Acridinium compounds having fused heterocycles

Abstract

Chemiluminescent acridinium conjugates and compounds capable of forming the conjugates are disclosed, which can be used as chemiluminescent tracers in immunoassays for the quantification and identification of specific analytes.

Description

[Technical Field]

[0001] The present disclosure relates to a class of chemiluminescent acridinium compounds containing one or more heterocyclyls, such as 2,3-cyclic alkylenedioxy, fused to the acridinium ring system. These substitutions result in high quantum yields and significantly improved chemiluminescence stability. The structural architecture of the acridinium compounds required for high quantum yields along with improved chemiluminescence stability is disclosed herein. [Background technology]

[0002] Acridinium-based chemiluminescence has been used in immunoassays for analytes in samples. By conjugating the acridinium system to a ligand or its binding partner, the chemiluminescence can be correlated with the presence or concentration of the analyte.

[0003] An early generation of stable acridiniums is the acridinium ester (AE) referred to as DMAE (dimethyl acridinium ester), shown in U.S. Patent No. 5,627,499 and U.S. Patent No. 5,627,499, which are incorporated herein by reference in their entireties, particularly with respect to the acridinium esters disclosed therein, such as DMAE. DMAE contains two methyl groups on the phenyl moiety, which flank the acridinium ester ring and stabilize the ester bond therebetween until, for example, chemiluminescence is triggered. DMAE-NHS labels can be used to form conjugates that can be used in immunoassays by conjugating them to reactive functional groups, such as N-hydroxysuccinimide (NHS) esters, which can be covalently attached to an analyte or its binding partner. DMAE-NHS has the following structure: [ka] wherein the numbering of the acridinium ring system is shown as used herein, and A - The same applies to the presence of counter ions. It has.

[0004] Modification of these acridinium compounds has resulted in changes in the properties of the compounds. For example, Patent Document 3, the entirety of which is incorporated herein by reference, includes a hydrophilic modification of DMAE (referred to as NSP-DMAE-NHS), in which the N-methyl group of DMAE is replaced with an N-sulfopropyl (NSP) group to form a zwitterionic compound. A zwitterion contains separated positive and negative charges within the same molecule with a net charge of zero. NSP-DMAE-NHS has the following structure: [ka] It has.

[0005] Similarly, U.S. Patent Nos. 5,999,049 and 5,999,052, both of which are incorporated herein by reference in their entireties, include AE ​​labels containing zwitterionic groups at one or more sites on the molecule, as well as other modifications to the acridinium ring system. Three labels of this AE group are referred to as ZAE, ISOZAE, and ISODIZAE, and have the structures: [ka] is.

[0006] US Patent Application Publication No. 2009 / 0129994, the entire contents of which are incorporated herein by reference, provides hydrophilic acridinium esters, such as HEGAE (1), in which a polyethylene glycol (PEG) moiety is introduced into the leaving group as a linker to a ligand or binding partner to detect an analyte. The incorporation of PEG increases the hydrophilicity of the label. HEGAE (1) has the following structure: [ka] It has.

[0007] US Patent Application Publication No. 2007 / 0129994, the entire contents of which are incorporated herein by reference, includes AEs that contain a branched PEG structure covalently attached to an acridinium ring system. The structural makeup of this molecular group is shown below: [ka]

[0008] U.S. Patent No. 5,999,222 and U.S. Patent No. 5,999,222, each of which is incorporated herein by reference in its entirety, detail acridinium compounds containing hydrophilic alkoxy groups at C2 and / or C7 of the acridinium ring, where the two oxygen atoms modify chemiluminescence output. The hydrophilic groups attached to the two oxygens also improve water solubility. In particular, the most useful labels, HQYAE (2) and TSPAE (3), possess essential parameters that make them superior to most other acridiniums in immunoassays. HQYAE contains two hydrophilic polyethylene glycol substituents. TSPAE contains two hydrophilic, negatively charged N-sulfopropyl groups at specific positions on the acridinium ring system. As a result, TSPAE is a water-soluble label that confers hydrophilicity to its conjugates, which can lower the isoelectric point (pI) of the labeled conjugates. TSPAE and HQYAE are acridinium labels that are increasingly being used in commercial immunoassays where high sensitivity is required. The structures of these compounds are as follows:

[0009] [ka]

[0010] HQYAE and TSPAE are increasingly being used in assays requiring high sensitivity, but suffer from stability issues. These labels must be formulated in slightly acidic pH buffers to maintain adequate chemiluminescence stability for long-term storage. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] U.S. Patent No. 4,918,192 [Patent Document 2] U.S. Patent No. 5,110,932 [Patent Document 3] U.S. Patent No. 5,656,426 [Patent Document 4] U.S. Patent No. 8,778,624 [Patent Document 5] U.S. Patent No. 9,575,062 [Patent Document 6] U.S. Patent No. 6,664,043 [Patent Document 7] U.S. Patent No. 11,332,445 [Patent Document 8] U.S. Patent No. 7,309,615 [Patent Document 9] U.S. Patent No. 7,785,904 Summary of the Invention [Problem to be solved by the invention]

[0012] There is a continuing need for chemiluminescent compounds that are stable while providing high sensitivity in the detection of analytes. [Means for solving the problem]

[0013] In accordance with the foregoing and other objectives, the present disclosure includes acridinium compounds that can be used in chemiluminescent assays. As shown herein, fusion of a heterocyclic group (e.g., a 5- to 10-membered heterocyclic group) to the acridinium ring system can affect the functionality of the compound, potentially improving stability and / or reaction rate and / or light output compared to other identical compounds with hydrogen at the fused positions (e.g., C2 and C3) or compounds with hydrogen and / or electron-donating groups at the fused positions. The chemiluminescent acridinium compounds of the present disclosure typically contain 2,3-cyclic alkylenedioxy (or dioxolo) substituents, which provide high quantum yields and significantly improved chemiluminescent stability. These compounds are useful in assays due to their high quantum yields and improved chemiluminescent stability.

[0014] For example, the structure of formula (I): [ka] wherein A is an analyte or a binding partner of an analyte; L is either absent (i.e., a bond) or a linker; Ψ has the following structure: [ka] wherein "j" is 1, 2, 3, 4, 5, or 6; R1 is hydrogen, -R, -X b , -R L -X b , -L1-R, -L1-X b , -Z, -R L -Z, -L1-Z, or -R L -L1-R L -Z; R2 and R3 are independently selected from hydrogen, -R, an electron donating group, or -Z, or R2 and R3 together form a 5- to 10-membered fused heterocyclyl group; Z, independently at each occurrence, has the structure: [ka] wherein "q" and "l" are independently 0 or 1; "r" is independently an integer from 0 to 10; L1 can be independently -O-, -S-, -NH-, -N(R N )-, -(CH2) 1~10 -, -S(=O) 1~2 -, -C=C-, -C=C-(CH2) 1~3 -, -C(O)-, -OC(O)-, -C(O)-(CH2) 1~4 -, -(CH2) 1~4 -C(O)-, -C(O)-O-, -C(O)-N(R N )-, -C(O)-NH-, -N(R N )-C(O)-, -NH-C(O)-, -C(O)-N(R N )-(CH2) 1~3 -, -(CH2) 1~3 -C(O)-N(R N)-, -NH-S(O) 1~2 -, -N(R N )-S(O) 1~2 -, -S(O) 1~2 -N(R N )-, -S(O) 1~2 -NH-, -(CH2) 1~3 -NH-S(O) 1~2 -, -(CH2) 1~3 -N(R N )-S(O) 1~2 -, -(CH2) 1~3 -S(O) 1~2 -N(R N )-, -(CH2) 1~3 -S(O) 1~2 -NH-, -O-(CH2) 1~4 -, -(CH2) 1~4 -O-, -S-(CH2) 1~4 -, -(CH2) 1~4 -S-, -NH-(CH2) 1~4 -, -N(R N )-(CH2) 1~4 -, -(CH2) 1~4 -N(R N )-, -(OCH2) 1~10 -, -(CHO) 1~10 -, -(OCH2CH2) 1~10 - or -(CH2CH2O) 1~10 - and; R L is independently C for each occurrence. 1~20 a divalent hydrocarbon (e.g., alkyl, alkenyl, aryl, phenyl, monoalkyl-substituted phenyl, dialkyl-substituted phenyl, alkynyl, arylalkyl), optionally having one or more (e.g., 1-10, 1-5) points of substitution (e.g., substituted with 1-10 heteroatoms and with 1-10 substituents); R is independently hydrogen or C at each occurrence. 1~35 is a hydrocarbon (e.g., alkyl, alkenyl, alkynyl, or aralkyl), optionally having one or more (e.g., 1-20, 1-10, 1-5) points of substitution (e.g., with 1-20 heteroatoms and with 1-20 substituents); R' and R" are independently hydrogen or C 1~10 is alkyl; X b is independently at each occurrence an anionic group; R N is, independently at each occurrence, hydrogen, or C 1~5 a chemiluminescent acridinium having a zwitterionic group selected from alkyl (e.g., methyl, ethyl, propyl) a compound having the formula: Salts thereof (e.g., halide salts such as chloride salts) are provided. In some embodiments, R3 is hydrogen. In various implementations, R2 is substituted with one or more independently selected substituents (e.g., -S(=O)) at one or more (e.g., 1, 2, 3) positions. 1-2 -R*, -OS(=O)2-R*, -S(=O)2-OR*, -O-SO 3、 -X such as -OS(=O)2-OR*, -OS(=O)-OR*, -OS(=O)-R*, -S(=O)-OR*, or -S(=O)-R* (wherein R* is H or C 1~10 is an alkoxy optionally substituted with a hydrocarbon;

[0015] Compounds for forming conjugates are also provided. For example, compounds (e.g., compounds for conjugating to an analyte or a binding partner of an analyte, such as a peptide, protein, or macromolecule, including an antibody) have the structure of formula (IV): [ka] where RFG is a reactive functional group for conjugating to an analyte or a binding partner of the analyte; L is either absent (i.e., a bond) or a linker; Ψ has the following structure: [ka] wherein "j" is 1, 2, 3, 4, 5, or 6; R1 is hydrogen, -R, -X, -R L -X b , -L1-R, -L1-X b , -Z, -R L -Z, -L1-Z, or -R L -L1-R L -Z; R2 and R3 are independently selected from hydrogen, -R, an electron donating group, or -Z, or R2 and R3 together form a 5- to 10-membered fused heterocyclyl group; Z, independently at each occurrence, has the structure: [ka] wherein "q" and "l" are independently 0 or 1; "r" is independently an integer from 0 to 10; L1 can be independently -O-, -S-, -NH-, -N(R N )-, -(CH2) 1~10 -, -S(=O) 1~2 -, -C=C-, -C=C-(CH2) 1~3 -, -C(O)-, -OC(O)-, -C(O)-(CH2) 1~4 -, -(CH2) 1~4 -C(O)-, -C(O)-O-, -C(O)-N(R N )-, -C(O)-NH-, -N(R N )-C(O)-, -NH-C(O)-, -C(O)-N(R N )-(CH2) 1~3 -, -(CH2) 1~3 -C(O)-N(R N )-, -NH-S(O) 1~2 -, -N(R N )-S(O) 1~2 -, -S(O) 1~2 -N(R N )-, -S(O) 1~2 -NH-, -(CH2) 1~3 -NH-S(O) 1~2 -, -(CH2) 1~3 -N(RN )-S(O) 1~2 -, -(CH2) 1~3 -S(O) 1~2 -N(R N )-, -(CH2) 1~3 -S(O) 1~2 -NH-, -O-(CH2) 1~4 -, -(CH2) 1~4 -O-, S-(CH2) 1~4 -, -(CH2) 1~4 -S-, -NH-(CH2) 1~4 -, -N(R N )-(CH2) 1~4 -, -(CH2) 1~4 -N(R N )-, -(OCH2) 1~10 -, -(CHO) 1~10 -, -(OCH2CH2) 1~10 - or -(CH2CH2O) 1~10 - and; R L is independently C for each occurrence. 1~20 a divalent hydrocarbon (e.g., alkyl, alkenyl, aryl, phenyl, monoalkyl-substituted phenyl, dialkyl-substituted phenyl, alkynyl, arylalkyl), optionally having one or more (e.g., 1-10, 1-5) points of substitution (e.g., substituted with 1-10 heteroatoms, substituted with 1-10 substituents); R is independently hydrogen or C at each occurrence. 1~35 is a hydrocarbon (e.g., alkyl, alkenyl, alkynyl, or aralkyl), optionally having one or more (e.g., 1-20, 1-10, 1-5) points of substitution (e.g., substituted with 1-20 heteroatoms, substituted with 1-20 substituents); R' and R" are independently hydrogen or C 1~10 is alkyl; X b is independently at each occurrence an anionic group; R N is, independently at each occurrence, hydrogen, or C 1~5In certain embodiments, R1 is selected from the group consisting of -R, -X ... b , -R L -X b , -L1-R, -L1-X b , -Z, -R L -Z, -L1-Z, or -R L -L1-R L -Z.

[0016] In various implementations, the compounds for forming the conjugate include the following: [ka] [ka] [ka] [ka] is selected from.

[0017] Methods for forming a conjugate are provided. In some embodiments, the methods may include reacting a compound for forming the conjugate with an analyte or a binding partner of the analyte (e.g., an antibody) to form the conjugate.

[0018] In another aspect of the present invention, a reagent for detecting an analyte is provided, comprising a detectable conjugate having a chemiluminescent acridinium with a heterocycle fused to an acridinium ring structure. The detectable conjugate may contain one or more (e.g., one, two) zwitterionic functional groups. The concentration of the reagent can be selected relative to the sensitivity of the assay, with higher concentrations being preferred for assays requiring greater sensitivity. For example, the concentration of the detectable analyte in a sample can be selected from 10 -3In some embodiments, the concentration of detectable conjugate in the sample may be less than 10 -15 M~10 -3 In various implementations, the concentration of detectable conjugate in the reagent may be 10 -3 In some embodiments, the concentration of detectable conjugate in the reagent may be less than 10 -15 ~10 -3 It may also be M.

[0019] In a further aspect of the invention, there is provided an assay for the detection or quantification of an analyte in a sample, the assay comprising: (a) providing a detectable conjugate having the structure of formula (I); (b) providing a solid support having immobilized thereon a molecule capable of forming a binding complex with the analyte and capable of forming a binding complex with the detectable conjugate; (c) mixing the compound, the solid support, and the sample; (d) separating the solid support from the mixture; (e) inducing chemiluminescence of any acridinium label complexed to the solid phase; (f) measuring the amount of light emitted with a luminometer; (g) detecting the presence of, or calculating the concentration of, an analyte by comparing the amount of light emitted with a standard dose-response curve relating the amount of light emitted to a known concentration of the analyte; Includes. In some embodiments, the sample is serum.

[0020] These and other aspects of the present invention will be better understood with reference to the following detailed description, including the appended claims. [Brief explanation of the drawings]

[0021] [Figure 1A]Figure 1 (1A-1E) are measured emission spectra of several acridinium compounds described herein. Figure 1A is the emission spectrum of ADOAE A (5). [Figure 1B] Figure 1 (1A-1E) shows the measured emission spectra of several acridinium compounds described herein. Figure 1B shows the emission spectra of ADOAE D (7) and ADOAE E (8). [Figure 1C] Figure 1 (1A-1E) are measured emission spectra of several acridinium compounds described herein. Figure 1C is the emission spectrum of ADOAE G(10). [Figure 1D] Figure 1 (1A-1E) are measured emission spectra of several acridinium compounds described herein. Figure 1D is the emission spectrum of ADOAE I (12). [Figure 1E] Figures 1 (1A-1E) show the measured emission spectra of several acridinium compounds described herein. Figure 1E shows the emission spectrum of ADOAE K (14). [Figure 2] Figure 1 shows the chemiluminescence kinetics of the acridinium esters HEGAE (1), HQYAE (2), and ADOAE D-L (7-15). As can be seen, ADOAE F (9), ADOAE H (11), ADOAE J (13), and ADOAE L (15) each have faster chemiluminescence kinetics than the other compounds tested. [Figure 3-1] Figure 3 (3A-3D) shows the structures of a comparative acridinium (Figure 3A) and exemplary ADOAEs (3B-3D) described and used in the synthesis, measurements, and analyses provided herein. [Figure 3-2] Same as above. [Figure 3-3] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0022] For convenience, certain terms employed herein, including the examples and appended claims, are collected here. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0023] Unless expressly defined otherwise, the following terms and phrases are intended to have the following meanings throughout this disclosure:

[0024] All percentages set forth herein refer to the weight percentage of the particular component relative to the total composition, including the carrier, unless otherwise indicated. It will be understood that the sum of the total weight percentages of the individual components within a composition does not exceed 100%.

[0025] As used herein, the terms "a" or "an" mean one or more. As used herein, the term "consisting essentially of" is intended to limit the invention to certain materials or steps, as will be understood upon reading the specification, and to those that do not materially affect the basic and novel characteristics of the claimed invention. The term "comprising" includes "consisting essentially of" and "consisting of." When a compound contains a designated chemical moiety, that chemical moiety is part of the compound and includes any number of substituents at any position occupied by hydrogen in the designated structure. For example, a compound containing a designated structure (e.g., structure A, L, Ψ of Formula (I)) may contain, for example, optionally substituted C1-C 35 The hydrocarbons may be independently substituted one or more times.

[0026] The following definitions of the various groups or substituents are used unless otherwise noted. The specific and general values ​​listed below for radicals, substituents, and ranges are for illustrative purposes only; they do not exclude other defined values ​​or other values ​​within defined ranges for radicals and substituents. Unless otherwise indicated, alkyl, alkenyl, alkynyl, alkoxy, etc., refer to straight-chain, branched, and cyclic groups, and any combination thereof.

[0027] The term hydrocarbon may refer to a radical or group containing carbon and hydrogen atoms that may be attached at the positions indicated (e.g., R, R', R", L, L C , R L , R C , R1, R2, R3, R4, R5, R6, R7, R8). Examples of hydrocarbon radicals include, but are not limited to, alkyl, alkenyl, alkynyl, aryl, aryl-alkyl, alkyl-aryl, and any combination thereof (e.g., alkyl-aryl-alkyl). As used herein, unless otherwise specified, hydrocarbon is a monovalent or polyvalent (e.g., divalent, trivalent) hydrocarbon radical. -(CH2) including methylene radical, i.e., -CH2-. n Radicals of the form - are considered alkyl radicals if there are no unsaturated bonds between carbon atoms. Unless otherwise specified, all hydrocarbon radicals (including substituted and unsubstituted alkyl, alkenyl, alkynyl, aryl, aryl-alkyl, and alkyl-aryl) may have 1 to 35 carbon atoms. In other embodiments, the hydrocarbon has 1 to 20, or 1 to 12, or 1 to 8, or 1 to 6, or 1 to 3 carbon atoms, including embodiments having, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. The hydrocarbon may have 2 to 70 atoms, or 4 to 40 atoms, or 4 to 20 atoms.

[0028] Substituted hydrocarbons may have one or more hydrocarbon radicals as substituents, may have substituted hydrocarbon radicals, or may contain one or more heteroatoms. Any of the hydrocarbon substituents disclosed herein (e.g., R, R', R", L, L) may be substituted or unsubstituted. C , R L , R C , R1, R2, R3, R4, R5, R6, R7, R8) may optionally contain 1 to 20 (e.g., 1 to 10, 1 to 5) heteroatoms. Examples of substituted hydrocarbon radicals include, but are not limited to, heterocycles such as heteroaryl. Unless otherwise specified, hydrocarbons substituted with one or more heteroatoms contain 1 to 20 heteroatoms. In other embodiments, hydrocarbons substituted with one or more heteroatoms contain 1 to 12, or 1 to 8, or 1 to 6, or 1 to 4, or 1 to 3, or 1 to 2 heteroatoms. Examples of heteroatoms include, but are not limited to, oxygen, nitrogen, sulfur, phosphorus, halogens (e.g., F, Cl, Br, I), boron, or silicon. In some embodiments, heteroatoms are selected from oxygen, nitrogen, sulfur, phosphorus, and halogens (e.g., F, Cl, Br, I). In certain embodiments, heteroatoms can be selected from O, N, or S. In some embodiments, a heteroatom or group may replace a carbon. In some embodiments, a heteroatom or group may replace a hydrogen. In some embodiments, a substituted hydrocarbon may contain one or more heteroatoms in the backbone or chain of the molecule (e.g., sandwiched between two carbon atoms, as in "oxa"). In some embodiments, a substituted hydrocarbon may contain one or more heteroatoms pendant from the backbone or chain of the molecule (e.g., covalently bonded to a carbon atom in the chain or backbone, as in "oxo").

[0029] Where a designated group is substituted with a designated substituent, the particular group can be substituted with one or more, or all, of any of the designated substituents. For example, when a group such as an alkyl group or heteroaryl group is described as "unsubstituted C1-C20 When "substituted with alkyl, or unsubstituted 2- to 20-membered heteroalkyl," the group is substituted with one or more unsubstituted C1-C 20 The term "R-substituted" refers to a group that is substituted with at least one R substituent, and each R substituent is optionally different. When a designated group occurs more than once in a chemical genus (e.g., an R group), it will be understood that each group is independently selected at each occurrence.

