Novel compounds and their applications

Abstract

The present invention relates to novel compounds and their applications, and more specifically to compounds capable of labeling biomolecules (e.g., nucleic acids, proteins, etc.), biomolecular labeling or detection compositions containing the compound, biomolecular labeling or detection supports containing the compound, and methods for labeling or detecting biomolecules using the compound.

Description

[Technical Field]

[0001] The present invention relates to novel compounds and their applications, and more specifically to compounds capable of labeling biomolecules (e.g., nucleic acids, proteins, etc.), biomolecular labeling or detection compositions containing the compound, biomolecular labeling or detection supports containing the compound, and methods for labeling or detecting biomolecules using the compound. [Background technology]

[0002] In molecular biology, cell biology, and molecular genetics, fluorescent dyes are used as detection labels to detect biomolecules in vivo and in vitro, or to observe biological phenomena at the cellular level.

[0003] The use of fluorescent dyes as detection labels for nucleic acids, proteins, and other substances is widely applied in various fields. Fluorescently labeled oligonucleotides, in particular, are useful in a variety of analyses, including DNA sequencing, fluorescence in situ hybridization (FISH), hybridization analysis of nucleic acid arrays, microarrays, and nucleic acid amplification analysis including real-time polymerase chain reaction. [Overview of the Initiative] [Problems that the invention aims to solve]

[0004] The present invention aims to provide novel compounds that can be widely used in molecular biology, cell biology, and genetics to observe the identification of biomolecules (e.g., nucleic acids, proteins, etc.) or to detect or diagnose analytes.

[0005] Furthermore, the present invention aims to provide, for various applications of the novel compound defined in this application, a biomolecular labeling or detection composition containing the compound, a biomolecular labeling or detection support containing the compound, and a method for labeling and detecting biomolecules using the compound.

Means for Solving the Problem

[0006] According to one aspect of the present invention for solving the above technical problem, a compound represented by the following Chemical Formula 1 is provided.

Chem.

[0007] Here, R1 to R4 are each independently hydrogen, optionally substituted C1-C 40 alkyl, optionally substituted C1-C 40 heteroalkyl, optionally substituted C2-C 40 alkenyl, optionally substituted C2-C 40 alkynyl, optionally substituted C3-C 20 cycloalkyl, optionally substituted C3-C 20 cycloalkenyl, optionally substituted C2-C 20 heterocycloalkyl, hydroxy, oxide (-O - )), optionally substituted C3-C 40 cycloalkyloxy, optionally substituted C5-C 40 aryloxy, optionally substituted C2-C 40 heteroaryloxy, thiol, optionally substituted C5-C 50 aralkyl, optionally substituted C1-C 40 alkylthio, optionally substituted C5-C 40 arylthio, optionally substituted C3-C 40 cycloalkylthio, optionally substituted C2-C 40Heteroarylthio, optionally substituted acylamino, acyloxy, sulfo group, optionally substituted ammonium, substituted sulfonic acid ester, optionally substituted sulfonamide, substituted ester, optionally substituted aminocarbonyl, nitroso (-N=O), optionally substituted silyl, optionally substituted amide, optionally substituted carbamate, phospho group, nitrile, acetal, ketal, optionally substituted amino, aminothiocarbonyl, aminocarbonyloxy, aminosulfonyloxy, amidino group, sulfonyloxy, isonitrile, cyanate, imide, iminium, ether, thioether, R x and R s Selected from, R5~R 13 Each of these is independently hydrogen, and C1-C which may be substituted. 40 Alkyl, possibly substituted C1-C 40 Heteroalkyl, possibly substituted C2-C 40 Alkenyl, may be substituted C2-C 40 Alkinyl, C3-C may be substituted. 20 Cycloalkyl, possibly substituted C3-C 20 Cycloalkenyl, C2-C may be substituted. 20 Heterocycloalkyl, hydroxy, oxide (-O - ), C1-C even if substituted 40 Alkoxy, possibly substituted C3-C 40 Cycloalkyloxy, possibly substituted C5-C 40 Aryloxy, possibly substituted C2-C 40 Heteroaryloxy, possibly substituted C5-C 50 Aryl, possibly substituted C2-C 50 Heteroaryl, thiol, and possibly substituted C5-C 50 Alalkyl, possibly substituted C1-C 40 Alkylthio, optionally substituted C5-C 40Arylthio, C3-C which may be substituted 40 Cycloalkylthio, C2-C may be substituted. 40 Heteroarylthio, optionally substituted acylamino, acyloxy, sulfo group, optionally substituted ammonium, substituted sulfonic acid ester, optionally substituted sulfonamide, substituted ester, optionally substituted aminocarbonyl, nitroso (-N=O), halogen, optionally substituted silyl, optionally substituted amide, optionally substituted carbamate, phospho group, nitrile, acetal, ketal, optionally substituted amino, aminothiocarbonyl, aminocarbonyloxy, aminosulfonyloxy, amidino group, sulfonyloxy, isonitrile, cyanate, imide, iminium, ether, thioether, R x and R s Selected from, R1~R 13 Two adjacent rings can bond to each other to form an optional aliphatic ring, an optional aromatic ring, or an optional mixed aliphatic and aromatic ring. A1 and A2 are, independently, hydrogen, halogen, or possibly substituted C1-C. 10 Alkyl, possibly substituted C3-C 10 Cycloalkyl, C1-C which may be substituted 10 Heterocycloalkyl, optionally substituted C1-C 10 Heteroalkyl, possibly substituted C2-C 10 Alkenyl, may be substituted C2-C 10 Alkinyl, C1-C which may be substituted. 10 Alkoxy, possibly substituted C5-C 40 Selected from aryloxy and cyano, except that A1 and A2 are hydrogen at the same time. R x This is a reactive group, or a group in which at least one reactive group is bonded to a backbone containing a hydrocarbon with 1 to 40 carbon atoms. The aforementioned reactive group is carboxyl, carboxyl derivative, carboxylate (-CO2 - ), functional groups selected from carboxylates, hydroxyls, diene derivatives, aldehydes, substituted ketones, sulfonyl halides, thiols, unsubstituted aminos, primary aminos, alkenes, alkynes, halogens, hydrazides, azides, imides, ketenes, isocyanates, thiocyanates, isothiocyanates, epoxides, maleimides, 1,2,4,5-tetrazine derivatives, cycloalkyne derivatives, cycloalkenes, triphosphates and phosphoramidites, substituted thioketones, haloformyl, formyl, acyl, acylamide, acyl azide, organic acid anhydrides (anhydride), aniline, aziridine, boronate, carbodiimide, diazoalkynes, haloacetamide, imide esters, glycols, halotriazines, hydrazines, acyl halides, alkyl halides and aryl halides, R s This is either a carrier molecule or a group in which at least one carrier molecule is bonded to a backbone containing a hydrocarbon with 1 to 40 carbon atoms. R1~R 13 At least one of them is R x or R s And, X is O, S, CR a R b , NR a or SiR a R b And, R a and R b C1-C may be substituted independently of each other. 40 Alkyl, possibly substituted C1-C 40 Heteroalkyl, possibly substituted C3-C 30 Cycloalkyl, possibly substituted C3-C 30 Heterocycloalkyl, possibly substituted C5-C 50 Aryl and possibly substituted C2-C 50The rings are selected from heteroaryls, or are optionally substituted aliphatic rings, optionally substituted aromatic rings, or optionally substituted mixed aliphatic and aromatic rings formed by bonding with each other.

[0008] Furthermore, according to another aspect of the present invention, a compound represented by the following chemical formula 2 is provided. [ka]

[0009] Here, R 21 ~R 24 Each of these is independently hydrogen, and C1-C which may be substituted. 40 Alkyl, possibly substituted C1-C 40 Heteroalkyl, possibly substituted C2-C 40 Alkenyl, may be substituted C2-C 40 Alkinyl, C3-C may be substituted. 20 Cycloalkyl, possibly substituted C3-C 20 Cycloalkenyl, C2-C may be substituted. 20 Heterocycloalkyl, hydroxy, oxide (-O - ), C3-C even if it is substituted. 40 Cycloalkyloxy, possibly substituted C5-C 40 Aryloxy, possibly substituted C2-C 40 Heteroaryloxy, thiol, possibly substituted C5-C 50 Alalkyl, possibly substituted C1-C 40 Alkylthio, optionally substituted C5-C 40 Arylthio, C3-C which may be substituted 40 Cycloalkylthio, C2-C may be substituted. 40heteroarylthio, optionally substituted acylamino, acyloxy, sulfo group, optionally substituted ammonium, substituted sulfonic acid ester, optionally substituted sulfonamide, substituted ester, optionally substituted aminocarbonyl, nitroso (-N=O), optionally substituted silyl, optionally substituted amide, optionally substituted carbamate, phospho group, nitrile, acetal, ketal, optionally substituted amino, aminothiocarbonyl, aminocarbonyloxy, aminosulfonyloxy, amidino group, sulfonyloxy, isonitrile, cyanate, imide, iminium, ether, thioether and -L3-R 36 selected from R 25 ~R 33 are each independently hydrogen, optionally substituted C1-C 40 alkyl, optionally substituted C1-C 40 heteroalkyl, optionally substituted C2-C 40 alkenyl, optionally substituted C2-C 40 alkynyl, optionally substituted C3-C 20 cycloalkyl, optionally substituted C3-C 20 cycloalkenyl, optionally substituted C2-C 20 heterocycloalkyl, hydroxy, oxide (-O - )), optionally substituted C1-C 40 alkoxy, optionally substituted C3-C 40 cycloalkyloxy, optionally substituted C5-C 40 aryloxy, optionally substituted C2-C 40 heteroaryloxy, optionally substituted C5-C 50 aryl, optionally substituted C2-C 50 heteroaryl, thiol, optionally substituted C5-C 50 aralkyl, optionally substituted C1-C 40 alkylthio, optionally substituted C5-C 40Arylthio, C3-C which may be substituted 40 Cycloalkylthio, C2-C may be substituted. 40 Heteroarylthio, optionally substituted acylamino, acyloxy, sulfo group, optionally substituted ammonium, substituted sulfonic acid ester, optionally substituted sulfonamide, substituted ester, optionally substituted aminocarbonyl, nitroso (-N=O), halogen, optionally substituted silyl, optionally substituted amide, optionally substituted carbamate, phospho group, nitrile, acetal, ketal, optionally substituted amino, aminothiocarbonyl, aminocarbonyloxy, aminosulfonyloxy, amidino group, sulfonyloxy, isonitrile, cyanate, imide, iminium, ether, thioether and -L3-R 36 Selected from, R 21 ~R 33 Two adjacent rings can bond to each other to form an optional aliphatic ring, an optional aromatic ring, or an optional mixed aliphatic and aromatic ring. A3 and A4 are, independently, hydrogen, halogen, and C1-C which may be substituted. 10 Alkyl, possibly substituted C3-C 10 Cycloalkyl, C1-C which may be substituted 10 Heterocycloalkyl, optionally substituted C1-C 10 Heteroalkyl, possibly substituted C2-C 10 Alkenyl, may be substituted C2-C 10 Alkinyl, C1-C which may be substituted. 10 Alkoxy, possibly substituted C5-C 40 Selected from aryloxy and cyano, except that A3 and A4 are hydrogen at the same time. X is O, S, CR a R b , NR a or SiR a R b And, R 21 ~R 33 At least one of them is -L3-R 36 And, L3 is a linker containing hydrocarbons with 1 to 40 carbon atoms. R 36 This is a nucleoside selected from chemical formulas 3 to 5 below, [ka] Here, * indicates the position where the nucleoside binds to L3. B is a nuclear base, R 40 is hydrogen, -P(OR 43 )(N(R 44 R 45 )) and -L4-R 46 Selected from, R 41 This refers to an alcohol protecting group, hydrogen or -P(OR 43 )(N(R 44 R 45 )) or may be a support or nucleic acid, R 43 ~R 45 Each of these is independently hydrogen, and C1-C which may be substituted. 10 Alkyl and optionally substituted C1-C 10 Selected from heteroalkyl groups, L4 may be a single bond or a phosphodiester bond between nucleotides, or it may be substituted C1-C 10 Alkyl and optionally substituted C1-C 10 Selected from heteroalkyl groups, R 46 is hydroxy or -P(OR 43 )(N(R 44 R 45 )) or may be a support or nucleic acid, R 42These are hydrogen, hydroxyl, alkoxy and -OR p It is one of the following that can be selected. R p This is a protecting group.

[0010] Furthermore, according to yet another aspect of the present invention, a conjugate (e.g., a nucleotide conjugate, probe, or primer) is provided that includes a compound represented by chemical formula 1 or chemical formula 2 as a labeling reporter.

[0011] Furthermore, according to yet another aspect of the present invention, a nucleic acid detection method is provided, comprising the steps of: (a) preparing a reaction mixture containing a target nucleic acid, reagents necessary for amplifying the target nucleic acid, and the nucleotide conjugate described in claim 12; (b) amplifying the target nucleic acid in the reaction mixture; and (c) measuring the fluorescence intensity of the reaction mixture. [Effects of the Invention]

[0012] The novel compounds according to the present invention have a lower detection limit compared to existing commercially available fluorescent substances, offering the advantage of easy detection even when target biomolecules are present in the sample at low concentrations.

[0013] As a result, the novel compounds according to the present invention can be applied to the field of labeling and detecting biomolecules (e.g., nucleic acids or proteins) as an alternative to existing commercially available fluorescent substances. [Brief explanation of the drawing]

[0014] [Figure 1] This graph shows the results of two Real-Time PCR tests performed on a BQCV (black queen cell virus) target using dual-labeled probes with EX 1-1 and EX 1-2. [Figure 2]This graph shows the results of two Real-Time PCR tests performed on a BQCV (black queen cell virus) target using double-labeled probes with EX 1-3 and EX 1-4. [Figure 3] This graph shows the results of two Real-Time PCR tests performed on a BQCV (black queen cell virus) target using double-labeled probes with EX 1-5 and EX 1-6. [Figure 4] This graph shows the results of Real-Time PCR performed twice on a BQCV (black queen cell virus) target using double-labeled probes with EX 1-7 and EX 1-8. [Figure 5] This figure shows the standard curves of Real-Time PCR experimental results using double-labeled probes EX 1-1 to EX 1-8, respectively. [Figure 6] This figure shows the standard curves of Real-Time PCR experimental results using double-labeled probes EX 1-1 to EX 1-8, respectively. [Figure 7] This figure shows the standard curves of Real-Time PCR experimental results using double-labeled probes EX 1-1 to EX 1-8, respectively. [Figure 8] This figure shows the standard curves of Real-Time PCR experimental results using double-labeled probes EX 1-1 to EX 1-8, respectively. [Figure 9] This figure shows the standard curves of Real-Time PCR experimental results using double-labeled probes EX 1-1 to EX 1-8, respectively. [Figure 10] This figure shows the standard curves of Real-Time PCR experimental results using double-labeled probes EX 1-1 to EX 1-8, respectively. [Figure 11] This figure shows the standard curves of Real-Time PCR experimental results using double-labeled probes EX 1-1 to EX 1-8, respectively. [Figure 12]This figure shows the standard curves of Real-Time PCR experimental results using double-labeled probes EX 1-1 to EX 1-8, respectively. [Figure 13] This graph shows the results of Real-Time PCR performed twice on a BQCV (black queen cell virus) target using dual-labeled probes with EX 2-1 and EX 2-2, respectively. [Figure 14] This graph shows the results of two Real-Time PCR tests performed on a BQCV (black queen cell virus) target using double-labeled probes with EX 2-3 and EX 2-4. [Figure 15] This graph shows the results of two Real-Time PCR tests performed on a BQCV (black queen cell virus) target using double-labeled probes with EX 2-5 and EX 2-6. [Figure 16] This graph shows the results of two Real-Time PCR tests performed on a BQCV (black queen cell virus) target using double-labeled probes with EX 2-7 and EX 2-8. [Figure 17] This figure shows the standard curves of Real-Time PCR experimental results using double-labeled probes EX 2-1 to EX 2-8, respectively. [Figure 18] This figure shows the standard curves of Real-Time PCR experimental results using double-labeled probes EX 2-1 to EX 2-8, respectively. [Figure 19] This figure shows the standard curves of Real-Time PCR experimental results using double-labeled probes EX 2-1 to EX 2-8, respectively. [Figure 20] This figure shows the standard curves of Real-Time PCR experimental results using double-labeled probes EX 2-1 to EX 2-8, respectively. [Figure 21]This figure shows the standard curves of Real-Time PCR experimental results using double-labeled probes EX 2-1 to EX 2-8, respectively. [Figure 22] This figure shows the standard curves of Real-Time PCR experimental results using double-labeled probes EX 2-1 to EX 2-8, respectively. [Figure 23] This figure shows the standard curves of Real-Time PCR experimental results using double-labeled probes EX 2-1 to EX 2-8, respectively. [Figure 24] This figure shows the standard curves of Real-Time PCR experimental results using double-labeled probes EX 2-1 to EX 2-8, respectively. [Modes for carrying out the invention]

[0015] For the convenience of understanding the present invention, certain terms are defined herein. Unless otherwise defined herein, scientific and technical terms used herein have the meanings generally understood by a person of ordinary skill in the art.

[0016] Furthermore, unless otherwise specified, singular terms shall include their plural forms, and plural terms shall include their singular forms.

[0017] Furthermore, unless otherwise defined herein, any functional group shall conform to the definition generally accepted in the art to which this invention pertains.

[0018] new compound According to one aspect of the present invention, a novel compound represented by the following chemical formula 1 is provided. [ka]

[0019] Here, R1~R4 are each independently hydrogen, or C1-C which may be substituted. 40Alkyl, possibly substituted C1-C 40 Heteroalkyl, possibly substituted C2-C 40 Alkenyl, may be substituted C2-C 40 Alkinyl, C3-C may be substituted. 20 Cycloalkyl, possibly substituted C3-C 20 Cycloalkenyl, C2-C may be substituted. 20 Heterocycloalkyl, hydroxy, oxide (-O - ), C3-C even if it is substituted. 40 Cycloalkyloxy, possibly substituted C5-C 40 Aryloxy, possibly substituted C2-C 40 Heteroaryloxy, thiol, possibly substituted C5-C 50 Alalkyl, possibly substituted C1-C 40 Alkylthio, optionally substituted C5-C 40 Arylthio, C3-C which may be substituted 40 Cycloalkylthio, C2-C may be substituted. 40 Heteroarylthio, optionally substituted acylamino, acyloxy, sulfo group, optionally substituted ammonium, substituted sulfonic acid ester, optionally substituted sulfonamide, substituted ester, optionally substituted aminocarbonyl, nitroso (-N=O), optionally substituted silyl, optionally substituted amide, optionally substituted carbamate, phospho group, nitrile, acetal, ketal, optionally substituted amino, aminothiocarbonyl, aminocarbonyloxy, aminosulfonyloxy, amidino group, sulfonyloxy, isonitrile, cyanate, imide, iminium, ether, thioether, R x and R s Selected from, R5~R 13 Each of these is independently hydrogen, and C1-C which may be substituted. 40 Alkyl, possibly substituted C1-C 40Heteroalkyl, possibly substituted C2-C 40 Alkenyl, may be substituted C2-C 40 Alkinyl, C3-C may be substituted. 20 Cycloalkyl, possibly substituted C3-C 20 Cycloalkenyl, C2-C may be substituted. 20 Heterocycloalkyl, hydroxy, oxide (-O - ), C1-C even if substituted 40 Alkoxy, possibly substituted C3-C 40 Cycloalkyloxy, possibly substituted C5-C 40 Aryloxy, possibly substituted C2-C 40 Heteroaryloxy, possibly substituted C5-C 50 Aryl, possibly substituted C2-C 50 Heteroaryl, thiol, and possibly substituted C5-C 50 Alalkyl, possibly substituted C1-C 40 Alkylthio, optionally substituted C5-C 40 Arylthio, C3-C which may be substituted 40 Cycloalkylthio, C2-C may be substituted. 40 Heteroarylthio, optionally substituted acylamino, acyloxy, sulfo group, optionally substituted ammonium, substituted sulfonic acid ester, optionally substituted sulfonamide, substituted ester, optionally substituted aminocarbonyl, nitroso (-N=O), halogen, optionally substituted silyl, optionally substituted amide, optionally substituted carbamate, phospho group, nitrile, acetal, ketal, optionally substituted amino, aminothiocarbonyl, aminocarbonyloxy, aminosulfonyloxy, amidino group, sulfonyloxy, isonitrile, cyanate, imide, iminium, ether, thioether, R x and R s Selected from, R1~R 13Two adjacent rings can bond to each other to form an optional aliphatic ring, an optional aromatic ring, or an optional mixed aliphatic and aromatic ring. A1 and A2 are, independently, hydrogen, halogen, or possibly substituted C1-C. 10 Alkyl, possibly substituted C3-C 10 Cycloalkyl, C1-C which may be substituted 10 Heterocycloalkyl, optionally substituted C1-C 10 Heteroalkyl, possibly substituted C2-C 10 Alkenyl, may be substituted C2-C 10 Alkinyl, C1-C which may be substituted. 10 Alkoxy, possibly substituted C5-C 40 Selected from aryloxy and cyano, except when A1 and A2 are both hydrogen.

[0020] Preferably, at least one of A1 and A2 may be selected from either a substituted C1-C3 alkyl group or a substituted C1-C5 alkoxy group.

