Chemiluminescent androstenedione conjugate

Abstract

Chemiluminescent compounds conjugated to androstenedione are provided that can be used in immunoassays to provide accurate and precise measurements of androstenedione concentrations in a sample. The present invention uses a conjugate having the structure of formula (I). TIFF2022008971000064.tif12128 wherein A is androstenedione or a derivative thereof, L is a linker, and Ψ is a chemiluminescent acridinium label.

Description

[Technical Field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 462,904, filed on 23 February 2017, which is incorporated in its entirety by reference herein.

[0002] This invention relates to chemiluminescent androstenedione conjugates. These chemiluminescent androstenedione conjugates are used as chemiluminescent tracers in immunoassays for the quantification and identification of specific analytes in a sample. [Background technology]

[0003] The concentrations of various androgens in circulating serum are directly related to various physiological and behavioral systems. In particular, serum concentrations of the androgen androstenedione are often measured to detect androgen-secreting tumors of ovarian and adrenal origin, to assess congenital abnormalities of sex steroid metabolism, or to evaluate adolescent disorders. Elevated androstenedione levels are associated with diseases such as masculinizing adrenal hyperplasia and polycystic ovary syndrome. Furthermore, measuring androstenedione in a sample provides greater accuracy in disease detection than measuring other androgens such as 17-hydroxyprogesterone. Also, androstenedione is banned by the World Anti-Doping Agency and in the Olympic Games. Therefore, measuring serum androgens is important in a variety of patients, including adult patients, elderly patients, pediatric patients with endocrine disorders, and oncologists.

[0004] The structure of the endogenous 4-androstenedione ("A4") hormone is shown below, with each of the 19 carbon atoms labeled. [ka]

[0005] Other androstenediones include the prohormones 5-androstenedione and 1-androstenedione, which are also on the World Anti-Doping Agency's list of banned substances. It should be understood that references to androstenedione include all androstenediones, including A4, 5-androstenedione, and 1-androstenedione.

[0006] Various techniques have been developed to detect androstenedione concentrations in samples. Analytical methods may include mass spectrometry (MS) in combination with gas chromatography (GC) or liquid chromatography (LC). For example, a liquid chromatography-tandem mass spectrometry ("LC-MS / MS") assay for androstenedione and other steroids is described in Non-Patent Literature 1, which is incorporated herein by reference in its entirety. However, the high cost of the equipment and the long execution time required for these methods limit their practicality for measuring multiple samples. Androstenedione concentrations have also been measured by immunoassays, which offer a cost-effective, simple, and rapid alternative to MS-based analysis. An alternative method is provided. These assays, such as the IMMULITE 2000 androstenedione assay or other enzyme-linked immunosorbent assays ("ELISA"), operate in a competitive binding format, where androstenedione in the sample to be measured competes with enzyme-conjugated androstenedione for binding to a limited number of antibodies. Typically, androstenedione is conjugated with alkaline phosphatase or horseradish peroxidase. After forming a binding complex with a solid phase containing a limited number of sites capable of conjugating either androstenedione or enzyme-linked androstenedione, the binding complex is separated and exhibits a measurable change (e.g., color). By removing the solid phase and observing the measurable change, the concentration of androstenedione in the sample can be estimated. However, as shown in Non-Patent Literature 2, which is incorporated in its entirety herein by reference, a comparison of the LC-MS / MS assay to the ELISA assay indicates that the androstenedione concentration measured by the ELISA method results in a nearly 2.5-fold increase in the measured androstenedione concentration compared to LC-MS / MS. Such overestimation is due to miscalibration and cross-reactivity of the enzyme-linked androstenedione conjugate used in the immunoassay, resulting in lower sensitivity and accuracy for the ELISA assay. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Kushnir, M. et al., Clin. Chem., 56, 1138 (2010) [Non-Patent Document 2] Fanelli, F. et al., Steroids, 76, 244 (2011) [Overview of the project] [Problems that the invention aims to solve]

[0008] Improvements to androstenedione immunoassays remain a persistent need in this field. Therefore, the object of the present invention is to provide a chemiluminescent compound conjugated with androstenedione that can be used in an immunoassay that provides accurate and highly precise measurement of androstenedione concentration in a sample. [Means for solving the problem]

[0009] Chemiluminescent acridinium compounds can be conjugated with androstenedione and have been found to provide more accurate results in immunoassays than ELISA measurements. Methods using detectable conjugates and detectable androstenedione conjugates are provided herein.

[0010] In one aspect of the present invention, the structure: [ka] [In the formula, A is androstenedione (e.g., A4, 5-androstenedione, 1-androstenedione, etc.); L is the linker; Ψ is the chemiluminescent part containing acridinium. A detectable androstenedione conjugate is provided, comprising an androstenedione conjugate to a chemiluminescent moiety having Ψ. In some embodiments, Ψ may be an acridinium ester or an acridinium sulfonamide. In this embodiment, A is a monovalent androstenedione group. In a preferred embodiment, A may be a 4-androstenedione ("A4") group. In most embodiments, A, L, and Ψ are covalently bonded to each other. In most embodiments, L is structural -L C -(Z L ) z - [In the formula, "z" is either 0 or 1; L Cis a divalent C optionally substituted with 1 to 20 heteroatoms 1~35 alkyl, alkenyl, alkynyl, aryl, or arylalkyl group;

[0011] Z L has the structure:

Chem.

[0012] Typically, Ψ is of formula (II):

Chem.

[0013] Z is structure: [ka] It is an amphoteric group having; "q" and "l" are independently either 0 or 1; "r" is an independent integer between 0 and 10; L1 is independently -O-, -S-, -NH-, -N(R) each time it appears. 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 -,-(CH2O) 1~10 -,-(OCH2CH2) 1~10 -, or -(CH2CH2O) 1~10 -and; R L Each instance is independently substituted with 1 to 10 heteroatoms, C 1~20 Divalent hydrocarbon groups (e.g., alkyl, alkenyl, aryl, phenyl, monoalkyl-substituted phenyl, dialkyl-substituted phenyl, alkynyl, arylalkyl, etc.); R is independently substituted with hydrogen, or possibly 1 to 20 heteroatoms, each instance of C 1~35 It is a hydrocarbon group (e.g., alkyl, alkenyl, alkynyl, or aralkyl); R' and R'' are, independently of each occurrence, hydrogen or C. 1~10 It is alkyl; X b Each instance is independently an anionic group; R N Each instance is independently either hydrogen or C 1~5 [Selected from alkyl groups (e.g., methyl, ethyl, propyl, etc.)] It has the structure of [the object].

[0014] In another aspect of the present invention, a reagent is provided for the detection of an analyte comprising a detectable conjugate of androstenedione bonded to chemiluminescent acridinium. The detectable conjugate may contain one or more (e.g., one, two, etc.) amphoteric functional groups. The reagent may contain a detectable conjugate at a concentration of 10-30 ng / mL.

[0015] In a further aspect of the present invention, an assay for the detection or quantification of an analyte in a sample, (a) A step of preparing a detectable conjugate having the structure of formula (I); (b) A step of preparing a solid support on which molecules capable of forming a binding complex with the analyte and capable of forming a binding complex with the detectable conjugate are immobilized; (c) A step of mixing the compound, the solid carrier, and the sample; (d) A step of separating the solid carrier from the mixture; (e) A step of inducing chemiluminescence of any acridinium label that has formed a composite with the solid phase; (f) the step of measuring the amount of light emitted with a luminometer; and (g) A step of detecting the presence of an analyte or calculating its concentration by comparing the amount of light emitted with a reference dose-response curve that relates the amount of light emitted to a known concentration of the analyte. The assay includes the following:

[0016] In some embodiments, the compound is supplied to a reagent further containing a buffer. Typically, the analyte to be detected or quantified is androstenedione (e.g., 4-androstenedione). In some embodiments, the sample is serum.

[0017] These and other aspects of the present invention will be better understood by referring to the following detailed description, including the appended claims. [Brief explanation of the drawing]

[0018] [Figure 1] This figure illustrates an example of a competitive androstenedione immunoassay using a chemiluminescent androstenedione conjugate. A sample with an unknown androstenedione concentration ("1") is mixed with a reagent containing a chemiluminescent androstenedione conjugate ("3", in this case an androstenedione-amphoterionic acridinium ester ("A4-ZAE") conjugate) and interacted with a solid phase ("2", "SP"). The solid phase has antibodies coated on it that can form binding complexes. Each antibody can form a binding complex with either androstenedione from the sample or the A4-ZAE conjugate. After the binding complexes are formed, the solid phase is removed from the sample and washed. The chemiluminescent light output from the solid phase that has complexed with both A4 and the A4 conjugate is measured through the use of a chemiluminescence triggering agent (in this case an acid / base). The intensity of the light output correlates with the number of chemiluminescent moieties (circles). Therefore, the amount of androstenedione in the sample can be estimated by the amount of light output. [Figure 2-1] Figures 2A to 2D are graphs showing the optical output of immunoassays for 3C3, 3H10, and 4G8 antibodies that form a complex with 4-androstenedione, respectively, using various A4 conjugates. [Figure 2-2] Continuation of Figure 2-1. [Figure 2-3] Figure 2E is a graph showing the optical output of the A4(6β)-hemisucinate conjugate initially and after 28 days of storage in buffer. [Figure 3A] This graph shows the optical output of immunoassays for samples containing androstenedione at various concentrations. The immunoassay includes an A4 conjugate conjugated at position 7 of the A4 portion. In the immunoassay measured for Figure 3A, the acridinium ester portion of each conjugate is the same (-Z-NSPDMAE). In the immunoassay measured for Figure 3B, the linker and acridinium ester portion are changed. [Figure 3B]This graph shows the optical output of immunoassays for samples containing androstenedione at various concentrations. The immunoassay includes an A4 conjugate conjugated at position 7 of the A4 portion. In the immunoassay measured for Figure 3A, the acridinium ester portion of each conjugate is the same (-Z-NSPDMAE). In the immunoassay measured for Figure 3B, the linker and acridinium ester portion are changed. [Figure 4] This graph compares LC-MS / MS assays of androstenedione samples with competitive assays using chemiluminescent androstenedione conjugates. The results for each assay are presented in terms of the measured concentration of androstenedione. The dotted line represents the Passing-Bablok regression analysis of the data comparing each assay method (slope = 1.02, intercept = -0.01). [Modes for carrying out the invention]

[0019] For convenience, certain terms used herein, including in the examples and appended claims, are summarized here. Unless otherwise defined, all scientific and technical terms used herein have the same meaning as those commonly understood by those skilled in the art of the field to which this disclosure belongs.

[0020] Unless otherwise clearly defined, the following terms and expressions are intended to have the following meanings throughout this disclosure:

[0021] Unless otherwise specified, all percentages given herein refer to the weight percentage of a particular component relative to the entire composition, including the carrier. It will be understood that the sum of the total weight percentages of individual components in the composition will not exceed 100%.