[0030] Unless otherwise specified, any compound disclosed herein having one or more chiral centers may be in the form of a racemic mixture with respect to each chiral center, or may be present as a pure or substantially pure (e.g., greater than 98% ee) R or S enantiomer with respect to each chiral center, or as a mixture of R or S enantiomers with respect to each chiral center, wherein the mixture contains an enantiomeric excess of one or the other configuration, for example, an enantiomeric excess of greater than 60%, or greater than 70%, or greater than 80%, or greater than 90%, or greater than 95%, or greater than 98%, or greater than 99% (R or S) enantiomeric excess. In some embodiments, any chiral center may be in the "S" or "R" configuration.

[0031] It will be understood that the description of compounds herein is limited by the principle of chemical bond.Therefore, when group is substituted by one or more of a large number of substituents, such substituents are selected, for example, according to the principle of chemical bond related to atomic valence, so as to obtain a compound that is not inherently unstable.For example, any carbon atom is bonded to two, three or four other atoms that correspond to the four valence electrons of carbon.

[0032] Substituent (radical) prefix names can be derived from the parent hydride by (i) replacing "ane" in the parent hydride with the suffix "yl," "diyl," "triyl," or "tetrayl," or (ii) replacing "e" in the parent hydride with the suffix "yl," "diyl," "triyl," or "tetrayl," where the atom with a free valence, if specified, is given the lowest possible number consistent with any established numbering of the parent hydride. Accepted abbreviations, such as adamantyl, naphthyl, anthryl, phenanthryl, furyl, pyridyl, isoquinolyl, quinolyl, and piperidyl, as well as trivial names, such as vinyl, allyl, phenyl, and thienyl, are also used throughout this specification.

[0033] Generally, anionic groups (X a and / or X b ) can provide an anionic charge to counterbalance any directly or indirectly covalently attached cationic charge and form a zwitterion. In some embodiments, X a and X b independently for each occurrence, a carboxylate (-C(O)O - ), sulfonate (-SO3 - ), sulfate (-OSO3 - ), phosphate (-OP(O)(OR P )O - ), or oxide (-O - ) and R P is hydrogen or C optionally substituted with up to 10 heteroatoms 1~12 It is a hydrocarbon.

[0034] An alkyl group typically refers to a saturated hydrocarbon chain, which may be straight or branched, containing the indicated number of carbon atoms. For example, C 1~C6 alkyl indicates that the group may have 1 to 6 (inclusive) carbon atoms therein. Any atom can be optionally substituted, for example, by one or more substituents. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, and tert-butyl. Any alkyl group referred to herein (e.g., R, R', R", L, L) can be substituted with any of the following: C , R L , R C , R1, R2, R3, R4, R5, R6, R7, R8) can have 1 to 35 carbon atoms. In other embodiments, the alkyl group has 1 to 20, or 1 to 12, or 1 to 8, or 1 to 6, or 1 to 3 carbon atoms, including embodiments having, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. The alkyl group can be a lower alkyl (e.g., C 1~ C alkyl). Alkyl groups substituted with one or more heteroatoms (e.g., N, O, halogen) include heteroalkyl groups, such as amino groups (e.g., alkylamino, dialkylamino), alkoxy groups, or haloalkyl groups.

[0035] A haloalkyl group is typically an alkyl group in which at least one hydrogen atom is replaced by halo. In some embodiments, two or more hydrogen atoms (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) are replaced by halo. In these embodiments, the hydrogen atoms can each be replaced by the same halogen (e.g., fluoro), or the hydrogen atoms can be replaced by a combination of different halogens (e.g., fluoro and chloro). Haloalkyl can also include an alkyl moiety in which all hydrogen atoms are replaced by halo (sometimes referred to herein as perhaloalkyl, including, for example, perfluoroalkyl such as trifluoromethyl). Haloalkyl groups can be optionally substituted.

[0036] Typically, an alkoxy group has the formula -O(alkyl). Alkoxy can be, for example, methoxy (-OCH), ethoxy, propoxy, isopropoxy, butoxy, iso-butoxy, sec-butoxy, pentoxy, 2-pentoxy, 3-pentoxy, or hexyloxy. Similarly, the term "thioalkoxy" refers to a group of the formula -S(alkyl). Finally, the terms "haloalkoxy" and "halothioalkoxy" refer to -O(haloalkyl) and -S(haloalkyl), respectively. The term "sulfhydryl" refers to -SH. As used herein, the term "hydroxyl," employed alone or in combination with other terms, refers to a group of the formula -OH. Any alkoxy, thioalkoxy, or haloalkoxy group referred to herein (e.g., R, R', R", L, L) is an alkyl group. C , R L , R C , R1, R2, R3, R4, R5, R6, R7, R8) can have 1 to 35 carbon atoms. In other embodiments, the alkoxy, thioalkoxy, or haloalkoxy group has 1 to 20, or 1 to 12, or 1 to 8, or 1 to 6, or 1 to 3 carbon atoms, including embodiments having, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. Alkoxy groups include lower alkoxy (e.g., C 1~ C4 alkoxy).

[0037] An aralkyl group typically refers to a group of an alkyl moiety in which an alkyl hydrogen atom is replaced by an aryl group. One of the carbon atoms of the alkyl moiety serves as the point of attachment of the aralkyl group to another moiety. Any ring or chain atom can be optionally substituted, for example, by one or more substituents. Non-limiting examples of aralkyls include benzyl, 2-phenylethyl, and 3-phenylpropyl groups. Aralkyl groups substituted with one or more heteroatoms (e.g., N, O, halogen) include heteroarylalkyl groups, such as amino groups (e.g., arylamino), aryloxy groups, or haloarylalkyl groups.

[0038] The term "alkenyl" can refer to a straight or branched hydrocarbon chain containing the indicated number of carbon atoms and having one or more carbon-carbon double bonds. Any atom can be optionally substituted, for example, by one or more substituents. Alkenyl groups include, for example, vinyl, allyl, 1-butenyl, and 2-hexenyl. One of the double-bonded carbons can optionally be the point of attachment of the alkenyl substituent. Any alkenyl group referred to herein (e.g., R, R', R", L, L) can be any of the following: C , R L , R C , R1, R2, R3, R4, R5, R6, R7, R8) can have 1 to 35 carbon atoms. In other embodiments, alkenyl groups have 1 to 20, or 1 to 12, or 1 to 8, or 1 to 6, or 1 to 3 carbon atoms, including embodiments having, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. Alkenyl groups substituted with one or more heteroatoms (e.g., N, O, halogen) include heteroalkenyl groups, such as amino groups (e.g., alkenylamino, alkenylalkylamino), alkenyloxy groups, or haloalkenyl groups.

[0039] The term alkynyl can refer to a straight or branched hydrocarbon chain containing the indicated number of carbon atoms and having one or more carbon-carbon triple bonds. Alkynyl groups (e.g., R, R', R", L, L) can be any group. C , R L , R C, R1, R2, R3, R4, R5, R6, R7, R8) can be optionally substituted, for example, by one or more substituents. Alkynyl groups include, for example, ethynyl, propargyl, and 3-hexynyl. One of the triple bond carbons can optionally be the attachment point of the alkynyl substituent. Alkynyl groups substituted with one or more heteroatoms (e.g., N, O, halogen) include heteroheteroalkynyl groups, for example, amino groups (e.g., alkynylamino, alkenylalkylamino), alkynyloxy groups, or haloalkynyl groups.

[0040] The term heterocyclyl typically refers to a fully saturated, partially saturated, or aromatic monocyclic, bicyclic, tricyclic, or other polycyclic ring system, which may contain one or two additional groups (e.g., R N It is understood that ) may be present), or S, and has one or more constituent heteroatom ring atoms independently selected from. The present disclosure is premised in part on the installation of one or more fused heterocyclyl groups on the acridinium. A heteroatom or ring carbon may be the point of attachment of the heterocyclyl substituent to another moiety. Any atom can be optionally substituted, for example, with one or more substituents (e.g., heteroatom or group X). Heterocyclyl groups include, for example, tetrahydrofuryl, tetrahydropyranyl, piperidyl (piperidino), piperazinyl, morpholinyl (morpholino), pyrrolinyl, and pyrrolidinyl. By way of example, the phrase "a heterocycle containing 5 to 6 ring atoms, wherein 1 to 2 ring atoms are independently selected from N, NH, N(C-C alkyl), NC(O)(C-C alkyl), O, and S; said heterocycle is optionally substituted with 1 to 3 independently selected R" includes, but is not limited to, tetrahydrofuryl, tetrahydropyranyl, piperidyl (piperidino), piperazinyl, morpholinyl (morpholino), pyrrolinyl, and pyrrolidinyl.

[0041] The term heterocycloalkenyl typically refers to a partially unsaturated monocyclic, bicyclic, tricyclic, or other polycyclic hydrocarbon group having one or more (e.g., 1 to 4) heteroatom ring atoms independently selected from O, N (it is understood that one or two additional groups may be present to satisfy the nitrogen valence and / or form salts), or S. A ring carbon (e.g., saturated or unsaturated) or heteroatom may be the point of attachment of the heterocycloalkenyl substituent. Any atom can be optionally substituted, for example, by one or more substituents. Heterocycloalkenyl groups include, for example, dihydropyridyl, tetrahydropyridyl, dihydropyranyl, 4,5-dihydrooxazolyl, 4,5-dihydro-1H-imidazolyl, 1,2,5,6-tetrahydro-pyrimidinyl, and 5,6-dihydro-2H-[1,3]oxazinyl.

[0042] A cycloalkyl group can be a fully saturated monocyclic, bicyclic, tricyclic, or other polycyclic hydrocarbon group. Any atom can be optionally substituted, for example, by one or more substituents. A ring carbon serves as the attachment point of the cycloalkyl group to another moiety. Examples of cycloalkyl moieties include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, and norbornyl (bicyclyl[2.2.1]heptyl). Cycloalkyl groups substituted with one or more heteroatoms (e.g., N, O, halogen) include heterocycloalkyl groups such as oxiranyl, oxetanyl, azetidinyl, aziridinyl, furanyl, pyranyl, pyrrolidinyl, piperidinyl, thiiranyl, thietanyl, tetrahydrothiphenyl, thiopyranyl, or halocycloakyl.

[0043] A cycloalkenyl group can be a partially unsaturated monocyclic, bicyclic, tricyclic, or other polycyclic hydrocarbon group. A ring carbon (e.g., saturated or unsaturated) is the point of attachment of the cycloalkenyl substituent. Any atom can be optionally substituted, for example, by one or more substituents. Examples of cycloalkenyl moieties include cyclohexenyl, cyclohexadienyl, or norbornenyl. Cycloalkenyl groups substituted with one or more heteroatoms (e.g., N, O, halogen) include heterocycloalkenyl groups such as oxiranyl, oxetanyl, azetidinyl, aziridinyl, furanyl, pyranyl, pyrrolidinyl, piperidinyl, thiiranyl, thietanyl, tetrahydrothiphenyl, thiopyranyl, or halocycloalkenyl.

[0044] Aryl groups are often aromatic monocyclic, bicyclic (two fused rings), tricyclic (three fused rings), or polycyclic (more than three fused rings) hydrocarbon ring systems. One or more ring atoms can be optionally substituted, for example, by one or more substituents. Aryl moieties include, for example, phenyl and naphthyl. Cycloalkenyl groups substituted with one or more heteroatoms (e.g., N, O, halogen) include heteroaryl or haloaryl groups.

[0045] Heteroaryl groups are typically aromatic monocyclic, bicyclic (two fused rings), tricyclic (three fused rings), or polycyclic (more than three fused rings) hydrocarbon groups having one or more heteroatom ring atoms independently selected from O, N (it being understood that one or two additional groups may be present to satisfy the valence of the nitrogen and / or form salts) in the ring, or S. One or more ring atoms can be optionally substituted, for example, by one or more substituents. Examples of heteroaryl groups include, but are not limited to, 2H-pyrrolyl, 3H-indolyl, 4H-quinolizinyl, acridinyl, benzo[b]thienyl, benzothiazolyl, β-carbolinyl, carbazolyl, coumarinyl, chromenyl, cinnolinyl, dibenzo[b,d]furanyl, furazanyl, furyl, imidazolyl, imidizolyl, indazolyl, indolyl, isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, naphthyl, and aryl. Examples include lysinyl, oxazolyl, perimidinyl, phenanthridinyl, phenanthrolinyl, phenarsazinyl, phenazinyl, phenothiazinyl, phenoxathiinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolyl, quinazolinyl, quinolyl, quinoxalinyl, thiadiazolyl, thianthrenyl, thiazolyl, thienyl, triazolyl, and xanthenyl.

[0046] In general, when the definition of a particular variable includes both hydrogen and non-hydrogen (halo, alkyl, aryl) possibilities, the term "non-hydrogen substituents" refers collectively to the non-hydrogen possibilities of that particular variable unless otherwise specified.

[0047] In general, the endpoints of any range described herein are to be understood as falling within the scope of the invention and as disclosed embodiments. Furthermore, half-integer values ​​within the range are also contemplated. For example, a range of 0 to 4 explicitly discloses 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, and any subset within that range (e.g., 1 to 2.5).

[0048] The term "substituent" can refer to a group that is "substituted" on a hydrocarbon (e.g., an alkyl, haloalkyl, cycloalkyl, heterocyclyl, heterocycloalkenyl, cycloalkenyl, aryl, or heteroaryl group) at any atom of that group, replacing one or more atoms. In one aspect, a substituent (e.g., R, R', R", L, L) of a group can be "substituted" on any atom of that group, replacing one or more atoms. C , R L , R C , R1, R2, R3, R4, R5, R6, R7, R8) are independently any single combination, or any combination of two or more, of the permissible atoms or groups of atoms described for that substituent. In another embodiment, a substituent can itself be substituted with any one of the above substituents. Furthermore, as used herein, the phrase "optionally substituted" means unsubstituted (e.g., substituted with H) or substituted. It is understood that substitution of a particular atom is limited by valence. Common substituents include halo (e.g., F), C, C- ... 1~12 Straight or branched chain alkyl, C 2~12 Alkenyl, C 2~12 Alkynyl, C 3~12 Cycloalkyl, C 6~12 Aryl, C 3~12 Heteroaryl, C 3~12 Heterocyclyl, C 1~12These include alkylsulfonyl, nitro, cyano, -COOR, -C(O)NRR', -OR, -SR, -NRR', and oxo, including mono-, di-, or tri-substituted moieties such as trifluoromethoxy, chlorine, bromine, fluorine, methyl, methoxy, pyridyl, furyl, triazyl, piperazinyl, pyrazoyl, imidazoyl, and the like, each optionally containing one or more heteroatoms such as halo, N, O, S, and P. R and R' are independently hydrogen, C 1~12 Alkyl, C 1~12 Haloalkyl, C 2~12 Alkenyl, C 2~12 Alkynyl, C 3~12 Cycloalkyl, C 4~24 Cycloalkylalkyl, C 6~12 Aryl, C 7~24 Aralkyl, C 3~12 Heterocyclyl, C 3~24 Heterocyclylalkyl, C3-12 heteroaryl, or C 4~24 Heteroarylalkyl. Unless otherwise specified, all groups described herein optionally contain one or more common substituents, as allowed by valence. Furthermore, as used herein, the phrase "optionally substituted" means unsubstituted (e.g., substituted with H) or substituted. As used herein, the term "substituted" means that a hydrogen and / or carbon atom has been removed and replaced with a substituent (e.g., a common substituent). The use of a prefix name of a substituent (radical), such as alkyl, without the modifier "optionally substituted" or "substituted" is understood to mean that the particular substituent is unsubstituted. However, the use of "haloalkyl" without the modifier "optionally substituted" or "substituted" is still understood to mean an alkyl group in which at least one hydrogen atom has been replaced with halo and, optionally, any other related substituents.

[0049] When a moiety of a compound of the present disclosure is described as an analyte or its binding partner, it is understood that a covalent bond is formed with the analyte or its binding partner (e.g., using a reactive functional group that forms a covalent bond), e.g., by replacing a hydrogen of the unconjugated analyte or its binding partner with a covalent bond to the indicated moiety. The covalent bond of the analyte or its binding partner can be formed, for example, with a group on the analyte, its binding partner, or with a derivatized version of the analyte that includes a group for forming a bond. This group can be, for example, an amine group, a thiol group, a carboxy group, a maleimidyl group, or a carbohydrate group. For example, when a covalent bond is formed through a primary amine on the analyte or its binding partner, the compound has the following structure: [ka] where the unconjugated analyte or binding partner A has the structure A'-NH. In some embodiments, when the covalent bond is formed through a thiol of the analyte or its binding partner, the compound may have the following structure: [ka] where the unconjugated analyte or binding partner A has the structure A'-SH. may have

[0050] In some embodiments, any hydrocarbon or substituted hydrocarbon (e.g., R, R′, R″, L, L) disclosed herein can be used. C , R L , R C, R1, R2, R3, R4, R5, R6, R7, R8) are one or more (e.g., 1 to 6, or 1 to 4, or 1 to 3, or 1, 2, or 3) substituents X, where X is independently selected at each occurrence from one or more (e.g., 1 to 20) heteroatoms or one or more (e.g., 1 to 10) heteroatom-containing groups, or X is independently selected at each occurrence from -F, -Cl, -Br, -I, -OH, -OR*, -NH2, -NHR*, -N(R*), -N(R*). + , -N(R*)-OH, -N(→O)(R*)2, -ON(R*)2, -N(R*)-OR*, -N(R*)-N(R*)2, -C=NR*, -N=C(R*)2, -C=NN(R*)2, -C(=NR*)(-N (R*)2), -C(H)(=N-OH), -SH, -SR*, -CN, -NC, -CHF2, -CCl3, -CF2Cl, -CFCl2, -C(=O)-R*, -CHO, -CO2H, -C(O)CH3, -CO2 - , -CO2R*, -C(=O)-SR*, -O-(C=O)-H, -O-(C=O)-R*, -SC(=O)-R*, -(C=O)-NH2, -C(=O)-N(R *)2, -C(=O)-NHNH2, -OC(=O)-NHNH2, -C(=S)-NH2, -(C=S)-N(R*)2, -N(R*)-CHO, -N(R*)- C(=O)-R*, -C(=NR)-OR*, -OC(=NR*)-R*, -SCN, -NCS, -NSO, -SSR*, -N(R*)-C(=O)-N(R*)2 , -CH3, -CH2-CH3, -CH2-CH2-CH3, -C(H)(CH2)2, -C(CH3)3, -N(R*)-C(=S)-N(R*)2, -S(=O) 1~2 -R*, -OS(=O)2-R*, -S(=O)2-OR*, -N(R*)-S(=O)2-R*, -S(=O)2-N(R*)2, -O-SO3, -OS(=O)2-OR*, -OS(=O)-OR*, -OS(=O)-R*, -S(= O)-OR*, -S(=O)-R*, -NO, -NO2, -NO3, -O-NO, -O-NO2, -N3, -N2-R*, -N(C2H4), -Si(R*)3, -CF3, -O-CF3, -O-CHF2, -O-CH3, -O-(CH2)1~6 and R* is selected from CH3, -OC(H)(CH2)2-OC(CH3)3, -PR*2, -OP(=O)(OR*)2, or -P(=O)(OR*)2, where, independently at each occurrence, R* is selected from H or C 1~10 Or C 1~8 Or C 1~6 Or C 1~4 X may be a hydrocarbon, including, but not limited to, alkyl, alkenyl, alkynyl, aryl (e.g., phenyl), alkyl-aryl (e.g., benzyl), and aryl-alkyl (e.g., tolyl). In some embodiments, X may comprise a C1-C8, C1-C6, or C2-C4 perfluoroalkyl. In some embodiments, X may comprise a C1-C8, C2-C6, or C3-C5 heterocycle (e.g., a heteroaryl radical). The term "halo" or "halogen" refers to any radical of fluorine, chlorine, bromine, or iodine. In some embodiments, X at each occurrence is independently selected from -OH, -SH, -NH2, -N(R*), -C(O)OR*, -C(O)NR*R*, -C(O)NR*R*, -C(O)OH, -C(O)NH2, F, or -Cl. In some embodiments, X is F. R and R*, independently at each occurrence, may be saturated or unsaturated alkyl (e.g., C1-C8 alkyl). In some embodiments, R and R* are independently selected from hydrogen, methyl, ethyl, propyl, or isopropyl. In some embodiments, R and R* are independently selected from hydrogen, methoxy, ethoxy, propoxy, or isopropoxy. In some embodiments, X is -CF3 or -O-CF3.

