1 2024P00697WO DESCRIPTION TIANEPTINE ANALOGS AND CONJUGATES AND USE OF THE SAME IN DETECTION OF TIANEPTINE AND ITS METABOLITES 5 CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of U.S. Provisional Application No. 63/636,143 filed on 19 April 2024. The entire disclosure of the above application is incorporated 10 herein by reference. FIELD [0002] This disclosure generally relates to methods, compositions, and kits for detecting the presence and/or amounts of tianeptine and/or metabolites of tianeptine in biological samples. In particular, the disclosure relates to haptens, conjugates, and assays for detection of tianeptine 15 and/or its metabolites. BACKGROUND [0003] This section provides background information related to the present disclosure which is not necessarily prior art. [0004] The clinical diagnostic field has seen a broad expansion in recent years, both as 20 to the variety of materials of interest that may be readily and accurately determined, as well as the methods for the determination. Over the last decade, testing for drugs of abuse has become commonplace. This testing is not only for the monitoring of criminal offenders and drug addicts, but employers also use it for the screening of workers. In recent years, immunoassays based on a reaction of an antibody with an antigen have been extensively investigated for this purpose. 25 Immunoassays may be roughly classified into a radioimmunoassay using a radioactive isotope, an enzyme-immunoassay (EIA) using an enzyme, and a luminescence assay using fluorescent labels, e.g., fluorescence polarization, and chemiluminescent labels. [0005] Tianeptine is an atypical tricyclic antidepressant which is used mainly in the treatment of major depressive disorder, but may also be used to treat anxiety, asthma, 30 and irritable bowel syndrome. As a μ-opioid agonist, tianeptine has potential for recreational use and abuse. Therefore, an assay for detecting tianeptine and/or a metabolite of tianeptine in a biological sample is needed.
2 2024P00697WO SUMMARY [0006] This section provides a general summary of the disclosure and is not a comprehensive disclosure of its full scope or all of its features. [0007] In certain aspects, the present disclosure provides a complex corresponding in 5 structure to a Formula (I): wherein:
“-----” represents a single bond or a double bond; 10 R1 is hydrogen, an alkyl, —(CH2)a(NH)(CO)(CH2)—halo, or —(CH2)b(NH)(CO)(CH2)S—Y1; a and b are each zero to 50; R2 is hydrogen, halo, —(C2H2)(CH2)c(NH)(CO)(CH2)—halo, —S(CH2)dNHCO(CH2)—halo, —(C2H2)(CH2)e(NH)(CO)(CH2)S—Y2, or —S(CH2)fNHCO(CH2)S—Y3; c, d, e, and f are each zero to 50; 15 when “-----” represents a single bond, then R3 is NR5R6, wherein R5 is hydrogen, –(CH2)g(NH)(CO)(CH2)—halo, —(CO)(CF3), or —(CH2)h(NH)(CO)(CH2)S—Y4; g and h are each zero to 50; 20 R6 is —(CO)(CH2)i(CO)(OH), —(CH2)j(CH2)(CO)-R7, , 2)p(CO)(NH)-Y6, or
i, j, k, l, m, n, o, p, and q are each zero to 50; 25 R7 is —OH, —(NH)(CH2)r(NH)(CO)(CH2)—halo,
3 2024P00697WO , —(NH)—Y9; r, s, t, and u are
each zero to 50; and 5 R8 is —(CH2)v(S)(CH2)w(CO)(NH)—Y10, —(CH2)xS—Y11, or —(CH2)y—halo; v, w, x, and y are each zero to 50; when “-----” represents a double bond, then R3 is NR4, wherein R4 is —O(CH2)x1(CO)(OH), —O(CH2)(CO)(NH)(CH2)x2(NH)(CO)(CH2)—halo, —O(CH2)x3(CO)(NH)—Y12 10 or —O(CH2)(CO)(NH)(CH2)x4(NH)(CO)(CH2)S—Y13; x1, x2, x3, and x4 are each zero to 50; and each of Y1, Y2, Y3, Y4, Y5, Y6, Y7, Y8, Y9, Y10, Y11, Y12, and Y13 is an 15 immunogenic carrier or a label; and wherein when R3 is NR5R6, R5 is hydrogen, R6 is (CH2)j(CH2)(CO)-R7, j is 1, 3, or 5, and R7 is —OH, then both R1 is not Cl and R2 is not methyl. [0008] In yet other aspects, the present disclosure provides a compound corresponding in structure to a Formula (Ia): 20 wherein: R1 is hydrogen or an 2
R is hydrogen or halo; 25 R3 is NR5R6,
4 2024P00697WO wherein R5 is hydrogen; R6 is —(CO)(CH2)i(CO)(OH), —(CH2)j(CH2)(CO)-R7, or 5
wherein when R6 is —(CH2)j(CH2)(CO)-R7, j is 1, 3, or 5, and R7 is —OH, then 10 both R1 is not Cl and R2 is not methyl. [0009] In yet other aspects, the present disclosure provides a conjugate corresponding in structure to a Formula (Ib): 15 wherein: “-----” represents a si
le bond; R1 is hydrogen, an alkyl, or —(CH2)b(NH)(CO)(CH2)S—Y1; b is zero to 50; R2 is hydrogen, halo, —(C2H2)(CH2)e(NH)(CO)(CH2)S—Y2, or —S(CH2)fNHCO(CH2)S—Y3; e and f are each zero to 50; 20 when “-----” represents a single bond, then R3 is NR5R6, wherein R5 is hydrogen, —(CO)(CF3), or —(CH2)h(NH)(CO)(CH2)S—Y4; h is zero to 50; R6 is —(CH2)j(CH2)(CO)-R7, –(CH2)nS(CH2)o(CO)(NH)—Y5,
5 2024P00697WO –(CO)(CH2)p(CO)(NH)-Y6, or —(CH2)q(NH)(CO)-R8; j, n, o, p, and q are each zero to 50; R7 is —OH, —(NH)(CH2)t(NH)(CO)(CH2)S—Y7, , or —(NH)—Y9; t and u are each
5 zero to fifty; R8 is —(CH2)v(S)(CH2)w(CO)(NH)—Y10 or —(CH2)xS—Y11; v, w, and x are each zero to 50; when “-----” represents a double bond, then R3 is NR4, wherein R4 is —O(CH2)x3(CO)(NH)—Y12 or 10 —O(CH2)(CO)(NH)(CH2)x4(NH)(CO)(CH2)S—Y13; x3 and x4 are each zero to 50; and each of Y1, Y2, Y3, Y4, Y5, Y6, Y7, Y8, Y9, Y10, Y11, Y12, and Y13 is an immunogenic carrier or a label; and wherein when R3 is NR5R6, R5 is hydrogen, R6 is (CH2)j(CH2)(CO)-R7, j is 1, 3, 15 or 5, and R7 is —OH, then both R1 is not Cl and R2 is not methyl. [0010] In yet other aspects, the present disclosure provides a conjugate corresponding in structure to a Formula (Ic): 20 wherein: R1 is hydrogen or an
R2 is hydrogen or halo; R3 is NR5R6, wherein 25 R5 is hydrogen, R6 is —(CH2)j(CH2)(CO)-R7, –(CH2)nS(CH2)o(CO)(NH)—Y5, –(CO)(CH2)p(CO)(NH)-Y6, or —(CH2)q(NH)(CO)-R8; j, n, o, p, and q are each zero to 50;
6 2024P00697WO R7 is —(NH)—Y9; and R8 is —(CH2)v(S)(CH2)w(CO)(NH)—Y10 or —(CH2)xS—Y11; v, w, and x are each zero to 50; and each of Y5, Y6, Y9, Y10, and Y11 is an immunogenic carrier or a label. 5 [0011] In yet other aspects, the present disclosure provides a kit including an anti- tianeptine antibody and a conjugate corresponding in structure to a Formula (Ib) as described herein or a Formula (Ic) as described herein. The anti-tianeptine antibody may be raised against a complex as described herein wherein the immunogenic carrier is present. [0012] In yet other aspects, the present disclosure provides a method for determining the 10 presence of tianeptine and/or a metabolite of tianeptine in a sample. The method includes combining the sample, a conjugate, and an anti-tianeptine antibody in a medium, and examining the medium for the presence of a complex comprising tianeptine and the anti-tianeptine antibody. The conjugate corresponds in structure to a Formula (Ib) as described herein or a Formula (Ic) as described herein. The anti-tianeptine antibody may be raised against a complex as described 15 herein wherein the immunogenic carrier is present. [0013] Further areas of applicability will become apparent from the description provided herein. The description and specific examples in this summary are intended for purposes of illustration only and are not intended to limit the scope of the present disclosure. DRAWINGS 20 [0014] The drawings described herein are for illustrative purposes only of selected embodiments and not all possible implementations and are not intended to limit the scope of the present disclosure. [0015] FIG.1 is a reaction scheme 1 depicting an example of a synthesis of compounds (XV), (XVI), and (XVII) and their corresponding conjugates and immunogens. 25 [0016] FIG.2 is a reaction scheme 2 depicting an example of a synthesis of compounds (III), (IV), (V), (VI), (VII), (VIII), (XII), (XIII), and (XIV) and their corresponding conjugates and/or immunogens as well as an example of a preparation of conjugate (G). [0017] FIG. 3 is a reaction scheme 3 depicting an example of a synthesis of compounds (IX), (X), and (XI) and their corresponding conjugates and/or immunogens. 30 [0018] FIG.4 is a reaction scheme 4 depicting an example of a synthesis of a compound (XVIII). [0019] FIG.5 is a reaction scheme 5 depicting an example of a synthesis of a compound (XIX).
7 2024P00697WO [0020] FIG.6 is a reaction scheme 6 depicting an example of a synthesis of a compound (XX). [0021] FIG. 7 is a reaction scheme 7 depicting an example of a synthesis of conjugates and/or immunogens of compound (XIX). 5 [0022] FIG.8 is a reaction scheme 8 depicting an example of a synthesis of compounds (XXI) and (XXII). [0023] FIG. 9 is a reaction scheme 9 depicting an example of a synthesis of conjugates and/or immunogens of compound (XXII) [0024] FIG.10 is a reaction scheme 10 depicting an example of a synthesis of compounds 10 (XXIII), (XXIV), (XXV), and (XXVI) and their corresponding conjugates and/or immunogens. [0025] FIG.11 is a reaction scheme 11 depicting an example of a synthesis of compound (XXVII). [0026] FIG.12 is a reaction scheme 12 depicting an example of a synthesis of a conjugate of compound (XXVII). 15 [0027] FIG.13 is a reaction scheme 13 depicting an example of a synthesis of conjugates and/or immunogens of compound (XXVII). [0028] FIG.14 is a reaction scheme 14 depicting an example of a synthesis of conjugates and/or immunogens of compound (XXVII). [0029] FIG.15 is a reaction scheme 15 depicting an example of a synthesis of compounds 20 (XXVIII), (XXIX), and (XXX) and their corresponding conjugates and/or immunogens. DETAILED DESCRIPTION [0030] Example embodiments will now be described more fully with reference to the accompanying drawings. [0031] Example embodiments are provided so that this disclosure will be thorough and 25 will fully convey the scope to those who are skilled in the art. Numerous specific details are set forth such as examples of specific compositions, components, devices, and methods, to provide a thorough understanding of embodiments of the present disclosure. It will be apparent to those skilled in the art that specific details need not be employed, that example embodiments may be embodied in many different forms and that neither should be construed to limit the scope of the 30 disclosure. In some example embodiments, well-known processes, well-known device structures, and well-known technologies are not described in detail. [0032] The terminology used herein is for the purpose of describing particular example embodiments only and is not intended to be limiting. As used herein, the singular forms “a,” “an,” and “the” may be intended to include the plural forms as well, unless the context clearly
8 2024P00697WO indicates otherwise. The terms “comprises,” “comprising,” “including,” and “having,” are inclusive and therefore specify the presence of stated features, elements, compositions, steps, integers, operations, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. 5 Although the open-ended term “comprising,” is to be understood as a non-restrictive term used to describe and claim various embodiments set forth herein, in certain aspects, the term may alternatively be understood to instead be a more limiting and restrictive term, such as “consisting of” or “consisting essentially of.” Thus, for any given embodiment reciting compositions, materials, components, elements, features, integers, operations, and/or process steps, the present 10 disclosure also specifically includes embodiments consisting of, or consisting essentially of, such recited compositions, materials, components, elements, features, integers, operations, and/or process steps. In the case of “consisting of,” the alternative embodiment excludes any additional compositions, materials, components, elements, features, integers, operations, and/or process steps, while in the case of “consisting essentially of,” any additional compositions, materials, 15 components, elements, features, integers, operations, and/or process steps that materially affect the basic and novel characteristics are excluded from such an embodiment, but any compositions, materials, components, elements, features, integers, operations, and/or process steps that do not materially affect the basic and novel characteristics can be included in the embodiment. [0033] Any method steps, processes, and operations described herein are not to be 20 construed as necessarily requiring their performance in the particular order discussed or illustrated, unless specifically identified as an order of performance. It is also to be understood that additional or alternative steps may be employed, unless otherwise indicated. [0034] It should be understood for any recitation of a method, composition, device, or system that “comprises” certain steps, ingredients, or features, that in certain alternative 25 variations, it is also contemplated that such a method, composition, device, or system may also “consist essentially of” the enumerated steps, ingredients, or features, so that any other steps, ingredients, or features that would materially alter the basic and novel characteristics of the invention are excluded therefrom. [0035] Throughout this disclosure, the numerical values represent approximate measures 30 or limits to ranges to encompass minor deviations from the given values and embodiments having about the value mentioned as well as those having exactly the value mentioned. Other than in the working examples provided at the end of the detailed description, all numerical values of parameters (e.g., of quantities or conditions) in this specification, including the appended claims, are to be understood as being modified in all instances by the term “about” whether or not “about”
9 2024P00697WO actually appears before the numerical value. “About” indicates that the stated numerical value allows some slight imprecision (with some approach to exactness in the value; approximately or reasonably close to the value; nearly). If the imprecision provided by “about” is not otherwise understood in the art with this ordinary meaning, then “about” as used herein indicates at least 5 variations that may arise from ordinary methods of measuring and using such parameters. For example, “about” may comprise a variation of less than or equal to 5%, optionally less than or equal to 4%, optionally less than or equal to 3%, optionally less than or equal to 2%, optionally less than or equal to 1%, optionally less than or equal to 0.5%, and in certain aspects, optionally less than or equal to 0.1%. 10 [0036] In addition, disclosure of ranges includes disclosure of all values and further divided ranges within the entire range, including endpoints and sub-ranges given for the ranges. [0037] Example embodiments will now be described more fully with reference to the accompanying drawings. A. Definitions 15 [0038] Throughout this description and in the appended claims, the following definitions are to be understood. [0039] The term “tianeptine” refers to the compound with the chemical structure shown below: . 20 Tianeptine also may be [(3-chloro-6-methyl-5,5-dioxo-11H-
benzo[c][2,1]benzothiazepin-11-yl)amino]heptanoic acid.” [0040] The term “metabolite of tianeptine” refers to a compound, such as an intermediate or a byproduct, formed by the metabolism of tianeptine. Exemplary metabolites of tianeptine are shown below: O O S N Cl OH 25 . [0041] The ter le of eliciting an immune response in an organis
10 2024P00697WO [0042] The term “conjugate” refers to any substance formed from the joining together of two parts. Representative conjugates in accordance with the present invention include those formed by the joining together of a small molecule and a large molecule, such as a protein. The term “conjugate” subsumes the term “immunogen.” 5 [0043] The term “hapten” refers to a portion of an immunogen that is typically low in molecular weight, which does not by itself stimulate antibody development. [0044] The phrase “activated hapten” refers to a hapten that has been provided with an available reaction site—for example, by the attachment of a linking group carrying a reactive moiety—that can be used to connect the hapten to a carrier, immunogen, label, tracer, or other 10 moiety. [0045] The term “linking group” (or “linker”) refers to a chemical moiety that is used to connect a hapten to a macromolecular carrier, immunogen, label, tracer, or other moiety. The use of a linking group may or may not be advantageous or needed, depending on the specific hapten and carrier and desired specificity of antibody. Suitable linkers include straight, branched, 15 saturated or unsaturated carbon chains, which may incorporate one or more heteroatoms—that is, atoms other than carbon (e.g., oxygen, nitrogen, sulfur, etc.)—within the chain or substituted onto and/or at a terminus thereof. [0046] The phrases “carrier” and “macromolecular carrier” refer to high molecular weight substances that can be coupled to haptens to form immunogens. Suitable macromolecular 20 carriers include but are not limited to proteins, glycoproteins, polymers, polysaccharides, polypeptides, and nucleic acids that are recognized as foreign and thereby elicit an immunologic response from a host. [0047] The term “polypeptide” refers to any compound formed by the linkage of two or more amino acids via an amide bond. Representative polypeptides include polymers of α-amino25 acids in which the α-amino group of each non-terminal amino acid residue is linked to the α- carboxyl group of an adjacent residue in a linear chain. High molecular weight polypeptides are referred to as “proteins.” [0048] The term “label” refers to a member of a signal producing system. The label is capable of being detected directly or is detectable through a specific binding reaction that 30 produces a detectable signal. For example, a label may be an identifying tag that can be attached to a carrier substance or molecule to detect an analyte. The labels generally are radioisotopic, luminescent, particulate, or enzymic. The label can be a poly(amino acid), or protein, or non- poly(amino acid), isotopic or non-isotopic, usually non-isotopic, and can be a catalyst, such as an enzyme (e.g., β-galactosidase, peroxidase, etc.), a polynucleotide coding for a catalyst,