[0021] R x This is a reactive group, or a group in which at least one reactive group is bonded to a backbone containing a hydrocarbon with 1 to 40 carbon atoms. The aforementioned reactive group is carboxyl, carboxyl derivative, carboxylate (-CO2 -), functional groups selected from carboxylates, hydroxyls, diene derivatives, aldehydes, substituted ketones, sulfonyl halides, thiols, unsubstituted aminos, primary aminos, alkenes, alkynes, halogens, hydrazides, azides, imides, ketenes, isocyanates, thiocyanates, isothiocyanates, epoxides, maleimides, 1,2,4,5-tetrazine derivatives, cycloalkyne derivatives, cycloalkenes, triphosphates and phosphoramidites, substituted thioketones, haloformyl, formyl, acyl, acylamide, acyl azide, organic acid anhydrides (anhydride), aniline, aziridine, boronate, carbodiimide, diazoalkynes, haloacetamide, imide esters, glycols, halotriazines, hydrazines, acyl halides, alkyl halides and aryl halides, R s This is either a carrier molecule or a group in which at least one carrier molecule is bonded to a backbone containing a hydrocarbon with 1 to 40 carbon atoms. R1~R 13 At least one of them is R x and R s And, X is O, S, CR a R b , NR a or SiR a R b And, R a and R b C1-C may be substituted independently of each other. 40 Alkyl, possibly substituted C1-C 40 Heteroalkyl, possibly substituted C3-C 30 Cycloalkyl, possibly substituted C3-C 30 Heterocycloalkyl, possibly substituted C5-C 50 Aryl and possibly substituted C2-C 50The rings are selected from heteroaryls, or are optionally substituted aliphatic rings, optionally substituted aromatic rings, or optionally substituted mixed aliphatic and aromatic rings formed by bonding with each other.

[0022] In one embodiment, R x and R s This may include the following structural formula 1. [ka]

[0023] Here, * indicates the position where structural formula 1 is attached to the compound represented by chemical formula 1. R 14 and R 15 Each of these is independently hydrogen, and C1-C which may be substituted. 20 Alkyl, possibly substituted C1-C 20 Heteroalkyl, possibly substituted C2-C 20 Alkenyl, may be substituted C2-C 20 Alkinyl, C3-C may be substituted. 20 Cycloalkyl, possibly substituted C3-C 20 Cycloalkenyl, C2-C may be substituted. 20 Heterocycloalkyl, hydroxy, oxide (-O - ), C1-C even if substituted 40 Alkoxy, possibly substituted C3-C 20 Cycloalkyloxy, possibly substituted C5-C 20 Aryloxy, possibly substituted C2-C 20 Heteroaryloxy, possibly substituted C5-C 20 Aryl, possibly substituted C2-C 20 Heteroaryl, thiol, and possibly substituted C5-C 20 Alalkyl, possibly substituted C1-C 20 Alkylthio, optionally substituted C5-C 20 Arylthio, C3-C which may be substituted20 Cycloalkylthio, C2-C may be substituted. 20 Selected from heteroarylthio, optionally substituted acylamino, acyloxy, sulfo group, optionally substituted ammonium, substituted sulfonic acid ester, optionally substituted sulfonamide, substituted ester, optionally substituted aminocarbonyl, nitroso (-N=O), halogen, optionally substituted silyl, optionally substituted amide, optionally substituted carbamate, phospho group, nitrile, acetal, ketal, optionally substituted amino, aminothiocarbonyl, aminocarbonyloxy, aminosulfonyloxy, amidino group, sulfonyloxy, isonitrile, cyanate, imide, iminium, ether and thioether, or bonded together to form optionally substituted aliphatic rings, optionally substituted aromatic rings or optionally substituted mixed aliphatic and aromatic rings, L1 is a linker that is either a single bond or a hydrocarbon containing 1 to 20 carbon atoms. R 14 and R 15 At least one of these is carboxyl, carboxyl derivative, carboxylate (-CO2 - The reaction group may be selected from carboxylates, hydroxyls, diene derivatives, aldehydes, substituted ketones, sulfonyl halides, thiols, unsubstituted aminos, primary aminos, alkenes, alkynes, halogens, hydrazides, azides, imides, ketenes, isocyanates, thiocyanates, isothiocyanates, epoxides, maleimides, 1,2,4,5-tetrazine derivatives, cycloalkyne derivatives, cycloalkenes, triphosphates and phosphoramidites, substituted thioketones, haloformyl, formyl, acyl, acylamide, acyl azide, organic acid anhydrides (anhydride), aniline, aziridine, boronate, carbodiimide, diazoalkynes, haloacetamide, imide esters, glycols, halotriazines, hydrazines, acyl halides, alkyl halides and aryl halides.

[0024] If L1 is a single bond, R 14 and R 15 One of them may be hydrogen.

[0025] In other embodiments, R x and R s This may include structural formula 2 shown below. [ka]

[0026] Here, * indicates the position where structural formula 2 is attached to the compound represented by chemical formula 1. R 16 C1-C may be hydrogen, or substituted. 20 Alkyl, possibly substituted C1-C 20 Heteroalkyl, possibly substituted C2-C 20 Alkenyl, may be substituted C2-C 20 Alkinyl, C3-C may be substituted. 20 Cycloalkyl, possibly substituted C3-C 20 Cycloalkenyl, C2-C may be substituted. 20 Heterocycloalkyl, hydroxy, oxide (-O - ), C1-C even if substituted 40 Alkoxy, possibly substituted C3-C 20 Cycloalkyloxy, possibly substituted C5-C 20 Aryloxy, possibly substituted C2-C 20 Heteroaryloxy, possibly substituted C5-C 20 Aryl, possibly substituted C2-C 20 Heteroaryl, thiol, and possibly substituted C5-C 20 Alalkyl, possibly substituted C1-C 20 Alkylthio, optionally substituted C5-C 20 Arylthio, possibly substituted C5-C 20 Cycloalkylthio, C2-C may be substituted.20 Selected from heteroarylthio, optionally substituted acylamino, acyloxy, sulfo group, optionally substituted ammonium, substituted sulfonic acid ester, optionally substituted sulfonamide, substituted ester, optionally substituted aminocarbonyl, nitroso (-N=O), halogen, optionally substituted silyl, optionally substituted amide, optionally substituted carbamate, phospho group, nitrile, acetal, ketal, optionally substituted amino, aminothiocarbonyl, aminocarbonyloxy, aminosulfonyloxy, amidino group, sulfonyloxy, isonitrile, cyanate, imide, iminium, ether and thioether, L2 is a linker that is either a single bond or a hydrocarbon containing 1 to 20 carbon atoms. The following describes in detail some of the functional groups referred to in this application. Functional groups not referred to below are defined according to the general definitions that are common in the art to which this invention pertains.

[0027] In this application, the term "may be substituted" means that any functional group may exist in an unsubstituted state, or may exist in a state substituted with at least one substituent, provided that this does not inhibit the effect of the compound as defined in this application or change it heterogeneously. The term "may be substituted" is a general expression used in the art when defining any functional group, and unless otherwise defined, the range of any functional group to be interpreted as "may be substituted" also follows the general definition used in the art to which this invention belongs.

[0028] If any functional group as defined in this application is a substituted functional group, any carbon contained in the functional group may be substituted with at least one substituent.

[0029] For example, the substituent is C1-C 40 Alkyl, C1-C 40 Heteroalkyl, C2-C40 Alkenyl, C2-C 40 Alkinyl, C3-C 20 Cycloalkyl, C3-C 20 Cycloalkenyl, C2-C 20 Heterocycloalkyl, hydroxy, oxide (-O - ), C1-C even if substituted 40 Alkoxy, C3-C 40 Cycloalkyloxy, C5-C 40 Aryloxy, C2-C 40 Heteroaryloxy, possibly substituted C5-C 50 Aryl, possibly substituted C2-C 50 Heteroaryl, thiol, C5-C 50 Alalkyl, C1-C 40 Alkylthio, C5-C 40 Arylthio, C3-C 40 Cycloalkylthio, C2-C 40 Heteroarylthio, acylamino, acyloxy, sulfo group, ammonium, sulfonic acid ester, sulfonamide, ester, aminocarbonyl, nitroso (-N=O), halogen, silyl, amide, optionally substituted carbamate, phospho group, nitrile, acetal, ketal, amino, aminothiocarbonyl, aminocarbonyloxy, aminosulfonyloxy, amidino group, sulfonyloxy, isonitrile, cyanate, imide, iminium, ether, thioether, R x and R s Selected from, The hydrogen used in this application may be light hydrogen (1H), deuterium (2H), or tritium (3H) with a mass number of 1.

[0030] In this application, heteroatoms refer to atoms other than carbon and hydrogen, and more specifically, the heteroatoms are atoms that can be used in place of carbon in a backbone structure made of carbon (more specifically, hydrocarbons), such as nitrogen, oxygen, sulfur, phosphorus, silicon, or selenium.

[0031] R x This is a reactive group, or a group in which at least one reactive group is bonded to a backbone containing a hydrocarbon with 1 to 40 carbon atoms.

[0032] The aforementioned backbone refers to a series of bonds that link a reactive group or carrier molecule to the matrix represented by chemical formula 1.

[0033] For example, the backbone may have an alkyl or heteroalkyl group containing 1 to 40 carbon atoms as its main chain. Furthermore, the main chain may also contain multiple alkyl or heteroalkyl groups linked together via intermediate functional groups such as amides or esters.

[0034] The backbone may contain at least one heteroatom selected from O, S, N, P, and Si. The backbone may also be linear or nonlinear and may consist of any combination of single, double, or triple bonds.

[0035] The aforementioned backbone may be, for example, alkyl, alkenyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, -C(O)-, -C(O)O-, or -NR. L -, -O-, -S-, -C(O)NR L -, -S(O) O -, -S(O)NR L -, S(O2)NR L -,-P(O) P -and may be selected from the following structural formulas (a) to (o), or composed of a combination thereof. [ka]

[0036] Here, R L is hydrogen, C1-C 40 Alkyl and C1-C 40Selected from heteroalkyls, each q is an integer from 0 to 10, which may be the same or different, each o is an integer from 1 to 2, and each p is an integer from 1 to 4.

[0037] The aforementioned reactive group is carboxyl, carboxyl derivative, carboxylate (-CO2 - The functional group is selected from carboxylates, hydroxyls, diene derivatives, aldehydes, substituted ketones, sulfonyl halides, thiols, unsubstituted aminos, primary aminos, alkenes, alkynes, halogens, hydrazides, azides, imides, ketenes, isocyanates, thiocyanates, isothiocyanates, epoxides, maleimides, 1,2,4,5-tetrazine derivatives, cycloalkyne derivatives, cycloalkenes, triphosphates and phosphoramidites, substituted thioketones, haloformyl, formyl, acyl, acylamide, acyl azide, organic acid anhydrides (anhydride), aniline, aziridine, boronate, carbodiimide, diazoalkynes, haloacetamide, imide esters, glycols, halotriazines, hydrazines, acyl halides, alkyl halides and aryl halides.

[0038] Furthermore, if necessary, the reactant group may be protected from participating in the reaction by the presence of a protecting group.

[0039] The protecting group is introduced by chemically modifying a reactive group in order to confer reaction selectivity to at least some of the reactive groups during a continuous chemical or biological reaction process.

[0040] Examples of protecting groups include hydroxyl protecting groups, amino protecting groups, carbonyl protecting groups, carboxyl protecting groups, thiol protecting groups, and phosphate protecting groups. In addition, unless otherwise defined in this application, functional groups other than those exemplified above that can be introduced and removed by specific reactive groups may be used as protecting groups.

[0041] For example, when the reactive group is hydroxyl, the protecting groups include alkanoyl groups such as acetyl, benzoyl, pivaloyl, chloroacetyl, trifluoroacetyl, and methoxyacetyl; alkyloxycarbonyl groups such as benzyloxycarbonyl, ethyloxycarbonyl, methoxybenzyloxycarbonyl, tert-butyloxycarbonyl, diphenylmethyloxycarbonyl, and 2,2,2-trichloroethyloxycarbonyl; alphaalkyl groups such as benzyl, nitrobenzyl, methoxybenzyl, trityl, methoxytrityl, 4,4-dimethoxytrityl (DMT), and diphenylmethyl; t-butyl, methoxymethyl, methoxyethyl (MOE), methyl, 2,2,2-trichloroethyl, 2-trimethylsilylethyl, methylthiomethyl, benzyloxymethyl, and 2-(trimethyl Alkyl groups such as lucilyl)-ethoxymethyl and 9-fluorenylmethyl; ether groups such as methoxymethyl ether (MOM), benzyl ether, tert-butyldimethylsilyl ether, p-methoxybenzyl ether (PMB), p-methoxyphenyl ether, β-methoxyethoxymethyl ether (MEM), methylthiomethyl ether, trimethylsilyl ether, methyl ether, and ethoxyethyl ether; ester groups such as acetate esters, methanesulfonate esters, and tert-butylcarbonate; silyl groups such as tert-butyldiphenylsilyl, trimethylsilyl, and triisopropylsilyl; sulfonyl groups such as methanesulfonyl; allyl; tetrahydropyranyl (THP); tetrahydrofuran (THF); or any of the following groups may be selected. [ka]

[0042] Furthermore, when the reactive group is amino, the protecting groups include carbamate groups such as tert-butylcarbamate and benzylcarbamate; amide groups such as acetamide, p-toluenesulfonamide, and diphenyl-phosphoroamide; phthalimide groups; 9-fluorenyl methoxycarbonyl (Fmoc), t-butyloxycarbonyl (Boc), t-amyloxycarbonyl, benzyloxycarbonyl (Cbz), 4-methoxybenzyloxycarbonyl, methyloxycarbonyl, ethyloxycarbonyl, nitrobenzyloxycarbonyl group, 2,2,2-trichloroethyloxycarbonyl group, aryloxycarbonyl, and isobornyloxycarbonyl groups. Carbonyl groups such as adamantyloxycarbonyl; sulfonyl groups such as 4-methylphenylsulfonyl, 2,4-dinitrobenzenesulfonyl, 2-trimethylsilylethanesulfonyl (SES); alphaalkyl groups such as benzyl, trityl, 1,1-bis-(4-methoxyphenyl)methyl, 2,4-dimethoxybenzyl (DMB), 4-methoxybenzyl (PMB); silyl groups such as trimethylsilyl (TMS), t-butyldimethylsilyl (TBS); alkanoyl groups such as acetyl, trifluoroacetyl, phthalyl, pivaloyl, benzoyl; allyl; 2-nitrophenylsulfenyl; or any of the following groups can be selected. [ka]

[0043] Furthermore, when the reactive group is carbonyl, the protecting group can be an acetal group such as dimethylacetal, 1,3-dioxane, or thioacetal; a ketal group such as dimethylketal or thioketal; an oxime group such as O-methyloxime; a hydrazone group such as N,N-dimethylhydrazone, or analogs thereof.

[0044] Furthermore, when the reactive group is a carboxyl group, the protecting group can be an ester group such as methyl esters, allyl esters, or benzyl esters; an amide group such as N,N-dimethylamide; an alkyl group such as methyl, ethyl, t-butyl, phenylsulfonylethyl, cyanoethyl, 2-trimethylsilylethyl, 2-trimethylsilylethoxymethyl, or nitroethyl; an alpha-alkyl group such as benzyl; an aryl group such as phenyl; an allyl group or analogues thereof.

[0045] Furthermore, when the reactive group is a thiol, the protecting group can be an ether group such as benzyl thioether; an ester group such as thioacetate ester, thiocarbonate, or thiocarbamate, or an analog thereof.

[0046] Furthermore, when the reactive group is phosphoric acid, the protecting group can be an alkyl group such as t-butyl, methyl, ethyl, cyanoethyl, trimethylsilylethyl, triphenylsilylethyl, or 2,2,2-trichloroethyl; an alkenyl group such as ethenyl, propenyl, butenyl, 2-cyanobutenyl, or 1-ethyl-2-butenyl; a cycloalkyl group such as cyclopropyl, cyclobutyl, or cyclohexyl; an alphaalkyl group such as benzyl, α-naphthylmethyl, trityl, dimethylphenyl, chlorobenzyl, or nitrobenzyl; an aryl group such as phenyl, naphthyl, methylphenyl, dimethylphenyl, or chlorophenyl; an allyl group, or analogs thereof.

[0047] Furthermore, examples of protecting groups can be found in the following reference (Greene's Protective Groups in Organic Synthesis, Fifth Edition, John Wiley & Sons, Inc. 2014; https: / / en.wikipedia.org / wiki / Protecting_group).

[0048] In this application, C a -C bA functional group refers to a functional group having a to b carbon atoms. For example, C a -C b Alkyl refers to a saturated aliphatic group having a to b carbon atoms, including linear and branched alkyl groups. Linear or branched alkyl groups have 40 or fewer carbon atoms in their main chain (for example, C1-C 10 A straight chain of C3-C 10 (This may also be a branched chain.)

[0049] Specifically, alkyl groups may be methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, i-butyl, t-butyl, pento-1-yl, pento-2-yl, pento-3-yl, 3-methylbutto-1-yl, 3-methylbutto-2-yl, 2-methylbutto-2-yl, 2,2,2-trimethyletho-1-yl, n-hexyl, n-heptyl, and n-octyl.

[0050] In this application, alkoxy means both an -O-(alkyl) group and an -O-(unsubstituted cycloalkyl) group, and is a linear or branched hydrocarbon having one or more ether groups and 1 to 10 carbon atoms.

[0051] Specifically, this includes, but is not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy, 1,2-dimethylbutoxy, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, and cyclohexyloxy.

[0052] In this application, amino acids can be classified into primary, secondary, and tertiary amino acids depending on the number of hydrogen atoms bonded to the nitrogen atom. Furthermore, if necessary, the amino acids may also be provided in quaternary ammonium form. Quaternary ammonium form refers to the case where the nitrogen atom in the amino group has a positive charge.

[0053] Non-restrictive examples of phospho groups usable in this application include, but are not limited to, substituted phosphine, substituted phosphinate, substituted phosphonate, substituted phosphite, substituted phosphate, phosphoric acid, phosphorous acid, and hypophosphorous acid. In one embodiment, the phospho group may be a substituted phosphonate, a substituted phosphate, and / or phosphoric acid.

[0054] Non-restrictive examples of sulfo groups usable in this application include, but are not limited to, substituted sulfonyl, sulfonium, sulfate, sulfonic acid, substituted sulfonate, substituted sulfinate, sulfite, and substituted sulfoxide. In one embodiment, sulfonic acid and / or substituted sulfonate can be used as the sulfo group.

[0055] In this application, halogen means fluoro(-F), chloro(-Cl), bromo(-Br), or iodine(-I), and haloalkyl means alkyl substituted with the aforementioned halogens. For example, halomethyl means methyl (-CH2X, -CHX2, or -CX3) in which at least one of the hydrogen atoms of methyl is substituted with a halogen.

[0056] In this application, alphaalkyl is a functional group in which an aryl is substituted on the alkyl carbon, -(CH2) n Ar is a general term for alkyl groups. Examples of alkyl groups include benzyl (-CH2C6H5) and phenethyl (-CH2CH2C6H5).

[0057] In this application, unless otherwise defined, aryl means an unsaturated aromatic ring containing a monocycle or a polycycle (preferably 1 to 4 rings) linked to one another by condensation or covalent bonds. Non-restrictive examples of aryl include phenyl, biphenyl, o-terphenyl, m-terphenyl, p-terphenyl, 1-naphthyl, 2-naphthyl, 1-anthryl, 2-anthryl, 9-anthryl, 1-phenanthrenyl, 2-phenantrenyl, 3-phenantrenyl, 4-phenantrenyl, 9-phenantrenyl, 1-pyrenyl, 2-pyrenyl, and 4-pyrenyl.

[0058] In this application, heteroaryl means a functional group in which one or more carbon atoms in the aryl group as defined above are substituted with non-carbon atoms such as nitrogen, oxygen, or sulfur. Non-restrictive examples of heteroaryls include furyl, tetrahydrofuryl, pyrrolyl, pyrrolidinyl, thienyl, tetrahydrothienyl, oxazolyl, isoxazolyl, triazolyl, thiazolyl, isothiazolyl, pyrazolyl, pyrazolidinyl, oxadiazolyl, thiadiazolyl, imidazolyl, imidazolinyl, pyridyl, pyridazinyl, triazinyl, piperidinyl, and morphol (inyl), thiomorpholinyl, pyrazinyl, piperazinyl, pyrimidinyl, naphthyridinyl, benzofuranyl, benzothienyl, indolyl, indolinyl, indazolyl, quinolidinyl, isoquinolinyl, cinolinyl Examples include cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, pteridinyl, quinuclidinyl, carbazoyl, acridinyl, phenadinyl, phenothizinyl, phenoxadinyl, purinyl, benzimidazolyl, and benzothiazolyl, as well as their condensed analogues.

[0059] In this application, unless otherwise defined, a hydrocarbon ring (cycloalkyl) or a hydrocarbon ring containing a heteroatom (heterocycloalkyl) can be understood as an alkyl or heteroalkyl cyclic structure, respectively.

[0060] Non-restrictive examples of hydrocarbon rings include cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, and cycloheptyl. Non-restrictive examples of hydrocarbon rings containing heteroatoms include 1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl, and 2-piperazinyl.

[0061] In this application, unless otherwise defined, a cycloalkene or cycloalkyne can be understood as a cyclic alkyl structure containing at least one unsaturated bond (double or triple bond). Furthermore, at least one of the carbon atoms of the cycloalkene or cycloalkyne may be substituted with a heteroatom. A cycloalkene or cycloalkyne is a C3-C 30 Or C3-C 20 It may have carbon atoms.

[0062] Furthermore, hydrocarbon rings or hydrocarbon rings containing heteroatoms may have a form in which a hydrocarbon ring, a hydrocarbon ring containing a heteroatom, an aryl or heteroaryl atom is condensed thereto or covalently linked thereto.

[0063] In this application, a derivative refers to an analog compound obtained by chemically altering a part of any compound.

[0064] The carboxyl derivative is a functional group that can be converted with carboxyl, and may be an N-hydroxysuccinimide ester, an N-hydroxybenzotriazole ester, an acyl halide, an acylimidazole, a thioester, an alkyl ester, an alkenyl ester, an alkynyl ester, or an aromatic ester.

[0065] The carboxyl derivative may be substituted, for example, trifluoromethyl ester, pentafluorophenyl ester, p-nitrophenyl ester, tetrafluorophenyl ester, sulfosuccinimidyl ester, sulfodichlorophenyl ester, or sulfotetrafluorophenyl ester.

[0066] Furthermore, in this application, the 1,2,4,5-tetrazine derivative may be 3,6-dimethyl-1,2,4,5-tetrazine, 3,6-diphenyl-1,2,4,5-tetrazine, or 3-methyl-6-phenyl-1,2,4,5-tetrazine.

