WO2024036295A1

WO2024036295A1 - Cross-reference to related applications

Info

Publication number: WO2024036295A1
Application number: PCT/US2023/072062
Authority: WO
Inventors: Khaledur Rashid; Michael Salvati; Barry A. Schoenfelner; Guoping Wang; Iaroslav KHOMUTNYK
Original assignee: Beckman Coulter, Inc.
Priority date: 2022-08-12
Filing date: 2023-08-11
Publication date: 2024-02-15

Abstract

The disclosure relates to conjugates of the formulae (I) and (II): wherein the variables R1, R2, R3, R17, R18, R19, R20, R20A, R21, and G are defined herein, as well as methods for the use of the conjugates, and kits including the conjugates.

Description

6-POSITION-MODIFIED STEROIDS AND USES THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application Nos. 63/397,586, filed August 12, 2022; and 63/436,829, filed January 3, 2023, both of which are incorporated by reference as if fully set forth herein.

BACKGROUND

[0002] In sandwich immunoassays, two independent epitopes bound by different antibodies provide certain advantages in terms of speed, sensitivity, and specificity. However, sandwich immunoassay formats have not been directly applicable to small molecular weight haptens. Haptens are not large enough to bind simultaneously to two antibodies independently. For these reasons, competitive immunoassays are the most widely used format for measurement of haptens.

[0003] To enhance assay sensitivities and specificities for haptens, noncompetitive methods have been used. For example, anti-immune complex assays (Proc. Natl. Acad. Sci. USA, 90, 1993, 1184-1189 and Clin. Chem. 40(11), 1994, 2035-2041) were successfully used for determinations of tetrahydrocannabinol (THC) and digoxin. Selective antibody or “idiometric” methodology (J Immunol Methods 181 , 1995, 83-90 and Steroids 60, 1995, 824-829) is another noncompetitive approach, which provided more sensitive assays for estradiol and progesterone than conventional competitive enzyme assays. However, these noncompetitive formats require unique antibodies and antiidiotypes that are potentially difficult to obtain.

[0004] Another noncompetitive two-site enzyme immunoassay format (hetero- two-site or immune complex transfer) (Biotechnology Annual Review 1 , 1995, 403- 451) has been also applied for small peptides or haptens with good detection levels. Unfortunately, a two-site enzyme immunoassay requires multiple steps including derivatization of the hapten of interest with another moiety that provides a second binding site. Multiple steps mean the assay is generally more expensive and time consuming than is desirable and the derivatization also involves the use of harsh chemicals and reaction conditions which can potentially damage sensitive biomolecules and complicate providing assays in non-laboratory settings.

[0005] Another non-competitive assay for small molecules has been employed for measurement of cortisol and estradiol is described in U.S. Patent No. 6,037,185. This assay permits the direct measurement of hapten-bound sites or initial amount of hapten in the sample. Unfortunately, the assay still requires multiple steps to perform, which is potentially costly and time consuming.

[0006] Optical immunosensors are popular for bio-analysis. The non-destructive nature of the technology permits multiple reuses of samples for other readings. Rapid signal generation and thus rapid result generation are also advantages of the system. Unfortunately, label-free optical immunosensors have relatively poor analytical sensitivities to haptens compared to traditional immunoassays such as ELISA. Despite significant developments in this field, optical immunosensors tend to be one magnitude less sensitive than commercial immunoassays for determining haptens.

[0007] There therefore continues to be a need for reagents and methods for accurate and sensitive determinations of small molecules (for example, steroids), including one or more steroid analytes of interest and analogs and metabolites thereof in samples.

SUMMARY

[0008] In some aspects, the steroids described in this disclosure may be used as immunogenic haptens (for example, when conjugated to protein carriers) to produce specific anti-steroid antibodies. The resulting antibodies may be used in immunoassays. Additionally or alternatively, the steroids described in this disclosure may be used in competitive immunoassays as the labeled, known antigen.

DESCRIPTION OF THE FIGURES

[0009] Aspects of the present disclosure may be better understood with reference to the following drawings. The components in the drawings are not necessarily to scale. Instead, emphasis is placed on illustrating the principles and methods underlying the present disclosure.

[0010] FIGS. 1-7 are cartoon depictions of immunoassays or steps thereof for detecting a steroid, as described herein.

[0011] Like reference numbers in the various figures indicate like elements. Some elements may be present in identical or equivalent multiples; in such cases only one or more representative elements may be designated by a reference number but it will be understood that such reference numbers apply to all such identical elements. Unless otherwise indicated, all figures and drawings in this document are not to scale and are chosen for the purpose of illustrating different embodiments of the invention. In particular, the dimensions of the various components are depicted in illustrative terms only, and no relationship between the dimensions of the various components should be inferred from the drawings, unless so indicated.

DESCRIPTION

[0012] Reference will now be made in detail to certain embodiments of the disclosed subject matter. While the disclosed subject matter will be described in conjunction with the enumerated claims, it will be understood that the exemplified subject matter is not intended to limit the claims to the disclosed subject matter.

[0013] The disclosure is generally directed to conjugates of the formula (I):

wherein:

R¹ is hydrogen, acyl, or alkoxy;

R² is the CD-ring skeleton of steroids of the androstane or the pregnane series;

R³ is -(CH₂)_n-, -(CH₂)n-S(O)t-aryl-X¹, -(CH₂)_n-S(O)t-(CH₂)_pX¹, or -(CH₂)_n-O- (CH₂)_PX¹ , wherein: t is 0, 1 or 2; n and p are each, independently, an integer from 1 to 10; and

X¹ is R^5a, -C(O)-, -C(O)NH-R⁴-, -NHC(O)-R⁴-R^5b or -C(O)NH-R⁴-R^5b wherein R⁴ is alkyl, R^5a is:

(wherein the SChNa group is representative of one of many examples of groups that can increase the solubility of the compounds described herein, including the solubility of compounds of the general formula (l)-(lll); other groups that can increase solubility are contemplated herein);

wherein in R^5a the wavy line on the left-hand side, adjacent to the carbonyl, represents a point of attachment to R³ and the wavy line on the right-hand side represents a point of attachment to G; and in R^5b, the wavy line on the left-hand side, adjacent to the the nitrogen atom represents a point of attachment to R⁴ and the wavy line on the right-hand side represents a point of attachment to G; and

G is or includes a magnetic particle (including, for example, a paramagnetic particle), a reporter, a carrier protein, or an immunogen.

[0014] In compounds of the formula (I), R² can be:

wherein: m is 1 or 2; wherein the dashed line, when present, represents a single or a double bond;

R⁴ is oxygen, hydrogen, trimethylsilyl, p-hydroxy, nitrooxy, p-halogen, a- or p- acyloxy, a- or p-alkoxy, a- or p-methoxymethoxy, or methylene;

R⁵ is methyl or ethyl;

R⁶ is hydrogen, acyl, halo, alkoxy or an acetal group of the formula -OCH(R⁹)(OR¹⁰), wherein R⁹ is hydrogen or alkyl and R¹⁰ is alkyl of up to 6 carbon atoms; and R⁷ is hydrogen, hydroxy, alkyl, alkoxy or acyloxy, nitrooxy, an acetal, or a hemithioacetal group of the formula -OCH(R⁹)(ZR¹⁰), wherein Z is oxygen or sulfur; and

R⁸ is hydrogen, a- or p-alkyl, alkylidene, a- or p-acyloxy, a- or p-alkoxy, or a- or p- OCH2OR¹⁰, or R⁷ and R⁸ together represent oxygen, methylene, or a group of the formula:

wherein R¹¹ and R¹² can be identical or different and each is hydrogen, alkyl, or alkoxy;

R¹³ is oxygen, ethylene dioxy, or =C-COOR¹⁰;

R¹⁴ is alkyl or haloalkyl; and

R¹⁵ is hydroxy, acyloxy, nitrooxy, alkoxy, or an acetal or hemithioacetal group of the formula -OCH(R⁹)(ZR¹⁰); and

R¹⁶ is alkyl, alkenyl or alkynyl, each of which can optionally be substituted by fluorine, chlorine or bromine.