11 2024P00697WO promoter, dye, fluorescent molecule (e.g., rhodamine, fluorescein isothiocyanate or FITC, etc.), chemiluminescent molecule (e.g., dioxetanes, luciferin, etc.), coenzyme, enzyme substrate, radioactive group (e.g., 125I), a protein-binding partner (e.g., biotin), a small organic molecule, amplifiable polynucleotide sequence, a particle such as latex or carbon particle, metal sol, 5 crystallite, liposome, cell, etc., which may or may not be further labeled with a dye, catalyst or other detectable group, and the like. [0049] The term “non-poly(amino acid) labels” refers to those labels that are not proteins such as enzymes. A non-poly(amino acid) label may be a member of a signal producing system. The non-poly(amino acid) label is capable of being detected directly or is detectable through a 10 specific binding reaction that produces a detectable signal. The non-poly(amino acid) labels generally are radioisotopic, luminescent, particulate, polynucleotidic, or the like. More particularly, the label can be isotopic or non-isotopic, usually non-isotopic, and can be a polynucleotide coding for a catalyst, promoter, dye, fluorescent molecule, chemiluminescent molecule, coenzyme, enzyme substrate, radioactive group, a small organic molecule, amplifiable 15 polynucleotide sequence, a particle such as latex or carbon particle, metal sol, crystallite, liposome, cell, etc., which may or may not be further labeled with a dye, catalyst or other detectable group, and the like. [0050] The signal producing system may have one or more components, at least one component being the label. The signal producing system generates a signal that relates to the 20 presence of an analyte in a sample. The signal producing system includes all of the reagents required to produce a measurable signal. Other components of the signal producing system may be included in a developer solution and can include substrates, enhancers, activators, chemiluminescent compounds, cofactors, inhibitors, scavengers, metal ions, specific binding substances required for binding of signal generating substances, and the like. Other components 25 of the signal producing system may be coenzymes, substances that react with enzymic products, other enzymes and catalysts, and the like. The signal producing system provides a signal detectable by external means, by use of electromagnetic radiation, desirably by visual examination. Exemplary signal-producing systems are described in U.S. Pat. No. 5,508,178 (Rose, et al.), the relevant disclosure of which is incorporated herein by reference. 30 [0051] The term “immunogenic carrier” refers to a group which, when conjugated to a hapten and injected into a mammal, will induce an immune response and elicit the production of antibodies that bind to the hapten. Haptens are compounds capable of binding specifically to corresponding antibodies, but do not themselves act as immunogens (or antigens) for preparation of the antibodies. Antibodies that recognize a hapten can be prepared against compounds
12 2024P00697WO comprised of the hapten linked to an immunogenic (or antigenic) carrier. Immunogenic carriers are also referred to as antigenic carriers. Typical immunogenic carriers include, without limitation, poly(amino acids), polysaccharides, nucleic acids, and particles (biologic and synthetic materials). A wide variety of such carriers are disclosed in Davalian, et al., U.S. Pat. 5 No. 5,089,390, column 4, line 57 to column 5, line 5, incorporated herein by reference. Immunogenic carriers include proteins such as, for example, albumins, serum proteins, e.g., globulins, ocular lens proteins and lipoproteins, and so forth. Illustrative proteins include bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), egg ovalbumin (OVA), bovine gamma-globulin (BGG), bovine thyroglobulin (BTG), glucose-6-phoshpate dehydrogenase 10 (G6PDH), and the like. [0052] The term “antibody” (abbreviated “Ab”) refers to a specific protein capable of binding an immunogen or portion thereof. An antibody may be produced in response to an immunogen, which may have been introduced into a host (e.g., an animal or a human) by injection. Alternatively, an antibody may be produced via hybridoma, phage display, transgenic 15 mice, and CRISPR/Cas9 technologies. The generic term “antibody” subsumes polyclonal antibodies, monoclonal antibodies, and antibody fragments. Monoclonal and polyclonal antibodies (abbreviated “mAb” and “pAb,” respectively) generally include the various classes and isotypes, including IgA, IgD, IgE, IgG1, IgG2A, IgG2B, IgG3, IgG4, IgM, and the like. Antibody fragments may include Fab, scFv, F(ab’)2, Fab’, and the like. “Antibody” may be used 20 in either a therapeutic or a diagnostic capacity. [0053] The term “analyte” refers to any substance, or group of substances, the presence or amount of which is to be determined. As used herein, the term “analyte” subsumes the term “antigen,” which refers to any compound that can bind to an antibody. Furthermore, as used herein, the term “analyte” refers to all manner of chemical substances including but not limited 25 to: conjugates; immunogens; drugs; drug metabolites; drug derivatives; hormones; proteins; antigens; oligonucleotides; and the like. [0054] The term “derivative” refers to a chemical compound made from a parent compound by one or more chemical reactions. [0055] The phrase “detecting an analyte” refers to any quantitative, semi-quantitative, or 30 qualitative method, as well as to all other methods for determining an analyte in general. For example, a method that merely detects the presence or absence of tianeptine and/or a metabolite of tianeptine in a sample lies within the scope of the present invention, as do methods that provide data as to the amount or concentration of the drug in the sample. The terms “detecting,”
13 2024P00697WO “determining,” “identifying,” and the like are used synonymously herein, and all lie within the scope of the present invention. [0056] The phrase “reagent kit” or term “kit” refers to an assembly of materials that are used in performing an assay. The reagents can be provided in packaged combination in the same 5 or in separate containers, depending on their cross-reactivities and stabilities, and in liquid or in lyophilized form. The amounts and proportions of reagents provided in the kit can be selected so as to provide optimum results for a particular application. A reagent kit embodying features of the present invention comprises antibodies specific for tianeptine, metabolites of tianeptine, conjugates of tianeptine analogs, and/or enzymes or proteins necessary for detecting the presence 10 and quantity of the antibody, tianeptine drug, and/or metabolite of tianeptine in a sample. The kit may further comprise calibration and control materials. The reagents may remain in liquid form or may be lyophilized. [0057] The phrase “calibration and control materials” refers to any standard or reference material containing a known amount of an analyte to be measured. A sample suspected of 15 containing an analyte and the corresponding calibration material are assayed under similar conditions. The concentration of analyte is calculated by comparing the results obtained for the unknown specimen with the results obtained for the standard. This is commonly done by constructing a calibration curve. [0058] The term “alkyl” (alone or in combination with another term(s)) refers to a 20 saturated hydrocarbon chain of 1 to about 12 carbon atoms in length, such as, but not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, decyl, and so forth. The alkyl group may be straight-chain, branched-chain. “Alkyl” is intended to embrace all structural isomeric forms of an alkyl group, cyclic, or acyclic. For example, as used herein, propyl encompasses both n- propyl and isopropyl; butyl encompasses n-butyl, sec-butyl, isobutyl, and tert-butyl; pentyl 25 encompasses n-pentyl, tert-pentyl, neopentyl, isopentyl, sec-pentyl and 3-pentyl. Further, as used herein, “Me” refers to methyl, “Et” refers to ethyl, “Pr” refers to propyl, “i-Pr” refers to isopropyl, “Bu” refers to butyl, “t-Bu” refers to tert-butyl, “iBu” refers to isobutyl, “Pn” refers to pentyl, and “NPn” refers to neopentyl. [0059] The term “halo” refers to fluoro (—F), chloro (—Cl), bromo (—Br), or iodo (— 30 I). [0060] The term “sample” refers to a composition to be tested for the presence of tianeptine and/or a metabolite of tianeptine. The sample may be organic or inorganic, biological (e.g., a “biosample”), non-biological, or environmental. Examples of a biological or biosample include, but are not limited to, urine, whole blood, plasma, serum, lymph, mucus, expressed
14 2024P00697WO breast milk, semen, stool, sputum, cerebral spinal fluid, tears, hair, saliva, cells, tissues, an organ, and/or a biopsy. [0061] The terms “identical,” “sequence identity,” or “percent identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or 5 subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence over a comparison window. The degree of amino acid or nucleic acid sequence identity for purposes of the present disclosure is determined using the BLAST algorithm, described in Altschul, S.F. et al. (1990) J. Mol. Biol. 215:403–10, which is incorporated herein by reference. The BLAST 10 algorithm is publicly available through software provided by the National Center for Biotechnology Information (at the web address www.ncbi.nlm.nih.gov). B. Tianeptine Analogs, Immunogens, and Conjugates [0062] Compounds, such as tianeptine analogs, which can be used for preparing immunogens, conjugates, and antibodies useful in immunoassays for the determination of 15 tianeptine and/or a metabolite of tianeptine, are provided herein. Immunogens and conjugates formed from the compounds are also provided herein. It is contemplated herein that tianeptine analogs include haptens and activated haptens. [0063] In any embodiment, such hapten, immunogen, and/or conjugate may be a complex corresponding in structure to a Formula (I): 20 wherein: “-----” represents a si
le bond; R1 is hydrogen, an alkyl, —(CH2)a(NH)(CO)(CH2)—halo, or 25 —(CH2)b(NH)(CO)(CH2)S—Y1; a and b are each zero to 50; R2 is hydrogen, halo, —(C2H2)(CH2)c(NH)(CO)(CH2)—halo, —S(CH2)dNHCO(CH2)—halo, —(C2H2)(CH2)e(NH)(CO)(CH2)S—Y2, or —S(CH2)fNHCO(CH2)S—Y3; c, d, e, and f are each zero to 50; when “-----” represents a single bond, then R3 is NR5R6, 30 wherein R5 is hydrogen, –(CH2)g(NH)(CO)(CH2)—halo,
15 2024P00697WO —(CO)(CF3), or —(CH2)h(NH)(CO)(CH2)S—Y4; g and h are each zero to 50; R6 is —(CO)(CH2)i(CO)(OH), —(CH2)j(CH2)(CO)-R7, 5 or alo, 10 are
R is —(CH2)v(S)(CH2)w(CO)(NH)—Y , —(CH2)xS—Y , or —(CH2)y—halo; v, w, x, and y are each zero to 50; 15 when “-----” represents a double bond, then R3 is NR4, wherein R4 is —O(CH2)x1(CO)(OH), —O(CH2)(CO)(NH)(CH2)x2(NH)(CO)(CH2)—halo, —O(CH2)x3(CO)(NH)—Y12 or —O(CH2)(CO)(NH)(CH2)x4(NH)(CO)(CH2)S—Y13; x1, x2, x3, and x4 are each 20 zero to
50; and each of Y1, Y2, Y3, Y4, Y5, Y6, Y7, Y8, Y9, Y10, Y11, Y12, and Y13 is an immunogenic carrier or a label. [0064] In any embodiment, when R3 is NR5R6, R5 is hydrogen, R6 is (CH2)j(CH2)(CO)- R7, j is 1, 3, or 5, and R7 is —OH, then both R1 is not Cl and R2 is not methyl. For example, a
16 2024P00697WO complex corresponding in structure to Formula (I) may not be one or more of the following com ounds: OH .
[0065] In any embod ment, R may be an a y , —(CH2)a(NH)(CO)(CH2)—halo, or 5 —(CH2)b(NH)(CO)(CH2)S—Y1 and R2 may be hydrogen, halo, —(C2H2)(CH2)c(NH)(CO)(CH2)—halo, —S(CH2)dNHCO(CH2)—halo, —(C2H2)(CH2)e(NH)(CO)(CH2)S—Y2, or —S(CH2)fNHCO(CH2)S—Y3. Additionally or alternatively, R1 may be an alkyl and R2 may be hydrogen or halo. [0066] In any embodiment, the alkyl may be C1-C12-alkyl, C1-C10-alkyl, C1-C8-alkyl, C1-10 C6-alkyl, C1-C4-alkyl, C1-C3-alkyl, C1-C2-alkyl, or methyl. For example, the alkyl may be C1-C6- alkyl, C1-C4-alkyl, C1-C3-alkyl, C1-C2-alkyl, or methyl. [0067] Additionally, each halo may be fluoro (F), chloro (Cl), bromo (Br), or iodo (I). For example, each halo may be Cl or Br. [0068] Additionally or alternatively, R1 may be C1-C4-alkyl, C1-C3-alkyl, C1-C2-alkyl, or 15 methyl and R2 may be Cl or Br. [0069] Additionally, a, b, c, d, e, f, g, h, i, j, k, l, m, n, o, p, q, r, s, t, u, v, w, x, y, z (a-z), x1, x2, x3, and x4 (x1-x4) each independently may be zero to 40, zero to 30, zero to 25, zero to 20, zero to 15, zero to 10, zero to 5, 1 to 40, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, or 1 to 5. For example, a-z and x1-x4 each may be 1 to 25, 1 to 10, or 1 to 5. 20 [0070] Suitable immunogenic carriers include, but are not limited to, a protein, a polypeptide, a polysaccharide, a nucleic acid, and a particle (e.g., biologic and synthetic materials). A wide variety of such carriers are disclosed U.S. Pat. No. 5,089,390, incorporated herein by reference. Examples of suitable proteins include, but are not limited to, albumins, serum proteins, e.g., globulins, ocular lens proteins and lipoproteins, and so forth. Nonlimiting examples 25 of proteins include the keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), bovine thyroglobulin (BTG), egg ovalbumin (OVA), bovine gamma globulin (BGG), and glucose-6- phoshpate dehydrogenase (G6PDH). A protein may be attached to a linking group by means of an amine group on the protein. [0071] In any embodiment, a label may be radioisotopic, luminescent, particulate or 30 enzymic. The label can be a poly(amino acid), or protein, or non-poly(amino acid), isotopic or
17 2024P00697WO non-isotopic, and can be a catalyst, such as an enzyme (e.g., β-galactosidase, peroxidase, etc.), a polynucleotide coding for a catalyst, promoter, dye, fluorescent molecule (e.g., rhodamine, fluorescein isothiocyanate or FITC, etc.), chemiluminescent molecule (e.g., dioxetanes, luciferin, etc.), coenzyme, enzyme substrate, radioactive group (e.g., 125I), a protein-binding partner, biotin 5 or another small organic molecule, amplifiable polynucleotide sequence, a particle such as latex or carbon particle, metal sol, crystallite, liposome, cell, etc., which may or may not be further labeled with a dye, catalyst or other detectable group, and the like. [0072] In any embodiment, R1 may be C1-C6 alkyl, C1-C4 alkyl, C1-C2 alkyl, or methyl. Alternatively, R1 may be —(CH2)a(NH)(CO)(CH2)—halo. Said halo may be Cl or Br and a may 10 be 1 to 25, 1 to 10, or 1 to 5, for example, said halo may be Br and a may be 1 or 2. [0073] In any embodiment, R2 may be hydrogen, halo, —(C2H2)(CH2)c(NH)(CO)(CH2)—halo, or —S(CH2)dNHCO(CH2)—halo. Each halo may be Cl or Br and c and d each may be 1 to 25, 1 to 10, or 1 to 5, for example, each halo may be Cl or Br, and c and d each may be 2 or 3. 15 [0074] In any embodiment, when “-----” represents a double bond, then R3 may be NR4. R4 may be —O(CH2)x1(CO)(OH) or —O(CH2)(CO)(NH)(CH2)x2(NH)(CO)(CH2)—halo. Each halo may be Cl or Br and x1 and x2 each may be 1 to 25, 1 to 10, or 1 to 5, for example, each halo may be Br, and x1 and x2 each may be 1 or 2. [0075] Alternatively, when “-----” represents a single bond, then R3 may be NR5R6. R5 20 may be hydrogen, —(CO)(CF3), or –(CH2)g(NH)(CO)(CH2)—halo. Said halo may be Cl or Br and g may be 1 to 25, 1 to 10, or 1 to 5, for example, said halo may be Br, and g may be 1 or 2. [0076] In any embodiment, R6 may be —(CO)(CH2)i(CO)(OH) and i may be 1 to 25, 1 to 10, or 1 to 5, for example, i may be 3 or 4. [0077] Alternatively, R6 may be (CH2)j(CH2)(CO)-R7. R7 may be —OH, 25 —(NH)(CH2)r(NH)(CO)(CH2)—halo . Said halo may be Cl or Br and j, r, and s each may b , said halo may be Br, j may be 1, 3, or 5, and r and s ea
[0078] In a further embodiment, R1 may be C1-C6 alkyl, C1-C4 alkyl or C1-C2 alkyl, R2 may be halo; R3 may be NR4; and R4 may be —O(CH2)x1(CO)(OH) or 30 —O(CH2)(CO)(NH)(CH2)x2(NH)(CO)(CH2)—halo. Each halo may be Cl or Br and x1 and x2 each may be 1 to 25 or 1 to 10. For example, R1 may be C1-C2 alkyl, such as methyl, R2 may be
18 2024P00697WO Cl, R4 may be —O(CH2)x1(CO)(OH) or —O(CH2)(CO)(NH)(CH2)x2(NH)(CO)(CH2)Br, and x1 and x2 each may be 1 to 5, such as 1 or 2. [0079] In a further embodiment, R1 may be —(CH2)a(NH)(CO)(CH2)—halo or C1-C6 alkyl, C1-C4 alkyl or C1-C2 alkyl; R2 may be hydrogen, halo, 5 —(C2H2)(CH2)c(NH)(CO)(CH2)—halo, or —S(CH2)dNHCO(CH2)—halo; R3 may be NR5R6, wherein R5 may be hydrogen, —(CO)(CF3), or —(CH2)g(NH)(CO)(CH2)—halo; R6 may be —(CO)(CH2)i(CO)(OH) or (CH2)j(CH2)(CO)-R7; and R7 may be —OH; —(NH)(CH2)r(NH)(CO)(CH2)—halo; , or
. Each halo may be Cl or Br and a, c, d, g, i, j, r, and s each may be 1 to 25. 10
In a further embodiment, R1 may be —(CH2)a(NH)(CO)(CH2)—halo or C1-C6 alkyl, C1-C4 alkyl or C1-C2 alkyl; R2 may be hydrogen or halo; R3 may be NR5R6, wherein R5 may be hydrogen or —(CO)(CF3); R6 may be —(CO)(CH2)i(CO)(OH) or (CH2)j(CH2)(CO)-R7; and R7 may be —OH; —(NH)(CH2)r(NH)(CO)(CH2)—halo or
. Each halo may be Cl or Br and a, i, j, r, and s each may be 1 to 25, 1 to 10, or 1 to 15 mple, R1 may be C1-C2 alkyl, such as methyl or —(CH2)a(NH)(CO)(CH2)Br; R2 may
be hydrogen or Cl; R5 may be hydrogen or —(CO)(CF3); R6 may be —(CO)(CH2)i(CO)(OH) or (CH2)j(CH2)(CO)-R7; R7 may be —OH, —(NH)(CH2)r(NH)(CO)(CH2)Br, d s each may be 1 to 25, for example,
19 2024P00697WO [0081] In a further embodiment, R1 may be C1-C6 alkyl, C1-C4 alkyl or C1-C2 alkyl; R2 may be halo; —(C2H2)(CH2)c(NH)(CO)(CH2)—halo, or —S(CH2)dNHCO(CH2)—halo; and R3 may be NR5R6, wherein R5 may be hydrogen or —(CH2)g(NH)(CO)(CH2)—halo; R6 may be (CH2)j(CH2)(CO)-R7; and R7 may be —OH. Each halo may be Cl or Br and c, d, g, and j each 5 may be 1 to 25, 1 to 10, or 1 to 5. For example, R1 may be C1-C2 alkyl, such as methyl; R2 may be Cl,—(C2H2)(CH2)c(NH)(CO)(CH2)Br, or —S(CH2)dNHCO(CH2)Br; R5 may be hydrogen or —(CH2)g(NH)(CO)(CH2)Br; R6 may be (CH2)j(CH2)(CO)-R7; R7 may be —OH; and c, d, g, and j each may be 1 to 25, for example, c, d, and g each may be 2 or 3 and j may be 1, 3, or 5. [0082] In a further embodiment, provided herein are tianeptine analog or hapten 10 compounds encompassed by Formula (I), which correspond in structure to a Formula (Ia): wherein: 1
R is hydrogen or an alkyl; 15 R2 is hydrogen or halo; R3 is NR5R6, wherein R5 is hydrogen; R6 is —(CO)(CH2)i(CO)(OH), —(CH2)j(CH2)(CO)-R7, 20 or
20 2024P00697WO [0083] In any embodiment, when R6 is —(CH2)j(CH2)(CO)-R7, j is 1, 3, or 5, and R7 is —OH, then both R1 is not Cl and R2 is not methyl. For example, a compound corresponding in structure to Formula (Ia) may not be one or more of compound (a), compound (b), and compound (c). 5 [0084] In any embodiment, R1 may be C1-C6 alkyl, C1-C4 alkyl, C1-C2 alkyl, or methyl. [0085] In any embodiment, R2 may be hydrogen or halo. Each halo may be Cl or Br. For example, R2 may be Cl. [0086] In any embodiment, R1 may be C1-C6 alkyl, C1-C4 alkyl, or C1-C2 alkyl, R2 may be halo, R5 is hydrogen, and R6 may be 10 . Each halo may be Cl or Br and k, l, and
ple, R1 may be C1-C2 alkyl, such as methyl, R2 may be Cl, R6 may be —(CH2)k(S2)(CH)3, and k may be 1 to 5, such as 1, 2, or 3. Alternatively, R1 may be C1-C2 alkyl, such as methyl, R2 may be Cl, R6 may be m may be 1 to 5, such as 1, 2, or 3. 15 1 may be C1-C6 alkyl, C1-C4 alkyl, or C1-C2 alkyl, R2
may be halo, R is hydrogen, R may be —(CH2)j(CH2)(CO)-R7, R7 may be —OH or , and j may be 1 to 25, 1 to 10, or 1 to 5. For example, R1 may be C1-C2 alkyl, , R2 may be Cl, R5 is hydrogen, R6 may be —(CH2)j(CH2)(CO)-R7, R7 may be
21 2024P00697WO —OH, and j may be 1 to 5, such as 1, 2, 3, or 4. Alternatively, R1 may be C1-C2 alkyl, such as methyl, R2 may be Cl, R5 is hydrogen, R6 may be —(CH2)j(CH2)(CO)-R7, R7 may be , and j may be 1 to 5, such as 3, 4, or 5.