[0067] Furthermore, in this application, the cycloalkyne derivative may be a compound that participates in bipolar cycloaddition, inverse-electron demand Diels-Alder, or strain-promoted azide-alkyne cycloaddition (SPAAC) reactions.

[0068] For example, cyclooctynes ​​such as OCT, COMBO(ALO), MOFO, DIFO, DIBO, BARAC, DIBAC(ADIBO), DBCO, DIMAC, BCN, or TMTH can be used as cycloalkyne derivatives participating in the SPAAC reaction.

[0069] Furthermore, any other examples of derivatives explicitly mentioned in this application can be found in the literature known in the art.

[0070] The parent diene is an alkene or alkyne that can undergo a Diels-Alder reaction with the diene, specifically, sp 2 -These may be alkenes or alkynes in which an electron-withdrawing group is directly linked to a hybrid carbon or sp-hybrid carbon.

[0071] An electron-withdrawing group is a functional group that tends to attract electrons through inductive or resonance effects, and can also be called a deactivating group. Examples of electron-withdrawing groups include trifluoromethylsulfonyl (-SO2CF3) and substituted or unsubstituted ammonium (-NR3). + Examples include nitro, sulfonic acid (-SO3H), sulfonyl (-SO2R), nitrile, trihalomethyl (-CF3, -CCl3, -CBr3, -CI3), haloformyl (-COCl, -COBr, -COI), formyl (-CHO), acyl (-COR), carboxyl (-CO2H), substituted ester (-CO2R), substituted or unsubstituted aminocarbonyl (-CONR2), and nitroso (-N=O). Furthermore, unless otherwise defined in this application, the electron-withdrawing groups may include other functional groups that tend to attract electrons, in addition to the functional groups exemplified above.

[0072] R1~R 13 These are the functional groups defined above, and may exist independently, but according to some embodiments, R1 to R1 are present within a range that maintains the properties of the compound as defined in this application. 13 Two adjacent rings can bond to each other to form an optionally substituted aliphatic ring, an optionally substituted aromatic ring, or an optionally substituted aliphatic-aromatic mixed ring.

[0073] R1~R 13When two adjacent ones of them form an aliphatic or aromatic ring, the aliphatic ring or the aromatic ring may be, for example, a monocyclic ring composed of 4 atoms, 5 atoms, 6 atoms or more atoms (generally, 7 atoms to 10 atoms), or may be a fused ring in which the monocyclic rings are condensed. Further, the ring may be a ring in which the aliphatic ring and the aromatic ring are mixed. At this time, the aliphatic ring and the aromatic ring can be in a form where they are condensed with each other or connected by a single bond. Further, the ring can also contain at least one heteroatom.

[0074] In one embodiment, at least one pair of R1 and R5, R2 and R6, R3 and R 10 and R4 and R9 can be bonded to each other to form an optionally substituted aliphatic ring, an optionally substituted aromatic ring, or an optionally substituted aliphatic and aromatic mixed ring. In other embodiments, at least one pair of R1 and R2 and R3 and R4 can be bonded to each other to form an optionally substituted aliphatic ring, an optionally substituted aromatic ring, or an optionally substituted aliphatic and aromatic mixed ring.

[0075] Also, R a and R b are functional groups defined as above and may each independently exist. However, according to some embodiments, within the range where the properties of the compounds defined in the present application are maintained, R a and R b can be bonded to each other to form an optionally substituted aliphatic ring, an optionally substituted aromatic ring, or an optionally substituted aliphatic and aromatic mixed ring.

[0076] R a and R bWhen they are bonded to each other to form an aliphatic or aromatic ring, the aliphatic ring or the aromatic ring may be, for example, a monocyclic ring composed of 4, 5, 6 or more atoms (generally, 7 to 10 atoms), or a condensed ring in which the monocyclic rings are condensed. Further, the ring may be a ring in which the aliphatic ring and the aromatic ring are mixed. At this time, the aliphatic ring and the aromatic ring can be condensed with each other or have a form connected by a single bond. Further, the ring can also contain at least one heteroatom.

[0077] In one embodiment, L1 and L2 may be linkers containing a hydrocarbon containing 1 to 40 carbon atoms.

[0078] The linker (including L3) can have a main chain of alkyl or heteroalkyl containing a carbon atom. Further, the main chain may contain a plurality of alkyls or heteroalkyls linked through an intermediate functional group such as an amide or an ester.

[0079] The linker may contain at least one heteroatom selected from O, S, N, P, and Si. Further, the linker may be linear or non-linear and may be composed of an arbitrary combination of single, double, and triple bonds.

[0080] The linker is, for example, alkyl, alkenyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, -C(O)-, -C(O)O-, -NR L -, -O-, -S-, -C(O)NR L -, -S(O) O -, -S(O)NR L -, S(O2)NR L -, -P(O) P - and may be selected from the following structural formulas (a) to (o) or composed of a combination thereof.

Chemical formula

[0081] Here, R L is hydrogen, C1-C 40 Alkyl and C1-C 40 Selected from heteroalkyls, each q is an integer from 0 to 10, which may be the same or different, each o is an integer from 1 to 2, and each p is an integer from 1 to 4.

[0082] R s This is a carrier molecule, or a group in which at least one carrier molecule is bonded to a backbone containing a hydrocarbon with 1 to 40 carbon atoms, and the definition of the backbone is R x It is identical to [the other one].

[0083] A carrier molecule refers to a substance that transports the compound represented by the chemical formula 1.

[0084] More specifically, carrier molecules include amino acids, amino acid polymers, peptides, proteins, neurotoxins, phallotoxins, cytokines, toxins, protease substrates, protein kinase substrates, enzymes, antibodies, antibody fragments, lectins, glycoproteins, histones, albumins, lipoproteins, avidin, streptavidin, protein A, protein G, protein L, phycobiliproteins, fluorescent proteins, hormones, growth factors, and nucleic acid bases. The base may be a nucleoside, nucleotide, nucleic acid polymer, nucleotide analog, nucleoside analog, nucleoside triphosphate, deoxynucleoside triphosphate (dNTP), dideoxynucleoside triphosphate (ddNTP), organic or inorganic nanoparticles, organic or inorganic microparticles, heptene, carbohydrates, polysaccharides, lipids, ion complexing moieties such as crown ethers, PEG groups, or organic or inorganic polymers.

[0085] Furthermore, the carrier molecule does not need to participate in any reaction if it is protected by a protecting group. In this case, protection of the carrier molecule by a protecting group means that the functional groups present in the carrier molecule are protected by the protecting group. The definition of the protecting group for the carrier molecule is the same as that of the protecting group for the reactant group described above.

[0086] Furthermore, the compound represented by chemical formula 1 may also contain a counterion. The counterion is an organic or inorganic anion and can be appropriately selected considering the solubility and stability of the compound.

[0087] Examples of counterions for compounds according to one embodiment of the present invention include anions derived from inorganic acids such as hexafluorophosphate ions, halogen ions, phosphate ions, perchlorate ions, periodate ions, hexafluoroantimonate ions, hexafluorostanate ions, fluoroborate ions, and tetrafluoroborate ions, as well as anions derived from organic acids such as thiocyanate ions, benzenesulfonate ions, naphthalenesulfonate ions, p-toluenesulfonate ions, alkylsulfonate ions, benzenecarboxylate ions, alkylcarboxylate ions, trihaloalkylcarboxylate ions, alkylsulfonate ions, trihaloalkylsulfonate ions, and nicotinate ions. In addition, metal complex ions such as bisphenyldithiol, thiobisphenol chelate, and bisdiol-α-diketone, metal ions such as sodium and potassium, and quaternary ammonium salts can also be selected as counterions.

[0088] Another aspect of the present invention provides a novel compound represented by the following chemical formula 2. Unless otherwise defined below, the definitions of the structure and functional groups in the compound represented by chemical formula 2, corresponding to the compound represented by chemical formula 1, are the same as those for chemical formula 1. [ka]

[0089] Here, R 21 ~R 24 Each of these is independently hydrogen, and C1-C which may be substituted. 40 Alkyl, possibly substituted C1-C 40 Heteroalkyl, possibly substituted C2-C 40 Alkenyl, may be substituted C2-C 40 Alkinyl, C3-C may be substituted. 20 Cycloalkyl, possibly substituted C3-C 20 Cycloalkenyl, C2-C may be substituted. 20Heterocycloalkyl, hydroxy, oxide (-O - ), C3-C even if it is substituted. 40 Cycloalkyloxy, possibly substituted C5-C 40 Aryloxy, possibly substituted C2-C 40 Heteroaryloxy, thiol, possibly substituted C5-C 50 Alalkyl, possibly substituted C1-C 40 Alkylthio, optionally substituted C5-C 40 Arylthio, C3-C which may be substituted 40 Cycloalkylthio, C2-C may be substituted. 40 Heteroarylthio, optionally substituted acylamino, acyloxy, sulfo group, optionally substituted ammonium, substituted sulfonic acid ester, optionally substituted sulfonamide, substituted ester, optionally substituted aminocarbonyl, nitroso (-N=O), optionally substituted silyl, optionally substituted amide, optionally substituted carbamate, phospho group, nitrile, acetal, ketal, optionally substituted amino, aminothiocarbonyl, aminocarbonyloxy, aminosulfonyloxy, amidino group, sulfonyloxy, isonitrile, cyanate, imide, iminium, ether, thioether and -L3-R 36 Selected from, R 25 ~R 33 Each of these is independently hydrogen, and C1-C which may be substituted. 40 Alkyl, possibly substituted C1-C 40 Heteroalkyl, possibly substituted C2-C 40 Alkenyl, may be substituted C2-C 40 Alkinyl, C3-C may be substituted. 20 Cycloalkyl, possibly substituted C3-C 20 Cycloalkenyl, C2-C may be substituted. 20 Heterocycloalkyl, hydroxy, oxide (-O -), C1-C even if substituted 40 Alkoxy, possibly substituted C3-C 40 Cycloalkyloxy, possibly substituted C5-C 40 Aryloxy, possibly substituted C2-C 40 Heteroaryloxy, possibly substituted C5-C 50 Aryl, possibly substituted C2-C 50 Heteroaryl, thiol, and possibly substituted C5-C 50 Alalkyl, possibly substituted C1-C 40 Alkylthio, optionally substituted C5-C 40 Arylthio, C3-C which may be substituted 40 Cycloalkylthio, C2-C may be substituted. 40 Heteroarylthio, optionally substituted acylamino, acyloxy, sulfo group, optionally substituted ammonium, substituted sulfonic acid ester, optionally substituted sulfonamide, substituted ester, optionally substituted aminocarbonyl, nitroso (-N=O), halogen, optionally substituted silyl, optionally substituted amide, optionally substituted carbamate, phospho group, nitrile, acetal, ketal, optionally substituted amino, aminothiocarbonyl, aminocarbonyloxy, aminosulfonyloxy, amidino group, sulfonyloxy, isonitrile, cyanate, imide, iminium, ether, thioether and -L3-R 36 Selected from, R 21 ~R 33 Two adjacent rings can bond to each other to form an optional aliphatic ring, an optional aromatic ring, or an optional mixed aliphatic and aromatic ring. A3 and A4 are, independently, hydrogen, halogen, and C1-C which may be substituted. 10 Alkyl, possibly substituted C3-C 10 Cycloalkyl, C1-C which may be substituted 10Heterocycloalkyl, optionally substituted C1-C 10 Heteroalkyl, possibly substituted C2-C 10 Alkenyl, may be substituted C2-C 10 Alkinyl, C1-C which may be substituted. 10 Alkoxy, possibly substituted C5-C 40 Selected from aryloxy and cyano, except that A3 and A4 are hydrogen at the same time. X is O, S, CR a R b , NR a or SiR a R b And, R a and R b C1-C may be substituted independently of each other. 40 Alkyl, possibly substituted C1-C 40 Heteroalkyl, possibly substituted C3-C 30 Cycloalkyl, possibly substituted C3-C 30 Heterocycloalkyl, possibly substituted C5-C 50 Aryl and possibly substituted C2-C 50 Selected from heteroaryls, or formed by bonding to each other, an optionally substituted aliphatic ring, an optionally substituted aromatic ring, or an optionally substituted aliphatic and aromatic mixed ring, R 21 ~R 33 At least one of them is -L3-R 36 And, L3 is a linker containing hydrocarbons with 1 to 40 carbon atoms. R 36 It is a nucleoside represented by one of the following chemical formulas 3 to 5: [ka] Here, * indicates the position where the nucleoside binds to L3. B is a nuclear base, R 40 is selected from hydrogen, -P(OR 43 )(N(R 44 R 45 )) and -L4-R 46 and is selected from, R 41 is an alcohol protecting group, hydrogen or -P(OR 43 )(N(R 44 R 45 )) or is an optionally substituted support or nucleic acid, R 43 ~R 45 are each independently selected from hydrogen, optionally substituted C1-C 10 alkyl and optionally substituted C1-C 10 heteroalkyl, L4 is a single bond or is selected from a phosphodiester bond between nucleotides, optionally substituted C1-C 10 alkyl and optionally substituted C1-C 10 heteroalkyl, R 46 is hydroxy or -P(OR 43 )(N(R 44 R 45 )) or is an optionally substituted support or nucleic acid, R 42 is any one selected from hydrogen, hydroxy, alkoxy and -OR p and, R p is a protecting group.

[0090] In one embodiment, said R 41 or R 46 may be an optionally substituted support or nucleic acid, The support or nucleic acid which may be substituted may be a support or nucleic acid substituted with coumarin, cyanine, bodypi, fluorescein, rhodamine, pyrene, carbopyronine, oxazine, xanthene, thioxanthene, acridine and / or derivatives thereof and at least one selected from chemical formula 1.

[0091] Specific examples of the compounds defined in this application are as follows. However, the following example compounds are provided to aid in understanding the compounds defined in this application and are not intended to limit the scope of the compounds defined in this application. Any of the following example compounds and compounds that are considered equivalent to the following example compounds within the scope of the compounds defined in this application can be expected to exhibit the effects of the compounds defined in this application. Accordingly, a compound according to any embodiment of the present invention may be at least one structure selected from the following compounds 1 to 47 or a derivative thereof.

[0092] Furthermore, compounds represented by chemical formula 1 or chemical formula 2 can also be synthesized by referring to the production examples of compounds 1 to 42 disclosed in detail in this application, or by known synthesis methods referring to the content defined in this application. [ka] JPEG2026514785000015.jpg237170JPEG2026514785000016.jpg207170JPEG2026514785000017.jpg146170

[0093] The compounds defined herein can be used as reporters for labeling or detecting biomolecules. These biomolecules include antibodies, lipids, proteins, peptides, carbohydrates, and / or nucleic acids (including DNA, RNA, or nucleotides).

[0094] Specific examples of lipids include fatty acids, phospholipids, and lipopolysaccharides, while specific examples of carbohydrates include monosaccharides, disaccharides, and polysaccharides (e.g., dextran).

[0095] Furthermore, the compounds defined in this application can be used to label or detect, in addition to biomolecules, drugs, hormones (including receptor ligands), receptors, enzymes or enzyme substrates, cells, cell membranes, toxins, microorganisms or nanobiomaterials (such as polystyrene microspheres), etc., which include at least one selected from amino, sulfhydryl, carbonyl, hydroxyl, carboxyl, phosphate and thiophosphate.

[0096] Conjugates containing novel compounds, compositions for nucleic acid detection, and supports for nucleic acid detection. According to another aspect of the present invention, a conjugate comprising a compound defined in this application as a nucleic acid labeling reporter is provided. The conjugate may be a probe or a primer, or a nucleotide conjugate.

[0097] If the conjugate is a probe, it is more preferable, but not limited to, that the probe is capable of binding complementarily to a target nucleic acid. Here, the probe may be selected from, but is not limited to, nucleic acids, peptides, saccharides, oligonucleotides, proteins, antibodies, or combinations thereof.

[0098] If the conjugate is a nucleotide conjugate, the conjugate may include an oligonucleotide.

[0099] The oligonucleotide refers to a polymer of 1 to several hundred nucleotides. The phosphodiester bond in the oligonucleotide can be modified or replaced with methylphosphonate, phosphorothioate (PTO), boranophosphate, phosphorylguanidine, guanidinopropylphosphoramidate, thiazole, guanidinium, etc. The oligonucleotide may also contain one or more modified nucleotides. For example, the modified nucleotide may include sugar-modified PNA, BNA (e.g., LNA, ENA, etc.), HNA, ANA, CeNA, and GNA, or cases where any substituent (e.g., MOE, alkyl, halogen, etc.) is introduced to the sugar.

[0100] Furthermore, modified nucleotides and nucleosides may also contain nuclear bases modified with adenine (A), guanine (G), thymine (T), cytosine (C), and uracil (U), for example, inosine, xanthine, hypoxanthine, nebularin, isoguanisine, tubercidine, 2-aminoadenine, 2-haloadenine, 2-alkyladenine, 2-methylaminoadenine, 6-methyladenine, 8-haloadenine, 8-aminoadenine, 8-thioadenine, diazaadenine, 8-hydroxyadenine and other substituted adenines, 5-halouracil, 4-thiouracil, 5-trifluoromethyluracil, shudouracil, 2- Thiouracil, 5-halouracil and other substituted uracils, 5-halocytosine, 5-trifluorocytosine, 6-azacytosine and other substituted cytosines, 2-thiothymine, 6-azacymine and other substituted thymines, 8-haloguanine, 8-aminoguanine, 8-thioguanine, 8-thioalkylguanine, 8-hydroxyguanine, 8-azacuanine, 7-deazaguanine, 7-methylguanine and other substituted guanines, pyrazolo[3,4-d]pyrimidine, 2,6-diaminopurine, phenoxazine, 2-aminopurine, 3-nitropyrrole, 5-hydroxybutynyluridine, Super A (registered trademark), Super G (registered trademark), Super T (registered trademark), Super D TM It may also include things like the following. Furthermore, for examples of modified nuclear bases, you can refer to the following reference (US Pat. Nos. 11, 155, 713; Current Topics in Medicinal Chemistry, Volume 7, Number 7, Wojciechowski, Filip, E. Hudson, Robert H, 2007, pp. 667-679).

[0101] In this application, the nuclear bases include modified nuclear bases.

[0102] The conjugate may include a minor groove binder (MGB) for selectively binding to the minor groove of the target nucleic acid. The minor groove binder is a crescent-shaped molecule capable of selectively noncovalently binding to minor groups contained within nucleic acids such as DNA (e.g., shallow furrow within the DNA helix).

[0103] Furthermore, the conjugate may include at least one exciter. In this case, the exciter may be located at the 5' end, the 3' end, or any other position inside the conjugate.

[0104] For example, the 5' end of the conjugate may be labeled with a compound represented by chemical formula 1 or chemical formula 2 (a reporter), and the 3' end may be labeled with a quencher. Between the 5' and 3' ends, a probe or minor group binder capable of binding complementaryly to the target nucleic acid may be located.

[0105] The absorbance range of the quencher usable in this application may be 400 to 800 nm. Furthermore, the absorbance range of the quencher can be appropriately selected considering the fluorescence characteristics of the reporter as defined in this application.

[0106] It is important that the probe is designed such that the reporter can be sufficiently quenched by the quencher while minimizing signal interference. Therefore, when designing the probe, it is necessary to confirm that the reporter and quencher labeled at the 5' and 3' ends of the probe, respectively, are interchangeable depending on the type of target biomolecule (e.g., nucleic acid).

[0107] As the aforementioned quencher, various known or commercially available quenchers (e.g., BHQ0, BHQ1, BHQ2, BHQ3, BBQ650, DABCYL, TAMRA, MGBEclipse, Atto540Q, Atto575Q, Atto612Q, QSY7, QSY21, etc.) can be used. Alternatively, as the aforementioned quencher, quenchers described in Korean Published Patent Publications No. 10-2020-0067733 and No. 10-2019-0062162 can be used.

[0108] The conjugates defined in this application can be used in a variety of applications in the chemical and biological fields. They are particularly useful in real-time polymerase chain reactions or microarrays, but are not limited to these applications.

[0109] Furthermore, according to another aspect of the present invention, a nucleic acid detection composition comprising the conjugate is provided.

[0110] A nucleic acid detection composition according to one embodiment of the present invention may further include an enzyme for reaction with a target biomolecule, a solvent (such as a buffer), and other reagents, along with a conjugate containing a compound defined in this application and a quencher simultaneously as a nucleic acid labeling reporter.

[0111] Here, the solvent can be a buffer selected from the group consisting of phosphate buffers, carbonate buffers, and Tris buffers, an organic solvent selected from dimethyl sulfoxide, dimethylformamide, dichloromethane, methanol, ethanol, and acetonitrile, or water, and the solubility can be adjusted by introducing various functional groups to the reporter depending on the type of solvent.

[0112] Furthermore, according to yet another aspect of the present invention, a nucleic acid detection support is provided, comprising a compound (reporter) as defined in this application; a support; and a connecting portion for connecting the probe and the support.

[0113] This allows biomolecules within the sample to be immobilized on the support through interaction with a reporter immobilized on the support.

[0114] The support can be manufactured from at least one selected from glass (e.g., CPG (controlled pore glass)), cellulose, nylon, acrylamide gel, dextran, polystyrene, resin, alginate, collagen, peptide, fibrin, hyaluronic acid, agarose, polyhydroxyethyl methacrylate, polyvinyl alcohol, polyethylene glycol, polyethylene oxide, polyethylene glycol diacrylate, gelatin, Matrigel®, polylactic acid, carboxymethylcellulose, dextran, chitosan, latex, or Sepharose®, but is not necessarily limited to these materials. The support may also be in the form of beads or membranes.

[0115] Here, the connecting portion is the part that connects the reporter and the support, and any material that can connect the reporter and the support can be used as the connecting portion intended in this application.

[0116] For example, the connecting portion C1-C may be replaced. 30Alkyl, possibly substituted C3-C 30 Cycloalkyl, optionally substituted C2-C 30 Heteroalkyl, possibly substituted C2-C 30 Heterocycloalkyl, optionally substituted C2-C 30 Alkenyl, may be substituted C5-C 50 Aryl, possibly substituted C2-C 50 You can choose from heteroaryls, amides (-CONH-), esters (-COO-), ketones (-CO-), nucleosides, and any combination thereof.