[0051] Structure of Formula (I): [ka] wherein A is an analyte or a binding partner of an analyte; L is either absent (i.e., a bond) or a linker; Ψ has the following structure: [ka] wherein "j" is 1, 2, 3, 4, 5, or 6; R1 is hydrogen, -R, -X b , -R L -X b , -L1-R, -L1-X b , -Z, -R L -Z, -L1-Z, or -R L -L1-R L -Z; R2 and R3 are independently selected from hydrogen, -R, an electron donating group, or -Z, or R2 and R3 together form a 5- to 10-membered fused heterocyclyl group; Z, independently at each occurrence, has the structure: [ka] wherein "q" and "l" are independently 0 or 1; "r" is independently an integer from 0 to 10; L1 can be independently -O-, -S-, -NH-, -N(R N )-, -(CH2) 1~10 -, -S(=O) 1~2 -, -C=C-, -C=C-(CH2) 1~3 -, -C(O)-, -OC(O)-, -C(O)-(CH2) 1~4 -, -(CH2) 1~4 -C(O)-, -C(O)-O-, -C(O)-N(R N )-, -C(O)-NH-, -N(R N )-C(O)-, -NH-C(O)-, -C(O)-N(R N )-(CH2) 1~3 -, -(CH2) 1~3 -C(O)-N(R N )-, -NH-S(O) 1~2 -, -N(R N )-S(O) 1~2 -, -S(O) 1~2 -N(R N )-, -S(O) 1~2 -NH-, -(CH2) 1~3-NH-S(O) 1~2 -, -(CH2) 1~3 -N(R N )-S(O) 1~2 -, -(CH2) 1~3 -S(O) 1~2 -N(R N )-, -(CH2) 1~3 -S(O) 1~2 -NH-, -O-(CH2) 1~4 -, -(CH2) 1~4 -O-, -S-(CH2) 1~4 -, -(CH2) 1~4 -S-, -NH-(CH2) 1~4 -, -N(R N )-(CH2) 1~4 -, -(CH2) 1~4 -N(R N )-, -(OCH2) 1~10 -, -(CHO) 1~10 -, -(OCH2CH2) 1~10 - or -(CH2CH2O) 1~10 - and; R L is independently C for each occurrence. 1~20 a divalent hydrocarbon group (e.g., alkyl, alkenyl, aryl, phenyl, monoalkyl-substituted phenyl, dialkyl-substituted phenyl, alkynyl, arylalkyl), optionally having one or more (e.g., 1-10, 1-5) points of substitution (e.g., with 1-10 heteroatoms such as a heteroalkyl, heteroalkenyl, heteroaryl, heteroalkynyl, or heteroarylalkyl group, with 1-10 substituents such as one or more groups -X); R is independently hydrogen or C at each occurrence. 1~35 a hydrocarbon (e.g., alkyl, alkenyl, alkynyl, or aralkyl) group, optionally having one or more (e.g., 1-20, 1-10, 1-5) points of substitution (e.g., with 1-20 heteroatoms such as a heteroalkyl, heteroalkenyl, heteroaryl, heteroalkynyl, or heteroarylalkyl group, with 1-20 substituents such as one or more groups -X); R' and R" are independently hydrogen or C 1~10 is alkyl; X b is independently at each occurrence an anionic group; R N is, independently at each occurrence, hydrogen, or C 1~5 a chemiluminescent acridinium having a zwitterionic group selected from alkyl (e.g., methyl, ethyl, propyl) or a compound (e.g., a detectable conjugate of an analyte or a binding partner of an analyte) having Salts thereof (e.g., halide salts such as chloride salts) are provided. In some embodiments, R3 is hydrogen. In various implementations, R2 is substituted with one or more independently selected substituents (e.g., -S(=O)) at one or more (e.g., 1, 2, 3) positions. 1-2 -R*, -OS(=O)2-R*, -S(=O)2-OR*, -O-SO 3、 -X such as -OS(=O)2-OR*, -OS(=O)-OR*, -OS(=O)-R*, -S(=O)-OR*, or -S(=O)-R* (wherein R* is H or C 1~10 In some embodiments, R is an alkoxy optionally substituted with -R, -X b , -R L -X b , -L1-R, -L1-X b , -Z, -R L -Z, -L1-Z, or -R L -L1-R L -Z.

[0052] The compound has the structure of formula (Ia): [ka] wherein "j" is 1, 2, 3, 4, 5, or 6; R1 is hydrogen, -R, -X b , -R L -X b , -L1-R, -L1-X b, -Z, -R L -Z, -L1-Z, or -R L -L1-R L -Z; R2 and R3 are independently selected from hydrogen, -R, an electron donating group, or -Z, or R2 and R3 together form a 5- to 10-membered fused heterocyclyl group; R4, independently at each occurrence, is selected from hydrogen and -R (e.g., R', R N , C 1~ (lower alkyl such as C4 alkyl); Ω is S, O, or N; Y is -R, -L1-R, -R L -Z, -L1-R L -Z, or when Ω is O or S, Y is absent; Y' is absent (i.e., a bond) or -L1-, -R L -, -R L -L1-, -L1-L1-, -L1-R L -, -L1-R L -L1 or -R L -L1-R L - selected from; Y' is L C or Z L containing one or more bonds to;

[0053] Z has the following structure: [ka] wherein "q" and "l" are independently 0 or 1; "r" is independently an integer from 0 to 10; L1 can be independently -O-, -S-, -NH-, -N(R N )-, -(CH2) 1~10 -, -S(=O) 1~2 -, -C=C-, -C=C-(CH2) 1~3 -, -C(O)-, -OC(O)-, -C(O)-(CH2) 1~4 -, -(CH2) 1~4 -C(O)-, -C(O)-O-, -C(O)-N(RN )-, -C(O)-NH-, -N(R N )-C(O)-, -NH-C(O)-, -C(O)-N(R N )-(CH2) 1~3 -, -(CH2) 1~3 -C(O)-N(R N )-, -NH-S(O) 1~2 -, -N(R N )-S(O) 1~2 -, -S(O) 1~2 -N(R N )-, -S(O) 1~2 -NH-, -(CH2) 1~3 -NH-S(O) 1~2 -, -(CH2) 1~3 -N(R N )-S(O) 1~2 -, -(CH2) 1~3 -S(O) 1~2 -N(R N )-, -(CH2) 1~3 -S(O) 1~2 -NH-, -O-(CH2) 1~4 -, -(CH2) 1~4 -O-, -S-(CH2) 1~4 -, -(CH2) 1~4 -S-, -NH-(CH2) 1~4 -, -N(R N )-(CH2) 1~4 -, -(CH2) 1~4 -N(R N )-, -(OCH2) 1~10 -, -(CHO) 1~10 -, -(OCH2CH2) 1~10 - or -(CH2CH2O) 1~10 - and; R L is independently C for each occurrence. 1~20a divalent hydrocarbon (e.g., alkyl, alkenyl, aryl, phenyl, monoalkyl-substituted phenyl, dialkyl-substituted phenyl, alkynyl, arylalkyl, combinations thereof), optionally having one or more (e.g., 1-10, 1-5) points of substitution (e.g., with 1-10 heteroatoms, such as a heteroalkyl, heteroalkenyl, heteroaryl, heteroalkynyl, or heteroarylalkyl group, with 1-10 substituents); R is independently hydrogen or C at each occurrence. 1~35 a hydrocarbon (e.g., alkyl, alkenyl, alkynyl, aralkyl, or combinations thereof) group, optionally having one or more (e.g., 1-20, 1-10, 1-5) points of substitution (e.g., with 1-10 heteroatoms, such as a heteroalkyl, heteroalkenyl, heteroaryl, heteroalkynyl, or heteroarylalkyl group, with 1-10 substituents); R' and R" are independently hydrogen or C 1~10 is alkyl; X b is independently at each occurrence an anionic group; R N is, independently at each occurrence, hydrogen, or C 1~5 alkyl (e.g., methyl, ethyl, propyl)) may have

[0054] Z, independently at each occurrence, has the structure: [ka] wherein "q" and "l" are independently 0 or 1; "r" is independently an integer from 0 to 10; L1 can be independently -O-, -S-, -NH-, -N(R N )-, -(CH2) 1~10 -, -S(=O) 1~2 -, -C=C-, -C=C-(CH2) 1~3 -, -C(O)-, -OC(O)-, -C(O)-(CH2)1~4 -, -(CH2) 1~4 -C(O)-, -C(O)-O-, -C(O)-N(R N )-, -C(O)-NH-, -N(R N )-C(O)-, -NH-C(O)-, -C(O)-N(R N )-(CH2) 1~3 -, -(CH2) 1~3 -C(O)-N(R N )-, -NH-S(O) 1~2 -, -N(R N )-S(O) 1~2 -, -S(O) 1~2 -N(R N )-, -S(O) 1~2 -NH-, -(CH2) 1~3 -NH-S(O) 1~2 -, -(CH2) 1~3 -N(R N )-S(O) 1~2 -, -(CH2) 1~3 -S(O) 1~2 -N(R N )-, -(CH2) 1~3 -S(O) 1~2 -NH-, -O-(CH2) 1~4 -, -(CH2) 1~4 -O-, -S-(CH2) 1~4 -, -(CH2) 1~4 -S-, -NH-(CH2) 1~4 -, -N(R N )-(CH2) 1~4 -, -(CH2) 1~4 -N(R N )-, -(OCH2) 1~10 -, -(CHO) 1~10 -, -(OCH2CH2) 1~10 - or -(CH2CH2O) 1~10 - and; R L is independently C for each occurrence. 1~20a divalent hydrocarbon radical (e.g., alkyl, alkenyl, aryl, phenyl, monoalkyl-substituted phenyl, dialkyl-substituted phenyl, alkynyl, arylalkyl), optionally having one or more (e.g., 1-10, 1-5) points of substitution (e.g., with 1-10 heteroatoms, such as a heteroalkyl, heteroalkenyl, heteroaryl, heteroalkynyl, or heteroarylalkyl group, with 1-10 substituents); R is independently hydrogen or C at each occurrence. 1~35 a hydrocarbon (e.g., alkyl, alkenyl, alkynyl, or aralkyl) radical, optionally having one or more (e.g., 1-20, 1-10, 1-5) points of substitution (e.g., with 1-20 heteroatoms, such as a heteroalkyl, heteroalkenyl, heteroaryl, heteroalkynyl, or heteroarylalkyl group, with 1-20 substituents); R' and R" are independently hydrogen or C 1~10 is alkyl; X b is independently at each occurrence an anionic group; R N is, independently at each occurrence, hydrogen, or C 1~5 alkyl (e.g., selected from methyl, ethyl, propyl) or In some embodiments, Ω is S, O, or N. In various implementations, R is -R, -X b , -R L -X b , -L1-R, -L1-X b , -Z, -R L -Z, -L1-Z, or -R L -L1-R L In certain embodiments, R is -R, -X b , -R L -X b , -L1-R, -L1-X b , -Z, -R L -Z, -L1-Z, or -R L -L1-RL -Z and Ω is S, O, or N. For example, each R4 in formula (Ia) can be hydrogen. In some embodiments, "j" is 2 or 3. In various implementations, the compound has the structure of formula (II): [ka] where Ω is S, O, or N; Y is -R, -L1-R, -R L -Z, -L1-R L -Z, or when Ω is O or S, Y is absent; Y' is absent (i.e., a bond) or -L1-, -R L -, -R L -L1-, -L1-L1-, -L1-R L -, -L1-R L -L1 or -R L -L1-R L -, and Y' is selected from A or L (e.g., L C Or Z L ) including one or more bonds to; R1 is hydrogen, -R, -X, -R L -X, -L1-R, -L1-X, -Z, -R L -Z, -L1-Z, or -R L -L1-R L -Z; R2 and R3 are independently selected from hydrogen, -R, an electron donating group, or -Z;

[0055] Z has the following structure: [ka] wherein "q" and "l" are independently 0 or 1; "r" is independently an integer from 0 to 10; L1 can be independently -O-, -S-, -NH-, -N(R N )-, -(CH2) 1~10 -, -S(=O) 1~2 -, -C=C-, -C=C-(CH2)1~3 -, -C(O)-, -OC(O)-, -C(O)-(CH2) 1~4 -, -(CH2) 1~4 -C(O)-, -C(O)-O-, -C(O)-N(R N )-, -C(O)-NH-, -N(R N )-C(O)-, -NH-C(O)-, -C(O)-N(R N )-(CH2) 1~3 -, -(CH2) 1~3 -C(O)-N(R N )-, -NH-S(O) 1~2 -, -N(R N )-S(O) 1~2 -, -S(O) 1~2 -N(R N )-, -S(O) 1~2 -NH-, -(CH2) 1~3 -NH-S(O) 1~2 -, -(CH2) 1~3 -N(R N )-S(O) 1~2 -, -(CH2) 1~3 -S(O) 1~2 -N(R N )-, -(CH2) 1~3 -S(O) 1~2 -NH-, -O-(CH2) 1~4 -, -(CH2) 1~4 -O-, -S-(CH2) 1~4 -, -(CH2) 1~4 -S-, -NH-(CH2) 1~4 -, -N(R N )-(CH2) 1~4 -, -(CH2) 1~4 -N(R N )-, -(OCH2) 1~10 -, -(CHO) 1~10 -, -(OCH2CH2) 1~10 - or -(CH2CH2O) 1~10 - and; R L is independently C for each occurrence. 1~20a divalent hydrocarbon (e.g., alkyl, alkenyl, aryl, phenyl, monoalkyl-substituted phenyl, dialkyl-substituted phenyl, alkynyl, arylalkyl), optionally having one or more (e.g., 1-10, 1-5) points of substitution (e.g., with 1-10 heteroatoms, such as a heteroalkyl, heteroalkenyl, heteroaryl, heteroalkynyl, or heteroarylalkyl group, with 1-10 substituents); R is independently hydrogen or C at each occurrence. 1~35 a hydrocarbon (e.g., alkyl, alkenyl, alkynyl, or aralkyl) radical, optionally having one or more (e.g., 1-20, 1-10, 1-5) points of substitution (e.g., with 1-10 heteroatoms, such as a heteroalkyl, heteroalkenyl, heteroaryl, heteroalkynyl, or heteroarylalkyl group, with 1-10 substituents); R' and R" are independently hydrogen or C 1~10 is alkyl; X b is independently at each occurrence an anionic group;

[0056] R N is, independently at each occurrence, hydrogen, or C 1~5 In certain embodiments, Ω is S, O, or N. In some embodiments, R is selected from -R, -X b , -R L -X b , -L1-R, -L1-X b , -Z, -R L -Z, -L1-Z, or -R L -L1-R L In certain embodiments, Ψ is the structure of formula (IIa): [ka] wherein "h" is 1, 2, 3, 4, 5, or 6. In some embodiments, the compound has the structure of formula (IIb): [ka] (wherein R5 to R8 are independently hydrogen or C 1~35 A hydrocarbon radical (e.g., alkyl, alkenyl, alkynyl, aryl, alkoxy, alkylthio, or amino); L1 is A (e.g., L is absent), optionally with one or more (e.g., 1 to 20 points of substitution), or L (e.g., L C Or Z L ) covalently bonded to L1 may be, for example, -NH-C(O)-, -C(O)-NH-, -C(O)-O-, or -OC(O)-. In certain embodiments, R5 and R6 are each lower alkyl (e.g., C1-C4 alkyl, methyl), and R7 and R8 are each hydrogen.

[0057] In some embodiments, the compound has the structure of Formula (IIc): [ka] (Wherein, Y" is absent or -L1-, -R L -, -L1-R L - or -R L -L1-, and Y" is A (e.g., L is absent) or L (e.g., L C Or Z L ) covalently bonded to may have

[0058] In some embodiments, the compound has the structure of Formula (IId): [ka] (Wherein, Y" is absent or -L1-, -R L -, -L1-R L - or -R L -L1-, and Y" is A (e.g., L is absent) or L (e.g., L C Or Z L ) covalently bonded to In some embodiments, R in Formula (IId) may be an optionally substituted aryl (e.g., C 6~ C 12 For example, the compound may have the structure of formula (IIe): [ka] (Wherein, Y" is absent or -L1-, -R L -, -L1-R L - or -R L -L1-, and Y" is A (e.g., L is absent) or L (e.g., L C Or Z L ) covalently bonded to may have

[0059] In some embodiments, L is the structure -L C -(Z L ) z - where "z" is 0 or 1; L C is a divalent C 1~35 an alkyl, alkenyl, alkynyl, aryl, or arylalkyl radical, optionally substituted with 1 to 20 heteroatoms; Z L is a zwitterionic linker group having the structure: [ka] "m" is 0 (i.e., a bond) or 1; "n" and "p" at each occurrence are independently an integer from 0 (i.e., a bond) to 10; X a is an anionic group; R L is independently C for each occurrence. 1~20a divalent hydrocarbon radical (e.g., alkyl, alkenyl, aryl, alkynyl, arylalkyl), optionally having one or more (e.g., 1-10, 1-5) points of substitution (e.g., substituted with 1-10 heteroatoms, substituted with 1-10 substituents); R' is hydrogen or C 1~10 It is alkyl. C has the following structure: -(X1) 0-1 -(R L ) 0-5 -(X2) 0-1 -(R L ) 0-5 -(X3) 0-1 -(R L ) 0-5 -(X4) 0-1 -(R L ) 0-5 - (wherein X1 is ═N—, —O—, —S—, or —NR N -Selected from; X2 to X4 are -O-, -S-, and -NR N -, -C(O)-, -NR N -C(O)-, -C(O)-NR N independently selected from -, -OC(O)-, or -C(O)-O-, -SC(O)-, or -C(O)-S-; R L is, independently for each occurrence, -(CH2) 1-5 -, -(CH2CH2O) 1-5 - or -(OCH2CH2) 1-5 - or C5-C6 optionally substituted cycloalkylene (e.g., cyclopentylene, cyclohexylene) In various embodiments, L C is between A and Ψ (or A and Z L In some embodiments, L comprises a linking moiety that is involved in conjugation to a thiol. For example, when the reactive functional group is a maleimide, X (or X 2~ X4) is: [ka] (In the formula, [ka] indicates the point of attachment to either adjacent group) For example, the attachment point marked with an "*" may be to a thiol of the analyte or its binding partner. In some embodiments, X a and X b may, for example, independently represent at each occurrence a carboxylate (-C(O)O - ), sulfonate (-SO3 - ), sulfate (-OSO3 - ), phosphate (-OP(O)(OR P )O - ), or oxide (-O - ), and R P is hydrogen or C 1~12 It is a hydrocarbon, optionally having one or more (e.g., 1-10, 1-5) points of substitution (e.g., with 1-10 heteroatoms and with 1-10 substituents). For example, R1 can be -R L -SO3 - (e.g., sulfopropyl) (or -R L -SO3 - In some embodiments, R1 comprises (or is) sulfopropyl. In some embodiments, R1 is -S(O)2-NH-Z or -(CH2) 1-3 In various implementations, R2 and R3, independently at each occurrence, are hydrogen, alkyl, or alkoxy (e.g., lower alkoxy such as C1-C4 alkoxy, methoxy, ethoxy, propoxy, isopropoxy). In some embodiments, R2 and R3 are each hydrogen. In other embodiments, one of R2 or R3 is hydrogen and the other of R2 or R3 is alkoxy (e.g., lower alkoxy such as C1-C4 alkoxy, methoxy, ethoxy, propoxy, isopropoxy). In some embodiments, ZL has the following structure: [ka] X, so that a is a sulfonate (-SO3 - ), m is 1, and R L is propyl, and n and p are each 3.

[0060] In certain embodiments, the compound has the structure of formula (IIIa) or (IIIb): [ka] It has. In certain embodiments, "j" is 2 or 3.

[0061] The compounds can be used to detect the presence of a substance in a sample, such as an analyte (e.g., a biomolecule). In some embodiments, the analyte is an antibody to a thyroid hormone (e.g., thyroid stimulating hormone, e.g., A is the binding partner, and thus an anti-thyroid stimulating hormone monoclonal antibody (AntiTSH-mAb)), an androgen, a steroid hormone (e.g., androstenedione, testosterone), a troponin, a thyroglobulin, an anti-thyroid peroxidase antibody, a triiodothyronine (T3) hormone, a thyroxine (T4) hormone, a thyroxine-binding globulin (TBG), a neurofilament light chain (e.g., serum neurofilament light chain), a vitamin (e.g., vitamin D, such as 25-hydroxyvitamin D), or a virus (e.g., hepatitis virus).

[0062] Compounds for forming conjugates are also provided. For example, compounds (e.g., compounds for conjugating to an analyte or a binding partner of an analyte, such as a peptide, protein, or macromolecule, including an antibody) have the structure of formula (IV): [ka] where RFG is a reactive functional group for conjugating to an analyte or a binding partner of the analyte; L is either absent (i.e., a bond) or a linker;

[0063] Ψ has the following structure: [ka] wherein "j" is 1, 2, 3, 4, 5, or 6; R1 is hydrogen, -R, -X b , -R L -X b , -L1-R, -L1-X b , -Z, -R L -Z, -L1-Z, or -R L -L1-R L -Z; R2 and R3 are independently selected from hydrogen, -R, an electron donating group, or -Z, or R2 and R3 together form a 5- to 7-membered fused heterocyclyl group;

[0064] Z, independently at each occurrence, has the structure: [ka] wherein "q" and "l" are independently 0 or 1; "r" is independently an integer from 0 to 10; L1 can be independently -O-, -S-, -NH-, -N(R N )-, -(CH2) 1~10 -, -S(=O) 1~2 -, -C=C-, -C=C-(CH2) 1~3 -, -C(O)-, -OC(O)-, -C(O)-(CH2) 1~4 -, -(CH2) 1~4 -C(O)-, -C(O)-O-, -C(O)-N(R N )-, -C(O)-NH-, -N(R N )-C(O)-, -NH-C(O)-, -C(O)-N(R N )-(CH2) 1~3-, -(CH2) 1~3 -C(O)-N(R N )-, -NH-S(O) 1~2 -, -N(R N )-S(O) 1~2 -, -S(O) 1~2 -N(R N )-, -S(O) 1~2 -NH-, -(CH2) 1~3 -NH-S(O) 1~2 -, -(CH2) 1~3 -N(R N )-S(O) 1~2 -, -(CH2) 1~3 -S(O) 1~2 -N(R N )-, -(CH2) 1~3 -S(O) 1~2 -NH-, -O-(CH2) 1~4 -, -(CH2) 1~4 -O-, S-(CH2) 1~4 -, -(CH2) 1~4 -S-, -NH-(CH2) 1~4 -, -N(R N )-(CH2) 1~4 -, -(CH2) 1~4 -N(R N )-, -(OCH2) 1~10 -, -(CHO) 1~10 -, -(OCH2CH2) 1~10 - or -(CH2CH2O) 1~10 - and; R L is independently C for each occurrence. 1~20 a divalent hydrocarbon radical (e.g., alkyl, alkenyl, aryl, phenyl, monoalkyl-substituted phenyl, dialkyl-substituted phenyl, alkynyl, arylalkyl), optionally having one or more (e.g., 1-10, 1-5) points of substitution (e.g., substituted with 1-10 heteroatoms such as heteroalkyl, heteroalkenyl, heteroaryl, heteroalkynyl, heteroarylalkyl, substituted with a hydrocarbon having 1-10 substituents, e.g., 1-10 substituents selected from one or more groups -X); R is independently hydrogen or C at each occurrence. 1~35a hydrocarbon (e.g., alkyl, alkenyl, alkynyl, or aralkyl) radical, optionally having one or more (e.g., 1-20, 1-10, 1-5) points of substitution (e.g., substituted with 1-20 heteroatoms, substituted with 1-20 substituents); R' and R" are independently hydrogen or C 1~10 is alkyl; X b is independently at each occurrence an anionic group; R N is, independently at each occurrence, hydrogen, or C 1~5 The reactive functional group may be a chemiluminescent acridinium containing a zwitterionic group having an alkyl group selected from the group consisting of:

[0065] [ka] [ka] You may choose from.