[0088] In a further embodiment, R1 may be C1-C6 alkyl, C1-C4 alkyl, or C1-C2 alkyl, R2 5 may be halo, R5 is hydrogen, R6 may be —(CO)(CH2)i(CO)(OH), and i may be 1 to 25, 1 to 10, or 1 to 5. For example, R1 may be C1-C2 alkyl, such as methyl, R2 may be Cl, R5 is hydrogen, R6 may be —(CO)(CH2)i(CO)(OH), and i may be 1 to 5, such as 1, 2, 3, or 4. [0089] In a further embodiment, R1 may be C1-C6 alkyl, C1-C4 alkyl, or C1-C2 alkyl, R2 may be halo, R5 is hydrogen, R6 may be —(CH2)q(NH)(CO)-R8; and R8 may be —(CH2)z—halo. 10 Each halo may be Cl or Br and q and z each may be 1 to 25 or 1 to 10. For example, R1 may be C1-C2 alkyl, such as methyl, R2 may be Cl, R5 is hydrogen, R6 may be —(CH2)q(NH)(CO)-R8; and R8 may be —(CH2)z—Br, and q and z each may be 1, 2, 3, or 4. [0090] Nonlimiting examples of compounds corresponding in structure to Formulas (I) and (Ia) are shown below in Table 1. 15 Table 1 O O S N
22 2024P00697WO
23 2024P00697WO
24 2024P00697WO
[0091] In a further embodiment, compounds corresponding in structure to Formulas (I) and (Ia) are shown below in Table 2. Table 2
25 2024P00697WO
mula (I), which correspond in structure to a Formula (Ib): 5 wherein:
26 2024P00697WO “-----” represents a single bond or a double bond; R1 is hydrogen, an alkyl, or —(CH2)b(NH)(CO)(CH2)S—Y1; b is zero to 50; R2 is hydrogen, halo, —(C2H2)(CH2)e(NH)(CO)(CH2)S—Y2, or —S(CH2)fNHCO(CH2)S—Y3; e and f are each zero to 50; 5 when “-----” represents a single bond, then R3 is NR5R6, wherein R5 is hydrogen, —(CO)(CF3), or —(CH2)h(NH)(CO)(CH2)S—Y4; h is zero to 50; R6 is —(CH2)j(CH2)(CO)-R7, –(CH2)nS(CH2)o(CO)(NH)—Y5, 10 –(CO)(CH2)p(CO)(NH)-Y6, or —(CH2)q(NH)(CO)-R8; j, n, o, p, and q are each zero to 50; R7 is —OH, —(NH)(CH2)t(NH)(CO)(CH2)S—Y7, , or —(NH)—Y9; t and u are each
15 R8 is —(CH2)v(S)(CH2)w(CO)(NH)—Y10 or —(CH2)xS—Y11; v, w, and x are each zero to 50; when “-----” represents a double bond, then R3 is NR4, wherein R4 is —O(CH2)x3(CO)(NH)—Y12 or —O(CH2)(CO)(NH)(CH2)x4(NH)(CO)(CH2)S—Y13; x3 and x4 are each zero to 20 50; and each of Y1, Y2, Y3, Y4, Y5, Y6, Y7, Y8, Y9, Y10, Y11, Y12, and Y13 is an immunogenic carrier or a label as described herein. [0093] In any embodiment, when R3 is NR5R6, R5 is hydrogen, R6 is (CH2)j(CH2)(CO)- R7, j is 1, 3, or 5, and R7 is —OH, then both R1 is not Cl and R2 is not methyl. For example, a 25 compound corresponding in structure to Formula (Ib) may not be one or more of: compound (a), compound (b), and compound (c). [0094] In any embodiment, R1 may be C1-C6 alkyl, C1-C4 alkyl, C1-C2 alkyl, or methyl. Alternatively, R1 may be —(CH2)b(NH)(CO)(CH2)S—Y1 and b may be 1 to 25, 1 to 10, or 1 to 5. Additionally or alternatively, Y1 may be keyhole limpet hemocyanin (KLH), bovine serum 30 albumin (BSA), bovine thyroglobulin (BTG), egg ovalbumin (OVA), bovine gamma globulin (BGG), or glucose-6-phosphate dehydrogenase (G6PDH) and b may be 1 or 2.
27 2024P00697WO [0095] In any embodiment, R2 may be halo, —(C2H2)(CH2)e(NH)(CO)(CH2)S—Y2, or S(CH2)fNHCO(CH2)S—Y3 and e and f each may be 1 to 25, 1 to 10, or 1 to 5. Additionally or alternatively, said halo may be Cl or Br, each Y2 and Y3 may be keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), bovine thyroglobulin (BTG), egg ovalbumin (OVA), 5 bovine gamma globulin (BGG), or glucose-6-phosphate dehydrogenase (G6PDH), and e and f each may be 2 or 3. [0096] In any embodiment, when “-----” represents a double bond, then R3 may be NR4. R4 may be —O(CH2)x3(CO)(NH)—Y12 or —O(CH2)(CO)(NH)(CH2)x4(NH)(CO)(CH2)S—Y13, and x3 and x4 each may be 1 to 25, 1 to 10, or 1 to 5. Additionally or alternatively, Y12 and Y13 10 each may be keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), bovine thyroglobulin (BTG), egg ovalbumin (OVA), bovine gamma globulin (BGG), or glucose-6- phosphate dehydrogenase (G6PDH) and x2 and x3 each may be 1 or 2. [0097] Alternatively, when “-----” represents a single bond, then R3 may be NR5R6. R5 may be hydrogen, —(CO)(CF3), or —(CH2)h(NH)(CO)(CH2)S—Y4; R6 may be 15 (CH2)j(CH2)(CO)-R7; R7 may be —OH; —(NH)(CH2)t(NH)(CO)(CH2)S—Y7; d u each may be 1 to 25, 1 to Y8 may be keyhole limpet
hemocyanin (KLH), bovine serum albumin (BSA), bovine thyroglobulin (BTG), egg ovalbumin (OVA), bovine gamma globulin (BGG), or glucose-6-phosphate dehydrogenase (G6PDH), and 20 h, t, and u each may be 1 or 2, and j may be 1, 3, or 5. [0098] In a further embodiment, R1 may be C1-C6 alkyl, C1-C4 alkyl or C1-C2 alkyl; R2 may be halo; R3 may be NR4; and R4 may be —O(CH2)x3(CO)(NH)—Y12 or —O(CH2)(CO)(NH)(CH2)x4(NH)(CO)(CH2)S—Y13. Said halo may be Cl or Br and x3 and x4 each may be 1 to 25 or 1 to 10. For example, R1 may be a C1-C2 alkyl, such as methyl, R2 may 25 be Cl, and x3 and x4 each may be 1 to 5. [0099] In various aspects, a conjugate may correspond in structure to:
28 2024P00697WO , wherein
each of Y and Y may be keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), bovine thyroglobulin (BTG), egg ovalbumin (OVA), bovine gamma globulin (BGG), or glucose-6-phosphate dehydrogenase (G6PDH). 5 [00100] In a further embodiment, R1 may be —(CH2)b(NH)(CO)(CH2)S—Y1 or a C1-C6 alkyl, C1-C4 alkyl or C1-C2 alkyl, R2 may be halo or —(C2H2)(CH2)e(NH)(CO)(CH2)S—Y2 or —S(CH2)fNHCO(CH2)S—Y3, and R3 may be NR5R6, wherein R5 may be hydrogen, — (CO)(CF3), or —(CH2)h(NH)(CO)(CH2)S—Y4, R6 may be (CH2)j(CH2)(CO)-R7, and R7 may be —OH; 10 Y9.
[00101] In a further embodiment, R1 may be —(CH2)b(NH)(CO)(CH2)S—Y1 or C1-C6 alkyl, C1-C4 alkyl or C1-C2 alkyl; R2 may be halo; and R3 maybe NR5R6, wherein R5 may be hydrogen or —(CO)(CF3); R6 may be (CH2)j(CH2)(CO)-R7; and R7 may be —OH; 15 Y9.
, -C6 alkyl, C1-C4 alkyl or C1-C2 alkyl; R2 may be halo; and R3 may be NR5R6, wherein R5 may be hydrogen or —(CO)(CF3); R6 may be (CH2)j(CH2)(CO)-R7; and R7 may be —OH;
29 2024P00697WO . Said halo may be
Cl or Br and b, j, t, and u each may be 1 to 25 or 1 to 10. For example, R1 may be C1-C2 alkyl, such as methyl, or —(CH2)b(NH)(CO)(CH2)S—Y1; R2 may be Cl; b, j, t, and u each may be 1 to 5; and each of Y1, Y7, and Y8 may be keyhole limpet hemocyanin (KLH), bovine serum albumin 5 (BSA), bovine thyroglobulin (BTG), egg ovalbumin (OVA), bovine gamma globulin (BGG), or glucose-6-phosphate dehydrogenase (G6PDH). [00103] In various aspects, a conjugate may correspond in structure to: Y7, 10 ine
thyroglobulin (BTG), egg ovalbumin (OVA), bovine gamma globulin (BGG), and glucose-6- phosphate dehydrogenase (G6PDH). [00104] In a further embodiment, R1 may be C1-C6 alkyl, C1-C4 alkyl or C1-C2 alkyl; R2 may be halo; and R3 may be NR5R6, wherein R5 may be hydrogen; R6 may be (CH2)j(CH2)(CO)- 15 R7; and R7 may be —(NH)—Y9. Said halo may be Cl or Br and j may be 1 to 25 or 1 to 10. For example, R1 may be a C1-C2 alkyl, such as methyl; R2 may be Cl; j may be 1 to 5; and Y9 may be keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), bovine thyroglobulin (BTG), egg ovalbumin (OVA), bovine gamma globulin (BGG), or glucose-6-phosphate dehydrogenase (G6PDH). 20 [00105] In various aspects, a conjugate may correspond in structure to:
30 2024P00697WO ,
wherein n may be 1, 3, or 5, and Y9 may be keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), bovine thyroglobulin (BTG), egg ovalbumin (OVA), bovine gamma globulin (BGG), or glucose-6-phosphate dehydrogenase (G6PDH). 5 [00106] In a further embodiment, R1 may be C1-C6 alkyl, C1-C4 alkyl or C1-C2 alkyl; R2 may be halo, —(C2H2)(CH2)e(NH)(CO)(CH2)S—Y2, or —S(CH2)fNHCO(CH2)S—Y3; and R3 may be NR5R6, wherein R5 may be hydrogen or —(CH2)h(NH)(CO)(CH2)S—Y4; R6 may be (CH2)j(CH2)(CO)-R7; and R7 may be —OH. Said halo may be Cl or Br and e, f, h, and j each may be 1 to 25 or 1 to 10. For example, R1 may be a C1-C2 alkyl, such as methyl; R2 may be Cl, 10 —(C2H2)(CH2)e(NH)(CO)(CH2)S—Y2, or —S(CH2)fNHCO(CH2)S—Y3; R5 may be hydrogen or —(CH2)h(NH)(CO)(CH2)S—Y4; R7 may be —OH; e, f, h, and j each may be 1 to 5; and each of Y2, Y3, and Y4 may be keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), bovine thyroglobulin (BTG), egg ovalbumin (OVA), bovine gamma globulin (BGG), and glucose-6-phosphate dehydrogenase (G6PDH). 15 [00107] In various aspects, a conjugate may correspond in structure to:
31 2024P00697WO wherein n may be 1, 3, or 5, and each of Y2, Y3, and Y4 may be keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), bovine thyroglobulin (BTG), egg ovalbumin (OVA), bovine gamma globulin (BGG), or glucose-6-phosphate dehydrogenase (G6PDH). [00108] In a further embodiment, provided herein are conjugates encompassed by Formula 5 (I), which correspond in structure to a Formula (Ic): wherein:
R1 is hydrogen or an alkyl; 10 R2 is hydrogen or halo; R3 is NR5R6, wherein R5 is hydrogen, R6 is —(CH2)j(CH2)(CO)-R7, –(CH2)nS(CH2)o(CO)(NH)—Y5, 15 –(CO)(CH2)p(CO)(NH)-Y6, or —(CH2)q(NH)(CO)-R8; j, n, o, p, and q are each zero to 50; R7 is —(NH)—Y9; and R8 is —(CH2)v(S)(CH2)w(CO)(NH)—Y10 or —(CH2)xS—Y11; v, w, and x are each zero to 50; and 20 each of Y5, Y6, Y9, Y10, and Y11 is an immunogenic carrier or a label. [00109] In any embodiment, R1 may be C1-C6 alkyl, C1-C4 alkyl, C1-C2 alkyl, or methyl. [00110] In any embodiment, R2 may be hydrogen or halo. Each halo may be Cl or Br. For example, R2 may be Cl. [00111] In any embodiment, R1 may be C1-C6 alkyl, C1-C4 alkyl, or C1-C2 alkyl, R2 may 25 be halo, R5 is hydrogen and R6 may be –(CH2)nS(CH2)o(CO)(NH)—Y5. Each halo may be Cl or Br, n and o each may be 1 to 25, 1 to 10, or 1 to 5, and the immunogenic carrier may be a protein, a polypeptide, and a polysaccharide. For example, R1 may be C1-C2 alkyl, such as methyl, R2 may be Cl, R6 may be –(CH2)nS(CH2)o(CO)(NH)—Y5, n and o each may be 1 to 5, such as 1, 2, or 3, and Y5 may be keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), bovine30 thyroglobulin (BTG), egg ovalbumin (OVA), bovine gamma globulin (BGG), or glucose-6- phosphate dehydrogenase (G6PDH).
32 2024P00697WO [00112] In various aspects, a conjugate may correspond in structure to: , where Y5 may be keyhole limpet hemocyanin (KLH), bovine serum
albumin (BSA), bovine thyroglobulin (BTG), egg ovalbumin (OVA), bovine gamma globulin (BGG), or glucose-6-phosphate dehydrogenase (G6PDH). 5 [00113] In any embodiment, R1 may be C1-C6 alkyl, C1-C4 alkyl, or C1-C2 alkyl, R2 may be halo, R5 is hydrogen and R6 may be –(CO)(CH2)p(CO)(NH)-Y6. Each halo may be Cl or Br, p may be 1 to 25, 1 to 10, or 1 to 5, and the immunogenic carrier may be a protein, a polypeptide, and a polysaccharide. For example, R1 may be C1-C2 alkyl, such as methyl, R2 may be Cl, R6 may be –(CO)(CH2)p(CO)(NH)-Y6, p may be 1 to 5, such as 2, 3, or 4, and Y6 may be keyhole 10 limpet hemocyanin (KLH), bovine serum albumin (BSA), bovine thyroglobulin (BTG), egg ovalbumin (OVA), bovine gamma globulin (BGG), or glucose-6-phosphate dehydrogenase (G6PDH). [00114] In various aspects, a conjugate may correspond in structure to: , where p may be 2, 3, 4, or 5, Y6 may be keyhole limpet 15 rum albumin (BSA), bovine thyroglobulin (BTG), egg ovalbumin
(OVA), bovine gamma globulin (BGG), or glucose-6-phosphate dehydrogenase (G6PDH). [00115] In any embodiment, R1 may be C1-C6 alkyl, C1-C4 alkyl, or C1-C2 alkyl, R2 may be halo, R5 is hydrogen, R6 may be —(CH2)q(NH)(CO)-R8, and R8 may be —(CH2)v(S)(CH2)w(CO)(NH)—Y10 or —(CH2)xS—Y11. Each halo may be Cl or Br, q, v, w, 20 and x each may be 1 to 25, 1 to 10, or 1 to 5, and the immunogenic carrier may be a protein, a polypeptide, and a polysaccharide. For example, R1 may be C1-C2 alkyl, such as methyl, R2 may be Cl, R6 may be —(CH2)q(NH)(CO)-R8, R8 may be —(CH2)v(S)(CH2)w(CO)(NH)—Y10, q, v, and w may be 1 to 5, such as 1, 2, or 3, and Y10 may be keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), bovine thyroglobulin (BTG), egg ovalbumin (OVA), bovine
33 2024P00697WO gamma globulin (BGG), or glucose-6-phosphate dehydrogenase (G6PDH). Alternatively, R1 may be C1-C2 alkyl, such as methyl, R2 may be Cl, R6 may be —(CH2)q(NH)(CO)-R8, R8 may be —(CH2)xS—Y11, q and x each may be 1 to 5, such as 1, 2, or 3, and Y11 may be keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), bovine thyroglobulin (BTG), egg ovalbumin 5 (OVA), bovine gamma globulin (BGG), or glucose-6-phosphate dehydrogenase (G6PDH) [00116] In various aspects, a conjugate may correspond in structure to: Y11
ine thyroglobulin (BTG), egg ovalbumin (OVA), bovine gamma globulin (BGG), or glucose-6- 10 phosphate dehydrogenase (G6PDH). [00117] In any embodiment, R1 may be C1-C6 alkyl, C1-C4 alkyl, or C1-C2 alkyl, R2 may be halo, R5 is hydrogen, R6 may be —(CH2)j(CH2)(CO)-R7, and R7 may be —(NH)—Y9. Each halo may be Cl or Br, j may be 1 to 25, 1 to 10, or 1 to 5, and the immunogenic carrier may be a protein, a polypeptide, and a polysaccharide. For example, R1 may be C1-C2 alkyl, such as 15 methyl, R2 may be Cl, R6 may be —(CH2)j(CH2)(CO)-R7, R7 may be —(NH)—Y9, j may be 1 to 5, such as 2, 3, 4, or 5, and Y9 may be keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), bovine thyroglobulin (BTG), egg ovalbumin (OVA), bovine gamma globulin (BGG), or glucose-6-phosphate dehydrogenase (G6PDH). [00118] In various aspects, a conjugate may correspond in structure to: 20 , where j may be 2 or 5, Y9 may be keyhole limpet m albumin (BSA), bovine thyroglobulin (BTG), egg ovalbumin
, (BGG), or glucose-6-phosphate dehydrogenase (G6PDH). [00119] In any embodiment, a conjugate may correspond in structure to:
34 2024P00697WO , wherei
ole limpet hemocyanin (KLH), bovine serum albumin (BSA), bovine thyroglobulin (BTG), egg 5 ovalbumin (OVA), bovine gamma globulin (BGG), or glucose-6-phosphate dehydrogenase (G6PDH). [00120] Nonlimiting examples of conjugates corresponding in structure to Formulas (I), (Ib), and (Ic) are shown below in Table 3. Table 3
35 2024P00697WO ne
5, j = 2 or 5, and p = 2, 3, 4, or 5. 5
36 2024P00697WO C. Synthesis [00121] The syntheses of representative examples of the above compounds (e.g., tianeptine analogs, haptens, activated haptens, immunogens, and conjugates) are discussed herein by way of illustration and not limitation. Other synthetic procedures will be suggested to those 5 skilled in the art in view of the disclosure herein. Other compounds within the scope of the present invention may be prepared using suitable variants of the reagents employed below. The reaction temperatures and time are those customary for the type of reactions conducted and should be evident to those skilled in the art. [00122] For immunogen and conjugate syntheses, a protein may be attached to a linking 10 group by means of an amine group or a thiol group on the protein. The formulas and compounds described herein may show the nitrogen atom of the amine group of the protein or the sulfur atom of the thiol group of the protein. In general, functional groups suitable for attaching a compound as described herein (e.g., hapten, activated hapten) to an immunogenic carrier (e.g., protein, enzyme) or label are usually an activated ester or alkylating agent when the amino acid(s) that 15 are to be conjugated on the enzyme have amino or hydroxyl groups and are usually alkylating agents or the like when the amino acid(s) that are to be conjugated on the immunogenic carrier comprise a sulfur atom such as, e.g., a cysteine. A large number of suitable functional groups are available for attaching to amino groups and alcohols such as activated esters including imidic esters, sulfonic esters and phosphate esters, activated nitrites, aldehydes, ketones, alkylating 20 agents and the like. Conjugation of haptens to proteins using these and other attaching groups are well known in the art and are described in reviews such as for example, Maggio, E. T. “Enzyme- Immunoassay” (CRC Press, Boca Raton, Fla., 1980), Chapter 4, which contains an assortment of conjugation techniques; pages 81–88 of which are incorporated herein by reference. [00123] Following reaction of the immunogenic carrier (e.g., protein, enzyme) with a 25 compound such as discussed above to form a conjugate, the product is then optionally purified as may be required. The purification and characterization of poly(amino acid)-hapten conjugates has been described in detail by Maggio, et al.; “Enzyme-immunoassay” (CRC Press, Boca Raton, Fla., 1980), Chapter 4, pages 86–88 of which are incorporated herein by reference. For example, the protein-hapten conjugate can be purified, for example, by dialysis against aqueous/organic 30 and aqueous solutions or by gel filtration chromatography on a support such as Sephadex®, and the like. [00124] As mentioned above, the conjugation can involve binding of a hapten to a free thiol group present on an amino acid side chain of the enzyme (e.g. cysteine). Such conjugation involves alkylation of the thiol sulfur atom by treatment with an electrophilic compound such as