[0015] Conjugates contemplated herein also include conjugates of the formula (II):

wherein:

R¹⁷ and R¹⁸ are each, independently, H, alklyl, alkenyl, alkynyl, acyl, or OR²² (wherein

R²² is H or alkyl), or R¹⁷ and R¹⁸, together with the carbon atom to which they are attached, form a carbonyl;

R¹⁹ is H, alkyl, or acyl;

R²⁰ and R^20A are each independently H or OR²², or R²⁰ and R^20A, together with the carbon atom to which they are attached, form a carbonyl;

R²¹ is H or alkyl;

R³ is -(CH₂)_n-, -(CH₂)n-S(O)t-aryl-X¹, -(CH₂)_n-S(O)t-(CH₂)_pX¹, or -(CH₂)_n-O-

(CH₂)_PX¹ , wherein: t is 0, 1 or 2; n and p are each, independently, an integer from 1 to 10; and X¹ is R^5a, -C(O)-, -C(O)NH-R⁴-, -NHC(O)-R⁴-R^5b or -C(O)NH-R⁴-R^5b wherein R⁴ is alkyl,

R^5a is:

(wherein the SOsNa group is representative of one of many examples of groups that can increase the solubility of the compounds described herein, including the solubility of compounds of the general formula (l)-(lll); other groups that can increase solubility are contemplated herein);

[0016] In some conjugates of the formula (II), R¹⁷ can be acyl, such as alkylacyl (for example, groups of the formula -C(O)-R²³-OR^24A or -C(O)R²³, wherein R²³ is alkyl (for example, (Ci-Ce)-alkyl) and R^24A is H or alkyl). Alternatively, or in addition, R¹⁹ can be Ci-Ce-alkyl or acyl (for example, -C(O)R^24B, wherein R^24B is H, alkyl, or OR²²). Alternatively, or in addition, R²⁰ can be H or OR²², wherein R²² can be H. Alternatively, or in addition, in some conjugates of the formula (II), R²¹ can be Ci- Ce-alkyl. Examples of acyl and alkylacyl groups for conjugates of the formula (II) include groups of the formulae -C(O)-R²³-OR^24A or -C(O)R²³, wherein R²³ is alkyl (for example, (Ci-Ce)-alkyl) and R^24A is H.

[0017] In the conjugates of the formula (II), R¹⁷ and R¹⁸, together with the carbon atom to which they are attached, can form a carbonyl. Alternatively, or in addition, R¹⁹ can be Ci-Ce-alkyl or acyl (for example, -C(O)R^24B, wherein R^24B is H, alkyl, or OR²²). Alternatively, or in addition, R²⁰ can be H or OR²², wherein R²² can be H. Alternatively, or in addition, R²¹ can be Ci-Ce-alkyl. Examples of acyl and alkylacyl groups include groups of the formulae -C(O)-R²³-OR^24A or -C(O)R²³, wherein R²³ is alkyl (for example, (Ci-Ce)-alkyl) and R^24A is H or alkyl.

[0018] Examples of conjugates of the formula (II) include conjugates of the formula:

[0019] Conjugates contemplated herein also include conjugates of the formula

wherein:

R²⁵ is a divalent linker of the formula -R²⁶-X²-R²⁷-X¹ , wherein R²⁶ is alkyl and R²⁷ is alkyl or aryl;

X² is O or S(O)t , wherein t is 0, 1 or 2; and

[0020] In the conjugates of the formula (III), R²⁶ can be alkyl, R²⁷ can be alkyl, and X² can be S. Alternatively, or in addition, R²⁵ can be -(CH2)n-S-(CH2)_PX¹ or -(CH2)n-O-(CH₂)pX¹, wherein n and p are each, independently, an integer from 1 to 10. Thus, for example, R²⁵ can include or can be of the formula:

,

For example, n and p can each independently be an integer of 1 to 10, or 1 to 9, or 1 to 8, or 1 to 7, or 1 to 6, or 1 to 5, or 1 to 4, or 1 to 3, or 1 to 2, or 2 to 10, or 2 to 9, or 2 to 8, or 2 to 7, or 2 to 6, or 2 to 5, or 2 to 4, or 2 to 3, or 3 to 10, or 3 to 9, or 3 to 8, or 3 to 7, or 3 to 6, or 3 to 5, or 3 to 4, or 4 to 10, or 4 to 9, or 4 to 8, or 4 to 7, or 4 to 6, or 4 to 5, or 5 to 10, or 5 to 9, or 5 to 8, or 5 to 7, or 5 to 6, or 6 to 10, or 6 to 9, or 6 to 8, or 6 to 7, or 7 to 10, or 7 to 9, or 7 to 8, or 8 to 10, or 8 to 9, or 9 to 10.

[0021] As described herein, G is or includes a magnetic particle, including, for example, a paramagnetic particle. Magnetic particles are commonly used in sample isolation or processing steps because they allow analytes of interest that are attached or bound to the magnetic particle to be easily separated from other (soluble) molecules or components in a sample solution. Such separation may occur by using magnetic forces to form a solid phase separate from a supernatant liquid phase. Pouring-off or aspirating-off the supernatant liquid effectively separates the analytes of interest associated with the particles from the other molecules, liquid, or components removed together with the supernatant. Several general types of magnetic particles have been used in the art. In some cases, paramagnetic, superparamagnetic, or ferromagnetic particles have been used. [0022] Alternatively, G can be a carrier protein. Carrier protein conjugates (for example, conjugates where G is or includes a carrier protein) contemplated herein can be used in various applications, including to generate “binding members” (for example, anti-steroid antibodies, such as anti-steroid monoclonal antibodies (mAbs)) specific for steroids in a sample. Exemplary steroids correspond to, for example, steroids of the formulae:

[0023] As is well known in the art, a given composition for immunization may have its immunogenicity boosted or enhanced by conjugating the composition (for example, a steroid) to a carrier protein so that the conjugate can be used to generate “binding members” (for example, antibodies) in a host (for example, in a rabbit). The binding members that are generated by the host immune system can then be used in the methods described herein. Exemplary carrier proteins include diphtheria toxin, a genetically modified cross-reacting material (CRM) of diphtheria toxin, tetanus toxoid (T), meningococcal outer membrane protein complex (OMPC), diphtheria toxoid (D), H. influenzae protein D (HiD), keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), and ovalbumin (OVA). Other albumins, such as mouse serum albumin or rabbit serum albumin, can also be used as carrier proteins. Additionally, as also is well known in the art, the immunogenicity of a particular composition (for example, the compounds described herein, wherein G is or includes a carrier protein) can be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants. Exemplary adjuvants include complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvants, and aluminum hydroxide adjuvant.