[0022] In this specification, the terms "a" or "an" mean one or more. As will be understood by reading this specification, the term "essentially consisting of" as used herein is intended to limit the present invention to specific materials or processes and to those that do not substantially affect the fundamental and novel features of the claimed invention.

[0023] Unless otherwise specified, the following definitions of groups and substituents shall apply. The specific and general meanings of groups, substituents, and ranges listed below are for illustrative purposes only and shall not exclude other defined meanings or other meanings within the defined ranges of groups and substituents. Unless otherwise indicated, alkyl, alkenyl, alkynyl, alkoxy, etc., refer to linear, branched, and cyclic groups, as well as any combination thereof.

[0024] The term "hydrocarbon" refers to a group (radical or group) containing carbon and hydrogen atoms. Examples of hydrocarbon groups include, but are not limited to, alkyl, alkenyl, alkynyl, aryl, arylalkyl, alkylaryl, and any combination thereof (e.g., alkylarylalkyl). As used herein, unless otherwise specified, hydrocarbons may be monovalent or polyvalent (e.g., divalent, trivalent, etc.) hydrocarbon groups. Methylene groups, i.e., -CH2-, and -(CH2) n A group of the form - is interpreted as an alkyl group if it does not have an unsaturated bond between carbon atoms. Unless otherwise specified, all hydrocarbon groups (including substituted and unsubstituted alkyl, alkenyl, alkynyl, aryl, arylalkyl, alkylaryl, etc.) may have 1 to 35 carbon atoms. In other embodiments, including embodiments having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms, hydrocarbons may have 1 to 20, or 1 to 12, or 1 to 8, or 1 to 6, or 1 to 3 carbon atoms. Hydrocarbons may have about 2 to about 70 atoms, or 4 to about 40 atoms, or 4 to about 20 atoms.

[0025] A "substituted" hydrocarbon may have one or more hydrocarbon groups, a substituted hydrocarbon group, or one or more heteroatoms as substituents. Any hydrocarbon substituent disclosed herein may optionally contain 1 to 20 heteroatoms (e.g., 1 to 10, 1 to 5, etc.). Examples of substituted hydrocarbon groups include, but are not limited to, heterocycles, such as heteroaryls. Unless otherwise specified, a hydrocarbon substituted with one or more heteroatoms will contain 1 to 20 heteroatoms. In other embodiments, a hydrocarbon substituted with one or more heteroatoms may contain 1 to 12, 1 to 8, 1 to 6, 1 to 4, 1 to 3, or 1 to 2 heteroatoms. Examples of heteroatoms include, but are not limited to, oxygen, nitrogen, sulfur, phosphorus, halogens (F, Cl, Br, I, etc.), boron, silicon, etc. In some embodiments, the heteroatoms will be selected from the group consisting of oxygen, nitrogen, sulfur, phosphorus, and halogens (F, Cl, Br, I, etc.). In preferred embodiments, the heteroatom may be selected from O, N, or S. In some embodiments, the heteroatom or group may substitute for carbon. In some embodiments, the heteroatom or group may substitute for hydrogen. In some embodiments, the substituted hydrocarbon may contain one or more heteroatoms in the molecular backbone or chain (e.g., between two carbon atoms, as in "oxa"). In some embodiments, the substituted hydrocarbon may contain one or more heteroatom side chains from the molecular backbone or chain (e.g., covalently bonded to carbon atoms in the chain or backbone, as in "oxo").

[0026] Furthermore, as used herein, the expression "substituted with one (a[n])~" means that a particular group may be substituted with any specified substituent or one or more of all of them. For example, a group such as an alkyl or heteroaryl group may be "unsubstituted C1~C 20If it is "substituted with alkyl or unsubstituted 2-20 member heteroalkyl," then the group is one or more unsubstituted C1-C 20 The group may contain alkyl groups and / or one or more unsubstituted 2- to 20-membered heteroalkyl groups. Furthermore, if a portion of the group is substituted with an R substituent, that group is called "R-substituted." If a portion is R-substituted, that portion is substituted with at least one R substituent, each R substituent being different in some cases.

[0027] Unless otherwise specified, any compound having one or more chiral centers disclosed herein may be in the form of a racemic mixture for each chiral center, or may exist as a pure or substantially pure (e.g., greater than about 98%ee) R or S enantiomer for each chiral center, or may exist as a mixture of R and S enantiomers for each chiral center, the mixture containing an enantiomer surplus of one or the other configuration, e.g., more than 60% (R or S) enantiomer surplus, or more than 70%, or more than 80%, or more than 90%, or more than 95%, or more than 98%, or more than 99% enantiomer surplus. In some embodiments, any chiral center may be in the "S" or "R" configuration. In preferred embodiments, the chiral center conjugated by the androstenedione may be in a single configuration. In a preferred embodiment, the indicated carbon positions (e.g., carbons 3, 6, 7, 17, 19, etc.) are conjugated only via the α (e.g., A(3α), A(6α), A(7α), A(17α), A(19α), etc.) or β (e.g., A(3β), A(6β), A(7β), A(17β), A(19β), etc.) directions.

[0028] It will be understood that the description of compounds herein is limited by the principles of chemical bonding known to those skilled in the art. Therefore, when a group is substituted by one or more substituents, such substituents are selected in accordance with the principles of chemical bonding regarding valence, etc., resulting in a compound that is not inherently unstable. For example, any carbon atom can be two, three, Alternatively, it would bond with four other atoms that match the four valence electrons of carbon.

[0029] In general, unless otherwise specified, the prefix names of substituents (groups) are derived from the parent hydride by either (i) replacing the "ane" of the parent hydride with the suffix "yl," "diyl," "triyl," "tetrayl," etc.; or (ii) replacing the "e" of the parent hydride with the suffix "yl," "diyl," "triyl," "tetrayl," etc. (where atoms with free valence are given the smallest number that matches any numbering set for the parent hydride, if specified). Throughout this specification, acceptable abbreviations, such as adamantyl, naphthyl, anthryl, phenanthryl, furyl, pyridyl, isoquinolyl, quinolyl, and piperidyl, as well as common names, such as vinyl, allyl, phenyl, and thienyl. Steroid groups may also be referred to with suffixes such as "yl," "diyl," "triyl," and "tetrayl." Acceptable abbreviations for steroids include androstenedionyl, 4-androstenedionyl, and androstenedion-7-yl. The conventional numbering / lettering system is also followed, with respect to the numbering of substituents and the naming of fused rings, spiro rings, bicyclic, tricyclic, and polycyclic rings.

[0030] The term "alkyl" refers to a saturated hydrocarbon chain, which may be linear or branched, containing a specified number of carbon atoms. For example, C1-C6 alkyl may have 1 to 6 carbon atoms (including 1 and 6) in its group. Any atom may be substituted, for example, by one or more substituents. Examples of alkyl groups, but not limited to, include methyl, ethyl, n-propyl, isopropyl, and tert-butyl. Any alkyl group referred to herein (e.g., R, R', R'', R1, R2, R3, R4, R5, etc.) may have 1 to 35 carbon atoms. In other embodiments, including embodiments having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms, the alkyl group may have 1 to 20, or 1 to 12, or 1 to 8, or 1 to 6, or 1 to 3 carbon atoms.

[0031] The term “haloalkyl” refers to an alkyl group in which at least one hydrogen atom is replaced by a halo. In some embodiments, more than one hydrogen atom (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14, etc.) is replaced by a halo. In these embodiments, each hydrogen atom can be replaced by the same halogen (e.g., fluoro), or by a combination of different halogens (e.g., fluoro and chloro). “Haloalkyl” also includes alkyl moieties in which all hydrogens are replaced by a halo (referred herein to as perhaloalkyl, e.g., perfluoroalkyl, e.g., trifluoromethyl). Any atom can optionally be replaced by, for example, one or more substituents.

[0032] As used herein, the term "alkoxy" refers to a group of the formula -O(alkyl). An alkoxy may be, for example, methoxy(-OCH3), ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, 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, either alone or in combination with other terms, the term "hydroxyl" refers to a group of the formula -OH. Any alkoxy, thioalkoxy, or haloalkoxy group referred to herein (e.g., R, R', R'', R1, R2, R3, R4, R5, etc.) may have 1 to 35 carbon atoms. In other embodiments, including embodiments having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms, the alkoxy, thioalkoxy, or haloalkoxy group may have 1 to 20, or 1 to 12, or 1 to 8, or 1 to 6, or 1 to 3 carbon atoms. It will probably happen.

[0033] The term "aralkyl" refers to an alkyl moiety in which an alkyl hydrogen atom is replaced by an aryl group. One of the carbon atoms in the alkyl moiety serves as a site for the aralkyl group to attach to another moiety. Any atom in the ring or chain can be substituted, for example, by one or more substituents. Non-limiting examples of "aralkyl" include benzyl, 2-phenylethyl, and 3-phenylpropyl groups.

[0034] The term “alkenyl” refers to a linear or branched hydrocarbon chain containing a specified number of carbon atoms and having one or more carbon-carbon double bonds. Any atom may be substituted, for example, by one or more substituents. Examples of alkenyl groups include vinyl, allyl, 1-butenyl, and 2-hexenyl. One of the double-bond carbons may optionally be the site to which an alkenyl substituent is added. Any alkenyl group referred to herein (e.g., R, R', R'', R1, R2, R3, R4, R5, etc.) may have 1 to 35 carbon atoms. In other embodiments, including embodiments having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms, the alkenyl group may have 1 to 20, or 1 to 12, or 1 to 8, or 1 to 6, or 1 to 3 carbon atoms.

[0035] The term "alkynyl" refers to a linear or branched hydrocarbon chain containing a specified number of carbon atoms and having one or more carbon-carbon triple bonds. Alkynyl groups can optionally be substituted with, for example, one or more substituents. Examples of alkynyl groups include ethynyl, propargyl, and 3-hexynyl. One of the triple-bond carbon atoms may optionally be the site of an alkynyl substituent.

[0036] The term "heterocyclyl" refers to a fully saturated, partially saturated, or aromatic compound containing one or two additional groups (e.g., R) to satisfy the valence of O, N (nitrogen) and / or to form a salt. NA heterocyclyl group is a monocyclic, dicyclic, tricyclic, or other polycyclic ring system having one or more atoms constituting a heteroatomic ring independently selected from S (it will be understood that a heteroatom may be present), or one or more atoms independently selected from S. The heteroatom or ring carbon can be a site where a heterocyclyl substituent is added to another part. Any atom may be substituted, for example, by one or more substituents (e.g., a heteroatom or an X group). Examples of heterocyclyl groups include tetrahydrofuryl, tetrahydropyranyl, piperidyl (piperidino), piperazinyl, morpholinyl (morpholino), pyrrolinyl, and pyrrolidinyl. Examples of the expression "heterocyclic ring containing 5-6 ring atoms (1-2 of the ring atoms independently selected from N, NH, N(C1-C6 alkyl), NC(O)(C1-C6 alkyl), O, and S; the heterocyclic ring may optionally be substituted with 1-3 independently selected R atoms)" include (but are not limited to) tetrahydrofuryl, tetrahydropyranyl, piperidyl (piperidino), piperazinyl, morpholinyl (morpholino), pyrrolinyl, and pyrrolidinyl.