37 2024P00697WO an alpha- or beta-unsaturated amide, ketone, ester, or the like, or an alkylating agent such as a reactive halide, e.g., bromide, or sulfonate or the like or reaction with an active disulfide such as a 2-nitro-4-carboxyphenyl disulfide. Specific examples by way of illustration and not limitation include alpha-bromoamides, maleimides, vinyl sulfones, alpha-iodoketones, and the like. 5 [00125] Conjugation reactions with proteins or enzymes can be affected by a number of factors. These include, but are not confined to, pH, temperature, buffer, ionic strength, substances which may protect the enzyme active site, amount and type of cosolvent, reaction time, and activation chemistry. A range of pH values from about 5.0 to about 9.5 can usually be used for conjugation reactions. These reactions are generally carried out at about 0ºC to about 40ºC, 10 preferably about 4ºC to about 20ºC. [00126] A number of buffers and salts, both alone and in combination, can be used for such reactions. These include Tris, bicarbonate, phosphate, pyrophosphate, ethylenediaminetetraacetic acid (EDTA), KCl, NaCl, and many others. The active site may be protected by substrates (i.e., glucose-6-phosphate and compounds that react reversibly with 15 lysine (i.e., pyridoxal) to reduce deactivation of the enzyme during conjugation. [00127] Co-solvents which may enhance hapten solubility include, but are not limited to, dimethylformamide, carbitol, dimethyl sulfoxide, 1-methyl-2-pyrrolidinone, and 1,3-dimethyl- 3,4,5,6-tetrahydro 2(1H)-pyrimidinone. These may be useful as about 1 to about 30% of the reaction volume. Reactions can vary from about 15 min to many days, depending on the20 activation chemistry. Carboxylic compounds may be activated to form esters with N- hydroxysuccinimide or its sulfo-analog, or to mixed anhydrides through reaction with carbitol chloroformate or t-butylchloroformate, or may be coupled directly using carbodiimides such as EDC. For reaction with cysteine thiols on the enzyme, the hapten should contain a good leaving group such as I, Br, or tosyl; alternatively, the hapten can contain a thiol, preferably activated 25 with 2,2′ dithiodipyridine, 5,5′dithiobis(2-nitrobenzoic acid) (DTNB), dithioerythritol (DTE), and the like. [00128] Another method of conjugation, described in Rowley, G. L., D. Leung, and P. Singh (U.S. Pat. No.4,220,722) involves modification of the immunogenic carrier (e.g., protein, enzyme) with bromoacetyl containing reactants; the bromo groups are subsequently reacted with 30 thiol-containing haptens. The reaction of the immunogenic carrier (e.g., protein, enzyme) with bromoacetyl modifier, and the bromoacetyl enzyme with the thiolated hapten, are subject to the same reaction condition variables described above. [00129] Referring to FIG. 1, the synthesis of compounds (XV), (XVI), and (XVII) may begin with reacting commercially available starting material 1 with compound 4 and a metal
38 2024P00697WO catalyst, such as a palladium complex (e.g., palladium-tetrakis(triphenylphosphine) (Pd(PPh3)4) or a nickel complex (e.g., dichloro[1,3-bis(diphenylphosphino)propane]nickel (NiCl2(dpp)) suitable for Suzuki coupling) to yield compound 5. Compound 5 may be combined with compound 6 and sodium cyanoborohydride (NaBH3CN) in an alcohol (e.g., MeOH) to yield 5 compound 7 (n = 1, 3, or 5). Compound 7 may be added to a suitable acid (e.g., trifluoroacetic acid (TFA)) and reacted with compound 8 to yield compounds (XV), (XVI) or (XVII). Conjugates and immunogens of compounds (XV), (XVI) or (XVII) can be produced by combining each of compounds (XV), (XVI) or (XVII) with a suitable buffer and a protein solution to yield conjugate (C) where Y2 is as described herein. Such buffer solutions include, 10 for example, phosphate buffer, e.g., a dihydrogen phosphate, a hydrogen phosphate, etc., and combinations thereof. Any suitable protein as described herein may be used (e.g., KLH, BSA, OVA, G6PDH, etc.). The resulting conjugates or immunogens may be purified by appropriate purification techniques such as, for example, dialysis or column chromatography, e.g., Sephadex®, etc., and the like using a suitable eluent, e.g., phosphate buffer, etc. 15 [00130] Referring to FIG. 2, the synthesis of compounds (XII), (XIII), and (XIV) may begin with reacting commercially available starting material 9 (n = 1, 3, or 5) with compound 10 followed by deprotection, e.g., using an acid (e.g., trifluoroacetic acid (TFA) and a solvent (e.g., dichloromethane (DCM)), and then reaction with compound 11 to yield compounds (XII), (XIII), and (XIV). Conjugates and immunogens of compounds (XII), (XIII), and (XIV) can be produced 20 by combining each of compounds (XII), (XIII), and (XIV) with a suitable buffer and a protein solution to yield conjugate (D) where Y4 is as described herein. Such buffer solutions include, for example, phosphate buffer, e.g., a dihydrogen phosphate, a hydrogen phosphate, etc., and combinations thereof. Any suitable protein as described herein may be used (e.g., KLH, BSA, OVA, G6PDH, etc.). The resulting conjugates or immunogens may be purified by appropriate 25 purification techniques such as, for example, dialysis or column chromatography, e.g., Sephadex®, etc., and the like using a suitable eluent, e.g., phosphate buffer, etc. [00131] Still referring to FIG. 2, the synthesis of compounds (VI), (VII), and (VIII) may begin with combining commercially available starting material 9 (n = 1, 3, or 5) with EDAC and NHS in N,N-dimethylformamide (DMF) followed by reaction with compound 12 to yield 30 compounds (VI), (VII), and (VIII). Conjugates and immunogens of compounds (VI), (VII), and (VIII) can be produced by combining each of compounds (VI), (VII), and (VIII) with a suitable buffer and a protein solution to yield conjugate (E) where Y7 is as described herein. Such buffer solutions include, for example, phosphate buffer, e.g., a dihydrogen phosphate, a hydrogen phosphate, etc., and combinations thereof. Any suitable protein as described herein may be used
39 2024P00697WO (e.g., KLH, BSA, OVA, G6PDH, etc.). The resulting conjugates or immunogens may be purified by appropriate purification techniques such as, for example, dialysis or column chromatography, e.g., Sephadex®, etc., and the like using a suitable eluent, e.g., phosphate buffer, etc. [00132] Still referring to FIG.2, the synthesis of compounds (III), (IV), and (V) may begin 5 with combining commercially available starting material 9 (n = 1, 3, or 5) with EDAC and NHS in DMF followed by reaction with compound 13 to yield compounds (III), (IV), and (V). Conjugates and immunogens of compounds (III), (IV), and (V) can be produced by combining each of compounds (III), (IV), and (V) with a suitable buffer and a protein solution to yield conjugate (F) where Y8 is as described herein. Such buffer solutions include, for example, 10 phosphate buffer, e.g., a dihydrogen phosphate, a hydrogen phosphate, etc., and combinations thereof. Any suitable protein as described herein may be used (e.g., KLH, BSA, OVA, G6PDH, etc.). The resulting conjugates or immunogens may be purified by appropriate purification techniques such as, for example, dialysis or column chromatography, e.g., Sephadex®, etc., and the like using a suitable eluent, e.g., phosphate buffer, etc. 15 [00133] Still referring to FIG. 2, synthesis of conjugate (G) may include combining commercially available starting material 9 (n = 1, 3, or 5) with a suitable buffer and a protein solution to yield conjugate (G) where Y9 is as described herein. Such buffer solutions include, for example, phosphate buffer, e.g., a dihydrogen phosphate, a hydrogen phosphate, etc., and combinations thereof. Any suitable protein as described herein may be used (e.g., KLH, BSA, 20 OVA, G6PDH, etc.). The resulting conjugates or immunogens may be purified by appropriate purification techniques such as, for example, dialysis or column chromatography, e.g., Sephadex®, etc., and the like using a suitable eluent, e.g., phosphate buffer, etc. [00134] Referring to FIG. 3, the synthesis of compounds (IX), (X), and (XI) may begin with combining commercially available starting material 9 (n=1, 3, or 5) with pyridine and a 25 suitable acid (e.g., HCl) at a suitable temperature followed by reacting with trifluoroacetic acid anhydride (TFAA), a solvent (e.g., dichloromethane (DCM)), and triethylamine (NEt3) to yield compound 14 (n=1, 3, or 5). Compound 14 may be reacted with compound 15 in a solvent (e.g., DMF) and K2CO3 solution and then combined with an acid (e.g., trifluoroacetic acid (TFA)) and a solvent (e.g., DCM) to yield compound 16 (n = 1, 3, or 5). Compound 16 may be reacted with 30 compound 11 and a solvent (e.g., DMF) and NEt3 to yield compounds (IX), (X), and (XI). Conjugates and immunogens of compounds (IX), (X), and (XI) can be produced by combining each of compounds (IX), (X), and (XI) with a suitable buffer and a protein solution to yield conjugate (H) where Y1 is as described herein. Conjugate (H) may be further reacted with a suitable basic buffer (e.g. at pH 8) to yield conjugate (I) where Y1 is as described herein. Such
40 2024P00697WO buffer solutions include, for example, phosphate buffer, e.g., a dihydrogen phosphate, a hydrogen phosphate, etc., and combinations thereof. Any suitable protein as described herein may be used (e.g., KLH, BSA, OVA, G6PDH, etc.). The resulting conjugates or immunogens may be purified by appropriate purification techniques such as, for example, dialysis or column chromatography, 5 e.g., Sephadex®, etc., and the like using a suitable eluent, e.g., phosphate buffer, etc. [00135] Referring to FIG. 4, the synthesis of compound (XVIII) may include reacting commercially available starting material 21 with dihydro-2H-pyran-2,6(3H)-dione and pyridine in a solvent (e.g., DMF) to yield compound (XVIII). [00136] Referring to FIG. 5, the synthesis of compound (XIX) may include reacting10 commercially available starting material 22 with N-(3-dimethylaminopropyl)-N’- ethylcarbodiimide hydrochloride (EDC∙HCl) and NHS in a solvent (e.g., DMF) at a suitable temperature and for a suitable amount of time to yield compound (XIX). [00137] Referring to FIG. 6, the synthesis of compound (XX) may begin with reacting commercially available starting material 23 with compound 24 in a solvent (e.g., DMF) and 15 K2CO3 solution at a suitable temperature to yield compound 25. Compound 25 may be combined with an acid (e.g., trifluoroacetic acid (TFA)) and a solvent (e.g., DCM) and then reacted with compound 26, NEt3, and an ether (e.g., tetrahydrofuran (THF)) solution to yield compound (XX). [00138] Referring to FIG. 7, conjugates and immunogens of tianeptine sodium salt (TNPT) (22) can be produced by suspending commercially available tianeptine sodium salt20 (TNPT) (22) in a solvent (e.g., DMF) and adding N-hydroxysuccinimide (NHS) and N-(3- dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC∙HCl) to give an NHS intermediate. Each protein (OVA, KLH) can be suspended in a buffer (e.g., phosphate buffer, tris buffer, NaCl) and then a solution of the NHS intermediate in a solvent (e.g., DMF) may be added to each protein solution to produce the desired conjugates or immunogens (TNPT-OVA 25 (28), TNPT-BSA (29), TNPT-KLH (30)). The resulting conjugates or immunogens may be purified by appropriate purification techniques such as, for example, dialysis or column chromatography, e.g., Sephadex®, etc., and the like using a suitable eluent, e.g., phosphate buffer, etc. [00139] Referring to FIG. 8, to prepare a TNPT002 hapten (compounds (XXI) and/or30 (XXII)), commercially available tianeptine-Cl (31) may be suspended in nitromethane, then 2- (methyldisulfanyl)ethan-1-amine hydrochloride (cystamineSMe*HCl) may be added followed by triethylamine (NEt3). The resulting reaction mixture may be heated at a suitable temperature (e.g., about 60 °C) for a suitable amount of time (e.g., 4 hours) and this reaction may be run once or twice (e.g., using the same amounts) and purified once. The volatiles may be removed (e.g.,
41 2024P00697WO on a rotatory evaporator) to give a yellow oil, which may be suspended in ethyl acetate (EtOAc) and washed with deionized water. The organic phase may be concentrated on a rotatory evaporator to give a yellow oil, which can be further dissolved in acetonitrile and purified via liquid chromatography (LC) and lyophilized to produce two fractions, one fraction contained 5 TNPT002 hapten as disulfide dimer (compound (XXII)) and the second fraction containing a mixture of 1:1 Tianeptine-disulfide (compound (XXII)): tianeptine-C2-SSMe (compound (XXI)) (TNPT002 hapten). Both fractions (dimer and/or mixture) can be used to prepare conjugates and immunogens. [00140] Referring to FIG. 9, TNPT002 hapten (compound XXII) may be activated by10 suspending the TNPT002 hapten (compound XXII) in a solvent (e.g., EtOH) and adding tris(2- carboxyethyl)phosphine (TCEP) to give activated hapten TNPT-CysSH (32). The amine group of each protein such as, for example, OVA, BSA, KLH, and the like may be treated with succinimidylbromo-acetate (BrAcSu) to introduce the bromo-acetamide functional group for thiol modification giving activated protein BrAc-OVA (36), activated protein BrAc-BSA (37), 15 and activated protein BrAc-KLH (38), and activated BrAc-G6PDH enzyme (43). Reaction of activated hapten TNPT-CysSH (32) with each activated protein BrAc-OVA (36), activated protein BrAc-BSA (37), and activated protein BrAc-KLH (38), and activated BrAc-G6PDH enzyme (43) gives the desired conjugates and immunogens (e.g., TNPT002-OVA conjugate (39), TNPT002-BSA conjugate (40), TNPT002-KLH immunogen (41), TNPT002-G6PDH-5x 20 conjugate (44)a, TNPT002-G6PDH-10x conjugate (44b), TNPT002-G6PDH-15x conjugate (44c), TNPT002-G6PDH-20x conjugate (44d), TNPT002-G6PDH-25x conjugate (44e)). Reaction conditions include, for example, a buffer solution at pH of about 7 to 9, about 7.5 to 8.5, about 8. Such buffer solutions include, for example, phosphate or borate buffer etc., and combinations thereof. The resulting immunogens and conjugates may be purified by appropriate 25 purification techniques such as, for example, dialysis or column chromatography, e.g., Sephadex®, etc., and the like using a suitable eluent, e.g., phosphate buffer, etc. The attachment of a protein to the linking moiety of the molecule can be through the amino group on a protein, where the nitrogen of the amino group may be the nitrogen of the linking group depicted above. [00141] Referring to FIG. 10, to prepare TNPT-NH-Glu-COOH haptens (compounds 30 XXIII), (XXIV), (XXV), and/or (XXVI)), a mixture of commercially available tianeptine-Cl (31) and ammonia may be heated to a suitable temperature (e.g. 70-80°C) for a suitable amount of time (e.g., 8-12 hours). The mixture solution then may be concentrated, e.g., on a rotavap under vacuum, to yield TNPT-NH2 (45) as a solid. TNPT-NH2 (45) solid may be dissolved in a solvent (e.g., DCM) and glutaric anhydride and pyridine may be added to the solution. The reaction
42 2024P00697WO mixture may be concentrated, e.g., on a rotavap under vacuum, to yield a crude product, which may be purified (e.g., via HPLC) to yield the desired product TPPT-NH-GLU-COOH (compound (XXIV)). As shown in FIG. 10, further haptens, compounds (XXIII), (XXV), and (XXVI), can be synthesized in a similar manner as shown in Scheme 10 depending on the shown variations in 5 the reactant compound (n = 1, 2, 3, or 4) as understood by a one of ordinary skill in the art. [00142] Still referring to FIG.10, TNPT-NH-Glu-COOH hapten (compound (XXIV)), N- hydroxysuccinimide (NHS), and EDAC may be dissolved in solvent (e.g., DMF) to produce desired TNPT-NH-Glu-NHS ester (47). NHS ester (47) in DMF may be added to protein KLH suspended in a buffer (e.g., phosphate or tris buffer). The resulting KLH immunogen and hapten 10 mixture may be purified by a Sephadex G-25 (medium) column using PBS buffer to produce TNPT-NH-Glu-KLH immunogen (51). As shown in FIG. 10, further activated haptens, compounds (46), (48), and (49), and further KLH immunogens, compounds (50), (52), and (53) can be synthesized in a similar manner as shown in Scheme 10 depending on the shown variations in the hapten compounds used as understood by a one of ordinary skill in the art. 15 [00143] Referring to FIG. 11, to prepare a TNPT-NHC2AcBr hapten (compound (XXVII)), commercially available tianeptine-Cl (31) may be dissolved in a solvent (e.g., EtOH) and ethylenediamine may be added. The resulting reaction mixture may be heated at suitable temperature (e.g., to 70 °C) for a suitable amount of time (e.g., for 6 hours). The volatiles may be removed, e.g., on a rotatory evaporator, to give a yellow oil. The oil may be dissolved in water 20 then Na2CO3 solution may be added, and the product may be extracted with EtOAc. The organic layer may be washed (e.g., 3 times) with deionized water to give TNPT-NHC2NH2 hapten (60). The hapten (60) may be purified via liquid chromatography. TNPT-NHC2NH2 hapten (60) as a TFA salt may be dissolved in a solvent (e.g., DMF) then NEt3 (40 µL) may be added followed by BrAcSu (e.g., dissolved in THF). The resulting reaction mixture may be purified via liquid 25 chromatography to yield TNPT-NHC2AcBr hapten (Compound (XXVII)). [00144] Referring to FIG. 12, to prepare an TNPT-C6-3K G6PDH conjugate (62) with TNPT-NHC2AcBr hapten (compound (XXVII)), a 3K G6PDH enzyme may be buffer exchanged with a buffer (e.g., phosphate or tris buffer). The 3K G6PDH enzyme (61) may be reduced by adding TCEP*HCl as TCEP-H2O solution. The TNPT-NHC2AcBr hapten (compound (XXVII)) 30 may be added to the enzyme to produce TNPT-C6-3K G6PDH conjugate (62). The resulting conjugate 62) may be purified by appropriate purification techniques such as, for example, dialysis or column chromatography, e.g., Sephadex®, etc., and the like using a suitable eluent, e.g., phosphate buffer, etc.