[0024] Alternatively, G can be a reporter. A reporter can include, for example, a reporter enzyme capable of reacting with a chromogenic, chemiluminescent or fluoregenic substrate. Examples of reporter enzymes include an alkaline phosphatase (ALP) or a horseradish peroxidase (HRP). Examples of chemiluminescent substrates for ALP include dioxetanes, such as those described in Published PCT Appl. No. WO 2021/086977. Example of a fluoregenic substrate for ALP include 4-methylumbelliferyl phosphate (4-MUP). Examples of chromogenic reporters include 3,3',5,5'-tetramethylbenzidine (TMB), which is a chromogenic substrate for HRP. A reporter can further include molecules which can act as a direct label including, for example, a fluorescent molecule or a triggerable chemiluminescent molecule.

[0025] Steroids are immunogenic haptens and, when conjugated to protein carriers, can be used to produce specific anti-steroid antibody responses in a host. The resulting antibodies may be used in immunoassays; examples of these immunoassays (or steps thereof) are shown in FIGS. 1-7. The resulting polyclonal antibodies may be used; or, in some cases, a monoclonal antibody (for example, a monoclonal antibody further selected from the resulting polyclonal antibodies) or a combination of monoclonal antibodies may be used.

[0026] The number of different, closely related steroid hormones in human serum is high, and their relative concentrations, which usually are very low, make accurate detection of steroids challenging. In addition, when steroids are conjugated to protein carriers, the location of the conjugation can affect the availability of certain epitopes on the steroid. Finally, when anti-steroid antibodies are raised against a particular steroid-protein conjugate, care must be taken that a resulting antibody or antibodies selected for detection of the steroid recognizes a steroid epitope and does not recognize the chemistry used to achieve the steroid- protein conjugate or the non-steroid portion of the steroid-protein conjugate.

[0027] One way to mitigate the effect of antibodies that recognize the chemistry used to achieve a steroid-protein conjugate on an assay intended to detect a steroid is to use a steroid conjugated to a protein carrier at a first location to immunize an animal and produce an anti-steroid antibody response; a steroid conjugated to a reporter molecule at a second location (as shown, for example, in FIGS. 1-3) or a steroid conjugated to a particle at a second location (as shown, for example, in FIGS. 4-6) may be used during an assay to determine the amount of steroid in a sample.

[0028] To achieve accurate detection of closely related steroids, additional locations on a steroid that can be used to conjugate a steroid to a protein carrier (allowing the exposure of different epitopes during antibody generation) or to conjugate a steroid to a reporter molecule or a particle (allowing the exposure of different epitopes during analyte detection) would be useful. To that end, the disclosure also relates to methods of determining one or both of the presence and the amount of a steroid in a sample, the method including: obtaining or providing in combination, in at least one medium,

(i) a sample suspected of containing a steroid, (ii) a first binding member specific for the steroid (for example, an antibody),

(iii) a particle including a second binding member (for example, an antibody) specific for the first binding member, and

(iv) a conjugate described herein, the conjugate including a reporter; subjecting the combination to conditions for forming a first complex including the first binding member and the steroid and a second complex including the first binding member and the conjugate, wherein the first complex and the second complex bind to the second binding member; washing to remove any conjugate from the combination that is not bound to the second binding member via the first binding member to produce a washed composition, and measuring the amount of the reporter present in the washed composition . The first binding member or the second binding member or both may be a monoclonal antibody. The particle may be a magnetic particle (including, for example, a paramagnetic particle). In one example, the steroid (for example, the steroid in the sample) is cortisol and the conjugate includes a cortisol radical:

[0029] The reporter used in the methods described herein can be any suitable reporter, including an enzyme reporter such as ALP.

[0030] The disclosure also relates to a method of determining one or both of the presence and the amount of a steroid in a sample, the method including: obtaining or providing in combination, in a medium,

(i) a sample suspected of containing a steroid,

(ii) a binding member (for example, an antibody) specific for the steroid, the binding member including a reporter, and

(iii) a conjugate described herein, the conjugate including a particle and a steroid; subjecting the combination to conditions for forming a first complex between a steroid of the conjugate and the binding member and a second complex between the steroid in the sample and the binding member; washing, wherein the washing step removes the second complex and produces a washed composition; and measuring the amount of reporter in the washed composition. The first binding member or the second binding member or both may be a monoclonal antibody. The particle may be a magnetic particle (including, for example, a paramagnetic particle).

[0031] In one example, the steroid is cortisol and the conjugate includes a cortisol radical:

[0032] The reporter used in the methods described herein can be any suitable reporter, including an enzyme reporter such as ALP.

[0033] FIGS. 1 and 2 are a schematic of an exemplary competitive binding assay to detect an analyte (for example, a steroid). In one example, the analyte is cortisol. Making reference to FIG. 1 , a sample is added to a reaction vessel (not shown, but can be any suitable vial) with first binding member 106 (for example, a rabbit antibody) specific to the analyte 116 (for example, cortisol), an analyte-reporter conjugate 108 (for example, a cortisol-alkaline phosphatase conjugate), which can include analyte 112 (for example, cortisol) conjugated to reporter 110 (for example, alkaline phosphatase) either directly or, optionally, via a suitable linker 114, and a plurality of particles 100 (for example, paramagnetic particles) coated with second binding member 102 (for example, a goat anti-rabbit capture antibody), optionally linked to the particles 100 via a suitable linker 104. Analyte 116 in the sample competes with the analyte-reporter conjugate 108 for binding sites on a limited amount of first binding member 106 (for example, anti-cortisol antibody). Making reference to FIG. 2, resulting first binding memberanalyte complexes 118 (including analyte 116) and first binding memberanalyte-reporter complexes 120 (including analyte-reporter conjugate 108) bind to the second binding member 102 (for example, goat anti-rabbit capture antibody). A particle 100, associated with second binding member 102 (for example, goat anti-rabbit capture antibody), with the second binding member 102 optionally linked to a particle 100 via a suitable linker 104, is an example of “the solid phase.”

[0034] Making reference to FIG. 2, after incubation in a reaction vessel, the bound phase 300 including materials (for example, analyte 116, analyte-reporter conjugate 108, and first binding member 106) bound to the solid phase 300 are retained (for example, held in a magnetic field) while unbound materials (not shown) are washed away. Unbound materials include any analyte-reporter complexes not bound to the second binding member via the first binding member. Unbound materials may be washed away by aspiration and, additionally by washing, with a suitable buffer. Making reference to FIG. 3, a substrate 302 (for example a chemiluminescent substrate) is added to the vessel (not shown) and light 304 generated by the reaction between the substrate 302 and the reporter from the cortisol-reporter conjugate 108 is measured (for example, with a luminometer 306). The light production 304 is inversely proportional to the concentration of analyte (in one example, cortisol) in the sample. The amount of analyte in the sample may be determined from a stored, multi-point calibration curve. If a different reporter is used, a color change, fluorescence, or other output may be detected instead of light.

