or a group represented by formula (L-2) may be bonded, and the group may have one or more substituents R ３ and / or S4 is an alkylene having 2 or more carbon atoms (preferably an alkylene having 2 to 30 carbon atoms) in which —NH— is bonded to the Tag side terminal and the E side terminal and / or one or more adjacent —CH ２ Between -O-, -CO-, -NR ａ -, -CO-NR ａ -, -NR ａ -CO-, a group represented by formula (L-1), or a group represented by formula (L-2) may be bonded, and the group may have one or more substituents R ３[Item 5] S3 is a branched alkylene having 2 or more carbon atoms (preferably a branched alkylene having 2 to 30 carbon atoms), and the R-terminal end of the main chain, the E-terminal end of the main chain, and / or one or more adjacent —CH ２ Between -O-, -CO-, -NR ａ -, -CO-NR ａ -, -NR ａ -CO-, a group represented by formula (L-1), or a group represented by formula (L-2) is bonded, and the end of the side chain of the group is a substituent R ３ and / or S4 is a branched alkylene having 2 or more carbon atoms (preferably a branched alkylene having 2 to 30 carbon atoms) having an E-side terminal of the main chain, a Tag-side terminal of the main chain, and / or one or more adjacent —CH ２ Between -O-, -CO-, -NR ａ -, -CO-NR ａ -, -NR ａ -CO-, a group represented by formula (L-1), or a group represented by formula (L-2) is bonded, and the end of the side chain of the group is a substituent R ３ [Item 6] The functional compound or salt thereof according to any one of Items 1 to 4, wherein -S3-E-S4- is -O-alkylene-(E-1)-(alkylene-O) ａ -Alkylene-NH-, -O-Alkylene-NH-CO-Alkylene-(E-1)-(Alkylene-O) ａ -Alkylene-NH-, -O-Alkylene-(E-1)-Alkylene-NH-CO-Alkylene-NH-CO-(Alkylene-O) ａ -Alkylene-NH-, -O-Alkylene-(E-1)-Alkylene-NH-CO-Alkylene-(L-1)-Alkylene-CO-NH-(Alkylene-O) ａ -Alkylene-NH-, -Alkylene-(E-1)-(Alkylene-O) ａ -Alkylene-NH-, -Alkylene-(E-1)-Alkylene-NH-CO-Alkylene-NH-CO-(Alkylene-O) ａ -Alkylene-NH-, -O-Alkylene-(E-6)-Alkylene-NH-CO-(Alkylene-O)ａ -Alkylene-NH-, and -O-alkylene-NH-CO-alkylene-(E-7)-alkylene-NH-CO-(alkylene-O) ａ -alkylene-NH-, each alkylene in the group is preferably C １－６ alkylene, and each alkylene in the group is a substituent R ３ The functional compound or salt thereof according to any one of items 1 to 5, wherein a is an integer of 1 or more (preferably an integer of 1 to 4). [Item 7] -S3-E-S4- is -O-alkylene-NH-CO-branched alkylene-(E-1)-(alkylene-O) ａ -Alkylene-NH-, -O-alkylene-(E-1)-alkylene-NH-CO-branched alkylene-NH-CO-(alkylene-O) ａ -Alkylene-NH-, and -Alkylene-(E-1)-Alkylene-NH-CO-Branched alkylene-NH-CO-(Alkylene-O) ａ -alkylene-NH-, wherein the alkylene and branched alkylene in the group are each preferably C １－６ Alkylene and branched chain C １－６ alkylene, and the branched alkylene of the group is a substituent R ３ Item 8: The functional compound or salt thereof according to Item 6, having R ３ is -N(R ３１ ) ２ and heteroaryl (preferably 5-15 membered heteroaryl) optionally substituted with -alkylene-SO3M, where M is H or an alkali metal, preferably H, Na, or K, more preferably Na, wherein the alkylene is preferably C １－６ alkylene, and two R ３１ each independently represents H, C optionally substituted with one or more substituents, １－６ Alkyl, -C(=O)R ３２ , and -C(=NH)NH ２ and R ３２[Item 9] The functional compound or salt thereof according to any one of Items 1 to 8, wherein the protein purification tag site Tag is selected from the group consisting of the following formulae (2-1), (2-2), (2-3), and (2-4): (wherein * represents a bond to the spacer S, and p is an integer of 1 to 10.) [Item 9a] R １ and R ２ are each independently H and C optionally substituted with one or more substituents. １－６ [Item 9b] The functional compound or salt thereof according to any one of Items 1 to 9 and Item 9a, wherein R is a group represented by the following formula (1A) or (1B): (In the formula, *, R １ , and R ２ has the same meaning as above.) [Item 9c] A composition comprising the functional compound or salt thereof according to any one of Items 1 to 9 and 9a, in which R is a group represented by the following formula (1A), and the functional compound or salt thereof according to any one of Items 1 to 9 and 9a, in which R is a group represented by the following formula (1B): (In the formula, *, R １ , and R ２ has the same meaning as above, and R in formula (1A) １ is R in formula (1B) １ is the same as R in Equation (1A). ２ is R in formula (1B) ２ ) [Item 9d] R １ and R ２ One of them is H and the other is C １－６ [Item 9e] The functional compound or salt thereof according to any one of items 1 to 9, 9a, and 9b, wherein R is alkyl. １ and R ２ [Item 9f] The functional compound or salt thereof according to any one of items 1 to 9, 9a, and 9b, wherein one of the following is H and the other is methyl. [Item 9f] S is -S3-E-S4-, and S3 is -O-C １－６Alkylene-, -O-C １－６ Alkylene-NH-CO-(NH ２ , NHT, NH(C １－６ alkyl), N(C １－６ alkyl) ２ , 1-(M-SO ３ -C １－６ (alkylene)-1,2,3-triazol-4-yl, and NH ２ C(=NH)NH ２－６ alkylene)- or C １－６ The functional compound or salt thereof according to any one of items 1 to 9, 9a, 9b, 9d, and 9e, wherein M is alkylene, M is H or an alkali metal, and T is any one of the following formulae (T-1), (T-2), (T-3), (T-4), (T-5), (T-6), (T-7), (T-8), (T-9), and (T-10): [Item 9g] S is -S3-E-S4- and S3 is -O-C ６ H １２ -, -O-C ６ H １２ -NH-CO-C ２ H ４ -, -O-C ６ H １２ -NH-CO-CH(-C ４ H ８ NH ２ )-, -O-C ６ H １２ -NH-CO-CH(-C ４ H ８ NHT)- or -C ２ H ４ [Item 9h] The functional compound or salt thereof according to Item 9f, wherein S is -S3-E-S4- and S4 is -(C １－６ Alkylene-O) １－４ -C １－６ Alkylene-NH-, -C １－６ Alkylene-NH-CO-(C １－６ Alkylene-O) １－４ -C １－６ Alkylene-NH-, -C １－６ Alkylene-NH-CO-(1-(M-SO ３ -C１－６ (alkylene)-1,2,3-triazol-4-yl, NH ２ , NH(C １－６ alkyl), N(C １－６ alkyl) ２ , N.H. ２ C(=NH)NH and C having a branched chain optionally having a substituent selected from NHT ２－６ Alkylene)-NH-CO-(C １－６ Alkylene-O) １－４ -C １－６ Alkylene-NH- or -C １－６ Alkylene-NH-CO-C １－６ Alkylene-(L-1)-C １－６ Alkylene-CO-NH-(C １－６ Alkylene-O) １－４ -C １－６ The functional compound or salt thereof according to any one of items 1 to 9, 9a, 9b, 9d, 9e, 9f, and 9g, wherein M is alkylene-NH-, M is H or an alkali metal, and T is any one of the following formulae (T-1), (T-2), (T-3), (T-4), (T-5), (T-6), (T-7), (T-8), (T-9), and (T-10): [Item 9i] S is -S3-E-S4- and S4 is -(C ２ H ４ O) ４ -C ２ H ４ -NH-, -C ２ H ４ -NH-CO-(C ２ H ４ O) ４ -C ２ H ４ -NH-, -CH(CH ３ ) ２ -CH ２ -NH-CO-(C ２ H ４ O) ４ -C ２ H ４ -NH-, -C ５ H １０ -NH-CO-(1-NaSO ３ C ３ H ６-1,2,3-triazol-4-yl-CH ２ - )CH-NH-CO-(C ２ H ４ O) ４ -C ２ H ４ -NH-、-CH ２ -NH-CO-(NH ２ C ４ H ８ - )CH-NH-CO-(C ２ H ４ O) ４ -C ２ H ４ -NH-、-CH ２ -NH-CO-(N(CH ３ ) ２ -C ４ H ８ - )CH-NH-CO-(C ２ H ４ O) ４ -C ２ H ４ -NH-、-CH ２ -NH-CO-(NH ２ -C(=NH)-NH-C ３ H ６ - )CH-NH-CO-(C ２ H ４ O) ４ -C ２ H ４ -NH-、或 -CH ２ -NH-CO-C ２ H ４ -(L-1)-C ２ H ４ -CO-NH-CH ２ -(C ２ H ４ O) ３ -C ３ H ６[Item 9j] A composition comprising the functional compound or salt thereof according to any one of Items 1 to 9, 9a, 9b, 9d, 9e, 9f, and 9g, wherein R is a group represented by the following formula (1A): [Item 9j] A composition comprising the functional compound or salt thereof according to any one of Items 9d, 9e, 9f, 9g, 9h, and 9i, wherein R is a group represented by the following formula (1B): [Item 9j] A composition comprising the functional compound or salt thereof according to any one of Items 9d, 9e, 9f, 9g, 9h, and 9i, wherein R is a group represented by the following formula (1B): (In the formula, *, R １ , and R ２ has the same meaning as above, and R in formula (1A) １ is R in formula (1B) １ is the same as R in Equation (1A). ２ is R in formula (1B) ２ (Item 10) The functional compound or salt thereof according to any one of Items 1 to 9, 9a, and 9b, selected from the group consisting of functional compounds represented by any one of the following formulae (P1), (P2), (P3), (P4), (P5), (P6), (P7), (P8), (P9), (P10), (P11), (P12), (P13), (P14), (P15), (P16), (P17), (P18), (P19), (P20), (P21), (P22), (P23), (P24), (P25), (P26), (P27), and (P28) and salts thereof. In (P5) and (P6), M is H or an alkali metal, and in (P9) and (P10), T is any one of the following formulae (T-1), (T-2), (T-3), (T-4), (T-5), (T-6), (T-7), (T-8), (T-9), and (T-10). [Item 10a] A composition comprising a functional compound represented by formula (P1) according to item 10 or a salt thereof, and a functional compound represented by formula (P2) according to item 10 or a salt thereof; a composition comprising a functional compound represented by formula (P3) according to item 10 or a salt thereof, and a functional compound represented by formula (P4) according to item 10 or a salt thereof; a composition comprising a functional compound represented by formula (P5) according to item 10 or a salt thereof, and a functional compound represented by formula (P6) according to item 10 or a salt thereof; a composition comprising a functional compound represented by formula (P7) according to item 10 or a salt thereof, and a functional compound represented by formula (P8) according to item 10 or a salt thereof; a composition comprising a functional compound represented by formula (P9) according to item 10 or a salt thereof, and a functional compound represented by formula (P10) according to item 10 or a salt thereof; The present invention includes a functional compound represented by formula (P11) or a salt thereof according to item 10 and a functional compound represented by formula (P12) or a salt thereof according to item 10; a functional compound represented by formula (P13) or a salt thereof according to item 10 and a functional compound represented by formula (P14) or a salt thereof according to item 10; a functional compound represented by formula (P15) or a salt thereof according to item 10 and a functional compound represented by formula (P16) or a salt thereof according to item 10; a functional compound represented by formula (P17) or a salt thereof according to item 10 and a functional compound represented by formula (P18) or a salt thereof according to item 10; a functional compound represented by formula (P19) or a salt thereof according to item 10 and a functional compound represented by formula (P20) or a salt thereof according to item 10; A composition comprising a functional compound represented by formula (P21) or a salt thereof according to item 10 and a functional compound represented by formula (P22) or a salt thereof according to item 10, or a functional compound represented by formula (P23) or a salt thereof according to item 10 and a functional compound represented by formula (P24) or a salt thereof according to item 10, or a functional compound represented by formula (P25) or a salt thereof according to item 10 and a functional compound represented by formula (P26) or a salt thereof according to item 10, or a functional compound represented by formula (P27) or a salt thereof according to item 10 and a functional compound represented by formula (P28) or a salt thereof according to item 10. [Item 11] A functional compound or a salt thereof according to any one of items 1 to 10 and items 9a and 9b, selected from the group consisting of: In (P9) and (P10), T is any one of the following formulas (T-1), (T-2), (T-3), (T-4), (T-5), (T-6), (T-7), (T-8), (T-9), and (T-10). [Item 11a] The composition includes a functional compound represented by formula (P1) according to item 11 and a functional compound represented by formula (P2) according to item 11; a functional compound represented by formula (P3) according to item 11 and a functional compound represented by formula (P4) according to item 11; a functional compound (salt) represented by formula (P5a) according to item 11 and a functional compound (salt) represented by formula (P6a) according to item 11; a functional compound (salt) represented by formula (P7a) according to item 11 and a functional compound (salt) represented by formula (P8a) according to item 11; a functional compound represented by formula (P9) according to item 11 and a functional compound represented by formula (P10) according to item 11; a functional compound represented by formula (P11) according to item 11 and a functional compound represented by formula (P12) according to item 11; The composition includes a functional compound (salt) represented by formula (P13a) according to item 11 and a functional compound (salt) represented by formula (P14a) according to item 11; a functional compound (salt) represented by formula (P15a) according to item 11 and a functional compound (salt) represented by formula (P16a) according to item 11; a functional compound (salt) represented by formula (P17) according to item 11 and a functional compound (salt) represented by formula (P18) according to item 11; a functional compound (salt) represented by formula (P19a) according to item 11 and a functional compound (salt) represented by formula (P20a) according to item 11; a functional compound (salt) represented by formula (P21a) according to item 11 and a functional compound (salt) represented by formula (P22a) according to item 11; A composition comprising a functional compound (salt) represented by formula (P23a) according to item 11 and a functional compound (salt) represented by formula (P24a) according to item 11, or a functional compound represented by formula (P25) according to item 11 and a functional compound represented by formula (P26) according to item 11, or a functional compound represented by formula (P27) according to item 11 and a functional compound represented by formula (P28) according to item 11.[Item 11b] A functional compound or a salt thereof according to any one of Items 1 to 10 and Items 9a and 9b, selected from the group consisting of the compounds of Examples 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14-1, 14-2, 14-3, 14-4, 14-5, 14-6, 14-7, 14-8, 14-9, and 14-10.

[0011] [Item 12] A method for detecting a protein having a tyrosine residue, comprising the following steps (1) and (2): (1) binding a tyrosine residue reactive site R in the functional compound or salt thereof contained in any of Items 1 to 11, 9a, 9b, 9d, 9e, 9f, 9g, 9h, 9i, and 11b, or in the composition described in any of Items 9c, 9j, 10a, and 11a, to a tyrosine residue in the protein; and (2) binding a labeling substance that binds to the protein purification tag site Tag in the functional compound or salt thereof bound to the protein, and detecting the label. [Item 12a] The method according to Item 12, wherein the functional compound or salt thereof is the functional compound or salt thereof described in any of Items 2 to 11, 9a, 9b, 9d, 9e, 9f, 9g, 9h, 9i, and 11b. [Item 13-1] A method for detecting or identifying a cell surface protein, comprising the following steps (A), (B), (C), (D), and (E): (A) reacting cells or tissues in a sample with the functional compound or a salt thereof according to any one of Items 2 to 11 and Items 9a, 9b, 9d, 9e, 9f, 9g, 9h, 9i, and 11b, or the functional compound or a salt thereof contained in the composition according to any one of Items 9c, 9j, 10a, and 11a, to bind a tyrosine residue reactive site R in the functional compound or a salt thereof to a tyrosine residue of a cell surface protein; (B) purifying a fraction containing the cell surface protein bound to the functional compound or a salt thereof; (C) binding a labeling substance to the protein purification tag site Tag in the functional compound or a salt thereof in the purified fraction to obtain a complex; (D) cleaving the complex at the cleavable site E; and (E) detecting or identifying the cell surface protein from the cell surface protein-binding fragments after cleavage. [Item 13-2] A method for detecting or identifying a cell surface protein, comprising the following steps (A), (B), (C), and (E):(A) reacting a functional compound or a salt thereof according to any one of Items 1 to 11 and Items 9a, 9b, 9d, 9e, 9f, 9g, 9h, 9i, and 11b, or a functional compound or a salt thereof contained in a composition according to any one of Items 9c, 9j, 10a, and 11a, with cells or tissues in a sample to bind a tyrosine residue reactive site R in the functional compound or a salt thereof to a tyrosine residue of a cell surface protein; (B) purifying a fraction containing a cell surface protein bound with the functional compound or a salt thereof; (C) binding a labeling substance to a protein purification tag site Tag in the functional compound or a salt thereof according to any one of Items 1 to 11 and Items 9a, 9b, 9d, 9e, 9f, 9g, 9h, 9i, and 11b in the purified fraction to obtain a complex; (E) A step of detecting or identifying a cell surface protein from the complex or its cell surface protein-binding fragment. [Item 14-1] The method of Item 13-1 or 13-2, wherein the step (A) is a step of reacting the functional compound or its salt with cells or tissues in the sample by activating it with hydrogen peroxide and peroxidase, thereby binding the tyrosine residue reactive site R in the functional compound or its salt to a tyrosine residue of the cell surface protein. [Item 14-2] The method of Item 13-1, 13-2, or 14-1, wherein the step (B) is a step of purifying a fraction containing the cell surface protein bound to the functional compound or its salt by lysing the cells. [Item 14-3] The method of Item 13-1, 13-2, 14-1, or 14-2, wherein the step (C) is a step of reacting a protein purification tag site Tag in the functional compound or its salt with a labeling substance that binds to Tag, and purifying the reaction product. [Item 14-4] The method according to Item 13-1, 14-1, 14-2, or 14-3, wherein step (D) is a step of chemically or biochemically cleaving the complex at cleavable site E. [Item 14-5] The method according to Item 13-1, 13-2, 14-1, 14-2, 14-3, or 14-4, wherein step (E) is a step of detecting or identifying a cell surface protein obtained by purifying and / or hydrolyzing the cell surface-binding domain fragment.[Item 15-1] Use of the functional compound or salt thereof according to any one of Items 1 to 11 and Items 9a, 9b, 9d, 9e, 9f, 9g, 9h, 9i, and 11b, or the composition according to any one of Items 9c, 9j, 10a, and 11a, for detecting or identifying a cell surface protein. [Item 15-2] Use of the functional compound or salt thereof according to any one of Items 1 to 11 and Items 9a, 9b, 9d, 9e, 9f, 9g, 9h, 9i, and 11b, or the composition according to any one of Items 9c, 9j, 10a, and 11a, in the method according to Items 13-1, 13-2, 14-1, 14-2, 14-3, or 14-4. [Item 16-1] The functional compound or salt thereof according to any one of Items 1 to 11 and Items 9a, 9b, 9d, 9e, 9f, 9g, 9h, 9i, and 11b, or the composition according to any one of Items 9c, 9j, 10a, and 11a, for use in detecting or identifying a cell surface protein. [Item 16-2] The functional compound or salt thereof according to any one of Items 1 to 11 and Items 9a, 9b, 9d, 9e, 9f, 9g, 9h, 9i, and 11b, or the composition according to any one of Items 9c, 9j, 10a, and 11a, for use in the method according to Items 13-1, 13-2, 14-1, 14-2, 14-3, or 14-4. [Item 17-1] Use of the functional compound or salt thereof according to any one of Items 1 to 11 and Items 9a, 9b, 9d, 9e, 9f, 9g, 9h, 9i, and 11b to produce a composition for use in detecting or identifying a cell surface protein. [Item 17-2] Use of the functional compound or salt thereof according to any one of Items 1 to 11 and Items 9a, 9b, 9d, 9e, 9f, 9g, 9h, 9i, and 11b to produce a composition for use in the method according to Items 13-1, 13-2, 14-1, 14-2, 14-3, or 14-4.

[0012] The functional compound or salt thereof of the present disclosure is useful in comprehensive analysis of cell surface proteins, enabling, for example, highly sensitive, simple, and comprehensive analysis of cell surface proteins. Furthermore, the functional compound or salt thereof of the present disclosure enables, for example, region-limited proximity-dependent labeling easily and relatively inexpensively. Furthermore, since the functional compound or salt thereof of the present disclosure allows for specific labeling sites, it can be applied to analyses that depend on peptide sequence identification, such as phosphorylation fluctuation analysis.

[0013] Figure 1 shows the distribution of the ratio of detected surface proteins compared to Comparative Example 1 in the Example. Figure 2 shows the distribution of detected surface and non-surface proteins compared to Comparative Example in the Example. Figure 3 shows the total number of cell surface proteins among the identified proteins. Figure 4 shows the total number of non-cell surface proteins among the identified proteins. Figure 5 shows the number of surface proteins in the top 100 identified proteins. Figure 6 shows the number of detected chemical probe modification sites. Figure 7 shows the intramolecular localization of amino acids modified with chemical probes (EGFR). Figure 8 shows cell staining. Figure 9 shows an example of a product ion spectrum. Figure 10 shows the TMT quantification value of CD7, which is highly expressed in Jurkat cells. Figure 11 shows the results of an analysis of interacting molecules using proximity labeling. Figure 12 shows the results of an analysis of receptor internalization following stimulation with EGF addition. Figure 13 shows the results of an analysis of changes in membrane protein phosphorylation following (1) stimulation with EGF addition and (2) stimulation with EGF and Gefitinib addition. Figure 14 shows the results of an analysis of labeled peptides following the addition of the anti-TFRC antibody OKT9. Figure 15 shows the labeling sites assigned to the known structural information of TFRC (PDB: 7ZQS).

[0014] (1) Explanation of Terms Used in This Specification Unless otherwise specified, the terms used in this specification have the meanings explained below.

[0015] In this specification, the term "comprise" encompasses the concepts of "essentially consist of" and "consist of."

[0016] The disclosures of all patent and non-patent publications cited herein are hereby incorporated by reference in their entirety.

[0017] As used herein, "alkyl" refers to a group of the formula "-C ｐ H ２ｐ＋１ Alkyl is a monovalent radical derived from a straight-chain or branched saturated aliphatic hydrocarbon alkane, represented by the formula (p) (where p is an integer of 1 or greater). Alkyl is a group containing 1 to 6 carbon atoms (C １－６ ) straight or branched alkyl. １－６ Specific examples of alkyl include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, isohexyl, 3-methylpentyl, etc. Alkyl also includes alkyl in which at least a portion of the hydrogen atoms has been substituted with deuterium atoms.

[0018] In the present specification, examples of the substituents in "alkyl optionally substituted with one or more substituents" include the following: (A) halogen (e.g., F, Cl, Br, I) (B) -CN (C) -NO2 (D) -NH2 (E) -OH (F) -CHO (G) -COOH or a salt or ester thereof (H) -S03H or a salt or ester thereof (I) -S02H (J) -SH (K) = O (L) = S (M) -PO3H2 or a salt or ester thereof (N) alkenyl (O) alkynyl (P) cycloalkyl (Q) cycloalkenyl (R) aryl (S) heterocyclyl (T) a group formed by combining two or more of these

[0019] As used herein, "alkylene" refers to a group having the formula "-[C(Z １ )(Z ２ )] ｐ -" (Z １ and Z ２ are each independently H or alkyl, and p is an integer of 1 or greater. １－３０), and straight-chain or branched-chain alkylene having 2 or more carbon atoms (particularly, 2 to 30 carbon atoms). Specific examples of alkylene (particularly, alkylene having 30 or less carbon atoms) include methylene, ethylene, 1-methylethylene, 2-methylethylene, trimethylene, 2-methyltrimethylene, 2,2-dimethyltrimethylene, 1-methyltrimethylene, methylmethylene, ethylmethylene, dimethylmethylene, tetramethylene, pentamethylene, and hexamethylene.

[0020] As used herein, "alkenyl" refers to a group of the formula "-C ｑ H ２ｑ－１ Alkenyl is a monovalent radical derived from an alkene of a straight-chain or branched unsaturated aliphatic hydrocarbon, represented by the formula (q) (where q is an integer of 2 or greater). Alkenyl is an alkyl group having 2 to 6 carbon atoms (C ２－６ ) Specific examples of alkenyl include vinyl and allyl.

[0021] As used herein, "alkynyl" refers to a group of the formula "-C ｑ H ２ｑ－３ Alkynyl is a monovalent radical derived from an alkyne of a straight-chain or branched unsaturated aliphatic hydrocarbon, represented by the formula (q) (where q is an integer of 2 or greater). Alkynyl is an alkyl group having 2 to 6 carbon atoms (C ２－６ ) Specific examples of alkynyl include ethynyl, propargyl, and the like.

[0022] As used herein, "cycloalkyl" refers to a monovalent radical derived from a cyclic saturated aliphatic hydrocarbon, cycloalkane. Cycloalkyl refers to a group having 3 to 18 carbon atoms (C 3－１８ ) Specific examples of cycloalkyl include cyclopropyl, cyclopentyl, cyclohexyl, and the like.

[0023] As used herein, "cycloalkenyl" refers to a monovalent radical derived from a cycloalkene, a cyclic unsaturated aliphatic hydrocarbon. Cycloalkenyl is a group of 5 to 18 carbon atoms (C ５－１８Specific examples of cycloalkenyl include cyclopentenyl, cyclohexynyl, and the like.