43 2024P00697WO [00145] Referring to FIG. 13, the amine group of a protein such as, for example, OVA, BSA, KLH, and the like may be treated with N-succinimidyl S-acetylthioacetate (SATA) in a solvent (e.g., DMF) to give activated SATA-OVA protein (66), activated SATA-BSA protein (67), and activated SATA-KLH protein (68). Reaction of TNPT-NHC2AcBr hapten (compound 5 (XXVII)) in a solvent (e.g., DMF) with each activated SATA-OVA protein (66), activated SATA-BSA protein (67), and activated SATA-KLH protein (68) gives the desired conjugates and immunogens (e.g., TNPT-C2Ac-SATA-OVA conjugate (69), TNPT-C2Ac-SATA-BSA conjugate (70), TNPT-C2Ac-SATA-KLH immunogen (71). Reaction conditions include, for example, a buffer solution at pH of about 7 to 9, about 7.5 to 8.5, about 8. Such buffer solutions 10 include, for example, phosphate or borate buffer etc., and combinations thereof. The resulting immunogens and conjugates may be purified by appropriate purification techniques such as, for example, dialysis or column chromatography, e.g., Sephadex®, etc., and the like using a suitable eluent, e.g., phosphate buffer, etc. [00146] Referring to FIG. 14, G6PDH enzyme may be buffer exchanged with a buffer 15 (e.g., PBS) and can be further diluted with the buffer. Glucose-6-phosphate di-sodium salt (G6PDNa2) and nicotinamide adenine dinucleotide (β-NADH) may be added to the enzyme solution. SATA (e.g., in DMF) then can be added to the enzyme solution. The resulting reaction mixtures may be further buffer exchanged to produce thioacetyl-G6PDH enzyme (73). Reaction of TNPT-NHC2AcBr hapten (compound (XXVII)) in a solvent (e.g., DMF) with the thioacetyl- 20 G6PDH enzyme (73) produces the desired conjugates (e.g., TNPT-C2Ac-SATA-G6PDH 5x (74a), TNPT-C2Ac-SATA-G6PDH 10x (74b), TNPT-C2Ac-SATA-G6PDH 15x (74c), TNPT- C2Ac-SATA-G6PDH 20x (74d), TNPT-C2Ac-SATA-G6PDH 25x (74e)). The resulting conjugates may be purified by appropriate purification techniques such as, for example, dialysis or column chromatography, e.g., Sephadex®, etc., and the like using a suitable eluent, e.g., 25 phosphate buffer, etc. [00147] Referring to FIG. 15, to prepare TNPT-NH-Hex-COOH haptens (compounds (XXVIII), (XXIX), and (XXX), a mixture of commercially available TNPT-Cl (31), methyl 6- aminohexanoate hydrochloride (75), a polymer-bound morpholine resin (e.g., PS-morpholine) and THF may be formed and heated (e.g., at 70-80 °C). The resin may then be removed and 30 washed with THF. The collected THF solution may be concentrated on a rotavap under vacuum to a white-yellow solid (76), and the solid (76) may be dissolved in THF-MeOH and treated with LiOH. The reaction mixture solution may undergo adjustment to pH 2-3 with an acid (e.g., 1 M HCl). The acidified mixture may then be treated with EtOAc and water. The EtOAc layer may be separated and washed with saturated brine solution, and the organic layer may be dried over
44 2024P00697WO anhydrous Na2SO4 then filtered. The collected EtOAc solution may concentrated on a rotavap under vacuum to give the crude TNPT-NH-Hex-COOH hapten products. The crude hapten product may be purified by HPLC to give the pure desired TNPT-NH-Hex-COOH hapten (compound (XXX)). As shown in FIG.15, compounds (XXVIII) and (XXIX) can be synthesized 5 in a similar manner as shown in Scheme 15 depending on the shown variations in compounds (75) and (76) as understood by a one of ordinary skill in the art. [00148] Still referring to FIG.15, TNPT-NH-Hex-COOH hapten (compound (XXX)), N- hydroxysuccinimide (NHS), and EDAC may be dissolved in solvent (e.g., DMF) to produce desired TNPT-NH-Hex-NHS ester (78). NHS ester (78) in DMF may be added to protein KLH 10 suspended in a buffer (e.g., phosphate or tris buffer). The resulting KLH immunogen and hapten mixture may be purified by a Sephadex G-25 (medium) column using PBS buffer to produce TNPT-NH-Hex-KLH immunogen (80). As shown in FIG. 15, further activated hapten compounds (77) and further KLH immunogen compound (79) can be synthesized in a similar manner as shown in Scheme 15 depending on the shown variations in the hapten compounds15 used as understood by a one of ordinary skill in the art. Additionally, BSA-contaning and OVA- containing immunogens of TNPT-NH-Hex-COOH haptens may be synthesized as shown in Scheme 15 in FIG.15 depending on the protein solution used as understoody by one of ordinary skill in the art. D. Antibodies 20 [00149] Antibodies raised against the compounds described above and useful in immunoassays for the determination of tianeptine are provided herein. [00150] In any embodiment, the antibodies may be raised against a complex of Formula (I) and Formula (Ia), wherein an immunogenic carrier is present (e.g., Y1, Y2, Y3, Y4, Y5, Y6, Y7, Y8, Y9, Y10, Y11, Y12, and/or Y13 may be KLH, BSA, BTG, OVA, BGG, or G6PDH). The 25 antibodies described herein may preferentially bind to tianeptine and/or a metabolite of tianeptine. The preferential binding of the antibody to tianeptine and/or a metabolite of tianeptine may be measured by any suitable means, including but not limited to a dissociation constant (KD) of the antibody for tianeptine and/or a metabolite of tianeptine. [00151] Additionally or alternatively, the preferential binding of an antibody may be 30 measured by an amount, e.g., a percentage, of tianeptine recovered from a sample, such as a sample from an immunoassay. In any example, an antibody may recover at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% of tianeptine.
45 2024P00697WO [00152] The immunoassay is not particularly limited, and may be an enzyme multiplied immunoassay technique (EMIT), an enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay (RIA), an enzyme channeling immunoassay (ECIA), a fluorescence polarization immunoassay (FPIA), an enzyme modulate mediated immunoassay (EMMIA), a 5 substrate labeled fluorescence immunoassay (SLFIA), a combined enzyme donor immunoassay (CEDIA), a particle enhanced turbidimetric inhibition immunoassay (PETINIA), a particle enhanced turbidimetric immunoassay (PETIA), a sol particle immunoassay (SPIA), a disperse dye immunoassay (DIA), a metalloimmunoassay (MIA), an enzyme membrane immunoassays (EMIA), or a luminoimmunoassays (LIA). In a specific embodiment, the immunoassay may be 10 an EMIT immunoassay. E. Nucleotide Sequences [00153] The antibodies described above can be prepared by conventional means known in the art. When monoclonal antibodies are desired, the amino acid sequence can be encoded by a nucleotide sequence (e.g., a DNA sequence) and provided to a cell (e.g., a hybridoma, bacteria, 15 yeast, etc.) which translates the nucleotide sequence to the antibody. The nucleotide sequences may be incorporated into a vector (e.g., a viral vector, a plasmid, etc.) for insertion into a cell (e.g., transformation, transfection, etc.) for subsequent production of the antibody. F. Kits [00154] Kits for determining the presence of tianeptine and/or a metabolite of tianeptine 20 in a sample, such as a biosample, are also provided herein. [00155] In any embodiment, the kit may include, for example, in packaged combination, an anti-tianeptine antibody described above and a conjugate of an enzyme and a tianeptine analog. For example, the conjugate may correspond in structure to a Formula (Ib): 25 wherein: “-----” represents a si le bond;
R1 is hydrogen, an alkyl, or —(CH2)b(NH)(CO)(CH2)S—Y1; b is zero to 50; R2 is hydrogen, halo, —(C2H2)(CH2)e(NH)(CO)(CH2)S—Y2, or 30 —S(CH2)fNHCO(CH2)S—Y3; e and f are each zero to 50; when “-----” represents a single bond, then R3 is NR5R6,
46 2024P00697WO wherein R5 is hydrogen, —(CO)(CF3), or —(CH2)h(NH)(CO)(CH2)S—Y4; h is zero to 50; R6 is —(CH2)j(CH2)(CO)-R7, –(CH2)nS(CH2)o(CO)(NH)—Y5, 5 –(CO)(CH2)p(CO)(NH)-Y6, or —(CH2)q(NH)(CO)-R8; j, n, o, p, and q are each zero to 50; R7 is —OH, —(NH)(CH2)t(NH)(CO)(CH2)S—Y7, , or —(NH)—Y9; t and u are each
zero o y; 10 R8 is —(CH2)v(S)(CH2)w(CO)(NH)—Y10 or —(CH2)xS—Y11; v, w, and x are each zero to 50; when “-----” represents a double bond, then R3 is NR4, wherein R4 is —O(CH2)x3(CO)(NH)—Y12 or —O(CH2)(CO)(NH)(CH2)x4(NH)(CO)(CH2)S—Y13; x3 and x4 are each zero to 15 50; and each of Y1, Y2, Y3, Y4, Y5, Y6, Y7, Y8, Y9, Y10, Y11, Y12, and Y13 is an enzyme. [00156] In any embodiment, the conjugate of (ii) is further defined wherein when R3 is NR5R6, R5 is hydrogen, R6 is (CH2)j(CH2)(CO)-R7, j is 1, 3, or 5, and R7 is —OH, then both R1 20 is not Cl and R2 is not methyl. For example, a compound corresponding in structure to Formula (Ib) may not be one or more of: compound (a), compound (b), and compound (c). [00157] Additionally or alternatively, in a kit as described herein, the conjugate may correspond in structure to a Formula (Ic): 25 wherein: R1 is hydrogen or an
47 2024P00697WO R2 is hydrogen or halo; R3 is NR5R6, wherein R5 is hydrogen, 5 R6 is —(CH2)j(CH2)(CO)-R7, –(CH2)nS(CH2)o(CO)(NH)—Y5, –(CO)(CH2)p(CO)(NH)-Y6, or —(CH2)q(NH)(CO)-R8; j, n, o, p, and q are each zero to 50; R7 is —(NH)—Y9; and R8 is —(CH2)v(S)(CH2)w(CO)(NH)—Y10 or —(CH2)xS—Y11; 10 v, w, and x are each zero to 50; and each of Y5, Y6, Y9, Y10, and Y11 is an enzyme, [00158] Examples of a suitable enzyme, when present, include, but are not limited to, glucose-6-phosphate dehydrogenase (G6PDH), an alkaline phosphatase, or horseradish peroxidase, or a functional isoform and/or analog thereof. For example, the G6PDH conjugated 15 to the conjugate may be a putative wild-type G6PDH or a mutant or recombinant form of G6PDH so long as the mutant or recombinant G6PDH can convert glucose-6-phosphate to 6-phospho-D- glucono-1,5-lactone. The functional isoform or analog of the alkaline phosphatase or horseradish peroxidase includes mutant or recombinant alkaline phosphatases or horseradish peroxidases that catalyze the same chemical reaction as wild-type or putative alkaline phosphatase (e.g., 20 converting p-nitrophenyl phosphate (PNP) to p-nitrophenol) or horseradish peroxidase (e.g., oxidation of 3,3’,5,5’-tetramethylbenzidine (TMB)). [00159] In any embodiment, the kit may comprise reagents sufficient for a single use assay or for a multiple use assay. For example, where a single use of the assay is warranted, the kit may comprise an individually packaged volume or amount of the antibody sufficient for a single run 25 of the assay and a volume or amount of the enzyme/tianeptine analog conjugate sufficient to detect the presence of tianeptine and/or a metabolite of tianeptine in the sample. Where multiples uses of the assay are warranted, the kit may comprise multiple, independently packaged volumes or amounts of the antibody and volumes or amounts of the enzyme/tianeptine analog conjugate. Alternatively, the components of the kit may be packaged in larger volumes of the antibody 30 and/or the conjugates where the artisan removes only a portion of said larger volume to run the assay to determine whether the sample contains tianeptine. The components of the kit may otherwise be packaged at a relatively higher concentration of the antibody and/or the conjugates where the artisan removes only a portion of said concentrated volume and dilutes said volume prior to performing the assay to determine whether the sample contains tianeptine. The antibody
48 2024P00697WO and/or conjugate may be provided in a powdered or lyophilized form for solubilization prior to use. The kit can further include a written description of a method in accordance with the present invention as described above. G. Methods and Assays 5 [00160] Complexes corresponding in structure to Formula (I), including where the compound comprises an enzyme, and antibodies raised against a complex corresponding in structure to Formulas (I) and (Ia) may be employed as reagents in all types of immunoassays to determine the amount (e.g., concentration) of tianeptine and/or a metabolite of tianeptine in samples. The reagents may also be employed in multi-analyte immunoassays wherein the 10 presence or absence of multiple analytes may be determined. [00161] Methods for determining the presence of tianeptine and/or a metabolite of tianeptine in a sample, such as a biosample, are provided herein. In any embodiment, the method for determining the presence of tianeptine and/or a metabolite of tianeptine in a sample includes combining the sample, a conjugate of an enzyme and tianeptine analog, and an anti-tianeptine 15 antibody described herein. The method further includes examining the medium for the presence of a complex comprising the anti-tianeptine antibody and tianeptine. In any embodiment of the method, the conjugate may correspond in structure to a Formula (Ib): 20 wherein: “-----” represents a si
le bond; R1 is hydrogen, an alkyl, or —(CH2)b(NH)(CO)(CH2)S—Y1; b is zero to 50; R2 is hydrogen, halo, —(C2H2)(CH2)e(NH)(CO)(CH2)S—Y2, or —S(CH2)fNHCO(CH2)S—Y3; e and f are each zero to 50; 25 when “-----” represents a single bond, then R3 is NR5R6, wherein R5 is hydrogen, —(CO)(CF3), or —(CH2)h(NH)(CO)(CH2)S—Y4; h is zero to 50; R6 is —(CH2)j(CH2)(CO)-R7, –(CH2)nS(CH2)o(CO)(NH)—Y5, 30 –(CO)(CH2)p(CO)(NH)-Y6, or —(CH2)q(NH)(CO)-R8; j, n, o, p, and q are each zero to 50;
49 2024P00697WO R7 is —OH, —(NH)(CH2)t(NH)(CO)(CH2)S—Y7, , or —(NH)—Y9; t and u are each
zero to fifty; R8 is —(CH2)v(S)(CH2)w(CO)(NH)—Y10 or —(CH2)xS—Y11; 5 v, w, and x are each zero to 50; when “-----” represents a double bond, then R3 is NR4, wherein R4 is —O(CH2)x3(CO)(NH)—Y12 or —O(CH2)(CO)(NH)(CH2)x4(NH)(CO)(CH2)S—Y13; x3 and x4 are each zero to 50; and 10 each of Y1, Y2, Y3, Y4, Y5, Y6, Y7, Y8, Y9, Y10, Y11, Y12, and Y13 is an enzyme. [00162] In some embodiments, the conjugate may be further defined wherein when R3 is NR5R6, R5 is hydrogen, R6 is (CH2)j(CH2)(CO)-R7, j is 1, 3, or 5, and R7 is —OH, then both R1 is not Cl and R2 is not methyl. For example, a compound corresponding in structure to Formula 15 (Ib) may not be one or more of: compound (a), compound (b), and compound (c). [00163] Additionally or alternatively, in a method as described herein, the conjugate may correspond in structure to a Formula (Ic): 20 wherein: R1 is hydrogen or an
R2 is hydrogen or halo; R3 is NR5R6, wherein 25 R5 is hydrogen, R6 is —(CH2)j(CH2)(CO)-R7, –(CH2)nS(CH2)o(CO)(NH)—Y5, –(CO)(CH2)p(CO)(NH)-Y6, or —(CH2)q(NH)(CO)-R8; i, n, o, p, and q are each zero to 50;
50 2024P00697WO R7 is —(NH)—Y9; and R8 is —(CH2)v(S)(CH2)w(CO)(NH)—Y10 or —(CH2)xS—Y11; v, w, and x are each zero to 50; and each of Y5, Y6, Y9, Y10, and Y11 is an enzyme 5 [00164] In any embodiment, the enzyme may be as described herein. For example, the enzyme may include, but is not limited to, glucose-6-phosphate dehydrogenase (G6PDH), an alkaline phosphatase, or horseradish peroxidase. [00165] Additionally or alternatively, the method may further include: incubating the sample, conjugate, and antibody for a time sufficient for the antibody to bind to the tianeptine 10 and/or metabolite of tianeptine in the sample; adding a substrate for the enzyme to the sample; and measuring the activity of the enzyme. The substrate for the enzyme may be G6PDH substrate (e.g., D-glucose 6-phosphate), an alkaline phosphatase substrate (e.g., p-nitrophenol (PNP)), or a horseradish peroxidase substrate (e.g., 3,3’,5,5’-tetramethylbenzidine (TMB)). In such a method, the presence of a complex comprising tianeptine and the anti-tianeptine antibody is 15 proportional to the activity of the enzyme. [00166] The sample tested in the method is not particularly limited. It may be organic or inorganic, biological (e.g., a “biosample”), non-biological, or environmental. Examples of a biological or biosample include, but are not limited to, urine, whole blood, plasma, serum, lymph, mucus, expressed breast milk, semen, stool, sputum, cerebral spinal fluid, tears, hair, saliva, cells, 20 tissues, an organ, and/or a biopsy. In particular, the sample may be urine, blood, plasma, mucus, or saliva. [00167] The assays described above may use various buffers to achieve and maintain a desired pH. The buffer is not particularly limited and may be borate, phosphate, carbonate, tris, barbital, and the like. Additional components, such as stabilizers for the medium, additional 25 proteins (e.g., albumins to block non-specific and/or off-target antibody binding), organic solvents (e.g., formamide), quaternary ammonium salts, polyanions, surfactants, and binding enhances may be used as necessary. Incubation times and temperatures (e.g., for antibody binding) are not particularly limited and may be adjusted as necessary. Incubation temperatures may be about 5°C to about 99°C, such as about 5°C, about 10°C, about 15°C, about 20°C, about 30 25°C, about 30°C, about 35°C, about 40°C, about 45°C, about 50°C, about 55°C, about 60°C, about 65°C, about 70°C, about 75°C, about 80°C, about 85°C, about 90°C, about 95°C, or about 99°C. Incubation times may be about 0.2 sec to about 6 h or overnight, for example about 5 min, about 10 min, about 15 min, about 30 min, about 45 min, about 1 h, about 1.5 h, about 2 h, about
51 2024P00697WO 3 h, about 4 h, about 5 h, or about 6 h. The specific time and temperature of the incubation may depend on the reagents used. [00168] The concentration of analyte that may be assayed generally varies from about 10−5 to about 10−9 M, more usually from about 10−6 to about 10−8 M. Considerations, such as 5 whether the assay is qualitative, semi-quantitative or quantitative (relative to the amount of analyte present in the sample), the particular detection technique and the concentration of the analyte will normally determine the concentrations of the various reagents. [00169] Binding of the antibody for tianeptine may result in the formation of an immune complex that can be detected directly or indirectly in numerous ways that are well known in the 10 art. The immune complexes are detected directly, for example, when the antibodies employed are conjugated to a label. The immune complex is detected indirectly by examining for the effect of immune complex formation in an assay medium on a signal producing system. [00170] Activation of the signal producing system depends on the nature of the signal producing system members. Activation methods include for example, light activation, addition 15 of base of pH systems, radioactivity, and addition of substrate, wherein a cofactor may be also added if necessary. [00171] In certain embodiments first and second labels may be employed and comprise a label pair. These label pairs may be, for example, a singlet oxygen generator or sensitizer and chemiluminescent reactant pair, an enzyme pair wherein a product of the first enzyme serves as 20 a substrate for the second enzyme and a luminescent energy donor and acceptor pair, e.g., an energy donor or acceptor and a fluorescent compound. The signal will usually be initiated by and/or detected as electromagnetic radiation and will preferably be luminescence such as chemiluminescence, fluorescence, electroluminescence, or phosphorescence. [00172] The examination for presence and level of the signal also includes the detection 25 of the signal, which is generally merely a step in which the signal is read. The signal is normally read using an instrument, the nature of which depends on the nature of the signal. The instrument may be a spectrophotometer, fluorometer, absorption spectrometer, luminometer, chemiluminometer, actinometer, photographic instrument, and the like. The presence and level of signal detected is related to the presence and amount of the entactogen/analyte present in a 30 sample above the predetermined cut-off level. Temperatures during measurements generally range from about 10°C to about 70°C, more usually from about 20°C to about 45°C, more usually about 20°C to about 25°C. In one approach standard curves are formed using known concentrations of the analytes to be screened. Calibrators and other controls may also be used.