[0117] Such a connection merely links the reporter and the support and does not affect any other reactions or fluorescence and quenching effects of the reporter or fluorophore.

[0118] Nucleic acid detection method A further aspect of the present invention provides a nucleic acid detection method comprising: (a) preparing a reaction mixture containing a target nucleic acid, reagents necessary for amplifying the target nucleic acid, and a nucleotide conjugate according to claim 12; (b) amplifying the target nucleic acid in the reaction mixture; and (c) measuring the fluorescence intensity of the reaction mixture.

[0119] Step (b) may include (b-1) a step in which the nucleotide conjugate hybridized to the target nucleic acid is extended by a polymerase; (b-2) a step in which the reporter and quencher of the nucleotide conjugate are separated from the target nucleic acid by the exonuclease activity of the polymerase; and (b-3) a step in which the reporter released from the reporter emits fluorescence.

[0120] Step (b) above involves Strand Displacement Amplification (SDA), Polymerase Chain Reaction (PCR), Reverse Transcription Polymerase Chain Reaction (RT-PCR), Real-time Polymerase Chain Reaction, Allele-specific Polymerase Chain Reaction, Ligase Chain Reaction (LCR), Rolling Circle Amplification (RCA), Isothermal Multiple Displacement Amplification (IMDA), Recombinase Polymerase Amplification (RPA), Self-Sustained Sequence Replication (3SR), Single Primer Isothermal Amplification (SPIA), and Multiple Displacement Amplification (MDA). Amplification, whole-genome amplification (WGA), cross-priming amplification (CPA), signal-mediated amplification of RNA technology (SMART), transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP), and helicase-dependent amplification (HDA)This can be done by a method selected from Amplification.

[0121] The method may further include a step (d) in which the amount of amplification of the target nucleic acid is measured from the fluorescence intensity measured in step (c).

[0122] DNA microarray method DNA microarray technology involves labeling a target nucleic acid with a dye while simultaneously preparing a single-stranded probe nucleic acid having a complementary base sequence to the target nucleic acid. The single-stranded modified target nucleic acid and the probe nucleic acid are then hybridized on a substrate, and the fluorescence of the target nucleic acid is measured.

[0123] In this labeling method, when investigating gene expression, the probe nucleic acid immobilized on the substrate can be a cDNA library, a genome library, or a probe prepared by amplified by PCR using the entire genome as a template.

[0124] Furthermore, when investigating gene mutations, it is possible to use various conjugates synthesized to correspond to mutations, based on already known standard sequences.

[0125] The method for immobilizing probe nucleic acids onto a substrate can be selected depending on the type of nucleic acid and the type of substrate. For example, one method can be used to electrostatically couple the DNA charge to a substrate surface-treated with cations such as polylysine.

[0126] A single-stranded, modified target nucleic acid is immobilized on a substrate and hybridized with a nucleotide conjugate. Here, the 5' end of the nucleotide conjugate is labeled with the compound defined in this application, and the 3' end is labeled with a quencher. A probe capable of binding complementaryly to the target nucleic acid may be positioned between the 5' and 3' ends.

[0127] Hybridization is preferably performed at room temperature to 70°C for 2 to 48 hours. Hybridization selectively binds the target nucleic acid, which has a base sequence complementary to the probe nucleic acid, to the probe nucleic acid. Afterwards, the substrate is washed and dried at room temperature.

[0128] In this process, the nucleotide conjugate is hybridized to the target nucleic acid by the probe, but the compound at the 5' end remains quenched by the quencher at the 3' end.

[0129] Next, the nucleotide conjugate hybridized to the target nucleic acid is extended by a polymerase. The nucleotide conjugate is then separated and degraded from the target nucleic acid by the exonuclease activity of the polymerase, and the compound at the 5' end and the quencher at the 3' end of the nucleotide conjugate separate from each other, thereby allowing the fluorophore to emit fluorescence.

[0130] In this process, the amount of fluorescence amplification of the target nucleic acid can be measured by measuring the intensity of the fluorescence generated.

[0131] PCR method PCR involves labeling the target nucleic acid to be labeled with a reporter probe that is complementary to its base sequence, and then reacting the target nucleic acid with the probe before or after amplification to measure the fluorescence of the target nucleic acid.

[0132] Specifically, the extension reaction of the target nucleic acid is carried out by enzymes (DNA polymerase, RNA polymerase). During this process, the enzymes recognize the double-stranded nucleic acid sequence formed by primers consisting of the target nucleic acid and oligonucleotides, and the extension reaction proceeds from this recognized position, amplifying only the desired gene region.

[0133] When enzymes synthesize, nucleotides (dNTPs, NTPs) are used as raw materials for the synthesis reaction.

[0134] In this process, by mixing a reporter-containing nucleotide with a normal nucleotide (dNTP, NTP) in any proportion, nucleic acids with that proportion of dye introduced can be synthesized.

[0135] Furthermore, it is possible to synthesize nucleic acids into which the reporter has been introduced by introducing nucleotides containing amino groups in any desired proportion using PCR, and then binding a reporter to them.

[0136] When enzymes synthesize substances, nucleotides are used as raw materials for the synthesis reaction. However, if the nucleotides used have the 3' OH group replaced with H, the nucleic acid extension reaction does not proceed any further, and the reaction terminates at that point.

[0137] This nucleotide, ddNTP (dideoxy nucleotide triphosphate), is called a terminator.

[0138] When nucleic acids are synthesized by mixing terminators with regular nucleotides, the terminator is introduced and the reaction terminates with a certain probability, resulting in the synthesis of nucleic acids of various lengths.

[0139] When these are separated by size using gel electrophoresis, the DNA is arranged in order of length. If each type of base in the terminator is labeled with a different reporter, a tendency can be observed where the endpoint of the synthesis reaction (3' end) depends on the base. By reading the fluorescence information from the reporter labeled on the terminator, the base sequence information of the target nucleic acid can be obtained.

[0140] Alternatively, instead of a terminator, a primer pre-labeled with a reporter can be used to hybridize with the target nucleic acid.

[0141] Furthermore, PNA (peptide nucleic acid) can also be used as a probe. PNA is a structure in which the pentose-phosphate skeleton, which is the basic skeletal structure of nucleic acids, is replaced with a polyamide skeleton with glycine units. It has a three-dimensional structure very similar to nucleic acids and binds very specifically and strongly to nucleic acids with complementary base sequences. Through this, it can be used not only in existing DNA analysis methods such as ISH (in-situ hybridization), but also as a reagent for telomere research by applying it to telomere PNA probes.

[0142] Labeling can be performed, for example, by hybridizing double-stranded DNA with PNA labeled with a reporter, which has a base sequence complementary to all or part of the DNA base sequence; heating the mixture to generate single-stranded DNA; slowly cooling the mixture to room temperature to prepare a PNA-DNA complex; and measuring its fluorescence. In the above example, a method was described in which a target nucleic acid is amplified by PCR and the fluorescence of the product is measured. However, with this method, it is necessary to confirm the size of the product by electrophoresis and then measure the fluorescence intensity to investigate the amount of amplified product.

[0143] For this purpose, the amount of product can be measured in real time using a probe designed to generate fluorescence by hybridizing with the PCR product using the energy transfer of a fluorescent dye.

[0144] For example, DNA labeled with donor and acceptor can be used. Specific labeling methods include molecular beaconization, TaqMan-PCR, and cycling probe techniques, which confirm the presence of specific nucleic acid sequences.

[0145] Other Signage Methods Furthermore, the reporter of the present invention can also be used in a method for labeling targets using specific bonding.

[0146] In other words, in labeling a subject containing a target or a subject modified with a modifying substance, one of the binding substances that specifically bind to the subject or the binding substance that specifically binds to the modifying substance can be labeled with a reporter, and the fluorescence from the labeled binding substance can be measured.

[0147] Here, the combination of the subject or modified substance and the binding substance can be an antigen-antibody, hapten-anti-hapten antibody, biotin-avidin, Tag antigen, Tag antibody, lectin-glycoprotein, or hormone-receptor.

[0148] Specifically, by reacting a binding substance, such as an antibody labeled with a reporter, with an antigen present in a substrate, solution, beads, or antibody, a particular antigen can be labeled through antigen-specific interactions of the antibody.

[0149] Possible antigens include proteins, polysaccharides, nucleic acids, and peptides. In addition to antigens, haptens such as FITC and dinitrophenyl groups (low molecular weight molecules) can also be used. Examples of antigen (or hapten)-antibody combinations include GFP and anti-GFP antibodies, and FITC and anti-FITC antibodies.

[0150] Labeled antigens can be used in various analytical methods such as immunohistochemistry, ELISA, Western blotting, or flow cytometry.

[0151] Furthermore, the reporter of the present invention can also be used to observe intracellular signaling phenomena. Various enzymes are involved in intracellular signaling or the resulting cellular responses. In typical signaling phenomena, it is known that a special protein kinase is activated, which induces protein phosphorylation and initiates signaling.

[0152] The binding and hydrolysis of nucleotides (e.g., ATP or ADP) play a crucial role in these activities, and by introducing a reporter into a nucleotide derivative, intracellular signaling phenomena can be observed with high sensitivity.

[0153] Furthermore, the reporter of the present invention can also be used to observe gene expression phenomena using RNA interference (RNAi).

[0154] RNAi involves introducing double-stranded RNA (dsRNA) into cells to degrade the mRNA of a target gene and suppress its expression. By labeling the designed dsRNA with a reporter, it is possible to observe the RNAi phenomenon.

[0155] Furthermore, the reporter of the present invention, by containing a reactive group capable of labeling target nucleic acids or target proteins within tissues or cells, can be used as a dye to confirm the transcription level of a target nucleic acid or the expression level of a target protein. [Examples]

[0156] Specific examples of the present invention are presented below. However, the examples described below are provided solely to illustrate or explain the present invention and should not be considered limiting. Furthermore, among the reporters defined in the claims and detailed description of this application, compounds whose synthesis methods are not disclosed in the following production examples may be synthesized by referring to the following production examples.

[0157] Synthesis Example 1. Synthesis of Compound 1 [ka]

[0158] Synthesis of intermediate 1-1 Intermediate 1-1 was synthesized by referring to US10,982,262B2.

[0159] Synthesis of intermediates 1-2 In a 500 mL four-neck reactor, intermediate 1-1 (10 g, 20.3 mmol), benzophenone imine (9.2 g, 50.7 mmol), cesium carbonate (19.8 g, 60.9 mmol), Pd2(dba)3 (0.37 g, 0.4 mmol), xanthophos (0.7 g, 1.2 mmol), and 1,4-dioxane (100 mL) were added and stirred at 100 °C for 24 hours. After cooling, the mixture was filtered over cellulite. The filtrate was concentrated and purified by column to synthesize intermediate 1-2.

[0160] Synthesis of intermediates 1-3 Intermediates 1-3 were synthesized by referring to Org.Lett.2019,21,5373.

[0161] Synthesis of Compound 1 In a 100 mL three-neck reactor, combine intermediates 1-3 (2.18 g, 8.11 mmol) and tetrahydrofuran (20 mL), and stir under a nitrogen stream at -78°C for 5 minutes. Slowly add 1.6 M n-butyllithium (5 mL, 8.11 mmol) dropwise to the reactor, then stir at -78°C for 1 hour. Dissolve intermediate 1-2 (1.5 g, 2.7 mmol) in tetrahydrofuran (15 mL) and add dropwise to the reactor, then stir at room temperature for 1 hour. Add 6 M hydrochloric acid (15 mL) to the reactor and stir at 80°C for 16 hours. After concentration, synthesize compound 1 by column purification (0.3 g). The obtained compound 1 1 The H-NMR results are as follows: 1 H-NMR (400MHz, MeOD) δ8.11(s, 1H), 8.11-8.09(d, 1H), 7.41-7.39(d, 1H), 7.10-7.06(m, 2H), 6.89-6.86(m, 4H), 2.14(s, 3H).

[0162] Synthesis Example 2. Synthesis of Compound 2 [ka]

[0163] Synthesis of intermediate 2-1 Intermediate 2-1 was synthesized by referring to Chemistry - An Asian Journal, 2014, vol.9, #12, pp.3586-3592.

[0164] Synthesis of intermediate 2-2 Intermediate 2-1 (2.67 g, 13.4 mmol), 4-formyl-3-methylbenzoic acid (1 g, 6.09 mmol), p-toluenesulfonic acid (0.21 g, 1.22 mmol), and toluene (30 mL) were placed in a 100 mL single-port reactor and stirred at 80°C for 16 hours. After cooling, the mixture was concentrated and then purified by column to synthesize intermediate 2-2.

[0165] Synthesis of Compound 2 In a 50 mL single-port reactor, combine intermediate 2-2 (1 g, 1.84 mmol) and concentrated hydrobromic acid (10 mL), and stir under reflux for 16 hours. After cooling, concentrate the mixture, then add chloranil (0.64 g, 2.62 mmol) and 1,4-dioxane (20 mL), and stir at 60°C for 4 hours. After cooling, filter the mixture through cellulite. Concentrate the filtrate and purify it by column to synthesize compound 2 (0.3 g). The obtained compound 2 1 The H-NMR results are as follows: 1 H-NMR (400MHz, MeOD) δ7.95(s, 1H), 7.90-7.88(d, 1H), 7.16-7.14(d, 1H), 6.98-6.96(d, 2H), 6.86-6.83(m, 2H), 2.05(s, 3H).

[0166] Synthesis Example 3. Synthesis of Compound 3 [ka]

[0167] Synthesis of intermediate 3-1 Intermediate 3-1 was synthesized by referring to EP3636637, 2020, A1.

[0168] Synthesis of Compound 3 In a 50 mL three-neck reactor, combine intermediate 3-1 (0.78 g, 2.7 mmol) and tetrahydrofuran (10 mL), and stir under a nitrogen stream at -78°C for 5 minutes. Slowly add 1.6 M n-butyllithium (1.6 mL, 2.7 mmol) dropwise to the reactor, then stir at -78°C for 1 hour. Dissolve intermediate 1-2 (0.5 g, 0.5 mmol) in tetrahydrofuran (5 mL) and add dropwise to the reactor, then stir at room temperature for 1 hour. After concentration, add trifluoroacetic acid (1.2 mL) and dichloromethane (10 mL) to the reactor, and stir at room temperature for 16 hours. After concentration, synthesize compound 3 by column purification (0.05 g). The obtained compound 3 1 The H-NMR results are as follows: 1 H-NMR (400MHz, MeOD) δ7.78(s, 4H, NH), 7.77-7.75(m, 2H), 7.44-7.40(m, 1H), 7.15-7.12(m, 2H), 6.88-6.79(m, 2H), 6.78(s, 2H), 3.74(s, 3H).

[0169] Synthesis Example 4. Synthesis of Compound 4 [ka]

[0170] Synthesis of intermediate 4-1 Intermediate 4-1 was synthesized by referring to US2010 / 0249094 A1.

[0171] Synthesis of intermediate 4-2 In a 100 mL three-neck reactor, combine intermediate 4-1 (1.24 g, 5.05 mmol) and tetrahydrofuran (15 mL), and stir under a nitrogen stream at -78°C for 5 minutes. Slowly add 2.5 M n-butyllithium (4 mL, 10.1 mmol) dropwise to the reactor, then stir at -78°C for 15 minutes. Dissolve intermediate 1-2 (2.0 g, 3.61 mmol) in tetrahydrofuran (20 mL) and add dropwise to the reactor, then stir at room temperature for 2 hours. After concentration, add trifluoroacetic acid (1.27 mL) and dichloromethane (8 mL) to the reactor and stir at room temperature for 16 hours. After concentration, column purification was performed to synthesize intermediate 4-2.

[0172] Synthesis of Compound 4 In a 50 mL single-port reactor, combine intermediate 4-2 (0.18 g, 0.48 mmol), TSTU (N,N,N',N'-Tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate) (0.22 g, 0.72 mmol), triethylamine (0.26 mL, 1.92 mmol), and dimethylformamide (3 mL). Stir at room temperature for 30 minutes. After concentration, synthesize compound 4 by column purification (0.1 g). The obtained compound 4 1 The H-NMR results are as follows: 1 H-NMR (400MHz, DMSO-d6) δ7.75(s, 1H), 7.52(s, 1H), 7.35-7.32(d, 2H), 7. 14-7.12(m, 2H), 7.04-7.02(d, 2H), 4.12(s, 3H), 2.9(s, 4H), 2.14(s, 3H).

[0173] Synthesis Example 5. Synthesis of Compound 5 [ka]

[0174] Synthesis of intermediate 5-1 Intermediate 5-1 was synthesized by referring to WO2018 / 042437A1.

[0175] Synthesis of intermediate 5-2 In a 100 mL single-port reactor, intermediate 5-1 (1.16 g, 6.09 mmol), 4-formyl-3-methylbenzoic acid (0.5 g, 3.05 mmol), and 60% sulfuric acid aqueous solution (10 mL) were added and stirred at 130 °C for 3 hours. After cooling, the mixture was concentrated and then purified by column. After cooling, the reaction mixture was poured into cold water, and the resulting solid was filtered to synthesize intermediate 5-2.

[0176] Synthesis of Compound 5 In a 100 mL single-port reactor, combine intermediate 5-2 (1.36 g, 2.67 mmol), HSTU (N,N,N',N'-Tetramethyl-O-(N-succinimidyl)uronium hexafluorophosphate) (1.44 g, 4.01 mmol), triethylamine (1.1 mL, 8.02 mmol), and dimethylformamide (15 mL). Stir at room temperature for 30 minutes. After concentration, synthesize compound 5 by column purification (0.4 g). The obtained compound 5 1 The H-NMR results are as follows: 1 H-NMR (400MHz, DMSO-d6) δ8.26(s, 1H), 8.19-8.17(d, 1H), 7.61-59(d, 1H), 7.34-7.13(m, 6H), 4.55-4.45(m, 4H), 2.94(s, 4H), 2.13(s, 3H)

[0177] Synthesis Example 6. Synthesis of Compound 6 [ka]

[0178] Synthesis of intermediate 6-1 Intermediate 6-1 was synthesized by referring to WO2018 / 042437A1.

[0179] Synthesis of intermediate 6-2 In a 100 mL single-port reactor, intermediate 6-1 (1.27 g, 6.07 mmol), 4-formyl-3-methylbenzoic acid (0.5 g, 3.05 mmol), and 60% sulfuric acid aqueous solution (10 mL) were added and stirred at 130 °C for 3 hours. After cooling, the mixture was concentrated and then purified by column. After cooling, the reaction mixture was poured into cold water, and the resulting solid was filtered to synthesize intermediate 6-2.

[0180] Synthesis of Compound 6 In a 100 mL single-port reactor, combine intermediate 6-2 (0.15 g, 0.28 mmol), HSTU (N,N,N',N'-Tetramethyl-O-(N-succinimidyl)uronium hexafluorophosphate) (0.3 g, 0.84 mmol), triethylamine (0.12 mL, 0.84 mmol), and dimethylformamide (6 mL). Stir at room temperature for 30 minutes. After concentration, column purification was performed to synthesize compound 6 (0.07 g). The obtained compound 6 1 The H-NMR results are as follows: 1 H-NMR (400MHz, DMSO-d6) δ8.24(s, 1H), 8.19-8.17(d, 1H), 7.64-7.63(d, 2H), 7. 56-7.54(d, 1H), 7.21-7.19(d, 2H), 4.53-4.50(m, 4H), 2.95(s, 4H), 2.15(s, 3H)

[0181] Synthesis Example 7. Synthesis of Compound 7 [ka]

[0182] Synthesis of intermediate 7-1 Intermediate 7-1 was synthesized by referring to Organic and Biomolecular Chemistry, 2017, vol.15, #19, pp. 4212-4217 and WO2018 / 042437A1.

[0183] Synthesis of intermediate 7-2 In a 100 mL single-port reactor, intermediate 7-1 (1.28 g, 6.07 mmol), 4-formyl-3-methylbenzoic acid (0.5 g, 3.05 mmol), and 60% sulfuric acid aqueous solution (10 mL) were added and stirred at 130 °C for 3 hours. After cooling, the mixture was concentrated and then purified by column. After cooling, the reaction mixture was poured into cold water, and the resulting solid was filtered to synthesize intermediate 7-2.

[0184] Synthesis of Compound 7 In a 100 mL single-port reactor, combine intermediate 7-2 (0.5 g, 0.86 mmol), HSTU (N,N,N',N'-Tetramethyl-O-(N-succinimidyl)uronium hexafluorophosphate) (0.62 g, 1.74 mmol), triethylamine (0.5 mL, 3.45 mmol), and dimethylformamide (8 mL). Stir at room temperature for 30 minutes. After concentration, synthesize compound 7 by column purification (0.07 g). The obtained compound 7 1 The H-NMR results are as follows: 1 H-NMR (400MHz, DMSO-d6) δ8.17(s, 1H), 8.11-8.09(d, 1H), 7.51-7.49(d, 1H), 6.39-6.36(d, 2H), 4.21-4.13(m, 4H), 2.93(s, 4H), 2.16(s, 3H).

[0185] Synthesis Example 8. Synthesis of Compound 8 [ka]

[0186] Synthesis of intermediate 8-1 Intermediate 8-1 was synthesized by referring to WO2022 / 226100,2022,A1.

[0187] Synthesis of intermediate 8-2 In a 100 mL single-port reactor, intermediate 6-1 (0.96 g, 4.58 mmol), intermediate 8-1 (0.5 g, 2.29 mmol), and 10 mL of 60% sulfuric acid aqueous solution were added, and the mixture was stirred at 130°C for 3 hours. After cooling, the mixture was concentrated and then purified by column. After cooling, the reaction mixture was poured into cold water, and the resulting solid was filtered to synthesize intermediate 8-2.

[0188] Synthesis of compound 8 In a 100 mL single-port reactor, combine intermediate 8-2 (0.6 g, 1.0 mmol), HSTU (N,N,N',N'-Tetramethyl-O-(N-succinimidyl)uronium hexafluorophosphate) (0.9 g, 2.51 mmol), triethylamine (0.5 mL, 4.0 mmol), and dimethylformamide (10 mL). Stir at room temperature for 30 minutes. After concentration, synthesize compound 8 by column purification (0.14 g). The obtained compound 8 1 The H-NMR results are as follows: 1 H-NMR (400MHz, DMSO-d6) δ8.51(s, 1H), 8.42-8.40(d, 1H), 7.59-7.57(d, 1H), 6.89-6.87(d, 2H), 6.68-6.65(d, 2H) 4.15-4.05(m, 4H), 2.94(s, 4H).