[0037] The term "heterocycloalkenyl" refers to a partially unsaturated monocyclic, dicyclic, tricyclic, or other polycyclic hydrocarbon group having one or more (e.g., 1 to 4) heteroatomic ring atoms independently selected from O, N (it will be understood that one or two additional groups may be present to satisfy the valence of nitrogen and / or to form a salt), or S. The ring carbon (e.g., saturated or unsaturated) or heteroatom can be the site of a heterocycloalkenyl substituent. Any atom may be substituted, for example, by one or more substituents. Examples include dihydropyridyl, tetrahydropyridyl, dihydropyranyl, 4,5-dihydroxazolyl, 4,5-dihydro-1H-imidazolyl, 1,2,5,6-tetrahydropyrimidinyl, and 5,6-dihydro-2H-[1,3]oxazinyl.

[0038] The term "cycloalkyl" refers to a fully saturated monocyclic, dicyclic, tricyclic, or other polycyclic hydrocarbon group. Any atom may be substituted, for example, by one or more substituents. The ring carbon serves as the site for the cycloalkyl group to attach to another part. Examples of cycloalkyl moieties include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, and norbornyl (bicyclo[2.2.1]heptyl).

[0039] The term "cycloalkenyl" refers to a partially unsaturated monocyclic, dicyclic, tricyclic, or other polycyclic hydrocarbon group. The ring carbon (e.g., saturated or unsaturated) is the site to which the cycloalkenyl substituent is added. Any atom can be substituted, for example, by one or more substituents. Examples of cycloalkenyl moieties include cyclohexenyl, cyclohexadienyl, or norbornenyl.

[0040] As used herein, the term "cycloalkylene" refers to a divalent monocyclic cycloalkyl group having the specified number of ring atoms.

[0041] As used herein, the term "heterocycloalkylene" refers to a divalent monocyclic heterocyclyl group having the specified number of ring atoms.

[0042] The term "aryl" refers to an aromatic monocyclic, dicyclic (two fused rings), tricyclic (three fused rings), or polycyclic (more than three fused rings) hydrocarbon ring system. One or more ring atoms may be substituted, for example, by one or more substituents. Examples of aryl moieties include phenyl and naphthyl.

[0043] The term "heteroaryl" refers to an aromatic monocyclic, dicyclic (two fused rings), tricyclic (three fused rings), or polycyclic (more than three fused rings) hydrocarbon group having one or more heteroatom ring atoms in the ring, independently selected from O, N (it will be understood that one or two additional groups may be present to satisfy the valence of nitrogen and / or to form a salt), or S. One or more ring atoms may be optionally substituted, for example, by one or more substituents. Examples of heteroaryl groups include 2H-pyrrolyl, 3H-indolyl, 4H-quinolidinyl, acridinyl, benzo[b]thienyl, benzothiazolyl, β-carbolinyl, carbazolyl, coumalinyl, clomenyl, sinnolinyl, dibenzo[b,d]furanyl, flazanyl, furyl, imidazolyl, imidizolyl, indazolyl, indolyl, isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, naphthilidinyl, oxazolyl, perimidinyl, and f Examples include, but are not limited to, phenanthrolinyl, phenanthrolinyl, phenarsazinyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl, prinyl, pyranyl, pyrazinyl, pyrazolyl, pyridadinyl, pyridyl, pyrimidinyl, pyrrolyl, quinazolinyl, quinolyl, quinoxalinyl, thiadiazolyl, thianthrenyl, thiazolyl, thienyl, triazolyl, and xanthenyl.

[0044] Generally, when the definition of a particular variable includes both hydrogen and non-hydrogen (halo, alkyl, aryl, etc.) possibilities, the term "non-hydrogen substituent" refers collectively to the non-hydrogen possibilities for that particular variable.

[0045] In general, any limits (endpoints) of any range cited herein should be understood to be within the scope of the invention and to be disclosed embodiments. Furthermore, any half-integer values ​​within that range are also considered. For example, the range of about 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., about 1 to 2.5).

[0046] The term “substituent” means a group that is “substituted” on an alkyl, haloalkyl, cycloalkyl, heterocyclyl, heterocycloalkenyl, cycloalkenyl, aryl, or heteroaryl group, where one or more hydrogen atoms of the group are replaced by any atom of that group. In one embodiment, a substituent on a group is independently any single, delimited, permissible atom or group of atoms, or any combination of two or more such atoms. In another embodiment, a substituent may itself be substituted by any one of the substituents described above. Furthermore, as used herein, the expression “optionally substituted” means either unsubstituted (e.g., substituted with H) or substituted. It will be understood that the substitution of a given atom is limited by its valence. Common substituents include halo (e.g., F), C 1~12 Linear or branched alkyl groups, C 2~12 Alkenil, C 2~12 Alkinyl, C 3~12 Cycloalkyl, C 6~12 Ariel, C 3~12 Heteroaryl, C 3~12 Heterocyclyl, C 1~12 Examples include alkylsulfonyl, nitro, cyano, -COOR, -C(O)NRR', -OR, -SR, -NRR', and oxo, and examples include monosubstituted, disubstituted, or trisubstituted portions of trifluoromethoxy, chlorine, bromine, fluorine, methyl, methoxy, pyridyl, furyl, triazyl, piperazinyl, pyrazoyl, imidazoyl, etc., each possibly 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, C2~12 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, C 3~12 Heteroaryl, or C 4~24 Heteroarylalkyl. Unless otherwise noted, all groups described herein optionally contain one or more conventional substituents within the scope permitted by valence. Further, as used herein, the expression "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 is removed and replaced by a substituent (e.g., a conventional substituent). It will be understood that the use of a prefix name for a substituent (group) such as alkyl without the modifying phrase "optionally substituted" or "substituted" means that the particular substituent is unsubstituted. However, the use of "haloalkyl" without the modifying phrase "optionally substituted" or "substituted" will further be understood to mean an alkyl group in which at least one hydrogen atom has been replaced by a halo.

[0047] "Conjugating via" with respect to a particular carbon refers to the formation of a linkage to the particular carbon where an atom of the linker group L is covalently bonded to the particular carbon. In some embodiments, the bond between carbon 17 or carbon 3 of androstenedione and the linker will include a double bond such as an oxime or imine in any geometric arrangement.

[0048] The "stability" of a chemiluminescent compound means that when the compound or conjugate is stored in an aqueous solution within a pH range of 6 - 9, typically within the physiological pH range, the chemiluminescent activity measured by the decrease in relative luminescence units ("RLU") is at a minimum decrease. An increase in the "instability" of a chemiluminescent compound compared to another compound will result in a greater decrease in chemiluminescent activity.

[0049] The main object of this invention is to disclose detectable labels for androstenedione. These compounds may be used in immunoassays for the identification or quantification of an analyte. The compounds of the invention have the structure of formula (I)

Chemical formula

[0050] In some embodiments, Ψ has the structure:

Chemical formula

[0051] Z is structure: [ka] It is an amphoteric group having; "q" and "l" are independently either 0 or 1; "r" is an independent integer between 0 and 10; L1 is independently -O-, -S-, -NH-, -N(R) each time it appears. 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 -,-(CH2O) 1~10 -,-(OCH2CH2) 1~10 -, or -(CH2CH2O) 1~10 -and; R L Each instance is independently substituted with 1 to 10 heteroatoms, C 1~20 Divalent hydrocarbon groups (e.g., alkyl, alkenyl, aryl, phenyl, monoalkyl-substituted phenyl, dialkyl-substituted phenyl, alkynyl, arylalkyl, etc.); R is independently substituted with hydrogen, or possibly 1 to 20 heteroatoms, each instance of C 1~35 It is a hydrocarbon group (e.g., alkyl, alkenyl, alkynyl, or aralkyl); R' and R'' are, independently of each occurrence, hydrogen or C. 1~10 It is alkyl; X b Each instance is independently an anionic group; R N Each instance is independently either hydrogen or C 1~5 [Selected from alkyl groups (e.g., methyl, ethyl, propyl, etc.)] It is a chemiluminescent acridinium having the structure. Typically, L is structure -L C -(Z L ) z - [In the formula, "z" is either 0 or 1; L C Divalent C is sometimes substituted with 1 to 20 heteroatoms. 1~35 It is an alkyl, alkenyl, alkynyl, aryl, or arylalkyl group;

[0052] Z L Structure: [ka] It is an amphoteric linker group having; "m" is either 0 (i.e., conjugate) or 1; Each instance of "n" and "p" is independently an integer between 0 (i.e., associative) and 10; X a It is an anionic group; R L C is sometimes substituted with 1 to 10 heteroatoms. 1~20 It is a divalent hydrocarbon group (e.g., alkyl, alkenyl, aryl, alkynyl, arylalkyl, etc.); R' is either hydrogen or C 1~10 It is alkyl. It has.

[0053] In a preferred embodiment, Ψ is given by equation (IIa) [ka] It has the structure of [the object].

[0054] Generally, anionic groups (X a and / or X b ) may balance any cationic charge directly or indirectly added covalently and supply an anionic charge to form an amphoteric ion. In some embodiments, X a and X b Each instance is independently represented by carboxylate (-C(O)O - ), sulfonate (-SO3 - ), sulfate (-OSO3- ), phosphate (-OP(O)(OR P )O - ), or oxide (-O - ) and R P C is substituted with hydrogen, or possibly up to 10 heteroatoms. 1~12 It is a hydrocarbon.

[0055] The R1 group attached to the positively charged nitrogen atom of the acridinium nucleus can be substituted with up to 20 heteroatoms (e.g., N, O, S, P, Cl, Br, F, etc.), and therefore, in combination with the positively charged acridinium nitrogen atom, can form an amphoteric group. Example For example, a sulfopropyl or sulfobutyl group attached to acridinium nitrogen may form an amphoteric pair. The R1 group is neutral (e.g., methyl) or is itself amphoteric (e.g., R1 is -Z, -R L -Z, -L8-Z, or -R L -L8-R M -Z) may also be the case. In some embodiments, R1 is structure: [ka] The compound has the following characteristics. If the compound is charged (for example, R1 has a net neutral charge), the compound may optionally contain a counterion to balance the positively charged nitrogen of the acridinium nucleus. There is essentially no limit to the choice of counterion, but in some embodiments, the counterion is CH3SO4 - FSO3 - CF3SO4 - , C4F9SO4 - CH3C6H4SO3 - , halide (e.g., Cl - F - , Br - etc.), CF3COO - CH3COO - , or NO3 - Selected from. In some embodiments, R1 is methyl, ethyl, propyl, or isopropyl. In other embodiments, R1 is -R L-X is a sulfonate (-SO3 - ) In some embodiments, R1 is -R L -X is a sulfonate (-SO3 - ) In some embodiments, R1 is -R L -X, or -L8-Z. In some embodiments, L8 is -S(O)2-NH-, or -(CH2) 1~3 It is -S(O)2-NH-. R1 is a sulfopropyl group (-(CH2)3-SO3 - ) may contain. In a preferred embodiment, R1 is sulfopropyl.