52 2024P00697WO [00173] In any embodiment, the method may be or utilize an immunoassay, such as enzyme multiplied immunoassay technique (EMIT), an enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay (RIA), an enzyme channeling immunoassay (ECIA), a fluorescence polarization immunoassay (FPIA), an enzyme modulate mediated immunoassay 5 (EMMIA), a substrate labeled fluorescence immunoassay (SLFIA), a combined enzyme donor immunoassay (CEDIA), a particle enhanced turbidimetric inhibition immunoassay (PETINIA), a particle enhanced turbidimetric immunoassay (PETIA), a sol particle immunoassay (SPIA), a disperse dye immunoassay (DIA), a metalloimmunoassay (MIA), an enzyme membrane immunoassays (EMIA), and a luminoimmunoassays (LIA). 10 [00174] The EMIT assay is a homogenous enzyme immunoassay based on competition between a drug (e.g., tianeptine) in the sample and the drug-conjugated to the enzyme (e.g., G6PDH). The method comprises the steps of: incubating the sample, drug-enzyme conjugate, and antibody for a time sufficient for the antibody to bind to the drug (e.g., tianeptine) in the sample; adding an enzyme substrate to the sample; and measuring the activity of the enzyme, 15 wherein the presence of the complex comprising tianeptine and the antibody is proportional to the activity of the enzyme. The unbound enzyme conjugate converts the oxidized nicotinamide adenine dinucleotide (NAD+) to NADH and a change in absorbance at 340 nm is measured. Enzyme activity decreases upon binding to the antibody, which allows the analyte concentration in the sample to be measured in terms of enzyme activity. Enzyme activity can be measured as 20 conventional in the art, such as measuring a change in fluorescence, radioactivity, or color of the sample. Measurement of the enzyme activity may be quantitative or qualitative. EXAMPLES General Synthesis Procedures [00175] Materials and Equipment: The compounds were purified on a Shimadzu HPLC 25 system (Riverwood, MD) equipped with a Silica-bond – C18 reverse phase column and Biotage LC (Charlotte, NC). The chemical reactions were monitored by TLC (thin layer chromatography) using Silica gel plates from Analtech Inc. (Newark, DE) and ESI-MS Waters HPLC (Milford, MA). The silica gel plates were visualized using UV short wave (254 nm). All chemicals were obtained from Sigma Aldrich (St. Louis, MO), Fluka (Waltham, MA), ThermoScientific 30 (Waltham, MA), VWR (Radnor, PA) and used as received. 1H NMR was recorded on a Bruker UltraShield™ 600 MHz spectrometer (Bruker, Billerica, MA). Chemical shifts were reported in parts per million (ppm, δ) and related to tetramethylsilane
with deuterated solvent as internal reference. NMR abbreviations used are: s (singlet), brs (broad singlet), d (doublet), t (triplet), q (quartet), dd (doublet of doublets), qui (quintet) J (coupling constant), Hz (Hertz). ESI-MS
53 2024P00697WO spectra were recorded on a Water UPLC (Milford, MA) instrument at Siemens Healthineers RD department (Newark, DE). UV: Carry 60 was used for OD280 and NanoDrop 2000. [00176] The following abbreviations have the meanings set forth below: ^-NADH – nicotinamide adenine dinucleotide 5 1H – proton (for NMR) 3K G6PDH – recombinant G6PDH enzyme ACN – acetonitrile AcOH – acetic acid BrAcSu - bromoacetic acid N-hydroxysuccinimide ester 10 BSA – bovine serum albumin CA – cellulose acetate filters calcd. – calculated CDCl3 – deuterated Chloroform (for NMR spectra) CDCN-d3 – deuterated Acetonitrile having 3 deuterium atoms (for NMR spectra) 15 conc. – concentration of enzyme/protein/conjugate/immunogen in mg/mL CV – column volume DCM – dichloromethane DI water – deionized water DIPEA - N,N-Diisopropylethylamine 20 DMF – N,N-dimethylformamide DTT – Dithiothreitol EDC, EDC∙HCl or EDAC– N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride EDTA – ethylenediaminetetraacetic acid eq. – molar equivalent 25 EtOAc – ethyl acetate EtOH - ethanol g – grams G6PDH – native glucose-6-phosphate dehydrogenase enzyme G6PDNa2 – glucose-6-phosphate disodium salt (substrate) 30 GMBS – N-γ-maleimidobutyryl-oxysuccinimide ester h – hour HPLC – high-performance liquid chromatography iPrOH - isopropanol KLH – keyhole limpet hemocyanin 35 LC – liquid chromatography LiOH – lithium hydroxide MeOD-d4 – deuterated methanol having 4 deuterium atoms (for NMR spectra) MeOH – methanol MeSH - Methanethiol 40 mg – milligram MHz – megahertz min – minute mL - milliliter
54 2024P00697WO mmol, mM – millimole MW CO – molecular weight cut-off NaOAc – sodium acetate NEt3 – triethylamine 5 NH2OH – hydroxylamine NMR – nuclear magnetic resonance OVA – ovalbumin isolated from chicken egg white (Sigma) PB – Phosphate buffer PS-Morpholine – polymer supported morpholine 10 RPM – rotations per minute rt – room temperature SATA - N-succinimidyl S-acetylthioacetate SuOH –N-hydroxysuccinimide TCEP∙HCl – tris(2-carboxyethyl)phosphine hydrochloride 15 TFA – trifluoroacetic acid THF - tetrahydrofuran Tianeptine-Cl – {8,11-Dichloro-6-methyl-6,11-dihydrodibenzyl[c,f][1,2]thiazepine5,5- dioxide} TRIS – tris(hydroxymethyl)aminomethane 20 UV – ultraviolet v/v – volume ratio μg – microgram μL – microliter [00177] Buffers - Buffer 1 – (50 mM PB pH 8.00, 2.5 mM EDTA); Buffer 2 – (50 mM PB 25 pH 7.00); Buffer 3 – 150 mM PB pH 7.88 +2.15 mM EDTA; Buffer 4 – 355 mM TRIS pH 7.88, EDTA 2.15 mM, PBS - 50 mM phosphate buffer, pH 7.00, 150 mM NaCl. Example 1: Synthesis of Compounds (XV), (XVI), and (XVII) [00178] Synthesis of compounds of (XV), (XVI), and (XVII) (FIG. 1, Scheme 1): Compound 1 is reacted with compound 4 and a metal catalyst to yield compound 5. Compound 30 5 is reacted with compound 6 and sodium cyanoborohydride (NaBH3CN) in MeOH to yield compound 7. Compound 7 is added to TFA and compound 8 to yield compounds (XV), (XVI) or (XVII). Example 2: Preparation of Immunogens and Conjugates of Compounds (XV), (XVI), and (XVII) 35 [00179] Preparation of the protein conjugate and immunogens (FIG. 1, Scheme 1): Each of compounds (XV), (XVI) or (XVII) are combined with a suitable buffer and a protein solution to yield conjugate (C). Such buffer solutions include, for example, phosphate buffer, e.g., a dihydrogen phosphate, a hydrogen phosphate, etc., and combinations thereof. Proteins, such as KLH, BSA, OVA and G6PDH, are used to prepare corresponding conjugates and 40 immunogens of compounds (XV), (XVI) or (XVII). The resulting conjugates or immunogens are
55 2024P00697WO purified by appropriate purification techniques such as, for example, dialysis or column chromatography, e.g., Sephadex®, etc., and the like using a suitable eluent, e.g., phosphate buffer, etc. Example 3: Synthesis of Compounds (XII), (XIII), and (XIV) 5 [00180] Synthesis of compounds of (XII), (XIII), and (XIV) (FIG. 2, Scheme 2): Compound 9 is reacted with compound 10, undergoes a deprotection step, and then is reacted with compound 11 to yield compounds (XII), (XIII), and (XIV). Example 4: Preparation of Immunogens and Conjugates of Compounds (XII), (XIII), and (XIV) 10 [00181] Preparation of the protein conjugate and immunogens (FIG. 2, Scheme 2): Each of compounds (XII), (XIII), and (XIV) are combined with a suitable buffer and a protein solution to yield conjugate (D). Such buffer solutions include, for example, phosphate buffer, e.g., a dihydrogen phosphate, a hydrogen phosphate, etc., and combinations thereof. Proteins, such as KLH, BSA, OVA and G6PDH, are used to prepare corresponding conjugates and 15 immunogens of compounds (XII), (XIII), and (XIV). The resulting conjugates or immunogens are purified by appropriate purification techniques such as, for example, dialysis or column chromatography, e.g., Sephadex®, etc., and the like using a suitable eluent, e.g., phosphate buffer, etc. Example 5: Synthesis of Compounds (VI), (VII), and (VIII) 20 [00182] Synthesis of compounds of (VI), (VII), and (VIII) (FIG. 2, Scheme 2): Compound 9 is combined with EDAC and NHS in DMF and then is reacted with compound 12 to yield compounds (VI), (VII), and (VIII). Example 6: Preparation of Immunogens and Conjugates of Compounds (VI), (VII), and (VIII) [00183] Preparation of the protein conjugate and immunogens (FIG. 2, Scheme 2): 25 Each of compounds (VI), (VII), and (VIII) are combined with a suitable buffer and a protein solution to yield conjugate (E). Such buffer solutions include, for example, phosphate buffer, e.g., a dihydrogen phosphate, a hydrogen phosphate, etc., and combinations thereof. Proteins, such as KLH, BSA, OVA and G6PDH, are used to prepare corresponding conjugates and immunogens of compounds (VI), (VII), and (VIII). The resulting conjugates or immunogens are 30 purified by appropriate purification techniques such as, for example, dialysis or column chromatography, e.g., Sephadex®, etc., and the like using a suitable eluent, e.g., phosphate buffer, etc. Example 7: Synthesis of Compounds (III), (IV), and (V) [00184] Synthesis of compounds of (III), (IV), and (V) (FIG.2, Scheme 2): Compound 35 9 is combined with EDAC and NHS in DMF and then is reacted with compound 13 to yield
56 2024P00697WO compounds (III), (IV), and (V). Example 8: Preparation of Immunogens and Conjugates of Compounds (III), (IV), and (V) [00185] Preparation of the protein conjugate and immunogens (FIG. 2, Scheme 2): Each of compounds (III), (IV), and (V) are combined with a suitable buffer and a protein solution 5 to yield conjugate (F). Such buffer solutions include, for example, phosphate buffer, e.g., a dihydrogen phosphate, a hydrogen phosphate, etc., and combinations thereof. Proteins, such as KLH, BSA, OVA and G6PDH, are used to prepare corresponding conjugates and immunogens of compounds ((III), (IV), and (V). The resulting conjugates or immunogens are purified by appropriate purification techniques such as, for example, dialysis or column chromatography, 10 e.g., Sephadex®, etc., and the like using a suitable eluent, e.g., phosphate buffer, etc. Example 9: Preparation of Immunogens and Conjugates (G) [00186] Preparation of the protein conjugate and immunogens (FIG. 2, Scheme 2): Compound 9 is combined with a suitable buffer and a protein solution to yield conjugate (G). Such buffer solutions include, for example, phosphate buffer, e.g., a dihydrogen phosphate, a 15 hydrogen phosphate, etc., and combinations thereof. Proteins, such as KLH, BSA, OVA and G6PDH, are used to prepare the conjugates and immunogens. The resulting conjugates or immunogens are purified by appropriate purification techniques such as, for example, dialysis or column chromatography, e.g., Sephadex®, etc., and the like using a suitable eluent, e.g., phosphate buffer, etc. 20 Example 10: Synthesis of Compounds (IX), (X), and (XI) [00187] Synthesis of compounds of (IX), (X), and (XI) (FIG.3, Scheme 3): Compound 9 is reacted with pyridine and HCl at a suitable temperature and then reacted with trifluoroacetic acid anhydride (TFAA), dichloromethane (DCM), and triethylamine (NEt3) to yield compound 14. Compound 14 is reacted with compound 15 in a DMF and K2CO3 solution and then is 25 combined with TFA and DCM to yield compound 16. Compound 16 is reacted with compound 11 and DMF and NEt3 to yield compounds (IX), (X), and (XI). Example 11: Preparation of Immunogens and Conjugates of Compounds (IX), (X), and (XI) [00188] Preparation of the protein conjugate and immunogens (FIG. 3, Scheme 3): Each of compounds (IX), (X), and (XI) are combined with a suitable buffer and a protein solution 30 to yield conjugate (H) Conjugate (H) is further reacted with a suitable basic buffer at a pH of 8 to yield conjugate (I). Such buffer solutions include, for example, phosphate buffer, e.g., a dihydrogen phosphate, a hydrogen phosphate, etc., and combinations thereof. Proteins, such as KLH, BSA, OVA and G6PDH, are used to prepare corresponding conjugates and immunogens of compounds (IX), (X), and (XI). The resulting conjugates or immunogens are purified by
57 2024P00697WO appropriate purification techniques such as, for example, dialysis or column chromatography, e.g., Sephadex®, etc., and the like using a suitable eluent, e.g., phosphate buffer, etc. Example 12: Synthesis of Compounds (XVIII) [00189] Synthesis of compound (XVIII) (FIG. 4, Scheme 4): Compound 21 is reacted 5 with dihydro-2H-pyran-2,6(3H)-dione and pyridine in DMF to yield compound (XVIII). Example 13: Synthesis of Compounds (XIX) [00190] Synthesis of compound (XIX) (FIG. 5, Scheme 5): Compound 22 is reacted with N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC∙HCl) (1.2 equivalent) and NHS (1.2 equivalent) in DMF at room temperature for 4 hrs to yield compound 10 (XIX). Example 14: Synthesis of Compound (XX) [00191] Synthesis of compound (XIX) (FIG. 6, Scheme 6): Compound 23 is reacted with compound 24 in a DMF and K2CO3 solution at a 110°C to 130°C to yield compound 25. Compound 25 is combined with TFA and DCM and then is reacted with compound 26, NEt3, 15 and THF solution to yield compound (XX). Example 15: Preparation of Immunogens and Conjugates of Tianeptine Sodium Salt (TNPT) [00192] Preparation of the protein conjugate and immunogens (FIG. 7, Scheme 7): To an 8 mL glass vial, tianeptine sodium salt (TNPT) (22) (10 mg, 0.021 mmol, 1 equiv.), EDC HCl (0.026 mmol, 5 mg, 1.2 equiv.) and N-hydroxysuccinimide (NHS) (0.026 mmol, 3 mg., 1.2 20 equiv.) were dissolved in anhydrous DMF (2 mL). The resulting reaction mixture was stirred at room temperature (rt) for 8 hours. No intermediate was isolated; however, the formation of the NHS ester was confirmed by Ultra-performance liquid chromatography-mass spectrometry (UPLC/MS), calculated for [C25H28ClN3O6S+ Na]: 556.13, found 556.2 [M+Na]. This solution was then added dropwise into a solution of the relevant carrier protein (ovalbumin 25 (OVA), bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH)) (at 10 mg/mL in PBS (50 mM phosphate buffer, pH 7.00, 150 mM NaCl) (see Table 4 for amounts). The reactions were stirred overnight at rt and the final conjugates (TNPT-OVA (28), TNPT-BSA (29), TNPT- KLH (30)) were purified via NAP25 column using the same reaction buffer as eluent. Table 4. TNPT-OVA, TNPT-BSA, TNPT-KLH Conjugates Hapten (TNPT Protein Protein quantity Na Salt) quantity Molar ratio BSA 20 mg 3.33 mg 20.8 30
58 2024P00697WO Example 16: Synthesis of TNPT002 Hapten (Compounds (XXI) and (XXII)) [00193] Synthesis of TNPT002 Hapten (Compounds (XXI) and (XXII)) (FIG. 8, Scheme 8): To a glass vial equipped with a magnetic stir bar, tianeptine-Cl (31) (150 mg, 0.45 mmol) was suspended in nitromethane (700 µL); then, cystamineSMe*HCl (86 mg, 1.17 mmol) 5 is added followed by NEt3 (133 µL). The resulting reaction mixture was heated to 60 °C for 4 hours. This reaction was run twice using the same amounts and purified once. The volatiles were removed on a rotatory evaporator to give a yellow oil, which was suspended in EtOAc (20 mL) and washed with DI water (2 x 20 mL). The organic phase was then concentrated on a rotatory evaporator to give a yellow oil. This oil was further dissolved in acetonitrile (2 mL) and injected 10 into a preparative liquid chromatography (LC) equipped with a C18 column and purified using Solvent A: Water + 0.1% AcOH, Solvent B: Acetonitrile + 0.1% AcOH. [00194] LC purification method: 0 – 2 mins isocratic Solvent B 20%, 2 – 20 mins gradient Solvent B 90%, 20 – 24 min isocratic solvent B 90%, 24 – 32 min gradient Solvent B 20%, 32 – 35 min isocratic Solvent B 20%, 35 min collection stopped. Some of the sample 15 formed the disulfide by-product. [00195] After purification and lyophilization, two fractions of 75 mg each were isolated, one fraction contained the hapten as disulfide dimer (compound (XXII)) and the second one a mixture of 1:1 Tianeptine-disulfide (compound (XXII)): tianeptine-C2-SSMe (compound (XXI)) (both fractions referred to as “TNPT002 hapten”). Both fractions can be used to prepare 20 conjugates, for example, as further described in Example 17 below. M/z calcd for [C32H33Cl2N4O4S4+] 735.08, 737.07, found 735.35, 737.37 and 415.25 and 415.23 (for SMe derivative). The average molecular weight of the hapten was 573. [00196] 1H NMR (600 MHz, CDCl3) 8.06 – 8.04 (m, 1H), 7.78 – 7.65 (m, 3H), 7.57 – 7.54 (m, 1H), 7.40 (t, J = 6.30 Hz, 1H), 7.36 (t, J = 6.30 Hz, 1H), 6.16 (s, 0.45H), 6.08 (s, 0.3H), 25 6.05 (s, 0.3H), 3.28 – 3.25 (m, 0.82H), 3.17 (s, 1H), 3.15(s, 1H), 3.12 (s, 1H), 2.97 – 2.85 (m, 2.33H), 2.72 – 2.66 (m, 1.35H), 2.26 (s, 1.17H). Example 17: Preparation of Immunogens and Conjugates of TNPT002 [00197] Preparation of the protein conjugate and immunogens of TNPT002 (FIG.9, Scheme 9): BrAcSu (10 mg) was dissolved in DMF (1mL) to make a 10 mg/mL solution. This 30 solution is used to introduce BrAc groups to the proteins (OVA, BSA, KLH) as well as the G6PDH enzyme (see below). [00198] Preparation of activated BrAc-OVA (36): Ovalbumin (33) (17 mg) was dissolved in buffer 1 (3 ml), the resulting reaction mixture was chilled on an ice bath. BrAcSu- DMF solution (2.21 mg, 221 µL) was added dropwise over 2 mins., then the resulting reaction
59 2024P00697WO mixture was allowed to warm up to rt and stirred for 90 mins to yield activated BrAc-OVA (36). The activated BrAc-OVA (36) was then loaded onto a dialysis bag and buffer exchanged with buffer 1 (3x250 mL) in a cold room. The activated protein was then placed in glass vial equipped with a stir bar. 5 [00199] Preparation of activated BrAc-BSA (37): BSA (34) (20 mg) was dissolved in buffer 1 (4 ml), the resulting reaction mixture was chilled on an ice bath. BrAcSu-DMF solution (3.00 mg, 300 µL) was added dropwise over 2 mins., then the resulting reaction mixture was allowed to warm up to rt and stirred for 90 mins to yield activated BrAc-BSA (37). The activated BrAc-BSA (37) was then loaded onto a dialysis bag buffer exchanged with buffer 1 (3x250 mL) 10 in a cold room. The activated protein was then placed in glass vial equipped with a stir bar. [00200] Preparation of activated BrAc-KLH (38): KLH (35) (20 mg) was dissolved in buffer 1 (4 ml), the resulting reaction mixture was chilled on an ice bath. BrAcSu-DMF solution (4.00 mg, 400 µL) was added dropwise over 2 mins., then the resulting reaction mixture was allowed to warm up to rt and stirred for 90 mins to yield activated BrAc-KLH (38). The activated 15 BrAc-KLH (38) was then loaded onto a dialysis bag and buffer exchanged with buffer 1 (3x250 mL) in a cold room. The activated protein was then placed in glass vial equipped with a stir bar. [00201] Activation of TNPT002 hapten: TNPT002 hapten (compound (XXII) (11.2 mg, 0.015 mmol) was suspended in EtOH (500 µL) then TCEP (1.1 eq.4.80 mg as NaOAc solution 10 mg/mL) was added. The resulting reaction mixture was blanketed with Ar and stirred 20 overnight at 400C. m/z calcd for [C16H17ClN2O2S2]+ 369.04, 371.04, found 369.12, 371.12. The volatiles are removed in vacuo to yield activated hapten TNPT-CysSH (32), which was re- suspended in EtOH (500 µL) to make a 20 mg/mL hapten-EtOH solution. [00202] Preparation of TNPT002-OVA conjugate (39): To BrAc-OVA (36) solution (17 mg in 4 mL buffer 1) chilled on an ice-bath, TNPT-CysSH (32) (3 mg, 150 µL) was added 25 dropwise. The resulting reaction mixture was then blanketed with argon and stirred overnight at rt to yield TNPT002-OVA conjugate (39), which further was purified by G25M (CV=60 mL) using buffer 2. A volume of 15 mL of conjugate (39) was collected, which was concentrated on an Amicon stir cell (10,000 MW CO) to a volume of 8.50 mL (conc ~ 2.35 mg/mL). [00203] Preparation of TNPT002-BSA conjugate (40): To BrAc-BSA (37) solution (20 30 mg in 4 mL buffer 1) chilled on an ice-bath, TNPT-CysSH (32) (4.26 mg, 213 µL) was added dropwise. The resulting reaction mixture was then blanketed with argon and stirred overnight at rt to yield TNPT002-BSA conjugate (40), which further was purified by G25M (CV=60 mL) using buffer 2. A volume of 15 mL of conjugate was collected, which was concentrated on an Amicon stir cell (10,000 MW CO) to a volume of 11 mL (conc ~ 1.81 mg/mL)