[0024] As used herein, "aryl" refers to a monovalent radical derived from a monocyclic or polycyclic, such as bicyclic or tricyclic, aromatic hydrocarbon. Aryl refers to an aromatic hydrocarbon having 6 to 18 carbon atoms (C ６－１８ aryl having 6 to 10 carbon atoms (C ６－１０ ) Specific examples of aryl include phenyl, naphthyl, biphenylyl, anthryl, phenanthryl, and terphenylyl. Aryl may have one or more substituents, and specific examples of the substituents include alkyl, the substituents (A), (B), (C), (D), (E), (F), (G), (H), (I), (J), (K), (L), (M), (N), (O), (P), and (Q) exemplified as alkyl substituents, and combinations thereof.

[0025] As used herein, "arylene" refers to a divalent radical derived from a monocyclic or polycyclic, such as bicyclic or tricyclic, aromatic hydrocarbon. Arylene is a group having 6 to 18 carbon atoms (C ６－１８ ) Specific examples of arylene include phenylene, naphthylene, biphenyldiyl, anthrylene, phenanthrylene, and terphenyldiyl. The arylene may have one or more substituents, and specific examples of the substituents include alkyl, the substituents (A), (B), (C), (D), (E), (F), (G), (H), (I), (J), (K), (L), (M), (N), (O), (P), and (Q) exemplified as alkyl substituents, and combinations thereof.

[0026] As used herein, "heterocyclyl" refers to a monovalent radical derived from a monocyclic or polycyclic (e.g., bicyclic, tricyclic) heterocyclic compound. Heterocyclyl encompasses heterocyclyls (3- to 15-membered) having 1 to 5 heteroatoms independently selected from the group consisting of N, O, and S as ring-membering atoms, and / or having 3 to 15 ring-membering atoms. Heterocyclyl includes heteroaryl. Heterocyclyl may have one or more substituents. Specific examples of the substituents include alkyl, the substituents (A), (B), (C), (D), (E), (F), (G), (H), (I), (J), (K), (L), (M), (N), (O), (P), (Q), and (R) exemplified as alkyl substituents, and combinations thereof.

[0027] As used herein, the term "heteroaryl" refers to a monovalent radical derived from a monocyclic or polycyclic (e.g., bicyclic, tricyclic) aromatic heterocyclic compound. Heteroaryl encompasses 5- to 15-membered heteroaryls having 1 to 5 heteroatoms independently selected from the group consisting of N, O, and S as ring-constituting atoms and / or 5 to 15 ring-constituting atoms. Specific examples of heteroaryls include pyrrolyl, pyrazolyl, imidazolyl, triazolyl, pyridyl, pyridazyl, pyrimidyl, pyrazyl, triazyl, indolyl, quinolinyl, oxazolyl, and thiazolyl. Heteroaryls may have one or more substituents. Specific examples of the substituents include alkyl, the substituents (A), (B), (C), (D), (E), (F), (G), (H), (I), (J), (K), (L), (M), (N), (O), (P), (Q), and (R) exemplified as alkyl substituents, and combinations thereof.

[0028] As used herein, the terms "protein" and "peptide" have substantially the same meaning and refer to amino acid polymers of any length (peptides are typically referred to as fragments of proteins). Amino acid polymers may be linear, branched, or cyclic. The amino acids may be natural or unnatural amino acids, or mutant amino acids. Examples of amino acids include glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I), serine (S), threonine (T), aspartic acid (D), glutamic acid (E), cysteine ​​(C), methionine (M), lysine (K), arginine (R), asparagine (N), glutamine (Q), proline (P), phenylalanine (F), tryptophan (W), histidine (H), tyrosine (Y), citrulline, ornithine, ε-acetyl-lysine, β-alanine, aminobenzoic acid, 6-aminocaproic acid, aminobutyric acid, hydroxyproline, mercaptopropionic acid, 3-nitrotyrosine, norleucine, pyroglutamic acid, etc. Amino acids may be, for example, L-amino acids, D-amino acids, or DL-amino acids.

[0029] The protein may be naturally occurring or synthetic, or may have a synthetically engineered sequence in a naturally occurring protein. The protein may be an intracellular protein, a cell surface protein (i.e., a protein bound to the surface of a cell), or a protein in solution (e.g., a protein secreted into the medium). The protein may also be a glycoprotein or a membrane protein. The protein may be any medicinally or commercially relevant protein with useful biological or chemical activity, such as a receptor, antibody, enzyme, hormone, regulator, antigen, or binding agent. The proteins listed below that may be used in the present disclosure are merely exemplary and are not intended to be limiting. One of ordinary skill in the art will appreciate that any protein containing a tyrosine residue may be a target protein for one or more functional compounds or salts thereof.

[0030] As used herein, a "target protein" may be a protein having tyrosine residues to which one or more functional compounds or salts thereof can bind. Such target proteins are preferably, but not limited to, those that can be present in biological fluids, on cells, or in tissues derived from mammals, including humans and non-human animals. More preferably, they are proteins that are expressed and present on living cells or living tissues. For example, if the target protein is a membrane protein expressed on the surface of a living cell, it will be associated with the cell membrane of the living cell, have at least one tyrosine residue exposed to the extracellular space, and be in a state where a functional compound or a salt thereof can bind.

[0031] The target protein is not particularly limited, but a membrane protein is preferred. A membrane protein is a protein that can directly or indirectly interact with a lipid membrane, particularly a lipid bilayer membrane, and in which one or more amino acid residues (at least tyrosine residues) that constitute the protein are present in the extracellular environment. Examples of membrane proteins include G protein-coupled receptors, seven-transmembrane receptors, receptor tyrosine kinases, the immunoglobulin superfamily and related proteins, scavenger receptors, and other receptors; transporters, ion channels, solution transporters, active transporters, auxiliary transporter proteins, enzymes, and others.

[0032] More specifically, examples of G protein-coupled receptors and seven-transmembrane receptors include Frizzled receptors, nucleic acid receptors, adenosine receptors, adrenergic receptors, angiotensin receptors, apelin receptors, vasopressin receptors, bradykinin receptors, bombesin receptors, chemokine receptors, cholecystokinin receptors, muscarinic acetylcholine receptors, cannabinoid receptors, cysteinyl leukotriene receptors, dopamine receptors, sphingolipid receptors, lysophosphatidin receptors, sphingosine monophosphate receptors, endothelin receptors, protease-activated receptors, free lipid receptors, galanin receptors, growth hormone secretagogue receptors, gonadotropin receptors, bile acid receptors, nicotinic acid receptors, lysophosphatidic acid receptors, anaferatoxin chemotaxis receptors, and gastrin receptors. Examples of the receptor include releasing peptide receptors, orexin receptors, histamine receptors, serotonin receptors, interleukin receptors, leucine-rich repeat-containing G protein-coupled receptors, leukotriene receptors, adrenocorticotropic hormone receptors, melanocortin receptors, melanin-concentrating hormone receptors, melatonin receptors, neuromedin receptors, neuropeptide receptors, neurotensin receptors, opioid receptors, nociceptin receptors, oxoglutarate receptors, oxytocin receptors, P2Y purinergic receptors, prostaglandin receptors, rhodopsin, relaxin receptors, somatostatin receptors, succinate receptors, substance P receptors, substance K receptors, thromboxane receptors, urotensin receptors, calcitonin receptors, taste receptors, metabotropic glutamate receptors, and olfactory receptors.

[0033] Examples of receptor tyrosine kinases include activin receptors, bone morphogenetic protein receptors, TNF-β receptors, AXL receptors, epidermal growth factor receptors (EGF receptors), ephrin receptors, insulin receptors, nerve growth factor receptors, discoidin domain receptors, vascular endothelial growth factor receptors, leukocyte receptors, hepatocyte growth factor receptors, macrophage-stimulating protein receptors, platelet-derived growth factor receptors, and enterotoxin receptors, as well as their precursors.

[0034] Examples of the immunoglobulin superfamily and its related proteins include immunoglobulin receptors, killer cell immunoglobulin-like receptors, leukocyte immunoglobulin-like receptors, netrin receptors, T cell receptors, various cytokine receptors, various fragments of the T cell surface glycoprotein CD, superfamilies, and their precursors.

[0035] Other receptors include, for example, adiponectin receptors, progestin receptors, contactin-associated proteins, delrins, integrins and their precursors, neurexins, neuropilins, Notch receptors, plexins and their precursors, receptor tyrosine phosphatases, selectins and their precursors, syndecan receptors, tumor necrosis factor (TNF) receptors, Toll-like receptors, transferrin receptors, and sortilin and its precursors.

[0036] Examples of transporters and ion channels include aquaporins, chloride ion channels, bestrophin, ryanodine receptors, voltage-gated potassium channels, cyclic nucleotide-gated channels, calcium-activated potassium channels, transient receptor potential channels (TRP channels), voltage-gated sodium channels, voltage-gated calcium channels, other voltage-gated channels, serotonin receptors, acetylcholine receptors, gamma-aminobutyric acid receptors, glycine receptors, ionotropic glutamate receptors, and P2X purinergic receptors.

[0037] Examples of solution transporters include various SLC family proteins, and examples of active transporters include ion-transporting ATPases and ABC transporters.

[0038] Examples of enzymes include NADH-ubiquinone oxidoreductase, cytochrome c, flavin-containing monooxygenase, cytochrome P450, and other oxidoreductases; acyltransferases, glucosetransferases, sulfotransferases, and other transferases; ligases, tyrosine phosphatases, phosphodiester hydrolases, glycosylases, serine peptide endohydrolases, metalloendopeptidases, nucleoside diphosphate phosphatases, and other hydrolases.

[0039] As used herein, the term "sample" or "biological sample" encompasses any cell (e.g., living cell) or tissue (e.g., living tissue), and any solid or fluid sample obtained from an organism. These include, for example, cell and tissue cultures, bioreactors, cells and tissues from humans or non-human animals, plants (including fruits and vegetables), unicellular microorganisms (e.g., bacteria, yeast), and multicellular organisms. Biological samples can be, for example, blood, plasma, serum, urine, bile, semen, cerebrospinal fluid, aqueous humor, or vitreous humor, or any bodily secretion, transudate, or exudate (e.g., fluid obtained from an abscess or any other site of infection or inflammation), or fluid obtained from a joint (e.g., biological fluid obtained from a normal joint or a joint affected by a disease such as rheumatoid arthritis, osteoarthritis, gout, or septic arthritis). Biological samples can also be, for example, samples obtained from any organ or tissue (including biopsy or autopsy specimens). Cells also include, for example, primary cells and cultured cells.

[0040] Preferably, the cells or tissues are living cells or tissues derived from a human or non-human animal.

[0041] "Cells" or "living cells" are not particularly limited as long as they express a protein, preferably a membrane protein, that can be detected using a functional compound or a salt thereof. Specific examples include, but are not limited to, CHO cells, MDCK cells, 3T3-L1 cells, 293 cells, MCF7 cells, A431 cells, 3T3 cells, CV-I cells, HeLa cells, L cells, BHK21 cells, HL-60 cells, U937 cells, HaK cells, Jurkat cells, THP-1 cells, and other cell lines obtained by transformation. Also included are established cell lines derived from mammalian or non-mammalian origin that have not been cultured and subjected to denaturing treatment; cells isolated from any organ or tissue under non-denaturing conditions (e.g., blood cells, organ-constituting cells, primary cultured cells, etc.); cell lines derived from in vitro cultures of primary tissues and primary grafts; and cells cultured in three-dimensional culture systems in addition to two-dimensional cultures (e.g., organoid cultures, etc.). Furthermore, cells into which a gene encoding a specific protein has been introduced using a vector or the like using these as a host may also be used, and examples thereof include, but are not limited to, CHO-K1 cells that stably express dopamine D2 receptors.

[0042] The "tissue" or "living tissue" is not particularly limited as long as it expresses a protein, preferably a membrane protein, that can be detected using a functional compound or a salt thereof. Specific examples include, but are not limited to, tissue slices and tissue culture samples (e.g., tissue slices and organ slice cultures of the brain, liver, kidney, etc.) isolated under non-denaturing conditions from any organ or tissue of a human or non-human mammal.

[0043] In this specification, the terms "alkylation reaction, hydrolysis reaction, amination reaction, esterification reaction, amidation reaction, etherification reaction, nucleophilic substitution reaction, addition reaction, oxidation reaction, and reduction reaction" refer to methods known per se. Examples of such methods include those described in "Experimental Chemistry Lectures" (5th edition, edited by the Chemical Society of Japan, Maruzen Co., Ltd.), "ORGANIC FUNCTIONAL GROUP PREPARATIONS" 2nd edition, Academic Press, Inc., 1989, "Comprehensive Organic Transformations" (VCH Publishers Inc., 1989), and "Greene's Protective Groups in Organic Synthesis" by P.G.M.Wuts and T.W.Greene, 4th edition, 2006; John Wiley & Sons, New York, 1991, p. 309.

[0044] In the present specification, protection and deprotection can be carried out by carrying out protection / deprotection reactions using commonly known protecting groups (see, for example, Protective Groups in Organic Synthesis, Fourth edition, T.W. Greene, John Wiley & Sons Inc. (2006)).

[0045] As used herein, the term "protecting group" includes, for example, a t-butoxycarbonyl (BOC) group, a 9-fluorenylmethyloxycarbonyl (Fmoc) group, a 2,2,2-trichloroethoxycarbonyl (Troc) group, a benzyloxycarbonyl (Z) group, an allyloxycarbonyl (Alloc) group, a trifluoroacetyl group, a phthaloyl group, a p-toluenesulfonyl (Ts) group, and a 2-nitrobenzenesulfonyl (Ns) group.

[0046] As used herein, the term "deprotecting agent" is not particularly limited as long as it can remove a protecting group, and examples thereof include bases such as piperidine and lithium diisopropylamide, zinc, acids such as TFA, nucleophiles in the presence of a zero-valent palladium catalyst, basic aqueous solutions, methylamine, hydrazine, one-electron reducing agents, and thiols.

[0047] As used herein, the term "solvent" refers to a solvent inert to the reaction, and examples thereof include water, ethers (e.g., linear ethers such as diethyl ether, 1,2-dimethoxyethane, diethylene glycol dimethyl ether, and ethylene glycol dimethyl ether; cyclic ethers such as dioxane and tetrahydrofuran), halohydrocarbons (e.g., methylene chloride, chloroform, 1,2-dichloroethane, and carbon tetrachloride), aromatic hydrocarbons (e.g., benzene, toluene, and xylene), alcohols (e.g., lower alcohols such as methanol, ethanol, and isopropanol), amides (e.g., N,N-dimethylformamide (DMF) and hexamethylphosphoric triamide), amines (e.g., cyclic amines such as N-methylpyrrolidone (NMP) and pyridine), sulfoxides (e.g., dimethyl sulfoxide (DMSO)), and nitriles (e.g., acetonitrile). These solvents may be used alone or in combination.

[0048] As used herein, the term "base" refers to, for example, inorganic bases, organic bases, etc. Examples of inorganic bases include alkali metal hydroxides (e.g., lithium hydroxide, sodium hydroxide, potassium hydroxide), alkaline earth metal hydroxides (e.g., magnesium hydroxide, calcium hydroxide, barium hydroxide), alkali metal carbonates (e.g., sodium carbonate, potassium carbonate, cesium carbonate), alkaline earth metal carbonates (e.g., magnesium carbonate, calcium carbonate, barium carbonate), alkali metal bicarbonates (e.g., sodium bicarbonate, potassium bicarbonate), alkali metal phosphates (e.g., sodium phosphate, potassium phosphate, cesium phosphate), alkaline earth metal phosphates (e.g., magnesium phosphate, calcium phosphate), alkali metal alkoxides (e.g., sodium methoxide, sodium ethoxide, potassium tert-butoxide), alkali metal hydrides (e.g., sodium hydride, potassium hydride), etc. Examples of organic bases include trialkylamines (e.g., trimethylamine, triethylamine (TEA), N,N-diisopropylethylamine (DIPEA)), dialkylamines (e.g., diethylamine, diisopropylamine), 4-dimethylaminopyridine (DMAP), N-methylmorpholine, picoline, 1,5-diazabicyclo[4.3.0]non-5-ene, 1,4-diazabicyclo[2.2.2]octane, and 1,8-diazabicyclo[5.4.0]-7-undecene (DBU). One or more of these may be appropriately selected and mixed for use.

[0049] As used herein, examples of the "condensing agent" include 1-hydroxybenzotriazole (HOBt), 3-hydroxy-3,4-dihydro-1,2,3-benzotriazin-4-one (HOOBt), N-hydroxysuccinimide (NHS), dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDAC), 2-(1H-7-azabenztriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate (HATU), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), 3,4-dihydro-1,2,3-benzotriazin-4-one-3-oxytetramethyluronium hexafluorophosphate (HDTU), benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP), benzotriazol-1-yloxytris-(pyrrolidino)-phosphonium hexafluorophosphate (PyBop), (3,4-dihydro-1,2,3-benzotriazin-4-one-3-oxy)diethylphosphate (DEPBt), 3,4-dihydro-1,2,3-benzotriazin-4-one-3-oxytris-(pyrrolidino)-phosphonium hexafluorophosphate (PDOP), 2-(benzotriazol-1-yloxy)-1,3 -dimethyl-2-pyrrolidin-1-yl-1,3,2-diazaphosphoridinium hexafluorophosphate (BOMP), 5-(1H-7-azabenzotriazol-1-yloxy)-3,4-dihydro-1-methyl 2H-pyrrolium hexachloroantimonate (AOMP), (1H-7-azabenzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (AOP), 5-(1H-benzotriazol-1-yl)-3,4-Dihydro-1-methyl 2H-pyrrolium hexachloroantimonate: N-oxide (BDMP), 2-bromo-3-ethyl-4-methylthiazolium tetrafluoroborate (BEMT), 2-bromo-1-ethylpyridinium tetrafluoroborate (BEP), 2-bromo-1-ethylpyridinium hexachloroantimonate (BEPH), benzotriazol-1-yloxy-N,N-dimethylmethaniminium hexachloroantimonate (BOMI), N,N'-bis(2-oxide) (iso-3-oxazolidinyl)phosphinic chloride (BOP-Cl), 1-(1H-benzotriazol-1-yloxy)phenylmethylenepyrrolidinium hexachloroantimonate (BPMP), 1,1,3,3-bis(tetramethylene)fluorouronium hexafluorophosphate (BTFFH), 4-(chloro-4-morpholinylmethylene)morpholinium hexafluorophosphate (CMMM), 2-chloro-1,3-dimethyl-1H-benzimidazolium hexafluorophosphate phosphate (CMBI), 2-fluoro-1-ethylpyridinium tetrafluoroborate (FEP), 2-fluoro-1-ethylpyridinium hexachloroantimonate (FEPH), 1-(1-pyrrolidinyl-1H-1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene)pyrrolidinium hexafluorophosphate N-oxide (HAPyU), O-(1H-benzotriazol-1-yl)-N,N,N',N'-bis-(pentamethylene)uronium hexafluorophosphine phosphate (HBPipU), O-(1H-benzotriazol-1-yl)N,N,N0,N0-bis(tetramethylene)urinium hexafluorophosphate (HBPyU), (1H-7-azabenzotriazol-1-yloxy)tris(pyrrolidino)phosphonium hexafluorophosphate (PyAOP), bromo-tripyrrolidinophosphonium hexafluorophosphate (PyBrOp), chloro-tripyrrolidinophosphonium hexafluorophosphate (PyClOP), 1,1,3,3-Bis(tetramethylene)chlorouronium hexafluorophosphate (PyClU), tetramethylfluoro-manmidinium hexafluorophosphate (TFFH), triphosgene, triazine reagents [cyanuric chloride, cyanuric fluoride, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM), 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT)], bis(2-chlorophenyl)phosphorochloridate, diphenylphosphorochloridate, diphenylphosphoroazide (DPPA), N-[1-(cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylamino(morpholino)]uronium Examples include hexafluorophosphate (COMU), N,N,N',N'-tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate (TSTU), etc.

[0050] (2) Functional Compound or Salt Thereof The functional compound or salt thereof of the present disclosure has a structure in which a tyrosine residue reactive site R is linked to a protein purification tag site Tag via a spacer S.

[0051] (2-1) Tyrosine Residue Reactive Site R In the functional compound or salt thereof of the present disclosure, the tyrosine residue reactive site R is not particularly limited as long as it has a structure capable of reacting with a tyrosine residue in a protein. Such a structure is preferably a luminol-derivative group or a group having a 2,3-dihydrophthalazine-1,4-dione skeleton, and is typically a group represented by the following formula (1):

[0052]

[0053] In formula (1), R １ and R ２ are each independently H, alkyl optionally substituted with one or more substituents (preferably C １－６ alkyl), aryl (preferably C) optionally substituted with one or more substituents ６－１０ aryl), and heteroaryl (preferably 5- to 15-membered heteroaryl) optionally substituted with one or more substituents. R １ and R２ are preferably each independently selected from the group consisting of H, alkyl which may be substituted with one or more substituents, and aryl which may be substituted with one or more substituents, and more preferably selected from the group consisting of H and alkyl which may be substituted with one or more substituents.

[0054] R １ and R ２ Suitable combinations of include: １ is alkyl optionally substituted with one or more substituents, R ２ Combination where is H ・R １ H, R ２ is alkyl optionally substituted with one or more substituents

[0055] In each of the above cases, R １ and / or R ２ The alkyl in is preferably C １－６ alkyl, more preferably C １－４ Alkyl, even more preferably C １－２ In each of the above cases, R １ and / or R ２ The alkyl may be unsubstituted or substituted, and in the case of a substituted alkyl, the number of substituents can be selected from the range of 1 or more to the maximum number of possible substituents, and can be, for example, 1 to 10, 1 to 8, 1 to 6, 1 to 4, 1 to 3, 1, or 2, etc.

[0056] In formula (1), * denotes a bond to the spacer S. The bonding position to the spacer S in formula (1) may be any of formulas (1A), (1B), (1C), and (1D) below, but is preferably formula (1A) or (1B) below.

[0057]

[0058] (2-2) Spacer S In the functional compound or a salt thereof of the present disclosure, the spacer S is not particularly limited as long as it has a structure that can link the tyrosine residue reactive site R and the protein purification tag site Tag. The type and length of the spacer can be selected so that steric congestion is minimized and the function of the functional group is not impaired. In one embodiment, S can be selected from the group consisting of -S1-, -S2-, and -S3-E-S4-.

[0059] S1 is one or more substituents R ３ In other words, S1 may be either an unsubstituted alkylene or a substituted alkylene. The alkylene of S1 is preferably a linear or branched alkylene, １－３０ Alkylene, C ２－３０ Alkylene, C ３－３０ Alkylene, C ４－３０ Alkylene, C ５－３０ Alkylene, or C ６－３０ It can be alkylene, and the like.

[0060] When S1 is substituted alkylene, R ３ The number of substitutions of R can be selected from the range of 1 to the maximum number of substitutions, for example, 1 to 10, 1 to 8, 1 to 6, 1 to 4, 1 to 3, 1, or 2. ３ If the number of substitutions is 2 or more, each R ３ may be the same or different from each other.

[0061] R ３ The substitution position of is not particularly limited and may be any position. For example, when the alkylene of S1 has a branched chain, R ３ is preferably substituted.

[0062] R ３ is -N(R ３１ ) ２ and heteroaryl (preferably 5-15 membered heteroaryl) optionally substituted with one or more substituents.

[0063] R ３ -N(R ３１ ) ２In the case of two R ３１ are each independently H, alkyl optionally substituted with one or more substituents (preferably C １－６ alkyl), substituted carbonyl, and -C(=NH)NH ２ It can be selected from the group consisting of:

[0064] The Two R's ３１ When at least one of is alkyl, the alkyl is preferably C １－６ alkyl, more preferably C １－４ Alkyl, even more preferably C １－２ Alkyl, particularly preferably methyl. The alkyl may be unsubstituted or substituted, and in the case of a substituted alkyl, the number of substituents may be selected from the range of 1 or more to the maximum number of possible substituents, for example, 1 to 10, 1 to 8, 1 to 6, 1 to 4, 1 to 3, 1, or 2.

[0065] The Two R's ３１ When at least one of the groups is a substituted carbonyl, the carbonyl substituent is preferably, but not limited to, a residue of a reagent. The reagent is typically a labeling reagent, examples of which include TMT (Tandem Mass Tag), fluorescein, Cy3, and TCO (transcyclooctene). Of these, TMT is preferred in terms of protein detection.

[0066]

[0067] The substituted carbonyl is preferably any one of the following formulae (T-1), (T-2), (T-3), (T-4), (T-5), (T-6), (T-7), (T-8), (T-9), and (T-10).

[0068]

[0069] R ３is a heteroaryl optionally substituted with one or more substituents, the heteroaryl is preferably a 5- or 6-membered heteroaryl, more preferably a 5- or 6-membered N-containing heteroaryl. Examples of the heteroaryl include pyrrolyl, pyrazolyl, imidazolyl, triazolyl, pyridyl, pyridazyl, pyrimidyl, pyrazyl, triazyl, etc.

[0070] R ３ The substituent that may be substituted on the heteroaryl of the formula (I) is preferably alkyl which may have one or more substituents, more preferably alkyl which may be substituted with an anionic group such as -SO3M (M is H or a monovalent metal, preferably an alkali metal), and even more preferably alkyl substituted with -SO3M. In each of the above cases, alkyl is preferably C １－６ alkyl, more preferably C １－４ The alkali metal is a Group 1 element other than hydrogen, and includes Li, Na, K, Rb, Cs, and Fr, preferably Li, Na, K, or Cs, and more preferably Na or K.