[0189] Synthesis Example 9. Synthesis of Compound 9 [ka]

[0190] Synthesis of intermediate 9-1 Intermediate 9-1 was synthesized by referring to WO2016 / 44641, 2016, A2.

[0191] Synthesis of intermediate 9-2 In a 1L four-port reactor, intermediate 9-1 (42g, 165 mmol), tert-butylcarbamate (21.3g, 182 mmol), cesium carbonate (108g, 331 mmol), Pd(OAc)2 (3.7g, 16.5 mmol), xanthophos (9.6g, 16.5 mmol), and 1,4-dioxane (420 mL) were added and stirred at 100°C for 24 hours. After cooling, the mixture was filtered over cellulite. The filtrate was concentrated and purified by column to synthesize intermediate 9-2.

[0192] Synthesis of intermediate 9-3 In a 250 mL single-port reactor, combine intermediate 9-2 (7 g, 28 mmol), trifluoroacetic acid (14 mL), and dichloromethane (56 mL), and stir at room temperature for 2 hours. After concentration, add dichloromethane (100 mL) and 5% sodium bicarbonate aqueous solution (100 mL), and stir vigorously. After separating the organic layer, add anhydrous sodium sulfate, stir for 5 minutes, and then filter. The filtrate is concentrated to synthesize intermediate 9-3.

[0193] Synthesis of intermediate 9-4 In a 500 mL single-port reactor, intermediate 9-3 (6.6 g, 23 mmol), boron tripromide (2.60 mL, 0.028 mmol), and dichloromethane (66 mL) were added and stirred at room temperature for 6 hours. 5% sodium bicarbonate aqueous solution (200 mL) was added and the mixture was vigorously stirred. After separating the organic layer, anhydrous sodium sulfate was added and the mixture was stirred for 5 minutes, then filtered. The filtrate was concentrated and purified by column to synthesize intermediate 9-4.

[0194] Synthesis of intermediates 9-5 Intermediate 9-5 was synthesized by referring to WO2018 / 042437A1.

[0195] Synthesis of compound 9 In a 100 mL single-port reactor, combine intermediate 9-5 (2.1 g, 8 mmol), 4-formyl-3-methylbenzoic acid (0.66 g, 4 mmol), and 60% sulfuric acid aqueous solution (5 mL). Stir at 130°C for 3 hours. After cooling, pour the reaction mixture into cold water for neutralization. Filter the resulting solid. Compound 9 was synthesized by vacuum drying at 60°C (1.5 g). The obtained compound 9 1 The H-NMR results are as follows: 1 H-NMR (400MHz, DMSO-d6) δ8.22(s, 1H), 8.16-8.11(d, 1H), 7.51-47(d, 1H), 7.44-7.23(m, 4H), 4.55-4.45(m, 4H), 2.13(s, 3H)

[0196] Synthesis Example 10. Synthesis of Compound 10 [ka]

[0197] Synthesis of intermediate 10-1 3-ethylamino-p-cresol (27.0 g, 0.178 mol), 4-formyl-3-methylbenzoic acid (14.7 g, 0.089 mol), and 70% sulfuric acid (110 mL) were added to a 250 mL single-port reactor and stirred at 130 °C for 3 hours. After cooling, the reaction mixture was poured into cold water and neutralized. The resulting solid was filtered. After drying, MeOH (1 L) and chloranil (11 g, 0.045 mol) were added and stirred under reflux for 1 hour. After cooling, the mixture was concentrated. After filtering the resulting solid, it was vacuum-dried at 60 °C to synthesize intermediate 10-1.

[0198] Synthesis of intermediate 10-2 In a 1L single-port reactor, intermediate 10-1 (23g, 0.054mol), HSTU (N,N,N',N'-Tetramethyl-O-(N-succinimidyl)uronium hexafluorophosphate) (23.1g, 0.06mol), triethylamine (22mL, 0.161mol), and dimethylformamide (230mL) were added and stirred at room temperature for 1 hour. After concentration, water was added to the reactor, and after vigorous stirring, the mixture was extracted with dichloromethane. Anhydrous sodium sulfate was added, the mixture was stirred for 5 minutes, and then filtered. The filtrate was concentrated and purified by column to synthesize intermediate 10-2.

[0199] Synthesis of intermediate 10-3 In a 500 mL single-port reactor, intermediate 10-2 (18.5 g, 0.0275 mol), 6-aminohexanoic acid (5.68 g, 0.055 mol), triethylamine (11.5 mL, 0.083 mol), and dimethylformamide (185 mL) were added and stirred at 40°C for 1 hour. After concentration, water was added to the reactor, and after vigorous stirring, the mixture was extracted with dichloromethane. Anhydrous sodium sulfate was added, the mixture was stirred for 5 minutes, and then filtered. The filtrate was concentrated and purified by column to synthesize intermediate 10-3.

[0200] Synthesis of compound 10 In a 250 mL single-port reactor, combine intermediate 10-3 (1 g, 1.5 mmol), HSTU (N,N,N',N'-Tetramethyl-O-(N-succinimidyl)uronium hexafluorophosphate) (0.64 g, 1.8 mmol), triethylamine (0.62 mL, 4 mmol), and dichloromethane (100 mL). Stir at room temperature for 1 hour. Add water to the reactor, stir vigorously, and then extract with dichloromethane. Add anhydrous sodium sulfate, stir for 5 minutes, and then filter. Concentrate the filtrate and purify by column to synthesize compound 10 (0.8 g). The obtained compound 10 1 The H-NMR results are as follows: 1H-NMR (400MHz, CDCl3) δ8.74(m, 1H), 8.01(s, 1H) 7.94(m, 1H), 7.78(t, 2H, J=6.0Hz), 7.37(d, 1H, J=7.6Hz), 6.95 (s, 2H), 6.84(s, 2H), 3.52-3.41(m, 6H), 2.8(m, 6H), 2.12(s, 6H), 2.05(s, 3H), 1.94(m, 6H), 1.27(t, 6H, J=6.8Hz)

[0201] Synthesis Example 11. Synthesis of Compound 11 [ka]

[0202] Synthesis of intermediate 11-1 3-ethylamino-p-cresol (25.0 g, 0.165 mol), 4-formyl-3,5-methylbenzoic acid (14.7 g, 0.083 mol), and 70% sulfuric acid (110 mL) were added to a 250 mL single-port reactor and stirred at 130 °C for 3 hours. After cooling, the reaction mixture was poured into cold water and neutralized. The resulting solid was filtered. After drying, MeOH (1 L) and chloranil (11 g, 0.045 mol) were added and stirred under reflux for 1 hour. After cooling, the mixture was concentrated. After filtering the resulting solid, it was vacuum-dried at 60 °C to synthesize intermediate 11-1.

[0203] Synthesis of intermediate 11-2 In a 1L single-port reactor, intermediate 11-1 (20g, 0.045mol), HSTU (N,N,N',N'-Tetramethyl-O-(N-succinimidyl)uronium hexafluorophosphate) (19.5g, 0.054mol), triethylamine (19mL, 0.136mol), and dimethylformamide (200mL) were added and stirred at room temperature for 1 hour. After concentration, water was added to the reactor, and after vigorous stirring, the mixture was extracted with dichloromethane. Anhydrous sodium sulfate was added, the mixture was stirred for 5 minutes, and then filtered. The filtrate was concentrated and purified by column to synthesize intermediate 11-2.

[0204] Synthesis of intermediate 11-3 In a 500 mL single-port reactor, intermediate 11-2 (17.0 g, 0.0275 mol), 4-aminohexanoic acid (5.11 g, 0.050 mol), triethylamine (10.4 mL, 0.074 mol), and dimethylformamide (170 mL) were added and stirred at 40°C for 1 hour. After concentration, water was added to the reactor, and after vigorous stirring, the mixture was extracted with dichloromethane. Anhydrous sodium sulfate was added, the mixture was stirred for 5 minutes, and then filtered. The filtrate was concentrated and purified by column to synthesize intermediate 11-3.

[0205] Synthesis of compound 11 In a 250 mL single-port reactor, combine intermediate 11-3 (9.0 g, 0.013 mol), HSTU (N,N,N',N'-Tetramethyl-O-(N-succinimidyl)uronium hexafluorophosphate) (7.2 g, 0.02 mol), triethylamine (5.5 mL, 0.04 mol), and dichloromethane (90 mL), and stir at room temperature for 1 hour. Add water to the reactor, stir vigorously, and then extract with dichloromethane. Add anhydrous sodium sulfate, stir for 5 minutes, and then filter. Concentrate the filtrate and purify by column to synthesize compound 11 (8.2 g). The obtained compound 11 1 The H-NMR results are as follows: 1 H-NMR (400MHz, DMSO-d6) δ8.77(m, 1H), 7.98(d, 2H, J=7.8Hz), 7.78(t, 2H, J=6.0Hz), 6.97(s, 2H), 6.8 2(s, 2H), 3.49-3.40(m, 6H), 2.77(m, 6H), 2.11(s, 6H), 2.06(s, 6H), 1.94(m, 2H), 1.28(t, 6H, J=6.8Hz)

[0206] Synthesis Example 12. Synthesis of Compound 12 [ka]

[0207] Synthesis of intermediate 12-1 3-ethylamino-p-cresol (25.0 g, 0.165 mol), 4-formyl-3-methoxymethylbenzoic acid (14.7 g, 0.083 mol), and 70% sulfuric acid (110 mL) were added to a 250 mL single-port reactor and stirred at 130 °C for 3 hours. After cooling, the reaction mixture was poured into cold water for neutralization. The resulting solid was filtered. After drying, MeOH (1 L) and chloranil (11.0 g, 0.045 mol) were added and stirred under reflux for 1 hour. After cooling, the mixture was concentrated. After filtering the resulting solid, it was vacuum-dried at 60 °C to synthesize intermediate 12-1.

[0208] Synthesis of intermediate 12-2 In a 1L single-port reactor, intermediate 12-1 (10.0g, 0.023mol), HSTU (N,N,N',N'-Tetramethyl-O-(N-succinimidyl)uronium hexafluorophosphate) (9.7g, 0.027mol), triethylamine (9.4mL, 0.067mol), and dimethylformamide (100mL) were added and stirred at room temperature for 1 hour. After concentration, water was added to the reactor, and after vigorous stirring, the mixture was extracted with dichloromethane. Anhydrous sodium sulfate was added, the mixture was stirred for 5 minutes, and then filtered. The filtrate was concentrated and purified by column to synthesize intermediate 12-2.

[0209] Synthesis of intermediate 12-3 In a 500 mL single-port reactor, intermediate 12-2 (10.0 g, 0.015 mol), 4-aminobutyric acid (3.07 g, 0.030 mol), triethylamine (6.22 mL, 0.045 mol), and dimethylformamide (100 mL) were added and stirred at 40°C for 1 hour. After concentration, water was added to the reactor, and after vigorous stirring, the mixture was extracted with dichloromethane. Anhydrous sodium sulfate was added, the mixture was stirred for 5 minutes, and then filtered. The filtrate was concentrated and purified by column to synthesize intermediate 12-3.

[0210] Synthesis of compound 12 In a 250 mL single-port reactor, combine intermediate 12-3 (5.0 g, 0.0074 mol), HSTU (N,N,N',N'-Tetramethyl-O-(N-succinimidyl)uronium hexafluorophosphate) (4.0 g, 0.011 mol), triethylamine (3.1 mL, 0.02 mol), and dichloromethane (50 mL). Stir at room temperature for 1 hour. Add water to the reactor, stir vigorously, and then extract with dichloromethane. Add anhydrous sodium sulfate, stir for 5 minutes, and then filter. Concentrate the filtrate and purify by column to synthesize compound 12 (4.0 g). The obtained compound 12 1 The H-NMR results are as follows: 1 H-NMR (400MHz, DMSO-d6) δ8.72 (m, 1H), 8.03 (s, 1H) 7.91 (m, 1H), 7.76 (t, 2H, J=6.0Hz), 7.34 (d, 1H, J=7.6Hz), 6.99 (s, 2H), 6.82(s, 2H), 3.89(s, 3H), 3.55-3.40(m, 6H), 2.77(m, 6H), 2.12(s, 6H), 1.94(m, 2H), 1.29(t, 6H, J=6.8Hz)

[0211] Synthesis Example 13. Synthesis of Compound 13 [ka]

[0212] Synthesis of intermediate 13-1 In a 250 mL single-port reactor, combine 3-ethylamino-p-cresol (25.0 g, 0.165 mol), 3-chloro-4-formylmethylbenzoic acid (15.3 g, 0.083 mol), and 70% sulfuric acid (110 mL), and stir at 130 °C for 3 hours. After cooling, pour the reaction mixture into cold water for neutralization. Filter the resulting solid. After drying, add MeOH (1 L) and chloranil (11.0 g, 0.045 mol), and stir under reflux for 1 hour. After cooling, concentrate. Filter the resulting solid and vacuum dry at 60 °C to synthesize intermediate 13-1.

[0213] Synthesis of intermediate 13-2 In a 1L single-port reactor, intermediate 13-1 (10.0g, 0.023mol), HSTU (N,N,N',N'-Tetramethyl-O-(N-succinimidyl)uronium hexafluorophosphate) (9.7g, 0.027mol), triethylamine (9.4mL, 0.067mol), and dimethylformamide (100mL) were added and stirred at room temperature for 1 hour. After concentration, water was added to the reactor, and after vigorous stirring, the mixture was extracted with dichloromethane. Anhydrous sodium sulfate was added, the mixture was stirred for 5 minutes, and then filtered. The filtrate was concentrated and purified by column to synthesize intermediate 13-2.

[0214] Synthesis of intermediate 13-3 In a 500 mL single-port reactor, intermediate 13-2 (10.0 g, 0.015 mol), 4-aminobutyric acid (3.07 g, 0.030 mol), triethylamine (6.22 mL, 0.045 mol), and dimethylformamide (100 mL) were added and stirred at 40°C for 1 hour. After concentration, water was added to the reactor, and after vigorous stirring, the mixture was extracted with dichloromethane. Anhydrous sodium sulfate was added, the mixture was stirred for 5 minutes, and then filtered. The filtrate was concentrated and purified by column to synthesize intermediate 13-3.

[0215] Synthesis of compound 13 In a 250 mL single-port reactor, combine intermediate 13-3 (5.0 g, 0.0074 mol), HSTU (N,N,N',N'-Tetramethyl-O-(N-succinimidyl)uronium hexafluorophosphate) (4.0 g, 0.011 mol), triethylamine (3.1 mL, 0.02 mol), and dichloromethane (50 mL). Stir at room temperature for 1 hour. Add water to the reactor, stir vigorously, and then extract with dichloromethane. Add anhydrous sodium sulfate, stir for 5 minutes, and then filter. Concentrate the filtrate and purify by column to synthesize compound 13 (4.2 g). The obtained compound 13 1 The H-NMR results are as follows: 1H-NMR (400MHz, DMSO-d6) δ8.73(m, 1H), 8.00(s, 1H) 7.87(m, 1H), 7.76(t, 2H, J=6.0Hz), 7.37(d, 1H, J=7.6Hz ), 6.90(s, 2H), 6.83(s, 2H), 3.50-3.42(m, 6H), 2.79(m, 6H), 2.11(s, 6H), 1.97(m, 2H), 1.27(t, 6H, J=6.8Hz)

[0216] Synthesis Example 14. Synthesis of Compound 14 [ka]

[0217] Synthesis of intermediate 14-1 3-ethylamino-p-cresol (5.0 g, 0.033 mol), 4-formyl-3-trifluorobenzoic acid (3.6 g, 0.016 mol), and 70% sulfuric acid (25 mL) were added to a 250 mL single-port reactor and stirred at 130 °C for 3 hours. After cooling, the reaction mixture was poured into cold water for neutralization. The resulting solid was filtered. After drying, MeOH (1 L) and chloranil (2.0 g, 0.008 mol) were added and stirred under reflux for 1 hour. After cooling, the mixture was concentrated. After filtering the resulting solid, it was vacuum-dried at 60 °C to synthesize intermediate 14-1.

[0218] Synthesis of intermediate 14-2 In a 1L single-port reactor, intermediate 14-1 (3.5g, 7.3 mmol), HSTU (N,N,N',N'-Tetramethyl-O-(N-succinimidyl)uronium hexafluorophosphate) (3.13g, 8.7 mmol), triethylamine (3.0 mL, 2.2 mmol), and dimethylformamide (35 mL) were added and stirred at room temperature for 1 hour. After concentration, water was added to the reactor, and after vigorous stirring, the mixture was extracted with dichloromethane. Anhydrous sodium sulfate was added, the mixture was stirred for 5 minutes, and then filtered. The filtrate was concentrated and purified by column to synthesize intermediate 14-2.

[0219] Synthesis of intermediate 14-3 In a 500 mL single-port reactor, intermediate 14-2 (4.0 g, 5.5 mmol), 6-aminohexanoic acid (1.14 g, 11.0 mmol), triethylamine (2.3 mL, 17.0 mmol), and dimethylformamide (40 mL) were added and stirred at 40°C for 1 hour. After concentration, water was added to the reactor, and after vigorous stirring, the mixture was extracted with dichloromethane. Anhydrous sodium sulfate was added, the mixture was stirred for 5 minutes, and then filtered. The filtrate was concentrated and purified by column to synthesize intermediate 14-3.

[0220] Synthesis of compound 14 In a 250 mL single-port reactor, combine intermediate 14-3 (3.0 g, 4.2 mmol), HSTU (N,N,N',N'-Tetramethyl-O-(N-succinimidyl)uronium hexafluorophosphate) (2.27 g, 6.3 mmol), triethylamine (1.8 mL, 12.6 mmol), and dichloromethane (30 mL). Stir at room temperature for 1 hour. Add water to the reactor, stir vigorously, and then extract with dichloromethane. Add anhydrous sodium sulfate, stir for 5 minutes, and then filter. Concentrate the filtrate and purify by column to synthesize compound 14 (2.2 g). The obtained compound 14 1 The H-NMR results are as follows: 1 H-NMR (400MHz, DMSO-d6) δ8.75(bs, 1H), 7.95(s, 1H) 7.86(m, 1H), 7.76(t, 2H, J=6.0Hz), 7.35(d, 1H, J=7.6Hz) ), 6.91(s, 2H), 6.82(s, 2H), 3.50-3.42(m, 6H), 2.79(m, 6H), 2.11(s, 6H), 1.97(m, 6H), 1.27(t, 6H, J=6.8Hz)

[0221] Synthesis Example 15. Synthesis of Compound 15 [ka]

[0222] Synthesis of intermediate 15-1 Intermediate 15-1 was synthesized by referring to WO2019 / 129590, 2019, A1.

[0223] Synthesis of intermediate 15-2 In a 50 mL single-port reactor, intermediate 15-1 (2.41 g, 0.016 mol), 3-ethylamino-p-cresol (2.5 g, 0.016 mol), o-tolualdehyde (1.92 g, 0.016 mol), and 70% sulfuric acid (15 mL) were added and stirred at 130 °C for 3 hours. After cooling, the reaction mixture was poured into cold water and neutralized. After filtering the resulting solid, intermediate 15-2 was synthesized by vacuum drying at 60 °C.

[0224] Synthesis of Compound 15 In a 25 mL single-port reactor, combine intermediate 15-2 (1.0 g, 1.8 mmol), 2-cyanoethyl N,N'-diisopropyl chlorophosphoramidate (0.56 g, 2.4 mmol), triethylamine (0.76 mL, 5.5 mmol), and dichloromethane (10 mL), and stir at room temperature for 1 hour. Add water to the reactor, stir vigorously, and then extract with dichloromethane. Add anhydrous sodium sulfate, stir for 5 minutes, and then filter. Concentrate the filtrate and purify by column to synthesize compound 15 (1.4 g). The obtained compound 15 1 The H-NMR results are as follows: 1 H-NMR (400MHz, DMSO-d6) δ7.76(bs, 2H), 7.35-7.10(m, 4H), 6.90(s, 2H), 6.77(s, 2H), 3.50-3.42( m, 10H), 2.69(t, 2H, J=5.6Hz), 2.15(s, 6H), 2.06(s, 3H), 1.48(t, 3H, J=6.8Hz), 1.20-1.18(m, 12H)

[0225] Synthesis Example 16. Synthesis of Compound 16 [ka]

[0226] Synthesis of intermediate 16-1 Intermediate 16-1 was synthesized by referring to WO2018 / 35128, 2018, A1.

[0227] Synthesis of intermediate 16-2 In a 1L single-port reactor, intermediate 16-1 (50.0g, 0.211mol), methyl 4-butyrate (45.5g, 0.253mol), potassium carbonate (58.3g, 0.422mol), and dimethylformamide (500mL) were added and stirred at 60°C for 24 hours. After cooling, the mixture was concentrated. Dichloromethane (500mL) and water (200mL) were added and stirred for 5 minutes, after which the organic layer was separated. 6N hydrochloric acid aqueous solution (100mL) was added and stirred for 5 minutes, after which the organic layer was separated. Anhydrous sodium sulfate was added and stirred for 5 minutes, after which the mixture was filtered. The filtrate was concentrated and purified by column to synthesize intermediate 16-2.

[0228] Synthesis of intermediate 16-3 In a 50 mL single-port reactor, intermediate 16-2 (3.57 g, 0.016 mol), 3-ethylamino-p-cresol (2.5 g, 0.016 mol), o-tolualdehyde (1.92 g, 0.016 mol), and 70% sulfuric acid (15 mL) were added and stirred at 130 °C for 3 hours. After cooling, the reaction mixture was poured into cold water and neutralized. After filtering the resulting solid, intermediate 16-3 was synthesized by vacuum drying at 60 °C.