60 2024P00697WO [00204] Preparation of TNPT002-KLH immunogen (41): To BrAc-KLH (38) solution (20 mg in 4 mL buffer 1) chilled on an ice-bath, TNPT-CysSH (32) (4.62 mg, 231 µL) was added dropwise. The resulting reaction mixture was then blanketed with argon and stirred overnight at rt to yield TNPT002-KLH immunogen (41), which further was purified by G25M (CV=60 mL) 5 using buffer 2. A volume of 15 mL of conjugate (41) was collected, which was concentrated on an Amicon stir cell (10,000 MW CO) to a volume of 10 mL (conc ~ 2 mg/mL). [00205] Preparation of TNPT002-G6PDH conjugate (FIG.9, Scheme 9): The G6PDH enzyme suspension (38 mg, 3.5 mL) was spun at 9g for 40 mins at 4 °C using a bench-top centrifuge. The resulting clear liquid was discarded, and the white precipitate was dissolved in 10 buffer 1 (4 mL). The resulting enzyme solution was dialyzed against buffer 1 (3x 250 mL, 4h in between the buffer exchanges in a cold room). The dialyzed enzyme was then collected v = 3.5 mL (conc 9.57 mg/mL). [00206] G6PDNa2 (40 mg) and β-NADH (40 mg) were added, and the enzyme was rocked until all solids were dissolved. The G6PDH (42) enzyme solution (38 mg) was placed in a glass 15 vial, then BrAcSu (2.45 mg, as 245 µL 10 mg/mL DMF solution, 30x mol excess) was added dropwise in a cold room. The resulting reaction mixture was stirred for 120 mins in a cold room, then purified by G-25M Sephadex column (CV = 60 mL). A fraction of 15 mL containing the activated BrAc-G6PDH enzyme (43) was collected and concentrated to 4.5 mL (c = 7.78 mg/mL). The concentration was then adjusted to 5 mg/mL by adding buffer 1 (2.5 mL). To 5 20 vials, 6 mg of the BrAc-G6PDH enzyme solution is placed. The vials were chilled on an ice bath. [00207] Activation of TNPT002 hapten: TNPT002 hapten (compound (XXII) (5.0 mg, MW 573) was suspended in iPrOH (200 µL), then TCEP*HCl (270 µL from a 10 mg/mL NaOAc-TCEP solution 1.1 eq.) was added. The resulting reaction mixture was blanketed with argon and stirred at 40 °C for 5h. The formation of the activated hapten (32) was confirmed by 25 uPLC. Volatiles were removed in vacuo (including the MeSH by-product) and the activated hapten (32) was resuspended in iPrOH (440 µL). The activated TNPT-CysSH hapten (32), corresponding for 5x to 25x, was added dropwise to each BrAc-G6PDH vial (column 2 in Tab le 5) on an ice bath. The resulting reaction mixtures were blanketed with argon and transferred to a cold room and stirred overnight. The resulting TNPT002-G6PDH conjugates (TNPT002-30 G6PDH-5x (44a), TNPT002-G6PDH-10x (44b), TNPT002-G6PDH-15x (44c), TNPT002- G6PDH-20x (44d), TNPT002-G6PDH-25x (44e), column 1, Table 5) were then purified on G- 25M Sephadex column (CV = 60 mL) using buffer 2. A fraction of 15 mL from each run was collected and concentrated using Amicon stir cells (MW CO 10,000 Da) to the volumes in column 3 and concentrations in column 4 in Table 4. These conjugates (TNPT002-G6PDH-5x
61 2024P00697WO (44a), TNPT002-G6PDH-10x (44b), TNPT002-G6PDH-15x (44c), TNPT002-G6PDH-20x (44d), TNPT002-G6PDH-25x (44e)) were then filtered through 0.20 µm CA filters. Table 5: TNPT-G6PDH conjugates Conju ate m | v Vol Conc 5
Example 18: Synthesis of TNPT-NH-Glu-COOH Haptens (Compounds (XXIII), (XXIV), (XXV), and (XXVI)) [00208] Synthesis of TNPT-NH-Glu-COOH Haptens (Compounds (XXIII), (XXIV), (XXV), and (XXVI)) (FIG. 10, Scheme 10): A mixture of TNPT-Cl (31) (83.1 mg) and 10 ammonia (7 M in MeOH solution, 5 mL) was heated at 70-80 °C overnight. The mixture solution was then concentrated on a rotavap under vacuum to give TNPT-NH2 (45) as a white-yellow solid. M/z calculated for [C14H14ClN2O2S+] 309.05, 311.04 found 309.11, 311.09. TNPT-NH2 (45) solid was dissolved in DCM (2 mL). Glutaric anhydride (58.2 mg) and pyridine (12 µL) were added to above DCM solution. The mixture solution was stirred overnight at RT. The 15 reaction mixture of DCM solution was concentrated on a rotavap under vacuum and the crude product was purified by HPLC to give the pure desired product TPPT-NH-GLU-COOH (compound (XXIV)) as a white solid (77.5 mg). M/z calculated for [C19H20ClN2O5S+] 423.08, 425.08 found 423.23, 425.21. As shown in FIG.10, further haptens, compounds (XXIII), (XXV), and (XXVI), can be synthesized in a similar manner as shown in Scheme 10 depending on the 20 shown variations in the reactant compound (n = 1, 2, 3, or 4) as understood by a one of ordinary skill in the art. [00209] 1H NMR (600 MHz, CD3OD) 7.92 (d, 1H, J = 2.2 Hz), 7.71 (dd, 1H, J = 8.3, 2.2 Hz), 7.66 (dd, 1H, J = 8.3, 2.2 Hz), 7.53 (m, 1H), 7.44 (m, 2H), 7.35 (m, 1H), 6.70 (s, 1H), 3.26 (s, 1H), 2.31 (t, 2H, J = 7.5 Hz), 2.26 (t, 3H, J = 7.4 Hz), 1.86 (p, 2H, J = 7.4 Hz). 25 Example 19: Preparation of Immunogens of TNPT-NH-Glu-COOH Haptens [00210] Preparation of TNPT-NH-Hex-KLH Immunogen (FIG. 10, Scheme 10): TNPT-NH-Glu-COOH (compound (XXIV)) (6.1 mg), N-hydroxysuccinimide (NHS, 4.9 mg), and EDAC (8.2 mg) were dissolved in DMF (0.3 mL) and stirred at rt. The NHS activation progress was monitored by LCMS. After 4 hours, LCMS indicated >90% of TNPT-NH-Glu-
62 2024P00697WO COOH was converted to desired NHS ester (TNPT-NH-Glu-NHS (47)). All the NHS ester (47) DMF solution was added dropwise to a KLH (6.0 mg/mL, 3.3 mL, 20 mg) in PBS buffer (50 mM sodium phosphate, pH 7.0, 150 mM NaCl). The resulting mixture solution was rocked overnight at RT. The KLH immunogen and hapten mixture was purified through a Sephadex G-25 medium 5 column using PBS buffer (pH 7.00) to afford the desired pure TNPT-NH-GLU-KLH immunogen (51). As shown in FIG.10, further activated haptens, compounds (46), (48), and (49), and further KLH immunogens, compounds (50), (52), and (53) can be synthesized in a similar manner as shown in Scheme 10 depending on the shown variations in the hapten compounds used as understood by a one of ordinary skill in the art. 10 Example 20: Synthesis of TNPT-NHC2AcBr Hapten (Compound (XXVII)) [00211] Synthesis of TNPT-NHC2AcBr Hapten (Compound (XXVII)) (FIG. 11, Scheme 11): Tianeptine-Cl (31) (160 mg, 0.489 mmol) was dissolved in EtOH (4 mL), then ethylenediamine (0.5 mL) was added, the resulting reaction mixture was heated to 70 °C for 6h. The volatiles were removed on a rotatory evaporator to give a yellow oil. The oil was dissolved 15 in water (10 mL) then Na2CO3 (3 ml) solution was added, and the product was extracted with EtOAc (30 mL), the organic layer was washed 3 times with DI water to give 203 mg of crude TNPT-NHC2NH2 hapten (60). The hapten (60) was then dissolved in ACN/Water (2 mL, 1/1 v/v) and purified by Shimadzu LC, equipped with a C18 column, to give 212 mg of product as TFA salt. m/z calcd for [C16H19ClN3OS2]+ 352.09, found 352.22 (1.87 mins). Purification LC 20 program: 0 – 1 min isocratic Solvent B10%, 1 – 20 min gradient Solvent B 70%, 20 – 28 min gradient solvent B 10%, 28 min isocratic Solvent B 10%. The product eluted at 9 mins. [00212] 1HNMR (600 MHz, CD3CN-d3) 7.86 (d, J = 2.12Hz, 1H), 7.62 (d, 8.37 Hz, 1H), 7.58 (dd, (J = 8.35, 2.12 Hz, 1H), 7.49 (dd, J = 7.67, 1.01 Hz, 1H), 7, 43 (td J= 7.67Hz, 1.01 Hz, 1H), 7.40 (td, J = 7.86, 1.19 Hz, 1H), 7.35 (td, J= 7.45, 1.27 Hz, 1H), 5.11 (s, 1H), 5.02 (s, 4H), 25 3.34 (s, 3H), 2.81 – 2.78 (m, 2H), 2.59 – 2.55 (m, 2H), 1.86 (s, 3H). (isolated as acetate salt) [00213] TNPT-NHC2NH2 hapten (60) as a TFA salt (50 mg, 0.142 mmol) was dissolved in DMF (0.33 ml), then NEt3 (40 µL) was added. The resulting reaction mixture was stirred for 5 mins on an ice bath, then BrAcSu (32.8 mg, dissolved in THF 300 µL) was added. The resulting reaction mixture was allowed to warm up to rt and stirred for 30 mins, then it was diluted with 30 ACN/Water 1/1 v/v to 2 mL and purified by a LC equipped with a C18 column, using Solvent A: Water + 0.1% TFA, Solvent B: Acetonitrile+0.1% TFA. [00214] After LC purification, 47.3 mg of TNPT-NHC2AcBr Hapten (Compound (XXVII)) in 76% yield was obtained as colorless powder. m/z calcd for [C18H20BrClN3O3S]+ 474.01, 472.01, found 474.32, 472.32. (2.63 mins). LC purification method: 0 – 1 min isocratic
63 2024P00697WO Solvent B10%, 1 – 20 min gradient Solvent B 60%, 20 – 28 min gradient solvent B 10%, 28 min isocratic Solvent B 10%. The product eluted at 15 mins. [00215] 1H NMR (600 MHz, CDCl3) 8.05 (s, 1H), 7.72 (s, 2H), 7.67 (d, J = 7.68 Hz, 1H), 7.56 (t, J = 7.32 Hz, 1H), 7.43 (s, 1H), 7.39 (t, J = 7.33 Hz, 1H), 7.35 (d, J = 8.07 Hz, 1H), 5.96 5 (s, 1H), 3.80 (q J = 3.32Hz, 2H), 3.50 (s, 2H), 3.15-3.14 (m, 3H), 3.04-3.02 (m, 2H), 2.81-2.79 (m, 2H). Example 21: Preparation of Conjugate of TNPT-NHC2AcBr Hapten [00216] Preparation of conjugate of TNPT-NHC2AcBr hapten (Figure 12, Scheme 12): The 3K G6PDH enzyme (6 mg, 0.555 mL) was loaded onto a dialysis bag and buffer 10 exchanged with buffer 4 (2 x 250 mL) in a cold room. After dialysis, 0.6 mL of enzyme (conc 8.71 mg/mL) was recovered. The enzyme (61) was then reduced using TCEP*HCl (0.11 mg, 8x mol excess) as TCEP-H2O solution (10 mg/mL). The resulting mixture was blanketed with Argon and rocked at rt for 19h. The TNPT-NHC2AcBr hapten (compound (XXVII)) (0.4 mg, 14x mol) was added and the resulting reaction mixture was blanketed with Ar and rocked for an additional 15 20h to produce TNPT-C6-3K G6PDH conjugate (62). The conjugate (62) was purified by G25M Sephadex column (CV 55 mL) using Buffer 2. A fraction of 15 mL of conjugate (62) was recovered which was then concentrated to 8.00 mL (c= 0.70 mg/mL) using an Amicon stir cell MW CO 10,000. Example 22: Preparation of Conjugates and Immunogens of TNPT-NHC2AcBr Hapten 20 [00217] Preparation of conjugates and immunogens of TNPT-NHC2AcBr hapten (Figure 13, Scheme 13): In a glass vial, SATA (8 mg) was dissolved in DMF (800 µL) to make a 10 mg/mL SATA solution. [00218] Preparation of activated SATA-OVA protein (66): OVA (63) (20 mg) was dissolved in buffer 3 (4 mL), then SATA-DMF (2.16 mg, 216 µL) corresponding to 20x mol 25 excess was added dropwise. The resulting mixture was stirred for 16h at rt. The next day, NH2OH (150 µL 80% wt.) was added, the reaction mixture was blanketed with argon, and stirred for 2.5h to yield activated SATA-OVA protein (66). The reaction mixture was then dialyzed against Buffer 3 (2x 250 mL) in a cold room 4h in between the buffer exchanges. [00219] Preparation of activated SATA-BSA protein (67): BSA (64) (20 mg) was 30 dissolved in buffer 3 (3mL), then SATA-DMF (2.77 mg, 277 µL) corresponding to 40x mol excess was added dropwise. The resulting mixture was stirred for 16h at rt. The next day, NH2OH (150 µL 80% wt.) was added, the reaction mixture was blanketed with Argon, and stirred for 2.5h to yield activated SATA-BSA protein (67). The reaction mixture was then dialyzed against Buffer 3 (2x 250 mL) in a cold room 4h in between the buffer exchanges.
64 2024P00697WO [00220] Preparation of activated SATA-KLH protein (68): KLH (65) (20 mg) was dissolved in buffer 3 (3mL), then SATA-DMF (3.00 mg, 300 µL) corresponding to 400x mol excess was added dropwise. The resulting mixture was stirred for 16h at rt. The next day, NH2OH (150 µL 80% wt.) was added, the reaction mixture was blanketed with Argon, and stirred for 5 2.5h to yield activated SATA-KLH protein (68). The reaction mixture was then dialyzed against buffer 3 (2x 250 mL) in a cold room 4h in between the buffer exchanges. [00221] TNPT-NHC2AcBr hapten (compound (XXVII)) (19 mg, 0.04 mmol) was dissolved in degassed DMF (1.9 mL) to make a 10 mg/mL hapten-DMF solution that was used immediately. 10 [00222] Preparation of TNPT-C2Ac-SATA-OVA conjugate (69): To the SATA activated OVA protein (66) solution, TNPT-NHC2AcBr hapten (compound (XXVII)) (4.41 mg, 441 µL DMF solution) was added dropwise. The resulting reaction mixture was stirred for 16h at rt to obtain TNPT-C2Ac-SATA-OVA conjugate (69). The conjugate (69) was then purified by Sephadex G-25M column (71 mL) using Buffer 2 and 20 mL of conjugate (69) was recovered, 15 which was concentrated to 3 mL using Amicon 10,000 MW CO stirring cells. The conjugate (69) was then filtered through 0.20 um filters. [00223] Preparation of TNPT-C2Ac-SATA-BSA conjugate (70): To the SATA activated BSA (67) protein solution, TNPT-NHC2AcBr hapten (compound (XXVII)) (7.08 mg, 708 µL DMF solution) was added dropwise. The resulting reaction mixture was stirred for 16h 20 at rt to obtain TNPT-C2Ac-SATA-BSA conjugate (70). The conjugate (70) was then purified by Sephadex G-25M column (71 mL) using buffer 2 and 20 mL of conjugate was recovered, which was concentrated to 3.5 mL using Amicon 30,000 MW CO stirring cells. The conjugate (70) was then filtered through CA 0.20 µm filters. [00224] Preparation of TNPT-C2Ac-SATA-KLH immunogen (71): To the SATA 25 activated KLH (68) protein solution, TNPT-NHC2AcBr hapten (compound (XXVII)) (7.41 mg, 741 µL DMF solution, 25 mol excess) was added dropwise. The resulting reaction mixture was stirred for 16h at rt to obtain TNPT-C2Ac-SATA-KLH immunogen (71). The immunogen (71) was then purified by Sephadex G-25M column (71 mL) using buffer 2 and 20 mL of immunogen (71) was recovered, which was concentrated to 3.5 mL using Amicon 10,000 MW CO stirring 30 cells. The immunogen (71) was then filtered through 0.20 µm filters Example 23: Preparation of Conjugates of TNPT-NHC2AcBr Hapten [00225] Preparation of conjugates of TNPT-NHC2AcBr hapten (Figure 14, Scheme 14): 30 mg of G6PDH enzyme (72) was dialyzed against buffer 3 (2 x 250 ml) in a cold room. Then G6PDNa2 (45 mg) was added. The resulting mixture was mixed until dissolved, then β-
65 2024P00697WO NADH (45 mg) was added and the resulting mixture was stirred until dissolved. In a separate vial, SATA (2.8 mg) was dissolved in DMF (0.56 mg) to make a 5 mg/mL stock solution. The G6PDH enzyme (30 mg) was chilled on an ice-bath, then 1.7 mg (340 µL SATA-DMF solution) was added dropwise. The resulting reaction mixture was stirred for 16h in a cold room. The next 5 day, NH2OH (80 wt.% 200 µL) was added and the reaction mixture was blanketed with Ar and stirred at rt for 2.5h to yield thioacetyl-G6PDH enzyme (73). The reaction mixture was then loaded onto a dialysis bag and buffer exchanged (2x 250 mL buffer 3). [00226] TNPT-NHC2AcBr hapten (compound (XXVII)) (3 mg) was dissolved in DMF (0.6 mL) to make a 5 mg/mL hapten-DMF solution. 10 [00227] The resulting thioacetyl-G6PDH enzyme (73) was then placed in 5 plastic tubes containing 4.7 mg of enzyme each, then, TNPT-C2SAcBr hapten-DMF solution corresponding to 5x to 25x was added dropwise. The resulting reaction mixtures were rocked for 16h at rt, then, they were quenched with iodoacetamide (3 mg, 30 µL DMF solution) at rt for 30 mins to obtain the following conjugates: TNPT-C2Ac-SATA-G6PDH 5x (74a), TNPT-C2Ac-SATA-G6PDH 15 10x (74b), TNPT-C2Ac-SATA-G6PDH 15x (74c), TNPT-C2Ac-SATA-G6PDH 20x (74d), TNPT-C2Ac-SATA-G6PDH 25x (74e). The conjugates (74a-74d) were then purified by Sephadex G-25M column (CV 55 mL), using buffer 2. A fraction of 15 mL was collected form each run. The conjugates (74a-74d) were then concentrated on Amicon stir cells MW CO 10,000 to the volumes indicated in the Table 6, then they were filtered through CA filters (0.2 um). 20 Table 6. TNPT-C2Ac-SATA-G6PDH Conjugates Conjugate G6PDH Hapten excess | mass Conjugate submitted mass |DMF solution volume mL | conc [mg/mL] XIX),
and (XXX)) [00228] Synthesis of TNPT-NH-Hex-COOH Haptens (Compounds (XXVIII),25 (XXIX), and (XXX) (FIG. 15, Scheme 15): A mixture of TNPT-Cl (31) (83.1 mg), methyl 6- aminohexanoate hydrochloride (75) (34.1 mg), polymer-bound morpholine resin (PS- morpholine, 205 mg), and THF (anhydrous, 10 mL) was heated at 70-80 °C overnight. The resin
66 2024P00697WO was then removed and washed with THF (2 x 5 mL). The collected THF solution was concentrated on a rotavap under vacuum to a white-yellow solid (76). This solid (76) was dissolved in THF-MeOH (1:1, 10 mL) and treated with LiOH (2 M, 2 mL) overnight at 40-50 °C. The reaction mixture solution was adjusted to pH 2-3 with 1 M HCl. The acidified mixture 5 was then treated with EtOAc (50 mL) and water (20 mL). The EtOAc layer was separated and washed with saturated brine solution (2 x 20 mL). The organic layer was dried over anhydrous Na2SO4 then filtered. The collected EtOAc solution was concentrated on a rotavap under vacuum to give the crude TNPT-NH-Hex-COOH hapten products. The crude hapten product was purified by HPLC (acetonitrile-water with 0.05% TFA) to give the pure desired TNPT-NH-Hex-COOH 10 hapten (compound (XXX)) as a white solid (68.3 mg). M/z calculated for [C20H24ClN2O4S+] 423.11, 425.11, found 423.20, 425.18. As shown in FIG.15, compounds (XXVIII) and (XXIX) can be synthesized in a similar manner as shown in Scheme 15 depending on the shown variations in compounds (75) and (76) as understood by a one of ordinary skill in the art. [00229] 1H NMR (600 MHz, CD3OD) 8.1 (d, J = 2.1 Hz, 1H), 7.86-7.92 (m, 2H), 7.73 15 (dd, J = 7.8, 1.3 Hz, 1H), 7.66 (m, 1H), 7.60 (dd, J = 8.2, 1.2 Hz, 1H), 7.50 (td., J = 7.5, 1.2 Hz, 1H), 5.95 (s, 1H), 3.26 (s, 1H), 2.98 (m, 1H), 2.85 (m, 1H), 2.29 (t, J = 7.3 Hz, 3H), 1.69 (m, 2H), 1.59 (m, 2H), 1.35 (m, 2H). Example 25: Preparation of Immunogens of TNPT-NH-Hex-COOH Haptens [00230] Preparation of immunogens of TNPT-NH-Hex-COOH haptens (Figure 15,20 Scheme 15): TNPT-NH-Hex-COOH hapten (compound (XXX)) (6.1 mg), and N- hydroxysuccinimide (NHS, 4.9 mg), and EDAC (8.2 mg) were dissolved in DMF (0.3 mL) and stirred at RT. The NHS activation progress was monitored by LCMS. After 4 hours, LCMS indicated >90% of TNPT-NH-Hex-COOH hapten (compound XXX) was converted to desired TNPT-NH-Hex-NHS ester (78). All the NHS ester (78) in DMF solution was added dropwise to 25 a KLH (6.0 mg/mL, 3.3 mL, 20 mg) in PBS buffer solution (pH 7.0 buffer, 50 mM sodium phosphate, 150 mM NaCl). The resulting mixture solution was rocked overnight at RT. The resulting KLH immunogen and hapten mixture was purified by a Sephadex G-25 (medium) column using PBS buffer (pH 7.00) to afford the desired pure TNPT-NH-Hex-KLH immunogen (80). As shown in FIG. 15, further activated hapten compounds (77) and further KLH 30 immunogen compound (79) can be synthesized in a similar manner as shown in Scheme 15 depending on the shown variations in the hapten compounds used as understood by a one of ordinary skill in the art. Additionally, BSA-contaning and OVA-containing immunogens of TNPT-NH-Hex-COOH haptens may be synthesized as shown in Scheme 15 in FIG. 15 depending on the protein solution used as understoody by one of ordinary skill in the art.