[0071] S2 is an alkylene having 2 or more carbon atoms (preferably an alkylene having 2 or more and 30 or less carbon atoms), and has at least one terminal and / or one or more adjacent —CH ２ Between -O-, -CO-, -NR ａ -, -CO-NR ａ -, -NR ａ -CO-, a group represented by formula (L-1), or a group represented by formula (L-2) is bonded to the group, and the group is one or more substituents R ３ may have

[0072]

[0073] In each of formulas (L-1) and (L-2), m is 0 or 1, and n is 0 or 1.

[0074] The group represented by formula (L-2) may be a group represented by formula (L-3).

[0075]

[0076] Examples of the group represented by formula (L-1) or formula (L-2) include the following.

[0077]

[0078] The alkylene of S2 is preferably a linear or branched alkylene of C ２－３０ Alkylene, C ３－３０ Alkylene, C ４－３０ Alkylene, C ５－３０ Alkylene, or C ６－３０ It can be alkylene, and the like.

[0079] At least one end of the alkylene of S2 may be -O-, -CO-, -NR ａ -, -CO-NR ａ - or -NR ａ -CO- may be bonded, and -NR may be bonded to the end of the tag side. ａ -or-CO-NR ａ -, especially -NR ａ The R-terminal of S2 is preferably -O-, -CO-, or -NR ａ - or -CO-NR ａ -, particularly -O- may be bonded.

[0080] At least one adjacent —CH ２ Between -, -O-, -CO-, -NR ａ -, -CO-NR ａ -, -NR ａ It is preferable that -CO-, a group represented by formula (L-1), or a group represented by formula (L-2) is bonded to S2. In one embodiment, S2 is -(alkylene-O) ａ -(a is an integer of 1 or more), -(O-alkylene) ａ -(a is an integer of 1 or more), -CO-NR ａ -, -NR ａ It is preferable that at least one selected from the group consisting of —CO—, a group represented by formula (L-1), and a group represented by formula (L-2) is contained.

[0081] Preferred examples of S2 include the following: In the following, it is preferred that the left end is bonded to R and the right end is bonded to Tag, at least one alkylene may be branched alkylene, and one or more substituents R ３ -O-alkylene-(L １ - alkylene) ｂ -(O-alkylene) ａ -NR ａ - (S21) - alkylene - (L １ - alkylene) ｂ -(O-alkylene) ａ -NR ａ - (S22) -O-Alkylene-NR ａ - (S23)

[0082] In each of S21 and S22, L １ is, independently at each occurrence, -CO-NR ａ -, -NR ａ -CO-, a group represented by formula (L-1), or a group represented by formula (L-2), and b is an integer of 1 or greater. b may preferably be 1 to 6, 1 to 5, or 1 to 4, etc.

[0083] -(alkylene-O) may be included in S2 ａ - and -(O-alkylene) ａ - is respectively -(C ２ H ４ -O) ａ -or-(O-C ２ H ４ ) ａ a is preferably 1 to 10, 1 to 8, 1 to 6, 1 to 4, 2 to 10, or 3 to 10, and is most preferably 1 to 4.

[0084] Can be included in S2 - NR ａ - In R ａ is H or alkyl optionally substituted with one or more substituents. ａ The alkyl in is preferably C １－６ alkyl, more preferably C １－４ Alkyl, even more preferably C１－２ Alkyl, particularly preferably methyl. R ａ The alkyl in R may be unsubstituted or substituted, and in the case of a substituted alkyl, the number of substituents can be selected from the range of 1 or more up to the maximum number of possible substituents, and can be, for example, 1 to 10, 1 to 8, 1 to 6, 1 to 4, 1 to 3, 1, or 2. ａ is preferably H.

[0085] S2 is one or more substituents R ３ If R ３ The number of substitutions of R can be selected from the range of 1 to the maximum number of substitutions, for example, 1 to 10, 1 to 8, 1 to 6, 1 to 4, 1 to 3, 1, or 2. ３ If the number of substitutions is 2 or more, each R ３ may be the same or different from each other.

[0086] R ３ The substitution position of is not particularly limited and may be any position. For example, when the alkylene contained in S2 has a branched chain, R ３ is preferably substituted.

[0087] S3 and S4 are each independently preferably selected from S1 and S2, more preferably selected from S2.

[0088] S3 is an alkylene having 2 or more carbon atoms (preferably an alkylene having 2 to 30 carbon atoms) in which —O— is bonded to the R-side terminal and —CH ２ Between -O-, -CO-, -NR ａ -, -CO-NR ａ -, -NR ａ It is preferably a group to which —CO—, a group represented by formula (L-1), or a group represented by formula (L-2) may be bonded, and the group may be bonded to one or more substituents R ３ In addition, S3 may be a branched alkylene having 2 or more carbon atoms (preferably a branched alkylene having 2 to 30 carbon atoms) in which the R-side terminal of the main chain, the E-side terminal of the main chain, and / or one or more adjacent —CH ２ Between -O-, -CO-, -NRａ -, -CO-NR ａ -, -NR ａ It is preferably a group to which —CO—, a group represented by formula (L-1), or a group represented by formula (L-2) is bonded, and the end of the side chain of the group is a substituent R ３ It is also preferable that the compound has the following structure:

[0089] Preferred examples of S3 include the following: In the following, it is preferred that the left end is bonded to R and the right end is bonded to E, at least one alkylene may be branched alkylene, and one or more substituents R ３ -O-alkylene-(L ２ - alkylene) ｃ - (S31) -Alkylene-(L ２ - alkylene) ｃ - (S32) -O-C １－６ Alkylene-(L ２ -C １－６ alkylene) ｃ - (S33) -C １－６ Alkylene-(L ２ -C １－６ alkylene) ｃ - (S34) -O-C ２－６ Alkylene-(L ２ - Branched chain C ２－６ alkylene) ｃ - (S35) -C ２－６ Alkylene-(L ２ - Branched chain C ２－６ alkylene) ｃ - (S36)

[0090] In each of S31, S32, S33, S34, S35, and S36, L ２ is, independently at each occurrence, -CO-NR ａ -or-NR ａ -CO-, and c is an integer of 0 or greater than 1. c is preferably 0 to 4, more preferably 0 to 3, even more preferably 0 to 2, and even more preferably 0 or 1.

[0091] In one embodiment, S3 is —O—C １－６ Alkylene-, -O-C １－６ Alkylene-NH-CO-(NH ２ , NHT, NH(C １－６ alkyl), N(C １－６ alkyl) ２ , 1-(M-SO ３ -C １－６ (alkylene)-1,2,3-triazol-4-yl, and NH ２ C(=NH)NH ２－６ alkylene)- or C １－６ Alkylene is preferred, and —O—C ６ H １２ -, -O-C ６ H １２ -NH-CO-C ２ H ４ -, -O-C ６ H １２ -NH-CO-CH(-C ４ H ８ NH ２ )-, -O-C ６ H １２ -NH-CO-CH(-C ４ H ８ NHT)- or -C ２ H ４ It is more preferable that it is -.

[0092] S4 is an alkylene having 2 or more carbon atoms (preferably an alkylene having 2 to 30 carbon atoms) in which —NH— is bonded to the Tag-side terminal and —CH— is bonded to the E-side terminal and / or one or more adjacent —CH ２ Between -O-, -CO-, -NR ａ -, -CO-NR ａ -, -NR ａ It is preferably a group to which —CO—, a group represented by formula (L-1), or a group represented by formula (L-2) may be bonded, and the group may be bonded to one or more substituents R ３ In addition, S4 may be a branched alkylene having 2 or more carbon atoms (preferably a branched alkylene having 2 to 30 carbon atoms) having an E-side terminal of the main chain, an Tag-side terminal of the main chain, and / or one or more adjacent —CH ２Between -O-, -CO-, -NR ａ -, -CO-NR ａ -, -NR ａ It is preferably a group to which —CO—, a group represented by formula (L-1), or a group represented by formula (L-2) is bonded, and the end of the side chain of the group is a substituent R ３ It is also preferable that the compound has the following structure:

[0093] Preferred examples of S4 include the following: In the following, it is preferred that the left end is bound to E and the right end is bound to a protein purification tag site Tag, at least one alkylene may be branched alkylene, and one or more substituents R ３ -(alkylene-L ３ ) ｄ -(Alkylene-O) ａ -Alkylene-NR ａ - (S41) - (C ２－６ Alkylene-L ３ ) ｄ -(C ２－６ Alkylene-O) ａ -C ２－６ Alkylene-NR ａ - (S42) - (C ２－５ Alkylene-L ３ ) ｄ -(C ２－５ Alkylene-O) ａ -C ２－５ Alkylene-NR ａ - (S43)

[0094] In each of S41, S42, and S43, L ３ is, independently at each occurrence, -CO-NR ａ -, -NR ａ -CO-, a group represented by formula (L-1), or a group represented by formula (L-2), and d is an integer of 0 or greater than 1. d is preferably 0 to 4, more preferably 0 to 3, and even more preferably 0 to 2.

[0095] -(Alkylene-O) included in S41 ａ -, included in S42-(C ２－６ Alkylene-O) ａ- and included in S43 - (C ２－５ Alkylene-O) ａ - is - (C ２ H ４ -O) ａ - is preferred. -(alkylene-O) included in S41 ａ -Alkylene-, included in S42-(C ２－６ Alkylene-O) ａ -C ２－６ Alkylene- and - included in S43 (C ２－５ Alkylene-O) ａ -C ２－５ Alkylene is -(C ２ H ４ -O) ａ -C ２ H ４ a can be 1 to 4, 2 to 4, 3 or 4, etc., and is most preferably 4.

[0096] In one embodiment, S4 is -(C １－６ Alkylene-O) １－４ -C １－６ Alkylene-NH-, -C １－６ Alkylene-NH-CO-(C １－６ Alkylene-O) １－４ -C １－６ Alkylene-NH-, -C １－６ Alkylene-NH-CO-(1-(M-SO ３ -C １－６ (alkylene)-1,2,3-triazol-4-yl, NH ２ , NH(C １－６ alkyl), N(C １－６ alkyl) ２ , N.H. ２ C(=NH)NH and C having a branched chain optionally having a substituent selected from NHT ２－６ Alkylene)-NH-CO-(C １－６ Alkylene-O) １－４ -C １－６ Alkylene-NH- or -C １－６ Alkylene-NH-CO-C １－６ Alkylene-(L-1)-C １－６ Alkylene-CO-NH-(C １－６Alkylene-O) １－４ -C １－６ Alkylene -NH- is preferred, -(C ２ H ４ O) ４ -C ２ H ４ -NH-, -C ２ H ４ -NH-CO-(C ２ H ４ O) ４ -C ２ H ４ -NH-, -CH(CH ３ ) ２ -CH ２ -NH-CO-(C ２ H ４ O) ４ -C ２ H ４ -NH-, -C ５ H １０ -NH-CO-(1-HSO ３ C ３ H ６ -1,2,3-triazol-4-yl-CH ２ -)CH-NH-CO-(C ２ H ４ O) ４ -C ２ H ４ -NH-, -C ５ H １０ -NH-CO-(1-NaSO ３ C ３ H ６ -1,2,3-triazol-4-yl-CH ２ -)CH-NH-CO-(C ２ H ４ O) ４ -C ２ H ４ -NH-, -C ５ H １０ -NH-CO-(1-KSO ３ C ３ H ６ -1,2,3-triazol-4-yl-CH ２ -)CH-NH-CO-(C ２ H ４ O) ４ -C ２ H ４-NH-, -CH ２ -NH-CO-(NH ２ C ４ H ８ -)CH-NH-CO-(C ２ H ４ O) ４ -C ２ H ４ -NH-, -CH ２ -NH-CO-(N(CH ３ ) ２ -C ４ H ８ -)CH-NH-CO-(C ２ H ４ O) ４ -C ２ H ４ -NH-, -CH ２ -NH-CO-(NH ２ -C(=NH)-NH-C ３ H ６ -)CH-NH-CO-(C ２ H ４ O) ４ -C ２ H ４ -NH- or -CH ２ -NH-CO-C ２ H ４ -(L-1)-C ２ H ４ -CO-NH-CH ２ -(C ２ H ４ O) ３ -C ３ H ６ It is more preferably —NH—.

[0097] E is a group (cleavable site) that can be cleaved under specific conditions. Such a group is not particularly limited as long as it has the above-mentioned effect, but is preferably a group that can be cleaved under mild reaction conditions. Examples of such groups include the following:

[0098] (wherein, one wavy line represents a bond with S3, the other wavy line represents a bond with S4, and Q １ , Q ２ , Q ３ , Q ４ , Q ５ , and Q６ are each independently H or alkyl (preferably C １－６ alkyl), and A １ , A ２ , A ３ , and A ４ are each independently an arylene (preferably C ６－１８ Pep is ENLYFQG (SEQ ID NO: 1), ENLYFQS (SEQ ID NO: 2), or X １ X ２ X ３ X ４ X ５ X ６ X ７ (X １ , X ２ , X ３ , X ５ , X ６ , and X ７ are each independently an amino acid residue other than K or R, and X ４ is K or R.)

[0099] Preferably Q １ and Q ２ is alkyl and Q ３ , Q ４ , Q ５ , and Q ６ is H. Here, alkyl is preferably C １－６ alkyl, more preferably C １－４ alkyl, and even more preferably C １－３ alkyl, particularly preferably C １－２ It is alkyl, and particularly preferably methyl.

[0100] A １ and A ２ is preferably phenylene or naphthylene, and more preferably phenylene. ３ and A ４ is preferably phenylene or naphthylene, more preferably phenylene (eg, 1,3-phenylene, 1,4-phenylene).

[0101] In the above, mild reaction conditions are preferably conditions under which the captured protein is not degraded and contaminating proteins nonspecifically bound to the avidin beads are not eluted. Specific examples include conditions under which the cleavable site E is chemically or biochemically cleaved, such as treatment with a dilute hydrazine solution, a dilute sodium periodate solution, a dilute sodium dithionite solution, a dilute formic acid solution, a tobacco etch virus (TEV) protease solution, a dilute tris(2-carboxyethyl)phosphine solution, or a dilute dithiothreitol solution. Treatment with a dilute hydrazine solution is more preferred. Here, "dilute" refers to a concentration of 5% by mass or less, preferably 3% by mass or less, although not limited thereto.

[0102] S is preferably -S2- or -S3-E-S4-. From the viewpoint of preventing nonspecific elution in protein purification, S is more preferably -S3-E-S4-, and even more preferably -S31-E-S41-, -S32-E-S41-, -S33-E-S42-, -S34-E-S42-, -S35-E-S42-, -S36-E-S42-, -S33-E-S43-, -S34-E-S43-, -S35-E-S43-, or -S36-E-S43-.

[0103] Preferred examples of S include the following: In the following, it is preferred that the left end is bonded to R and the right end is bonded to Tag, at least one alkylene may be branched alkylene, and one or more substituents R ３ -O-alkylene-(E-1)-(alkylene-O) ａ -Alkylene-NH- (S-1) -O-Alkylene-NH-CO-Alkylene-(E-1)-(Alkylene-O) ａ -Alkylene-NH- (S-2) -O-Alkylene-(E-1)-Alkylene-NH-CO-Alkylene-NH-CO-(Alkylene-O) ａ-Alkylene-NH- (S-3) -O-Alkylene-(E-1)-Alkylene-NH-CO-Alkylene-(L-1)-Alkylene-CO-NH-(Alkylene-O) ａ -Alkylene-NH- (S-4) -Alkylene-(E-1)-(Alkylene-O) ａ -Alkylene-NH- (S-5) -Alkylene-(E-1)-Alkylene-NH-CO-Alkylene-NH-CO-(Alkylene-O) ａ -Alkylene-NH- (S-6) -O-Alkylene-(E-6)-Alkylene-NH-CO-(Alkylene-O) ａ -Alkylene-NH- (S-7) -O-Alkylene-NH-CO-Alkylene-(E-7)-Alkylene-NH-CO-(Alkylene-O) ａ -Alkylene-NH- (S-8) -O-Alkylene-NH- (S-9) In each of the above, alkylene is a straight or branched C ２－６ Alkylene is preferred, (alkylene-O) ａ The alkylene in ２－５ Alkylene is preferred, and C ２－４ More preferably, it is alkylene, and C ２－３ More preferably, it is alkylene, and C ２ It may also be alkylene.

[0104] More preferred examples of S include the following: In the following, it is preferred that the left end is bonded to R and the right end is bonded to Tag, and the branched alkylene (particularly its end) may contain one or more substituents R ３ It is preferable that the formula is: -O-alkylene-NH-CO-branched alkylene-(E-1)-(alkylene-O) ａ -Alkylene-NH- (S-10), -O-Alkylene-(E-1)-Alkylene-NH-CO-Branched alkylene-NH-CO-(Alkylene-O) ａ -Alkylene-NH- (S-11) -Alkylene-(E-1)-Alkylene-NH-CO-Branched alkylene-NH-CO-(Alkylene-O) ａ-Alkylene-NH- (S-12) In each of the above, alkylene is a straight-chain or branched C ２－６ Preferably, the branched alkylene is C ２－６ Alkylene is preferred, (alkylene-O) ａ The alkylene in ２－５ Alkylene is preferred, and C ２－４ More preferably, it is alkylene, and C ２－３ More preferably, it is alkylene, and C ２ It may also be alkylene.

[0105] (2-3) Protein Purification Tag The protein purification tag is not particularly limited as long as it can purify the protein. Examples of such tags include biotin tags, imidazolidinone tags, His tags, Halo tag ligands, Flag™ tags, Spot tags, C tags, Strep tags, GST (Glutathione-S-Transferase) tags, and MBP (Maltose binding protein) tags. Among these, biotin tags, imidazolidinone tags, His tags, and Halo tag ligands are preferred, and specifically, the following formulas (2-1), (2-2), (2-3), and (2-4) are preferred, the following formulas (2-1), (2-2), and (2-3) are more preferred, and the following formula (2-1) is even more preferred.

[0106]

[0107] In each of the formulas (2-1), (2-2), (2-3), and (2-4), * represents a bond to the spacer S. In the formula (2-3), p is an integer of 1 to 10.

[0108] (2-4) RS-Tag RS-Tag can be any combination of the above R, S, and Tag. Suitable examples of RS-Tag include the following: (1A)-S3-E-S4-(2-1) (1B)-S3-E-S4-(2-1) (1A)-S31-E-S41-(2-1) (1B)-S31-E-S41-(2-1) (1A)-S32-E-S41-(2-1) (1B)-S32-E-S41-(2-1) (1A)-S33-E-S42-(2-1) (1A)-S34-E-S42-(2-1) (1A)-S35-E-S42-(2-1) (1A)-S36-E-S42-(2-1) (1A)-S33-E-S43-(2-1) (1A)-S34-E-S43-(2-1) (1A)-S35-E-S43-(2-1) (1A)-S36-E-S43-(2-1) (1A)-S2-(2-2) (1B)-S2-(2-2) (1A)-S2-(2-3) (1B)-S2-(2-3)

[0109] More preferred examples of RS-Tags include the following: (1A)-(S-1)-(2-1) (1B)-(S-1)-(2-1) (1A)-(S-2)-(2-1) (1B)-(S-2)-(2-1) (1A)-(S-3)-(2-1) (1B)-(S-3)-(2-1) (1A)-(S-4)-(2-1) (1B)-(S-4)-(2-1) (1A)-(S-5)-(2-1) (1B)-(S-5)-(2-1) (1A)-(S-6)-(2-1) (1B)-(S-6)-(2-1) (1A)-(S-7)-(2-1) (1B)-(S-7)-(2-1) (1A)-(S-8)-(2-1) (1B)-(S-8)-(2-1) (1A)-(S-10)-(2-1) (1B)-(S-10)-(2-1) (1A)-(S-11)-(2-1) (1B)-(S-11)-(2-1) (1A)-(S-12)-(2-1) (1B)-(S-12)-(2-1) (1A)-(S-9)-(2-2) (1B)-(S-9)-(2-2) (1A)-(S-9)-(2-3) (1B)-(S-9)-(2-3)

[0110] (2-5) Salt Form The functional compound having an RS-Tag may be in the form of a salt. The salt may be a salt with an acid or a salt with a base.

[0111] The acid is not particularly limited, and examples thereof include inorganic acids and organic acids. Examples of inorganic acids include hydrochloric acid, sulfuric acid, nitric acid, hydrobromic acid, and phosphoric acid. Examples of organic acids include acetic acid, trifluoroacetic acid, oxalic acid, phthalic acid, fumaric acid, tartaric acid, maleic acid, citric acid, succinic acid, methanesulfonic acid, p-toluenesulfonic acid, and 10-camphorsulfonic acid. The acids may be used alone or in combination of two or more.

[0112] The base is not particularly limited, and examples thereof include inorganic bases and organic bases. Examples of inorganic bases include alkali metal hydroxides (e.g., sodium hydroxide, potassium hydroxide), alkaline earth metal hydroxides (e.g., magnesium hydroxide, calcium hydroxide), alkali metal carbonates (e.g., sodium carbonate, potassium carbonate), alkaline earth metal carbonates (e.g., magnesium carbonate, calcium carbonate), alkali metal bicarbonates (e.g., sodium bicarbonate, potassium bicarbonate), alkali metal phosphates (e.g., sodium phosphate, potassium phosphate), alkaline earth metal phosphates (e.g., magnesium phosphate, calcium phosphate), etc. Examples of organic bases include trialkylamines (e.g., trimethylamine, triethylamine, diisopropylethylamine), picoline, 1,5-diazabicyclo[4.3.0]non-5-ene, 1,4-diazabicyclo[2.2.2]octane, 1,8-diazabicyclo[5.4.0]undec-7-ene, etc.

[0113] (2-6) Suitable Examples Suitable examples of the functional compound or salt thereof of the present disclosure include functional compounds represented by any of the following formulae (P1), (P2), (P3), (P4), (P5), (P6), (P7), (P8), (P9), (P10), (P11), (P12), (P13), (P14), (P15), (P16), (P17), (P18), (P19), (P20), (P21), (P22), (P23), (P24), (P25), (P26), (P27), and (P28) and salts thereof.

[0114]

[0115] In (P5) and (P6), M is preferably H or an alkali metal, more preferably H, Na, or K, and more preferably Na. In (P9) and (P10), T is preferably a substituted carbonyl, more preferably any one of the following formulae (T-1), (T-2), (T-3), (T-4), (T-5), (T-6), (T-7), (T-8), (T-9), and (T-10).

[0116]

[0117] Suitable examples of the salt of the functional compound of the present disclosure include a salt of a functional compound represented by formula (P5a) or (P6a), and a trifluoroacetic acid (TFA) salt of a functional compound represented by formula (P7), (P8), (P13), (P14), (P15), (P16), (P19), (P20), (P21), (P22), (P23), or (P24). Specific examples include the following.

[0118]

[0119] Suitable examples of salts of functional compounds of the present disclosure include the compounds of Example 1, Example 2, Example 3, Example 4, Example 5, Example 6, Example 7, Example 8, Example 9, Example 10, Example 11, Example 12, Example 13, Example 14-1, Example 14-2, Example 14-3, Example 14-4, Example 14-5, Example 14-6, Example 14-7, Example 14-8, Example 14-9, and Example 14-10.

[0120] (2-7) Production Method The production method of the functional compound or salt thereof of the present disclosure can be, for example, based on the production method shown below, but is not limited thereto.

[0121] The starting compounds may be commercially available or may be synthesized according to a method known per se or a method similar thereto.

[0122] The solvents, acids, bases, protecting groups, and leaving groups that may be appropriately used in the production of the functional compounds or salts thereof of the present disclosure are not particularly limited, as long as they are commonly used in the field of organic synthetic chemistry.

[0123] In the production of the functional compound or salt thereof of the present disclosure, the product can be used in the next reaction as a reaction solution or as a crude product, or it can be isolated from the reaction mixture according to a conventional method and easily purified by conventional separation means, such as filtration, extraction, concentration, evaporation, crystallization, recrystallization, reprecipitation, distillation, chromatography, and optical resolution.

[0124] In the general method for producing a functional compound or a salt thereof according to the present disclosure, the starting compounds, intermediate compounds, and final compounds may be in the form of salts, and the target compounds obtained in each reaction may also form salts. When each compound is a free compound, it can be converted into the target salt by a method known per se, and when the compound is a salt, it can be converted into the free form or another target salt by a method known per se.

[0125] The method for producing a functional compound or a salt thereof of the present disclosure typically includes a step of reacting R-SA with SB-Tag to obtain R-S-Tag. SA and SB are precursor groups of S, and are not particularly limited as long as they are groups that link to form S upon reaction. In the above step, the step of producing R-SA, or the step of producing SB-Tag, at least one reaction selected from, for example, alkylation reaction, hydrolysis reaction, amination reaction, esterification reaction, amidation reaction, etherification reaction, nucleophilic substitution reaction, addition reaction, oxidation reaction, and reduction reaction may be used. In each reaction, protection with a protecting group and deprotection with a deprotecting agent may be carried out as necessary.

[0126] When R-S-Tag is R-S3-(E-1)-S4-Tag, for example, as shown in the following reaction scheme, it can be obtained by reacting a compound represented by formula (R1) with a compound represented by formula (R2) in the presence of a base (e.g., a trialkylamine such as DIPEA) in a solvent (e.g., an amide solvent such as DMF).

[0127]

[0128] When R-S-Tag is R-S3-(E-6)-S4-Tag, for example, as shown in the following reaction scheme, it can be obtained by reacting a compound represented by formula (R3) with a compound represented by formula (R4) in the presence of a compound represented by formula (R5) and a base (e.g., a trialkylamine such as TEA) in a solvent (e.g., a halohydrocarbon solvent such as DCM, or an amine solvent such as pyridine).