[0056] The properties of chemiluminescent acridinium are not particularly limited and include, but are not limited to, chemiluminescent acridinium esters and sulfonamides. In a preferred embodiment, chemiluminescent acridinium Ψ is an acridinium ester. For example, Ψ has the following structure: [ka] It may have. In a preferred embodiment, Ψ is given by equation (IIb): [ka] [In the formula, R5 to R7 are independently hydrogen or C 1~35 It is alkyl, alkenyl, alkynyl, aryl, alkoxy, alkylthio, or amino; L1 is L (for example L C or Z L (covalently bonded to) It has the following structure. In some embodiments, L1 in formula (IIa) is -C(O)-NH-. In some embodiments, R5 and R6 are methyl, and R7 and R8 are hydrogen.

[0057] The substituents of the chemiluminescent acridinium ester may be modified to alter the rate and yield of luminescence, reduce nonspecific binding to improve stability, or improve hydrophilicity. However, as those skilled in the art will recognize, the important point of the present invention is that the chemiluminescent acridinium ester does not substantially hinder the binding of the androstenedione compound (A) to the antibody, particularly the binding of the corresponding unconjugated androstenedione compound. An example of substituent variability is disclosed in U.S. Patent No. 7,309,615 by Natrajan et al., incorporated herein by reference, which describes a high quantum yield acridinium compound containing an alkoxy group (OR*) at C2 and / or C7, where R* is a group comprising a sulfopropyl moiety, an ethylene glycol moiety, or a combination thereof. In some embodiments, R2 and / or R3 may be an alkoxy group (e.g., OR and / or OR*). Natrajan et al. also describe hydrophilic, high quantum yield chemiluminescent acridinium esters having certain electron-donating functional groups at the C2 and / or C7 positions in International Publication WO2015 / 006174, which is incorporated herein in its entirety by reference. These electron-donating groups of R1 and / or R2 are structured as follows: [ka] [In the formula, R9~R 14 Each occurrence is independently either a methyl group or -(CH2CH2O) a Selected from CH3 groups, where a is an integer between 1 and 5. It may have the following characteristics. In some embodiments, R2 and R3 are independently hydrogen, alkyl (e.g., methyl, ethyl, propyl, isopropyl, etc.), or alkoxy (e.g., methoxy, ethoxy, propoxy, or isopropoxy, etc.) depending on their appearance. In some embodiments, R2 and R3 are each hydrogen. In other embodiments, either R2 or R3 is hydrogen, and the other of R2 or R3 is an alkoxy or an electron-donating group. The detectable conjugate may contain a chemiluminescent acridinium sulfonamide. For example, Ψ has the structure of formula (IIc): [ka] [In the formula, Y'' does not exist, or -L1-, -R L -, or -R L -L1- is one of the following, where Y'' is L (for example, L C or Z L It may have [a covalent bond] attached to it.

[0058] In some embodiments, R L R is a 5-membered or 6-membered divalent aromatic hydrocarbon, which may be substituted in some cases. For example, any R L Structure: [ka] [In the formula, R 14 Each instance is independently hydrogen, halogen, or R. It may have R L Structure: [ka] [In the formula, R5 to R7 are independent of C 1~35 [It is alkyl, alkenyl, alkynyl, aryl, alkoxy, alkylthio, or amino.] It has the following characteristics. In some embodiments, R7 and R8 are hydrogen atoms, and R5 and R6 are methyl atoms, respectively. In some embodiments, as disclosed in Law et al., Journal of Bioluminescence and Chemiluminescence 4: pp. 88-89 (1989), which is incorporated herein by reference in whole, Ψ contains two adjacent methyl groups on the phenol ester to stabilize the bond. In some embodiments, Ψ has the following structure: [ka] It has.

[0059] In some embodiments, A, L, and Ψ are each covalently linked. The portion of the covalent linkage between A and Ψ may be formed from a reactive functional group for forming a covalent linkage with a peptide, protein, or polymer, and the functional group may include an electrophilic group, a nucleophilic group, or a photoreactive group. The reactive functional group may be an amine-reactive group, a thiol-reactive group, a carboxyl-reactive group, a maleimidyl-reactive group, or a carbohydrate-reactive group. For example, the linkage may be: [ka] It may be formed from a reactive group selected from. In some embodiments, the compound includes a linker group having the structure -NH-C(O)- or -C(O)-NH-. In preferred embodiments, the compound (e.g., L C (Ψ, etc.) contains at least one -NH-C(O)- or -C(O)-NH-linker group.

[0060] The covalent linkage between A and Ψ (e.g., L) is sometimes substituted with up to 20 heteroatoms (e.g., N, O, S, P, Cl, F, Br, etc.), and divalent C 1~20 It may contain alkyl, alkenyl, alkynyl, aryl, or arylalkyl groups. In several embodiments, L includes an amphoteric ion linker. L is structure-L C -(Z L ) z -[wherein z is 0 or 1] may be included. C is 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 - [In the formula, X1 is =N-, -O-, -S-, or -NR N- Selected from; X2~X4 are independent of -O-, -S-, and -NR. N -, -C(O)-, -NR N -C(O)-, -C(O)-NR N -, -OC(O)-, or -C(O)-O-, -SC(O)-, or -C(O)-S-; R L Each instance is independently selected from -CH2-, -(CH2CH2O)-, or -(OCH2CH2)-; However, L C is between A and Ψ (or A and Z) L [The chain between the two contains at least one atom (or at least two atoms)] It may have.

[0061] In some embodiments, L and / or Ψ include -C(O)-NH-. In some embodiments, L C Structure: [ka] It has.

[0062] The detectable conjugate has the following structure: [ka] It may have.

[0063] The detectable label may include an amphoteric linker comprising a dimethylacridinium ester (DMAE) moiety and an amphoteric linker or a polyethylene glycol-derived linker to improve the properties of the androstenedione compound (A). When Ψ contains an amphoteric linker, a polyethylene glycol-derived linker, or a dimethylphenyl ester, properties such as nonspecific bonding, hydrophilicity, or compound stability may be improved. In some embodiments, Z L Structure: [ka] It has the following characteristics. In most embodiments, R' is hydrogen or a lower alkyl group (e.g., methyl, ethyl, propyl, etc.).

[0064] In some embodiments, the detectable conjugate has the structure of equation (III): [ka] It may have.

[0065] The detectable conjugate consists of the dimethylacridinium ester (DMAE) moiety and the structure of formula (IIIa): [ka] It may include an amphoteric linker having L. In some embodiments, the structure in formula (IIIa) does not include an amphoteric linker (for example, L is L C ). In some embodiments, L is L C And L C ha-(CH2CH2O) 1~10 -(e.g., -(CH2CH2O)5-), or -(OCH2CH2) 1~10 This includes -(for example, -(OCH2CH2)5-, etc.)

[0066] In a preferred embodiment, the compound is of formula (IIIb) [ka] It has the structure of [the object].

[0067] The detectable label androstenedione is typically a monovalent group of androstenedione (e.g., 4-androstenedionyl). If the conjugation site is carbon 3 or 17, the added =O may be replaced by =N to form an oxime group. The oxime group may be in one geometric configuration (i.e., E or Z) or a mixture of the two. In some embodiments, androstenedione conjugates with the compound via any one of carbons 3, 6, 7, 19, or 17 of the androstenedione moiety. In some embodiments, androstenedione conjugates via any one of carbons 4-16. In preferred embodiments, androstenedione conjugates via carbon 6 or 7 of the androstenedione. In some embodiments, A4 conjugates via the α position of a particular carbon (e.g., 6α, 7α, etc.). In some embodiments, A4 conjugates via the β position of a particular carbon (e.g., 6β, 7β, etc.). In some embodiments, the compound is provided as a racemic mixture of α and β configurations.

[0068] Exemplary compounds are disclosed in Table 1. When used to describe conjugates, "Z" refers to an amphoteric linker, "CMO" refers to a carboxymethyl oxime linker, "CME" refers to a carboxymethyl ether linker, "CETE" refers to a carboxyethyl thioether, "ZAE" refers to an amphoteric acridinium ester (typically N-sulfopropyldimethylacridinium ester ("NSP-DMAE") in the sample shown), and "ISODIZAE" refers to a compound with an isopropoxy functional group added, forming a complete amphoteric group (N + and X - This refers to an acridinium nucleus in which (including both) is added to the positive nitrogen of an acridinium nucleus.

[0069] [Table 1] [Table 2] [Table 3] [Table 4] [Table 5]

[0070] Compounds can be prepared from commercially available starting materials, compounds known in the literature, or readily available intermediates by using standard synthetic methods and procedures known to those skilled in the art. Standard synthetic methods and procedures for the preparation of organic molecules, as well as the transformation and manipulation of functional groups, can be readily obtained from the relevant scientific literature or from standard textbooks in the art. Where typical or preferred process conditions (i.e., reaction temperature, time, molar ratio of reactants, solvent, pressure, etc.) are given, it will be understood that other process conditions may also be used unless otherwise stated. Optimal reaction conditions may vary depending on the specific reactants or solvent used, but such conditions can be determined by those skilled in the art through predetermined optimization procedures. Those skilled in organic synthesis will recognize that the nature and order of the presented synthetic steps may be altered to optimize the formation of the compounds described herein.

[0071] Useful synthetic chemical transformations (including protecting group methodologies) in the synthesis of the compounds described herein are known in the art, for example, as described in RCLarock, Comprehensive Organic Transformations, 2nd Ed., Wiley-VCH Publishers (1999); PGMWuts and TW Greene, Protective Groups in Organic Synthesis, 4th Ed., 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 thereof.

[0072] The processes described herein can be monitored by any suitable method known in the art, for example, nuclear magnetic resonance spectroscopy (e.g., 1 H or 13 C) Product formation can be monitored by spectroscopic means such as infrared spectroscopy (FT-IR), spectrophotometry (e.g., UV-visible light), or mass spectrometry (MS), or by chromatography such as high-performance liquid chromatography (HPLC) or thin-layer chromatography (TLC).