[0066] The compound has the structure of formula (IVa): [ka] wherein "j" is 1, 2, 3, 4, 5, or 6; R1 is hydrogen, -R, -X b , -R L -X b , -L1-R, -L1-X b , -Z, -R L -Z, -L1-Z, or -R L -L1-R L -Z; R2 and R3 are independently selected from hydrogen, -R, an electron donating group, or -Z, or R2 and R3 together form a 5- to 10-membered fused heterocyclyl group; R4, independently at each occurrence, is selected from hydrogen and -R (e.g., R', R N , C 1~(lower alkyl such as C4 alkyl); Ω is S, O, or N; Y is -R, -L1-R, -R L -Z, -L1-R L -Z, or when Ω is O or S, Y is absent; Y' is absent (i.e., a bond) or -L1-, -R L -, -R L -L1-, -L1-L1-, -L1-R L -, -L1-R L -L1 or -R L -L1-R L -, and Y' is selected from L C or Z L containing one or more bonds to;

[0067] Z has the following structure: [ka] wherein "q" and "l" are independently 0 or 1; "r" is independently an integer from 0 to 10; L1 can be independently -O-, -S-, -NH-, -N(R N )-, -(CH2) 1~10 -, -S(=O) 1~2 -, -C=C-, -C=C-(CH2) 1~3 -, -C(O)-, -OC(O)-, -C(O)-(CH2) 1~4 -, -(CH2) 1~4 -C(O)-, -C(O)-O-, -C(O)-N(R N )-, -C(O)-NH-, -N(R N )-C(O)-, -NH-C(O)-, -C(O)-N(R N )-(CH2) 1~3 -, -(CH2) 1~3 -C(O)-N(R N )-, -NH-S(O) 1~2 -, -N(R N )-S(O) 1~2 -, -S(O) 1~2 -N(RN )-, -S(O) 1~2 -NH-, -(CH2) 1~3 -NH-S(O) 1~2 -, -(CH2) 1~3 -N(R N )-S(O) 1~2 -, -(CH2) 1~3 -S(O) 1~2 -N(R N )-, -(CH2) 1~3 -S(O) 1~2 -NH-, -O-(CH2) 1~4 -, -(CH2) 1~4 -O-, -S-(CH2) 1~4 -, -(CH2) 1~4 -S-, -NH-(CH2) 1~4 -, -N(R N )-(CH2) 1~4 -, -(CH2) 1~4 -N(R N )-, -(OCH2) 1~10 -, -(CHO) 1~10 -, -(OCH2CH2) 1~10 - or -(CH2CH2O) 1~10 - and; R L is independently C for each occurrence. 1~20 a divalent hydrocarbon (e.g., alkyl, alkenyl, aryl, phenyl, monoalkyl-substituted phenyl, dialkyl-substituted phenyl, alkynyl, arylalkyl, combinations thereof), optionally substituted with 1 to 10 heteroatoms and / or substituents; R is independently hydrogen or C at each occurrence. 1~35 a hydrocarbon (e.g., alkyl, alkenyl, alkynyl, aralkyl, or combinations thereof) radical, optionally substituted with 1 to 20 heteroatoms and / or substituents; R' and R" are independently hydrogen or C 1~10 is alkyl; X b is independently at each occurrence an anionic group; R N is, independently at each occurrence, hydrogen, or C 1~5alkyl (e.g., methyl, ethyl, propyl)) may have

[0068] Z, independently at each occurrence, has the structure: [ka] wherein "q" and "l" are independently 0 or 1; "r" is independently an integer from 0 to 10; L1 can be independently -O-, -S-, -NH-, -N(R N )-, -(CH2) 1~10 -, -S(=O) 1~2 -, -C=C-, -C=C-(CH2) 1~3 -, -C(O)-, -OC(O)-, -C(O)-(CH2) 1~4 -, -(CH2) 1~4 -C(O)-, -C(O)-O-, -C(O)-N(R N )-, -C(O)-NH-, -N(R N )-C(O)-, -NH-C(O)-, -C(O)-N(R N )-(CH2) 1~3 -, -(CH2) 1~3 -C(O)-N(R N )-, -NH-S(O) 1~2 -, -N(R N )-S(O) 1~2 -, -S(O) 1~2 -N(R N )-, -S(O) 1~2 -NH-, -(CH2) 1~3 -NH-S(O) 1~2 -, -(CH2) 1~3 -N(R N )-S(O) 1~2 -, -(CH2) 1~3 -S(O) 1~2 -N(R N )-, -(CH2) 1~3 -S(O) 1~2 -NH-, -O-(CH2) 1~4 -, -(CH2) 1~4 -O-, -S-(CH2)1~4 -, -(CH2) 1~4 -S-, -NH-(CH2) 1~4 -, -N(R N )-(CH2) 1~4 -, -(CH2) 1~4 -N(R N )-, -(OCH2) 1~10 -, -(CHO) 1~10 -, -(OCH2CH2) 1~10 - or -(CH2CH2O) 1~10 - and; R L is independently C for each occurrence. 1~20 a divalent hydrocarbon radical (e.g., alkyl, alkenyl, aryl, phenyl, monoalkyl-substituted phenyl, dialkyl-substituted phenyl, alkynyl, arylalkyl), optionally having one or more (e.g., 1-10, 1-5) points of substitution (e.g., substituted with 1-10 heteroatoms, substituted with 1-10 substituents); R is independently hydrogen or C at each occurrence. 1~35 a hydrocarbon (e.g., alkyl, alkenyl, alkynyl, or aralkyl) radical, optionally having one or more (e.g., 1-20, 1-10, 1-5) points of substitution (e.g., substituted with 1-20 heteroatoms, substituted with 1-20 substituents); R' and R" are independently hydrogen or C 1~10 is alkyl; X b is independently at each occurrence an anionic group; R N is, independently at each occurrence, hydrogen, or C 1~5 alkyl (e.g., selected from methyl, ethyl, propyl) or a zwitterionic group having a salt thereof (e.g., a halide salt such as a chloride salt). In some embodiments, Ω is S, O, or N. In various implementations, R is -R, -X b , -R L -X b , -L1-R, -L1-X b , -Z, -R L-Z, -L1-Z, or -R L -L1-R L In certain embodiments, R is -R, -X b , -R L -X b , -L1-R, -L1-X b , -Z, -R L -Z, -L1-Z, or -R L -L1-R L -Z and Ω is S, O, or N. For example, each R in formula (IVa) can be hydrogen. In some embodiments, "j" is 2 or 3.

[0069] Compounds for forming conjugates may contain any of the relevant groups described herein (e.g., Ω, Y, Y', Y", R, R', R", L, L C , R L , R C , R1, R2, R3, R4, R5, R6, R7, R8, X, X a , X b For example, the compound may have the structure of formula (V), (Va), (Vb), (Vc), (Vd), (Ve), (VIa), (VIb), (VIc), (VId), or (VIe): [ka] [ka] [ka] [ka] may have

[0070] In various implementations, the compound is: [ka] [ka] [ka] is selected from.

[0071] In some embodiments, compounds of the present disclosure may be zwitterionic or may contain one or more zwitterionic groups. For example, the R group attached to the positively charged acridinium nitrogen can be optionally substituted with up to 20 heteroatoms (e.g., N, O, S, P, Cl, Br, F), and thus, in combination with the positively charged acridinium nitrogen atom, can comprise a zwitterionic group. For example, a sulfopropyl or sulfobutyl group attached to the acridinium nitrogen can form a zwitterionic ion pair. The R group can be neutral (e.g., methyl) or itself zwitterionic (e.g., R can be -Z, -R L -Z, -L8-Z, or -R L -L8-R M In some embodiments, R1 may have the following structure: [ka] It has.

[0072] If the compound is charged (e.g., R1 has a net neutral charge), the compound may be in its salt form, optionally including a counterion to balance the positively charged nitrogen of the acridinium nucleus. The counterion may be CH3SO4 - , FSO3 - , CF3SO4 - , C4F9SO4 - , CH3C6H4SO3 - , halides (e.g., Cl - , F - , Br - ), CF3COO - , CH3COO - , or NO3 -In some embodiments, R1 is methyl, ethyl, propyl, or isopropyl. In some embodiments, the acridinium compound may be zwitterionic through a covalent bond to an anion. For example, R1 is -R L -X, where -X is a sulfonate (-SO - In some embodiments, R is -R L -X, where -X is a sulfonate (-SO3 - In some embodiments, R is -R L In some embodiments, L is -X or -L-Z. In some embodiments, L is -S(O)-NH- or -(CH) 1-3 -S(O)2-NH-. R1 is a sulfopropyl group (-(CH2)3-SO3 - In certain embodiments, R1 is sulfopropyl.

[0073] In some implementations, the chemiluminescent acridinium Ψ is an acridinium ester. For example, Ψ has the following structure: [ka] where Ω is S, O, or N; Y is -R, -L1-R, -R L -Z, -L1-R L -Z, or when Ω is O or S, Y is absent; Y' is absent (i.e., a bond) or -L1-, -R L -, -R L -L1-, -L1-L1-, -L1-R L -, -L1-R L -L1 or -R L -L1-R L -, and Y' is selected from L C or Z L containing one or more bonds to; R1 is hydrogen, -R, -X b , -R L -X b, -L1-R, -L1-X b , -Z, -R L -Z, -L1-Z, or -R L -L1-R L -Z; R2 and R3 are independently selected from hydrogen, -R, an electron donating group, or -Z;

[0074] Z has the following structure: [ka] wherein "q" and "l" are independently 0 or 1; "r" is independently an integer from 0 to 10; L1 can be independently -O-, -S-, -NH-, -N(R N )-, -(CH2) 1~10 -, -S(=O) 1~2 -, -C=C-, -C=C-(CH2) 1~3 -, -C(O)-, -OC(O)-, -C(O)-(CH2) 1~4 -, -(CH2) 1~4 -C(O)-, -C(O)-O-, -C(O)-N(R N )-, -C(O)-NH-, -N(R N )-C(O)-, -NH-C(O)-, -C(O)-N(R N )-(CH2) 1~3 -, -(CH2) 1~3 -C(O)-N(R N )-, -NH-S(O) 1~2 -, -N(R N )-S(O) 1~2 -, -S(O) 1~2 -N(R N )-, -S(O) 1~2 -NH-, -(CH2) 1~3 -NH-S(O) 1~2 -, -(CH2) 1~3 -N(R N )-S(O) 1~2 -, -(CH2) 1~3 -S(O) 1~2 -N(R N )-, -(CH2) 1~3 -S(O)1~2 -NH-, -O-(CH2) 1~4 -, -(CH2) 1~4 -O-, -S-(CH2) 1~4 -, -(CH2) 1~4 -S-, -NH-(CH2) 1~4 -, -N(R N )-(CH2) 1~4 -, -(CH2) 1~4 -N(R N )-, -(OCH2) 1~10 -, -(CHO) 1~10 -, -(OCH2CH2) 1~10 - or -(CH2CH2O) 1~10 - and; R L is independently C for each occurrence. 1~20 a divalent hydrocarbon (e.g., alkyl, alkenyl, aryl, phenyl, monoalkyl-substituted phenyl, dialkyl-substituted phenyl, alkynyl, arylalkyl) optionally substituted with 1 to 10 heteroatoms (e.g., heteroalkyl, heteroalkenyl, heteroaryl, heteroalkynyl, heteroarylalkyl) and / or substituents; R is independently hydrogen or C at each occurrence. 1~35 a hydrocarbon (e.g., alkyl, alkenyl, alkynyl, or aralkyl) radical, optionally substituted with 1 to 20 heteroatoms (e.g., heteroalkyl, heteroalkenyl, heteroaryl, heteroalkynyl, heteroarylalkyl) and / or substituents; R' and R" are independently hydrogen or C 1~10 is alkyl; X b is independently at each occurrence an anionic group; R N is, independently at each occurrence, hydrogen, or C 1~5 alkyl (e.g., methyl, ethyl, propyl)) In certain embodiments, Ω is S, O, or N, and / or R is —R, —X b , -R L -Xb , -L1-R, -L1-X b , -Z, -R L -Z, -L1-Z, or -R L -L1-R L In some embodiments, Ψ has the following structure: [ka] wherein "h" is 1, 2, 3, 4, 5, or 6. In some embodiments, Ψ may be a chemiluminescent acridinium having the following structure: [ka] It is a chemiluminescent acridinium comprising:

[0075] Substituents on chemiluminescent acridinium esters can be modified to alter the rate and yield of light emission, reduce nonspecific binding, increase stability, or increase hydrophilicity. Typically, these modifications have minimal interference with the binding of the analyte and its binding partner. Examples of substituent variability are disclosed in U.S. Pat. No. 7,309,615 to Natrajan et al., incorporated herein by reference, which describes high-quantum-yield acridinium compounds containing alkoxy groups (OR*) at C2 and / or C7, where R* is a group containing a sulfopropyl moiety, an ethylene glycol moiety, or a combination thereof. In some embodiments, R2 and / or R3 may be alkoxy groups (e.g., OR and / or OR*). WO 2015 / 006174 to Natrajan et al., incorporated herein by reference in its entirety, also describes hydrophilic, high-quantum-yield chemiluminescent acridinium esters with specific electron-donating functional groups at C2 and / or C7. These electron donating groups of R1 and / or R2 have the following structure: [ka] (In the formula, R 9~R 14 is, independently at each occurrence, a methyl group or the group -(CH2CH2O) a CH3 (wherein a is an integer from 1 to 5) In some embodiments, R2 and R3, independently at each occurrence, are hydrogen, alkyl (e.g., methyl, ethyl, propyl, isopropyl), or alkoxy (e.g., methoxy, ethoxy, propoxy, or isopropoxy). In some embodiments, R2 and R3 are each hydrogen. In other embodiments, R2 or R3 is hydrogen, and the other of R2 or R3 is an alkoxy or an electron-donating group.

[0076] The detectable conjugate or the compound for forming the detectable conjugate may comprise a chemiluminescent acridinium sulfonamide. For example, Ψ in the conjugate or the compound for forming the detectable conjugate may have the structure of formula (IIa), (IIb), (IIc), (IId), or (IIe): [ka] (wherein "h" is 1, 2, 3, 4, 5, or 6); [ka] (wherein R5 to R8 are independently hydrogen or C 1~35 is alkyl, alkenyl, alkynyl, aryl, alkoxy, alkylthio, or amino; L is A (e.g., L is absent) or L (e.g., L C Or Z L ) covalently bonded to

[0077] [ka] [ka] (Wherein, Y" is absent or -L1-, -R L -, -L1-R L- or -R L -L1-, and Y" is L (e.g., L C or Z L ) covalently bonded to In some embodiments, R L is an optionally substituted 5- or 6-membered divalent aromatic hydrocarbon. For example, any R L has the following structure: [ka] (In the formula, R 15 is independently hydrogen, halogen, or R at each occurrence) In some embodiments, R L has the following structure: [ka] (In the formula, R 5~ R8 is independently C 1~35 alkyl, alkenyl, alkynyl, aryl, alkoxy, alkylthio, or amino) In some embodiments, R7 and R8 are each hydrogen, and R5 and R6 are each methyl. In some embodiments, Ψ includes two adjacent methyl groups on the phenolic ester to stabilize the bond, as disclosed in Law et al., Journal of Bioluminescence and Chemiluminescence 4:88-89 (1989), which is incorporated herein by reference in its entirety. In some embodiments, Ψ has the following structure: [ka] It has.

[0078] In some embodiments, A, L, and Ψ are each covalently bonded. A portion of the covalent bond between A and Ψ can be formed from a reactive functional group for forming a covalent bond with a peptide, protein, or macromolecule, and the functional group can include an electrophilic group, a nucleophilic group, or a photoreactive group. The reactive functional group can be an amine-reactive group, a thiol-reactive group, a carboxy-reactive group, a maleimidyl-reactive group, or a carbohydrate-reactive group. In some embodiments, the reactive functional group can react with a functional group of an analyte or binding partner, such as a primary amine. The reactive functional group can include (or be) an isothiocyanate, an isocyanate, an acyl azide, an NHS ester, a sulfonyl chloride, an aldehyde, a glyoxal, an epoxide, an oxirane, a carbonate, an aryl halide, a maleimidyl, an imidoester, a carbodiimide, an anhydride, a fluorophenyl ester, or a combination thereof. In various implementations, the reactive functional group labels the analyte or its binding partner through acylation or alkylation. For example, the bond can be: [ka] [ka] The reactive group may be selected from the group consisting of: In some embodiments, the compound comprises a linker group having the structure -NH-C(O)- or -C(O)-NH-. In preferred embodiments, the compound or portion thereof (e.g., L C , Ψ) comprises at least one -NH-C(O)- or -C(O)-NH- linker group.

[0079] The covalent bond between A and Ψ (e.g., L) or between RFG and Ψ (e.g., L) is a divalent C 1~20It may comprise (or be) an alkyl, alkenyl, alkynyl, aryl, or arylalkyl radical, optionally substituted with up to 20 heteroatoms (e.g., N, O, S, P, Cl, F, Br). In some embodiments, L comprises a zwitterionic linker. L has the structure -L C -(Z L ) z - (wherein z is 0 or 1). C has the following structure: -(X1) 0-1 -(R L ) 0-5 -(X2) 0-1 -(R L ) 0-5 -(X3) 0-1 -(R L ) 0-5 -(X4) 0-1 -(R L ) 0-5 - (wherein X1 is —O—, —S—, —NR N -, -C(O)-, -NR N -C(O)-, -C(O)-NR N selected from -, -OC(O)-, or -C(O)-O-, -SC(O)-, or -C(O)-S-, ═N-, -O-, or -S-; X2 to X4 are -O-, -S-, and -NR N -, -C(O)-, -NR N -C(O)-, -C(O)-NR N independently selected from -, -OC(O)-, or -C(O)-O-, -SC(O)-, or -C(O)-S-; R L is independently for each occurrence, --(CH2) 1-5 -, -(CH2CH2O) 1-5 - or -(OCH2CH2) 1-5 - or, for example, L C between A and Ψ (or A and Z L For example, alkyl (e.g., C 1~ C alkyl), alkoxy (e.g., C1~ optionally substituted cycloalkylene (e.g., C C alkoxy) or halo (e.g., F) at one or more positions; 5~ C6 cycloalkylene).

[0080] In some embodiments, L and / or Ψ comprises -C(O)-NH-. C has the following structure: [ka] [ka] It has.

[0081] The detectable label may include a dimethyl acridinium ester (DMAE) moiety and a zwitterionic linker, including a zwitterionic linker or a polyethylene glycol-derived linker, to improve the properties of the compound. Properties such as non-specific binding, hydrophilicity, or compound stability can be improved when Ψ includes a zwitterionic linker, a polyethylene glycol-derived linker, or a dimethyl phenyl ester. In some embodiments, Z L has the following structure: [ka] It has. In some embodiments, R' is hydrogen or lower alkyl (eg, methyl, ethyl, propyl).

[0082] In some embodiments, the detectable conjugate has the structure of formula (III): [ka] (wherein z is 0 (i.e., a bond) or 1). In some embodiments, the compound for forming the conjugate may have the following structure: [ka] may have

[0083] Exemplary compounds for forming the conjugates are disclosed in Table 1. In some embodiments, the detectable conjugate is formed by reacting a compound (e.g., a compound of Formula (IV), (Va), (Vb), (Vc), (Vd), (Ve), (VIa), (VIb), a compound in Table 1) with an analyte, its binding partner, or a derivatized version of the aforementioned analyte capable of reacting with a reactive functional group. As used to describe the compounds, they are generally acridinium-containing 2,3-cyclic alkylenedioxy ("ADO") compounds. The compound may also be an acridinium ester ("AE"). Compound designations include "Z", which can refer to a zwitterionic linker; "CMO", which can refer to a carboxymethyl oxime linker; "CME", which can refer to a carboxymethyl ether linker; "CETE", which can refer to a carboxyethyl thioether; "ZAE", which can refer to a zwitterionic acridinium ester (typically N-sulfopropyldimethylacridinium ester ("NSP-DMAE") in the example shown); and "ZAE", which can refer to a zwitterionic acridinium ester (typically N-sulfopropyldimethylacridinium ester ("NSP-DMAE") in the example shown; and "ZAE", which can refer to a zwitterionic linker with an isopropoxy functional group attached, resulting in a complete zwitterionic group (N + and X - and "ISODIZAE," which may refer to the acridinium nucleus attached to the positive N of acridinium.