[0129]

[0130] When R-S-Tag is R-S3-(E-7)-S41-Tag, for example, as shown in the following reaction scheme, it can be obtained by reacting a compound represented by formula (R6) with a compound represented by formula (R7) in the presence of a condensing agent (e.g., COMU) and a base (e.g., a trialkylamine such as DIPEA) in a solvent (e.g., an amide solvent such as DMF).

[0131]

[0132] Even if S is other than those, a functional compound having an R-S-Tag or a salt thereof can be produced in the same manner as in the above reaction scheme or according to a reaction scheme that can be appropriately designed based on common knowledge in chemical synthesis.

[0133] (3) Composition The composition of the present disclosure contains a functional compound in which R is a group represented by formula (1A) or a salt thereof, and a functional compound in which R is a group represented by formula (1B) or a salt thereof. Here, the functional compound in which R is a group represented by formula (1B) is an isomer of the functional compound in which R is a group represented by formula (1A), that is, R in formula (1B) １ is R in formula (1A). １ is the same as R in formula (1B) ２ is R in formula (1A). ２ is preferably the same as

[0134] In the composition of the present disclosure, the mass ratio (or molar ratio) of the functional compound in which R is a group represented by formula (1A) or a salt thereof to the functional compound in which R is a group represented by formula (1B) or a salt thereof is, for example, 10 / 90 to 90 / 10, preferably 20 / 80 to 80 / 20, and more preferably 30 / 70 to 70 / 30.

[0135] Suitable examples of the compositions of the present disclosure include the following compositions:A composition comprising a functional compound represented by formula (P1) or a salt thereof, and a functional compound represented by formula (P2) or a salt thereof. A composition comprising a functional compound represented by formula (P3) or a salt thereof, and a functional compound represented by formula (P4) or a salt thereof. A composition comprising a functional compound represented by formula (P5) or a salt thereof (preferably (P5a)) and a functional compound represented by formula (P6) or a salt thereof (preferably (P6a)). A composition comprising a functional compound represented by formula (P7) or a salt thereof (preferably a TFA salt or (P7a)) and a functional compound represented by formula (P8) or a salt thereof (preferably a TFA salt or (P8a)). A composition comprising a functional compound represented by formula (P9) or a salt thereof and a functional compound represented by formula (P10) or a salt thereof. A composition comprising a functional compound represented by formula (P11) or a salt thereof and a functional compound represented by formula (P12) or a salt thereof. A composition comprising a functional compound represented by formula (P13) or a salt thereof (preferably a TFA salt or (P13a)) and a functional compound represented by formula (P14) or a salt thereof (preferably a TFA salt or (P14a)). A composition comprising a functional compound represented by formula (P15) or a salt thereof (preferably a TFA salt or (P15a)) and a functional compound represented by formula (P16) or a salt thereof (preferably a TFA salt or (P16a)). A composition comprising a functional compound represented by formula (P17) or a salt thereof and a functional compound represented by formula (P18) or a salt thereof. A composition comprising a functional compound represented by formula (P19) or a salt thereof (preferably a TFA salt or (P19a)) and a functional compound represented by formula (P20) or a salt thereof (preferably a TFA salt or (P20a)). A composition comprising a functional compound represented by formula (P21) or a salt thereof (preferably a TFA salt or (P21a)) and a functional compound represented by formula (P22) or a salt thereof (preferably a TFA salt or (P22a)). A composition comprising a functional compound represented by formula (P23) or a salt thereof (preferably a 2TFA salt or (P23a)) and a functional compound represented by formula (P24) or a salt thereof (preferably a 2TFA salt or (P24a)). A composition comprising a functional compound represented by formula (P25) or a salt thereof and a functional compound represented by formula (P26) or a salt thereof. A composition comprising a functional compound represented by formula (P27) or a salt thereof and a functional compound represented by formula (P28) or a salt thereof.

[0136] The composition of the present disclosure can be produced, for example, as an intermediate product (or crude product) in the production method described in (2-7) above. The composition of the present disclosure can be suitably used as a chemical probe, and further can be suitably used for the detection or identification of cell surface proteins, comprehensive analysis of cell surface proteins, or proximity-dependent labeling, as described below.

[0137] (4) Comprehensive Analysis of Cell Surface Proteins Using the functional compound, its salt, or composition described above as a chemical probe makes it possible to comprehensively identify, for example, proteins containing tyrosine residues, particularly cell surface proteins present in living cells or living tissues. Using the functional compound, its salt, or composition described above as a chemical probe also enables proximity-dependent labeling using commonly available reagents, for example, peroxidase and hydrogen peroxide. Furthermore, using the functional compound or its salt can also analyze changes in protein phosphorylation (e.g., changes in EGFR phosphorylation caused by EGFR tyrosine kinase inhibitors such as gefinitib).

[0138] Protein detection or identification using a chemical probe can be carried out by a method including, but not limited to, the following steps (1) and (2): (1) binding a tyrosine residue reactive site R in the chemical probe to a tyrosine residue in the protein, and (2) binding a labeling substance that binds to the protein purification tag site Tag in the chemical probe bound to the protein, and detecting the label.

[0139] The concentration of the chemical probe used in step (1) is not particularly limited, but is preferably 1 μg / mL or more, more preferably 5 μg / mL or more, and even more preferably 10 μg / mL or more. The reaction temperature in step (1) is not particularly limited, but is, for example, 1 to 35°C. The reaction time in step (1) may be, for example, 10 seconds or more, 30 seconds or more, or 1 minute or more, or 30 minutes or less, 20 minutes or less, 10 minutes or less, or 5 minutes or less.

[0140] The detection or identification of cell surface proteins using chemical probes is preferably carried out by a method comprising the following steps (A), (B), (C), and, if necessary, steps (D) and (E). (A) reacting a chemical probe with cells or tissues in a sample (preferably by activating it with hydrogen peroxide and peroxidase) to bind the tyrosine residue reactive site R in the chemical probe to the tyrosine residue of the cell surface protein; (B) purifying a fraction containing the cell surface protein bound to the chemical probe (preferably by lysing the cells); (C) binding the protein purification tag site Tag in the chemical probe to a labeling substance (for example, binding a biotin site, which is an example of T, to avidin, which is an example of a labeling substance) to form a chemical probe-labeling substance complex; (D) cleaving (preferably chemically or biochemically cleaving) the formed chemical probe-labeling complex at the cleavable site E in the chemical probe; and (E) detecting or identifying the cell surface protein from the complex or a fragment of its cell surface protein-binding region.

[0141] Each step is described below: (A) A step of binding the tyrosine residue reactive site R to a tyrosine residue on a cell surface protein. This step can be performed, for example and without limitation, by culturing cells at 37°C in a 5% CO2 atmosphere and confirming that the cells are confluent. The medium is then removed and the cells are washed with PBS or similar. After this, hydrogen peroxide (e.g., 0.00003% or more) and peroxidase (e.g., 0.0016% or more) diluted in PBS are added, and a chemical probe adjusted to a desired concentration in DMSO (e.g., 1 μg / mL or more, preferably 5 μg / mL or more, and more preferably 10 μg / mL or more) is added. The mixture is then left to stand at approximately 1 to 35°C for approximately 1 to 5 minutes. After labeling, the reaction solution is removed, and unreacted probe can be removed by washing with a medium such as DMEM. Furthermore, washing with a medium such as DMEM can inactivate unreacted probe due to the high content of phenol red and tyrosine as an amino acid.

[0142] (B) Step of purifying fractions containing cell surface proteins bound to chemical probes: This step can be performed, but is not limited to, by the following method: First, RIPA buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% SDS, 0.5% SDC, 1% Triton-X-100, Complete protease inhibitor (Roche)) is added to the cells, vortexed, incubated on ice for 30 minutes, and then centrifuged at 16,000 × g at 4°C for 20 minutes to recover the supernatant, allowing the fraction containing the proteins bound to the chemical probes to be recovered.

[0143] (C) A step of forming a chemical probe-labeled substance complex by binding a protein purification tag site (e.g., biotin site) in the chemical probe to a labeling substance (e.g., avidin). This step is not limited to, but may involve, for example, adding labeling substance magnetic particles (e.g., streptavidin magnetic particles) to the fraction collected in (B) above. In this way, the biotin site, which is an example of a protein purification tag site (e.g., avidin), binds to the labeling substance, and magnetic separation is then performed according to a standard method. The supernatant is then discarded, allowing the chemical probe-labeled substance complex to be purified and collected.

[0144] (D) A step of cleaving the formed chemical probe-labeled substance complex at the cleavable site E in the chemical probe. For example, if the compound recovered in step (C) is a chemical probe-avidin complex (magnetic particle complex), this step can be performed by adding an aqueous solution of SDS (sodium dodecyl sulfate) and hydrazine to the complex and allowing it to stand at room temperature (1-30°C) for 30 minutes. This reaction cleaves the cleavable site in the chemical probe, separating the chemical probe-avidin complex into the target protein binding domain and the biotin-avidin binding domain. The reaction solution is magnetically separated to obtain the supernatant, allowing the cell surface protein binding domain fragment to be recovered.

[0145] (E) Step of detecting or identifying cell surface proteins When detecting or identifying cell surface proteins, the previously collected complex or its cell surface protein-binding domain fragment may be subjected to purification and / or hydrolysis, as necessary. Specifically, but not limited to, for example, dithiothreitol is added to the obtained supernatant and the mixture is left to stand at 70°C for 15 minutes. Next, iodoacetamide is added and the mixture is stirred at room temperature for 30 minutes. SP3 beads are added to the resulting solution, followed by MeCN and incubation for 18 minutes. After incubation, magnetic separation is performed and the supernatant is discarded. For example, peptide degradation is performed by adding a trypsin solution to the residue collected above and incubating at 37°C for 12 to 16 hours. MeCN is added, followed by incubation for 18 minutes, magnetic separation is performed, and the supernatant is discarded. Aqueous TFA solution is added to the residue and the mixture is incubated for 3 minutes. After magnetic separation, the supernatant is obtained.

[0146] The peptides thus prepared are desalted and resuspended in an appropriate buffer solution for analysis using a high-mass accuracy mass spectrometer. The mass spectrometer is operated in a data-dependent acquisition mode, where, for example, but not limited to, an ion signal above a predetermined threshold automatically triggers the instrument to switch from MS to MS / MS mode to generate a collision-induced dissociation (CID) spectrum or a higher-energy collisional dissociation (HCD) spectrum of the peptide. It may also be operated in a data-independent acquisition mode, but not limited to this. All MS / MS spectra are searched against a standard protein database.

[0147] The peptides thus obtained are analyzed. Any method for analyzing such compounds available in the art can be used, with mass spectrometry being the preferred method. Mass spectrometry methods are well known to those skilled in the art (see, for example, Yates, J. Mass Spect. 33:1-19 (1998); Kinter and Sherman, Protein Sequencing and Identification Using Tandem Mass Spectrometry, John Wiley and Sons, New York (2000); Aebersold and Goodlett, Chem. Rev. 101:269-295 (2001)). For high-resolution polypeptide fragment separation, liquid chromatography ESI-MS / MS or automated LC-MS / MS can be used, using capillary reversed-phase chromatography as the separation method (Yates et al., Methods Mol. Biol. 112:553-569 (1999)). Preferably, data-dependent collision-induced dissociation (CID) or higher-energy collision-induced dissociation (HCD) with dynamic exclusion is used as the mass spectrometry method of choice (Goodlett et al., Anal. Chem. 72:1112-1118 (2000)). For such analyses, mass spectrometers are typically operated in a data-dependent acquisition mode, in which ion signals above a predetermined threshold automatically trigger the instrument to switch from MS to MS / MS mode to generate a collision-induced dissociation (CID) or higher-energy collision-induced dissociation (HCD) spectrum of the peptide.

[0148] For protein identification, all MS / MS spectra are searched against standard protein databases using standard algorithms (SEQUEST, Mascot, X!tandem, MS Aanda, etc.), with typical filtering to limit the false-positive protein identification rate to less than 1%.

[0149] In one embodiment, the concentration of membrane proteins in a sample can be quantitatively compared with that of a control sample. This allows for the detection of specific enrichment of target membrane protein receptors. For this label-free mass analysis, reversed-phase chromatography immediately prior to mass analysis can be displayed as an MS feature map, plotting retention time features against mass / charge ratio. As detected by the mass spectrometer, peptides in such a map appear with distinct isotopic patterns over a given time period and with defined ion current intensities depending on their abundance in the sample. Once peptides are identified through fragmentation and MS / MS analysis, this information can be assigned to specific peptide features in the MS map and combined with semi-quantitative data analysis using open-source or commercial algorithms such as MaxQuant (Cox et al., Nature Biotechnology (2008) vol. 26 pp. 1367-1372) or Proteome Discoverer (Thermo Fischer Scientific). MS feature maps of different samples (e.g., sample vs. control) can then be overlaid and compared to obtain peptide abundance ratios. For stochastically labeled peptides derived from membrane proteins, these ratios should be approximately 1, with membrane protein peptides that are specifically captured based on the ligand obtaining higher values ​​compared to the control sample.

[0150] In other embodiments, alternative mass spectrometry-based quantification methods can be used, such as single reaction monitoring (SRM), stable isotope labeling with amino acids in cell culture (SILAC; see, e.g., Nilsson et al., Nat Methods (2010) vol. 7(9) pp. 681-5), data-independent acquisition of mass spectra (SWATH MS; see, e.g., Gillet et al., (Targeted Data Extraction of the MS / MS Spectra Generated by Data-independent Acquisition: A New Concept for Consistent and Accurate Proteome Analysis)), tandem mass tags (TMT; see, e.g., Dayon et al., (Relative Quantification of Proteins in Human Cerebrospinal Fluids by MS / MS using 6-Plex Isobaric Tags)), etc.

[0151] It is contemplated that MS analysis may be substituted with other analytical methods, and such other methods are included as part of the present method.

[0152] By using the chemical probe of the present invention to identify peptide sequences to which a part of the probe structure has been added by mass spectrometry, the topology of membrane proteins can be determined. Furthermore, the solvent-exposed sites of proteins can be identified, which can be used as structural information.

[0153] (5) Region-restricted proximity-dependent labeling By using the above-described functional compound or a salt thereof as a chemical probe, it is possible to perform region-restricted proximity-dependent labeling of, for example, proteins having tyrosine residues, particularly surface proteins of cells present in living cells or living tissues.

[0154] By confining the peroxidase used for labeling to a specific region, region-restricted proximity-dependent labeling can be achieved. For example, a ligand or antibody specific to a membrane protein can be used as a means for confining the peroxidase to a specific region. For example, instead of adding peroxidase, which can be used for global cell surface labeling, proximity-dependent labeling can be achieved by adding a peroxidase-labeled ligand or peroxidase-labeled antibody, or by fusing peroxidase to a specific membrane protein through genetic engineering and expressing it.

[0155] Protein detection or identification by proximity-dependent labeling using chemical probes can be carried out by a method comprising, but not limited to, the following steps (1a), (1b), and (2): (1a) binding a peroxidase-labeled ligand or a peroxidase-labeled antibody to cells or tissues (e.g., living cells or living tissues), (1b) binding a tyrosine residue reactive site R in the chemical probe to a tyrosine residue of a cell surface protein in the presence of hydrogen peroxide, and (2) binding a labeling substance that binds to the protein purification tag site Tag in the chemical probe bound to the protein, and detecting the label.

[0156] The concentration of the chemical probe used in step (1b) is not particularly limited, but is preferably 1 μg / mL or more, more preferably 5 μg / mL or more, and even more preferably 10 μg / mL or more. The concentration of hydrogen peroxide used in step (1b) is not particularly limited, but may be, for example, 0.00003% or more, 0.00004% or more, or 0.00005% or more. The reaction temperature in step (1b) is not particularly limited, but may be, for example, 1 to 35°C. The reaction time in step (1b) may be, for example, 10 seconds or more, 30 seconds or more, or 1 minute or more, or 30 minutes or less, 20 minutes or less, 10 minutes or less, or 5 minutes or less.

[0157] Proximity-dependent labeling using a chemical probe is preferably carried out by a method comprising the following steps (A1), (A2), (B), (C), (D), and (E). (A1) reacting a peroxidase-labeled ligand or a peroxidase-labeled antibody with cells or tissues in a sample to bring peroxidase into close proximity with a specific cell surface protein; (A2) reacting a chemical probe with cells or tissues in a sample by activating it with hydrogen peroxide to bind a tyrosine residue reactive site R in the chemical probe to a tyrosine residue of the specific cell surface protein and its neighboring proteins; (B) purifying (preferably by lysing the cells) a fraction containing the cell surface protein bound to the chemical probe; (C) binding the protein purification tag site Tag in the chemical probe to a labeling substance (for example, binding a biotin site, which is an example of T, to avidin, which is an example of a labeling substance) to form a chemical probe-labeling substance complex; (D) cleaving (preferably chemically or biochemically cleaving) the formed chemical probe-labeling complex at a cleavable site E in the chemical probe; (E) detecting or identifying a cell surface protein from the complex or a fragment of the cell surface protein-binding domain thereof.

[0158] Each of these steps is explained below: (A1) A step in which a peroxidase-labeled ligand or peroxidase-labeled antibody is reacted with cells or tissues in a sample, bringing peroxidase into close proximity with a specific membrane protein. This step is not limited to one specific step, but as an example, it can be performed by culturing cells at 37°C in a 5% CO2 atmosphere, confirming that the cells are confluent, removing the medium, washing with PBS or similar containing 2% FCS (fetal calf serum), adding the peroxidase-labeled ligand or peroxidase-labeled antibody, and allowing to stand on ice for approximately 20 minutes. After the peroxidase-labeled ligand or peroxidase-labeled antibody binds to a specific receptor, unbound ligand can be removed by washing with a buffer such as PBS.

[0159] (A2) A step of binding a tyrosine residue reactive site R to a tyrosine residue of a cell surface protein in the presence of hydrogen peroxide. This step can be carried out by adding hydrogen peroxide diluted with PBS (e.g., 0.00003% or more), adding a chemical probe adjusted to a desired concentration with DMSO (e.g., 1 μg / mL or more, preferably 5 μg / mL or more, and more preferably 10 μg / mL or more), and allowing to stand at about 1 to 35°C for about 1 to 5 minutes. After labeling, the reaction solution can be removed, and unreacted probe can be removed by washing with a medium such as DMEM. Furthermore, washing with a medium such as DMEM can inactivate unreacted probe, since it contains a large amount of phenol red and tyrosine as an amino acid.

[0160] The subsequent steps can be carried out in accordance with the method described in the method for comprehensive analysis of cell surface proteins.

[0161] The present invention will be further illustrated by the following examples, which are not intended to limit the scope of the present invention and may be modified without departing from the scope of the present invention.

[0162] In the following examples, "room temperature" generally refers to about 10°C to about 35°C. Ratios shown for mixed solvents are by volume unless otherwise specified. % refers to % by weight unless otherwise specified.

[0163] H NMR (proton nuclear magnetic resonance) spectra were measured using a Fourier transform NMR (Bruker AVANCE III 400 (400 MHz), Bruker AVANCE III HD (500 MHz), BRUKER AVANCE 400 (400 MHz), or BRUKER AVANCE III 300N (300 MHz)). Spectra were analyzed using either MestReNova version 14.1.2 (Mestrelab Research) or ACD / Spectrus Processor™ (ACD). MS (mass spectra) were measured using LC / MS (either ACQUITY UPLC H-Class or Shimadzu LCMS2020). Electrospray ionization (ESI) was used, and data reported are actual values. Molecular ion peaks (e.g., [M+H]+, [MH]-) are typically observed. In the case of salts, free molecular ion peaks or fragment ion peaks are typically observed. In silica gel column chromatography, when basic is mentioned, aminopropylsilane-bonded silica gel was used.

[0164] In the examples, the following abbreviations may be used: AcOEt: Ethyl acetate AcOH: Acetic acid COMU: (1-cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylaminomorpholinocarbenium hexafluorophosphate DCM: Dichloromethane DIPEA: N,N-Diisopropylethylamine DMAP: 4-Dimethylaminopyridine DMEM: Dulbecco's modified Eagle's medium DMF: N,N-Dimethylformamide DTT: Dithiothreitol EtOH: Ethanol FCS: Fetal bovine serum Fmoc: 9-Fluorenylmethyloxycarbonyl HOBt: 1-Hydroxybenzotriazole HRP: Horseradish peroxidase MeCN: Acetonitrile MeOH: Methanol NHS: N-Hydroxysuccinimide ODS: Octadecylsilyl Pbf: 2,2,4,6,7-Pentamethyldihydrobenzofuran-5-sulfonyl PBS: Phosphate buffered saline Ph: Phenyl RIPA: Radioimmunoprecipitation assay RPMI: Roswell Park Memorial Institute SDC: sodium deoxycholate TBS: Tris-buffered saline TEAB: triethylammonium bicarbonate TFA: trifluoroacetic acid THF: tetrahydrofuran TMT: tandem mass tag Tris: trishydroxymethylaminomethane WSC: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride

[0165] [Example 1]

[0166] In the 2,3-dihydrophthalazine-1,4-dione of M1-1, the constituent atoms of the ring are numbered for convenience in explaining the substitution position. M1-1 and Example 1 have a methyl (R １ ), and third place is H (R ２ ), and its isomers (methyl (R １ ), and third place is H (R ２ ), and a compound having a substituent at the 7-position.

[0167] Intermediate M1-1 (75 mg) and Intermediate M1-2 (140 mg) were dissolved in DMF (5 mL), and DIPEA (159 μL) was added, followed by stirring for 2 hours at 60° C. The solvent was evaporated, and the residue was purified by column chromatography (ODS, water / MeCN) to obtain Example 1 (165 mg). 1 H NMR (400 MHz, DMSO-d6) δ 13.48 (t, J = 4.8 Hz, 1H), 11.55 (br, 1H), 8.11 (d, J = 8.8 Hz, 0.5H), 7.88 (d, J = 8.8 Hz, 0.5H), 7.82 (t, J = 5.5 Hz, 1H), 7.58 (d, J = 2.6 Hz, 0.5H), 7.45-7.38 (m, 1H), 7.29 (d, J = 2.2 Hz, 0.5H), 6.41 (s, 1H), 6.35 (s, 1H), 4.31-4.28 (m, 1H), 4.17-4.10 (m, 3H), 3.61 (t, J = 6.2 Hz, 2H), 3.56-3.47 (m, 17H), 3.39-3.23 (m, overlapped with water signal), 3.17 (q, J = 5.8 Hz, 2H), 3.10-3.05 (m, 1H), 2.81 (dd, J = 12.4, 5.1 Hz, 1H), 2.57 (d, J = 12.4 Hz, 1H), 2.26 (s, 4H), 2.05 (t, J = 7.4 Hz, 2H), 1.81-1.78 (m, 2H), 1.66-1.40 (m, 10H), 1.36-1.23 (m, 2H), 0.93 (s, 6H);LC-MS: [M+H] + = 887.29.

[0168] [Example 2]

[0169] In the 2,3-dihydrophthalazine-1,4-dione of M2-1, the constituent atoms of the ring are numbered for convenience in explaining the substitution position. M2-1, M2-3, M2-4, and Example 2 have a methyl group (R １ ), and third place is H (R２ ), and its isomers (methyl (R １ ), and third place is H (R ２ ), and a compound having a substituent at the 7-position.

[0170] (Synthesis of M2-3) M2-1 (17 mg) and M2-2 (16 mg) were dissolved in DMF (1 mL), and COMU (37 mg) and DIPEA (30 μL) were added and stirred at room temperature for 5 hours. The solvent was evaporated, and the residue was purified by column chromatography (ODS, water / MeCN) to obtain M2-3 (19 mg). 1 H NMR (400 MHz, CDCl3) δ 8.31 (d, J = 8.8 Hz, 0.4H), 8.02 (d, J = 8.8 Hz, 0.6H), 7.76 (d, J = 2.6 Hz, 0.6H), 7.41 (s, 0.4H), 7.35-7.31 (m, 1H), 6.21-6.13 (m, 1H), 5.09-5.02 (m, 1H), 4.15-4.10 (m, 2H), 3.72-3.69 (m, 3H), 3.44 (dd, J = 12.6, 6.3 Hz, 2H), 3.33-3.27 (m, 2H), 3.04-2.97 (m, 2H), 2.80 (t, J = 6.6 Hz, 2H), 2.60 (t, J = 6.9 Hz, 2H), 1.88-1.81 (m, 2H), 1.60-1.44 (m, 15H);LC-MS: [M+H] + = 555.32.

[0171] Synthesis of Example 2: To a mixture of the above-prepared M2-3 (19 mg), DCM (0.5 mL), and MeOH (0.1 mL), 4 M HCl ethyl acetate solution (428 μL) was added and stirred at room temperature for 2 hours. The solvent was evaporated, and M2-5 (17 mg), COMU (22 mg), and DIPEA (30 μL) shown in the formula above were added to the residue, followed by stirring at room temperature for 3 hours. The solvent was evaporated, and the residue was purified by column chromatography (ODS, water / MeCN) to obtain Example 2 (15 mg). 1H NMR (400 MHz, DMSO-d6) δ 8.10-8.03 (m, 1+0.4H), 7.94-7.82 (m, 2+0.6H), 7.56 (d, J = 2.5 Hz, 0.6H), 7.41 (dd, J = 8.8, 2.4 Hz, 0.6H), 7.35-7.31 (m, 2x0.4H), 6.41 (s, 1H), 6.35 (s, 1H), 4.30 (dd, J = 7.6, 5.2 Hz, 1H), 4.14-4.09 (m, 3H), 3.58 (t, J = 6.5 Hz, 2H), 3.53-3.03 (m, overlapped with water signal), 2.88 (t, J = 7.1 Hz, 2H), 2.81 (dd, J = 12.4, 5.1 Hz, 1H), 2.75 (t, J = 6.8 Hz, 2H), 2.59-2.43 (m, overlapped with DMSO signal), 2.31 (t, J = 6.6 Hz, 2H), 2.06 (t, J = 7.4 Hz, 2H), 1.80-1.72 (m, 2H), 1.65-1.56 (m, 1H), 1.53-1.23 (m, 11H);LC-MS: [M+H] + = 928.42.