[0073] 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 Ed., Wiley & Sons, 1991, which is incorporated herein by reference in its entirety.

[0074] The reactions of the processes described herein can be carried out in suitable solvents that can be readily selected by those skilled in the art of organic synthesis. Suitable solvents can be substantially inactive with the starting materials (reactants), intermediates, or products at the temperature in which the reaction is carried out, i.e., a temperature ranging from the solidification temperature 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. Depending on the specific reaction step, a suitable solvent can be selected for a particular step.

[0075] The separation of racemic mixtures of compounds can be carried out by any of the many methods known in the art. For example, the absolute configuration of stereoisomers may 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): pp. 239-252, or Bowler, J, Steroids, 54 / 1(1989): pp. 71-99, respectively, which are incorporated herein by reference in their entirety. Another exemplary method is the preparation of moscher ester or amide derivatives of the corresponding alcohol or amine, respectively. In this case, the absolute configuration of the ester or amide is determined by protons and / or 19 The fraction is determined by 1F NMR spectroscopy. An exemplary method is fractional recrystallization using optically active salt-forming organic acids, known as "chiral splitting acids." Suitable splitting agents for fractional recrystallization are optically active acids such as D and L tartaric acid, diacetyltartaric acid, dibenzoyl tartaric acid, mandelic acid, malic acid, lactic acid, or various optically active camphor sulfonic acids. Separation of racemic mixtures can also be performed by elution using a column packed with an optically active splitting agent (e.g., dinitrobenzoylphenylglycine). The liquid composition can be determined by a person skilled in the art.

[0076] Exemplary androstenedione derivatives may be obtained from Steraloids, Inc. (Newport, RI). Four androstenedione derivatives used in the synthesis are shown below (wavy bonds indicate one of the configuration isomers or combinations of each isomer).

[0077] [ka]

[0078] This specification also provides androstenedione conjugates useful for synthesizing detectable conjugates. These conjugates have the following structure: [ka] It may have.

[0079] As described in U.S. Patent No. 6,664,043, No. 7,309,615, No. 9,575,062, or No. 9,487,480 to Natrajan et al., which are incorporated herein by reference in their entirety, amphoteric acridinium esters and their synthesis described herein, amphoteric acridinium esters ("ZAEs") typically containing reactive functional groups for forming covalent links may be used to synthesize the compounds of the present invention. For example, the amphoteric acridinium ester starting material may contain an N-sulfopropyl ("NSP") group in the amphoteric moiety, and / or a charged nitrogen atom bonded to a charged acridinium nucleus ("DIZAE"), and / or a sterically stabilized dimethylacridinium ester ("DMAE"), and / or an isopropoxy-functionalized acridinium nucleus ("ISO"), and / or an amphoteric ("Z") and / or a ("HEG") derived from hexa(ethylene) glycol and / or a (e.g., -C(O)-(CH2)3-C(O)-) linkage between the acridinium ester and the reactive functional group. The reactive functional group may be NH2 or N-hydroxysuccinimidyl ester ("NHS"). Exemplary acridinium esters that may be used as starting materials are given in Table 2 below.

[0080] [Table 6] [Table 7]

[0081] Chemiluminescent androstenedione conjugates may also be synthesized through the use of acridinium sulfonamide reaction products. For example, the acridinium sulfonamide disclosed in U.S. Patent No. 5,543,524 to Mattingly et al., which is incorporated herein in whole by reference, is a useful starting material for the production of the chemiluminescent androstenedione conjugates disclosed herein.

[0082] Chemiluminescent androstenedione conjugates are useful as labels in assays for the determination or quantification of certain analytes that may compete for binding with androstenedione's binding partner. Most typically, the analyte being measured is 4-androstenedione.

[0083] The assay may be a “competitive” immunoassay, typically involving the detection of large molecules, also known as macromolecular analytes, and using binding molecules such as antibodies. The antibody is immobilized or attached to a solid phase, such as particles, beads, membranes, microtiter plates, or any other solid surface. A schematic diagram of a competitive assay for androstenedione using the compounds described herein is shown in Figure 1.

[0084] In an example of a competitive heterogeneous assay, a carrier containing an antibody against the bound analyte (e.g., a 3C3, 3H10, 4G8 bovine monoclonal antibody) is brought into contact with a sample suspected of containing the analyte and a medium containing a chemiluminescent androstenedione conjugate (or "labeled analogue") as described herein. The analyte in the sample competes with the labeled analogue for binding to the analyte antibody. After separating the carrier and the medium, the labeling activity of the carrier or medium is determined by conventional techniques, but the labeling activity is related to the amount of analyte in the sample. In a variation of the above competitive heterogeneous assay, the carrier contains an analyte analogue and competes with the analyte in the sample for binding to the antibody reagent according to the principle described herein. The labeled analyte analogue may be covalently attached to a chemiluminescent or fluorescent molecule, often called a label or tracer.

[0085] When a solid phase containing immobilized antibodies is mixed with a sample containing the analyte and / or labeled analyte, a binding complex is formed between the analyte and the labeled analyte. This type of assay is often called a heterogeneous assay because of the involvement of the solid phase. The chemiluminescent signal associated with the binding complex can then be measured to infer the presence or absence of the analyte in the sample. Typically, the binding complex is separated from the remaining binding reaction components, such as excess labeled analyte, before signal generation. For example, if the binding complex is associated with magnetic beads, magnetism can be used to separate the binding complex associated with the beads from the bulk solution.

[0086] By using a set of "references," i.e., known concentrations of the analyte, a "dose-response" curve can be created for a known labeled analyte. Thus, the dose-response curve correlates a certain amount of measured signal with a specific concentration of the analyte. In competitive assays, when measuring chemiluminescence from a bound complex, the amount of signal decreases as the concentration of the analyte increases. The concentration of the analyte in the unknown sample can then be calculated by comparing the signal generated by an unknown sample containing a high-molecular-weight analyte with the dose-response curve.

[0087] Methodologies for attaching binding molecules such as antibodies to a solid phase are well known in the prior art. For example, antibodies can be covalently attached to particles containing amines on their surface by using crosslinking molecules such as glutaraldehyde. The attachment may also be noncovalent, and may 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 usually called a conjugation reaction, with labeled antibodies often being called conjugates. Typically, the amine-reactive portion of the label reacts with the amine in the antibody to form an amide linkage. Other linkages between the antibody and the label, such as thioethers, esters, and carbamates, are also well known in the prior art.

[0088] In another aspect of the present invention, a reagent for detecting an analyte containing a chemiluminescent acridinium compound bound to androstenedione is provided. The reagent may contain about 0.1 to about 100 ng / mL of the chemiluminescent acridinium compound, or about 1 to about 50 ng / mL of the chemiluminescent acridinium compound, or about 5 to about 30 ng / mL of the chemiluminescent acridinium compound. In some embodiments, the compound is supplied to a reagent further containing a buffer.

[0089] Typically, assays for the detection or quantification of analytes in a sample are: (a) the process of preparing a detectable conjugate; (b) A step of preparing a solid support on which molecules capable of forming a binding complex with the analyte and capable of forming a binding complex with the detectable conjugate are immobilized; (c) A step of mixing the compound, the solid carrier, and the sample; (d) A step of separating the solid carrier from the mixture; (e) A step of inducing chemiluminescence of any acridinium label that has formed a composite with the solid phase; (f) the step of measuring the amount of light emitted with a luminometer; and (g) A step of detecting the presence of an analyte or calculating its concentration by comparing the amount of light emitted with a reference dose-response curve that relates the amount of light emitted to a known concentration of the analyte. Includes.

[0090] Typically, the analyte to be detected or quantified is androstenedione (e.g., 4-androstenedione). In some embodiments, the sample is derived from a mammal (e.g., human). In some embodiments, the sample is saliva and / or blood and / or serum. In some embodiments, the sample is saliva and / or blood and / or serum.

[0091] In some assays, the sample to be analyzed is pre-treated to release the analyte from endogenous binding substances, such as plasma or serum proteins that bind to the analyte. Release of the analyte from endogenous binding substances may be achieved, for example, by adding a warming agent or a release agent, or by a combination of a warming agent and a release agent used in succession. The warming agent decomposes the endogenous binding substances so that they can no longer bind to the analyte.

[0092] Conditions for performing an assay on a portion of a sample according to the principles described herein may include performing the assay in an aqueous buffer medium of a moderate pH that generally yields optimal assay sensitivity. The aqueous medium may be water alone or may contain 0.1 to about 40% by volume of a cosolvent. The pH of the medium may be in the range of about 4 to about 11, or about 5 to about 10, or about 6.5 to about 9.5. Typically, the pH value of the solution will be a compromise between the optimal binding of the binding member of any specific binding pair and the optimal pH for other reagents in the assay, such as members of the signal-generating system. Various buffers may be used to achieve the desired pH and maintain the pH during the assay. Examples of buffers include, for example, borate, phosphate, carbonate, TRIS, barbital, PIPES, HEPES, MES, ACES, MOPS, and BICINE.

[0093] Various auxiliary materials may be used in the assay method. For example, the medium may include a buffer in addition to stabilizers for the medium or for the reagents used. 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 glycol), polysaccharides (e.g., dextran, trehalose, etc.), and combinations thereof.

[0094] The chemiluminescence of analogs may be induced by adding a known amount of a triggering reagent. The triggering reagent may be acidic or basic. Multiple triggering reagents may be added sequentially. For example, an acidic solution may be added first, followed by a basic solution. In some embodiments, the triggering reagent includes hydrogen peroxide, hydrogen peroxide salts, nitric acid, nitrates, sodium hydroxide, ammonium salts, or combinations thereof. [Examples]

[0095] The following examples illustrate the synthesis of several representative compounds and their use in the measurement of androstenedione samples in heterogeneous competitive assays. Therefore, the examples are for illustrative purposes only and not intended to limit the disclosure. Additional compounds not specifically illustrated may be synthesized using conventional methods in combination with the methods described herein. [Examples]

[0096] Synthesis of A4(7)-propionic acid intermediate [ka]

[0097] A stirring bar, magnesium shavings (164 mg, 6.82 mmol), and anhydrous THF (6 mL) were placed in a round-bottom flask (RBF). (3-bromopropoxy)-tert-butyldimethylsilane (1654 mg, 6.53 mmol) was added while stirring at 40°C. The resulting mixture was stirred at 40°C until most of the magnesium shavings had disappeared. The reaction mixture was cooled to room temperature (RT). Additional THF (10 mL) was added to dissolve all the precipitate and form a homogeneous Grignard reagent solution. This Grignard reagent solution (16 mL) was added to a suspension of CuI (597 mg, 3.14 mmol) in THF (10 mL) over 15 minutes at -40°C to -30°C. The resulting mixture was stirred at -40°C to -30°C for 10 minutes to form an organic cuprate solution. Next, 4,6-androstadiene-3,17-dione (150 mg, 0.53 mmol in 1 mL of THF solution) was added to the organic cuprate solution. The resulting mixture was stirred at -40°C to -30°C for 1 hour. Glacial acetic acid (AcOH, 2 mL) was added while stirring the reaction mixture at -40°C to -30°C. The resulting mixture was stirred for 30 minutes. Saturated aqueous NH4Cl solution (30 mL) was added, and the mixture was stirred at RT for a further 20 minutes. The resulting mixture was extracted three times with methyl t-butyl ether (MTBE). The combined MTBE extracts were washed twice with saturated brine, dried over Na2SO4, filtered, and concentrated to obtain the crude product. This crude product was purified by silica gel flash chromatography (hexane / ethyl acetate) to obtain the pure desired product A4-7-PrOTBS as a mixture of α and β (181 mg, 74%). MS (M+H, 459.3).