[0035] FIG. 4 shows a schematic of an alternative format for a competitive binding assay to detect an analyte (for example, a steroid). In one example, the analyte is cortisol. A sample is added to a reaction vessel with a complex 120 including a binding member 106 specific for the analyte (for example, an antibody specific to cortisol) conjugated to a reporter 110 (for example, ALP) either directly or optionally via a suitable linker 121 ; and a plurality of particles 100 coated with analyte (for example, cortisol 112), optionally linked to the particles 100 via a suitable linker 204. Analyte 116 in the sample competes with the analyte 112 bound to particles 100 for binding sites on a limited amount of reporter-binding member conjugates 120 (for example ALP conjugated-anti-cortisol antibody), as shown in FIG. 5. As shown in FIG. 5, a first complex 500 is formed between the analyte 112 coated on the particle and the binding member 106 conjugated to a reporter, and a second complex 502 is formed between the analyte 116 in the sample and the binding member 106 conjugated to a reporter. A particle 100, associated with analyte 112 and, optionally, with the binding member 106 conjugated to a reporter, is an example of “the solid phase.”

[0036] Making reference to FIG. 6, after incubation in a reaction vessel, materials bound to the solid phase 400 are retained (for example, held in a magnetic field) while unbound materials are washed away. Then, as shown in FIG. 7, a substrate 302 (for example a chemiluminescent substrate) is added to the vessel (not shown) and light 304 generated by the reaction between the substrate 302 and the reporter from the binding member-reporter conjugate 120 is measured (for example, with a luminometer 306). The light production 304 is inversely proportional to the concentration of cortisol 116 in the sample. The amount of analyte (in one example, cortisol) in the sample may be determined from a stored, multi-point calibration curve. If a different reporter is used, a color change, fluorescence, or other output may be detected instead of light.

[0037] As used herein, “first binding member” and “second binding member” are members of a specific binding pair in which two different molecules, having an area on their surface or in a cavity, which specifically binds to and is thereby defined as complementary with a particular spatial and polar organization of the other molecule. The first binding member and the second binding member may each include an antibody or a fragment thereof.

[0038] Specific binding involves the specific recognition of one molecule over another. On the other hand, non-specific binding involves non-covalent binding between molecules that is relatively independent of specific surface structures. Non-specific binding may result from several factors including hydrophobic and hydrophilic interactions between molecules. Preferred binding partners are antibodies.

[0039] Examples of assays contemplated herein include those where the binding member specific for steroid analytes of interest is an antibody for steroid analytes of interest, which may be a complete immunoglobulin molecule (antibody) or a fragment thereof. Antibodies include various classes and isotypes, such as IgA, IgD, IgE, lgG1 , lgG2a, lgG2b and lgG3, and IgM, and combinations thereof, for example. An antibody of the present disclosure may include antibody composition that is monoclonal or polyclonal.

[0040] Antibody fragments may include Fab, Fv and F(ab'), Fab', a diabody, a triabody, a mini body, a single-domain antibody, a single-chain variable fragment (scFv), a VHH antibody (or nanobody), a domain antibody (dAb) fragment that consists of a VH domain, an Fd fragment (consisting of the VH and CH1 domains), and the like. In addition, aggregates, polymers, and conjugates of immunoglobulins or their fragments can be used where appropriate so long as binding affinity for steroid analytes of interest is retained. Antibodies for steroid analytes of interest may be prepared by techniques including, but not limited to, immunization of a host and collection of sera (polyclonal), preparing hybridomas and collecting the secreted protein (monoclonal) or cloning and expressing nucleotide sequences or mutagenized versions thereof coding at least for the amino acid sequences required for specific binding of natural antibodies, for example.

[0041] The sample to be analyzed is typically one that is suspected of containing steroid analytes of interest. The samples may be biological samples or non- biological samples. Biological samples may be from a mammalian subject or a non-mammalian subject. Mammalian subjects may be, for example, humans or other animal species. Biological samples include biological fluids such as whole blood, serum, plasma, sputum, lymphatic fluid, semen, vaginal mucus, feces, urine, spinal fluid, saliva, stool, cerebral spinal fluid, tears, mucus, and the like; biological tissue such as hair, skin, sections or excised tissues from organs or other body parts; and so forth. In many instances, the sample is whole blood, plasma or serum. Non-biological samples including, but not limited to, waste streams, for example, may also be analyzed using the conjugates described herein (for example, conjugates of the formula (I), (II) or (III)), or antibodies generated using a conjugate of the formula (I), (II) or (III).

[0042] The sample can be prepared in any convenient medium, which may be, for example, an assay medium. In some aspects, the combination in the medium is subjected to conditions for binding of the conjugates described herein (for example, conjugates of the formula (I), (II) or (III)) to the binding member specific for a steroid analyte of interest to form a complex. Additionally or alternatively, the combination in the medium is subjected to conditions for binding of a binding member generated using a conjugate of the formula (I), (II) or (III), wherein the binding member and a steroid analyte of interest to form a complex. The amount of a complex including a reporter is measured, and the amount of the complex measured is related to one or both of the presence and amount of the steroid analytes in the sample.

[0043] In some examples, a sample to be analyzed is combined in an assay medium with an antibody for steroid analytes of interest and a conjugate described herein. The medium is examined for one or both of the presence and amount of a complex including the conjugate and the antibody for steroid analytes of interest where the presence and/or the amount of such complex indicates the presence and/or amount of steroid analytes of interest in the sample.

[0044] In some examples, the sample to be analyzed is subjected to a pretreatment to release steroid analytes of interest from endogenous binding, using substances such as, for example, plasma or serum proteins that bind steroid analytes of interest, such as, for example, globulins and/or albumin. The release of steroid analytes of interest from endogenous binding substances may be carried out, for example, by increasing the temperature of the sample. For example, the temperature of the sample may be increased to 55°C to 65°C for a period of 0.5 hours to 2 hours or 1 hour to 1.5 hours. In another approach, addition of a releasing agent to the sample may be employed to release steroid analytes of interest from endogenous binding substances. Releasing agents include, but are not limited to, sodium salicylate and danazol, for example.

[0045] The conditions such as, for example, duration, temperature, pH, and concentration of the releasing agent in the medium for carrying out the releasing actions are dependent on the nature of the endogenous binding substances, the nature of the sample, and the nature of the releasing agent, for example. In general, the conditions are sufficient to achieve the desired effect or function. An example of an effective concentration of releasing agent is 0.01 mg/mL to 20 mg/mL, or 0.01 mg/mL to 10 mg/mL, or 0.01 mg/mL to 5 mg/mL, or 0.1 mg/mL to 20 mg/mL, or 0.1 mg/mL to 10 mg/mL, or 0.1 mg/mL to 5 mg/mL, or 0.1 mg/mL to 1 mg/mL. The pretreatment of the sample to release steroid analytes of interest from endogenous binding substances may be carried out as a separate step prior to conducting an assay or as a first step in an assay. In either case, one or more reagents may be required to stop the action of the releasing agent.

[0046] The conditions for conducting assays for one or more steroid analytes of interest include carrying out the assay in an aqueous buffered medium at a moderate pH, generally that which provides optimum assay sensitivity. The aqueous medium may be solely water or may include from 0.1 to 40 volume percent of a cosolvent. The pH for the medium will be in the range of 4 to 11 , or in the range of 5 to 10, in the range of 6.5 to 9.5, or in the range of 6.5 to 8, for example. The pH will usually be a compromise between optimum binding of the binding members of any specific binding pairs, the pH optimum for other reagents of the assay, and so forth. Various buffers may be used to achieve the desired pH and maintain the pH during the assay. Illustrative buffers include, by way of illustration and not limitation, borate, phosphate, carbonate, TRIS, barbital, PIPES, HEPES, MES, ACES, MOPS, and BICINE, for example. The particular buffer employed is not critical, but in an individual assay one or another buffer may be preferred.