[0172] [Example 3]

[0173] In the 2,3-dihydrophthalazine-1,4-dione of M3-12, the constituent atoms of the ring are numbered for convenience in explaining the substitution position. M3-12, M3-13, and Example 3 have a methyl group (R １ ), and third place is H (R ２ ), and its isomers (methyl (R １ ), and third place is H (R ２ ), and a compound having a substituent at the 7-position.

[0174] (Synthesis of M3-3) To a mixture of M3-1 (300 mg) and DMF (5 mL), M3-2 (allyl bromide) (168 μL) and DIPEA (340 μL) were added and stirred at room temperature for 7 hours. M3-2 (allyl bromide) (168 μL) and DIPEA (340 μL) were added, and the mixture was stirred at room temperature for 13 hours. The solvent was evaporated, and the residue was purified by column chromatography (silica gel, hexane / AcOEt) to obtain M3-3 (329 mg). 1 H NMR (400 MHz, CDCl3) δ 5.97-5.87 (m, 1H), 5.31 (ddt, J = 17.2, 1.4, 1.4 Hz, 1H), 5.23 (ddt, J = 10.4, 1.4, 1.4 Hz, 1H), 4.57 (dt, J = 5.8, 1.4 Hz, 2H), 4.52 (br, 1H), 3.11 (q, J = 6.4 Hz, 2H), 2.34 (t, J = 7.5 Hz, 2H), 1.69-1.62 (m, 2H), 1.53-1.44 (m, 11H), 1.39-1.31 (m, 2H);LC-MS: [M+Na] + = 294.34.

[0175] (Synthesis of M3-6) 4 M HCl ethyl acetate solution (921 μL) was added to M3-3 (20 mg) prepared above and stirred at room temperature for 1 hour. The solvent was evaporated, and DMF (1 mL), M3-5 (N-(tert-butoxycarbonyl)-L-propargylglycine) (16 mg), COMU (47 mg), and DIPEA (64 μL) were added to the residue and stirred at room temperature for 3 hours. The solvent was evaporated, and the residue was purified by column chromatography (ODS, water / MeCN) to give M3-6 (22 mg). 1H NMR (400 MHz, CDCl3) δ 6.29 (br, 1H), 5.97-5.87 (m, 1H), 5.34-5.22 (m, 3H), 4.58 (dt, J = 5.8, 1.36 Hz, 2H), 4.24-4.22 (m, 1H), 3.29 (q, J = 6.6 Hz, 2H), 2.80 (ddd, J = 16.8, 5.3, 2.7 Hz, 1H), 2.58 (ddd, J = 16.8, 7.0, 2.6 Hz, 1H), 2.34 (t, J = 7.4 Hz, 2H), 2.08 (t, J = 2.6Hz, 1H), 1.69-1.62 (m, 2H), 1.58-1.50 (m, 2H), 1.46 (s, 9H), 1.41-1.33 (m, 2H); LC-MS: [M+H] + = 367.34.

[0176] (Synthesis of M3-9) 4 M HCl ethyl acetate solution (1 mL) was added to M3-6 (22 mg) prepared above and stirred at room temperature for 1 hour. The solvent was evaporated, and DMF (1 mL), M3-8 (30 mg) shown in the formula above, COMU (39 mg), and DIPEA (52 μL) were added to the residue and stirred at room temperature for 2 hours. The solvent was evaporated, and the residue was purified by column chromatography (ODS, water / MeCN) to obtain M3-9 (39 mg). 1H NMR (400 MHz, CDCl3) δ 7.48 (d, J = 8.0 Hz, 1H), 7.10 (t, J = 5.3 Hz, 1H), 7.00 (t, J = 5.0 Hz, 1H), 6.31 (br, 1H), 5.96-5.87 (m, 1H), 5.31 (ddt, J = 17.2, 1.5, 1.5 Hz, 1H), 5.23 (ddt, J = 10.4, 1.3, 1.3 Hz, 1H), 5.15 (br, 1H), 4.63-4.56 (m, 3H), 4.53-4.50 (m, 1H), 4.36-4.33 (m, 1H), 3.76 (t, J = 6.0 Hz, 2H), 3.65-3.64 (m, 12H), 3.58 (t, J = 4.9 Hz, 2H), 3.50-3.38 (m, 2H), 3.36-3.27 (m, 1H), 3.21-3.13 (m, 2H), 2.92 (dd, J = 12.8, 4.9 Hz, 1H), 2.76-2.61 (m, 3H), 2.60-2.46 (m, 2H), 2.34 (t, J = 7.4 Hz, 2H), 2.20 (t, J = 7.5 Hz, 2H), 2.08 (t, J = 2.6 Hz, 1H), 1.78-1.32 (m, 12H); LC-MS: [M+H] + = 740.84.

[0177] (Synthesis of M3-10) To a mixture of M3-9 (39 mg) and DCM (2 mL), Pd(PPh3)4 (6.1 mg) and 1,3-dimethylbarbituric acid (16 mg) were added and stirred at room temperature for 2 hours. The solvent was evaporated, and the residue was purified by column chromatography (ODS, water / MeCN) to give M3-10 (31 mg). 1H NMR (400 MHz, CD3OD) δ 8.05-8.02 (m, 1H), 4.53-4.47 (m, 2H), 4.33 (dd, J = 7.8, 4.5 Hz. 1H), 3.82-3.73 (m, 2H), 3.66-3.62 (m, 12H), 3.56 (t, J = 5.4 Hz, 2H), 3.39 (q, J = 5.0 Hz, 2H), 3.28-3.16 (m, 3H), 2.95 (dd, J = 12.8, 5.0 Hz, 1H), 2.74-2.59 (m, 3H), 2.55 (t, J = 6.1 Hz, 2H), 2.43 (t, J = LC-MS: [M+H] + = 700.73.

[0178] (Synthesis of M3-11) To a mixture of M3-10 (31 mg) and DMF (1 mL), dimedone (6.8 mg), WSC.HCl (8.9 mg), and DMAP (5.4 mg) were added and stirred at room temperature for 20 hours. The solvent was removed by evaporation, and the residue was purified by column chromatography (ODS, water / MeCN, containing 0.1% AcOH) to obtain M3-11 (24 mg). 1H NMR (400 MHz, CDCl3) δ 7.36 (d, J = 8.0 Hz, 1H), 7.12 (t, J = 5.5 Hz, 1H), 7.07 (t, J = 5.3 Hz, 1H), 6.32 (s, 1H), 5.26 (s, 1H), 4.61 (q, J = 7.2 Hz, 1H), 4.54-4.51 (m, 1H), 4.36-4.32 (m, 1H), 3.76 (t, J = 6.0 Hz, 2H), 3.65-3.64 (m, 12H), 3.58 (t, J = 5.0 Hz, 2H), 3.50-3.38 (m, 2H), 3.36-3.27 (m, 1H), 3.22-3.13 (m, 2H), 3.01 (t, J = 7.5 Hz, 2H), 2.92 (dd, J = 12.8, 4.9 Hz, 1H), 2.76-2.61 (m, 3H), 2.60-2.47 (m, 4H), 2.35 (s, 2H), 2.20 (t, J = 7.5 Hz, 2H), 2.11 (t, J = 2.6 Hz, 1H), 1.78-1.37 (m, 12H), 1.08 (s, 6H);LC-MS: [M+H] + = 823.14.

[0179] (Synthesis of M3-13) To a mixture of M3-11 (24 mg) prepared above and DMF (1 mL), M3-12 (8.5 mg) shown in the formula above and DIPEA (20 μL) were added, followed by stirring for 2 hours at 60° C. The solvent was evaporated, and the residue was purified by column chromatography (ODS, water / MeCN) to obtain M3-13 (28 mg). 1H NMR (400 MHz, CDCl3) δ 13.46-13.45 (m, 1H), 8.28 (d, J = 8.8 Hz, 0.4H), 8.01 (d, J = 8.8 Hz, 0.6H), 7.72 (d, J = 1.8 Hz, 0.6H), 7.60 (d. J = 8.2 Hz, 0.4H), 7.53 (d, J = 8.0 Hz, 0.6H), 7.42 (d, J = 2.3 Hz, 0.4H), 7.33-7.21 (m, 2H), 7.06-6.98 (m, 1H), 6.56 (d, J = 8.6 Hz, 1H), 5.96-5.82 (m, 1H), 4.71-4.62 (m, 1H), 4.55-4.52 (m, 1H), 4.37-4.34 (m, 1H), 4.16-4.10 (m, 2H), 3.78-3.75 (m, 2H), 3.67-3.56 (m, 17H), 3.45-3.16 (m, 7H), 2.94-2.91 (m, 3H), 2.77-2.48 (m, 5H), 2.34 (d, J = 5.2 Hz, 4H), 2.21 (t, J = 6.7 Hz, 2H), 2.15-2.14 (m, 1H), 1.90-1.84 (m, 2H), 1.73-1.47 (m, 18H), 1.02 (d, J = 3.8 Hz, 6H);LC-MS: [M+H] + = 1096.21.

[0180] Synthesis of Example 3: The M3-13 prepared above and M3-14 (3.1 mg) represented by the formula above were dissolved in THF / HO (3:1, 1 mL), and copper(II) sulfate pentahydrate (0.7 mg) and sodium L-ascorbate (1.1 mg) were added. The mixture was stirred at room temperature for 16 hours. M3-14 (5.1 mg), copper(II) sulfate pentahydrate (1.7 mg), and sodium L-ascorbate (2.7 mg) were added, and the mixture was stirred at room temperature for 4 hours. M3-14 (5.1 mg), copper(II) sulfate pentahydrate (1.7 mg), and sodium L-ascorbate (2.7 mg) were added, and the mixture was stirred at room temperature for 2 hours. The reaction mixture was purified by column chromatography (ODS, water / MeCN) to obtain Example 3 (11 mg). 1 H NMR (400 MHz, DMSO-d6) δ 13.35 (s, 1H), 11.57-11.46 (m, 1H), 8.12-8.06 (m, 1+0.4H), 7.87-7.82 (m, 2+0.6H), 7.75 (s, 1H), 7.58 (s, 0.6H), 7.42 (t, J = 11.0 Hz, 1H), 7.28 (s, 0.4H), 6.41 (s, 1H), 6.34 (s, 1H), 4.47-4.38 (m, 3H), 4.32-4.28 (m, 1H), 4.14-4.11 (m, 3H), 3.56-3.27 (m, overlapped with water signal), 3.17 (q, J = 5.7 Hz, 2H), 3.11-2.79 (m, 8H), 2.59-2.49 (m, overlapped with DMSO signal), 2.39-2.32 (m, 4H), 2.25 (s, 4H), 2.08-204 (m, 4H), 1.79 (br, 1H), 1.64-1.24 (m, 18H), 0.92 (s, 6H); LC-MS: [M+H] + = 1260.72.

[0181] [Example 4]

[0182] In the 2,3-dihydrophthalazine-1,4-dione of M4-1, the constituent atoms of the ring are numbered for convenience in explaining the substitution position. M4-1, M4-3, M4-4, M4-6, and Example 4 have a methyl group (R １ ), and third place is H (R ２ ), and its isomers (methyl (R １ ), and third place is H (R ２ ), and a compound having a substituent at the 7-position.

[0183] (Synthesis of M4-3) M4-1 (20 mg), M4-2 (N ε -(tert-Butoxycarbonyl)-N α To a mixture of (9H-fluoren-9-ylmethoxy)carbonyl]-L-lysine (32 mg) and DMF (2 mL), WSC·HCl (16 mg) and HOBt·HO (13 mg) were added and stirred at room temperature for 15 hours. The solvent was evaporated, water was added, and the mixture was extracted with ethyl acetate. The organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. After filtration, the solvent in the filtrate was evaporated, and the residue was purified by column chromatography (silica gel, hexane / AcOEt→AcOEt / MeOH) to give M4-3 (34 mg). 1 H NMR (400 MHz, CD3OD) δ 8.14 (d, J = 8.7 Hz, 0.5H), 7.93-7.85 (m, 1+0.5H), 7.75-7.72 (m, 2H), 7.64-7.61 (m, 2+0.5H), 7.55-7.45 (m, 0.5H), 7.37-7.26 (m, 5+0.5H), 7.20 (d, J = 6.5 Hz, 0.5H), 4.39-4.31 (m, 2H), 4.18 (t, J = 6.7 Hz, 1H), 4.12-4.02 (m, 3H), 3.64-3.63 (m, 3H), 3.24-3.19 (m, 2H), 3.02 (t, J = 6.8 Hz, 2H), 1.84-1.71 (m, 3H), 1.66-1.28 (m, 20H); LC-MS: [M+H] + = 742.46.

[0184] (Synthesis of M4-6) Piperidine (9 μL) was added to a mixture of M4-3 (34 mg) and DMF (2 mL) prepared above, and the mixture was stirred at room temperature for 16 hours. The solvent was evaporated, and the residue was purified by column chromatography (ODS, water / MeCN). The obtained M4-4 (containing impurities) was dissolved in DMF (2 mL), and M4-5 (32 mg) and DIPEA (24 μL) shown in the formula above were added, followed by stirring at 60 °C for 2 hours. The solvent was evaporated, and the residue was purified by column chromatography (ODS, water / MeCN) to obtain M4-6 (38 mg). 1 H NMR (400 MHz, DMSO-d6) δ 13.62 (d. J = 8.2 Hz, 1H), 8.28-8.23 (m, 1H), 8.08 (d, J = 8.8 Hz, 0.5H), 7.88 (d, J = 8.8 Hz, 0.5H), 7.84-7.80 (m, 1H), 7.56 (d, J = 2.6 Hz, 0.5H), 7.41 (dd, J = 8.8, 2.6 Hz, 0.5H), 7.35 (d, J = 8.1 Hz, 0.5 H), 7.30 (d, J = 2.4 Hz, 0.5H), 6.76-6.73 (m, 1H), 6.40 (s, 1H), 6.34 (s, 1H), 4.50 (q, J = 6.9 Hz, 1H), 4.31-4.28 (m, 1H), 4.14-4.10 (m, 3H), 3.56-3.02 (m, overlapped with water signal), 2.87 (q, J = 6.4 Hz, 2H), 2.81 (dd, J = 12.4, 5.1 Hz, 1H), 2.57 (d, J = 12.4 Hz, 1H), 2.29 (s, 4H), 2.07-2.04 (m, 2H), 1.76-1.55 (m, 6H), 1.49-1.19 (m, 23H), 0.94 (s, 6H); LC-MS: [M+H] + = 1115.74.

[0185] (Synthesis of Example 4) To a mixture of the above-prepared M4-6 (14 mg) and DCM (1 mL), TFA (0.3 mL) was added and stirred at room temperature for 2 hours. The solvent was evaporated, and the residue was purified by column chromatography (ODS, water / MeCN) to give Example 4 (4.2 mg). 1 H NMR (400 MHz, DMSO-d6) δ 8.24 (t, J = 5.3 Hz, 1H), 7.98 (d, J = 8.7 Hz, 0.1H), 7.90-7.84 (m, 1+0.9H), 7.53 (d, J = 2.6 Hz, 0.9H), 7.38-7.35 (m, 1H), 7.19 (dd, J = 8.8, 2.7 Hz, 0.1H), 6.41 (s, 1H), 6.35 (s, 1H), 4.48 (t, J = 6.4 Hz, 1H), 4.31-4.28 (m, 1H), 4.14-4.11 (m, 3H), 3.57-2.99 (m, overlapped with water signal), 2.81 (dd, J = 12.4, 5.1 Hz, 1H), 2.59-2.49 (m, overlapped with DMSO signal), 2.29 (s, 4H), 2.06 (t, J = 7.4 Hz, 2H), 1.77-1.23 (m, 20H), 0.94 (s, 6H);LC-MS: [M+H] + = 1016.13.

[0186] [Example 5]

[0187] In the 2,3-dihydrophthalazine-1,4-dione of M5-9, the constituent atoms of the ring are numbered for convenience in explaining the substitution position. M5-9, M5-10, and Example 5 have a methyl group (R １ ), and third place is H (R ２ ), and its isomers (methyl (R １ ), and third place is H (R ２ ), and a compound having a substituent at the 7-position.

[0188] (Synthesis of M5-3) To a mixture of M5-1 (51 mg), M5-2 ((S)-Allyl 2-amino-6-[(tert-butoxycarbonyl)amino]hexanoate hydrochloride) (34 mg), and DMF (2 mL), WSC·HCl (30 mg), HOBt·HO (24 mg), and DIPEA (36 μL) were added and stirred at room temperature for 2 days. The solvent was evaporated, and the residue was purified by column chromatography (ODS, water / MeCN) to obtain M5-3 (73 mg). 1 H NMR (400 MHz, CDCl3) δ 7.32 (br, 1H), 6.84 (br, 1H), 5.95-5.85 (m, 2H), 5.33 (ddt, J = 17.2, 1.5, 1.5 Hz, 1H), 5.25 (dd, J = 10.4, 1.2 Hz, 1H), 5.04-4.85 (br, 2H), 4.63-4.49 (m, 4H), 4.34-4.32 (m, 1H), 3.79-3.74 (m, 2H), 3.65-3.63 (m, 12H), 3.57 (t, J = 5.0 Hz, 2H), 3.46-3.44 (m, 2H), 3.18-3.08 (m, 3H), 2.92 (dd, J = 12.9, 5.0 Hz, 1H), 2.74 (d, J = 12.8 Hz, 1H), 2.54 (br, 2H), 2.22 (t, J = 7.7 Hz, 2H), 1.83-1.44 (m, 21H); LC-MS: [M+H] + = 760.40.

[0189] (Synthesis of M5-4) To a mixture of M5-3 (73 mg) and DCM (2 mL), Pd(PPh3)4 (11 mg) and 1,3-dimethylbarbituric acid (30 mg) were added and stirred at room temperature for 2 hours. The solvent was evaporated, and the residue was purified by column chromatography (ODS, water / MeCN) to give M5-4 (49 mg). 1H NMR (400 MHz, CDCl3) δ 7.26-7.23 (m, 1H), 7.06-7.03 (m, 2H), 5.07 (br, 1H), 4.83 (br, 1H), 4.57-4.52 (m, 2H), 4.38-4.35 (m, 1H), 3.80-3.70 (m, 2H), 3.65-3.58 (m, 14H), 3.51-3.40 (m, 2H), 3.21-3.16 (m, 1H), 3.13-3.07 (m, 2H), 2.94 (dd, J = 12.9, 4.9 Hz, 1H), 2.73 (d, J = 12.9 Hz, 1H), 2.53-2.51 (m, 2H), 2.33-2.21 (m, 2H), 1.94-1.85 (m, 1H), 1.79-1.66 (m, 5H), 1.53-1.43 (m, 15H);LC-MS: [M+H] + = 720.74.

[0190] (Synthesis of M5-6) To a mixture of M5-4 (78 mg), M5-5 (shown in the formula above), and DMF (2 mL), WSC·HCl (31 mg), HOBt·HO (25 mg), and DIPEA (38 μL) were added and stirred at room temperature for 17 hours. The solvent was evaporated, and the residue was purified by column chromatography (ODS, water / MeCN) to obtain M5-6 (88 mg). 1H NMR (400 MHz, CD3OD) δ 5.99-5.89 (m, 1H), 5.33 (ddt, J = 17.2, 1.5, 1.5 Hz, 1H), 5.23 (ddt, J = 10.5, 1.2, 1.2 Hz, 1H), 4.63 (dt, J = 5.6, 1.3 Hz, 2H), 4.51-4.47 (m, 1H), 4.37 (dd, J = 8.7, 5.3 Hz, 1H), 4.30 (dd, J = 7.9, 4.5 Hz, 1H), 4.01 (d, J = 17.6 Hz, 1H), 3.94 (d, J = 17.6 Hz, 1H), 3.75 (td, J = 6.1, 1.2 Hz, 2H), 3.63-3.61 (m, 12H), 3.54 (t, J = 5.5 Hz, 2H), 3.36 (t, J = 5.4 Hz, 2H), 3.23-3.18 (m, 1H), 3.04 (t, J = 6.5 Hz, 2H), 2.93 (dd, J = 12.7, 5.0 Hz, 1H), 2.70 (d, J = 12.7 Hz, 1H), 2.52 (t, J = 6.1 Hz, 2H), 2.22 (t, J = 7.4 Hz, 2H), 1.89-1.55 (m, 6H), 1.51-1.38 (m, 15H); LC-MS: [M+H] + = 817.79.

[0191] (Synthesis of M5-7) To a mixture of M5-6 (29 mg) and DCM (1 mL), Pd(PPh3)4 (4.1 mg) and 1,3-dimethylbarbituric acid (11 mg) were added and stirred at room temperature for 1 hour. The solvent was evaporated, and the residue was purified by column chromatography (ODS, water / MeCN) to give M5-7 (26 mg). 1H NMR (400 MHz, CD3OD) δ 8.15 (d, J = 7.6 Hz, 1H), 8.02 (t, J = 5.1 Hz, 1H), 4.49 (dd, J = 7.8, 4.4 Hz, 1H), 4.41-4.36 (m, 1H), 4.30 (dd, J = 7.9, 4.5 Hz, 1H), 3.96-3.86 (m, 2H), 3.77-3.72 (m, 2H), 3.64-3.59 (m, 12H), 3.54 (t, J = 5.4 Hz, 2H), 3.38-3.34 (m, 2H), 3.23-3.18 (m, 1H), 3.03 (t, J = 6.7 Hz, 2H), 2.93 (dd, J = 12.7, 5.0 Hz, 1H), 2.70 (d, J = 12.7 Hz, 1H), 2.52 (t, J = 6.1 Hz, 2H), 2.22 (t, J = 7.4 Hz, 2H), 1.90-1.81 (m, 1H), 1.78-1.53 ​​(m, 5H), 1.49-1.37 (m, 15H);LC-MS: [M+H] + = 777.72.

[0192] (Synthesis of M5-8) Dimedone (4.8 mg), WSC.HCl (6.5 mg), and DMAP (3.8 mg) were added to a mixture of M5-7 (24 mg) and DMF (1 mL), and the mixture was stirred at room temperature for 5 hours. The solvent was removed by evaporation, and the residue was purified by column chromatography (ODS, water / MeCN, containing 0.1% AcOH) to give M5-8 (8.3 mg). 1H NMR (400 MHz, CDCl3) δ 7.52-7.46 (m, 2H), 7.07 (br, 1H), 6.31 (br, 1H), 5.21 (br, 1H), 4.89 (br, 1H), 4.76-4.60 (m, 2H), 4.56-4.50 (m, 2H), 4.36-4.33 (m, 1H), 3.77 (t, J = 5.8 Hz, 2H), 3.64-3.63 (m, 12H), 3.57 (t, J = 5.0 Hz, 2H), 3.44-3.41 (m, 2H), 3.18-3.09 (m, 3H), 2.91 (dd, J = 12.8, 4.9 Hz, 1H), 2.72 (d, J = 12.8 Hz, 1H), 2.54-2.45 (m, 6H), 2.20 (t, J = 7.5 Hz, 2H), 1.89-1.58 (m, 6H), 1.52-1.43 (m, 15H), 1.08 (s, 6H); LC-MS: [M+H] + = 899.63.

[0193] (Synthesis of M5-10) DIPEA (3.7 μL) was added to a mixture of M5-8 (4.8 mg), M5-9 (1.6 mg) represented by the formula above, and DMF (0.5 mL), and the mixture was stirred at 60° C. for 3 hours. The reaction mixture was purified by column chromatography (ODS, water / MeCN) to obtain M5-10 (6.0 mg). 1H NMR (400 MHz, CD3OD) δ 8.18 (d, J = 8.8 Hz, 0.4H), 8.00 (d, J = 8.8 Hz, 0.6H), 7.68 (d, J = 2.5 Hz, 0.6H), 7.45-7.36 (m, 1.4H), 6.52 (br, 1H), 4.52-4.43 (m, 3H), 4.31-4.25 (m, 2H), 4.19-4.16 (m, 2H), 3.84-3.78 (m, 2H), 3.76-3.69 (m, 2H), 3.68-3.61 (m, 15H), 3.53 (t, J = 5.4 Hz, 2H), 3.35 (t, J = 5.4 Hz, 2H), 3.22-3.17 (m, 1H), 3.00 (t, J = 6.1 Hz, 2H), 2.92 (dd, J = 12.7, 4.9 Hz, 1H), 2.70 (d, J = 12.7 Hz, 1H), 2.56-2.43 (m, 2H), 2.37 (s, 4H), 2.21 (t, J = 7.3 Hz, 2H), 1.92-1.88 (m, 2H), 1.83-1.54 (m, 12H), 1.47-1.30 (m, 15H), 1.02 (s, 6H);LC-MS: [M+H] + = 1173.01.