[0098] To a solution of A4-7-PrOTBS (176 mg, 0.384 mmol) in THF (2 mL), 1.0 M THF solution (1.5 mL, 1.5 mmol) was added while stirring at 0°C (ice bath). The resulting mixture was removed from the ice bath and stirred at RT for 1 hour. Saturated saline (8 mL) was added to the reactant. The resulting mixture was stirred for 30 minutes and then extracted three times with siRNA. The combined siRNA extract was washed twice with saturated saline, dried over Na2SO4, filtered, and concentrated to obtain the crude product. This crude product was purified by silica gel flash chromatography (hexane / siRNA) to obtain the pure desired product A4-7-PrOH as a mixture of α and β (92.9 mg, 70%). MS(M+ Na, 367.2).

[0099] A4-7-PrOH (87.5 mg, 0.255 mmol) was dissolved in MeCN (3 mL). Tetrapropylammonium perruthenate ("TPAP", 18.9 mg, 0.0538 mmol) was dissolved in MeCN (2 mL). The TPAP MeCN solution was added to N-methylmorpholine N-oxide ("NMO") monohydrate (366 mg, 2.71 mmol) with stirring until all solids were dissolved. The NMO-TPAP solution was then added to the A4-7-PrOH solution with stirring. The resulting mixture was stirred at RT for 1 hour. After 1 hour, 2-PrOH (1 mL) was added to the mixture and stirred for 1 hour to quench the excess NMO. The reaction mixture solution was directly loaded into a dry silica gel cartridge. The silica gel cartridge was washed with 5% HOAc in toluene. The recovered 5% HOAc in toluene solution was concentrated to obtain the crude product. This crude product was purified by HPLC (0.05% TFA in water / 0.05% TFA in MeCN) to obtain the pure desired product A4-7-propionic acid as a mixture of α and β (51.6 mg, 57%). MS (M + Na, 367.2). [Examples]

[0100] Synthesis of A4(3)-CMO-Z1-ZAE conjugate The A4(3)-carboxymethyloxime ("CMO")-Z-ZAE conjugate was synthesized using the synthetic scheme described below.

[0101] [ka]

[0102] A4(3)-CMO-Z-ZAE was also synthesized by reacting 4-androsten-3,17-dione 3-O-carboxymethyloxime sodium salt with NSP-DMAE-Z-NH2 in the presence of (benzotriazole-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate ("BOP"), N,N-diisopropylethylamine ("(iPR)2NEt"), and N,N-dimethylformamide ("DMF"). A 10% H2O:90% DMF solution (vol:vol) was prepared by mixing 100 μL of H2O and 900 μL of DMF. A mixture of 3,17-dione 3-O-carboxymethyloxime sodium salt (1.1 mg, 2.9 μmol), NSP-DMAE-Z-NH2 (1.3 mg, 1.7 μmol), and BOP (1.7 mg, 3.8 μmol) was added to 333 μL of H2O:DMF solution. The solution was thoroughly mixed, and (iPr)2NEt (0.9 μL, 0.7 mg, 5.4 μmol) was added. Upon addition of the (iPr)2NEt base, the solution changed from yellow to colorless. The reaction mixture was stirred overnight at room temperature for 19.1 hours.

[0103] After stirring overnight, the reaction mixture was treated with Phenomenex Bondclone C. 18Analytical high-performance liquid chromatography (HPLC) was performed using a 10 μm, 300 × 3.9 mm column and a multi-stage gradient of eluents (A = water containing 0.05% trifluoroacetic acid and TFA; B = acetonitrile containing 0.05% TFA) at a flow rate of 1.0 mL / min, injection loop size of 250 μL, and detection wavelength of 260 nm. The reaction mixture was stored at -70°C for 72 hours. The reaction mixture was purified using semi-preparative HPLC. The reaction mixture was then purified using YMC-Pack ODS-A, No.3025000136(W), C 18Preparative HPLC was performed using a 10 μm, 250 × 30 mm column and a 10% B → 70% B gradient over 50 minutes with eluents (A = water containing 0.05% trifluoroacetic acid and TFA, B = acetonitrile containing 0.05% TFA), at a flow rate of 20.0 mL / min, injection loop size of 5 mL, and detection wavelength of 262 nm. The product eluted as two isomer peaks with retention times of 34.1–34.9 minutes. The peak fractions were collected, frozen at -70°C, and lyophilized to produce two separate yellow powders (total yield 65%). Each fraction was characterized by HPLC and matrix-assisted laser desorption / ionization (TOF-MS) ("MALDI TOF-MS"). For MALDI TOF-MS analysis, each sample was dissolved at concentrations of 0.05–2.00 mg / mL in a 1:1 mixture of acetonitrile + 0.05% trifluoroacetic acid:water + 0.05% trifluoroacetic acid. This mixture was mixed with α-cyano-4-hydroxycinnamic acid ("HCCA") matrix solution in approximately a 1:1 ratio and spotted onto a target plate. Concentrations were adjusted as necessary to produce a good signal against noise. MALDI TOF-MS was performed in linear or reflector mode for positive ions. Fractions with a retention time of 34.15 minutes (93.54% purity) had MALDI TOF-MS peaks of 1085.426 (linear positive mode) and 1084.990 (reflector positive mode). The fraction with a retention time of 34.05 minutes (89.63% purity) had MALDI TOF-MS peaks 1084.782 (linear positive mode) and 1084.704 (reflector positive mode). [Examples]

[0104] Synthesis of A4(17)-CMO-Z-ZAE conjugate The A4(17)-CMO-Z-ZAE conjugate was synthesized using the synthesis schema described below.

[0105] [ka]

[0106] The A4(17)-CMO-Z-ZAE conjugate was also synthesized by reacting 4-androsten-3,17-dione 17-carboxymethoxyloxime with NSP-DMAE-Z-NH2 in the presence of (benzotriazole-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate ("BOP"), N,N-diisopropylethylamine ("(iPR)2NEt"), and N,N-dimethylformamide ("DMF"). A 10% H2O:90% DMF solution (vol:vol) was prepared by mixing 100 μL of H2O with 900 μL of DMF. A mixture of 4-androsten-3,17-dione 17-carboxymethoxyloxime (1.1 mg, 3.1 μmol), NSP-DMAE-Z-NH2 (1.3 mg, 1.7 μmol), and BOP (1.9 mg, 4.3 μmol) was added to 333 μL of H2O:DMF solution. The solution was thoroughly mixed, and (iPr)2NEt (0.9 μL, 0.7 mg, 5.4 μmol) was added. Upon addition of the (iPr)2NEt base, the solution changed from yellow to colorless. The reaction mixture was stirred overnight at room temperature for 20.2 hours.

[0107] After stirring overnight, the reaction mixture was treated with Phenomenex Bondclone C. 18 Analytical high-performance liquid chromatography (HPLC) was performed using a 10 μm, 300 × 3.9 mm column and a multi-stage gradient of eluents (A = water containing 0.05% trifluoroacetic acid and TFA; B = acetonitrile containing 0.05% TFA) at a flow rate of 1.0 mL / min, injection loop size of 250 μL, and detection wavelength of 260 nm. The reaction mixture was stored at -70°C for 72 hours. The reaction mixture was purified using semi-preparative HPLC. The reaction mixture was then purified using YMC-Pack ODS-A, No.3025000136(W), C 18, a 10 μm, 250×30 mm column, and a 50-minute gradient of 10%B→70%B of eluent (A = water containing 0.05% trifluoroacetic acid, TFA; B = acetonitrile containing 0.05% TFA) were used at a flow rate of 20.0 mL / min, an injection loop size of 5 mL, and a detection wavelength of 262 nm for preparative HPLC. The product eluted as a peak at a retention time of 32.7 - 34.0 minutes. The peak fractions were collected and - frozen at -70 °C and lyophilized to produce a yellow powder (1.2 mg, total yield 65%). The fractions were characterized using HPLC and MALDI TOF-MS. The fractions had MALDI TOF-MS peaks at 1084.718 (linear positive mode), and 1084.722 (reflector positive mode).

Example

[0108] Synthesis of A4(19)-CME-Z-ZAE conjugate The synthesis of A4(19)-carboxymethyl ether (“CME”)-Z-ZAE conjugate was carried out using the synthetic scheme described below.

[0109]

Chemical formula

[0110] (Benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (“BOP”), N,N-diisopropylethylamine (“(iPR)2NEt”), and N,N-dimethylformamide (“DMF”), 3,17-dioxo-4-androsten-19-yl carboxymethyl ether was reacted with NSP-DMAE-Z-NH2 to also synthesize A4(17)-CMO-Z-ZAE conjugate. RA4, Pemmaraju Narashima et al., Journal of Steroid Biochemistry, regarding androstenedione conjugate synthesis, which is incorporated herein by reference in its entirety 3,17-dioxo-4-androsten-19-ylcarboxymethyl ether was synthesized as disclosed in 17(5), pp. 523-527 (1982). A 10% H2O:90% DMF solution (vol:vol) was prepared by mixing 100 μL of H2O with 900 μL of DMF. A mixture of 3,17-dioxo-4-androsten-19-ylcarboxymethyl ether (6.8 mg, 1.9 μmol), NSP-DMAE-Z-NH2 (13.8 mg, 18.6 μmol), and BOP (13.3 mg, 30.1 μmol) was added to 952 μL of the H2O:DMF solution. The solution was thoroughly mixed, and (iPr)2NEt (9.6 μL, 7.1 mg, 5.5 μmol) was added. Upon addition of the (iPr)2NEt base, the solution changed from yellow to colorless.