[0047] Various ancillary materials may be employed in the assay methods. For example, in addition to buffers the medium may include stabilizers for the medium and for the reagents employed. In some embodiments, in addition to these additives, proteins may be included. Such as, for example, albumins; organic solvents such as, for example, formamide; quaternary ammonium salts; polyanions such as, for example, dextran sulfate; binding enhancers, for example, polyalkylene glycols; polysaccharides such as, for example, dextran or trehalose. The medium may also include agents for preventing the formation of blood clots. Such agents are well known in the art and include, but are not limited to, EDTA, EGTA, citrate, heparin, for example. The medium may also include one or more preservatives such as, but not limited to, sodium azide, neomycin sulfate, PROCLINR 300, streptomycin, for example. The medium may additionally include one or more surfactants. Any of the above materials, if employed, is present in a concentration or amount sufficient to achieve the desired effect or function.

[0048] One or more incubation periods may be applied to the medium at one or more intervals including any intervals between additions of various reagents employed in an assay including those mentioned above. The medium is usually incubated at a temperature and for a time sufficient for binding of various components of the reagents and binding of steroid analytes of interest in the sample to occur. In some aspects, the temperature may preferably be held constant for a time sufficient for binding of various components of the reagents and binding of steroid analytes of interest in the sample to occur. In some examples, incubation temperatures range from 5°C to 99°C, or from 15°C to 70°C, from 20°C to 45°C, from 30°C to 40°C, from 35°C to 38°C, or from 36°C to 37°C for example. The time period for the incubation, in some examples, is 0.2 seconds to 24 hours, or 1 second to 6 hours, or 2 seconds to 1 hour, 1 minute to 15 minutes, 1 minute to 10 minutes, or 1 minute to 5 minutes, for example. The time period depends on the temperature of the medium and the rate of binding of the various reagents, which is determined by the association rate constant, the concentration, the binding constant and dissociation rate constant.

[0049] The concentration of steroid analytes of interest in a sample that may be assayed generally varies from 10^-5 M to 10'⁷ M, or from 10'⁶ M to 10'¹⁴ M, for example. Considerations such as whether the assay is qualitative, semiquantitative or quantitative (relative to the amount of steroid analytes of interest present in the sample), the particular detection technique and the expected concentration of steroid analytes of interest normally determine the concentrations of the various reagents.

[0050] The concentrations of the various reagents in the assay medium will generally be determined by the concentration range of interest of steroid analytes of interest, the nature of the assay, and the like. However, the final concentration of each of the reagents is normally determined empirically to optimize the sensitivity of the assay over the range of interest. That is, a variation in concentration of steroid analytes of interest that is of significance should provide an accurately measurable signal difference. Considerations such as the nature of the signal producing system and the nature of the analytes normally determine the concentrations of the various reagents. [0051] As mentioned herein, the sample and reagents are provided in combination in the medium. While the order of addition to the medium may be varied, there will be certain preferences for some embodiments of the assay formats described herein. The simplest order of addition, of course, is to add all the materials simultaneously and determine the effect that the assay medium has on the signal as in a homogeneous assay. Alternatively, each of the reagents, or groups of reagents, can be combined sequentially. In some embodiments, an incubation step may be involved subsequent to each addition as discussed above. In heterogeneous assays, washing steps may also be employed after one or more incubation steps.

[0052] In many embodiments the examination of the medium involves detection of a signal from the medium. The presence and/or amount of the signal is related to the presence and/or amount of steroid analytes of interest in the sample. The particular mode of detection depends on the nature of the signal producing system. As discussed herein, there are numerous methods by which a label of a signal producing signal can produce a signal detectable by external means. Activation of a signal producing system depends on the nature of the signal producing system members. Temperatures during measurements generally range from 10°C to 70°C, from 20°C to 45°C from 20°C to 45°C, from 20°C to 25°C, from 30°C to 40°C, from 35°C to 38°C, or from 36°C to 37°C, for example. In one approach standard curves are formed using known concentrations of steroid analytes of interest. Calibrators and other controls may also be used.

[0053] An output from a reporter including, for example, luminescence or light produced from a label can be measured visually, photographically, actinometrically, spectrophotometrically, such as by using a photomultiplier or a photodiode, or by any other convenient means to determine the amount thereof, which is related to the amount of steroid analytes of interest in the medium. The examination for presence and/or amount of the signal also includes the detection 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, but is not limited to, a spectrophotometer, fluorometer, absorption spectrometer, luminometer, and chemiluminometer, for example.

[0054] A reagent including a conjugate, for example, conjugates of the formula (I), (II) or (III), to an assay molecule and other reagents for conducting a particular assay for steroid analytes of interest may be present in a kit useful for conveniently performing an assay for the determination of steroid analytes of interest. Additionally or alternatively, such a kit may include a binding member generated using a conjugate of the formula (I), (II) or (III), The kit may further include other reagents for performing the assay, the nature of which depend upon the particular assay format.

[0055] The reagents may each be in separate containers or various reagents can be combined in one or more containers depending on the cross-reactivity and stability of the reagents. The kit can further include other separately packaged reagents for conducting an assay such as additional specific binding pair members, signal producing system members, and ancillary reagents, for example. [0056] The relative amounts of the various reagents in the kits can be varied widely to provide for concentrations of the reagents that substantially optimize the reactions that need to occur during the present methods and further to optimize substantially the sensitivity of an assay. Under appropriate circumstances one or more of the reagents in the kit can be provided as a dry powder, lyophilized if necessary or unlyophilized, including excipients, which on dissolution will provide for a reagent solution having the appropriate concentrations for performing a method or assay using a compound reagent described herein. The kit can further include a written description of a method for utilizing reagents that include a compound described herein.