[0194] Synthesis of Example 5 To a mixture of M5-10 (6.0 mg) prepared above and DCM (0.5 mL), TFA (0.2 mL) was added and stirred at room temperature for 1 hour. The solvent was evaporated, and the residue was purified by column chromatography (ODS, water / MeCN, containing 0.1% TFA) to give Example 5 (4.9 mg). 1H NMR (400 MHz, DMSO-d6) δ 13.29 (t, J = 5.2 Hz, 1H), 11.54 (br, 1H), 8.13-8.10 (m, 1+0.5H), 7.91-7.87 (m, 1+0.5H), 7.82 (t, J = 5.7 Hz, 1H), 7.60-7.58 (m, 3+0.5H), 7.45-7.39 (m, 1H), 7.29 (s, 0.5H), 6.40 (s, 1H), 6.35 (s, 1H), 4.43-4.29 (m, 3H), 4.25-4.20 (m, 1H), 4.17-4.11 (m, 3H), 3.67 (q, J = 6.3 Hz, 2H), 3.61-3.06 (m, overlapped with water signal), 2.82 (dd, J = 12.5, 5.1 Hz, 1H), 2.77-2.69 (m, 2H), 2.58 (d, J = 12.5 Hz, 1H), 2.43-2.29 (m, 6H), 2.06 (t, J = 7.4 Hz, 2H), 1.81-1.76 (m, 2H), 1.65-1.56 (m, 4H), 1.51-1.39 (m, 10H), 1.35-1.23 (m, 4H), 0.95 (s, 6H); LC-MS: [M+H] + = 1072.86.

[0195] [Example 6]

[0196] In the 2,3-dihydrophthalazine-1,4-dione of M6-5, the constituent atoms of the ring are numbered for convenience in explaining the substitution position. M6-5, M6-6, and Example 6 have a methyl group (R １ ), and third place is H (R ２ ), and its isomers (methyl (R １ ), and third place is H (R ２ ), and a compound having a substituent at the 7-position.

[0197] (Synthesis of M6-3) A mixture of nickel(II) chloride, dimethoxyethane adduct (0.8 mg), 4,4'-Di-tert-butyl-2,2'-dipyridyl (1.0 mg), and trifluoromethylbenzene (1.2 mL) was added with tris(trimethylsilyl)silane (0.23 mL) and (4,4'-Di-tert-butyl-2,2'-bipyridine)bis[3,5-difluoro-2-[5-trifluoromethyl-2-pyridinyl-κN)phenyl-κC]iridium(III) hexafluorophosphate (8.2 mg). The solution was added to M6-1 (200 mg), shown in the formula above. Trifluoromethylbenzene (6.1 mL) and M6-2 (tert-butyl aziridine-1-carboxylate) (0.19 mL) were added, and the mixture was irradiated with blue LED light for 2 hours. The solvent was evaporated, and the residue was purified by column chromatography (silica gel, hexane / AcOEt) to obtain the desired product (104 mg). 1 H NMR (400 MHz, CDCl3) δ 7.70 (d, J = 7.9 Hz, 1H), 7.52 (d, J = 1.4 Hz, 1H), 7.37 (dd, J = 7.9, 1.4 Hz, 1H), 4.55 (br, 1H), 3.91 (s, 3H), 3.90 (s, 3H), 3.39 (dt, J = 6.7, 6.7 Hz, 2H), 2.87 (t, J = 6.7 Hz, 2H), 1.43 (s, 9H);LC-MS: [M+Na] + = 360.30.

[0198] (Synthesis of M6-4) To a mixture of the prepared M6-3 (52 mg) and MeOH (1 mL), 5 M aqueous sodium hydroxide solution (0.15 mL) was added and stirred at room temperature for 7 hours. 5 M aqueous sodium hydroxide solution (0.15 mL) was added, and the mixture was stirred at room temperature for 16 hours. Hydrochloric acid was added to neutralize the mixture, and the reaction mixture was purified by column chromatography (ODS, water / MeCN) to obtain M6-4 (35 mg).1 H NMR (400 MHz, CDCl3) δ 7.72 (d, J = 7.9 Hz, 1H), 7.58 (s, 1H), 7.42 (d, J = 7.9 Hz, 1H), 3.34-3.28 (m, 2H), 2.85 (t, J = 7.1 Hz, 2H), 1.41 (s, 9H);LC-MS: [M+Na] + = 332.23.

[0199] (Synthesis of M6-5) To a mixture of M6-4 (35 mg) and THF (1 mL), acetic anhydride (53 μL) was added and the mixture was stirred under reflux for 4 hours. EtOH (1 mL) and methylhydrazine (30 μL) were added and the mixture was stirred under reflux for 4 hours. The solvent was evaporated, and the residue was purified by column chromatography (ODS, water / MeCN) to give M6-5 (32 mg). 1 H NMR (400 MHz, CD3OD) δ 8.21 (d, J = 8.2 Hz, 0.5H), 8.13 (d, J = 1.1 Hz, 0.5H), 8.00 (d, J = 8.2 Hz, 0.5H), 7.91 (d, J = 1.3 Hz, 0.5H), 7.76-7.71 (m, 1H), 6.69 (br, 1H), 3.66-3.65 (m, 3H), 3.37-3.30 (m, 2H), 2.98 (t, J = 7.0 Hz, 2H), 1.38 (s, 9H); LC-MS: [M+H] + = 320.30.

[0200] (Synthesis of M6-6) To a mixture of M6-5 (32 mg) and MeOH (0.5 mL), 4 M HCl 1,4-dioxane solution (2 mL) was added and stirred at room temperature for 2 hours. The solvent was evaporated to give M6-6 (26 mg). 1H NMR (400 MHz, CD3OD) δ 8.28 (d, J = 8.2 Hz, 0.5H), 8.19 (s, 0.5H), 8.08 (d, J = 8.2 Hz, 0.5H), 8.00 (s, 0.5H), 7.83-7.77 (m, 1H), 3.67-3.66 (m, 3H), 3.31-3.27 (m, 2H), 3.18 (t, J = 7.4 Hz, 2H); LC-MS: [M+H] + = 220.20.

[0201] (Synthesis of Example 6) DIPEA (13 μL) was added to a mixture of M6-6 (4.6 mg) prepared above, M6-7 (11 mg) represented by the formula above, and DMF (1 mL), and the mixture was stirred for 2 hours at 60° C. The reaction solution was purified by column chromatography (ODS, water / MeCN) to obtain Example 6 (13 mg). 1 H NMR (400 MHz, DMSO-d6) δ 13.45 (t, J = 5.3 Hz, 1H), 8.16-8.14 (m, 1H), 7.91 (d, J = 8.1 Hz, 0.5H), 7.87 (s, 0.5), 7.83-7.78 (m, 2H), 6.40 (s, 1H), 6.34 (s, 1H), 4.31-4.28 (m, 1H), 4.14-4.10 (m, H), 3.89 (q, J = 5.9 Hz, 2H), 3.60-3.06 (m, overlapped with water signal), 2.81 (dd, J = 12.4, 5.1 Hz, 1H), 2.57 (d, J = 12.4 Hz, 1H), 2.25 (s, 4H), 2.05 (t, J = 7.4 Hz, 2H), 1.64-1.56 (m, 1H), 1.52-1.40 (m, 3H), 1.34-1.23 (m, 2H), 0.93 (s, 6H);LC-MS: [M+H] + = 815.63.

[0202] [Example 7]

[0203] In the 2,3-dihydrophthalazine-1,4-dione of M7-1, the constituent atoms of the ring are numbered for convenience in explaining the substitution position. M7-1, M7-3, and Example 7 have a methyl (R １ ), and third place is H (R ２ ), and its isomers (methyl (R １ ), and third place is H (R ２ ), and a compound having a substituent at the 7-position.

[0204] (Synthesis of M7-3) DIPEA (6.5 μL) was added to a mixture of M7-1 (M6-6 synthesized in Example 6) (2.4 mg), M7-2 (M5-8 synthesized in Example 5) (8.3 mg), and DMF (0.5 mL), and the mixture was stirred at 60° C. for 2 hours. The reaction solution was purified by column chromatography (ODS, water / MeCN) to obtain M7-3 (10 mg). 1H NMR (400 MHz, CDCl3) δ 13.53 (t, J = 5.5 Hz, 1H), 8.31 (d, J = 8.1 Hz, 0.5H), 8.23 ​​(s, 0.5H), 8.03-8.01 (m, 1H), 7.71 (dd, J = 8.1, 1.2 Hz, 0.5), 7.66 (dd, J = 8.2, 1.5 Hz, 0.5H), 7.54 (br, 1H), 7.41-7.34 (m, 1H), 6.98-6.89 (m, 1H), 6.66 (s, 1H), 5.64 (br, 1H), 4.95-4.91 (m, 1H), 4.53 (t, J = 5.6 Hz, 1H), 4.42-4.31 (m, 4H), 4.27-4.14 (m, 2H), 3.75-3.54 (m, 18H), 3.45-3.41 (m, 2H), 3.18-3.05 (m, 6H), 2.91 (dd, J = 12.9, 4.9 Hz, 1H), 1.10 (dd, J = 12.7, 4.4 Hz, 1H), 2.53-2.49 (m, 2H), 2.38-2.35 (m, 4H), 2.26-2.19 (m, 2H), 1.78-1.58 (m, 6H), 1.43-1.23 (m, 15H), 1.02 (s, 6H); LC-MS: [M+H] + = 1100.82.

[0205] Synthesis of Example 7 To a mixture of M7-3 (10 mg) prepared above and DCM (0.5 mL), TFA (0.1 mL) was added and stirred at room temperature for 1 hour. The solvent was evaporated, and the residue was purified by column chromatography (ODS, water / MeCN, containing 0.1% TFA) to give Example 7 (8.4 mg). 1H NMR (400 MHz, DMSO-d6) δ 13.23 (t, J = 5.3 Hz, 1H), 11.62 (br, 1H), 8.17-8.08 (m, 2H), 7.92-7.78 (m, 4H), 7.59 (br, 3H), 6.40 (s, 1H), 6.35 (s, 1H), 4.40-4.29 (m, 3H), 4.27-4.19 (m, 1H), 4.14-4.11 (m, 1H), 4.05-3.97 (m, 2H), 3.62-3.06 (m, overlapped with water signal), 2.82 (dd, J = 12.4, 5.1 Hz, 1H), 2.77-2.71 (m, 2H), 2.57 (d, J = 12.4 Hz, 1H), 2.42-2.29 (m, 6H), 2.06 (t, J = 7.4 Hz, 2H), 1.65-1.56 (m, 2H), 1.54-1.40 (m, 6H), 1.37-1.24 (m, 4H), 0.95 (s, 6H);LC-MS: [M+H] + = 1000.67.

[0206] [Example 8]

[0207] In the 2,3-dihydrophthalazine-1,4-dione of M8-8, the constituent atoms of the ring are numbered for convenience in explaining the substitution position. M8-8 and Example 8 have a methyl (R １ ), and third place is H (R ２ ), and its isomers (methyl (R １ ), and third place is H (R ２ ), and a compound having a substituent at the 7-position.

[0208] (Synthesis of M8-2) Dimedone (93 mg), WSC·HCl (122 mg), and DMAP (74 mg) were added to a mixture of M8-1 (N-(tert-butoxycarbonyl)glycine) (106 mg) and DMF (6 mL), and the mixture was stirred at room temperature for 15 hours. The solvent was evaporated, and the residue was purified by column chromatography (ODS, water / MeCN, containing 0.1% AcOH). 4 M HCl in 1,4-dioxane (3 mL) was added to the resulting compound (containing impurities), and the mixture was stirred at room temperature for 30 minutes. The solvent was evaporated, and the residue was purified by column chromatography (ODS, water / MeCN, containing 0.1% AcOH) to give M8-2 (26 mg). 1 H NMR (400 MHz, DMSO-d6) δ 8.14 (br, 3H), 4.28 (s, 2H), 2.52-2.49 (m, 4H), 1.02 (s, 6H); LC-MS: [M+H] + = 198.27.

[0209] (Synthesis of M8-4) To a mixture of M8-2 (24 mg), M8-3 (28 mg), and DMF (1 mL), WSC·HCl (23 mg), HOBt·HO (19 mg), and DIPEA (35 μL) were added and stirred at room temperature for 3 hours. The solvent was removed, and the residue was purified by column chromatography (ODS, water / MeCN, containing 0.1% AcOH) to obtain M8-4 (27 mg). 1 H NMR (400 MHz, DMSO-d6) δ 7.80 (br, 1H), 6.76 (d, J = 9.6 Hz, 1H), 4.28 (dd, J = 18.4, 6.4 Hz, 1H), 3.96 (dd, J = 18.4, 4.4 Hz, 2H), 2.88-2.82 (m, 2H), 2.62 (s, 6H), 2.07 (s, 4H), 1.66-1.61 (m, 2H), 1.53-1.48 (m, 2H), 1.42-1.28 (m, 11H);LC-MS: [M+H] + = 455.01.

[0210] Synthesis of Example 8: 4 M HCl 1,4-dioxane solution (1 mL) was added to M8-4 (27 mg) prepared above and stirred at room temperature for 30 minutes. The solvent was evaporated, and DMF (1 mL), M8-6 (29 mg) represented by the formula above, WSC·HCl (14 mg), HOBt·HO (11 mg), and DIPEA (52 μL) were added to the residue and stirred at room temperature for 3 hours. DIPEA (21 μL) was added and stirred at room temperature for 1 hour. The reaction solution was purified by column chromatography (ODS, water / MeCN, containing 0.1% AcOH). DMF (0.5 mL), M8-8 (1.0 mg) represented by the formula above, and DIPEA (2.1 μL) were added to the obtained M8-7 (containing impurities) and stirred at 60 °C for 2 hours. The reaction mixture was purified by column chromatography (ODS, water / MeCN, containing 0.1% TFA) to obtain Example 8 (0.8 mg). 1 H NMR (400 MHz, CD3OD) δ 8.18 (d, J = 8.7 Hz, 0.5H), 8.00 (d, J = 8.9 Hz, 0.5H), 7.68 (d, J = 2.5 Hz, 0.5H), 7.45-7.42 (m, 1H), 7.38 (dd, J = 8.9, 1.9 Hz, 0.5H), 4.50-4.47 (m, 3H), 4.34-4.28 (m, 2H), 4.20-4.16 (m, 2H), 3.82-3.69 (m, 4H), 3.66-3.61 (m, 15H), 3.53 (t, J = 5.4 Hz, 2H), 3.36-3.17 (m, overlapped with methanol signal), 3.00-2.96 (m, 2H), 2.92 (dd, J = 12.8, 4.9 Hz, 1H), 2.79 (s, 6H), 2.70 (d, J = 12.8 Hz, 1H), 2.53-2.46 (m, LC-MS: [M+H] + = 1101.61.

[0211] [Example 9]

[0212] In the 2,3-dihydrophthalazine-1,4-dione of M9-8, the constituent atoms of the ring are numbered for convenience in explaining the substitution position. M9-8, M9-9, and Example 9 have a methyl (R １ ), and third place is H (R ２ ), and its isomers (methyl (R １ ), and third place is H (R ２ ), and a compound having a substituent at the 7-position.

[0213] (Synthesis of M9-3) To a mixture of M9-1 (Fmoc-Arg(Pbf)-OH) (100 mg), M9-2 (23 mg) represented by the formula above, and DMF (3 mL), WSC·HCl (36 mg), HOBt·HO (28 mg), and DIPEA (54 μL) were added and stirred at room temperature for 3 hours. The solvent was evaporated, and the residue was purified by column chromatography (ODS, water / MeCN). DMF (2 mL) and piperidine (15 μL) were added to the resulting compound (containing impurities), and the mixture was stirred at room temperature for 15 hours. The solvent was evaporated, and the residue was purified by column chromatography (ODS, water / MeCN) to obtain M9-3 (19 mg). 1 H NMR (400 MHz, CD3OD) δ 5.98-5.88 (m, 1H), 5,32 (ddt, J = 17.2, 1.4, 1.4 Hz, 1H), 5.22 (ddt, J = 10.4, 1.4, 1.4 Hz, 1H), 4.62 (dt, J = 2.57 (s, 3H), 2.51 (s, 3H), 2.08 (s, 3H), 1.70-1.63 (m, 1H), 1.61-1.51 (m, 3H), 1.45 (s, 6H); LC-MS: [M+H] + = 524.67.

[0214] (Synthesis of M9-5) To a mixture of M9-3 (19 mg), M9-4 (18 mg) and DMF (1 mL), WSC·HCl (8.4 mg) and HOBt·HO (6.7 mg) were added and stirred at room temperature for 4 hours. The solvent was removed by evaporation, and the residue was purified by column chromatography (ODS, water / MeCN) to give M9-5 (28 mg). 1 H NMR (400 MHz, CD3OD) δ 5.98-5.88 (m, 1H), 5.32 (ddt, J = 17.2, 1.4, 1.4 Hz, 1H), 5.22 (ddt, J = 10.5, 1.4, 1.4 Hz, 1H), 4.62 (dt, J = 5.6, 1.4 Hz, 2H), 4.48 (dd, J = 7.8, 4.3 Hz, 1H), 4.42 (dd, J = 8.6, 5.1 Hz, 1H), 4.29 (dd, J = 7.8, 4.5 Hz, 1H), 4.01 (d, J = 17.5 Hz, 1H), 3.93 (d, J = 17.5 Hz, 1H), 3.73 (t, J = 6.0 Hz, 2H), 3.63-3.58 (m, 12H), 3.53 (t, J = 5.5 Hz, 2H), 3.35 (t, J = 5.5 Hz, 2H), 3.22-3.15 (m, 3H), 3.00 (s, 2H), 2.91 (dd, J = 12.7, 5.0 Hz, 1H), 2.69 (d, J = 12.7 Hz, 1H), 2.58 (s, 3H), 2.51-2.48 (m, 5H), 2.21 (t, J = 7.4 Hz, 2H), 2.08 (s, 3H), 1.92-1.82 (m, 1H), 1.78-1.54 (m, 7H), 1.47-1.40 (m, 8H);LC-MS: [M+H] + = 998.28.

[0215] (Synthesis of M9-6) To a mixture of M9-5 (28 mg) and DCM (1 mL), Pd(PPh3)4 (3.2 mg) and 1,3-dimethylbarbituric acid (8.8 mg) were added and stirred at room temperature for 2 hours. The solvent was evaporated, and the residue was purified by column chromatography (ODS, water / MeCN) to give M9-6 (22 mg). 1 H NMR (400 MHz, CD3OD) δ 4.48 (dd, J = 7.9, 4.3 Hz, 1H), 4.43-4.40 (m, 1H), 4.29 (dd, J = 7.9, 4.5 Hz, 1H), 3.95-3.85 (m, 2H), 3.73 (t, J = 6.0 Hz, 2H), 3.64-3.58 (m, 12H), 3.53 (t, J = 5.5 Hz, 2H), 3.37-3.33 (m, 2H), 3.24-3.17 (m, 3H), 3.00 (s, 2H), 2.91 (dd, J = 12.8, 5.1 Hz, 1H), 2.69 (d, J = 12.8 Hz, 1H), 2.57 (s, 3H), 2.51-2.48 (m, 5H), 2.21 (t, J = 7.4 Hz, 2H), 2.08 (s, 3H), 1.91-1.83 (m, 1H), 1.78-1.54 (m, 7H), 1.47-1.39 (m, 8H);LC-MS: [M+H] + = 958.18.

[0216] (Synthesis of M9-7) Dimedone (3.5 mg), WSC.HCl (4.9 mg), and DMAP (2.8 mg) were added to a mixture of M9-6 (22 mg) and DMF (1 mL), and the mixture was stirred at room temperature for 4 hours. The reaction mixture was purified by column chromatography (ODS, water / MeCN, containing 0.1% AcOH) to give M9-7 (11 mg). 1H NMR (400 MHz, CD3OD) δ 8.13-8.11 (m, 1H), 7.97 (t, J = 4.7 Hz, 1H), 4.66-4.55 (m, 2H), 4.49-4.44 (m, 2H), 4.29 (dd, J = 7.8, 4.5 Hz, 1H), 3.73 (t, J = 6.0 Hz, 2H), 3.63-3.58 (m, 12H), 3.53 (t, J = 5.4 Hz, 2H), 3.37-3.33 (m, 2H), 3.21-3.17 (m, 3H), 3.00 (s, 3H), 2.91 (dd, J = 12.7, 5.0 Hz, 1H), 2.69 (d, J = 12.7 Hz, 1H), 2.57-2.47 (m, 12H), 2.21 (t, J = 7.4 Hz, 2H), 2.08 (s, 3H), 1.93-1.85 (m, 1H), 1.77-1.54 (m, 7H), 1.47-1.39 (m, 8H), 1.08 (s, 6H);LC-MS: [M+H] + = 1080.71.

[0217] (Synthesis of M9-9) DIPEA (5.3 μL) was added to a mixture of M9-7 (11 mg), M9-8 (3.0 mg) represented by the formula above, and DMF (1 mL), and the mixture was stirred at 60° C. for 2 hours. The reaction mixture was purified by column chromatography (ODS, water / MeCN) to obtain M9-9 (10 mg). 1H NMR (400 MHz, CD3OD) δ 8.18 (d, J = 8.8 Hz, 0.4H), 7.97 (d, J = 8.8 Hz, 0.6H), 7.68 (d, J = 2.5 Hz, 0.6H), 7.43-7.37 (m, 1.4H), 4.50-4.43 (m, 3H), 4.31-4.27 (m, 2H), 4.18-4.14 (m, 2H), 3.85-3.55 (m, 19H), 3.52 (t, J = 5.4 Hz, 2H), 3.34 (t, J = 5.5 Hz, 2H), 3.20-3.13 (m, 3H), 2.98 (s, 2H), 2.90 (dd, J = 12.7, 5.0 Hz, 1H), 2.69 (d, J = 12.7 Hz, 1H), 2.58-2.40 (m, 8H), 2.06 (s, 4H), 1.95-1.85 (m, 2H), 1.81-1.51 (m, 14H), 1.45-1.38 (m, 8H), 1.00 (s, 6H);LC-MS: [M+H] + = 1354.17.

[0218] (Synthesis of Example 9) To a mixture of the above-prepared M9-9 (10 mg) and DCM (0.5 mL), TFA (0.5 mL) was added and stirred at room temperature for 1 hour. The solvent was evaporated, and the residue was purified by column chromatography (ODS, water / MeCN, containing 0.1% TFA) to obtain Example 9 (6.3 mg). 1H NMR (400 MHz, DMSO-d6) δ 8.16-8.11 (m, 1.4H), 7.94 (t, J = 6.3 Hz, 1H), 7.88 (d, J = 8.8 Hz, 0.6H), 7.83 (t, J = 5.5 Hz, 1H), 7.59 (d, J = 2.6 Hz, 0.6H), 7.45-7.39 (m, 1.4H), 7.00 (br, 4H), 6.41 (s, 1H), 6.36 (s, 1H), 4.44-4.26 (m, 4H), 4.17-4.11 (m, 3H), 3.68-3.05 (m, overlapped with water signal), 2.81 (dd, J = 12.4, 5.1 Hz, 1H), 2.59-2.35 (m, overlapped with DMSO signal), 2.29 (s, 4H), 2.06 (t, J = 7.4 Hz, 2H), 1.83-1.75 (m, 2H), 1.68-1.56 (m, 4H), 1.53-1.37 (m, 10H), 1.35-1.23 (m, 2H), 0.95 (s, 6H); LC-MS: [M+H] + = 1101.14.

[0219] [Example 10]

[0220] In the 2,3-dihydrophthalazine-1,4-dione of M10-8, the constituent atoms of the ring are numbered for convenience in explaining the substitution position. M10-8 and Example 10 have a methyl (R １ ), and third place is H (R ２ ), and its isomers (methyl (R １ ), and third place is H (R ２ ), and a compound having a substituent at the 7-position.

[0221] (Synthesis of M10-2) To a mixture of M10-1 (283 mg), THF (2 mL), and HO (20 mL), 0.25 M aqueous sodium hydroxide solution (5.3 mL) was added and stirred under ice-cooling for 1 hour. Hydrochloric acid was added to neutralize the mixture, and the solvent was evaporated. The residue was purified by column chromatography (ODS, water / MeCN) to obtain M10-2 (79 mg). 1 H NMR (400 MHz, CD3OD) δ 3.70 (s, 3H), 3.21-3.16 (m, 6H), 2.81-2.76 (m, 6H), 2.59-2.53 (m, 4H); LC-MS: [M+H] + = 245.33.

[0222] (Synthesis of M10-4) To a mixture of M10-2 (10 mg), M10-3 (20 mg), and DMF (1 mL), WSC·HCl (9.4 mg), HOBt·HO (7.5 mg), and DIPEA (21 μL) were added and stirred at room temperature for 16 hours. The solvent was evaporated, and the residue was purified by column chromatography (ODS, water / MeCN) to give M10-4 (29 mg). 1 H NMR (400 MHz, CD3OD) δ 4.50 (dd, J = 7.9, 4.3 Hz, 1H), 4.31 (dd, J = 7.9, 4.5 Hz, 1H), 3.70 (s, 3H), 3.65-3.63 (m, 4H), 3.60-3.58 (m, 4H), 3.54-3.50 (m, 4H), 3.31-3.08 (m, overlapped with methanol signal), 2.93 (dd, J = 12.8, 5.0 Hz, 1H), 2.75-2.69 (m, 3H), 2.63-2.58 (m 2H), 2.20 (t, J = 7.4 Hz, 2H), 1.80-1.55 (m, 8H), 1.48-1.40 (m, 2H); LC-MS: [M+H] + = 245.33.

[0223] (Synthesis of M10-5) To a mixture of M10-4 (28 mg) and MeOH (0.5 mL), 5 M aqueous sodium hydroxide solution (41 μL) was added and stirred at room temperature for 3 days. Hydrochloric acid was added to neutralize the mixture, and the reaction mixture was purified by column chromatography (ODS, water / MeCN) to obtain M10-5 (21 mg). 1 H NMR (400 MHz, CD3OD) δ 4.50-4.47 (m, 1H), 4.30 (dd, J = 7.9, 4.4 Hz, 1H), 3.65-3.63 (m, 4H), 3.60-3.58 (m, 4H), 3.54-3.50 (m, 4H), 3.31-3.14 (m, overlapped with methanol signal), 2.93 (dd, J = 12.8, 4.9 Hz, 1H), 2.77-2.69 (m, 7H), 2.53 (t, J = 6.7 Hz, 2H), 2.39 (t, J = 6.9 Hz, 2H), 2.20 (t, J = 7.4 Hz, 2H), 1.79-1.55 (m, 8H), 1.49-1.40 (m, 2H); LC-MS: [M+H] + = 660.05.