[0229] Synthesis of intermediate 16-4 In a 100 mL single-port reactor, combine intermediate 16-3 (3.19 g, 0.0070 mol), HSTU (N,N,N',N'-Tetramethyl-O-(N-succinimidyl)uronium hexafluorophosphate) (3.0 g, 0.0084 mol), triethylamine (3.0 mL, 0.021 mol), and dimethylformamide (32 mL), and stir at room temperature for 1 hour. Add 2-(methylamino)ethanol (1.0 g, 0.014 mol) and stir at room temperature for 2 hours. After concentration, add water to the reactor, stir vigorously, and extract with dichloromethane. Add anhydrous sodium sulfate, stir for 5 minutes, and then filter. The filtrate is concentrated and purified by column to synthesize intermediate 16-4.

[0230] Synthesis of compound 16 In a 25 mL single-port reactor, combine intermediate 16-4 (1.0 g, 1.5 mmol), 2-cyanoethyl N,N'-diisopropyl chlorophosphoramidate (0.47 g, 2.0 mmol), triethylamine (0.86 mL, 4.5 mmol), and dichloromethane (10 mL). Stir at room temperature for 1 hour. Add water to the reactor, stir vigorously, and then extract with dichloromethane. Add anhydrous sodium sulfate, stir for 5 minutes, and then filter. Concentrate the filtrate and purify by column to synthesize compound 16 (0.7 g). The obtained compound 16 1 The H-NMR results are as follows: 1 H-NMR (400MHz, DMSO-d6) δ7.86(bs, 2H), 7.42(d, 2H, J=7.8Hz), 7.32(t, 1H, J=7.8Hz), 6.90(s, 2H), 6.77(s, 2H), 3.50 -3.42(m, 15H), 2.69(t, 2H, J=5.6Hz), 2.48(t, 2H, J=6.0Hz) 2.11(s, 6H), 2.07(s, 6H), 1.48(m, 5H), 1.20-1.18(m, 12H)

[0231] Synthesis Example 17. Synthesis of Compound 17 [ka]

[0232] Synthesis of intermediate 17-1 In a 100 mL single-port reactor, intermediate 12-1 (3.19 g, 0.0070 mol), HSTU (N,N,N',N'-Tetramethyl-O-(N-succinimidyl)uronium hexafluorophosphate) (3.0 g, 0.0084 mol), triethylamine (3.0 mL, 0.021 mol), and dimethylformamide (32 mL) were added and stirred at room temperature for 1 hour. 6-amino-1-hexanol (1.0 g, 0.014 mol) was added and stirred at room temperature for 2 hours. After concentration, water was added to the reactor, and after vigorous stirring, the mixture was extracted with dichloromethane. Anhydrous sodium sulfate was added and stirred for 5 minutes, then filtered. The filtrate was concentrated and purified by column to synthesize intermediate 17-1.

[0233] Synthesis of Compound 17 In a 25 mL single-port reactor, combine intermediate 17-1 (1.0 g, 1.5 mmol), 2-cyanoethyl N,N'-diisopropyl chlorophosphoramidate (0.48 g, 2.0 mmol), triethylamine (0.65 mL, 4.5 mmol), and dichloromethane (10 mL). Stir at room temperature for 1 hour. Add water to the reactor, stir vigorously, and then extract with dichloromethane. Add anhydrous sodium sulfate, stir for 5 minutes, and then filter. Concentrate the filtrate and purify by column to synthesize compound 17 (0.8 g). The obtained compound 17 1 The H-NMR results are as follows: 1 H-NMR (400MHz, DMSO-d6) δ8.72(bs, 1H), 8.03(s, 1H) 7.91(m, 1H), 7.76(t, 2H, J=6.0Hz), 7.34(d, 1H, J=7.6Hz), 6.99(s, 2H) , 6.82(s, 2H), 3.89(s, 3H), 3.55-3.40(m, 12H), 2.77(t, 2H, J=5.8Hz), 2.12(s, 6H), 1.28-1.22(m, 14H), 1.20-1.18(m, 12H)

[0234] Synthesis Example 18. Synthesis of Compound 18 [ka]

[0235] Synthesis of intermediate 18-1 In a 100 mL single-port reactor, combine intermediate 13-1 (2.0 g, 4.5 mmol), HSTU (N,N,N',N'-Tetramethyl-O-(N-succinimidyl)uronium hexafluorophosphate) (2.4 g, 6.7 mmol), triethylamine (1.2 mL, 8.9 mmol), and dimethylformamide (32 mL), and stir at room temperature for 1 hour. Add 2-(2-aminoethoxy)ethanol (0.5 g, 4.5 mmol) and stir at room temperature for 2 hours. After concentration, add water to the reactor, stir vigorously, and extract with dichloromethane. Add anhydrous sodium sulfate, stir for 5 minutes, and then filter. The filtrate is concentrated and purified by column to synthesize intermediate 18-1.

[0236] Synthesis of compound 18 In a 25 mL single-port reactor, combine intermediate 18-1 (1.0 g, 1.5 mmol), 2-cyanoethyl N,N'-diisopropyl chlorophosphoramidate (0.45 g, 2.0 mmol), triethylamine (0.61 mL, 4.5 mmol), and dichloromethane (10 mL). Stir at room temperature for 1 hour. Add water to the reactor, stir vigorously, and then extract with dichloromethane. Add anhydrous sodium sulfate, stir for 5 minutes, and then filter. Concentrate the filtrate and purify by column to synthesize compound 18 (0.9 g). The obtained compound 18 1 The H-NMR results are as follows: 1 H-NMR (400MHz, DMSO-d6) δ8.74(m, 1H), 8.02(s, 1H) 7.90(m, 1H), 7.76(t, 2H, J=6.0Hz), 7.36(d, 1H, J=7.6Hz), 6.91(s, 2 H), 6.82(s, 2H), 3.63-3.40(m, 16H), 2.79(t, 2H, J=6.0Hz), 2.11(s, 6H), 1.28-1.22(t, 6H, J=5.8Hz), 1.20-1.18(m, 12H)

[0237] Synthesis Example 19. Synthesis of Compound 19 [ka]

[0238] Synthesis of intermediate 19-1 In a 100 mL single-port reactor, combine intermediate 14-1 (3.0 g, 6.7 mmol), HSTU (N,N,N',N'-Tetramethyl-O-(N-succinimidyl)uronium hexafluorophosphate) (3.6 g, 10.0 mmol), triethylamine (1.8 mL, 13.4 mmol), and dimethylformamide (30 mL), and stir at room temperature for 1 hour. Add 4-aminobutyric acid (0.5 g, 4.5 mmol) and stir at room temperature for 12 hours. After concentration, add water to the reactor, stir vigorously, and extract with dichloromethane. Add anhydrous sodium sulfate, stir for 5 minutes, and then filter. The filtrate is concentrated and purified by column to synthesize intermediate 19-1.

[0239] Synthesis of intermediate 19-2 In a 100 mL single-port reactor, combine intermediate 19-1 (2.5 g, 3.5 mmol), HSTU (N,N,N',N'-Tetramethyl-O-(N-succinimidyl)uronium hexafluorophosphate) (1.9 g, 5.3 mmol), triethylamine (1.0 mL, 7.0 mmol), and dimethylformamide (25 mL), and stir at room temperature for 1 hour. Add 2-(methylamino)ethanol (0.3 g, 3.5 mmol) and stir at room temperature for 2 hours. After concentration, add water to the reactor, stir vigorously, and extract with dichloromethane. Add anhydrous sodium sulfate, stir for 5 minutes, and then filter. The filtrate is concentrated and purified by column to synthesize intermediate 19-2.

[0240] Synthesis of compound 19 In a 25 mL single-port reactor, combine intermediate 19-2 (1.0 g, 1.3 mmol), 2-cyanoethyl N,N'-diisopropyl chlorophosphoramidate (0.40 g, 1.7 mmol), triethylamine (0.54 mL, 3.9 mmol), and dichloromethane (10 mL), and stir at room temperature for 1 hour. Add water to the reactor, stir vigorously, and then extract with dichloromethane. Add anhydrous sodium sulfate, stir for 5 minutes, and then filter. Concentrate the filtrate and purify by column to synthesize compound 19 (0.7 g). The obtained compound 19 1 The H-NMR results are as follows: 1 H-NMR (400MHz, DMSO-d6) δ8.71(bs, 1H), 7.94(s, 1H) 7.84(m, 1H), 7.79(t, 2H, J=6.0Hz), 7.38(d, 1H, J=7.6Hz), 6.90(s, 2H), 6. 84(s, 2H), 3.66-3.38(m, 17H), 2.78(t, 2H, J=6.0Hz), 2.42(t, 2H, J=6.0Hz)2.10(s, 6H), 1.29-1.23(m, 8H), 1.20-1.16(m, 12H)

[0241] Synthesis Example 20. Synthesis of Compound 20 [ka]

[0242] Synthesis of intermediate 20-1 In a 100 mL single-port reactor, combine intermediate 18-1 (2.0 g, 2.8 mmol), HSTU (N,N,N',N'-Tetramethyl-O-(N-succinimidyl)uronium hexafluorophosphate) (1.5 g, 4.2 mmol), triethylamine (0.8 mL, 5.6 mmol), and dimethylformamide (20 mL), and stir at room temperature for 1 hour. Add 4-hydroxypiperidine (0.2 g, 2.8 mmol) and stir at room temperature for 1 hour. After concentration, add water to the reactor, stir vigorously, and extract with dichloromethane. Add anhydrous sodium sulfate, stir for 5 minutes, and then filter. The filtrate is concentrated and purified by column to synthesize intermediate 20-1.

[0243] Synthesis of compound 20 In a 25 mL single-port reactor, combine intermediate 20-1 (1.0 g, 1.3 mmol), 2-cyanoethyl N,N'-diisopropyl chlorophosphoramidate (0.40 g, 1.6 mmol), triethylamine (0.52 mL, 3.8 mmol), and dichloromethane (10 mL). Stir at room temperature for 1 hour. Add water to the reactor, stir vigorously, and then extract with dichloromethane. Add anhydrous sodium sulfate, stir for 5 minutes, and then filter. Concentrate the filtrate and purify by column to synthesize compound 20 (0.9 g). The obtained compound 20 1 The H-NMR results are as follows: 1 H-NMR (400MHz, DMSO-d6) δ8.70(bs, 1H), 7.91(s, 1H) 7.87(m, 1H), 7.75(t, 2H, J=6.0Hz), 7.37(d, 1H, J=7.6Hz), 6.92(s, 2H), 6.83(s, 2H), 4.13( m, 1H), 3.76-3.48(m, 14H), 2.77(t, 2H, J=6.0Hz), 2.41(t, 2H, J=6.0Hz) , 2.10(s, 6H), 2.07-1.77(m, 4H), 1.29-1.23(m, 8H), 1.20-1.16(m, 12H)

[0244] Synthesis Example 21. Synthesis of Compound 21 [ka]

[0245] Synthesis of intermediate 21-1 In a 3L four-neck reactor, 3-bromoanisole (150g, 0.802mol), benzophenone hydrazone (189g, 0.962mol), sodium tert-butoxide (108g, 1.12mol), Pd2(dba)3 (29.4g, 0.032mol), BINAP (20.0g, 0.032mol), and toluene (1500mL) were added and stirred at 100°C for 24 hours. After cooling, the mixture was filtered over cellulite. The filtrate was concentrated and purified by column to synthesize intermediate 21-1.

[0246] Synthesis of intermediate 21-2 Intermediate 21-1 (200g, 0.661mol), 3-methyl-2-butanone (85.5g, 0.992mol), concentrated hydrochloric acid (400mL), and ethanol (1600mL) were placed in a 3L four-port reactor and stirred at 100°C for 24 hours. After cooling, the mixture was concentrated and then purified by column to synthesize intermediate 21-2.

[0247] Synthesis of intermediates 21-3 In a 1L four-port reactor, intermediate 21-2 (40.0g, 0.211mol), boron tripromide (40.7mL, 0.423 mmol), and dichloromethane (400mL) were added and stirred at room temperature for 4 hours. The reaction mixture was poured into 1L of 5% sodium bicarbonate aqueous solution and stirred vigorously. After separating the organic layer, anhydrous sodium sulfate was added and stirred for 5 minutes, then filtered. The filtrate was concentrated and purified by column to synthesize intermediate 21-3.

[0248] Synthesis of intermediates 21-4 In a 1L four-port reactor, intermediate 21-3 (40.0g, 0.211mol), boron tripromide (40.7mL, 0.423 mmol), and dichloromethane (400mL) were added and stirred at room temperature for 4 hours. The reaction mixture was poured into 1L of 5% sodium bicarbonate aqueous solution and stirred vigorously. After separating the organic layer, anhydrous sodium sulfate was added and the mixture was stirred for 5 minutes, then filtered. The filtrate was concentrated and purified by column to synthesize intermediate 21-4.

[0249] Synthesis of Compound 21 In a 250 mL single-port reactor, combine intermediate 21-4 (27.9 g, 0.157 mol), 4-formyl-3-methylbenzoic acid (12.9 g, 0.079 mol), and 70% sulfuric acid (90 mL), and stir at 130 °C for 3 hours. After cooling, pour the reaction mixture into cold water for neutralization. Filter the resulting solid. After drying, add MeOH (1 L) and chloranil (9.7 g, 0.039 mol), and stir under reflux for 1 hour. After cooling, concentrate. Filter the resulting solid, and vacuum dry at 60 °C to synthesize compound 21 (12.0 g). The obtained compound 21 1 The H-NMR results are as follows: 1 H-NMR (400MHz, DMSO-d6) δ8.01(s, 1H), 7.93(d, 1H, J=7.8Hz), 7.37(d, 1H, J=7.8 Hz), 6.91(s, 2H), 3.81(q, 2H, J=6.0Hz), 2.06(s, 3H), 1.20(s, 12H), 1.04(s, 6H)

[0250] Synthesis Example 22. Synthesis of Compound 22 [ka]

[0251] Synthesis of compound 22 In a 50 mL single-port reactor, combine intermediate 21-4 (5.0 g, 0.028 mol), 4-formyl-3,5-methylbenzoic acid (2.51 g, 0.014 mol), and 70% sulfuric acid (10 mL), and stir at 130 °C for 3 hours. After cooling, pour the reaction mixture into cold water for neutralization. Filter the resulting solid. After drying, add MeOH (50 mL) and chloranil (1.7 g, 0.007 mol), and stir under reflux for 1 hour. After cooling, concentrate. Filter the resulting solid, and vacuum dry at 60 °C to synthesize intermediate compound 22 (2.3 g). The obtained compound 22 1 The H-NMR results are as follows: 1 H-NMR (400MHz, DMSO-d6) δ8.05(s, 2H), 6.90(s, 2H), 6.87(s, 2H), 3.81(q, 2H, J=6.0Hz), 2.07(s, 6H), 1.20(s, 12H), 1.04(s, 6H)

[0252] Synthesis Example 23. Synthesis of Compound 23 [ka]

[0253] Synthesis of intermediate 23-1 Intermediate 23-1 was synthesized by referring to Angew. Chem., 2013, vol.125, #2, pp.678-682.

[0254] Synthesis of compound 23 Compound 23 was synthesized by referring to the method in Synthesis Example 21. The obtained compound 23 1 The H-NMR results are as follows: 1 H-NMR (400MHz, DMSO-d6) δ8.03(s, 1H), 7.95(d, 1H, J=7.8Hz), 7.36(d, 1H, J=7.8H z), 6.91(s, 2H), 6.84(s, 2H), 3.56(m, 4H), 2.70(m, 4H), 2.07(s, 3H), 1.78(m, 4H)

[0255] Synthesis Example 24. Synthesis of Compound 24 [ka]

[0256] Synthesis of intermediate 24-1 Intermediate 24-1 was synthesized by referring to US2016 / 312033, 2016, A1.

[0257] Synthesis of Compound 24 Compound 24 was synthesized by referring to the method in Synthesis Example 21. The obtained compound 24 1 The H-NMR results are as follows: 1 H-NMR (400MHz, DMSO-d6) δ8.03(s, 1H), 7.92(d, 1H, J=7.8Hz), 7.34(d, 1H, J=7.8Hz), 6.90(s, 2H), 6.8 2(s, 2H), 2.86(m, 2H), 2.05(s, 3H), 1.73-1.55(m, 4H), 1.30(s, 6H), 1.24(d, 6H, J=6.4Hz), 1.15(s, 6H)

[0258] Synthesis Example 25. Synthesis of Compound 25 [ka]

[0259] Synthesis of intermediate 25-1 Intermediate 25-1 was synthesized by referring to US2016 / 312033, 2016, A1.

[0260] Synthesis of Compound 25 Compound 25 was synthesized by referring to the method in Synthesis Example 21. The obtained compound 25 1 The H-NMR results are as follows: 1H-NMR (400MHz, DMSO-d6) δ8.05(s, 1H), 7.97(d, 1H, J=7.8Hz), 7.39(d, 1H, J=7.8H z), 6.94(s, 2H), 6.87(s, 2H), 5.31(s, 2H), 2.07(s, 3H), 1.94(s, 6H), 1.26(s, 12H)

[0261] Synthesis Example 26. Synthesis of Compound 26 [ka]

[0262] Synthesis of intermediate 26-1 Intermediate 26-1 was synthesized by referring to Chemistry-A European Journal, 2022, vol.28, #35, art.no.E202200647.

[0263] Synthesis of Compound 26 Compound 26 was synthesized by referring to the method in Synthesis Example 21. The obtained compound 26 1 The H-NMR results are as follows: 1 H-NMR (400MHz, DMSO-d6) δ8.01(s, 1H), 7.93(d, 1H, J=7.8Hz), 7.31(d, 1H, J=7.8Hz), 6.92(s, 2H), 6.80(s, 2H), 3.86-3.50( m, 4H), 2.86(m, 2H), 2.05(s, 3H), 1.73-1.55(m, 4H), 1.36(t, 6H, J=6.8Hz), 1.29(s, 6H), 1.22(d, 6H, J=6.4Hz), 1.17(s, 6H)

[0264] Synthesis Example 27. Synthesis of Compound 27 [ka]

[0265] Synthesis of Compound 27 Compound 27 was synthesized by referring to the method in Synthesis Example 21. The obtained compound 27 1 The H-NMR results are as follows: 1 H-NMR (400MHz, DMSO-d6) δ8.07(s, 1H), 7.96(d, 1H, J=7.8Hz), 7.33(d, 1H, J=7.8Hz), 6.92(s, 2H), 6.86 (s, 2H), 5.33(s, 2H), 3.88-3.54(m, 4H), 2.05(s, 3H), 1.97(s, 6H), 1.35(t, 6H, J=6.8Hz), 1.22(s, 12H)

[0266] Synthesis Example 28. Synthesis of Compound 28 [ka]

[0267] Synthesis of compound 28 Compound 28 was synthesized by referring to the method in Synthesis Example 21. The obtained compound 28 1 The H-NMR results are as follows: 1 H-NMR (400MHz, DMSO-d6) δ7.91(s, 1H), 7.83(d, J=7.60Hz, 1H), 7.21(d, J=8.00Hz, 1H), 6.65(s , 2H), 3.58-3.48(m, 8H), 3.09-3.06(m, 4H), 2.82-2.76(m, 4H), 2.21-1.85(m, 8H), 2.08(s, 3H)

[0268] Synthesis Example 29. Synthesis of Compound 29 [ka]

[0269] Synthesis of compound 29 Compound 29 was synthesized by referring to the method in Synthesis Example 21. The obtained compound 29 1 The H-NMR results are as follows: 1H-NMR (400MHz, DMSO-d6) δ8.03(s, 1H), 7.93(d, 1H, J=7.8Hz), 7.31(d, 1H, J=7.8 Hz), 6.92(s, 1H), 6.79(s, 1H), 6.65(s, 1H), 3.86-3.50(m, 6H), 3.09-2.86(m, 3H) , 3.09-3.06(m, 2H), 2.82-2.76(m, 2H), 2.21-1.85(m, 2H), 2.05(s, 3H), 1.73-1. 55(m, 2H), 1.36(t, 3H, J=6.8Hz), 1.29(s, 3H), 1.22(d, 3H, J=6.4Hz), 1.17(s, 3H)

[0270] Synthesis Example 30. Synthesis of Compound 30 [ka]

[0271] Synthesis of intermediate 30-1 Intermediate 30-1 was synthesized by referring to WO2020 / 132607,2020,A1.

[0272] Synthesis of intermediate 30-2 In a 500 mL single-port reactor, intermediate 30-1 (10 g, 0.04 mol), 10% Pd / C (0.5 g), and MeOH (100 mL) were added and stirred at room temperature under a hydrogen stream for 24 hours. The mixture was filtered through cellulite. The filtrate was concentrated to synthesize intermediate 30-2.

[0273] Synthesis of compound 30 In a 50 mL single-port reactor, combine intermediate 30-2 (3.0 g, 0.013 mol), 4-formyl-3-methylbenzoic acid (1.06 g, 0.0065 mol), and 70% sulfuric acid (7 mL), and stir at 130 °C for 3 hours. After cooling, pour the reaction mixture into cold water for neutralization. Filter the resulting solid. After drying, add MeOH (30 mL) and chloranil (0.8 g, 0.005 mol), and stir under reflux for 1 hour. After cooling, concentrate. Filter the resulting solid, and vacuum dry at 60 °C to synthesize compound 30 (1.7 g). The obtained compound 301 The H-NMR results are as follows: 1 H-NMR (400MHz, DMSO-d6) δ8.03(s, 1H), 7.94(d, 1H, J=7.8Hz), 7.33(d, 1H, J=7.8Hz), 6.81(s, 2H), 3.59-3.47(m, 4H), 3.1 0-3.06(m, 2H), 2.81-2.75(m, 4H), 2.20-1.83(m, 7H), 1.74-1.51(m, 4H), 1.29(s, 6H), 1.22(d, 6H, J=6.4Hz), 1.14(s, 6H)

[0274] Synthesis Example 31. Synthesis of Compound 31 [ka]

[0275] Synthesis of compound 31 Compound 31 was synthesized by referring to the method of Synthesis Example 21. The obtained compound 31 1 The H-NMR results are as follows: 1 H-NMR (400MHz, DMSO-d6) δ8.07(s, 1H), 7.96(d, 1H, J=7.8Hz), 7.33(d, 1H, J=7.8Hz), 6.64(s, 2H), 5.33(s, 2H), 3.88-3.54( m, 4H), 3.56-3.27(m, 2H), 3.09-3.06(m, 2H), 2.82-2.76(m, 2H), 2.21-1.85(m, 2H), 2.05(s, 3H), 1.97(s, 6H), 1.22(s, 12H)

[0276] Synthesis Example 32. Synthesis of Compound 32 [ka]

[0277] Synthesis of intermediate 32-1 Intermediate 32-1 was synthesized by referring to Org.Lett.2020,2,381-385.