[0084] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5]

[0085] In some embodiments, the detectable conjugate is: [ka] wherein z is independently at each occurrence 0 or 1; y is independently 0, 1, 2, 3, 4, or 5 at each occurrence; A' is an analyte or its binding partner conjugated via a primary amine of the unconjugated analyte or its binding partner A; where: X2 to X4 are -O-, -S-, and -NR N -, -C(O)-, -NR N -C(O)-, -C(O)-NR N independently selected from -, -OC(O)-, or -C(O)-O-, -SC(O)-, or -C(O)-S-; R L is, independently for each occurrence, -(CH2) 1-5 -, -(CH2CH2O) 1-5 - or -(OCH2CH2) 1-5 -, or optionally substituted C5-C6 cycloalkylene. For example, the conjugate may have one or more structures of the following formula: [ka] may have

[0086] In various implementations, the compound has the following structure: [ka] wherein z is independently at each occurrence 0 or 1; y is independently 0, 1, 2, 3, 4, or 5 at each occurrence; A' is the unconjugated analyte or its binding partner conjugated via the thiol of its binding partner A; Here, X2 to X4 are -O-, -S-, -NR N -, -C(O)-, -NR N -C(O)-, -C(O)-NR N independently selected from -, -OC(O)-, or -C(O)-O-, -SC(O)-, or -C(O)-S-; R L is, independently for each occurrence, -(CH2) 1-5 -, -(CH2CH2O) 1-5 - or -(OCH2CH2) 1-5 or optionally substituted C selected from 5~ C cycloalkylene (e.g., cyclohexane) It has.

[0087] In various implementations, the linker moiety may reduce the rate of hydrolysis of the reactive functional group, for example, compared to an otherwise identical compound having an alkyl linkage proximal to the reactive functional group. For example, particularly in embodiments including a maleimide reactive functional group, the linker may include a moiety such as an optionally saturated cycloalkyl group proximal to the reactive functional group. For example, the compound may have the following structure: [ka] may have

[0088] Compounds of the present disclosure can be characterized by their stability. For example, a compound or conjugate can be considered stable if it experiences minimal loss of chemiluminescent activity, as measured by loss of relative light units ("RLU"), when stored in aqueous solution, typically at a pH range of 6 to 9. A compound with increased instability relative to another compound may experience a greater loss of chemiluminescent activity. For example, a compound of the present disclosure (e.g., a compound having the structure of Formulas (I)-(VI)) can be characterized as having improved stability at pH 6 and / or 7 and / or 8 at 4°C (common reagent storage temperature) and / or 37°C (accelerated temperature) for 33 days. This compound may have improved stability compared to an otherwise identical compound without the fused heterocycle conjugated to the acridinium system. In some embodiments, the compounds can be characterized as having less than a 40% (or between 1% and 40%) change in chemiluminescent activity (e.g., less than 30%, less than 20%, 10% and 40%, 10% and 30%, 10% and 20%) after storage at 37°C and pH 7 and / or pH 8 for 33 days.

[0089] Comparative chemiluminescence quantum yields can also be used to characterize compounds of the present disclosure. Quantum yields can be measured as the amount of observable chemiluminescence per defined mass of a compound. The increased quantum yield of acridinium esters is one of several advantageous properties of the acridinium compounds of the present disclosure, which exhibit higher quantum yields compared to other acridinium compounds. Chemiluminescence can be measured as relative light units (RLU) in a luminometer. Acridinium quantum yield can be measured as the amount of chemiluminescence (RLU) per mole of acridinium. Increasing the quantum yield of acridinium esters increases the likelihood of detection even when only low masses of acridinium esters are required for detection, such as when using small amounts of analyte in immunoassays. Increasing the quantum yield of acridinium esters can result in improved sensitivity of immunoassays employing high-quantum-yield acridiniums. Relative quantum yields can be calculated as the ratio of a compound of the present disclosure to HEGAE. A relative quantum yield greater than 1 indicates improved quantum yield relative to HEGAE. In some embodiments, the compounds have a relative quantum yield to HEGAE greater than 1.0 (or up to 5) (e.g., 1-6, 1-5, greater than 1.5, 1.5-4, 1.7-3.8, greater than 2, greater than 3). In some embodiments, the compounds have a relative quantum yield to HQYAE greater than 1 (or up to 3) (e.g., 1.1-1.5).

[0090] The compounds can also be characterized by the wavelength of chemiluminescence. For example, the compounds of the present disclosure can have a maximum emission wavelength (λmax) of 430 nm to 460 nm.

[0091] In some embodiments, the compounds can be characterized by their light-emission rate. The compounds of the present disclosure generally complete their light emission within 5 seconds of the onset of chemiluminescence. In some embodiments, the compounds of the present disclosure can have a faster light-emission rate than other acridinium compounds, for example, which emit 90% of their light within 2 seconds, measured over a 5-second period.

[0092] Table 2 shows exemplary characterization of compounds for some measured species (dashed double bonds indicate fusion to the acridinium ring).

[0093] [Table 2]

[0094] Compounds can also be characterized by their light output or signal-to-noise ratio in a chemiluminescent assay. For example, compounds can be characterized by a thyroid-stimulating hormone assay having a greater signal-to-noise ratio than HQYAE or TSPAE, acridinium conjugates, which have similar signal-to-noise ratios as shown in U.S. Patent Nos. 7,309,615 and 7,785,904, each of which is incorporated herein by reference in its entirety, particularly with respect to the quantum yield of TSPAE and HQYAE. In some embodiments, compounds can be characterized as having a relative signal-to-noise ratio greater than 1 (e.g., 1-2) compared to HQYAE or TSPAE (e.g., in a TSH assay). Typically, a greater signal-to-noise ratio for the same amount of analyte indicates better assay sensitivity.

[0095] The compounds can be prepared by employing standard synthetic methods from commercially available starting materials (in addition to those provided herein), compounds known in the literature, or readily prepared intermediates. Standard synthetic methods and procedures for the preparation of organic molecules and functional group transformations and manipulations are readily available from the relevant scientific literature or standard textbooks in the field. Unless otherwise specified, where typical or preferred process conditions (e.g., reaction temperature, time, molar ratios of reactants, solvents, pressure) are given, it will be understood that other process conditions can also be used. Optimum reaction conditions may vary with the particular reactants or solvents used; however, such conditions can be determined by one skilled in the art by routine optimization procedures. Those skilled in the art of organic synthesis will recognize that the nature and order of the synthetic steps presented can be varied for the purpose of optimizing the formation of the compounds described herein.

[0096] Synthetic chemical transformations (including protecting group methodology) useful in synthesizing the compounds described herein are known in the art and include, for example, R.C. Larock, Comprehensive Organic Transformations, 2nd Edition, Wiley-VCH Publishers (1999); P.G.M. Buts and T.W. Greene, Protective Groups in Organic Synthesis, 4th Edition, John Wiley and Sons (2007); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995), and subsequent editions, each of which is incorporated herein by reference in its entirety.

[0097] The processes described herein can be monitored according to any suitable method known in the art. For example, product formation can be monitored by spectroscopy, such as nuclear magnetic resonance spectroscopy (e.g., 1 H or 13 C), infrared spectroscopy (FT-IR), spectrophotometry (e.g., UV-visible spectroscopy), or mass spectrometry (MS), or chromatography, e.g., high pressure liquid chromatography (HPLC), or thin layer chromatography (TLC).

[0098] The preparation of compounds may involve the protection and deprotection of various chemical groups.The need for protection and deprotection, and the selection of appropriate protecting groups can be easily determined by those skilled in the art.The chemical properties of protecting groups can be found, for example, in Greene et al., Protective Groups in Organic Synthesis, 2nd Edition, Wiley & Sons, 1991, which is incorporated herein by reference in its entirety.

[0099] The reactions of the processes described herein can be carried out in a suitable solvent, which can be easily selected by one skilled in the art of organic synthesis. A suitable solvent can be substantially non-reactive with the starting materials (reactants), intermediates, or products at the temperature at which the reaction is carried out, i.e., at temperatures ranging from the freezing temperature of the solvent to the boiling temperature of the solvent. A given reaction can be carried out in one solvent or a mixture of two or more solvents. A suitable solvent for a particular reaction step can be selected depending on the particular reaction step.

[0100] Resolution of racemic mixtures of compounds can be carried out by any of a number of methods known in the art. For example, the absolute configuration of stereoisomers can be determined by 1D and 2D NMR techniques, such as COSY, NOESY, HMBC, and HSQC. Specific implementations of these NMR techniques can be found in Hauptmann, H et al., Bioconjugate Chem. 11 (2000): 239-252 or Bowler, J., Steroids 54 / 1 (1989): 71-99, each of which is incorporated herein by reference in its entirety. Another exemplary method involves the preparation of Mosher ester or amide derivatives of the corresponding alcohol or amine, respectively. The absolute configuration of the ester or amide can be determined by the proton and / or 19 The chirality is determined by F NMR spectroscopy. An exemplary method includes fractional crystallization using a "chiral resolving acid," which is an optically active, salt-forming organic acid. Suitable resolving agents for fractional crystallization are, for example, optically active acids, such as D- and L-tartaric acid, diacetyltartaric acid, dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid, or various optically active camphorsulfonic acids. Resolution of racemic mixtures can also be achieved by eluting with a column packed with an optically active resolving agent (e.g., dinitrobenzoylphenylglycine). Suitable elution solvent compositions can be determined by those skilled in the art.

[0101] Typically, zwitterionic acridinium esters ("ZAEs") containing reactive functional groups for forming covalent bonds, as described in U.S. Pat. Nos. 6,664,043 to Natrajan et al., 7,309,615 to Natrajan et al., 9,575,062 to Natrajan et al., or 9,487,480 to Natrajan et al., each of which is incorporated herein by reference in its entirety, and are specifically incorporated herein with respect to the zwitterionic acridinium esters and their synthesis described therein. For example, the zwitterionic acridinium ester starting material may contain an N-sulfopropyl ("NSP") group in the zwitterionic moiety, and / or a charged nitrogen atom attached to a charged acridinium nucleus ("DIZAE"), and / or a sterically stabilized dimethyl acridinium ester ("DMAE"), and / or an isopropoxy-functionalized acylidinium nucleus ("ISO"), and / or a zwitterionic ("Z") and / or hexa(ethylene)glycol-derived ("HEG") and / or glutaric acid-derived (e.g., -C(O)-(CH)-C(O)-) linkage between the acridinium ester and the reactive functionality. The reactive functionality may be NH, or N-hydroxysuccinimidyl ester ("NHS"), or may be maleimide-derived. For example, a compound (e.g., a compound for conjugation to an analyte or a binding partner of an analyte, such as a peptide, protein, or macromolecule, including an antibody) can have the structure of formula (IV): [ka] where RFG is a reactive functional group for conjugating to an analyte or a binding partner of the analyte; L is either absent (i.e., a bond) or a linker;

[0102] Ψ has the following structure: [ka] wherein "j" is 1, 2, 3, 4, 5, or 6; R1 is hydrogen, -R, -X, -R L -X b , -L1-R, -L1-X b , -Z, -R L -Z, -L1-Z, or -R L -L1-R L -Z; R2 and R3 are independently selected from hydrogen, -R, an electron donating group, or -Z, or R2 and R3 together form a 5- to 7-membered fused heterocyclyl group;

[0103] Z, independently at each occurrence, has the structure: [ka] wherein "q" and "l" are independently 0 or 1; "r" is independently an integer from 0 to 10; L1 can be independently -O-, -S-, -NH-, -N(R N )-, -(CH2) 1~10 -, -S(=O) 1~2 -, -C=C-, -C=C-(CH2) 1~3 -, -C(O)-, -OC(O)-, -C(O)-(CH2) 1~4 -, -(CH2) 1~4 -C(O)-, -C(O)-O-, -C(O)-N(R N )-, -C(O)-NH-, -N(R N )-C(O)-, -NH-C(O)-, -C(O)-N(R N )-(CH2) 1~3 -, -(CH2) 1~3 -C(O)-N(R N )-, -NH-S(O) 1~2 -, -N(R N )-S(O) 1~2 -, -S(O) 1~2 -N(R N )-, -S(O) 1~2 -NH-, -(CH2) 1~3 -NH-S(O) 1~2 -, -(CH2) 1~3-N(R N )-S(O) 1~2 -, -(CH2) 1~3 -S(O) 1~2 -N(R N )-, -(CH2) 1~3 -S(O) 1~2 -NH-, -O-(CH2) 1~4 -, -(CH2) 1~4 -O-, S-(CH2) 1~4 -, -(CH2) 1~4 -S-, -NH-(CH2) 1~4 -, -N(R N )-(CH2) 1~4 -, -(CH2) 1~4 -N(R N )-, -(OCH2) 1~10 -, -(CHO) 1~10 -, -(OCH2CH2) 1~10 - or -(CH2CH2O) 1~10 - and; R L is independently C for each occurrence. 1~20 divalent hydrocarbon radicals (e.g., alkyl, alkenyl, aryl, phenyl, monoalkyl-substituted phenyl, dialkyl-substituted phenyl, alkynyl, arylalkyl), optionally having one or more (e.g., 1-10, 1-5) points of substitution (e.g., with 1-10 heteroatoms, such as heteroalkyl, heteroalkenyl, heteroaryl, heteroalkynyl, heteroarylalkyl groups, with 1-10 substituents); R is independently hydrogen or C at each occurrence. 1~35 a hydrocarbon (e.g., alkyl, alkenyl, alkynyl, or aralkyl) radical, optionally having one or more (e.g., 1-20, 1-10, 1-5) points of substitution (e.g., with 1-20 heteroatoms, such as a heteroalkyl, heteroalkenyl, heteroaryl, heteroalkynyl, or heteroarylalkyl group, with 1-20 substituents); R' and R" are independently hydrogen or C 1~10 is alkyl; X b is independently at each occurrence an anionic group; R N is, independently at each occurrence, hydrogen, or C 1~5 a chemiluminescent acridinium having a zwitterionic group selected from alkyl (e.g., methyl, ethyl, propyl) may have

[0104] Chemiluminescent conjugates or compounds for forming this conjugate can also be synthesized using acridinium sulfonamide reactants. For example, the acridinium sulfonamides disclosed in U.S. Patent No. 5,543,524 to Mattingly et al., which is incorporated herein by reference in its entirety, are useful starting materials for the production of the chemiluminescent compounds disclosed herein.

[0105] Chemiluminescent conjugates are useful as labels in assays to measure or quantify specific analytes that can compete for binding to a binding partner.

[0106] The assay may be, for example, a competitive immunoassay, which typically involves the detection of a macromolecule, also referred to as a macromolecular analyte, using a binding molecule such as an antibody. The antibody is immobilized or attached to a solid phase, such as a particle, bead, membrane, microtiter plate, or other solid surface. The analyte typically measured in such an assay is often a substance of some clinical relevance and can range from a wide range of molecules, from macromolecules such as proteins, nucleic acids, viruses, and bacteria to small molecules such as valproate, vitamins, steroids, hormones, and therapeutic drugs.

[0107] The compounds of the present disclosure can be used in sandwich immunoassays, which typically involve the detection of macromolecules, also referred to as macromolecular analytes, using two binding molecules, such as antibodies. One antibody is immobilized or attached to a solid phase, such as a particle, bead, membrane, microtiter plate, or other solid surface. The compounds can also be used in competitive assays. In one example of a competitive heterogeneous assay, a support bearing an analyte-binding antibody (e.g., 3C3, 3H10, or 4G8 bovine monoclonal antibody) is contacted with a sample suspected of containing the analyte and a medium containing a chemiluminescent conjugate (or "labeled analog") described herein. The sample analyte competes with the labeled analog for binding to the analyte antibody. After separation of the support and medium, the label activity of the support or medium is measured by conventional techniques and related to the amount of analyte in the sample. In a variation of the above competitive heterogeneous assay, the support contains an analyte analog, which competes with the sample analyte for binding to the antibody reagent according to the principles described herein. Labeled analyte analogs can be covalently attached to a chemiluminescent or fluorescent molecule, often referred to as a label or tracer.

[0108] A solid phase containing an immobilized antibody or other binding agent is mixed with a sample containing an analyte and a labeled analyte, typically resulting in the formation of a binding complex between the analyte or the labeled analyte. The binding agent may be, for example, an antibody, antibody fragment, nucleic acid, peptide, binding protein, or synthetic binding polymer. In some embodiments, the binding agent may be a protein, such as intrinsic factor, which binds vitamin B12, or folate-binding protein, which binds folate. This type of assay is often referred to as a heterogeneous assay because of the solid phase involved. The chemiluminescent signal associated with the binding complex can then be measured, and the presence or absence of the analyte in the sample can be inferred. Typically, the binding complex is separated from the remaining binding reaction components, such as excess labeled analyte, prior to signal generation. For example, if the binding complex is bound to magnetic beads, a magnet can be used to separate the bead-bound binding complex from the bulk solution.

[0109] By using a series of "standards," i.e., known concentrations of analyte, a "dose-response" curve can be generated for a known labeled analyte. The dose-response curve thus correlates a fixed amount of measured signal with a particular concentration of analyte. In a competitive assay, when measuring chemiluminescence from a binding complex, the amount of signal decreases as the concentration of analyte increases. The concentration of analyte in an unknown sample can be calculated by comparing the dose-response curve to the signal generated by an unknown sample containing the macromolecular analyte.

[0110] The methodology for binding a binding molecule, such as an antibody, to a solid phase typically involves mixing the components necessary to induce binding. For example, antibodies can be covalently bound to particles containing amines on their surfaces by using a cross-linking molecule such as glutaraldehyde. Binding can also be non-covalent and involve simple adsorption of the binding molecule to the surface of a solid phase, such as polystyrene beads and microtiter plates. Labeling of binding molecules, such as antibodies and other binding proteins, is also well known in the prior art and is generally referred to as a conjugation reaction, with labeled antibodies often referred to as conjugates. Typically, an amine-reactive moiety on the label reacts with an amine on the antibody to form an amide bond. Other bonds between the antibody and the label, such as thioethers, esters, carbamates, etc., can also be used.

[0111] In another aspect of the invention, a reagent can be provided for the detection of an analyte comprising a chemiluminescent acridinium compound conjugated to the analyte or a binding partner. The concentration of detectable conjugate in the reagent is 10 -3 In some embodiments, the concentration of the reagent may be less than 10 -3 M (e.g., 10 -15 M~10 -3 A chemiluminescent acridinium compound of M. In some embodiments, the compound can be provided in a reagent that further comprises a buffer.

[0112] Typically, an assay for the detection or quantification of an analyte in a sample comprises: (a) providing a detectable conjugate; (b) providing a solid support having immobilized thereon a molecule capable of forming a binding complex with the analyte and capable of forming a binding complex with the detectable conjugate; (c) mixing the compound, the solid support, and the sample; (d) separating the solid support from the mixture; (e) inducing chemiluminescence of any acridinium label complexed to the solid phase; (f) measuring the amount of light emitted with a luminometer; (g) detecting the presence of, or calculating the concentration of, the analyte by comparing the amount of light emitted to a standard dose-response curve relating the amount of light emitted to a known concentration of the analyte.

[0113] In some embodiments, the sample is from a mammal (e.g., a human). In some embodiments, the sample comprises saliva and / or blood and / or serum. In some embodiments, the sample is saliva and / or blood and / or serum.

[0114] In some assays, the sample to be analyzed is pretreated to release the analyte from endogenous binding substances, such as plasma or serum proteins, that bind to the analyte. Release of the analyte from the endogenous binding substances can be achieved, for example, by adding a digestion agent or a release agent, or a combination of a digestion agent and a release agent used sequentially. A digestion agent breaks down the endogenous binding substance so that it can no longer bind to the analyte.

[0115] Conditions for performing an assay on a portion of a sample according to the principles described herein can include performing the assay in an aqueous buffered medium at a moderate pH, generally a pH that provides optimal assay sensitivity. The aqueous medium may be water alone or may contain 0.1-40% by volume of a cosolvent. The pH of the medium may range from 4-11, 5-10, 6.5-9.5, or 7-8. Typically, the pH value of the solution is a compromise between optimal binding of the binding members of any particular binding pair and the optimal pH for other reagents in the assay, such as members of the signal generation system. A variety of buffers can be used to achieve the desired pH and maintain it during the assay. Exemplary buffers include borate, phosphate, carbonate, TRIS, barbital, PIPES, HEPES, MES, ACES, MOPS, and BICINE.

[0116] A variety of auxiliary materials may be employed in the assay method. For example, in addition to a buffer, the medium may include stabilizers for the medium and the reagents employed. In some embodiments, the medium may include proteins (e.g., albumin), organic solvents (e.g., formamide), quaternary ammonium salts, polyanions (e.g., dextran sulfate), binding enhancers (e.g., polyalkylene glycols), polysaccharides (e.g., dextran, trehalose), and combinations thereof.