[0057] The term “alkyl” as used herein refers to substituted or unsubstituted straight chain, branched and cyclic, saturated mono- or bi-valent groups having from 1 to 20 carbon atoms, 10 to 20 carbon atoms, 12 to 18 carbon atoms, 6 to 10 carbon atoms, 1 to 10 carbons atoms, 1 to 8 carbon atoms, 2 to 8 carbon atoms, 3 to 8 carbon atoms, 4 to 8 carbon atoms, 5 to 8 carbon atoms, 1 to 6 carbon atoms, 2 to 6 carbon atoms, 3 to 6 carbon atoms, or 1 to 3 carbon atoms. Examples of straight chain mono-valent (Ci-C2o)-alkyl groups include those with from 1 to 8 carbon atoms such as methyl (i.e. , CH3), ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl groups. Examples of branched mono-valent (Ci-C2o)-alkyl groups include isopropyl, iso-butyl, sec-butyl, t-butyl, neopentyl, and isopentyl. Examples of straight chain bi-valent (Ci-C2o)alkyl groups include those with from 1 to 6 carbon atoms such as -CH₂-, -CH2CH2-, -CH₂CH₂CH₂-, -CH2CH2CH2CH2-, and - CH2CH2CH2CH2CH2-. Examples of branched bi-valent alkyl groups include - CH(CH3)CH2- and -CH2CH(CH3)CH2-. Examples of cyclic alkyl groups include cyclopropyl, cyclobutyl, cyclopently, cyclohexyl, cyclooctyl, bicyclo[1 .1 .1 ]pentyl, bicyclo[2.1.1]hexyl, and bicyclo[2.2.1]heptyl. Cycloalkyl groups further include polycyclic cycloalkyl groups such as, but not limited to, norbornyl, adamantyl, bornyl, camphenyl, isocamphenyl, and carenyl groups, and fused rings such as, but not limited to, decalinyl, and the like. In some embodiments, alkyl includes a combination of substituted and unsubstituted alkyl. As an example, alkyl, and also (Ci)alkyl, includes methyl and substituted methyl. As a particular example, (Ci)alkyl includes benzyl. As a further example, alkyl can include methyl and substituted (C2-Cs)alkyl. Alkyl can also include substituted methyl and unsubstituted (C2-Cs)alkyl. In some embodiments, alkyl can be methyl and C2-C8 linear alkyl. In some embodiments, alkyl can be methyl and C2-C8 branched alkyl. The term methyl is understood to be -CH3, which is not substituted. The term methylene is understood to be -CH2-, which is not substituted. For comparison, the term (Ci)alkyl is understood to be a substituted or an unsubstituted -CH3 or a substituted or an unsubstituted -CH2-. Representative substituted alkyl groups can be substituted one or more times with any of the groups listed herein, for example, cycloalkyl, heterocyclyl, aryl, amino, haloalkyl, hydroxy, cyano, carboxy, nitro, thio, alkoxy, and halogen groups. As further example, representative substituted alkyl groups can be substituted one or more fluoro, chloro, bromo, iodo, amino, amido, alkyl, alkoxy, alkylamido, alkenyl, alkynyl, alkoxycarbonyl, acyl, formyl, arylcarbonyl, aryloxycarbonyl, aryloxy, carboxy, haloalkyl, hydroxy, cyano, nitroso, nitro, azido, trifluoromethyl, trifluoromethoxy, thio, alkylthio, arylthiol, alkylsulfonyl, alkylsulfinyl, dialkylaminosulfonyl, sulfonic acid, carboxylic acid, dialkylamino, and dialkylamido. In some embodiments, representative substituted alkyl groups can be substituted from a set of groups including amino, hydroxy, cyano, carboxy, nitro, thio and alkoxy, but not including halogen groups. Thus, in some embodiments alkyl can be substituted with a non-halogen group. For example, representative substituted alkyl groups can be substituted with a fluoro group, substituted with a bromo group, substituted with a halogen other than bromo, or substituted with a halogen other than fluoro. In some embodiments, representative substituted alkyl groups can be substituted with one, two, three, or more fluoro groups or they can be substituted with one, two, three, or more non-fluoro groups. For example, alkyl can be trifluoromethyl, difluoromethyl, or fluoromethyl, or alkyl can be substituted alkyl other than trifluoromethyl, difluoromethyl or fluoromethyl. Alkyl can be haloalkyl or alkyl can be substituted alkyl other than haloalkyl. The term “alkyl” also generally refers to alkyl groups that can include one or more heteroatoms in the carbon chain. Thus, for example, “alkyl” also encompasses groups such as - [(CH₂)_PO]_qH and the like.

[0058] The term “alkenyl” as used herein refers to substituted or unsubstituted straight chain, branched and cyclic, saturated mono- or bi-valent groups having at least one carbon-carbon double bond and from 2 to 20 carbon atoms, 10 to 20 carbon atoms, 12 to 18 carbon atoms, 6 to 10 carbon atoms, 2 to 10 carbons atoms, 2 to 8 carbon atoms, 3 to 8 carbon atoms, 4 to 8 carbon atoms, 5 to 8 carbon atoms, 2 to 6 carbon atoms, 3 to 6 carbon atoms, 4 to 6 carbon atoms, 2 to 4 carbon atoms, or 2 to 3 carbon atoms. The double bonds can be trans or cis orientation. The double bonds can be terminal or internal. The alkenyl group can be attached via the portion of the alkenyl group containing the double bond, for example, vinyl, propen-1-yl and buten-1-yl, or the alkenyl group can be attached via a portion of the alkenyl group that does not contain the double bond, for example, penten-4-yl. Examples of mono-valent (C2-C2o)-alkenyl groups include those with from 1 to 8 carbon atoms such as vinyl, propenyl, propen-1 -yl, propen- 2-yl, butenyl, buten-1-yl, buten-2-yl, sec-buten-1-yl, sec-buten-3-yl, pentenyl, hexenyl, heptenyl, and octenyl groups. Examples of branched mono-valent (C2- C2o)-alkenyl groups include isopropenyl, iso-butenyl, sec-butenyl, t-butenyl, neopentenyl, and isopentenyl. Examples of straight chain bi-valent (C2-C2o)alkenyl groups include those with from 2 to 6 carbon atoms such as -CHCH-, -CHCHCH2-, -CHCHCH2CH2-, and -CHCHCH2CH2CH2-. Examples of branched bi-valent alkyl groups include -C(CH3)CH- and -CHC(CH3)CH2-. Examples of cyclic alkenyl groups include cyclopentenyl, cyclohexenyl, and cyclooctenyl. It is envisaged that alkenyl can also include masked alkenyl groups, precursors of alkenyl groups or other related groups. As such, where alkenyl groups are described it, compounds are also envisaged where a carbon-carbon double bond of an alkenyl is replaced by an epoxide or aziridine ring. Substituted alkenyl also includes alkenyl groups which are substantially tautomeric with a non-alkenyl group. For example, substituted alkenyl can be 2-aminoalkenyl, 2-alkylaminoalkenyl, 2-hydroxyalkenyl, 2-hydroxyvinyl, 2-hydroxypropenyl, but substituted alkenyl is also understood to include the group of substituted alkenyl groups other than alkenyl which are tautomeric with non-alkenyl containing groups. In some embodiments, alkenyl can be understood to include a combination of substituted and unsubstituted alkenyl. For example, alkenyl can be vinyl and substituted vinyl. For example, alkenyl can be vinyl and substituted (C3-Cs)alkenyl. Alkenyl can also include substituted vinyl and unsubstituted (C3-Cs)alkenyl. Representative substituted alkenyl groups can be substituted one or more times with any of the groups listed herein, for example, monoalkylamino, dialkylamino, cyano, acetyl, amido, carboxy, nitro, alkylthio, alkoxy, and halogen groups. As further example, representative substituted alkenyl groups can be substituted one or more fluoro, chloro, bromo, iodo, amino, amido, alkyl, alkoxy, alkylamido, alkenyl, alkynyl, alkoxycarbonyl, acyl, formyl, arylcarbonyl, aryloxycarbonyl, aryloxy, carboxy, haloalkyl, hydroxy, cyano, nitroso, nitro, azido, trifluoromethyl, trifluoromethoxy, thio, alkylthio, arylthiol, alkylsulfonyl, alkylsulfinyl, dialkylaminosulfonyl, sulfonic acid, carboxylic acid, dialkylamino and dialkylamido. In some embodiments, representative substituted alkenyl groups can be substituted from a set of groups including monoalkylamino, dialkylamino, cyano, acetyl, amido, carboxy, nitro, alkylthio and alkoxy, but not including halogen groups. Thus, in some embodiments alkenyl can be substituted with a non-halogen group. In some embodiments, representative substituted alkenyl groups can be substituted with a fluoro group, substituted with a bromo group, substituted with a halogen other than bromo, or substituted with a halogen other than fluoro. For example, alkenyl can be 1 -fluorovinyl, 2-fluorovinyl, 1 ,2-difluorovinyl, 1 ,2,2- trifluorovinyl, 2,2-difluorovinyl, trifluoropropen-2-yl, 3,3,3-trifluoropropenyl, 1- fluoropropenyl, 1 -chlorovinyl, 2-chlorovinyl, 1 ,2-dichlorovinyl, 1 ,2,2-trichlorovinyl or 2,2-dichlorovinyl. In some embodiments, representative substituted alkenyl groups can be substituted with one, two, three or more fluoro groups or they can be substituted with one, two, three or more non-fluoro groups.