[0111] After 1.5 hours, analytical HPLC showed 94% conversion to the product. For the reaction mixture, Phenomenex Bondclone C 18 A 10 μm, 300 × 3.9 mm column and a multi-stage gradient of eluents (A = water containing 0.05% trifluoroacetic acid and TFA; B = acetonitrile containing 0.05% TFA) (0% B for 1 minute; 0% B for 30 minutes) Analytical HPLC was performed using %→100%B (holding 100%B for 6 minutes) at a flow rate of 1.0 mL / min, injection loop size of 250 μL, and detection wavelength of 260 nm. Analytical HPLC showed a 94% conversion to product. The mixture was stored at -70°C for 24 hours. The reaction mixture was purified by semi-preparative HPLC. The reaction mixture was purified using YMC-Pack ODS-A, No.3025000136(W), C 18Preparative HPLC was performed on two injections using a 10 μm, 250 × 30 mm column and a 10% B → 70% B gradient over 50 minutes with eluents (A = water containing 0.05% trifluoroacetic acid and TFA, B = acetonitrile containing 0.05% TFA), at a flow rate of 20.0 mL / min, injection loop size of 5 mL, and detection wavelength of 261 nm. The product was eluted at a retention time of 21.9–23.8 minutes. The peak fraction was collected, frozen at -70°C, and lyophilized to produce a yellow powder (7.0 mg, total yield 35%). The fraction was characterized by HPLC and MALDI TOF-MS. The fraction had MALDI TOF-MS peaks 1085.400 (linear positive mode) and 1085.382 (reflector positive mode). [Examples]

[0112] Synthesis of A4(6β)-hemisucinate-Z-ZAE conjugate The A4(6β)-hemisuccinate-Z-ZAE conjugate was synthesized using the synthesis schema described below.

[0113] [ka] [Examples]

[0114] Synthesis of A4(6β)-carbamate-Z1-ZAE conjugate The A4(6β)-carbamate-Z-ZAE conjugate was synthesized using the synthesis scheme described below.

[0115] [ka] [Examples]

[0116] Synthesis of A4(7α)-CETE-ZAE conjugate The synthesis of A4(7α)-carboxyethylthioether (「CETE」)-NH-Z-ZAE conjugate was carried out using the synthetic scheme described below.

[0117]

Chemical formula

Example

[0118] Synthesis of A4(7)-propionamide-Z-ZAE conjugate The synthesis of A4(7)-propionamide-Z-ZAE conjugate was carried out using the synthetic scheme described below.

[0119]

Chemical formula

[0120] Into a vial charged with NSP-DMAE-Z-NH2 (5.0 mg, 6.73 μmol), A4 7-propionic acid (2.51 mg, 7.01 μmol), and (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (「BOP」, 5.97 mg, 13.5 μmol), a DMF-water mixed solvent (9 / 1, 0.6 mL) and N,N-diisopropylethylamine (「DIPEA」, 4.7 μL, 26.9 μmol) were added with stirring. The resulting mixture was stirred at RT for 4 hours. This reaction mixture was purified directly by HPLC (0.05% TFA in water / 0.05% TFA in MeCN) to obtain pure desired product A4-7-propionic acid as a mixture of α and β (4.36 mg, 60%). MS (M+Na, 1105.5).

Example

[0121] Synthesis of A4(7)-propionamide-HEG-ZAE conjugate The synthesis of A4(7)-propionamide-HEG-Z-ZAE conjugate was carried out using the synthetic scheme described below.

[0122] [ka] [Examples]

[0123] Synthesis of A4(7)-propionamide-et-NH-glutarate-NH-Z-ISODIZAE conjugate NSP-DMAE-HEG-NH2 (1.5 mg, 1.98 μmol), A4 7-propionic acid (5.4 mg, 14 μmol), and (7-azabenzotriazole-1-yloxy)tripyrrolidino-phosphonium hexafluorophosphate ("PyA4P", 23.1 mg, 44.3 μmol) were added to a vial, to which DMSO (1.0 mL) and TEA (30 μL) were added with stirring. The resulting mixture was stirred at RT for 4 hours. This reaction mixture was directly purified by HPLC (0.05% TFA in water / 0.05% TFA in MeCN) to obtain the pure desired product A4-7-propionic acid as a mixture of α and β (1.55 mg, 40%). MS (M+Na, 1118.6).

[0124] The A4(7)-propionamide-et-NH-glutarate-NH-Z-ISODIZAE conjugate was synthesized using the synthetic schema described below.

[0125] [ka] [Examples]

[0126] Synthesis of A4(7)-et-NH-glutarate-NH-Z-ISODIZAE conjugate The A4(7)-et-NH-glutarate-NH-Z-ISODIZAE conjugate was synthesized using the synthesis schema described below.

[0127] [ka] [Examples]

[0128] Evaluation of A4-AE Conjugate The A4-AE conjugate was evaluated using a competitive assay format utilizing solid-phase reagents and Wright reagents, employing an ADVIA Centaur family immunoassay analyzer (available from Siemens Healthcare Diagnostics Inc., Tarrytown, NY). The Wright reagent contains 20 ng / mL of the A4-AE tracer compound. The solid reagent includes superparamagnetic beads (Dynabeads® M-270 Streptavidin, available from ThermoFisher Scientific) labeled with a biotinylated monoclonal anti-A4 antibody. To generate antibody-attached solid-phase particles, biotinylated sheep monoclonal androstenedione antibodies 3C3, 3H10, or 4G8 (available from Bioventix, UK) were introduced onto streptavidin-coated superparamagnetic beads. In the absence of A4, solid-phase particles mixed with A4-AE formed solid-phase complexes with the A4-AE compound. When the Wright reagent contains androstenedione, competition arises between A4 and the A4-AE conjugate for the formation of binding complexes with each antibody. As can be seen in Figure 1, solid-phase particles ("SP") with more androstenedione binding complexes are more It will likely contain a small amount of chemiluminescent acridinium moiety (circled in Figure 1). Therefore, an increase in androstenedione concentration in the sample correlates with a decrease in chemiluminescence of the separated and washed solid-phase particle-bound complex. The chemiluminescence of the separated solid-phase particle-bound complex was induced and measured by adding an acidic solution (0.5% peroxide / 0.45% nitric acid (vol / vol)), followed by a basic solution (0.25N NA4H / 0.45% ARQUAD® 16-50 (w / v)). The volume, concentration, and assay parameters of the reagents were the same in each experiment, except for the change in the A4-AE tracer conjugate being measured. The androstenedione samples used in the Wright reagent were prepared using a human serum albumin-based matrix (available from Bioresource Technology, Inc.) at the androstenedione sample concentrations (ng / mL) shown in Figures 2A-2D.

[0129] Chemiluminescence was monitored for various A4-AE conjugates. Table 3 and corresponding Figures 2A-2D below summarize the results of chemiluminescence experiments for several A4-AE conjugates synthesized as described in the indicated example numbers. In Table 3, "+" indicates evaluable binding of a particular A4-AE conjugate to a particular antibody, and "-" indicates that no evaluable binding was observed.

[0130] [Table 8]

[0131] As can be seen, conjugations at position 6 or 17 of the androstenedione moiety with a carbamate (-OC(O)NH-) linkage ("A4-6-AE" or "A4-17-AE", respectively) did not show any evaluable binding. The A4-3-AE conjugate with a carboxymethyl oxime linkage ("CME", =NO-CH2-C(O)-NH-) bound to the 3C3 and 3H10 antibodies, but with low affinity to each antibody (determined by signal separation, shown in Figure 2A). As shown in Figure 2B, the tracer with a conjugation at position 19 bound strongly to the 3H10 and 4G8 antibodies, but showed no evaluable binding to the 3C3 antibody. Conjugations at the 6β or 7α position with hemisuccinate (-OC(O)CH2CH2C(O)NH-) or carboxyethyl thioether ("CETE", -SCH2CH2C(O)NH-) binding moieties showed strong binding affinity for all three antibodies. Furthermore, these binding moieties at these positions have a different affinity order for each antibody / A4-AE complex. As can be seen, the conjugate position and binding moiety of an A4-AE tracer are related to the binding affinity of that tracer to a particular antibody. For example, the A4-6-AE conjugate containing a carbamate binding moiety did not show any evaluable binding, while the A4-6-AE conjugate containing a hemisuccinate binding moiety showed affinity for two of the three antibodies measured.

[0132] To determine the stability of the compound by measuring the change in chemiluminescence after storage, aqueous solutions of the tracer were stored in an aqueous buffer at pH 6.5 at 4°C for 28 days, and the immunoassay was repeated. These conditions were met using the ADVIA Centaur immunoassay analyzer. This is typical of commercial automated instruments, such as those in the family. As shown in Figure 2E, assays with A4-AE tracers containing a conjugation at the 6β position and having a hemisuccinate linker showed a decrease in measured optic output when the assay was repeated 28 days after the initial assay. As can be seen, A4-AE tracers containing a conjugation at the 6β position show a considerable decrease in chemiluminescence after 28 days in a buffer system. Measurements performed with androstenedione conjugated at the 7 position (A4-7-AE) did not show similar instability. [Examples]

[0133] Shape of the curve of the A4-7-AE conjugate The chemiluminescence curve shapes for multiple A4-7-AE conjugates were further evaluated using a 3C3 antibody. The effect of various linkages on the curve shape of the optical output was measured as a function of androstenedione (ng / mL). The curve shapes were prepared from assays using A4-AE tracers that have an androstenedione conjugation at position 7 and have a -SCH2CH2C(O)NH- or -CH2CH2C(O)NH- linkage. Figure 3A shows the signals obtained from assays using three different A4-AE conjugates, normalized as the percentage of the signal generated from a light reagent without androstenedione. In these experiments, [A4] = 10 ng / mL was selected as the 0% optical output. As can be seen, these A4-7-AE conjugates, which have conjugations at positions 7α and 7β and in which each linkage is measured, showed similar curve shapes.

[0134] To determine the impact on performance in the assay, the AE and / or ligation moieties, which possess various chemical properties and optical output, were also measured. The ligation moieties were conjugated with androstenedione at position 7. The measured ZAE conjugate moieties had the structures shown in Table 3. AE moieties in which the amphoteric linker was replaced with an HEG linker were also compared (HEG-ZAE). Figure 3B shows the signals generated and obtained from assays using A4-7-AE conjugates, including the A4-AE conjugates described in Table 3. [Examples]

[0135] Nonspecific binding of A4-7-AE conjugate The interference of biotin in the nonspecific binding of A4-7-AE conjugates was measured. ADVIA Centaur assays were performed on natural serum containing approximately 2.2 ng / mL of androstenedione and multiple A4-7-AE conjugates using a solid phase containing 3C3 antibody. Biotin may interfere with the binding of conjugates to the ANDRO solid phase by altering the surface of streptavidin-coated beads. Therefore, to determine which conjugates were more affected by the presence of biotin during measurement, the same assay was performed for each conjugate, with serum samples having a biotin concentration of 100 ng / mL, and the results were compared. Table 4 shows the androstenedione concentrations measured in assays using biotin-free and biotin-added samples. The change in androstenedione concentration between biotin-free and biotin-added samples indicates biotin interference in the nonspecific binding of A4-7-AE conjugates. As can be seen, conjugates containing the CETE linker (-SCH2CH2C(O)NH-) exhibit greater biotin-induced nonspecific binding than conjugates without a sulfur-binding moiety ("carbon"; -CH2CH2C(O)NH-). This tendency is observed in both 7α and 7β conjugation configurations.