[0117] The chemiluminescence of the analog may be induced by the addition of a chemiluminescence inducing reagent. The chemiluminescence inducing reagent may be acidic or basic. Multiple chemiluminescence inducing reagents may be added sequentially. For example, an acidic solution (e.g., an acidic solution containing hydrogen peroxide) may be added first, followed by a basic solution (e.g., an alkali hydroxide solution containing a surfactant). In some embodiments, the chemiluminescence inducing reagent includes hydrogen peroxide, a hydrogen peroxide salt, nitric acid, a nitrate salt, sodium hydroxide, an ammonium salt, a surfactant, or a combination thereof. [Example]

[0118] The following examples illustrate the synthesis of a representative number of compounds, the characterization of parameters involved in assay development, and the use of these compounds in measuring samples in heterogeneous competitive assays. Thus, the examples are intended to illustrate, but not limit, the present disclosure. Additional compounds not specifically exemplified may be synthesized using conventional methods in combination with the methods described herein. [Example]

[0119] quantum yield Comparative chemiluminescence quantum yields were measured for new acridinium ester structures. The increased quantum yield of acridinium esters is one of several advantageous properties of acridinium esters that exhibit high quantum yields compared to those exhibiting low quantum yields. Chemiluminescence is measured as relative light units (RLU) in a luminometer. The quantum yield of an acridinium ester is measured as the amount of chemiluminescence (RLU) per mole of acridinium ester. Quantum yield, therefore, is the amount of observable chemiluminescence per defined mass of acridinium ester. Increasing the quantum yield of an acridinium ester increases the possibility of detection, for example, using small amounts of analyte in immunoassays, especially with acridinium esters with significantly lower masses. Increasing the quantum yield of an acridinium ester may consequently improve the sensitivity of immunoassays employing acridinium esters with high quantum yields. The relative quantum yield was calculated as a ratio of the quantum yield of HEGAE. A relative quantum yield with a value greater than 1 indicates an improvement in quantum yield relative to HEGAE.

[0120] Table 3 lists the relative quantum yields of the novel ADO acridinium esters of the present invention, along with HEGAE and HQYAE for comparison. The measured quantum yields of the new ADOAEs are significantly higher (1.7-3.8 times) than that of HEGAE, except for ADOAE A (4).

[0121] [Table 3]

[0122] Compounds ADOAE A (4) and ADOAE C (6) are structurally very similar. The only difference is that the former contains a five-membered ADO ring at the 2- and 3-positions, while the latter contains a six-membered ADO ring. However, the quantum yield of ADOAE A (4) is only one-fifth that of ADOAE C (6). This demonstrates the finding that ring size is involved in the performance of the acridinium ester and does not interfere with chemiluminescence. Without wishing to be bound by theory, increasing the ring size of the 2,3-cyclic substituent (e.g., six or seven members) increases the viability of the compound in immunoassays.

[0123] [ka] [Example]

[0124] Emission wavelength The emission spectra of the new compounds were measured using a PR-740 FSSS Spectro camera, which can measure emission intensities in the wavelength range of 380-780 nm. For comparison, the emission wavelengths of HEGAE and HQYAE were also measured. The maximum emission wavelength (λmax) of the new acridinium esters was found to be 440-450 nm, which is longer than that of HEGAE (425 nm) and shorter than that of HQYAE (475 nm).

[0125] All acridinium compounds were diluted in DMF to prepare a 1 mg / mL stock solution. 20 μL aliquots were placed in glass tubes and further diluted with 250 μL of DMF. Next, 300 μL of Flash Reagent 1 was added to the sample, and the glass tube was placed in front of a PR-740 FSSS Spectro camera. Emission spectra for all compounds were recorded over a 5-second time window after the addition of 300 μL of Flash Reagent 2. The emission spectra are shown in Figures 1A-E. Table 4 provides the measured λmax for several acridinium esters of the present disclosure.

[0126] [Table 4] [Example]

[0127] Light emission speed The chemiluminescence rates of the new acridinium esters were measured using a Berthold Technologies AutoLumat Plus LB953 luminometer (LB953). HEGAE and HQYAE were included in the measurements for comparison. Aliquots of ADOAE and TSPAE were initially diluted in DMF to produce a concentration of 1 mg / mL. The concentrations were diluted 100-fold with DMF to produce a 10 -2 This solution was diluted to 10 mg / mL in flush buffer using 100 μL of solution per dilution. -2 times to 10 -8 Ten microliters of the diluted sample was used to measure the chemiluminescence rate for a total of 5 seconds with 50 data points on an AutoLumat Plus LB953. The chemiluminescence values ​​were 10 5 ~10 6 Good linearity was obtained in the range of RLU / 10 μL and was acceptable.

[0128] The results are shown in Figure 2. As can be seen, all acridinium esters tested completed their emission within 5 seconds. The new compounds ADOAE F (9), ADOAE H (11), ADOAE J (13), and ADOAE L (15) emitted approximately 90% of their light within 2 seconds, which was significantly faster than HEGAE and HQYAE. The fast emission rate is suitable for short-cycle photodetection, which is desirable for high-throughput instruments. [Example]

[0129] Thyroid-stimulating hormone (TSH) assay AntiTSH-mAb conjugates of the new acridinium esters 7-11 and 13-15 were prepared, along with TSPAE (3) for comparison. TSPAE (3) is one of the best high-quantum-yield AEs for use in assays, with light output similar to that of HQYAE, U.S. Patent Nos. 7,309,615 and 7,785,904, which are incorporated herein by reference in their entireties, particularly with respect to the light output of HQYAE in immunoassay measurements. Conjugate concentrations were determined by Micro BCA™ Protein assay. Acridinium ester incorporation into antiTSH mAb was measured by MALDI-TOF mass spectrometry.

[0130] The immunoassay functionality of the conjugates was evaluated using a Siemens-Healthineers Centaur TSH3 UL in an ADVIA Centaur XPT assay, substituting the Lite reagent with the experimental antiTSH-mAb conjugate, while all other test reagents remained the same. The relative light output of each tested compound was measured with 10 different standards, each with a known concentration of TSH.

[0131] Briefly, anti-TSH conjugates of several TSH conjugates AE were diluted to 0.3 mg / mL in TSH3-UL Lite reagent buffer (SAP procedure 42196). Commercially available TSH3-UL reagent (REF06491072 Lot 332) was used for this study. Anti-FITC solid phase and FITC auxiliary reagents were recovered from Lot 332 and combined with each TSH-AELite reagent. The reagents were then assayed on an ADVIA Centaur XPT (Instrument ID: B1072). This instrument automatically performs the following actions: Dispense 100 μL of sample (standard substance) into a cuvette. Dispense 50 μL of Supplementary Reagent and 50 μL of Lite Reagent and incubate at 37°C for 2.75 minutes. Dispense 200 μL of solid phase and incubate at 37°C for 5.5 minutes. Wash1 separates, aspirates, and washes the cuvette. Dispense 300 μL each of Acid Reagent (Flash Reagent 1) and Base Reagent (Flash Reagent 2) to initiate the chemiluminescence reaction. Internal TSH3-UL master curve standard lot 19031 was used as the sample and average RLU was calculated.

[0132] As shown in Tables 5 and 6, the conjugates tested were functional in the assay.

[0133] [Table 5]

[0134] [Table 6]

[0135] The signal-to-noise ratios (SN ratios) for each compound under each test condition were also calculated based on the RLU values ​​in Tables 5 and 6. Tables 7 and 8 provide the SN ratios of the measured conjugates. Most conjugates had low background signals with the zero-dose TSH standard. Compounds 7, 9, 11, and 15 showed comparable or higher signals at the high dose end compared to TSPAE (3) (e.g., S10). The overall SN ratios for compounds 9, 13, and 15 at zero dose were higher than those of TSPAE. A higher SN ratio typically correlates with better assay sensitivity provided by the compounds of the present disclosure.

[0136] [Table 7]

[0137] [Table 8] [Example]

[0138] Chemiluminescence stability Excellent chemiluminescence stability (negligible instability) is one of several advantageous properties of acridinium esters used as labels in immunoassays, ensuring that assay-derived clinical data will remain unchanged and not be invalidated over the lifetime of the test kit. For example, the primary mechanism of chemiluminescence instability of acridinium esters in aqueous solution is hydrolysis of the phenolic ester by hydroxide anion and other nucleophiles. High-quantum-yield acridinium esters, such as HQYAE and TSPAE, which contain two hydrophilic alkoxy groups at the 2- and 7-positions, have been observed to be less stable than unsubstituted acridinium esters, likely due to an additional mechanism of chemiluminescence instability.

[0139] Comparative chemiluminescence stability was measured for new ADO acridinium esters conjugated to anti-hTSH monoclonal antibodies via N-hydroxysuccinimide activation of the acridinium ester benzoic acid group. The rate of chemiluminescence instability of the acridinium ester was measured by loss of chemiluminescence over a set period of time under conditions approximating the expected storage and handling conditions of the assay test kit.

[0140] Furthermore, isolating and heating acridinium esters at temperatures exceeding the recommended storage conditions was intended to estimate the long-term instability of the product. In addition to evaluating the stability of the newly generated acridinium ester conjugates, we also tested one control conjugate from TSPAE, representing commercially available high-quantity acridinium esters. The incubation buffers were prepared to narrow the interpretation of results to those related to loss of chemiluminescence due to temperature and pH alone. Therefore, these buffers did not contain complex biological components or significant amounts of detergents and other potential stabilizers that could complicate the interpretation of results.

[0141] The chemiluminescence instability of the comparative acridinium esters was measured at two temperature ranges: 4°C (standard refrigeration) and 37°C (accelerated heating), and at three pH values: 6.0, 7.0, and 8.0, within each of these two temperature ranges. The buffer used to measure the chemiluminescence stability of the comparative acridinium ester antibody conjugates consisted of 0.10 M sodium phosphate (pH buffer), 0.15 M sodium chloride (ionic strength agent), 7.7 mM sodium azide (antimicrobial preservative), and 0.1% (w / v) bovine serum albumin (protein conjugate stabilizer). Three volumes of this buffer were then separately adjusted to the three pH values ​​indicated. Chemiluminescence was measured as relative light units (RLU) in a luminometer. Measurement of the residual chemiluminescence of each acridinium ester antibody conjugate was performed using an ADVIA Centaur XPT, with the conjugates initially diluted to approximately 5 x 10 6 The solution was diluted to a target chemiluminescence concentration of RLU / 25 μL. This level of chemiluminescence provided a high enough starting value to allow for measurable decay and was well within the linear range of the Centaur luminometer. Chemiluminescence was measured periodically over approximately one month on the Centaur using five 25 μL replicates at each time point. The chemiluminescence reaction was initiated in the cuvette by sequentially adding 0.30 mL of Flash Reagent 1, followed 60 seconds later by adding 0.30 mL of Flash Reagent 2. The chemiluminescence acquisition time was nominally 3,500 seconds. The dark count time was 2,000 seconds. Chemiluminescence was reported as net chemiluminescence, calculated by subtracting the adjusted dark counts from the total chemiluminescence. The percentage of remaining chemiluminescence was calculated relative to the initial chemiluminescence from the average of the five replicates collected from each time point and tabulated.

[0142] Two set-term experiments were performed. Tables 9 and 10 show the stability of several representative ADO acridinium esters at three pH conditions (pH 6, 7, and 8) at 4°C (common reagent storage temperature, Table 9) and 37°C (accelerated temperature, Table 10) for 33 days in the first set-term experiment and 35 days in the second set-term experiment. All ADOAEs exhibited better chemiluminescence stability than TSPAE when stored at the nominal 4°C. At 37°C, although the elevated temperature accelerated the instability of the acridinium esters, all new ADOAEs demonstrated significantly better chemiluminescence stability than TSPAE. This was particularly evident at pH 7 and pH 8, the pH range in which most immunoassays are performed. For example, at 37°C on day 33, the chemiluminescence activity of TSPAE decreased to less than 1% of its original chemiluminescence at pH 7 and pH 8, while the chemiluminescence activity of the new ADOAEs remained high at 73% and 85%, respectively.

[0143] [Table 9]

[0144] [Table 10] [Example]

[0145] Synthesis of ADOAE A (4) and ADOAE B (5) [ka]

[0146] The synthesis of compounds ADOAE A (4) and ADOAE B (5) began with the known starting material, 5-methoxyisatin (4B). N-Arylation of 5-methoxyisatin was carried out in a 2 g scale with 12 (2.1 g, 11 mmol) in DMF at 150 °C for 8 h using NaH (11 mmol) as the base and CuI (22 mmol) as the coupling agent. LC / MS analysis indicated that 70% of the N-arylisatin had undergone further rearrangement to acridine 9-carboxylic acid (4C). At this stage, DMF was removed from the reaction mixture under high vacuum at 60 °C, and 10% KOH solution was added. The mixture was refluxed at 120 °C for 2 h. LCMS analysis confirmed the complete conversion of the reaction intermediate to 4C. The mixture was filtered through a sintered funnel, and the resulting filtrate was cooled to room temperature and acidified to pH 2 with concentrated HCl. The orange precipitate was isolated at 5 °C, filtered, and dried under vacuum to give acridine 9-carboxylic acid (4C) in 85% yield in two steps. Esterification of acridine 9-carboxylic acid (4C) (1.32 g, 4.44 mmol) with the phenol derivative 4D (400 mg, 2.22 mmol) and tosyl chloride in pyridine was completed at 35 °C for 8 h to give acridine 9-carboxylate 4E in 70% yield. N-Sulfopropanation of 4E (50 mg, 0.11 mmol) was carried out in a microwave reactor at 155 °C using 10 equivalents of 1,3-propane sultone in the ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate [BMIM][PF6] and 2,6-di-tert-butylpyridine as the base. The reaction was 60% complete in 6 hours, at which point 2N HCl was added to the reaction mixture and stirring was continued at 120 °C for 2 hours to give ADOAE A (4) in 14% overall yield. Next, the activated NHS ester compound was prepared for protein conjugation. ADOAE A (9 mg, 0.015 mmol) was treated with TSTU and N,N-diisopropylethylamine in DMF at room temperature for 30 minutes to give the final ADOAE B (5) as a yellow solid. [Example]

[0147] Synthesis of ADOAE C (6) and ADOAE D (7) [ka]

[0148] The synthesis of ADOAE C (6) and ADOAE D (7) was initiated from the starting materials 5-methoxyisatin (4B) and bromo derivative 6A. Coupling of 4B (2 g, 11 mmol) and bromo derivative 6A (2.1 g, 11 mmol) was completed in 8 h at 150 °C in DMF using NaH (11 mmol) as the base and CuI (22 mmol) as the coupling agent. LC / MS analysis confirmed that 50% of the N-arylisatin product had been rearranged to acridine-9-carboxylic acid 6B. At this stage, DMF was removed from the reaction mixture under high vacuum at 60 °C, and 10% KOH solution was added. The mixture was refluxed at 120 °C for 2 h. The N-arylisatin was completely converted to acridine-9-carboxylic acid, as confirmed by LC / MS analysis. The mixture was filtered through a sintered funnel, and the resulting filtrate was cooled to room temperature and further acidified to pH 2 with concentrated HCl. After 30 min at 5 °C, the orange-yellow precipitate was filtered and dried under vacuum to afford acridine 9-carboxylic acid 6B as an orange powder in 86% yield in two steps. Esterification of acid 6B (1.44 g, 4.44 mmol) with the phenol derivative 4D (400 mg, 2.22 mmol) and tosyl chloride in pyridine was completed in 8 h at room temperature to give acridine ester 6C in 70% yield. N-Sulfopropanation of 6C (50 mg, 0.11 mmol) was carried out in a microwave reactor at 155 °C using 10 molar equivalents of 1,3-propane sultone in the ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate [BMIM][PF6] and 2,6-ditert-butylpyridine as the base. The reaction was 80% complete in 6 hours. At this stage, 2N HCl was added to the reaction mixture and stirring was continued at 105 °C for 2 hours, affording ADOAE C (6) in 50% overall yield. The activated NHS ester was then prepared. Compound 6 (10 mg, 0.017 mmol) was treated with TSTU (0.051 mmol) and N,N-diisopropylethylamine (0.034 mmol) in DMF for 30 minutes, affording ADOAE D (7) in 52% yield after prep-HPLC purification. [Example]

[0149] Synthesis of ADOAE E (8) [ka]

[0150] The synthesis of ADOAE E (8) began with the intermediate acridine 9-ester (6C). Methyl ether cleavage of 6C (400 mg, 0.084 mmol) was carried out using 10 equivalents of BBr3 (1 M, CHCl2) at 0 °C for 5 h to give the hydroxy derivative 8A in 75.5% yield. Dialkylation was then carried out at elevated temperature using 1,3-propane sultone in ionic liquid (IL). Compound 8A (50 mg, 0.11 mmol) was reacted with 20 equivalents of 1,3-propane sultone, 10 equivalents of 2,6-di-tert-butylpyridine, and 2 equivalents of KCO3 in 1-butyl-3-methylimidazolium hexafluorophosphate [BMIM][PF6] in a microwave reactor at 160 °C for 6 h. 80% of the reaction was complete within 6 h. At this stage, the crude reaction mixture was hydrolyzed with 2N HCl at 120 °C for 2 h. The resulting acid compound was directly purified by prep-HPLC. The lyophilized HPLC fraction afforded pure acid 8B (17 mg, 23% yield). Finally, NHS ester synthesis was carried out using 3 equivalents of TSTU and 2 equivalents of N,N-diisopropylethylamine in DMF. After prep-HPLC purification of the crude reaction mixture, 8 mg of ADOAE E (8) was obtained. [Example]

[0151] Synthesis of ADOAE F(9) [ka]

[0152] The acid derivative 6 (10 mg, 0.015 mmol) was directly coupled to the HEG amine spacer in one step for 30 min by in situ activation of the acid with TSTU. Subsequently, the reaction mixture was added to diamino HEG in DMF. After 2 h, the reaction mixture was purified by prep-HPLC, and the prep-HPLC fraction was lyophilized for 48 h to give the amine compound 9A in 79% yield. The resulting amine derivative (10 mg, 0.11 mmol) was converted to the NHS ester using 3 equivalents of DSG and pH 7.2 phosphate buffer in DMF at room temperature. The reaction mixture was directly purified by HPLC, and after lyophilization of the preparative fraction, 6.2 mg of ADOAE F (9) was obtained as a yellow compound. [Example]

[0153] Synthesis of ADOAE G (10) and ADOAE H (11) [ka]

[0154] The synthesis of symmetric-dioxane-AE was initiated from the known, commercially available (5,6)-ethylenedioxy-isatin 10A. Isatin (1 g, 4.47 mmol) and bromo derivative 6A (1.83 g, 8.94 mmol) were coupled in the presence of CuI and NaH in dry DMF at 155 °C for 12 h, whereupon the reaction proceeded further to rearrange to acridine-9-carboxylic acid. The crude product was purified using an acid / base extraction to give the desired pure acid 10B in 30% yield. Next, in an esterification reaction, acid derivative 10B (200 mg, 0.59 mmol) was reacted with phenol derivative 4D (106 mg, 0.59 mmol) using tosyl chloride in a solvent mixture of CHCl and pyridine (9:1, 10 mL) to give 10C in 70% yield. The synthesis of NSP-AE-acid involved two reactions in a one-pot synthesis. First step: Acridine 9-carboxylate 10C (80 mg, 0.16 mmol) was N-alkylated with 1,3-propane sultone in a microwave reactor. The reaction was monitored by LCMS, and the N-alkylation was complete in 8 h. Second step: Methyl ester hydrolysis was carried out with 2 N HCl at 120 °C for 2 h, and the acid compound was purified using prep-HPLC to give 19 mg of 10D in 20% yield. Finally, for the NHS ester synthesis, HPLC-purified 10D (6 mg, 0.001 mmol) was treated with 3 equivalents of TSTU and 2 equivalents of N,N-diisopropylethylamine in DMF. After 30 min, the reaction mixture was purified by prep-HPLC to give 8 mg of ADOAE G (10).