67 2024P00697WO Example 26: EMIT Tianeptine Assay [00231] An anti-tianeptine antibody as described herein and a G6PDH conjugate as described herein are evaluated in an EMIT assay format. The EMIT format assay is a homogenous enzyme immunoassay technique used for the analysis of specific compounds in a 5 biological sample. The assay is based on competition for antibody binding sites between a drug in a sample and the drug labelled with a marker, such as G6PDH. Enzyme activity decreases upon binding to the antibody, so the drug concentration in the sample can be measured in terms of enzyme activity. When the marker is G6PDH, the enzyme converts nicotinamide adenine dinucleotide (NAD) to NADH in the presence of glucose-6-phosphate (G6P), resulting in an 10 absorbance change that is measured spectrophotometrically. Endogenous G6PDH does not interfere with the assay because the coenzyme NAD functions only with the bacterial enzyme (from Leuconostoc mesenteroides) employed in the assay. The assay reactions are shown below: . [00232] The EMIT reagents include one G6PDH conjugate and one anti-tianeptine 15 antibody (Ab) reagent. The G6PDH conjugate is labeled with a hapten as described herein through a linkage. In the absence of tianeptine and its metabolites (“analyte”), the G6PDH conjugate is bound by the antibody (see above). The bound G6PDH in the reaction vessel generates enzymatic inhibition and decreases the signal (NADH absorption in 340 nm). The addition of tianeptine and/or its metabolites from a sample to the reaction competes with the 20 binding of the G6PDH conjugate to the antibody, resulting in disrupting enzymatic inhibition with an increase in signal generation. This increase in (NADH) signal is a direct function of the amount of tianeptine and/or its metabolites in the sample when measured against a calibration curve. [00233] The foregoing description of the embodiments has been provided for purposes of 25 illustration and description. It is not intended to be exhaustive or to limit the disclosure. Individual elements or features of a particular embodiment are generally not limited to that particular embodiment, but, where applicable, are interchangeable and can be used in a selected
68 2024P00697WO embodiment, even if not specifically shown or described. The same may also be varied in many ways. Such variations are not to be regarded as a departure from the disclosure, and all such modifications are intended to be included within the scope of the disclosure. NON-LIMITING ILLUSTRATIVE EMBODIMENTS 5 [00234] Illustrative embodiment 1. A complex corresponding in structure to a Formula (I):
le bond; 10 R1 is hydrogen, an alkyl, —(CH2)a(NH)(CO)(CH2)—halo, or —(CH2)b(NH)(CO)(CH2)S—Y1; a and b are each zero to 50; R2 is hydrogen, halo, —(C2H2)(CH2)c(NH)(CO)(CH2)—halo, —S(CH2)dNHCO(CH2)—halo, —(C2H2)(CH2)e(NH)(CO)(CH2)S—Y2, or —S(CH2)fNHCO(CH2)S—Y3; c, d, e, and f are each zero to 50; 15 when “-----” represents a single bond, then R3 is NR5R6, wherein R5 is hydrogen, –(CH2)g(NH)(CO)(CH2)—halo, —(CO)(CF3), or —(CH2)h(NH)(CO)(CH2)S—Y4; g and h are each zero to 50; 20 R6 is —(CO)(CH2)i(CO)(OH), —(CH2)j(CH2)(CO)-R7, or
25 R7 is —OH, —(NH)(CH2)r(NH)(CO)(CH2)—halo,
69 2024P00697WO , —(NH)—Y9; r, s, t, and u are
each zero to 50; and 5 R8 is —(CH2)v(S)(CH2)w(CO)(NH)—Y10, —(CH2)xS—Y11, or —(CH2)y—halo; v, w, x, and y are each zero to 50; when “-----” represents a double bond, then R3 is NR4, wherein R4 is —O(CH2)x1(CO)(OH), —O(CH2)(CO)(NH)(CH2)x2(NH)(CO)(CH2)—halo, —O(CH2)x3(CO)(NH)—Y12 10 or —O(CH2)(CO)(NH)(CH2)x4(NH)(CO)(CH2)S—Y13; x1, x2, x3, and x4 are each zero to 50; and each of Y1, Y2, Y3, Y4, Y5, Y6, Y7, Y8, Y9, Y10, Y11, Y12, and Y13 is an 15 immunogenic carrier or a label; and wherein when R3 is NR5R6, R5 is hydrogen, R6 is (CH2)j(CH2)(CO)-R7, j is 1, 3, or 5, and R7 is —OH, then both R1 is not Cl and R2 is not methyl. [00235] Illustrative Embodiment 2. The complex of illustrative embodiment 1, wherein the alkyl is a C1-C4 alkyl, preferably a C1-C2 alkyl; each halo is Cl or Br; a-y and x1-x4 are each 20 1 to 25, preferably 1 to 10, more preferably 1 to 5; and the immunogenic carrier is selected from the group consisting of a protein, a polypeptide, and a polysaccharide, wherein the protein is preferably selected from the group consisting of keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), bovine thyroglobulin (BTG), egg ovalbumin (OVA), bovine gamma globulin (BGG), and glucose-6-phosphate dehydrogenase (G6PDH) 25 [00236] Illustrative Embodiment 3. The complex of illustrative embodiment 1 or 2, wherein the complex is one of the following compounds:
70 2024P00697WO
71 2024P00697WO
72 2024P00697WO rein
5 wherein: R1 is hydrogen or an 2
R is hydrogen or halo; R3 is NR5R6,
73 2024P00697WO wherein R5 is hydrogen; R6 is —(CO)(CH2)i(CO)(OH), —(CH2)j(CH2)(CO)-R7, or 5
wherein when R6 is —(CH2)j(CH2)(CO)-R7, j is 1, 3, or 5, and R7 is —OH, then 10 both R1 is not Cl and R2 is not methyl. [00238] Illustrative Embodiment 5. The complex of illustrative embodiment 4, wherein R1 is C1-C4 alkyl, preferably C1-C2 alkyl; R2 is halo, preferably Cl; and R6 is —(CH2)k(S2)(CH)3, m are each 1 to 25, 15
. p bodiment 4, wherein R1 is C1-C4 alkyl, preferably C1-C2 alkyl; R2 is halo, preferably Cl; and R6 is —(CH2)j(CH2)(CO)- R7 or —(CH2)q(NH)(CO)-R8; R7 is —OH o ; R8 is —(CH2)z
—halo; halo is Cl or Br, preferably Br; and j, q, and z each are 1 to 25 bly 1 to 10, more preferably 1 to 5.
74 2024P00697WO [00240] Illustrative Embodiment 7. The complex of illustrative embodiment 1, wherein the complex is a conjugate corresponding in structure to a Formula (Ib): 5 wherein:
“-----” represents a single bond or a double bond; R1 is hydrogen, an alkyl, or —(CH2)b(NH)(CO)(CH2)S—Y1; b is zero to 50; R2 is hydrogen, halo, —(C2H2)(CH2)e(NH)(CO)(CH2)S—Y2, or —S(CH2)fNHCO(CH2)S—Y3; e and f are each zero to 50; 10 when “-----” represents a single bond, then R3 is NR5R6, wherein R5 is hydrogen, —(CO)(CF3), or —(CH2)h(NH)(CO)(CH2)S—Y4; h is zero to 50; R6 is —(CH2)j(CH2)(CO)-R7, –(CH2)nS(CH2)o(CO)(NH)—Y5, 15 –(CO)(CH2)p(CO)(NH)-Y6, or —(CH2)q(NH)(CO)-R8; j, n, o, p, and q are each zero to 50; R7 is —OH, —(NH)(CH2)t(NH)(CO)(CH2)S—Y7, ach 20
s — 2 v 2 w — or — 2 x — ; v, w, and x are each zero to 50; when “-----” represents a double bond, then R3 is NR4, wherein R4 is —O(CH2)x3(CO)(NH)—Y12 or —O(CH2)(CO)(NH)(CH2)x4(NH)(CO)(CH2)S—Y13; x3 and x4 are each zero to 25 50; and each of Y1, Y2, Y3, Y4, Y5, Y6, Y7, Y8, Y9, Y10, Y11, Y12, and Y13 is an immunogenic carrier or a label; and
75 2024P00697WO wherein when R3 is NR5R6, R5 is hydrogen, R6 is (CH2)j(CH2)(CO)-R7, j is 1, 3, or 5, and R7 is —OH, then both R1 is not Cl and R2 is not methyl. [00241] Illustrative Embodiment 8. The complex of illustrative embodiment 1, wherein the complex is a conjugate corresponding in structure to a Formula (Ic): 5
y g y; R2 is hydrogen or halo; 10 R3 is NR5R6, wherein R5 is hydrogen, R6 is —(CH2)j(CH2)(CO)-R7, –(CH2)nS(CH2)o(CO)(NH)—Y5, –(CO)(CH2)p(CO)(NH)-Y6, or —(CH2)q(NH)(CO)-R8; 15 j, n, o, p, and q are each zero to 50; R7 is —(NH)—Y9; and R8 is —(CH2)v(S)(CH2)w(CO)(NH)—Y10 or —(CH2)xS—Y11; v, w, and x are each zero to 50; and each of Y5, Y6, Y9, Y10, and Y11 is an immunogenic carrier or a label. 20 [00242] Illustrative Embodiment 9. The complex of illustrative embodiment 8, wherein R1 is C1-C4 alkyl, preferably C1-C2 alkyl, R2 is halo, preferably Cl; R6 is –(CH2)nS(CH2)o(CO)(NH)—Y5 or –(CO)(CH2)p(CO)(NH)-Y6; n, o, and p each are 1 to 25, preferably 1 to 10, more preferably 1 to 5; and the immunogenic carrier is selected from the group consisting of a protein, a polypeptide, and a polysaccharide. 25 [00243] Illustrative Embodiment 10. The complex of illustrative embodiment 8, wherein R1 is C1-C4 alkyl, preferably C1-C2 alkyl, R2 is halo, preferably Cl; R6 is —(CH2)q(NH)(CO)-R8 or —(CH2)j(CH2)(CO)-R7; R8 is —(CH2)v(S)(CH2)w(CO)(NH)—Y10 or —(CH2)xS—Y11; R7 is —(NH)—Y9; and j, q, v, w, and x are each 1 to 25, preferably 1 to 10, more preferably 1 to 5; and the immunogenic carrier is selected from the group consisting of a protein, a polypeptide, 30 and a polysaccharide.
76 2024P00697WO [00244] Illustrative Embodiment 11. The complex of any one of illustrative embodiments 8 to 10, wherein the conjugate corresponds in structure to: , 5
from the group consisting of keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), bovine thyroglobulin (BTG), egg ovalbumin (OVA), bovine gamma globulin (BGG), and glucose-6- phosphate dehydrogenase (G6PDH). [00245] Illustrative Embodiment 12. A kit comprising: 10 (i) an anti-tianeptine antibody, preferably wherein the anti-tianeptine antibody is raised against the complex of illustrative embodiments 1, 2 and 7-11 wherein the immunogenic carrier is present; and (ii) a conjugate corresponding in structure to a Formula (Ib): 15 wherein: “-----” represents a si le bond; R1
is hydrogen, an alkyl, or —(CH2)b(NH)(CO)(CH2)S—Y1; b is zero to 50; R2 is hydrogen, halo, —(C2H2)(CH2)e(NH)(CO)(CH2)S—Y2, or 20 —S(CH2)fNHCO(CH2)S—Y3; e and f are each zero to 50;
77 2024P00697WO when “-----” represents a single bond, then R3 is NR5R6, wherein R5 is hydrogen, —(CO)(CF3), or —(CH2)h(NH)(CO)(CH2)S—Y4; h is zero to 50; 5 R6 is —(CH2)j(CH2)(CO)-R7, –(CH2)nS(CH2)o(CO)(NH)—Y5, –(CO)(CH2)p(CO)(NH)-Y6, or —(CH2)q(NH)(CO)-R8; j, n, o, p, and q are each zero to 50; R7 is —OH, —(NH)(CH2)t(NH)(CO)(CH2)S—Y7, , or —(NH)—Y9; t and u are each 10
y; R8 is —(CH2)v(S)(CH2)w(CO)(NH)—Y10 or —(CH2)xS—Y11; v, w, and x are each zero to 50; when “-----” represents a double bond, then R3 is NR4, wherein R4 is —O(CH2)x3(CO)(NH)—Y12 or 15 —O(CH2)(CO)(NH)(CH2)x4(NH)(CO)(CH2)S—Y13; x3 and x4 are each zero to 50; and each of Y1, Y2, Y3, Y4, Y5, Y6, Y7, Y8, Y9, Y10, Y11, Y12, and Y13 is an enzyme, wherein the enzyme is selected from the group consisting of glucose-6- phosphate dehydrogenase (G6PDH), an alkaline phosphatase, and horseradish 20 peroxidase, preferably glucose-6-phosphate dehydrogenase (G6PDH); and wherein when R3 is NR5R6, R5 is hydrogen, R6 is (CH2)j(CH2)(CO)-R7, j is 1, 3, or 5, and R7 is —OH, then both R1 is not Cl and R2 is not methyl. [00246] Illustrative Embodiment 13. The kit of illustrative embodiment 12, wherein the conjugate corresponds in structure a Formula (Ic): 25 wherein: R1 is hydrogen or an
78 2024P00697WO R2 is hydrogen or halo; R3 is NR5R6, wherein R5 is hydrogen, 5 R6 is —(CH2)j(CH2)(CO)-R7, –(CH2)nS(CH2)o(CO)(NH)—Y5, –(CO)(CH2)p(CO)(NH)-Y6, or —(CH2)q(NH)(CO)-R8; j, n, o, p, and q are each zero to 50; R7 is —(NH)—Y9; and R8 is —(CH2)v(S)(CH2)w(CO)(NH)—Y10 or —(CH2)xS—Y11; 10 v, w, and x are each zero to 50; and each of Y5, Y6, Y9, Y10, and Y11 is an enzyme, wherein the enzyme is selected from the group consisting of glucose-6-phosphate dehydrogenase (G6PDH), an alkaline phosphatase, and horseradish peroxidase, preferably glucose-6-phosphate dehydrogenase (G6PDH). 15 [00247] Illustrative Embodiment 14. A method for determining the presence of tianeptine and/or a metabolite of tianeptine in a sample, the method comprising: combining the sample, a conjugate, and an anti-tianeptine antibody in a medium, preferably wherein the anti-tianeptine antibody is raised against the complex of illustrative embodiments 1, 2, and 7-11 wherein the immunogenic carrier is present; and 20 examining the medium for the presence of a complex comprising tianeptine and the anti- tianeptine antibody, wherein the conjugate corresponds in structure to a Formula (Ib): 25 “-----” represe a double bond; R1 is hydrogen, an al )(CO)(CH2)S—Y1; b is zero to 50;
R2 is hydrogen, halo, —(C2H2)(CH2)e(NH)(CO)(CH2)S—Y2, or —S(CH2)fNHCO(CH2)S—Y3; e and f are each zero to 50; when “-----” represents a single bond, then R3 is NR5R6, 30 wherein R5 is hydrogen, —(CO)(CF3), or
79 2024P00697WO —(CH2)h(NH)(CO)(CH2)S—Y4; h is zero to 50; R6 is —(CH2)j(CH2)(CO)-R7, –(CH2)nS(CH2)o(CO)(NH)—Y5, –(CO)(CH2)p(CO)(NH)-Y6, or —(CH2)q(NH)(CO)-R8; j, n, o, p, and q are each zero to 50; 5 R7 is —OH, —(NH)(CH2)t(NH)(CO)(CH2)S—Y7, , or —(NH)—Y9; t and u are each
zero to fifty; R8 is —(CH2)v(S)(CH2)w(CO)(NH)—Y10 or —(CH2)xS—Y11; v, w, and x are each zero to 50; 10 when “-----” represents a double bond, then R3 is NR4, wherein R4 is —O(CH2)x3(CO)(NH)—Y12 or —O(CH2)(CO)(NH)(CH2)x4(NH)(CO)(CH2)S—Y13; x3 and x4 are each zero to 50; and each of Y1, Y2, Y3, Y4, Y5, Y6, Y7, Y8, Y9, Y10, Y11, Y12, and Y13 is an15 enzyme, wherein the enzyme is selected from the group consisting of glucose-6- phosphate dehydrogenase (G6PDH), an alkaline phosphatase, and horseradish peroxidase, preferably glucose-6-phosphate dehydrogenase (G6PDH); and wherein when R3 is NR5R6, R5 is hydrogen, R6 is (CH2)j(CH2)(CO)-R7, j is 1, 3, or 5, and R7 is —OH, then both R1 is not Cl and R2 is not methyl. 20 [00248] Illustrative Embodiment 15. The method of illustrative embodiment 14, wherein the conjugate corresponds in structure a Formula (Ic): wherein: 25 R1 is hydrogen or an
R2 is hydrogen or halo; R3 is NR5R6, wherein
80 2024P00697WO R5 is hydrogen, R6 is —(CH2)j(CH2)(CO)-R7, –(CH2)nS(CH2)o(CO)(NH)—Y5, –(CO)(CH2)p(CO)(NH)-Y6, or —(CH2)q(NH)(CO)-R8; i, n, o, p, and q are each zero to 50; 5 R7 is —(NH)—Y9; and R8 is —(CH2)v(S)(CH2)w(CO)(NH)—Y10 or —(CH2)xS—Y11; v, w, and x are each zero to 50; and each of Y5, Y6, Y9, Y10, and Y11 is an enzyme, wherein the enzyme is selected from the group consisting of glucose-6-phosphate dehydrogenase 10 (G6PDH), an alkaline phosphatase, and horseradish peroxidase, preferably glucose-6-phosphate dehydrogenase (G6PDH).