[0059] The term “alkynyl” as used herein, refers to substituted or unsubstituted straight and branched chain alkyl groups, except that at least one triple bond exists between two carbon atoms. Thus, alkynyl groups have from 2 to 50 carbon atoms, 2 to 20 carbon atoms, 10 to 20 carbon atoms, 12 to 18 carbon atoms, 6 to 10 carbon atoms, 2 to 10 carbons atoms, 2 to 8 carbon atoms, 3 to 8 carbon atoms, 4 to 8 carbon atoms, 5 to 8 carbon atoms, 2 to 6 carbon atoms, 3 to 6 carbon atoms, 4 to 6 carbon atoms, 2 to 4 carbon atoms, or 2 to 3 carbon atoms. Examples include, but are not limited to ethynyl, propynyl, propyn-1-yl, propyn-2-yl, butynyl, butyn-1-yl, butyn-2-yl, butyn-3-yl, butyn-4-yl, pentynyl, pentyn-1-yl, hexynyl, Examples include, but are not limited to -C=CH, -C=C(CH3), -C=C(CH₂CH3), -CH₂C=CH, -CH₂C=C(CH₃), and -CH₂C=C(CH₂CH₃) among others.

[0060] The term “aryl” as used herein refers to substituted or unsubstituted univalent groups that are derived by removing a hydrogen atom from an arene, which is a cyclic aromatic hydrocarbon, having from 6 to 20 carbon atoms, 10 to 20 carbon atoms, 12 to 20 carbon atoms, 6 to 10 carbon atoms or 6 to 8 carbon atoms. Examples of (Ce-C₂o)aryl groups include phenyl, napthalenyl, azulenyl, biphenylyl, indacenyl, fluorenyl, phenanthrenyl, triphenylenyl, pyrenyl, naphthacenyl, chrysenyl, anthracenyl groups. Examples include substituted phenyl, substituted napthalenyl, substituted azulenyl, substituted biphenylyl, substituted indacenyl, substituted fluorenyl, substituted phenanthrenyl, substituted triphenylenyl, substituted pyrenyl, substituted naphthacenyl, substituted chrysenyl, and substituted anthracenyl groups. Examples also include unsubstituted phenyl, unsubstituted napthalenyl, unsubstituted azulenyl, unsubstituted biphenylyl, unsubstituted indacenyl, unsubstituted fluorenyl, unsubstituted phenanthrenyl, unsubstituted triphenylenyl, unsubstituted pyrenyl, unsubstituted naphthacenyl, unsubstituted chrysenyl, and unsubstituted anthracenyl groups. Aryl includes phenyl groups and also non-phenyl aryl groups. From these examples, it is clear that the term (Ce-C2o)aryl encompasses mono- and polycyclic (Ce-C2o)aryl groups, including fused and non-fused polycyclic (Ce-C2o)aryl groups.

[0061] The term “heterocyclyl” as used herein refers to substituted aromatic, unsubstituted aromatic, substituted non-aromatic, and unsubstituted non-aromatic rings containing 3 or more atoms in the ring, of which, one or more is a heteroatom such as, but not limited to, N, O, and S. Thus, a heterocyclyl can be a cycloheteroalkyl, or a heteroaryl, or if polycyclic, any combination thereof. In some embodiments, heterocyclyl groups include 3 to 20 ring members, whereas other such groups have 3 to 15 ring members. In some embodiments, heterocyclyl groups include heterocyclyl groups that include 3 to 8 carbon atoms (Cs-Cs), 3 to 6 carbon atoms (Cs-Ce) or 6 to 8 carbon atoms (Ce-Cs). A heterocyclyl group designated as a C2-heterocyclyl can be a 5-membered ring with two carbon atoms and three heteroatoms, a 6-membered ring with two carbon atoms and four heteroatoms and so forth. Likewise, a C4-heterocyclyl can be a 5-membered ring with one heteroatom, a 6-membered ring with two heteroatoms, and so forth. The number of carbon atoms plus the number of heteroatoms equals the total number of ring atoms. A heterocyclyl ring can also include one or more double bonds. A heteroaryl ring is an embodiment of a heterocyclyl group. The phrase “heterocyclyl group” includes fused ring species including those that include fused aromatic and non-aromatic groups. Representative heterocyclyl groups include, but are not limited to piperidynyl, piperazinyl, morpholinyl, furanyl, pyrrolidinyl, pyridinyl, pyrazinyl, pyrimidinyl, triazinyl, thiophenyl, tetrahydrofuranyl, pyrrolyl, oxazolyl, imidazolyl, triazyolyl, tetrazolyl, benzoxazolinyl, and benzimidazolinyl groups. For example, heterocyclyl groups include, without limitation:

(Ci-C2o)alkyl, (Ce-C2o)aryl, or an amine protecting group (for example, a t- butyloxycarbonyl group) and wherein the heterocyclyl group can be substituted or unsubstituted. A nitrogen-containing heterocyclyl group is a heterocyclyl group containing a nitrogen atom as an atom in the ring. In some embodiments, the heterocyclyl is other than thiophene or substituted thiophene. In some embodiments, the heterocyclyl is other than furan or substituted furan.

[0062] The term “alkoxy” as used herein refers to an oxygen atom connected to an alkyl group, including a cycloalkyl group, as are defined herein. Examples of linear alkoxy groups include but are not limited to methoxy, ethoxy, propoxy, butoxy, pentyloxy, hexyloxy, and the like. Examples of branched alkoxy include but are not limited to isopropoxy, sec-butoxy, tert-butoxy, isopentyloxy, isohexyloxy, and the like. Examples of cyclic alkoxy include but are not limited to cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, and the like. An alkoxy group can include one to 12 to 20 or 12 to 40 carbon atoms bonded to the oxygen atom, and can further include double or triple bonds, and can also include heteroatoms. Thus, alkyoxy also includes an oxygen atom connected to an alkyenyl group and oxygen atom connected to an alkynyl group. For example, an allyloxy group is an alkoxy group within the meaning herein. A methoxyethoxy group is also an alkoxy group within the meaning herein, as is a methylenedioxy group in a context where two adjacent atoms of a structure are substituted therewith.

[0063] The term “aryloxy” as used herein refers to an oxygen atom connected to an aryl group as are defined herein.