[0155] The synthesis of ADOAE H (11) was initiated from the acid derivative 10D. The acid derivative (10 mg, 0.016 mmol) was coupled with HEG-diamine (13.4 mg, 0.048 mmol) via acid activation with TSTU, followed by amide formation with HEG-diamine. The reaction was complete in 30 min. The crude product was directly purified by preparative HPLC to give 2 mg of the terminal amine 11A. The final NHS ester synthesis was achieved by treating 11A (2 mg, 0.0028 mmol) with DSG in DMF and pH 7.5 phosphate buffer at room temperature for 30 min. The crude product was purified by preparative HPLC to give 2 mg of ADOAE H (11) in 64% yield. [Example]

[0156] Synthesis of ADOAE I(12) and J(13) [ka]

[0157] The synthesis of ADOAEs I (12) and J (13) began with commercially available 5-methoxyisatin. 5-Methoxyisatin 4B (2 g, 13.6 mmol) was N-arylated with bromo 12A (1 g, 5.64 mmol) using CuI and NaH in dry DMF at 150 °C for 12 h, where the maximum reaction (90%) proceeded further to rearrange to acridine-9-carboxylic acid. At this stage, DMF was removed from the reaction mixture under reduced pressure at 60 °C, and the crude mixture was refluxed in 10% KOH (10 mL) for 30 min. The crude acid product was acidified with concentrated hydrochloric acid to give 1.2 g of acridine-9-carboxylic acid 12B in 67% overall yield. Next, the esterification reaction of 12B (0.5 g, 1.53 mmol) with the phenol derivative 4D (221 mg, 1.23 mmol) was carried out overnight at 35 °C using tosyl chloride in a solvent mixture of CHCl:pyridine (9:1), affording acridine 9-phenylcarboxylate 12C in 67% yield. The synthesis of NSP-AE-acid 12D involved two reactions using a one-pot synthesis protocol. First step: Acridine 9-carboxylate 12C (100 mg, 0.205 mmol) was N-alkylated with 10 equivalents of 1,3-propane sultone in a microwave reactor. The reaction was monitored by LCMS, and the N-alkylation was complete within 8 h. Second step: Methyl ester hydrolysis was carried out in a microwave reactor with 2N HCl (10 mL) at 105 °C for 2 h. The acid compound was purified using prep-HPLC to give acridinium NSP-DMAE-acid 12D in 17% yield. The HPLC-purified 12D (10 mg, 0.017 mmol) was then treated with 3 equivalents of TSTU and 2 equivalents of N,N-diisopropylethylamine in DMF. After 30 min, the reaction mixture was purified by prep-HPLC to give 8 mg of ADOAE I (12).

[0158] The synthesis of HEG-amine was carried out in two steps from acid 12D. Step 1: Acid 12D (8 mg, 0.015 mmol) was treated with TSTU (7.7 mg, 0.026 mmol) and 2 equivalents of DIPEA in DMF. The reaction was complete in 30 min by LCMS mass spectrometry. At this stage, the NHS ester intermediate mixture was transferred to a stirred mixture of Diamino-PEG6 and 4 equivalents of DIPEA in DMF at room temperature. After 2 h, the mixture was purified by HPLC to give 3 mg of HEG-amine 13A in 26% yield. The resulting HEG-amine derivative 13A (3 mg, 0.0035 mmol) was reacted with DSG (3.4 mg) in DMF at room temperature and 5 equivalents of pH 7.5 buffer as a base. After stirring for 30 min at room temperature, the final product was purified by HPLC to give 2.8 mg of ADOAE J (13) in 76% yield. [Example]

[0159] Synthesis of ADOAE K(14) and L(15) [ka]

[0160] The synthesis of ADOAE K (14) began with the known, commercially available isatin (14A) and 6-bromo-1,4-benzodioxane 6A. Isatin (2 g, 13.6 mmol) was N-arylated with bromo 6A (4.36 g, 20.4 mmol) using CuI and NaH in dry DMF at 150 °C for 12 h, where the maximum reaction proceeded further and resulted in complete rearrangement to acridine-9-carboxylic acid. At this stage, DMF was removed from the reaction mixture under reduced pressure at 60 °C, and the crude acid product was acidified with concentrated hydrochloric acid to give 2 g of acridine-9-carboxylic acid 14B in 52% overall yield. The subsequent esterification reaction of 14B (0.5 g, 1.77 mmol) with the phenol derivative 4D (256 mg, 1.54 mmol) using tosyl chloride in CHCl:pyridine (9:1) at 35 °C overnight gave acridine 9-methylcarboxylate 14C in 76% yield. The synthesis of NSP-AE-acid involved two reactions in one pot. First, acridine 9-carboxylate 14C (120 mg, 0.127 mmol) was N-alkylated with 10 equivalents of 1,3-propane sultone in a microwave reactor. The reaction was monitored by LCMS and was complete within 8 h. Second, the methyl ester hydrolysis was carried out in a microwave reactor with 2 N HCl (10 mL) at 110 °C for 2 h. The resulting crude product was filtered through a sintered funnel, and the filtrate was purified by prep-HPLC to give acridinium NSP-AE acid 14D in 56% yield. The HEG-amine synthesis was carried out in two steps from acid 14D (30 mg, 0.054 mmol). Step 1: 20 mg of the acid derivative was treated with TSTU (24 mg, 0.082 mmol) and 2 equivalents of DIPEA base in DMF. The reaction was complete in 30 min, as confirmed by LCMS. At this stage, 50% of the reaction mixture was directly purified by prep-HPLC, and 6 mg of ADOAE K (14) was isolated. Step 2: The remaining 50% of the reaction mixture (Step 1) was reacted with Diamino-PEG6 and 4 equivalents of DIPEA in DMF. The mixture was purified by HPLC, and after lyophilization of the preparative fractions, 5 mg of pure HEG-amine (15A) was obtained.The final NHS ester synthesis was carried out with DSG in DMF, 5 equivalents of pH 7.5 buffer as the base, and the final product was purified by HPLC to give 3 mg of ADOAE L (15). [Example]

[0161] Synthesis of ADOAE M (16), ADOAE N (17), ADOAE P (19), and ADOAE Q (20) [ka]

[0162] General synthesis: The synthesis of ADOAE M (16), ADOAE N (17), ADOAE P (19), and ADOAE Q (20) was accomplished in two steps from the AE acid intermediates 14D, 10D, ADOAE C (6), and 12D.

[0163] Step 1: 3 mg of the AE acid of each compound was separately activated with 2 equivalents of TSTU and 2 equivalents of DIPEA in DMF. The reaction was complete in 30 minutes by LCMS mass spectrometry. At this stage, the NHS-activated mixture (AE-NHS ester) was transferred dropwise to a stirred mixture of 1.5 equivalents of HEG-diamine and 2 equivalents of DIPEA in DMF at 0 °C. The reaction temperature was slowly raised to room temperature over 30 minutes. After 2 hours at room temperature, LCMS indicated complete conversion of the AE-NHS ester to the AE-HEG-amine product. Step 2: 2 equivalents of (sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) sulfo-SMCC were added to the reaction mixture, the temperature was raised to 40 °C, and the reaction was complete in 1 hour by LCMS mass spectrometry. The resulting crude mixture was directly purified by prep-HPLC. Lyophilized HPLC fractions yielded pure ADOAE M (16): 3 mg (53%), ADOAE N (17): 2 mg (37.2%), ADOAE P (19): 1.5 mg (28.3%), and ADOAE Q (20): 2 mg (37%). [Example]

[0164] Synthesis of ADOAE O(18) [ka]

[0165] The synthesis of ADOAE-O(18) was carried out in a single step from acid 8B. Acid 8B (3.5 mg, 0.015 mmol) was treated with TSTU (2.3 mg, 0.076 mmol) and DIPEA in DMF, and the reaction was complete in 30 min by LCMS mass spectrometry. At this stage, aminoethylmaleimide (3.81 mg, 0.015 mmol) was added to the reaction mixture at room temperature. After 2 h, the mixture was purified by HPLC to produce 2.5 mg of ADOAE-O(18) in 60.6% yield. [Example]

[0166] Synthesis of ADOAE R(21) [ka]

[0167] The synthesis of ADOAE® (21) was initiated from the acid derivative 8B. The acid derivative (5 mg, 0.009 mmol) was coupled with HEG-diamine (3.64 mg, 0.013 mmol) via acid activation with TSTU, followed by amide formation with HEG-diamine. The reaction was complete in 30 min. The crude product was directly purified by preparative HPLC to give 3 mg of the terminal amine 21A in 43% yield. The final acridinium ester-maleimide cyclohexanecarboxylate (AE-MCC) synthesis was achieved by reacting 21A (3 mg, 0.0028 mmol) with 1.5 equivalents of SULFO-SMCC in DMF / pH 7.5 phosphate buffer at room temperature for 30 min. The crude product was directly purified by preparative HPLC to give 2 mg of ADOAE® (21) in 54% yield. [Example]

[0168] Preparation of acridinium ester-antiTSH antibody conjugate The following procedure was typical for producing the conjugates described herein. A 1 mL solution of AntiTSH-mAb (2 mg) in 0.1 M phosphate buffer (pH = 8) was treated with 10 equivalents of acridinium esters (TSPAE (2), ADOAE D (7), ADOAE E (8), ADOAE F (9), ADOAE G (10), ADOAE H (11), ADOAE J (13), ADOAE K (14), and ADOAE L (15) (structures shown in Figures 3A-3C), which were added as a solution in DMSO (0.033 mL of a 4 mmole / L solution in DMSO). The reaction was stirred for 16 hours in the dark at room temperature between 2 and 5°C. The labeling reaction was prepared using Amicon The conjugate was transferred to an Ultra-430 kDa molecular weight cutoff filter and diluted with 3 mL of deionized water. The filter was centrifuged at 5000 × G for 10 minutes to reduce the volume to approximately 0.2 mL. This process was repeated four more times. Approximately 0.2 mL of the final conjugate was brought to a total of 1 mL with deionized water to yield a 2 mg / mL solution. AE-antiTSH mAb protein concentration was measured by microBCA assay. Acridinium ester incorporation into the antiTSH mAb was measured by MALDI-TOF mass spectrometry.

[0169] All references cited herein, including patent applications and publications, are incorporated herein by reference and for all purposes to the same extent as if each publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Many modifications and variations of the present invention can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific embodiments described herein are provided by way of example only, and the present invention is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled.

Claims

1. Structure of Formula (I): 【Chemical 1】 wherein A is an analyte or a binding partner of the analyte; L is either absent (i.e., a bond) or a group L Ｃ or Z Ｌ and optionally comprising: Ψ has the following structure: 【Chemistry 2】 wherein "j" is 1, 2, 3, 4, 5, or 6; R １ is hydrogen, -R, -X, -R Ｌ -X ｂ , -L １ -R, -L １ -X ｂ , -Z, -R Ｌ -Z, -L １ -Z or -R Ｌ -L １ -R Ｌ -Z; R ２ and R ３ are independently selected from hydrogen, —R, an electron donating group, or —Z, or R ２ and R ３ together form a 5- to 7-membered fused heterocyclic group; Z independently at each occurrence has the structure: 【Chemistry 3】 wherein "q" and "l" are independently 0 or 1; "r" is independently an integer from 0 to 10 (e.g., 1 to 10); L １ is independently, for each occurrence, -O-, -S-, -NH-, -N(R Ｎ ), -(CH ２ ) １～１０ -, -S(=O) １～２ -, -C=C-, -C=C-(CH ２ ) １～３ -, -C(O)-, -O-C(O)-, -C(O)-(CH ２ ) １～４ -, -(CH ２ ) １～４ -C(O)-, -C(O)-O-, -C(O)-N(R Ｎ ), -C(O)-NH-, -N(R Ｎ )-C(O)-, -NH-C(O)-, -C(O)-N(R Ｎ )-(CH ２ ) １～３ -, -(CH ２ ) １～３ -C(O)-N(R Ｎ ), -NH-S(O) １～２ -, -N(R Ｎ )-S(O) １～２ -, -S(O) １～２ -N(R Ｎ ), -S(O) １～２ -NH-, -(CH ２ ) １～３ -NH-S(O) １～２ -, -(CH ２ ) １～３ -N(R Ｎ )-S(O) １～２ -, -(CH ２ ) １～３ -S(O) １～２ -N(R Ｎ ), -(CH ２ ) １～３ -S(O) １～２ -NH-, -O-(CH ２ ) １～４ -, -(CH ２ ​​​​​​​​​​​​​​​​)-(CH ２ ) １～４ -, -(CH ２ ) １～４ -N(R Ｎ ) -, -(OCH ２ ) １～１０ -, -(CH ２ O) １～１０ -, -(OCH ２ CH ２ ) １～１０ - or -(CH ２ CH ２ O) １～１０ - and; R Ｌ is independently C for each occurrence. １～２０ divalent hydrocarbon (e.g., alkyl, alkenyl, aryl, phenyl, monoalkyl-substituted phenyl, dialkyl-substituted phenyl, alkynyl, arylalkyl), optionally having one or more (e.g., 1-10, 1-5) points of substitution (e.g., with 1-10 heteroatoms such as heteroalkyl, heteroalkenyl, heteroaryl, heteroalkynyl, heteroarylalkyl groups, with 1-10 substituents such as groups -X); R is independently at each occurrence hydrogen or C １～３５ a hydrocarbon (e.g., alkyl, alkenyl, alkynyl, or aralkyl) radical, optionally having one or more (e.g., 1-20, 1-10, 1-5) points of substitution (e.g., with 1-20 heteroatoms such as heteroalkyl, heteroalkenyl, heteroaryl, heteroalkynyl, heteroarylalkyl groups, with 1-20 substituents such as groups -X); R′ and R″ at each occurrence are independently hydrogen or C １～１０ is alkyl; X ｂ is independently at each occurrence an anionic group; R Ｎ is independently at each occurrence hydrogen, or C １～５ a chemiluminescent acridinium containing a zwitterionic group having an alkyl group selected from alkyl (e.g., methyl, ethyl, propyl); L Ｃ is a divalent C optionally having one or more (e.g., 1-10, 1-5) points of substitution, one or more (e.g., 1-20, 1-10, 1-5) points of substitution (e.g., with 1-20 heteroatoms such as heteroalkyl, heteroalkenyl, heteroaryl, heteroalkynyl, heteroarylalkyl groups, with 1-20 substituents); １～３５ is a hydrocarbon; Z Ｌ is a zwitterionic linker group having the structure: 【Chemistry 4】 "m" is 0 (i.e., a bond) or 1; "n" and "p" at each occurrence are independently an integer from 0 (i.e., a bond) to 10; X ａ is independently at each occurrence an anionic group; R Ｌ is independently C for each occurrence. １～２０ a divalent hydrocarbon radical (e.g., alkyl, alkenyl, aryl, alkynyl, arylalkyl), optionally having one or more (e.g., 1-10, 1-5) points of substitution (e.g., substituted with 1-10 heteroatoms, substituted with 1-10 substituents); R' is hydrogen or C １～１０ alkyl) or a salt thereof (e.g., a halide salt such as a chloride salt).

2. R １ But -R, -X, -R Ｌ -X ｂ , -L １ -R, -L １ -X ｂ , -Z, -R Ｌ -Z, -L １ -Z or -R Ｌ -L １ -R Ｌ The compound of claim 1, wherein the group is -Z.

3. R ３ 3. The compound of claim 1 or 2, wherein is hydrogen.

4. R ２ is at one or more (e.g., 1, 2, 3) positions independently selected from one or more substituents (e.g., —S(═O) １－２ -R*, -O-S (=O) ２ -R*, -S (=O) ２ -OR*, -O-SO ３、 -O-S(=O) ２ -X such as -OR*, -O-S(=O)-OR*, -O-S(=O)-R*, -S(=O)-OR*, or -S(=O)-R* (wherein R* is H or C １～１０ The compound of any one of claims 1 to 3, wherein the alkoxy is optionally substituted with a hydrocarbon.

5. Ψ has the following structure: 【Chemistry 5】 wherein "h" is 1, 2, 3, 4, 5, or 6.

2. The compound of claim 1, comprising:

6. Ψ has the following structure: 【Chemistry 6】 The compound according to any one of claims 1 to 5, comprising:

7. Ψ is a structure of formula (II) 【Chemistry 7】 where Ω is S, O, or N; Y is -R, -L １ -R, -R Ｌ -Z, -L １ -R Ｌ -Z, or when Ω is O or S, Y is absent; Y' is absent (i.e., a bond) or -L １ -, -R Ｌ -, -R Ｌ -L １ -, -L １ -L １ -, -L １ -R Ｌ -, -L １ -R Ｌ -L １ , or -R Ｌ -L １ -R Ｌ -, and Y' is selected from A (e.g., when L is absent) or L (e.g., when L Ｃ Or Z Ｌ ) ; R １ is hydrogen, -R, -X, -R Ｌ -X, -L １ -R, -L １ -X, -Z, -R Ｌ -Z, -L １ -Z or -R Ｌ -L １ -R Ｌ -Z; R ２ and R ３ is independently selected from hydrogen, —R, an electron donating group, or —Z; Z has the following structure: 【Chemistry 8】 wherein "q" and "l" are independently 0 or 1; "r" is independently an integer from 0 to 10; L １ is, independently for each occurrence, —O—, —S—, —NH—, —N(R Ｎ ), —(CH ２ ), １～１０ —S(=O) １～２ —, —C═C—, —C═C—(CH ２ ), １～３ —, —C(O)—, —O—C(O)—, —C(O)—(CH ２ ), １～４ —, —(CH ２ ), １～４ —C(O)—, —C(O)—O—, —C(O)—N(R Ｎ ), —C(O)—NH—, —N(R Ｎ ), —C(O)—, —NH—C(O)—, —C(O)—N(R Ｎ ), —(CH ２ ), １～３ —, —(CH ２ ), １～３ —C(O)—N(R Ｎ ), —NH—S(O) １～２ —, —N(R Ｎ ), —S(O) １～２ —, —S(O) １～２ —N(R Ｎ ), —S(O) １～２ —NH—, —(CH ２ ), １～３ —NH—S(O) １～２ —, —(CH ２ ),[[ID=5​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​)-(CH ２ ) １～４ -, -(CH ２ ) １～４ -N(R Ｎ ) -, -(OCH ２ ) １～１０ -, -(CH ２ O) １～１０ -, -(OCH ２ CH ２ ) １～１０ - or -(CH ２ CH ２ O) １～１０ - and; R Ｌ is independently C for each occurrence. １～２０ a divalent hydrocarbon (e.g., alkyl, alkenyl, aryl, phenyl, monoalkyl-substituted phenyl, dialkyl-substituted phenyl, alkynyl, arylalkyl), optionally substituted with 1 to 10 heteroatoms; R is independently at each occurrence hydrogen or C １～３５ a hydrocarbon (e.g., alkyl, alkenyl, alkynyl, or aralkyl) radical, optionally substituted with 1 to 20 heteroatoms; R′ and R″ at each occurrence are independently hydrogen or C １～１０ is alkyl; X ｂ is independently at each occurrence an anionic group; R Ｎ is independently at each occurrence hydrogen, or C １～５ alkyl (e.g., methyl, ethyl, propyl)) The compound according to any one of claims 1 to 6, having the formula:

8. Ω is S, O, or N, and R １ But -R, -X, -R Ｌ -X, -L １ -R, -L １ -X, -Z, -R Ｌ -Z, -L １ -Z or -R Ｌ -L １ -R Ｌ The compound of claim 7, wherein the group is -Z.

9. Ψ is a structure of formula (IIa) 【Chemistry 9】 wherein "h" is 1, 2, 3, 4, 5, or 6. The compound according to any one of claims 6 to 8, having the formula:

10. Ψ is a structure of formula (IIb): 【Chemistry 10】 (In the formula, R ５ ~R ８ are independently hydrogen or C １～３５ alkyl, alkenyl, alkynyl, aryl, alkoxy, alkylthio, or amino; L １ is A (e.g., L is absent) or L (e.g., L Ｃ Or Z Ｌ ) covalently bonded to The compound according to any one of claims 1 to 9, having the formula:

11. L １ The compound according to claim 10, wherein is —NH—C(O)—, —C(O)—NH—, —C(O)—O—, or —O—C(O)—.

12. R ５ and R ６ are each lower alkyl (e.g., C １ ~C ４ alkyl, methyl), and R ７ and R ８ 12. The detectable conjugate of claim 10 or 11, wherein each is hydrogen.

13. Ψ is a structure of formula (IIc): 【Chemistry 11】 (wherein Y" is absent or -L １ -, -R Ｌ -, -L １ -R Ｌ - or -R Ｌ -L １ - and Y" is A (e.g., L is absent) or L (e.g., L Ｃ Or Z Ｌ ) covalently bonded to The compound according to any one of claims 1 to 9, having the formula:

14. Ψ is a structure of formula (IId): 【Chemistry 12】 (wherein Y" is absent or -L １ -, -R Ｌ -, -L １ -R Ｌ - or -R Ｌ -L １ - and Y" is A (e.g., L is absent) or L (e.g., L Ｃ Or Z Ｌ ) covalently bonded to The compound according to any one of claims 1 to 9, having the formula:

15. Ψ is a structure of formula (IIe): 【Chemistry 13】 (wherein Y" is absent or -L １ -, -R Ｌ -, -L １ -R Ｌ - or -R Ｌ -L １ - and Y" is A (e.g., L is absent) or L (e.g., L Ｃ Or Z Ｌ ) covalently bonded to The compound of any one of claims 1 to 9 and 14, having the formula:

16. L is the structure -L Ｃ -(Z Ｌ ) ｚ -, where "z" is 0 or 1; L Ｃ is a divalent C １～３５ an alkyl, alkenyl, alkynyl, aryl, or arylalkyl radical, optionally substituted with 1 to 20 heteroatoms; Z Ｌ is a zwitterionic linker group having the structure: 【Chemistry 14】 "m" is 0 (i.e., a bond) or 1; "n" and "p" at each occurrence are independently an integer from 0 (i.e., a bond) to 10; X ａ is an anionic group; R Ｌ is independently C for each occurrence. １～２０ a divalent hydrocarbon radical (e.g., alkyl, alkenyl, aryl, alkynyl, arylalkyl), optionally having one or more (e.g., 1-10, 1-5) points of substitution (e.g., substituted with 1-10 heteroatoms, substituted with 1-10 substituents); R' is hydrogen or C １～１０ alkyl) The compound according to any one of claims 1 to 15, having the formula:

17. L Ｃ but has the following structure: -(X １ ) ０－１ -(R Ｌ ) ０－５ -(X ２ ) ０－１ -(R Ｌ ) ０－５ -(X ３ ) ０－１ -(R Ｌ ) ０－５ -(X ４ ) ０－１ -(R Ｌ ) ０－５ - (In the formula, X １ is ═N—, —O—, —S—, or —NR Ｎ -, -C(O)-, -NR Ｎ -C(O)-, -C(O)-NR Ｎ -, -O-C(O)-, or -C(O)-O-, -S-C(O)-, or -C(O)-S-, or X １ is the base 【Chemistry 15】 (In the formula, 【Chemistry 16】 indicates the point of attachment to any adjacent group; X ２ ~X ４ is -O-, -S-, -NR Ｎ -, -C(O)-, -NR Ｎ -C(O)-, -C(O)-NR Ｎ independently selected from —, —O—C(O)—, or —C(O)—O—, —S—C(O)—, or —C(O)—S—; R Ｌ independently for each occurrence, -(CH ２ ) １－５ -, -(CH ２ CH ２ O) １－５ - or -(OCH ２ CH ２ ) １－５ - is selected from 17. The compound of claim 16, having the formula:

18. X ａ and X ｂ may independently represent at each occurrence a carboxylate (-C(O)O － ), sulfonate (-SO ３ － ), sulfate (-OSO ３ － ), phosphate (-OP(O)(OR Ｐ ) O － ), or oxide (—O － ) and R Ｐ is hydrogen or C １～１２ 18. A compound according to any one of claims 1 to 17, which is hydrocarbon and optionally has one or more (e.g. 1-10, 1-5) points of substitution (e.g. with 1-10 heteroatoms and with 1-10 substituents).