[0278] Synthesis of compound 32 In a 100 mL three-neck reactor, combine intermediate 32-1 (2.58 g, 0.0102 mol) and tetrahydrofuran (26 mL), and stir under a nitrogen stream at -78°C for 5 minutes. Slowly add 2.5 M n-butyllithium (4 mL, 0.0102 mol) dropwise to the reactor, then stir at -78°C for 1 hour. Dissolve intermediate 32-1 (1.5 g, 0.0034 mol) in tetrahydrofuran (30 mL) and add dropwise to the reactor, then stir at room temperature for 2 hours. After concentration, add 6 N hydrochloric acid aqueous solution (38 mL) to the reactor and stir at 80°C for 16 hours. After concentration, column purification was performed to synthesize compound 32 (2.1 g). The obtained compound 32 1 The H-NMR results are as follows: 1 H-NMR (400MHz, DMSO-d6) δ8.06(s, 1H), 7.35(s, 1H), 7.33(s, 1H), 6.81(s, 1H), 6.79(s, 1H), 6.75(s, 1H), 3.86-3.50(m, 6H), 3.09-2.86(m, 3H), 3.09-3.06(m, 2H), 2.82-2.76(m, 2H), 2.21-1.85(m, 2H), 2.05(s, 3H), 1.96(s, 6H), 1.73-1.5 5(m, 2H), 1.36(t, 3H, J=6.8Hz), 1.29(s, 3H), 1.22(d, 3H, J=6.4Hz), 1.17(s, 3H)

[0279] Synthesis Example 33. Synthesis of Compound 33 [ka]

[0280] Synthesis of intermediate 33-1 Intermediate 33-1 was synthesized by referring to Org.Lett.2019,21,5373.

[0281] Synthesis of compound 33 In a 100 mL three-neck reactor, combine intermediate 33-1 (1.13 g, 4 mmol) and tetrahydrofuran (8 mL), and stir under a nitrogen stream at -78°C for 5 minutes. Slowly add 2.5 M n-butyllithium (1.6 mL, 4 mmol) dropwise to the reactor, then stir at -78°C for 1 hour. Dissolve intermediate 30-1 (0.7 g, 1.6 mmol) in tetrahydrofuran (10 mL) and add dropwise to the reactor, then stir at room temperature for 2 hours. After concentration, add 6 N hydrochloric acid aqueous solution (20 mL) to the reactor and stir at 80°C for 16 hours. After concentration, column purification was performed to synthesize compound 33 (0.6 g). The obtained compound 33 1 The H-NMR results are as follows: 1 H-NMR (400MHz, DMSO-d6) δ8.08(s, 1H), 7.33(s, 1H), 7.30(s, 1H), 6.80(s, 1H), 6.75(s, 1H), 6.71(s, 1H), 3.88-3.49(m, 6H), 3.09-2.86(m, 3H), 3.09-3.06(m, 2H), 2.82-2.76(m, 2H), 2.21-1.85(m, 2H), 2.06(s, 3H), 1.96(s, 6H), 1.75-1.5 0(m, 2H), 1.34(t, 3H, J=6.8Hz), 1.30(s, 3H), 1.21(d, 3H, J=6.4Hz), 1.16(s, 3H)

[0282] Synthesis Example 34. Synthesis of Compound 34 [ka]

[0283] Synthesis of intermediate 34-1 Intermediate 34-1 was synthesized by referring to US11,155,713,B2.

[0284] Synthesis of compound 34 In a 250 mL single-port reactor, combine intermediate 34-1 (12.0 g, 0.0257 mol), 4-formyl-3-methylbenzoic acid (4.22 g, 0.0257 mol), and 70% sulfuric acid (20 mL), and stir at 130 °C for 3 hours. After cooling, pour the reaction mixture into cold water for neutralization. Filter the resulting solid. After drying, add MeOH (1 L) and chloranil (3.1 g, 0.0128 mol), and stir under reflux for 1 hour. After cooling, concentrate. Filter the resulting solid, and vacuum dry at 60 °C to synthesize compound 34 (1.3 g). The obtained compound 34 1 The H-NMR results are as follows: 1 H-NMR (400MHz, DMSO-d6) δ8.09(s, 1H), 7.95(d, 1H, J=7.8Hz), 7.35(d, 1H, J=7.8Hz), 6.65(s, 2H), 5.31(s, 2H), 3.89-3.52(m, 4H) , 3.56-3.27(m, 2H), 3.09-3.06(m, 2H), 2.82-2.76(m, 2H), 2.20-1.84(m, 2H), 2.06(s, 3H), 1.96(s, 6H), 1.88(s, 6H), 1.21(s, 12H)

[0285] Synthesis Example 35. Synthesis of Compound 35 [ka]

[0286] Synthesis of intermediate 35-1 Intermediate 35-1 was synthesized by referring to US11,155,713,B2.

[0287] Synthesis of Compound 35 In a 250 mL single-port reactor, combine intermediate 35-1 (7.0 g, 0.018 mol), 4-formyl-3-methylbenzoic acid (2.95 g, 0.018 mol), and 70% sulfuric acid (100 mL), and stir at 130 °C for 3 hours. After cooling, pour the reaction mixture into cold water for neutralization. Filter the resulting solid. After drying, add MeOH (1 L) and chloranil (1.1 g, 0.005 mol), and stir under reflux for 1 hour. After cooling, concentrate. Filter the resulting solid, and vacuum dry at 60 °C to synthesize compound 35 (1.6 g). The obtained compound 35 1 The H-NMR results are as follows: 1 H-NMR (400MHz, DMSO-d6) δ8.03(s, 1H), 7.78(d, 1H, J=7.60Hz), 7.30(d, 1H, J=8.00Hz), 6.52(s, 2H), 3.45-3 .23(m, 8H), 3.02-2.92(m, 4H), 2.50-2.39(m, 4H), 2.13(s, 3H), 2.04(s, 3H), 2.00-1.88(m, 7H), 1.86(m, 4H)

[0288] Synthesis Example 36. Synthesis of Compound 36 [ka]

[0289] Synthesis of intermediate 36-1 Intermediate 36-1 was synthesized by referring to US2020 / 247831,2020,A1.

[0290] Synthesis of Compound 36 In a 100 mL three-neck reactor, combine intermediate 36-1 (2.58 g, 0.0102 mol) and tetrahydrofuran (26 mL), and stir under a nitrogen stream at -78°C for 5 minutes. Slowly add 2.5 M n-butyllithium (4 mL, 0.0102 mol) dropwise to the reactor, then stir at -78°C for 1 hour. Dissolve intermediate 33-1 (1.5 g, 0.0034 mol) in tetrahydrofuran (30 mL), add dropwise to the reactor, and stir at room temperature for 2 hours. After concentration, add 6 N hydrochloric acid aqueous solution (38 mL) to the reactor and stir at 80°C for 16 hours. After concentration, column purification was performed to synthesize compound 36 (3.0 g). The obtained compound 36 1 The H-NMR results are as follows: 1 H-NMR (400MHz, DMSO-d6) δ7.93(s, 1H), 7.81(d, J=7.60Hz, 1H), 7.20(d, J=8.00Hz, 1H), 6.66(s, 2H), 3.68-3.47(m, 8H), 3.10-3.06(m, 4H), 2.87-2.74(m, 4H), 2.24-1.84(m, 8H), 2.08(s, 3H), 0.59(s, 6H)

[0291] Synthesis Example 37. Synthesis of Compound 37 [ka]

[0292] Synthesis of Compound 37 Compound 1 (1.0 g, 2.9 mmol) and 20% fuming sulfuric acid (6 mL) were placed in a 50 mL single-port reactor and stirred at room temperature for 12 hours. The reaction mixture was poured into ethyl ether (60 mL) and stirred vigorously. The supernatant was discarded, and the mixture was dissolved in water and purified by reverse-phase to synthesize Compound 37 (0.2 g). The obtained compound 37 1 The H-NMR results are as follows: 1H-NMR (400MHz, D2O) δ8.67(s, 1H), 8.29(d, 1H, J=8.0Hz), 7.41(d, 1H, J=8.0Hz), 7.00(d, 2H, J=8.0Hz), 6.79(d, 2H, J=8.0Hz), 2.07(s, 3H)

[0293] Synthesis Example 38. Synthesis of Compound 38 [ka]

[0294] Synthesis of compound 38 Compound 21 (1.0 g, 2.1 mmol) and 20% fuming sulfuric acid (6 mL) were placed in a 50 mL single-port reactor and stirred at room temperature for 12 hours. The reaction mixture was poured into ethyl ether (60 mL) and stirred vigorously. The supernatant was discarded, and the mixture was dissolved in water and purified by reverse-phase to synthesize compound 38 (0.5 g). The obtained compound 38 1 The H-NMR results are as follows: 1 H-NMR (400MHz, D2O) δ8.65(s, 1H), 8.27(d, 1H, J=7.8Hz), 7.37(d, 1H, J=7.8Hz) ), 6.85(s, 2H), 3.83(q, 2H, J=6.0Hz), 2.06(s, 3H), 1.19(s, 12H), 1.06(s, 6H)

[0295] Synthesis Example 39. Synthesis of Compound 39 [ka]

[0296] Synthesis of compound 39 Compound 24 (1.0 g, 1.9 mmol) and 20% fuming sulfuric acid (6 mL) were placed in a 50 mL single-port reactor and stirred at room temperature for 12 hours. The reaction mixture was poured into ethyl ether (60 mL) and stirred vigorously. The supernatant was discarded, and the mixture was dissolved in water and purified by reverse-phase purification to synthesize compound 39 (0.2 g). The obtained compound 39 1 The H-NMR results are as follows: 1 H-NMR (400MHz, D2O) δ8.60(s, 1H), 8.17(d, 1H, J=7.8Hz), 7.35(d, 1H, J=7.8Hz), 6.75(s, 2H), 2.93(m, 2H), 2.09(s, 3H), 1.78-1.57(m, 4H), 1.33(s, 6H), 1.26(d, 6H, J=6.4Hz), 1.17(s, 6H)

[0297] Synthesis Example 40. Synthesis of Compound 40 [ka]

[0298] Synthesis of compound 40 Compound 27 (1.0 g, 1.8 mmol) and 20% fuming sulfuric acid (6 mL) were placed in a 50 mL single-port reactor and stirred at room temperature for 12 hours. The reaction mixture was poured into ethyl ether (60 mL) and stirred vigorously. The supernatant was discarded, and the mixture was dissolved in water and purified by reverse-phase purification to synthesize compound 40 (0.3 g). The obtained compound 40 1 The H-NMR results are as follows: 1 H-NMR (400MHz, D2O) δ8.67(s, 1H), 8.13(d, 1H, J=7.8Hz), 7.33(d, 1H, J=7.8Hz), 6.98(s, 2H), 6.88(s , 2H), 5.33(s, 2H), 3.78-3.54(m, 4H), 2.08(s, 3H), 2.03(m, 4H), 1.33(t, 6H, J=6.8Hz), 1.20(s, 12H)

[0299] Synthesis Example 41. Synthesis of Compound 41 [ka]

[0300] Synthesis of intermediate 41-1 Intermediate 41-1 was synthesized by referring to WO2016 / 100401.

[0301] Synthesis of intermediate 41-2 In a 100 mL single-port reactor, combine intermediate 41-1 (2.21 g, 0.0032 mol), compound 10 (2.0 g, 0.0026 mol), triethylamine (1.1 mL, 0.0079 mol), tetrahydrofuran (30 mL), and dichloromethane (30 mL), and stir at room temperature for 1 hour. Add water to the reactor, stir vigorously, and then extract with dichloromethane. Add anhydrous sodium sulfate, stir for 5 minutes, and then filter. The filtrate is concentrated and purified by column to synthesize intermediate 41-2.

[0302] Synthesis of compound 41 In a 50 mL single-port reactor, combine intermediate 41-2 (1.0 g, 0.0007 mol), 2-cyanoethyl N,N'-diisopropyl chlorophosphoramidate (0.23 g, 0.001 mol), triethylamine (1.1 mL, 0.0022 mol), and dichloromethane (15 mL). Stir at room temperature for 1 hour. Add water to the reactor, stir vigorously, and then extract with dichloromethane. Add anhydrous sodium sulfate, stir for 5 minutes, and then filter. Concentrate the filtrate and purify by column to synthesize compound 41 (0.5 g). The obtained compound 41 1 The H-NMR results are as follows: 1 H-NMR (400MHz, CDCl3) δ8.68(m, 1H), 8.09-7.77(m, 6H), 7.48-7.03(m, 13H) , 6.95(s, 2H), 6.87-6.82(m, 6H), 6.21-6.11(m, 1H), 4.47(m, 1H), 4.26(m, 1H) ), 3.71(m, 8H), 3.66-3.43(m, 7H), 3.27-3.02(m, 6H), 2.76-2.61(m, 2H), 2.1 6-2.03(m, 19H), 1.81(m, 2H), 1.41(m, 4H), 1.26(m, 11H), 1.17-0.96(m, 15H)

[0303] Synthesis Example 42. Synthesis of Compound 42 [ka]

[0304] Synthesis of intermediate 42-1 Intermediate 42-1 was synthesized by referring to WO2016 / 100401.

[0305] Synthesis of intermediate 42-2 In a 250 mL single-port reactor, intermediate 42-1 (3.83 g, 0.0055 mol), compound 28 (3.8 g, 0.0046 mol), triethylamine (1.7 mL, 0.0114 mol), tetrahydrofuran (57 mL), and dichloromethane (57 mL) were added and stirred at room temperature for 2 hours. Water was added to the reactor and stirred vigorously, then extracted with dichloromethane. Anhydrous sodium sulfate was added and stirred for 5 minutes, then filtered. The filtrate was concentrated and purified by column to synthesize intermediate 42-2.

[0306] Synthesis of compound 42 In a 50 mL single-port reactor, combine intermediate 42-2 (1.0 g, 0.0007 mol), 2-cyanoethyl N,N'-diisopropyl chlorophosphoramidate (0.4 g, 0.0017 mol), N-methylmorpholine (0.2 mL, 0.00176 mol), and dichloromethane (20 mL). Stir at room temperature for 1 hour. Add water to the reactor, stir vigorously, and then extract with dichloromethane. Add anhydrous sodium sulfate, stir for 5 minutes, and then filter. Concentrate the filtrate and purify by column to synthesize compound 42 (0.6 g). 1H-NMR (400MHz, DMSO-d6) δ11.60(s, 1H), 8.60-8.70(m, 1H), 7.80-8.10(m, 5H), 7.08-7.40(m, 13H), 6.80-6. 88(m, 4H), 6.56(s, 2H), 6.10-6.20(m, 1H), 4.40(s, 1H), 3.95-4.05(m, 1H), 3.72(s, 7H), 3.40-3.60(m, 11H) , 3.32-3.33(m, 4H), 3.00-2.18(m, 8H), 2.71-2.80(m, 1H), 2.59-2.70(m, 5H), 2.15-2.22(m, 2H), 1.98-2.10 (m, 9H), 1.72-1.91(m, 6H), 1.31-1.42(m, 4H), 1.15-1.30(m, 4H), 1.08-1.15(m, 12H), 0.97(d, 2H, J=6.80Hz)

[0307] Manufacturing Example 1. Synthesis of a double-labeled probe (oligonucleotide) Using Universal UnyLinker Support (Chemgene, 500 Å), 5'-SEQ 1-3' was synthesized as a forward primer and 5'-SEQ 2-3' as a reverse primer against BQCV (black queen cell virus) on a 1 μmol scale. The primers were then cleaved from the support using ammonium hydroxide deprotection and purified by RP-HPLC.

[0308] First, using excretion 1-CPG and excretion 2-CPG, 5'-SEQ 3-excretion 1-3' and 5'-SEQ 3-excretion 2-3' are synthesized as single-labeled probes (synthesis is performed referring to Korean Published Patent Publications No. 10-2019-0062162 and No. 10-2020-0067733), MerMade TM Using a 48X DNA Synthesizer, compound 3, compound 22, compound 30, and compound 35, as defined in this application, and commercially available fluorescent substances Cy5, ROX, HEX, and FAM were each labeled at the 5' end in phosphoramidite form to synthesize double-labeled probes.

[0309] The structures of extensor 1 and extensor 2, which were used in the synthesis of the double-labeled probe, are as follows. [ka]

[0310] The synthesized double-labeled probe was separated from the CPG and then purified by RP-HPLC.

[0311] The morphology of the synthesized double-labeled probes is shown in Table 1 below. [Table 1]

[0312] Manufacturing Example 2. Synthesis of a double-labeled probe (oligonucleotide) Using Universal UnyLinker Support (Chemgene, 500 Å), 5'-SEQ 1-3' was synthesized as a forward primer and 5'-SEQ 2-3' as a reverse primer against BQCV (black queen cell virus) on a 1 μmol scale. The primers were then cleaved from the support using a deprotection process with ammonium hydroxide, and subsequently purified by RP-HPLC.

[0313] First, using excretion 1-CPG and excretion 2-CPG, 5'-SEQ 3-excretion 1-3' and 5'-SEQ 3-excretion 2-3' are synthesized as single-labeled probes (synthesis is performed referring to Korean Published Patent Publications No. 10-2019-0062162 and No. 10-2020-0067733), MerMade TM Using a 48X DNA Synthesizer, compound 3, compound 22, compound 30, and compound 35, as defined in this application, were labeled at their 5' ends in phosphoramidite form as reporters, and Dye A, Dye B, Dye C, and Dye D, designed as control groups for the compounds defined in this application, were labeled at their 5' ends to synthesize double-labeled probes.

[0314] The structures of Dye A to Dye D, designed as the control group, are as follows: [ka]

[0315] The synthesized double-labeled probe was separated from the CPG and then purified by RP-HPLC.

[0316] The morphology of the synthesized double-labeled probes is shown in Table 2 below. [Table 2]

[0317] Experimental Example 1. Real-Time PCR experiment using a dual-labeled probe. Real-Time PCR against BQCV (black queen cell virus) plasmid DNA was performed twice using each of the dual-labeled probes synthesized according to Manufacturing Example 1, with the compositions listed in Table 3 below (Biorad, CFX-96). TM (using Touch). PCR Protocol: 95℃, 3min-[95℃, 10s-60℃, 30s]×45cycles [Table 3]

[0318] The Real-Time PCR results are shown in Table 4. [Table 4]

[0319] Referring to Table 4 and Figures 1-4, EX 1-1, 1-3, 1-5, and 1-7, which used the compounds defined in this application as reporters, showed relatively lower Ct and higher RFU compared to EX 1-2, 1-4, 1-6, and 1-8, which used commercially available fluorescent substances as reporters.

[0320] Furthermore, looking at the results in Figures 5 to 12, where standard curves were created using Ct values ​​and concentrations, the R values ​​for EX 1-1, 1-3, 1-5, and 1-7 are as follows: 2 The values ​​are all 0.98 or higher, confirming that linearity is maintained.

[0321] Furthermore, considering the perspective of molecular diagnostics, the detection limit (LoD) of the compounds defined in this application is lower than that of existing commercially available fluorescent substances. Therefore, when using the compounds defined in this application as reporters (fluorescent substances) for a dual-labeled probe, it is expected that detection will be easier and more accurate than with commercially available fluorescent substances, even if the target gene in the sample is present at a relatively low concentration.

[0322] Therefore, the nucleic acid labeling reporters defined in this application can be applied to existing nucleic acid labeling and detection fields (e.g., PCR experiments) or can adequately replace existing commercially available fluorescent substances.

[0323] Experimental Example 2. Real-Time PCR experiment using a dual-labeled probe. Real-Time PCR against BQCV (black queen cell virus) plasmid DNA was performed twice using each of the double-labeled probes synthesized according to Manufacturing Example 2, with the compositions listed in Table 5 below (Biorad, CFX-96). TM (using Touch). PCR Protocol: 95℃, 3min-[95℃, 10s-60℃, 30s]×45cycles [Table 5]

[0324] The Real-Time PCR results are shown in Table 6. [Table 6]

[0325] Referring to Table 6 and Figures 13-16, EX 2-1, 2-3, 2-5, and 2-7, which used the compounds defined in this application as reporters, showed relatively lower Ct and higher RFU compared to Dye A-Dye D, where both A1 and A2 in chemical formula 1 are hydrogen.

[0326] Furthermore, looking at the results in Figures 17-24, where standard curves were created using Ct values ​​and concentrations, the R values ​​for EX 2-1, 2-3, 2-5, and 2-7 are as follows: 2 The values ​​are all 0.98 or higher, confirming that linearity is maintained. From the above results, it can be confirmed that the value of the reagent as a reporter is enhanced because one or more of A1 and A2 in chemical formula 1 are not hydrogen.

[0327] Therefore, the compounds defined in this application are expected to be applicable as nucleic acid labeling reporters in existing nucleic acid labeling and detection fields (e.g., PCR experiments) or to adequately replace existing commercially available fluorescent substances.

[0328] Experimental Example 3. Evaluation of the optical properties of nucleic acid labeling reporters. [Table 7]

[0329] Referring to Table 7, it can be confirmed that in phenyl-substituted xanthene fluorescent dyes, such as Dye B used in Production Example 2, when substituents are present at the meta or para position, the rotation of phenyl is not blocked, resulting in a functional defect that inhibits the fluorescence properties of the dye. On the other hand, compounds with substituents at the ortho position, such as compound 30, experience steric hindrance, blocking the rotation of phenyl, and as a result, the fluorescence properties are significantly improved. Furthermore, the substituent effect at the ortho position allows for fine-tuning of the absorption or emission spectrum of the compound, which offers the possibility of developing fluorescent compounds in various wavelength bands.