[0136] [Table 9] [Examples]

[0137] Precision measurement of A4-7-AE conjugate To determine the accuracy of the A4-7-AE conjugate across various androstenedione concentrations and test periods, nine serum samples containing various androstenedione concentrations (and other analytes) were assayed using the ADVIA Centaur assay with a 3C3 antibody. The samples contained two levels of commercially available quality control materials, available as Liquichek® Immunoassay Plus Quality Control ("QC2" and "QC3" from Bio-Rad, Hercules, CA). The other seven conjugated native or serum patient samples were provided by BioreclamationIVT. Five repeated measurements were performed on each sample within a single day. These five repeated measurements were also repeated over five different days. The overall mean A4 concentration (over all 25 measurements for each sample) using A4-carbon-AE is shown in Table 5. The standard deviation ("SD") and percent covariance ("%CV") of the ANOVA parameters are also shown, ranging from five repeated measurements per day to the average measurements over five days. As can be seen, the A4-carbon-AE conjugate provides highly accurate results over repeated measurements. Similar results were obtained for the A4-7-ISO-DIZAE and A4-7-HEG-ZAE conjugates.

[0138] [Table 10] [Examples]

[0139] Linearity performance in assays using A4-7-AE conjugate For the A4-7-AE conjugate, seven different samples with known A4 concentrations were prepared and analyzed using weighted linear fitting to determine the deviation between the observed and predicted androstenedione concentrations. A linear model was then used to fit the predicted A4 concentration, A4 concentration, and each predicted androstenedione concentration when using the A4-carbon-AE conjugate. The percentage bias is shown in Table 6. As can be seen, across the range of measured androstenedione concentrations, the conjugation at position 7 resulted in assays with less bias from the linear model. Typically, the androstenedione concentrations of serum-derived samples will fall within these ranges.

[0140] [Table 11] [Examples]

[0141] Bias in A4-7-AE conjugate assays using patient samples To determine the bias of assay measurements compared to liquid chromatography / tandem mass spectrometry (LC-MS / MS) measurements of each sample (ARUP Laboratories, Salt Lake City, Utah), a series of patient samples obtained from Bioreclamation IVT were also measured for androstenedione concentration using the ADVIA Centaur immunoassay containing an A4-carbon-AE conjugate. Patient samples were either native or contained additional androstenedione. Percentage bias is reported as the difference between assay and LC-MS / MS measurements compared to LC-MS / MS measurements. Table 7 shows the measurement results. The A4-AE conjugate can be used to determine the concentration of androstenedione in patient samples using an immunoassay with low bias across the entire range of measured androstenedione concentrations.

[0142] [Table 12]

[0143] Figure 4 shows a comparison of concentrations obtained from immunoassays using the A4-AE conjugate with concentrations obtained from LC-MS / MS assays. As can be seen, the A4-AE conjugate can measure the concentration of A4 more accurately.

[0144] All references, including patent applications and publications cited herein, are incorporated by reference in whole for any purpose, to the same extent as when specifically and individually directed, that individual publications or patents or patent applications may be incorporated in whole for any purpose. Incorporated herein by reference. Numerous 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 for illustrative purposes only, and the present invention should be limited solely by the terms of the appended claims, along with the entire scope of equivalents granted by such claims.

Claims

1. Androstenegion conjugate, 【Chemistry 1】 [In the formula, L-Ψ is -L C - (Z L ) z -Ψ is; L C teeth, In the left formula, the structure: -(X 1 ) 1 -(CH 2 ) 1 to 5 -C(O)-NR N -(R L ) 0 to 5 -(X 3 ) 0 to 1 -(R L ) 0 to 5 -(X 4 ) 0 to 1 -(R L ) 0 to 5 has In the equation on the right, structure: -(X 1 ) 1 -C(O)-(CH 2 ) 2 ~ 5 -C(O)-NR N - (R L ) 0 ~ 5 - (X 4 ) 0 ~ 1 - (R L ) 0 ~ 5 - has; X 1 is selected from -O- or -S-; X 3 and X 4 These are independently -O-, -S-, and -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-, selected; -L C - R L Independently, -CH 2 -, - (CH 2 CH 2 O) -, or - (OCH 2 CH 2 ) - Selected from; R N These are independently hydrogen or C 1 ~ 5 Selected from alkyl groups, "z" is 1; Z L Structure: 【Chemistry 2】 It is an amphoteric linker group having; "m" is either 0 (i.e., a combination) or 1; "n" and "p" are independently integers between 0 (i.e., associative) and 10; X a These are independently carboxylates (-C(O)O - ), sulfonate (-SO 3 - ), sulfate (-OSO 3 - ), phosphate (-OP(O)(OR P ) O - ), or oxide (-O - ) and R P C is a carbon atom in which hydrogen, or in some cases up to 10 carbon atoms, is replaced by heteroatoms. 1 ~ 12 It is a hydrocarbon; Z L In R L In some cases, C has 1 to 10 carbon atoms substituted with heteroatoms. 1 ~ 20 It is a divalent hydrocarbon group; R' is hydrogen or C 1 ~ 10 It is alkyl; Ψ is a chemiluminescent acridinium label. The conjugate having the structure of the above.

2. Ψ is given by equation (IIa) 【Transformation 3】 [In the formula, Ω is CH, O, or N; Y is -R or -R L - If selected from Z, or if Ω is O, then Y does not exist; Y' does not exist (i.e., it is a bond), or Ω-Y' is Ω-L 1 -, Ω-R L -, Ω-R L -L 1 -, Ω-L 1 -L 1 -, Ω-L 1 -R L -, Ω-L 1 -R L -L 1 , or Ω- R L -L 1 -R L - is selected from any one of; 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 These are independently selected from hydrogen, -R, or -Z; Z is structure: 【Chemistry 4】 It is an amphoteric group having; "q" and "l" are independently either 0 or 1; "r" is an independent integer between 0 and 10; L 1 These are independently -O-, -S-, -NH-, -N(R N )-,-(CH 2 ) 1 ~ 10 -, -S (=O) 1 ~ 2 -, -C=C-, -C=C-(CH 2 ) 1 ~ 3 -, -C(O)-, -OC( 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 -、-(C H 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 in Ψ L Independently, 1 to 10 carbon atoms are substituted with heteroatoms in some cases. Yes, C 1 ~ 20 It is a divalent hydrocarbon group; R is independently a hydrogen atom, or optionally 1 to 20 carbon atoms substituted with heteroatoms, C 1 ~ 35 It is a hydrocarbon group; R' and R'' are independently hydrogen or C 1 ~ 10 It is alkyl; X b These are independently carboxylates (-C(O)O - ), sulfonate (-SO 3 - ), sulfate (-OSO 3 - ), phosphate (-OP(O)(OR P ) O - ), or oxide (-O - ) and R P C is a carbon atom in which hydrogen, or in some cases up to 10 carbon atoms, is replaced by heteroatoms. 1 ~ 12 It is a hydrocarbon; R N These are independently hydrogen or C 1 ~ 5 Selected from alkyl groups] The conjugate according to claim 1, having the structure described above.

3. Ψ is given by equation (IIb): 【Transformation 5】 [In the formula, R 5 ~R 7 R is independently hydrogen, or alkyl, alkenyl, alkynyl, aryl, alkoxy, alkylthio, or amino; 8 is hydrogen; L 1 [It is covalently bonded to L] The conjugate according to claim 2, having the structure described above.

4. L in equation (IIb) 1 The conjugate according to claim 3, wherein is -C(O)-NH-.

5. R 5 and R 6 Each of them is methyl, and R 7 is hydrogen, as described in claim 3 or 4. Conjugate.

6. Ψ is given by equation (IIc): 【Transformation 6】 [In the formula, Y'' does not exist, or S (= O) 2 -Y'' is S (=O) 2 -L 1 -, S (= O ) 2 -R L - or S (=O) 2 -R L -L 1 - and here Y'' is covalently bonded to L [Added] The conjugate according to claim 2, having the structure described above.

7. The conjugate according to any one of claims 2 to 6, wherein the bond of L to androstenedione is a 6β covalent bond.

8. X 1 The conjugate according to any one of claims 2 to 7, wherein is -O-.

9. L C any of claims 2 to 8, wherein -C(O)-NH- or -NH-C(O)- The conjugate described in item 1.

10. L C Structure: 【Transformation 7】 A conjugate according to any one of claims 2 to 9, having the following characteristics.

11. R 1 Ha-R L -SO 3 - A conjugate according to any one of claims 2 to 10, including the conjugate described in any one of claims 2 to 10.

12. R 1 The conjugate according to any one of claims 2 to 10, comprising sulfopropyl. 。

13. R 1 ha-S(O) 2 -NH-Z, or -(CH 2 ) 1 ~ 3 -S(O) 2 A conjugate according to any one of claims 2 to 10, wherein the conjugate is -NH-Z.

14. R 2 and R 3 The conjugate according to any one of claims 2 to 13, wherein is independently hydrogen, alkyl, or alkoxy.

15. R 2 and R 3 The conjugate according to any one of claims 2 to 13, wherein each of them is hydrogen.

16. R 2 or R 3 One of them is hydrogen, R 2 or R 3 The conjugate according to any one of claims 2 to 13, wherein the other is an alkoxy.

17. Z L X in a is sulfonate (-SO 3 - ) and m is 1, R L It is propyl Yes, n and p are 3, and as a result Z L Structure: 【Transformation 8】 A conjugate according to any one of claims 1 to 16, having the following characteristics.

18. Structure of equation (III): 【Chemistry 9】 The conjugate according to any one of claims 2 to 5, wherein A has a structure in which androstenedione.

19. A reagent composition for detecting an analyte, wherein the reagent composition comprises a conjugate according to any one of claims 1 to 18 in a pH buffer medium.

20. An assay method for detecting or quantifying an analyte in a sample, (a) A step of preparing the conjugate according to any one of claims 1 to 18; (b) A step of preparing a solid support on which molecules capable of forming a binding complex with the analyte and capable of forming a binding complex with the conjugate are immobilized; (c) A step of mixing the conjugate, the solid carrier, and the sample; (d) A step of separating the solid carrier from the mixture; (e) A step of inducing chemiluminescence of the acridinium label that has formed a composite with the solid phase; (f) the step of measuring the amount of light emitted with a luminometer; and (g) A step of detecting the presence of an analyte or calculating its concentration by comparing the amount of light emitted with a reference dose-response curve that relates the amount of light emitted to a known concentration of the analyte. The assay method, including the above.

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