19. R １ But, -R Ｌ -SO ３ － The compound according to any one of claims 1 to 18, comprising:

20. R １ The compound of any one of claims 1 to 18, wherein comprises (or is) sulfopropyl.

21. R １ But -S(O) ２ -NH-Z or -(CH ２ ) １－３ -S(O) ２ The compound of any one of claims 1 to 18, which is -NH-Z.

22. R ２ and R ３ is independently at each occurrence hydrogen, alkyl, or alkoxy (e.g., C １～ C ４ 22. The compound of any one of claims 1 to 21, wherein the alkoxy is alkoxy, methoxy, ethoxy, propoxy, or lower alkoxy such as isopropoxy.

23. R ２ and R ３ The compound of any one of claims 1 to 21, wherein each is hydrogen.

24. R ２ or R ３ one of R is hydrogen; ２ or R ３ the other of which is alkoxy (e.g., C １～ C ４ 22. The compound of any one of claims 1 to 21, wherein the alkoxy is alkoxy, methoxy, ethoxy, propoxy, or lower alkoxy such as isopropoxy.

25. Z Ｌ has the following structure: 【Chemistry 17】 X so that ａ is a sulfonate (-SO ３ － ), m is 1, R Ｌ The compound according to any one of claims 16 to 24, wherein is propyl, and n and p are each 3.

26. Structure of formula (IIIa) or (IIIb): 【Chemistry 18】 (wherein "z" is 0 or 1) The compound according to any one of claims 1 to 25, having the formula:

27. 27. The compound of any one of claims 1 to 26, wherein the analyte is an antibody to a thyroid hormone (e.g., thyroid stimulating hormone, e.g., A is a binding partner and thus an anti-thyroid stimulating hormone monoclonal antibody (AntiTSH-mAb)), troponin, a steroid hormone (e.g., androstenedione, testosterone), thyroglobulin, an anti-thyroid peroxidase antibody, triiodothyronine (T3) hormone, thyroxine (T4) hormone, thyroxine-binding globulin (TBG), neurofilament light chain (e.g., serum neurofilament light chain), a vitamin (e.g., vitamin D such as 25-hydroxyvitamin D), or a virus (e.g., hepatitis virus).

28. The analyte or its binding partner may be: 【Chemistry 19-1】 【Chemistry 19-2】 【Chemistry 19-3】 28. The compound of any one of claims 1-12 and 16-27, formed by reacting

29. A reagent composition for detecting an analyte comprising a compound according to any one of claims 1 to 28 in a pH buffered medium.

30. 1. An assay for the detection or quantification of an analyte in a sample comprising: (a) providing a detectable conjugate according to any one of claims 1 to 28; (b) providing a solid support having immobilized thereon a molecule capable of forming a binding complex with the analyte and / or capable of forming a binding complex with the detectable conjugate; (c) mixing the compound, the solid support, and the sample; (d) separating the solid support from the mixture; (e) inducing chemiluminescence of any acridinium label complexed to the solid phase; (f) measuring the amount of light emitted with a luminometer; (g) detecting the presence of, or calculating the concentration of, an analyte by comparing the amount of light emitted with a standard dose-response curve relating the amount of light emitted to a known concentration of the analyte; An assay comprising:

31. Structure of formula (IV): 【Chemistry 20】 where RFG is a reactive functional group for conjugating to an analyte or a binding partner of the analyte; L is either absent (i.e., a bond) or a group Z Ｌ Or L Ｃ and optionally comprising: Ψ has the following structure: 【Chemical 21】 wherein "j" is 1, 2, 3, 4, 5, or 6; R １ is hydrogen, -R, -X ｂ , -R Ｌ -X ｂ , -L １ -R, -L １ -X ｂ , -Z, -R Ｌ -Z, -L １ -Z or -R Ｌ -L １ -R Ｌ -Z; R ２ and R ３ are independently selected from hydrogen, —R, an electron donating group, or —Z, or R ２ and R ３ together form a 5- to 7-membered fused heterocyclyl group; Z independently at each occurrence has the structure: 【Chemical 22】 wherein "q" and "l" are independently 0 or 1; "r" is independently an integer from 0 to 10; L １ is, independently for each occurrence, -O-, -S-, -NH-, -N(R Ｎ ), -(CH ２ ) １～１０ -, -S(=O) １～２ -, -C=C-, -C=C-(CH ２ ) １～３ -, -C(O)-, -O-C(O)-, -C(O)-(CH ２ ) １～４ -, -(CH ２ ) １～４ -C(O)-, -C(O)-O-, -C(O)-N(R Ｎ ), -C(O)-NH-, -N(R Ｎ )-C(O)-, -NH-C(O)-, -C(O)-N(R Ｎ )-(CH ２ ) １～３ -, -(CH ２ ) １～３ -C(O)-N(R Ｎ ), -NH-S(O) １～２ -, -N(R Ｎ )-S(O) １～２ ]>-, -S(O) １～２ -N(R Ｎ ), -S(O) １～２ -NH-, -(CH ２ ) １～３ -NH-S(O) １～２ -, -(CH ２ ) １～３ -N(R Ｎ )-S(O) １～２ -, -(CH ２ ) １～３ -S(O) １～２ -N(R Ｎ ), -(CH ２ ) １～３ -S(O) １～２ -NH-, -O-(CH ２ ) １～４ -, -(CH ２ ) １～４ -O-, -S-(CH ２ ) １～４ -, -(CH ２ ) １～４ -S-, -NH-(CH ２ ) １～４ -, -N(R Ｎ )-(CH ２ ) １～４ -, -(CH ２ ) １～４ -N(R Ｎ ) -, -(OCH ２ ) １～１０ -, -(CH ２ O) １～１０ -, -(OCH ２ CH ２ ) １～１０ - or -(CH ２ CH ２ O) １～１０ - and; R Ｌ is independently C for each occurrence. １～２０ divalent hydrocarbon (e.g., alkyl, alkenyl, aryl, phenyl, monoalkyl-substituted phenyl, dialkyl-substituted phenyl, alkynyl, arylalkyl), optionally having one or more (e.g., 1-10, 1-5) points of substitution (e.g., with 1-10 heteroatoms, such as heteroalkyl, heteroalkenyl, heteroaryl, heteroalkynyl, heteroarylalkyl, with 1-10 substituents); R is independently at each occurrence hydrogen or C １～３５ a hydrocarbon (e.g., alkyl, alkenyl, alkynyl, or aralkyl) radical, optionally having one or more (e.g., 1-20, 1-10, 1-5) points of substitution (e.g., with 1-20 heteroatoms, such as heteroalkyl, heteroalkenyl, heteroaryl, heteroalkynyl, heteroarylalkyl, with 1-20 substituents); R′ and R″ at each occurrence are independently hydrogen or C １～１０ is alkyl; X ｂ is independently at each occurrence an anionic group; R Ｎ is independently at each occurrence hydrogen, or C １～５ a chemiluminescent acridinium containing a zwitterionic group having an alkyl group selected from alkyl (e.g., methyl, ethyl, propyl), L Ｃ is a divalent C having one or more (e.g., 1-10, 1-5) points of substitution (e.g., with 1-10 heteroatoms, such as heteroalkyl, heteroalkenyl, heteroaryl, heteroalkynyl, heteroarylalkyl, with 1-10 substituents). １～３５ is a hydrocarbon (e.g., alkyl, alkenyl, alkynyl, aryl, or arylalkyl); Z Ｌ is a zwitterionic linker group having the structure: 【Chemical 23】 "m" is 0 (i.e., a bond) or 1; "n" and "p" at each occurrence are independently an integer from 0 (i.e., a bond) to 10; X ａ is an anionic group; R Ｌ is independently C for each occurrence. １～２０ a divalent hydrocarbon radical (e.g., alkyl, alkenyl, aryl, alkynyl, arylalkyl), optionally having one or more (e.g., 1-10, 1-5) points of substitution (e.g., with 1-10 heteroatoms, such as heteroalkyl, heteroalkenyl, heteroaryl, heteroalkynyl, heteroaryl, heteroarylalkyl, with 1-10 substituents); R' is hydrogen or C １～１０ alkyl) or a salt thereof (e.g., a halide salt such as a chloride salt).

32. R １ But -R, -X, -R Ｌ -X ｂ , -L １ -R, -L １ -X ｂ , -Z, -R Ｌ -Z, -L １ -Z or -R Ｌ -L １ -R Ｌ 30. The compound of claim 29, wherein:

33. R ３ 33. The compound of claim 31 or 32, wherein is hydrogen.

34. R ２ is at one or more (e.g., 1, 2, 3) positions independently selected from one or more substituents (e.g., —S(═O) １－２ -R*, -O-S (=O) ２ -R*, -S (=O) ２ -OR*, -O-SO ３、 -O-S(=O) ２ -X such as -OR*, -O-S(=O)-OR*, -O-S(=O)-R*, -S(=O)-OR*, or -S(=O)-R* (wherein R* is H or C １～１０ The compound of any one of claims 31 to 35, wherein the alkoxy is optionally substituted with a hydrocarbon.

35. Ψ has the following structure: 【Chemistry 24】 (wherein "h" is 1, 2, 3 or 4) 32. The compound of claim 31 , comprising:

36. Ψ has the following structure: 【Chemistry 25】 The compound according to any one of claims 31 to 35, comprising:

37. Ψ is a structure of formula (V) 【Chemical 26】 where Ω is S, O, or N; Y is -R, -L １ -R, -R Ｌ -Z, -L １ -R Ｌ -Z, or when Ω is O or S, Y is absent; Y' is absent (i.e., a bond) or -L １ -, -R Ｌ -, -R Ｌ -L １ -, -L １ -L １ -, -L １ -R Ｌ -, -L １ -R Ｌ -L １ , or -R Ｌ -L １ -R Ｌ -, and Y' is selected from A (e.g., when L is absent) or L (e.g., when L Ｃ Or Z Ｌ ) ; R １ is hydrogen, -R, -X, -R Ｌ -X, -L １ -R, -L １ -X, -Z, -R Ｌ -Z, -L １ -Z or -R Ｌ -L １ -R Ｌ -Z; R ２ and R ３ is independently selected from hydrogen, —R, an electron donating group, or —Z; Z has the following structure: 【Chemical 27】 wherein "q" and "l" are independently 0 or 1; "r" is independently an integer from 0 to 10; L １ is, independently for each occurrence, —O—, —S—, —NH—, —N(R Ｎ ), —(CH ２ ) １～１０ —, —S(=O) １～２ —, —C═C—, —C═C—(CH ２ ) １～３ —, —C(O)—, —O—C(O)—, —C(O)—(CH ２ ) １～４ —, —(CH ２ ) １～４ —C(O)—, —C(O)—O—, —C(O)—N(R Ｎ ), —C(O)—NH—, —N(R Ｎ )—C(O)—, —NH—C(O)—, —C(O)—N(R Ｎ )—(CH ２ ) １～３ —, —(CH ２ ) １～３ —C(O)—N(R Ｎ ), —NH—S(O) １～２ —, —N(R Ｎ )—S(O) １～２ —, —S(O) １～２ —N(R Ｎ ), —S(O) １～２ —NH—, —(CH ２ ) １～３ —NH—S(O) １～２ —, —(CH ２ ) １～３ —N(R Ｎ )—S(O) １～２ —, —(CH ２ ) １～３ —S(O) １～２ —N(R Ｎ ), —(CH ２ ) １～３ —S(O) １～２ —NH—, —O—(CH ２ ) １～４ —, —(CH ２ ) １～４ —O—, —S—(CH ２ ) １～４ —, —(CH ２ ) １～４ —S—, —NH—(CH ２ ) １～４ —, —N(R Ｎ )-(CH ２ ) １～４ -, -(CH ２ ) １～４ -N(R Ｎ ) -, -(OCH ２ ) １～１０ -, -(CH ２ O) １～１０ -, -(OCH ２ CH ２ ) １～１０ - or -(CH ２ CH ２ O) １～１０ - and; R Ｌ is independently C for each occurrence. １～２０ divalent hydrocarbon (e.g., alkyl, alkenyl, aryl, phenyl, monoalkyl-substituted phenyl, dialkyl-substituted phenyl, alkynyl, arylalkyl), optionally having one or more (e.g., 1-10, 1-5) points of substitution (e.g., with 1-10 heteroatoms, such as heteroalkyl, heteroalkenyl, heteroaryl, heteroalkynyl, heteroarylalkyl, with 1-10 substituents); R is independently at each occurrence hydrogen or C １～３５ a hydrocarbon (e.g., alkyl, alkenyl, alkynyl, or aralkyl) radical, optionally having one or more (e.g., 1-10, 1-5) points of substitution (e.g., with 1-20, or 1-20 heteroatoms, such as heteroalkyl, heteroalkenyl, heteroaryl, heteroalkynyl, heteroarylalkyl, with 1-20, or 1-10 substituents); R′ and R″ at each occurrence are independently hydrogen or C １～１０ is alkyl; X ｂ is independently at each occurrence an anionic group; R Ｎ is independently at each occurrence hydrogen, or C １～５ alkyl (e.g., methyl, ethyl, propyl)) The compound according to any one of claims 31 to 36, having the formula:

38. Ω is S, O, or N, and R １ But -R, -X, -R Ｌ -X, -L １ -R, -L １ -X, -Z, -R Ｌ -Z, -L １ -Z or -R Ｌ -L １ -R Ｌ 38. The compound of claim 37, wherein:

39. Ψ is a structure of formula (Va) 【Chemical Formula 28】 wherein "h" is 1, 2, 3, 4, 5, or 6. The compound according to any one of claims 35 to 38, having the formula:

40. Ψ is a structure of formula (Vb): 【Chemical 29】 (In the formula, R ５ ~R ８ are independently hydrogen or C １～３５ hydrocarbon (e.g., alkyl, alkenyl, alkynyl, aryl, alkoxy, alkylthio, or amino); L １ is A (e.g., L is absent) or L (e.g., L Ｃ Or Z Ｌ ) covalently bonded to The compound according to any one of claims 31 to 39, having the formula:

41. L １ is —NH—C(O)—, —C(O)—NH—, —C(O)—O—, or —O—C(O)—.

42. R ５ and R ６ are each lower alkyl (e.g., C １ ~C ４ alkyl, methyl), and R ７ and R ８ 42. The compound of claim 40 or 41, wherein each is hydrogen.

43. Ψ is a structure of formula (Vc): 【Chemistry 30】 (wherein Y" is absent or -L １ -, -R Ｌ -, -L １ -R Ｌ - or -R Ｌ -L １ - and Y" is A (e.g., L is absent) or L (e.g., L Ｃ Or Z Ｌ ) covalently bonded to The compound according to any one of claims 31 to 39, having the formula:

44. Ψ is a structure of formula (Vd): 【Chemical Formula 31】 (wherein Y" is absent or -L １ -, -R Ｌ -, -L １ -R Ｌ - or -R Ｌ -L １ - and Y" is A (e.g., L is absent) or L (e.g., L Ｃ Or Z Ｌ ) covalently bonded to The compound according to any one of claims 31 to 39, having the formula:

45. Ψ is a structure of formula (Ve): 【Chemical 32】 (wherein Y" is absent or -L １ -, -R Ｌ -, -L １ -R Ｌ - or -R Ｌ -L １ - and Y" is A (e.g., L is absent) or L (e.g., L Ｃ Or Z Ｌ ) covalently bonded to 45. The compound of any one of claims 31 to 39 and 44, having the formula:

46. L is the structure -L Ｃ -(Z Ｌ ) ｚ -, where "z" is 0 or 1; L Ｃ is a divalent C １～３５ an alkyl, alkenyl, alkynyl, aryl, or arylalkyl radical, optionally substituted with 1 to 20 heteroatoms; Z Ｌ is a zwitterionic linker group having the structure: 【Chemical 33】 "m" is 0 (i.e., a bond) or 1; "n" and "p" at each occurrence are independently an integer from 0 (i.e., a bond) to 10; X ａ is an anionic group; R Ｌ is independently C for each occurrence. １～２０ a divalent hydrocarbon radical (e.g., alkyl, alkenyl, aryl, alkynyl, arylalkyl), optionally having one or more (e.g., 1-10, 1-5) points of substitution (e.g., substituted with 1-10 heteroatoms, substituted with 1-10 substituents); R' is hydrogen or C １～１０ alkyl) The compound according to any one of claims 31 to 45, having the formula:

47. L Ｃ but has the following structure: -(X １ ) ０－１ -(R Ｌ ) ０－５ -(X ２ ) ０－１ -(R Ｌ ) ０－５ -(X ３ ) ０－１ -(R Ｌ ) ０－５ -(X ４ ) ０－１ -(R Ｌ ) ０－５ - (In the formula, X １ is ═N—, —O—, —S—, or —NR Ｎ -, -C(O)-, -NR Ｎ -C(O)-, -C(O)-NR Ｎ -, -O-C(O)-, or -C(O)-O-, -S-C(O)-, or -C(O)-S-; X ２ ~X ４ is -O-, -S-, -NR Ｎ -, -C(O)-, -NR Ｎ -C(O)-, -C(O)-NR Ｎ independently selected from —, —O—C(O)—, or —C(O)—O—, —S—C(O)—, or —C(O)—S—; R Ｌ independently for each occurrence, -(CH ２ ) １－５ -, -(CH ２ CH ２ O) １－５ -, -(OCH ２ CH ２ ) １－５ - or optionally substituted cycloalkylene (e.g., C ５ Cycloalkylene, C ６ cycloalkylene) 47. The compound of claim 46, having the formula:

48. X ａ and X ｂ may independently represent at each occurrence a carboxylate (-C(O)O － ), sulfonate (-SO ３ － ), sulfate (-OSO ３ － ), phosphate (-OP(O)(OR Ｐ ) O － ), or oxide (—O － ) and R Ｐ is hydrogen or C １～１２ 48. A compound according to any one of claims 31 to 47, which is hydrocarbon and optionally has one or more (e.g. 1-10, 1-5) points of substitution (e.g. with 1-10 heteroatoms and with 1-10 substituents).

49. R １ But, -R Ｌ -SO ３ － The compound of any one of claims 31 to 48, comprising:

50. R １ The compound of any one of claims 31 to 49, wherein comprises (or is) sulfopropyl.

51. R １ But -S(O) ２ -NH-Z or -(CH ２ ) １－３ -S(O) ２ The compound of any one of claims 31 to 50, which is -NH-Z.

52. R ２ and R ３ is independently at each occurrence hydrogen, alkyl, or alkoxy (e.g., C １～ C ４ 52. The compound of any one of claims 31 to 51, which is alkoxy, methoxy, ethoxy, propoxy, lower alkoxy such as isopropoxy.

53. R ２ and R ３ The compound of any one of claims 31 to 51, wherein each is hydrogen.

54. R ２ or R ３ one of R ２ or R ３ the other of which is alkoxy (e.g., C １～ C ４ 52. The compound of any one of claims 31 to 51, which is alkoxy, methoxy, ethoxy, propoxy, lower alkoxy such as isopropoxy.

55. Z Ｌ has the following structure: 【Chemical 34】 X so that ａ is a sulfonate (-SO ３ － ), m is 1, R Ｌ The compound of any one of claims 47 to 54, wherein is propyl, and n and p are each 3.

56. Structure of formula (VIa) or (VIb): 【Chemistry 35】 The compound of any one of claims 31 to 42 and 47 to 55,

57. The reactive functional group is: 【Hua 36-1】 【Hua 36-2】 57. The compound according to any one of claims 31 to 56, selected from:

58. The compound is selected from the group consisting of: 【Hua 37-1】 【Hua 37-2】 【Hua 37-3】 The compound according to any one of claims 31 to 42 and 46 to 57,

59. A method comprising reacting a compound according to any one of claims 31 to 58 with an analyte or a binding partner of the analyte (eg an antibody).

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