[0330] Although several embodiments of the present invention have been described above, any person with ordinary skill in the art can modify or change the present invention in various ways by adding, changing, deleting, or adding components, without departing from the spirit of the invention as described in the claims, and these modifications are also included within the scope of the rights of the present invention.

Claims

1. The compound represented by the following chemical formula 1: 【Chemistry 1】 Here, R １ ~R ４ are each independently hydrogen, optionally substituted C １ -C ４０ alkyl, optionally substituted C １ -C ４０ heteroalkyl, optionally substituted C ２ -C ４０ alkenyl, optionally substituted C ２ -C ４０ alkynyl, optionally substituted C ３ -C ２０ cycloalkyl, optionally substituted C ３ -C ２０ cycloalkenyl, optionally substituted C ２ -C ２０ heterocycloalkyl, hydroxy, oxide (-O － ), optionally substituted C ３ -C ４０ cycloalkyloxy, optionally substituted C ５ -C ４０ aryloxy, optionally substituted C ２ -C ４０ heteroaryloxy, thiol, optionally substituted C ５ -C ５０ aralkyl, optionally substituted C １ -C ４０ alkylthio, optionally substituted C ５ -C ４０ arylthio, optionally substituted C ３ -C ４０ cycloalkylthio, optionally substituted C ２ -C ４０ Heteroarylthio, optionally substituted acylamino, acyloxy, sulfo group, optionally substituted ammonium, substituted sulfonic acid ester, optionally substituted sulfonamide, substituted ester, optionally substituted aminocarbonyl, nitroso (-N=O), optionally substituted silyl, optionally substituted amide, optionally substituted carbamate, phospho group, nitrile, acetal, ketal, optionally substituted amino, aminothiocarbonyl, aminocarbonyloxy, aminosulfonyloxy, amidino group, sulfonyloxy, isonitrile, cyanate, imide, iminium, ether, thioether, R ｘ and R ｓ Selected from, R ５ ~R １３ These are, independently, hydrogen and possibly substituted C. １ -C ４０ Alkyl, optionally substituted C １ -C ４０ Heteroalkyl, optionally substituted C ２ -C ４０ Alkenyl, C may be substituted. ２ -C ４０ Alkynyl, C may be substituted. ３ -C ２０ Cycloalkyl, optionally substituted C ３ -C ２０ Cycloalkenyl, optionally substituted C ２ -C ２０ Heterocycloalkyl, hydroxy, oxide (-O － ), C may be substituted. １ -C ４０ Alkoxy, possibly substituted C ３ -C ４０ Cycloalkyloxy, optionally substituted C ５ -C ４０ Aryloxy, possibly substituted C ２ -C ４０ Heteroaryloxy, possibly substituted C ５ -C ５０ Aryl, C may be substituted. ２ -C ５０ Heteroaryl, thiol, and possibly substituted C ５ -C ５０ Alalkyl, optionally substituted C １ -C ４０ Alkylthio, optionally substituted C ５ -C ４０ Arylthio, C may be substituted. ３ -C ４０ Cycloalkylthio, optionally substituted C ２ -C ４０ Heteroarylthio, optionally substituted acylamino, acyloxy, sulfo group, optionally substituted ammonium, substituted sulfonic acid ester, optionally substituted sulfonamide, substituted ester, optionally substituted aminocarbonyl, nitroso (-N=O), halogen, optionally substituted silyl, optionally substituted amide, optionally substituted carbamate, phospho group, nitrile, acetal, ketal, optionally substituted amino, aminothiocarbonyl, aminocarbonyloxy, aminosulfonyloxy, amidino group, sulfonyloxy, isonitrile, cyanate, imide, iminium, ether, thioether, R ｘ and R ｓ Selected from, R １ ~R １３ Two adjacent rings can bond to each other to form an optional aliphatic ring, an optional aromatic ring, or an optional mixed aliphatic and aromatic ring. A １ and A ２ are each independently selected from hydrogen, halogen, optionally substituted C １ -C １０ alkyl, optionally substituted C ３ -C １０ cycloalkyl, optionally substituted C １ -C １０ heterocycloalkyl, optionally substituted C １ -C １０ heteroalkyl, optionally substituted C ２ -C １０ alkenyl, optionally substituted C ２ -C １０ alkynyl, optionally substituted C １ -C １０ alkoxy, optionally substituted C ５ -C ４０ aryloxy and cyano, provided that the case where A １ and A ２ are simultaneously hydrogen is excluded. R ｘ This is a reactive group, or a group in which at least one reactive group is bonded to a backbone containing a hydrocarbon having 1 to 40 carbon atoms. The aforementioned reactive group is carboxyl, carboxyl derivative, carboxylate (-CO ２ － ), carboxylates, hydroxyl, diene derivatives, aldehydes, substituted ketones, sulfonyl halides, thiols, unsubstituted aminos, primary aminos, alkenes, alkynes, halogens, hydrazides, azides, imides, ketenes, isocyanates, thiocyanates, isothiocyanates, epoxides, maleimides, 1,2,4,5-tetrazine derivatives, cycloalkyne derivatives, cycloalkenes, triphosphates and phosphoramidites, substituted thioketones, haloformyl, formyl, acyl, acylamide, acyl azide, organic acid anhydrides (anhydride), aniline, aziridine, boronate, carbodiimide, diazoalkynes, haloacetamides, imide esters, glycols, halotriazines, hydrazines, acyl halides, alkyl halides and aryl halides. R ｓ This is a carrier molecule, or a group in which at least one carrier molecule is bonded to a backbone containing a hydrocarbon with 1 to 40 carbon atoms. R １ ~R １３ At least one of them is R ｘ or R ｓ And, X is O, S, CR ａ R ｂ , NR ａ or SiR ａ R ｂ And, R ａ and R ｂ C may be substituted independently of each other. １ -C ４０ Alkyl, optionally substituted C １ -C ４０ Heteroalkyl, optionally substituted C ３ -C ３０ Cycloalkyl, optionally substituted C ３ -C ３０ Heterocycloalkyl, optionally substituted C ５ -C ５０ Aryl and possibly substituted C ２ -C ５０ The rings are selected from heteroaryls, or are optionally substituted aliphatic rings, optionally substituted aromatic rings, or optionally substituted mixed aliphatic and aromatic rings formed by bonding with each other.

2. R ｘ or R ｓ The compound according to claim 1, characterized by containing the following structural formula 1: 【Chemistry 2】 Here, * indicates the position where structural formula 1 is bonded to the compound represented by chemical formula 1. R １４ and R １５ These are, independently, hydrogen and possibly substituted C. １ -C ２０ Alkyl, optionally substituted C １ -C ２０ Heteroalkyl, optionally substituted C ２ -C ２０ Alkenyl, C may be substituted. ２ -C ２０ Alkynyl, C may be substituted. ３ -C ２０ Cycloalkyl, optionally substituted C ３ -C ２０ Cycloalkenyl, optionally substituted C ２ -C ２０ Heterocycloalkyl, hydroxy, oxide (-O － ), C may be substituted. １ -C ４０ Alkoxy, possibly substituted C ３ -C ２０ Cycloalkyloxy, optionally substituted C ５ -C ２０ Aryloxy, possibly substituted C ２ -C ２０ Heteroaryloxy, possibly substituted C ５ -C ２０ Aryl, C may be substituted. ２ -C ２０ Heteroaryl, thiol, and possibly substituted C ５ -C ２０ Alalkyl, optionally substituted C １ -C ２０ Alkylthio, optionally substituted C ５ -C ２０ Arylthio, C may be substituted. ３ -C ２０ Cycloalkylthio, optionally substituted C ２ -C ２０ Selected from heteroarylthio, optionally substituted acylamino, acyloxy, sulfo group, optionally substituted ammonium, substituted sulfonic acid ester, optionally substituted sulfonamide, substituted ester, optionally substituted aminocarbonyl, nitroso (-N=O), halogen, optionally substituted silyl, optionally substituted amide, optionally substituted carbamate, phospho group, nitrile, acetal, ketal, optionally substituted amino, aminothiocarbonyl, aminocarbonyloxy, aminosulfonyloxy, amidino group, sulfonyloxy, isonitrile, cyanate, imide, iminium, ether and thioether, or bonded to each other to form optionally substituted aliphatic rings, optionally substituted aromatic rings or optionally substituted mixed aliphatic and aromatic rings, L １ It is a linker that is either a single bond or a hydrocarbon containing 1 to 20 carbon atoms.

3. R １４ and R １５ At least one of these is a carboxyl, a carboxyl derivative, or a carboxylate (-CO ２ － The compound according to claim 2, characterized by comprising a reactive group selected from ), carboxylate salts, hydroxyl, diene derivatives, aldehydes, substituted ketones, sulfonyl halides, thiols, unsubstituted aminos, primary aminos, alkenes, alkynes, halogens, hydrazides, azides, imides, ketenes, isocyanates, thiocyanates, isothiocyanates, epoxides, maleimides, 1,2,4,5-tetrazine derivatives, cycloalkyne derivatives, cycloalkenes, triphoates and phosphoramidites, substituted thioketones, haloformyl, formyl, acyl, acylamide, acyl azide, organic acid anhydrides (anhydride), aniline, aziridine, boronate, carbodiimide, diazoalkynes, haloacetamide, imide esters, glycols, halotriazines, hydrazines, acyl halides, alkyl halides and aryl halides.

4. R ｘ or R ｓ The compound according to claim 1, characterized by containing the following structural formula 2: 【Transformation 3】 Here, * indicates the position where structural formula 2 is bonded to the compound represented by chemical formula 1. R １６ C may be hydrogen or substituted. １ -C ２０ Alkyl, optionally substituted C １ -C ２０ Heteroalkyl, optionally substituted C ２ -C ２０ Alkenyl, C may be substituted. ２ -C ２０ Alkynyl, C may be substituted. ３ -C ２０ Cycloalkyl, optionally substituted C ３ -C ２０ Cycloalkenyl, optionally substituted C ２ -C ２０ Heterocycloalkyl, hydroxy, oxide (-O － ), C may be substituted. １ -C ４０ Alkoxy, possibly substituted C ３ -C ２０ Cycloalkyloxy, optionally substituted C ５ -C ２０ Aryloxy, possibly substituted C ２ -C ２０ Heteroaryloxy, possibly substituted C ５ -C ２０ Aryl, C may be substituted. ２ -C ２０ Heteroaryl, thiol, and possibly substituted C ５ -C ２０ Alalkyl, optionally substituted C １ -C ２０ Alkylthio, optionally substituted C ５ -C ２０ Arylthio, C may be substituted. ３ -C ２０ Cycloalkylthio, optionally substituted C ２ -C ２０ Selected from heteroarylthio, optionally substituted acylamino, acyloxy, sulfo group, optionally substituted ammonium, substituted sulfonic acid ester, optionally substituted sulfonamide, substituted ester, optionally substituted aminocarbonyl, nitroso (-N=O), halogen, optionally substituted silyl, optionally substituted amide, optionally substituted carbamate, phospho group, nitrile, acetal, ketal, optionally substituted amino, aminothiocarbonyl, aminocarbonyloxy, aminosulfonyloxy, amidino group, sulfonyloxy, isonitrile, cyanate, imide, iminium, ether and thioether, L ２ It is a linker containing hydrocarbons with 1 to 20 carbon atoms.

5. The compound contains at least one R ｘ or at least one R ｓ There exists at least one R ｘ or at least one R ｓ The compound according to claim 1, characterized in that it is protected with a protecting group.

6. A １ and A ２ At least one of these may be substituted C １ -C ３ Alkyl and optionally substituted C １ -C ５ The compound according to claim 1, characterized in that it is selected from alkoxy groups.

7. R １ and R ５ , R ２ and R ６ , R ３ and R １０ and R ４ and R ９ The compound according to claim 1, characterized in that at least one pair of these rings are bonded to each other to form an optionally substituted aliphatic ring, an optionally substituted aromatic ring, or an optionally substituted aliphatic and aromatic mixed ring.

8. R １ and R ２ and R ３ and R ４ The compound according to claim 1, characterized in that at least one pair of these rings are bonded to each other to form an optionally substituted aliphatic ring, an optionally substituted aromatic ring, or an optionally substituted aliphatic and aromatic mixed ring.

9. The compound represented by the following chemical formula 2: 【Chemistry 4】 Here, R ２１ ~R ２４ These are, independently, hydrogen and possibly substituted C. １ -C ４０ Alkyl, optionally substituted C １ -C ４０ Heteroalkyl, optionally substituted C ２ -C ４０ Alkenyl, C may be substituted. ２ -C ４０ Alkynyl, C may be substituted. ３ -C ２０ Cycloalkyl, optionally substituted C ３ -C ２０ Cycloalkenyl, optionally substituted C ２ -C ２０ Heterocycloalkyl, hydroxy, oxide (-O － ), C may be substituted. ３ -C ４０ Cycloalkyloxy, optionally substituted C ５ -C ４０ Aryloxy, possibly substituted C ２ -C ４０ Heteroaryloxy, thiol, and possibly substituted C ５ -C ５０ Alalkyl, optionally substituted C １ -C ４０ Alkylthio, optionally substituted C ５ -C ４０ Arylthio, C may be substituted. ３ -C ４０ Cycloalkylthio, optionally substituted C ２ -C ４０ Heteroarylthio, optionally substituted acylamino, acyloxy, sulfo group, optionally substituted ammonium, substituted sulfonic acid ester, optionally substituted sulfonamide, substituted ester, optionally substituted aminocarbonyl, nitroso (-N=O), optionally substituted silyl, optionally substituted amide, optionally substituted carbamate, phospho group, nitrile, acetal, ketal, optionally substituted amino, aminothiocarbonyl, aminocarbonyloxy, aminosulfonyloxy, amidino group, sulfonyloxy, isonitrile, cyanate, imide, iminium, ether, thioether and -L ３ -R ３６ Selected from, R ２５ ~R ３３ These are, independently, hydrogen and possibly substituted C. １ -C ４０ Alkyl, optionally substituted C １ -C ４０ Heteroalkyl, optionally substituted C ２ -C ４０ Alkenyl, C may be substituted. ２ -C ４０ Alkynyl, C may be substituted. ３ -C ２０ Cycloalkyl, optionally substituted C ３ -C ２０ Cycloalkenyl, optionally substituted C ２ -C ２０ Heterocycloalkyl, hydroxy, oxide (-O － ), C may be substituted. １ -C ４０ Alkoxy, possibly substituted C ３ -C ４０ Cycloalkyloxy, optionally substituted C ５ -C ４０ Aryloxy, possibly substituted C ２ -C ４０ Heteroaryloxy, possibly substituted C ５ -C ５０ Aryl, C may be substituted. ２ -C ５０ Heteroaryl, thiol, and possibly substituted C ５ -C ５０ Alalkyl, optionally substituted C １ -C ４０ Alkylthio, optionally substituted C ５ -C ４０ Arylthio, C may be substituted. ３ -C ４０ Cycloalkylthio, optionally substituted C ２ -C ４０ Heteroarylthio, optionally substituted acylamino, acyloxy, sulfo group, optionally substituted ammonium, substituted sulfonic acid ester, optionally substituted sulfonamide, substituted ester, optionally substituted aminocarbonyl, nitroso (-N=O), halogen, optionally substituted silyl, optionally substituted amide, optionally substituted carbamate, phospho group, nitrile, acetal, ketal, optionally substituted amino, aminothiocarbonyl, aminocarbonyloxy, aminosulfonyloxy, amidino group, sulfonyloxy, isonitrile, cyanate, imide, iminium, ether, thioether and -L ３ -R ３６ Selected from, R ２１ ~R ３３ Two adjacent rings can bond to each other to form an optional aliphatic ring, an optional aromatic ring, or an optional mixed aliphatic and aromatic ring. A ３ and A ４ These are, independently, hydrogen, halogen, and a substituted C. １ -C １０ Alkyl, optionally substituted C ３ -C １０ Cycloalkyl, optionally substituted C １ -C １０ Heterocycloalkyl, optionally substituted C １ -C １０ Heteroalkyl, optionally substituted C ２ -C １０ Alkenyl, C may be substituted. ２ -C １０ Alkynyl, C may be substituted. １ -C １０ Selected from alkoxy, optionally substituted aryloxy and cyano, however A ３ and A ４ Except when both are hydrogen, X is O, S, CR ａ R ｂ , NR ａ or SiR ａ R ｂ And, R ａ and R ｂ C may be substituted independently of each other. １ -C ４０ Alkyl, optionally substituted C １ -C ４０ Heteroalkyl, optionally substituted C ３ -C ３０ Cycloalkyl, optionally substituted C ３ -C ３０ Heterocycloalkyl, optionally substituted C ５ -C ５０ Aryl and possibly substituted C ２ -C ５０ Selected from heteroaryls, or formed by bonding to each other, an optionally substituted aliphatic ring, an optionally substituted aromatic ring, or an optionally substituted aliphatic and aromatic mixed ring, R ２１ ~R ３３ At least one of them is -L ３ -R ３６ And, L ３ It is a linker containing hydrocarbons with 1 to 40 carbon atoms, R ３６ This is a nucleoside selected from chemical formulas 3 to 5 below, 【Transformation 5】 Here, * indicates that the nucleoside is L ３ This indicates the position where it will connect. B is a nuclear base, R ４０ is hydrogen, -P(OR ４３ ) (N(R ４４ R ４５ )) and -L ４ -R ４６ Selected from, R ４１ This refers to an alcohol protecting group, hydrogen or -P (OR ４３ ) (N(R ４４ R ４５ A support or nucleic acid which may be substituted, R ４３ ~R ４５ These are, independently, hydrogen and possibly substituted C. １ -C １０ Alkyl and optionally substituted C １ -C １０ Selected from heteroalkyl groups, L ４ The C atoms may be single bonds, phosphodiester bonds between nucleotides, or substituted. １ -C １０ Alkyl and optionally substituted C １ -C １０ Selected from heteroalkyl groups, R ４６ is hydroxy or -P(OR ４３ ) (N(R ４４ R ４５ A support or nucleic acid which may be substituted, R ４２ are hydrogen, hydroxyl, alkoxy and -OR ｐ It is one of the following that can be selected. R ｐ This is a protecting group.

10. The compound according to claim 9, characterized in that the optionally substituted support or nucleic acid is a support or nucleic acid substituted with coumarin, cyanine, bolus, furosein, rhodamine, pyrene, carbopyronine, oxazine, xanthene, thioxanthene, acridine and / or derivatives thereof and at least one selected from chemical formula 1.

11. A compound characterized in that the compound described in any one of claims 1 to 10 is a labeling reporter.

12. A conjugate comprising a compound according to any one of claims 1 to 10 as a labeling reporter.

13. The conjugate according to claim 12, characterized in that the conjugate is a nucleotide conjugate.

14. The conjugate according to claim 12, characterized in that the conjugate is a probe or a primer.

15. The conjugate according to claim 12, further comprising a minor groove binder (MGB).

16. The compound according to any one of claims 1 to 10; and Extinction photon; A conjugate that includes this.

17. The compound according to any one of claims 1 to 10; and Two or more extinction photons; A conjugate that includes this.

18. The conjugate according to claim 16, characterized in that the quencher is at least one selected from azo, coumarin, cyanine, bolus, furosein, rhodamine, pyrene, carbopyronine, oxazine, xanthene, thioxanthene, acridine, and derivatives thereof.

19. A nucleic acid detection composition comprising the conjugate described in claim 12.

20. The compound according to any one of claims 1 to 10; Support; and A connecting portion that connects the reporter and the support; including, Support for nucleic acid detection.

21. The nucleic acid detection support according to claim 20, characterized in that the support is glass, cellulose, nylon, acrylamide gel, dextran, polystyrene, or resin.

22. The linker may be replaced by C １ -C ３０ Alkyl, optionally substituted C ３ -C ３０ Cycloalkyl, optionally substituted C ２ -C ３０ Heteroalkyl, optionally substituted C ２ -C ３０ Heterocycloalkyl, optionally substituted C ２ -C ３０ Alkenyl, C may be substituted. ５ -C ５０ Aryl, C may be substituted. ２ -C ５０ A nucleic acid detection support according to claim 20, characterized by being selected from heteroaryls, amides (-CONH-), esters (-COO-), ketones (-CO-), nucleosides, and any combination thereof.

23. (a) A step of preparing a reaction mixture comprising a target nucleic acid, reagents necessary for amplifying the target nucleic acid, and the nucleotide conjugate according to claim 13; (b) a step of amplifying the target nucleic acid in the reaction mixture; and (c) A step of measuring the fluorescence intensity of the reaction mixture; including, Nucleic acid detection methods.

24. The aforementioned step (b) is, (b-1) A step in which the nucleotide conjugate hybridized to the target nucleic acid is extended by polymerase; (b-2) The step in which the reporter and quencher of the nucleotide conjugate are separated from the target nucleic acid by the exonuclease activity of the polymerase; and (b-3) The reporter released from the reporter emits fluorescence; The nucleic acid detection method according to claim 23, characterized by including the following:

25. The nucleic acid detection method according to claim 23, further comprising step (d) of measuring the amount of amplification of a target nucleic acid from the fluorescence intensity measured in step (c).

26. Step (b) above involves Strand Displacement Amplification (SDA), Polymerase Chain Reaction (PCR), Reverse Transcription Polymerase Chain Reaction (RT-PCR), Real-Time Polymerase Chain Reaction (Real-time Polymerase Chain Reaction), Allele-Specific Polymerase Chain Reaction (Allele-specific Polymerase Chain Reaction), and Ligase Chain Reaction (LCR). Reaction, Rolling Circle Amplification (RCA), Isothermal Multiple Displacement Amplification (IMDA), Recombinase Polymerase Amplification (RPA), Self-Sustained Sequence Replication (3SR), Single Primer Isothermal Amplification (SPIA), Multiple Displacement Amplification (MDA) Amplification, Whole Genome Amplification (WGA), Cross-priming Amplification (CPA), RNA Signal-Mediated Amplification of RNA Technology (SMART), Transcription-Mediated Amplification (TMA), Nucleic Acid Sequence-Based Amplification (NASBA), Loop-Mediated Isothermal Amplification (LAMP) Amplification, and helicase-dependent amplification (HDA).The nucleic acid detection method according to claim 23, characterized in that it is performed by a method selected from Amplification.

Images