[0224] (Synthesis of M10-7) To a mixture of M10-5 (21 mg), M10-6 (M8-2 synthesized in Example 8) (7.5 mg), and DMF (1 mL), WSC·HCl (7.3 mg), HOBt·HO (5.9 mg), and DIPEA (11 μL) were added and stirred at room temperature for 4 hours. The reaction mixture was purified by column chromatography (ODS, water / MeCN, containing 0.1% AcOH) to obtain M10-7 (12 mg). 1H NMR (400 MHz, CDCl3) δ 8.53 (t, J = 4.9 Hz, 1H), 7.81 (t, J = 5.0 Hz, 1H), 6.68 (t, J = 5.1 Hz, 1H), 6.09 (s, 1H), 5.60 (s, 1H), 4.72 (d, J = 5.3 Hz, 2H), 4.53-4.50 (m, 1H), 4.34-4.31 (m, 1H), 3.65-3.52 (m, 12H), 3.37-3.29 (m, 4H), 3.19-3.14 (m, 1H), 2.92 (dd, J = 12.8, 5.0Hz, 1H), 2.80-2.43 (m, 18H), 2.21-2.17 (m, 2H), 2.04 (s, 4H), 1.81-1.60 (m, 8H), 1.49-1.41 (m, 2H), 1.09 (s, 6H);LC-MS: [M+H] + = 839.39.

[0225] (Synthesis of Example 10) DIPEA (7.5 μL) was added to a mixture of M10-7 (12 mg) prepared above, M10-8 (4.2 mg) represented by the above formula, and DMF (1 mL), and the mixture was stirred for 4 hours at 60° C. The reaction solution was purified by column chromatography (ODS, water / MeCN, containing 0.1% TFA) to obtain Example 10 (5.1 mg). 1H NMR (400 MHz, DMSO-d6) δ 13.27 (t, J = 5.5 Hz, 1H), 11.61 (br, 1H), 8.27 (br, 1H), 8.12 (d, J = 8.8 Hz, 0.5H), 8.04 (br, 1H), 7.88 (d, J = 9.0 Hz, 0.5H), 7.74 (t, J = 5.7 Hz, 1H), 7.58 (d, J = 2.6 Hz, 0.5H), 7.45-7.39 (m, 1H), 7.29 (d, J = 2.1 Hz, 0.5H), 6.41 (s, 1H), 6.36 (s, 1H), 4.41-4.39 (m, 2H), 4.31 (dd, J = 7.6, 4.7 Hz, 1H), 4.17-4.11 (m, 3H), 3.71-3.04 (m, overlapped with water signal), 2.82 (dd, J = 12.4, 5.1 Hz, 1H), 2.59-2.45 (m, overlapped with DMSO LC-MS: [M+H] + = 1112.74.

[0226] [Example 11]

[0227] In the 2,3-dihydrophthalazine-1,4-dione of M11-5, the constituent atoms of the ring are numbered for convenience in explaining the substitution position. M11-5 and Example 11 have a methyl (R １ ), and third place is H (R ２ ), and its isomers (methyl (R １ ), and third place is H (R ２ ), and a compound having a substituent at the 7-position.

[0228] (Synthesis of M11-3) Potassium carbonate (493 mg) was added to a mixture of M11-1 (500 mg), M11-2 (710 mg), and DMF (10 mL), followed by stirring at 50°C for 7 hours. The solvent was removed by distillation, water was added, and the mixture was extracted with ethyl acetate. The organic layer was washed with saturated brine and then dried over anhydrous sodium sulfate. After filtration, the solvent in the filtrate was removed by distillation, and the residue was purified by column chromatography (silica gel, hexane / AcOEt) to obtain M11-3 (828 mg). 1 H NMR (400 MHz, CDCl3) δ 7.79 (d, J = 8.7 Hz, 1H), 7.36-7.32 (m, 4H), 7.30-7.25 (m, 1H), 7.04 (d, J = 2.6 Hz, 1H), 6.96 (dd, J = 8.7, 2.6 Hz, 1H), 4.50 (s, 2H), 4.00 (t, J = 6.5 Hz, 2H), 3.91 (s, 3H), 3.87 (s, 3H), 3.48 (t, J = 6.5 Hz, 2H), 1.83-1.77 (m, 2H), 1.68-1.62 (m, 2H), 1.52-1.40 (m, 4H);LC-MS: [M+H] + = 401.29.

[0229] (Synthesis of M11-4) To a mixture of M11-3 (828 mg) prepared above and MeOH (20 mL), 5 M aqueous sodium hydroxide solution (4.1 mL) was added and stirred at room temperature for 20 hours. 5 M aqueous sodium hydroxide solution (1.2 mL) was added, and the mixture was stirred at room temperature for 4 hours. Hydrochloric acid was added for neutralization, and the mixture was extracted with ethyl acetate. The organic layer was washed with saturated brine and then dried over anhydrous sodium sulfate. After filtration, the solvent in the filtrate was evaporated to give M11-4 (968 mg). 1H NMR (400 MHz, DMSO-d6) δ 7.78 (d, J = 8.6 Hz, 1H), 7.36-7.25 (m, 5H), 7.12 (s, 1H), 7.06 (dd, J = 8.6, 2.6 Hz), 4.44 (s, 2H), 4.06-4.00 (m, 2H), 3.44-3.41 (m, 2H), 1.75-1.69 (m, 2H), 1.59-1.53 ​​(m, 2H), 1.45-1.34 (m, 4H);LC-MS: [M+H] + = 373.26.

[0230] (Synthesis of M11-5) Acetic anhydride (971 μL) was added to a mixture of M11-4 (770 mg) and THF (20 mL) prepared above, and the mixture was stirred under reflux for 4 hours. EtOH (20 mL) and methylhydrazine (550 μL) were added, and the mixture was stirred under reflux for 4 hours. The solvent was evaporated, and the residue was purified by column chromatography (silica gel, hexane / AcOEt → AcOEt / MeOH). EtOH (150 mL) was added to the resulting compound (containing impurities), and 10% Pd / C (440 mg) was added under ice-cooling and nitrogen atmosphere. The mixture was purged with hydrogen and stirred at room temperature for 6 hours. After filtration, the solvent was evaporated from the filtrate, and the residue was washed with MeOH and filtered to obtain a solid. The solvent was evaporated from the filtrate, and the residue was purified by column chromatography (ODS, water / MeCN) to obtain a solid. The obtained solids were combined, MeOH was added, and the mixture was stirred, and the solvent was evaporated to obtain M11-5 (475 mg). 1 H NMR (400 MHz, DMSO-d6) δ 8.11 (d, J = 8.8 Hz, 0.5H), 7.88 (d, J = 8.8 Hz, 0.5H), 7.57 (d, J = 2.6 Hz, 0.5H), 7.45-7.38 (m, 1H), 7.29 (d, J = 2.4 Hz, 0.5H), 4.35 (br, 1H), 4.13 (dt, J = 6.1 Hz, 2H), 3.55-3.53 (m, 3H), 3.41-3.38 (m, 2H), 1.80-1.73 (m, 2H), 1.48-1.34 (m, 6H); LC-MS: [M+H]+ = 293.32.

[0231] (Synthesis of Example 11) Triethylamine (18 μL) and dichlorodiphenylsilane (10 μL) were added to a mixture of M11-6 shown in the formula above and DCM (1 mL), and the mixture was stirred at room temperature for 3 hours. A mixture of M11-5 (34 mg) prepared above and pyridine (0.5 mL) was added to the reaction mixture, and the mixture was stirred at room temperature for 15 hours. The solvent was evaporated, and the residue was purified by column chromatography (silica gel, DCM / MeOH and ODS, water / MeCN) to obtain Example 11 (2.1 mg). 1 H NMR (400 MHz, CD3OD) δ 8.14 (d, J = 8.8 Hz, 0.5H), 8.01 (d, J = 8.7 Hz, 0.5H), 7.65-7.61 (m, 4+0.5H), 7.49 (s, 0.5H), 7.42-7.33 (m, 5+0.5H), 7.29 (dd, J = 8.8, 2.4 Hz, 0.5H), 4.48-4.45 (m, 1H), 4.28 (dd, J = 7.7, 4.5 Hz, 1H), 4.11 (t, J = 6.3 Hz, 2H), 3.79 (t, J = 6.3 Hz, 2H), 3.70 (t, J = 6.1 Hz, 2H), 3.64-3.48 (m, 17H), 3.35-3.30 (m, 4H), 3.20-3.15 (m, 1H), 2.90 (dd, J = 12.7, 5.0 Hz, 1H), 2.68 (d, J = 12.7 Hz, 1H), 2.45 (t, J = 6.0 Hz, 2H), 2.20 (t, J = 7.3 Hz, 2H), 1.84-1.38 (m, 14H), 1.25 (s, 6H); LC-MS: [M+H] + = 1035.88.

[0232] [Example 12]

[0233] In the 2,3-dihydrophthalazine-1,4-dione of M12-1, the constituent atoms of the ring are numbered for convenience in explaining the substitution position. M12-1 and Example 12 have a methyl (R１ ), and third place is H (R ２ ), and its isomers (methyl (R １ ), and third place is H (R ２ ), and a compound having a substituent at the 7-position.

[0234] (Synthesis of Example 12) To a mixture of M12-1 (7.6 mg), M12-2 (d-desthiobiotin) (5.6 mg), and DMF (1 mL), WSC·HCl (6.0 mg) and HOBt·HO (4.8 mg) were added and stirred at room temperature for 15 hours. The solvent was evaporated, and the residue was purified by column chromatography (ODS, water / MeCN) to obtain Example 12 (8.9 mg). 1 H NMR (400 MHz, DMSO-d6) δ 8.09 (d, J = 8.8 Hz, 0.4H), 7.88 (d, J = 8.8 Hz, 0.6H), 7.72 (t, J = 5.4 Hz, 1H), 7.57 (d, J = 2.6 Hz, 0.6H), 7.42 (dd, J = 8.8, 2.6 Hz, 0.6H), 7.37 (dd, J = 8.8, 2.4 Hz, 0.4H), 7.29 (d, J = 2.4 Hz, 0.4H), 6.28 (s, 1H), 6.10 (s, 1H), 4.12 (dt, J = 6.3Hz, 2H), 3.62-3.42 LC-MS: [M+H] + = 488.59.

[0235] [Example 13]

[0236] In the 2,3-dihydrophthalazine-1,4-dione of M13-1, the constituent atoms of the ring are numbered for convenience in explaining the substitution position. M13-1 and Example 13 have a methyl (R １ ), and third place is H (R２ ), and its isomers (methyl (R １ ), and third place is H (R ２ ), and a compound having a substituent at the 7-position.

[0237] Synthesis of Example 13: M13-2 (N-hydroxysuccinimide) (13 mg) and WSC·HCl (22 mg) were added to a mixture of M13-1 (9.1 mg) and DMF (0.5 mL), followed by stirring at room temperature for 10 hours. The reaction mixture was purified by column chromatography (ODS, water / MeCN, containing 0.1% TFA). DMF (1 mL), M13-4 (Hexa-His) (6.7 mg), and DIPEA (4.2 μL) were added to the resulting M13-3 (containing impurities), followed by stirring at room temperature for 2 hours. DIPEA (13.9 μL) was added, followed by stirring at room temperature for 2 hours. The reaction mixture was purified by column chromatography (ODS, water / MeCN, containing 0.1% TFA) to obtain Example 13 (2.8 mg). 1 H NMR (400 MHz, DMSO-d6) δ 8.81-8.63 (m, 7H), 8.48-8.41 (m, 2H), 8.32-8.28 (m, 2H), 8.23-8.21 (m, 1H), 8.12 (d, J = 8.8 Hz, 0.6H), 7.95-7.93 (m, 1H), 7.89 (d, J = 8.8 Hz, 0.4H), 7.57 (d, J = 2.6 Hz, 0.4H), 7.42 (dd, J = 8.8, 2.6 Hz, 0.4H), 7.38 (dd, J = 8.8, 2.6 Hz, 0.6H), 7.32 (s, 1H), 7.28-7.21 (m, 5+0.6H), 4.54-4.45 (m, 6H), 4.14-4.10 (m, 2H), 3.55-2.83 (m, overlapped with water signal), 2.53-2.32 (m, overlapped with DMSO signal), 1.76-1.72 (m, 2H), 1.45-1.24 (m, 6H);LC-MS: [M+H] + = 1214.97.

[0238] [Example 14]

[0239] It should be noted that Example 14 is the same isomer mixture as Example 4.

[0240] Synthesis of Example 14-2: A DMSO solution (10 mg / mL, 1 μL) of Example 4 shown in the formula above, an acetonitrile solution (60 mM, 2.5 μL) of M14-1 from the TMT reagent TMT10plex™ Isobaric Label Reagents (cat: 90110, Thermo Scientific™), and 1 μL of 1 M TEAB (triethylammonium bicarbonate) were mixed and reacted at room temperature for 1 hour. The production of the target product, Example 14-2, was confirmed by LC-MS. In Test Example 3, 10 labeled adducts were produced in the same manner. LC-MS: [M+H]+ = 1244.91.

[0241] Example 14 used in Test Example 3 was prepared as follows: The TMT reagent shown in the table below, i.e., TMT10plex TM Isobaric Label Reagents (cat:90110, Thermo Scientific TM ) was dissolved in acetonitrile to make a 50 mM solution. To 2.5 μL of this TMT solution, 1 μL each of Example 4 (10 mg / mL) dissolved in DMSO and 1 M TEAB were added, and the mixture was allowed to react at room temperature for 1 hour. After the reaction, PBS was added to adjust the concentration of the TMT-labeled probe to 100 μg / mL, and this was used for cell labeling in Test Example 3.

[0242] [Comparative example] Comparative example 1: N-(15-(4,4-dimethyl-2,6-dioxocyclohexylidene)-18-(4-hydroxyphenyl)-3,6,9,12-tetraoxa -16-azaoctadecyl)-5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamide

[0243] DIPEA (24 μL) was added to a mixture of 21 mg of compound RM-1 (cas no. 2567570-42-5) represented by the formula above, 6 mg of tyramine hydrochloride, and 1 mL of DMF, and the mixture was stirred at 60° C. for 4 hours. The reaction mixture was purified by column chromatography (ODS, water / MeCN) to obtain Comparative Example 1 (21 mg). 1 H NMR (400 MHz, DMSO-d6) δ 13.40 (t, J = 5.1 Hz, 1H), 9.25 (s, 1H), 7.82 (t, J = 5.7 Hz, 1H), 7.06 (d, J = 8.5 Hz, 2H), 6.68 (dt, J = 8.5, 2.3 Hz, 2H), 6.41 (s, 1H), 6.35 (s, 1H), 5.47 (dd, J = 7.6, 5.1 Hz, 1H), 4.14-4.10 (m, 1H), 3.72 (q, 6.5 Hz, 2H), 3.56 (t, J = 6.2 Hz, 2H), 3.48-3.46 (m, 12H), 3.40-3.33 (m, overlapped with water signal), 3.20-3.15 (m, 4H), 3.11-3.06 (m, 1H), 2.83-2.76 (m, 3H), 2.57 (d, J = 12.4 Hz, 1H), 2.26 (s, 4H), 2.06 (t, J = 7.3 Hz, 2H), 1.65-1.40 (m, 4H), 1.35-1.24 (m, 2H), 0.93 (s, 1H);LC-MS: [M+H] + = 733.06.

[0244] Comparative example 2: sodium 2,5-dioxo-1-((3-((2-(5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)ethyl)disulfaneyl)propanoyl)oxy)pyrrolidine-3-sulfonate

[0245] Comparative Example 2: EZ-LinkTM Sulfo-NHS-SS-Biotin (Thermo Scientific TM , product number A39258).

[0246] [Test Examples] The effects of the compounds of the present invention will be explained below by showing test results using representative compounds among the chemical probes of the present invention, although the present invention is not limited to these test examples.

[0247] Test Example 1: Comprehensive analysis of cell surface proteins using chemical probes 1. Cell culture and treatment of cultured cells with the compound A549 cells (ATCC® registration number CCL185 TM ) into a 24-well plate at 2 x 10 ５ Cells were seeded and cultured overnight at 37°C under a 5% CO2 atmosphere. After washing with PBS, the cells were immersed in a 0.2% HRP solution prepared in PBS. A labeling solution containing 0.006% hydrogen peroxide and 20 μg / mL of the probe shown in Examples 1-13 or Comparative Example 1, prepared in PBS, was then added in an amount equal to the HRP solution added earlier. The final concentrations were 10 μg / mL probe, 0.003% hydrogen peroxide, and 0.1% HRP. After gentle stirring, the mixture was placed on ice for 5 minutes, discarded, and washed with PBS. The cells were harvested using a cell scraper and frozen at -80°C. For amine labeling, A549 cells cultured in a 24-well plate were washed with PBS, and the probe from Comparative Example 2, dissolved in PBS and adjusted to 0.25 mg / mL, was added. The cells were then incubated on ice for the specified time. The labeling solution was then removed, and the cells were washed with TBS(+) and harvested using a cell scraper. The collected cell pellet was frozen and stored at -80°C.

[0248] 2. Cell lysis RIPA buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% SDS, 0.5% SDC, 1% Triton-X-100, Complete protease inhibitor (Roche)) was added to the cells, vortexed, and incubated on ice for 30 minutes. After centrifugation at 16,000 × g and 4°C for 20 minutes, the supernatant was collected and used as the cell lysate.

[0249] 3. Purification of biotinylated proteins To recover biotinylated proteins from cell lysates, Pierce TM Streptavidin Magnetic Beads (#88817, Thermo Scientific TM The purification was carried out using a KingFisher TM Flex (Thermo Scientific TM ) was used. After the protein was adsorbed onto the beads, they were washed with RIPA buffer (50 mM Tris-HCl pH 7.5 / 150 mM NaCl / 0.1% SDS / 0.5% SDC / 1% Triton X-100) containing 8 M urea and 400 mM arginine. 2% hydrazine / 0.05% SDS solution was added to the resulting beads and incubated at room temperature for 1 hour (two 30-minute incubations, with magnetic separation after each incubation to recover the supernatant). This separated the protein from the biotin, and the solution was then recovered. When Sulfo-NHS-SS-Biotin was used as the probe, the protein was separated from the biotin by reductive elution with DTT according to the manufacturer's instructions, and the solution was recovered by magnetic separation. The recovered solution was stored in a Savant TM SpeedVac TM Centrifugal concentrator (Thermo Scientific TM The resulting solution was concentrated using a 5000-kJ / mL PBS, then reduced with 5 mM DTT (incubated at 70°C for 15 minutes) and alkylated with 50 mM iodoacetamide (incubated at room temperature with shaking for 30 minutes in the dark). TMProteins were purified using SpeedBead Carboxylate-Modified [E3, E7] Magnetic Particles (#65152105050250, #45152105050250, Cytiva) on a Bravo (Agilent) automated purification system, followed by overnight digestion at 37°C with 0.025 μg of Trypsin / Lys-C Mix, Mass Spec Grade (#V5073, Promega). The digested peptides were purified using the same SpeedBeads on a Bravo and subjected to LC-MS analysis.

[0250] 4. LC-MS analysis and target protein identification LC-MS was performed using the nano-HPLC instrument UltiMate TM 3000 (Thermo Scientific TM ) and FAIMS Pro TM Orbitrap Eclipse mass spectrometer with interface TM (Thermo Scientific TM The nano-HPLC system was equipped with a trap column (C18 Acclaim TM PepMap TM , Thermo Scientific TMThe peptides were analyzed by reversed-phase chromatography using a gradient analysis system consisting of a column (stationary phase) consisting of a 75 μm id column and a tip column (75 μm id, 12 cm long, 3 μm C18 particles, Nikkyo Technos) and a 0.1% formic acid-acetonitrile mobile phase. The flow rate was 300 nL / min, and peptides were eluted with a linear gradient increasing the acetonitrile concentration from 4% to 35%. Mass spectrometry was performed using either data-dependent acquisition (DDA) or data-independent acquisition (DIA), both of which were performed using nanoelectrospray ionization in positive ion mode. For DDA, MS1 spectra were measured from m / z 375 to 1500, and precursors were data-dependently selected and fragmented by HCD to obtain MS2 spectra. FAIMS was performed under three conditions: CV = -40 V, -60 V, and -80 V. The data obtained by DDA was analyzed using the Proteome Discoverer 3.0 (PD 3.0, Thermo Scientific) software. TM) for analysis. Proteins were identified by searching against a database downloaded from Uniprot, and proteins were identified if the false-positive protein identification rate was less than 1%. Protein quantification was calculated using label-free quantification in PD3.0. For the data-independent acquisition method DIA, MS1 spectra were measured from m / z 495 to 745, and this range was divided into sections with an isolation width of 4 Th, and sequential fragmentation was performed using HCD to obtain MS2 spectra. FAIMS was performed at a CV of -45 V. The obtained data was analyzed using DIA-NN 1.8.1 for protein identification and quantification. Proteins were identified by searching against a database downloaded from Uniprot, and proteins were identified if the false-positive protein identification rate was less than 1%. The identified proteins were identified as surface proteins by matching them with the in silico surfaceome database (Bausch-Fluck, D., Goldmann, U., Muller, S., van Oostrum, M., Muller, M., Schubert, O.T., & Wollscheid, B. (2018). The in silico human surfaceome. Proceedings of the National Academy of Sciences, 115(46), E10988-E10997.).

[0251] 5. Results The amount of surface protein detected relative to Comparative Example 1 was determined from the surface protein identification results obtained with each probe. Specifically, the ratio of each Comparative Example to Comparative Example 1 was expressed as a logarithmic value with the base 2, and the distribution is shown in the histogram in Figure 1. When the average value of the distribution was calculated, 29.04 times more surface protein was detected on average in Example 1, 36.76 times more in Example 2, 41.64 times more in Example 5, and 35.02 times more in Example 6 than in Comparative Example 1. The average ratio of the amount of surface protein detected in each Example to Comparative Example 1 was as shown in Table 1.

[0252]

[0253] Next, the amounts of surface and non-surface proteins detected were determined for Comparative Example 2. Specifically, the ratio of each Comparative Example to Comparative Example 2 was expressed as a logarithmic value with the base 2, and the average value was calculated from the distribution. The labeling time was 5 minutes for both the Comparative Examples and the Examples. The results are shown in the histograms in Figure 2. For surface proteins, Example 2 averaged 1.27 times and Example 6 averaged 1.10 times compared to Comparative Example 1, showing nearly equivalent results (Figures 2(a) and (b)). For non-surface proteins, Example 2 averaged -2.14 times and Example 6 averaged -1.77 times compared to Comparative Example 1 (Figures 2(c) and (d)). This indicates that the Examples have higher selectivity for surface proteins than Comparative Example 2.

[0254] Next, the number of identified proteins in each Comparative Example and each Example is shown in Figure 3. Compared to Comparative Example 1, nearly twice as many types of surface proteins were identified in each Example. The number of identified non-surface proteins is shown in Figure 4. In Comparative Example 1, the number of detected total proteins was low, and therefore the number of identified non-surface proteins was also low. Compared to Comparative Example 2, the number of identified non-surface proteins was low in each Example, suggesting high selectivity for surface proteins.

[0255] Figure 5 shows the number of surface proteins for the top 100 identified proteins in Comparative Example 1, Comparative Example 2, and each Example. In Comparative Example 1, 30 of the top 100 were surface proteins, while in each Example, 57 to 64 were surface proteins. Furthermore, in Comparative Example 2, the number of surface proteins was 50, but this decreased to 44 after 2 hours of labeling. Comparative Example 2 is currently widely used, but its stability is low due to hydrolysis, making long-term storage in solution difficult. Furthermore, since it remains reactive at all times, the reaction cannot be essentially controlled. In each Example, activation relies on HRP, so it activates and reacts only in the vicinity of HRP, i.e., outside the cells, and does not enter the cells. Inactivation can be easily achieved by washing away extracellular HRP and hydrogen peroxide, and even if there is residual probe present during cell lysis, it will not be labeled. These findings suggest that highly selective cell surface protein labeling is possible.

[0256] When analyzing the data obtained by LC-MS in Test Example 1 using PD3.0, identification was performed assuming mass variation due to chemical modification in order to detect traces of probe compound binding. That is, for Examples 1, 6, 9, 10, and 11, the mass variation due to chemical modification was set to +289.1426 (non-fixed) relative to tyrosine, +435.2376 (Example 4), +217.0851 (Example 5 and Example 7), and +434.1624 (including alkylation) (Comparative Example 1). For Comparative Example 2, the mass variation due to lysine was set to +145.0197 (non-fixed). As a result, the number of modifications detected by each probe was as shown in Figure 6.

[0257] Using the identification in Example 6, the modification positions were analyzed using Protter 1.0 (https: / / wlab.ethz.ch / protter / #), which visualizes the locations of chemically modified residues in membrane proteins based on predicted membrane topology information. As an example, the results for the EGF receptor are shown in Figure 7. As a result, the modification positions in Example 6 selectively modified tyrosine residues in the extracellular region, and no traces of chemical modification by the probe were detected in tyrosine residues in the intracellular region. Because this compound reacts with extracellular tyrosine residues, it suggests that it is possible to detect and recover cell surface proteins with high selectivity.

[0258] Test Example 2: A549 cells were labeled using Example 6 according to the method of Test Example 1. After labeling, 4% paraformaldehyde was added to the cells, and the cells were fixed by standing on ice for 20 minutes. The cells were then incubated in FCSB (eBioscience TM After washing with Flow Cytometry Staining Buffer (# 00-4222-26), fluorescent reagents (Streptavidin-Alexa 10 μg / mL, DAPI 500 ng / mL) were added and incubated at room temperature for 20 minutes. Finally, the cells were washed once with FCSB and twice with PBS, and then analyzed using a CellVoyager TM Imaging was performed using a CV8000 (Yokogawa Electric).

[0259] The results of cell staining are shown in Figure 8. The area stained with Streptavidin-AlexaFluor 647 was the cell periphery, suggesting that biotinylation according to Example 6 selectively labels cell surface proteins.

[0260] Test Example 3: [TMT Labeling of Probe] TMT labeling of Example 4 was carried out as described in Example 14.

[0261] [Labeling of cells with TMT probes] Jurkat cells (ATCC® accession number TIB-152 TM ), THP-1 cells (ATCC® accession number TIB-202 TM ), K562 cells (ATCC® registration number CCL-243 TM) were cultured at 37°C in a 5% CO2 atmosphere. They were washed with PBS and immersed in a 0.2% HRP solution prepared with PBS. Then, hydrogen peroxide diluted with PBS and the 10 types of TMT-labeled probes prepared above in Examples 14-1 to 14-10 were added. The TMT-labeled probes and the cell numbers were 2 x 10 for Examples 14-1, 14-2, and 14-3. ６ Jurkat cells were labeled, and in Examples 14-4, 14-5, and 14-6, 2 × 10 ６ K562 cells were labeled, and Examples 14-7, 14-8, and 14-9 were labeled with 2 × 10 ６ In Example 14-10, 0.7 × 10 Jurkat cells, 0.7 × 10 K562 cells, and 0.7 × 10 THP-1 cells were labeled. ６ The mixture was labeled. The final concentrations of each reagent were 10 μg / mL for TMT Label Example 4, 0.003% for hydrogen peroxide, and 0.1% for HRP. The mixture was left on ice for 5 minutes, and the cells were centrifuged, after which the labeling solution was discarded and the cells were washed with PBS.

[0262] [Purification of labeled peptides] The 10 types of cells labeled with the TMT-labeled probes from Example 14 and other sources were pooled together, and the cells were lysed and biotinylated proteins were purified according to the method of Test Example 1. The digested peptides were dissolved in TBS. Immobilized Anti-TMT TM Resin (# 90076, Thermo Scientific TM TMT-labeled peptides were purified using a centrifuge tube. First, the Anti-TMT Resin was washed three times with TBS, and the peptide solution was added and allowed to react at room temperature for two hours. The resin was then washed five times with 0.1% NP-40 solution, three times with TBS, and three times with MilliQ water, and then eluted from the resin using a 40% MeCN / 2% TFA solution. The eluted solution was dried using a centrifugal evaporator, dissolved in 0.1% TFA, and subjected to LC-MS analysis.

[0263] [LC-MS analysis and target protein identification] LC-MS was performed using a nano-HPLC instrument, UltiMate TM 3000 (Thermo ScientificTM ) and FAIMS Pro TM Orbitrap Exploris Mass Spectrometer with Interface TM 480 (Thermo Scientific TM The nano-HPLC system was equipped with a trap column (C18 Acclaim TM PepMap TM , Thermo Scientific TM The analysis was performed by gradient analysis using reversed-phase chromatography with a column (stationary phase) consisting of a 75 μm id column and a tip column (75 μm id, 12 cm long, 3 μm C18 particles, Nikkyo Technos) and a 0.1% formic acid-acetonitrile mobile phase. The flow rate was 300 nL / min, and peptides were eluted with a linear gradient increasing the acetonitrile concentration from 4% to 35%. Mass spectrometry was performed using data-dependent acquisition, with nanoelectrospray ionization in positive ion mode. MS1 spectra were measured from m / z 375 to 1500, and precursors were data-dependently selected and fragmented by HCD to obtain MS2 spectra. FAIMS was performed under two conditions: CV = -50V and -70V. Data obtained by DDA were analyzed using the Proteome Discovere 3.0 (PD 3.0, Thermo Scientific) analysis software. TM Proteins were identified by searching a database downloaded from Uniprot, and proteins were considered to be identified if the false positive protein identification rate was less than 1%. Protein quantification was calculated using TMT label quantification in PD3.0.

[0264] [Results] As a result of the analysis, 540 types of TMT-labeled peptides were identified, and the detection of TMT reporter ions was confirmed. An example of a product ion spectrum is shown in Figure 9. The peptide shown is LENLEPEHEYK (SEQ ID NO: 3), which begins at the 356th amino acid of receptor-type tyrosine-protein phosphatase C, and was identified as the peptide in which TMT labeling Example 4 was added to the tyrosine residue in this sequence. The reporter ions of the TMT reagent were measured in the m / z region from 126 to 131 in the product ion spectrum.

[0265] Quantitation of these TMT reporter ions allows for the identification of cell surface proteins characteristic of each cell type. As an example, Figure 10 shows the TMT quantification values ​​for CD7, which is highly expressed in Jurkat cells. The peptide shown is QLGPQPQDIIYYEDGVVPTTDRR (SEQ ID NO: 4) derived from the CD7 protein, with TMT conjugation Example 4 attached to the 11th tyrosine residue. Although CD7 is a protein highly expressed in Jurkat cells, the signals from the TMT reporter ions 126, 127N, and 127C labeled in Jurkat cells were high, while the signals from reporter ions derived from other cells were low. Furthermore, the reporter ion 131 derived from a sample containing three types of cells exhibited a value close to the average signal value of the other nine samples. These results suggest that TMT conjugation Example 4 can be used to directly isobarically label surface proteins in live cells, enabling quantitative analysis.

[0266] Tagging reagents such as TMT reagents are generally applied to peptides and are subject to sample preparation errors during the multi-step process of protein extraction from cells, affinity purification, trypsin digestion, and peptide purification. The method of directly labeling the cell surface of living cells with an isobaric reagent, as in this example, allows multiple samples to be mixed and performed simultaneously from the protein extraction stage, enabling quantitative analysis that is not affected by sample preparation.

[0267] Test Example 4: Proximity Labeling Jurkat cells were cultured at 37°C under a 5% CO atmosphere. After harvesting, the cells were washed with PBS and then with SB (staining buffer: PBS / 2% FCS). Next, Fc block (Fc Receptor Binding Inhibitor Polyclonal Antibody, eBioscience) was added. TM ) was added, the mixture was left to stand at 4°C for 15 minutes, and then centrifuged to remove the supernatant. The cell pellet was then incubated with a primary antibody (OKT3, eBioscience TM An antibody solution containing Goat anti-mIgG-HRP (SouthernBiotech, #1030-05) (antibody 10 μg / mL, 1% Fc block, in SB) was added and incubated at 4°C for 20 minutes. Mouse IgG2a (BioLegend™, Cat# 401508) was used as the primary antibody for the negative control control IgG. After washing with SB, an antibody solution containing Goat anti-mIgG-HRP (SouthernBiotech, #1030-05) (antibody 1 μg / mL, 1% Fc block, in SB) was added and incubated at 4°C for 20 minutes. The cells were washed with SB and PBS and suspended in PBS. A hydrogen peroxide solution and probe mixture dissolved in PBS was then added to the cell suspension to achieve a final concentration of 10 μg / mL probe (Comparative Example 1, Example 1, or Example 6) and 0.003% hydrogen peroxide. After gentle stirring and incubation on ice for 5 minutes, DMEM was added, the cells were centrifuged, the supernatant was removed, washed with PBS, and centrifuged to obtain a cell pellet. Using this cell pellet, purification using streptavidin beads and protein analysis by LC-MS were performed according to the method of Test Example 1. Three samples were prepared for each of the OKT3 and Control IgG groups, and the abundance ratio of each protein between the groups and the significant difference p-value were calculated.

[0268] Results The results of the analysis of interacting molecules using proximity labeling are shown in Figure 11. The graph is a volcano plot, with the horizontal axis representing the abundance ratio of each protein between groups (OKT3 / Control IgG) and the vertical axis representing the p-value indicating significance. Volcano plots were created individually for the results using (a) Comparative Example 1, (b) Example 1, or (c) Example 6 as the chemical probe. As a result, the known CD3 complex was detected in all results when OKT3 was used. However, when the tyramide-type Comparative Example 1 was used, many nonspecific membrane proteins other than the CD3 complex were detected, making it difficult to say that the overall analysis results selectively identified a specific complex. On the other hand, when Examples 1 and 6 were used, there were fewer membrane proteins other than the CD3 complex, and the constituent proteins of the TCR / CD3 complex were detected more specifically than in Comparative Example 1. (Probes such as tyramide and luminol are normally inactive and become activated in the presence of peroxidase or hydrogen peroxide. Peroxidase can be localized by expressing it with an antibody or through genetic manipulation, allowing the probe to be activated only around the peroxidase, enabling localized labeling. This is not possible with normally active probes such as N-hydroxysuccinimide.)

[0269] Test Example 5: Internalization of EGF receptor A549 cells seeded on a 24-well plate were washed twice with PBS(-). The cells were then cultured in serum-free RPMI 1640 medium (#11875, Thermo Scientific) at 37°C. TM) was added and cultured at 37°C for 3 hours. Next, 4°C serum-free RPMI 1640 medium was added, and after EGF was added, the cells were incubated at 4°C for 15 minutes. The medium containing the EGF solution was removed, and the cells were rinsed with PBS(-). PBS(-) was then added and allowed to stand at room temperature for 5 to 30 minutes. For the negative control, PBS was used instead of the EGF solution. Next, after removing the PBS(-), the cells were rinsed with 4°C PBS(-). A labeling solution containing 0.1% HRP solution, 0.003% hydrogen peroxide, and 10 μg / mL of Example 6 prepared in 4°C PBS was added, and the cells were allowed to stand on ice for several minutes (5 minutes). After discarding the labeling solution, the cells were washed with PBS. The cells were harvested using a cell scraper and purified using streptavidin beads and analyzed for proteins by LC-MS according to the method described in Test Example 1. Three samples were prepared for each of the EGF and control groups, and the abundance ratios of each protein and the p-values ​​for the significant differences between the groups were calculated.

[0270] Results: Figure 12 shows the analysis results of receptor internalization following EGF stimulation. The horizontal axis of the graph represents the abundance ratio of each protein between groups (EGF / Control), and the vertical axis is a volcano plot showing the p-value indicating significance. Each detected protein is plotted, with black dots indicating those specifically annotated as cell surface proteins. EGF stimulation was performed for 5, 15, and 30 minutes, and EGF receptor (EGFR) specifically decreased at all stimulation times, while no significant changes were observed in other membrane proteins. These results suggest that membrane protein analysis using the probe from Example 6 is quantitative and capable of capturing changes occurring on the cell membrane within a short stimulation time of 5 minutes. In particular, membrane proteins normally undergo dynamic localization and fluctuate between the cell membrane and cytoplasm. Unless this recycling phenomenon is suppressed by low temperature, it is difficult to capture ligand-specific receptor internalization. This method can be performed in a short time under low-temperature conditions, making it possible to label even when recycling is suppressed by low temperature. Moreover, since labeling is completed in a short time, it is possible to perform profiling analysis of receptor internalization with low false positives.

[0271] Test Example 6: Analysis of membrane protein phosphorylation A431 cells seeded on a 12-well plate were washed twice with PBS(-). The cells were then incubated in serum-free RPMI 1640 medium (#11875, Thermo Scientific) at 37°C. TM ) was added and cultured at 37°C for 3 hours. Next, 4°C serum-free RPMI 1640 medium was added, and 1 μM gefitinib (sc-202166, Santa Cruz) was added, followed by incubation at 4°C for 60 minutes. DMSO was used instead of gefitinib for the negative control. EGF was then added and incubated at 4°C for 10 minutes. PBS was used instead of the EGF solution for the negative control. The medium was removed, rinsed with PBS(-), and then PBS(-) was added and the cells were left to stand at room temperature for 5 minutes. Next, the PBS(-) was removed and the cells were rinsed with 4°C PBS(-). A labeling solution containing 0.1% HRP solution, 0.003% hydrogen peroxide, and 10 μg / mL of Example 6 prepared in 4°C PBS was added, and the cells were left to stand on ice for 5 minutes. The labeling solution was discarded, and the cells were washed with PBS. The cells were collected using a cell scraper and purified with streptavidin beads and digested with trypsin according to the method of Test Example 1. After digestion, the phosphorylated peptides were purified using MagReSyn® Ti-IMAC HP (#MR-THP005, ReSyn Biosciences). TM A Flex (Thermo Fisher Scientific) was used. The digested peptides were prepared as an 80% acetonitrile / 5% TFA solution and reacted with Ti-IMAC for 20 minutes. Afterwards, the column was washed three times with washing buffer. The first wash was 80% MeCN / 5% TFA, the second wash was 80% MeCN / 1% TFA, and the third wash was 10% MeCN / 0.2% TFA. Elution from Ti-IMAC was performed with a 1% ammonia solution. The recovered 1% ammonia solution was dried in a centrifuge and dissolved in 0.1% TFA for LC-MS analysis.

[0272] Results: Figure 13 shows the results of the analysis of membrane protein phosphopeptides. Figure 13 plots membrane protein-derived phosphopeptides. The horizontal axis of the graph represents the abundance ratio of each protein between groups, and the vertical axis is a volcano plot showing the p-value indicating significance. Among the detected phosphopeptides, EGFR-derived phosphopeptides are indicated by black dots. Figure 13 (1) shows that several phosphorylations were increased or decreased after 5 minutes of stimulation with EGF compared to the control. In particular, phosphorylation of EGFR, the ligand for EGF, was significantly increased, including phosphopeptides containing tyrosine 1172, which is phosphorylated by EGF stimulation. Figure 13 (2) also shows the effect of gefitinib, an EGFR tyrosine kinase inhibitor, on EGF-induced phosphorylation. Gefitinib treatment significantly suppressed EGF-induced phosphorylation. These results demonstrate that membrane protein analysis using the probes of Example 6 can be used to analyze changes in membrane protein phosphorylation. Because protein phosphorylation by kinases occurs rapidly, maintaining a certain phosphorylation state requires rapid transition to a low-temperature state and suppression of kinase activity. This method allows rapid labeling even under low-temperature conditions that suppress kinase activity. This could potentially be a useful method for phosphorylation profiling of membrane proteins that maintain a transient phosphorylation state.

[0273] Test Example 7: Analysis of membrane proteins by labeling A549 cells cultured in a 12-well plate were washed twice with PBS(+), then 37°C serum-free RPMI 1640 medium was added and cultured at 37°C for 3 hours. The culture plate was placed on ice and washed with 4°C PBS(-). 4°C serum-free RPMI 1640 medium was added, and an antibody against the transferrin receptor (anti-TFRC antibody, OKT9) was added to a concentration of 0.5 mg / mL. The cells were incubated on ice for 10 minutes, after which the antibody solution was removed and washed with cold PBS(+). PBS was added instead of the anti-TFRC antibody solution as a negative control. 1.5 mM Sulfo-NHS Acetate (Thermo Scientific TM , #26777) was added and incubated on ice for 1 hour. The reaction solution was discarded, washed with cold PBS (+), and then a labeling solution containing 0.1% HRP solution, 0.003% hydrogen peroxide, and 10 μg / mL of probe prepared in 4°C PBS was added. The mixture was left on ice for 5 minutes, discarded, and then washed with PBS. Cells were collected with a cell scraper and purified using streptavidin beads and analyzed by LC-MS according to the method described in Test Example 1. When analyzing using PD3.0, mass shifts due to chemical modification were assumed to be involved in the detection of labeling reagents. Specifically, acetylation with Sulfo-NHS Acetate was analyzed with a mass shift of +42.0106 for lysine, arginine, histidine, serine, threonine, and tyrosine, while the chemical modification according to Example 6 was analyzed with a mass shift of +289.1426 for tyrosine. Four samples were prepared for each of the OKT9 and control groups, and the abundance ratio of each modified peptide between the groups and the significant difference p-value were calculated.

[0274] Results: Figure 14 shows the results of analyzing labeled peptides after addition of the anti-TFRC antibody OKT9. The horizontal axis of the graph represents the ratio of peptide abundance between groups (anti-TFRC antibody / control), and the vertical axis is a volcano plot showing the p-value indicating significance. The graph plots labeled peptides derived from membrane proteins, with TFRC-derived labeled peptides specifically indicated by black circles. The numbers in the figure represent the amino acid residue positions in the TFRC sequence. Addition of the antibody significantly altered the labeled peptides of membrane proteins, all of which were derived from TFRC. Lysines at positions 205, 231, 371, and 374 in the TFRC sequence showed particularly significant changes. These results suggest that acetylation was reduced by the binding of the OKT9 antibody to TFRC. Figure 15 shows the assignment of these labeled sites to the known TFRC structure (PDB: 7ZQS). The lysines at positions 205, 231, 371, and 374, which showed reduced labeling, were identified as being in the same steric region. This region has been reported to be the OKT9 antigen epitope region on TFRC (J Virol. 2021 Sep; 95(17): e01868-20).

Claims

1. A functional compound or a salt thereof, in which a tyrosine residue reactive site R is linked to a protein purification tag site Tag via a spacer S, wherein the tyrosine residue reactive site R is a group represented by the following formula (1): (wherein * means a bond to the spacer S, R １ and R ２ each independently represents H, C optionally substituted with one or more substituents, １－６ alkyl, optionally substituted with one or more substituents; ６－１０ aryl, and 5- to 15-membered heteroaryl optionally substituted with one or more substituents. The spacer S is selected from the group consisting of -S1-, -S2-, and -S3-E-S4-, and S1 is selected from the group consisting of one or more substituents R ３ C optionally substituted with １－３０ is alkylene, and R ３ is -N(R ３１ ) ２ and 5-15 membered heteroaryl optionally substituted with one or more substituents, ３１ each independently represents H, C optionally substituted with one or more substituents, １－６ Alkyl, substituted carbonyl, and -C(=NH)NH ２ S2 is an alkylene having 2 to 30 carbon atoms, and is selected from the group consisting of -CH ２ Between -O-, -CO-, -NR ａ -, -CO-NR ａ -, -NR ａ -CO-, a group represented by formula (L-1), or a group represented by formula (L-2) is bonded to the group, and the group is one or more substituents R ３ R ａ is H or C optionally substituted with one or more substituents １－６ alkyl, and the formulas (L-1) and (L-2) are the following formulas: (wherein m is 0 or 1, and n is 0 or 1); S3 and S4 are each independently selected from the group consisting of S1 and S2; and E is a cleavable site; or a salt thereof.

2. The functional compound or salt thereof according to claim 1, wherein S is -S3-E-S4-.

3. The functional compound or salt thereof according to claim 1 or 2, wherein E is selected from the group consisting of the following formulae (E-1), (E-2), (E-3), (E-4), (E-5), (E-6), (E-7), (E-8), and (E-9). (wherein, one wavy line represents a bond with S3, the other wavy line represents a bond with S4, and Q １ , Q ２ , Q ３ , Q ４ , Q ５ , and Q ６ are each independently H or C １－６ is alkyl, and A １ , A ２ , A ３ , and A ４ are each independently ６－１８ Pep is ENLYFQG (SEQ ID NO: 1), ENLYFQS (SEQ ID NO: 2), or X １ X ２ X ３ X ４ X ５ X ６ X ７ (X １ , X ２ , X ３ , X ５ , X ６ , and X ７ are each independently an amino acid residue other than K or R, and X ４ is K or R.) 4. The functional compound or salt thereof according to claim 1 or 2, wherein S3 and S4 are each independently selected from S2.

5. S3 is a branched alkylene having 2 to 30 carbon atoms, and is selected from the group consisting of an R-terminal group of the main chain, an E-terminal group of the main chain, and / or one or more adjacent —CH ２ Between -O-, -CO-, -NR ａ -, -CO-NR ａ -, -NR ａ -CO-, a group represented by formula (L-1), or a group represented by formula (L-2) is bonded, and the end of the side chain of the group is a substituent R ３ and / or S4 is a branched alkylene having 2 to 30 carbon atoms, and has an E-terminus of the main chain, a Tag-terminus of the main chain, and / or one or more adjacent —CH ２ Between -O-, -CO-, -NR ａ -, -CO-NR ａ -, -NR ａ -CO-, a group represented by formula (L-1), or a group represented by formula (L-2) is bonded, and the end of the side chain of the group is a substituent R ３ 3. The functional compound or salt thereof according to claim 1 or 2, having the following structure:

6. -S3-E-S4- is -O-alkylene-(E-1)-(alkylene-O) ａ -Alkylene-NH-, -O-Alkylene-NH-CO-Alkylene-(E-1)-(Alkylene-O) ａ -Alkylene-NH-, -O-Alkylene-(E-1)-Alkylene-NH-CO-Alkylene-NH-CO-(Alkylene-O) ａ -Alkylene-NH-, -O-Alkylene-(E-1)-Alkylene-NH-CO-Alkylene-(L-1)-Alkylene-CO-NH-(Alkylene-O) ａ -Alkylene-NH-, -Alkylene-(E-1)-(Alkylene-O) ａ -Alkylene-NH-, -Alkylene-(E-1)-Alkylene-NH-CO-Alkylene-NH-CO-(Alkylene-O) ａ -Alkylene-NH-, -O-Alkylene-(E-6)-Alkylene-NH-CO-(Alkylene-O) ａ -Alkylene-NH-, and -O-alkylene-NH-CO-alkylene-(E-7)-alkylene-NH-CO-(alkylene-O) ａ -alkylene-NH-, each alkylene in the group being a substituent R ３ 3. The functional compound or salt thereof according to claim 1 or 2, wherein a is an integer of 1 or more.

7. -S3-E-S4- is -O-alkylene-NH-CO-branched alkylene-(E-1)-(alkylene-O) ａ -Alkylene-NH-, -O-alkylene-(E-1)-alkylene-NH-CO-branched alkylene-NH-CO-(alkylene-O) ａ -Alkylene-NH-, and -Alkylene-(E-1)-Alkylene-NH-CO-Branched alkylene-NH-CO-(Alkylene-O) ａ -alkylene-NH-, wherein the branched alkylene in the group is selected from the group consisting of the substituent R ３ The functional compound or salt thereof according to claim 6, having the formula:

8. R ３ is -N(R ３１ ) ２ , and -C １－６ 5-15 membered heteroaryl optionally substituted with alkylene-SO3M, where M is H or an alkali metal; ３１ each independently represents H, C optionally substituted with one or more substituents, １－６ Alkyl, -C(=O)R ３２ , and -C(=NH)NH ２ and R ３２ 3. The functional compound or salt thereof according to claim 1 or 2, wherein is a residue of a reagent selected from the group consisting of TMT, fluorescein, Cy3, and transcyclooctene.

9. The functional compound or salt thereof according to claim 1 or 2, wherein the protein purification tag site Tag is selected from the group consisting of the following formulae (2-1), (2-2), (2-3), and (2-4): (wherein * represents a bond to the spacer S, and p is an integer of 1 to 10.) 10. The functional compound or salt thereof according to claim 1 or 2, which is selected from the group consisting of functional compounds represented by any one of the following formulas (P1), (P2), (P3), (P4), (P5), (P6), (P7), (P8), (P9), (P10), (P11), (P12), (P13), (P14), (P15), (P16), (P17), (P18), (P19), (P20), (P21), (P22), (P23), (P24), (P25), (P26), (P27), and (P28) and salts thereof. In (P5) and (P6), M is H or an alkali metal, and in (P9) and (P10), T is any one of the following formulae (T-1), (T-2), (T-3), (T-4), (T-5), (T-6), (T-7), (T-8), (T-9), and (T-10).

11. The functional compound or salt thereof according to claim 10, selected from the group consisting of: In (P9) and (P10), T is any one of the following formulas (T-1), (T-2), (T-3), (T-4), (T-5), (T-6), (T-7), (T-8), (T-9), and (T-10).

12. A method for detecting a protein having a tyrosine residue, comprising the following steps (1) and (2): (1) binding the tyrosine residue reactive site R in the functional compound or salt thereof according to claim 1 to a tyrosine residue of the protein, and (2) binding a labeling substance that binds to the protein purification tag site Tag in the functional compound or salt thereof bound to the protein, and detecting the label.

13. A method for detecting or identifying a cell surface protein, comprising the following steps (A), (B), (C), and (E): (A) reacting the functional compound or salt thereof according to claim 1 with cells or tissues in a sample to bind the tyrosine residue reactive site R in the functional compound or salt thereof to a tyrosine residue of the cell surface protein, (B) purifying a fraction containing the cell surface protein bound to the functional compound or salt thereof, (C) binding the protein purification tag site Tag in the functional compound or salt thereof in the purified fraction with a labeling substance to obtain a complex, and (E) detecting or identifying the cell surface protein from the complex or its cell surface protein-binding fragment.

14. A method for detecting or identifying a cell surface protein, comprising the following steps (A), (B), (C), (D), and (E): (A) reacting the functional compound or salt thereof according to claim 2 with cells or tissues in a sample to bind a tyrosine residue reactive site R in the functional compound or salt thereof to a tyrosine residue of the cell surface protein, (B) purifying a fraction containing the cell surface protein bound to the functional compound or salt thereof, (C) binding a labeling substance to the protein purification tag site Tag in the functional compound or salt thereof in the purified fraction to obtain a complex, (D) cleaving the complex at the cleavable site E, and (E) detecting or identifying the cell surface protein from the cell surface protein-binding fragments after cleavage.

15. Use of the functional compound or salt thereof according to claim 1 for detecting or identifying a cell surface protein.

Images