[0064] The term “aralkyl” and “arylalkyl” as used herein refers to alkyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to an aryl group as defined herein. Representative aralkyl groups include benzyl, biphenylmethyl and phenylethyl groups and fused (cycloalkylaryl)alkyl groups such as 4-ethyl-indanyl. Aralkenyl groups are alkenyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to an aryl group as defined herein.

[0065] The terms “halo,” “halogen,” or “halide” group, as used herein, by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom.

[0066] The term “amine” and “amino” as used herein refers to a substituent of the form -NH2, -NHR, -NR2, -NRs⁺, wherein each R is independently selected, and protonated forms of each, except for -NRs⁺, which cannot be protonated. Accordingly, any compound substituted with an amino group can be viewed as an amine. An “amino group” within the meaning herein can be a primary, secondary, tertiary, or quaternary amino group. An “alkylamino” group includes a monoalkylamino, dialkylamino, and trialkylamino group.

[0067] The term “acyl” as used herein refers to a group containing a carbonyl moiety wherein the group is bonded via the carbonyl carbon atom. The carbonyl carbon atom is also bonded to another carbon atom, which can be part of a substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocyclyl, group, or the like.

[0068] The term “formyl” as used herein refers to a group containing a carbonyl moiety wherein the group is bonded via the carbonyl carbon atom. The carbonyl carbon atom is also bonded to a hydrogen atom.

[0069] The term “alkoxycarbonyl” as used herein refers to a group containing a carbonyl moiety wherein the group is bonded via the carbonyl carbon atom. The carbonyl carbon atom is also bonded to an oxygen atom which is further bonded to an alkyl group. Alkoxycarbonyl also includes the group where a carbonyl carbon atom is also bonded to an oxygen atom which is further bonded to an alkyenyl group. Alkoxycarbonyl also includes the group where a carbonyl carbon atom is also bonded to an oxygen atom which is further bonded to an alkynyl group. In a further case, which is included in the definition of alkoxycarbonyl as the term is defined herein, and is also included in the term “aryloxycarbonyl,” the carbonyl carbon atom is bonded to an oxygen atom which is bonded to an aryl group instead of an alkyl group.

[0070] The term “arylcarbonyl” as used herein refers to a group containing a carbonyl moiety wherein the group is bonded via the carbonyl carbon atom. The carbonyl carbon atom is also bonded to an aryl group.

[0071] The term “alkylamido” as used herein refers to a group containing a carbonyl moiety wherein the group is bonded via the carbonyl carbon atom. The carbonyl carbon atom is also bonded to a nitrogen group which is bonded to one or more alkyl groups. In a further case, which is also an alkylamido as the term is defined herein, the carbonyl carbon atom is bonded to a nitrogen atom which is bonded to one or more aryl group instead of, or in addition to, the one or more alkyl group. In a further case, which is also an alkylamido as the term is defined herein, the carbonyl carbon atom is bonded to a nitrogen atom which is bonded to one or more alkenyl group instead of, or in addition to, the one or more alkyl and or/aryl group. In a further case, which is also an alkylamido as the term is defined herein, the carbonyl carbon atom is bonded to a nitrogen atom which is bonded to one or more alkynyl group instead of, or in addition to, the one or more alkyl, alkenyl and/or aryl group.

[0072] The term “carboxy” as used herein refers to a group containing a carbonyl moiety wherein the group is bonded via the carbonyl carbon atom. The carbonyl carbon atom is also bonded to a hydroxy group or oxygen anion so as to result in a carboxylic acid or carboxylate. Carboxy also includes both the protonated form of the carboxylic acid and the salt form. For example, carboxy can be understood as COOH or CO₂H.

[0073] The term “amido” or “amide” as used herein refers to a group having the formula C(O)NRR, wherein R is defined herein and can each independently be, for example, hydrogen, alkyl, aryl or each R, together with the nitrogen atom to which they are attached, form a heterocyclyl group.

[0074] The term “alkylthio” as used herein refers to a sulfur atom connected to an alkyl, alkenyl, or alkynyl group as defined herein.

[0075] The term “arylthio” as used herein refers to a sulfur atom connected to an aryl group as defined herein.

[0076] The term “alkylsulfonyl” as used herein refers to a sulfonyl group connected to an alkyl, alkenyl, or alkynyl group as defined herein.

[0077] The term “alkylsulfinyl” as used herein refers to a sulfinyl group connected to an alkyl, alkenyl, or alkynyl group as defined herein.

[0078] The term “dialkylaminosulfonyl” as used herein refers to a sulfonyl group connected to a nitrogen further connected to two alkyl groups, as defined herein, and which can optionally be linked together to form a ring with the nitrogen. This term also includes the group where the nitrogen is further connected to one or two alkenyl groups in place of the alkyl groups.

[0079] The term “dialkylamino” as used herein refers to an amino group connected to two alkyl groups, as defined herein, and which can optionally be linked together to form a ring with the nitrogen. This term also includes the group where the nitrogen is further connected to one or two alkenyl groups in place of the alkyl groups.

[0080] The term “dialkylamido” as used herein refers to an amido group connected to two alkyl groups, as defined herein, and which can optionally be linked together to form a ring with the nitrogen. This term also includes the group where the nitrogen is further connected to one or two alkenyl groups in place of the alkyl groups.

[0081] The term “substituted” as used herein refers to a group that is substituted with one or more groups including, but not limited to, the following groups: halogen (for example, F, Cl, Br, and I), R, OR, ROH (for example, CH₂OH), OC(O)N(R)₂ (also known as carbamate), CN, NO, NO₂, ONO₂, azido, CF3, OCF3, methylenedioxy, ethylenedioxy, (C3-C₂o)heteroaryl, N(R)₂, Si(R)s, SR, SOR, SO₂R, SO₂N(R)₂, SO3R, P(O)(OR)₂, OP(O)(OR)_2I C(O)R, C(O)C(O)R, C(O)CH₂C(O)R, C(S)R, C(O)OR, OC(O)R, C(O)N(R)₂, C(O)N(R)OH, OC(O)N(R)₂, C(S)N(R)₂, (CH₂)_0-2N(R)C(O)R, (CH₂)O-₂N(R)N(R)₂, N(R)N(R)C(O)R, N(R)N(R)C(O)OR, N(R)N(R)CON(R)₂, N(R)SO₂R, N(R)SO₂N(R)₂, N(R)C(O)OR, N(R)C(O)R, N(R)C(S)R, N(R)C(O)N(R)₂, N(R)C(S)N(R)₂, N(COR)COR, N(OR)R,

C(=NH)N(R)₂, C(O)N(OR)R, or C(=NOR)R wherein R can be hydrogen, (C1- C₂o)alkyl, (C6-C₂o)aryl, heterocyclyl or polyalkylene oxide groups, such as polyalkylene oxide groups of the formula -(CH₂CH₂O)f-R-OR, -(CH₂CH₂CH₂O)_g-R- OR, -(CH₂CH₂O)f(CH₂CH₂CH₂O)g-R-OR each of which can, in turn, be substituted or unsubstituted and wherein f and g are each independently an integer from 1 to 50 (for example, 1 to 10, 1 to 5, 1 to 3 or 2 to 5). Substituted also includes a group that is substituted with one or more groups including, but not limited to, the following groups: fluoro, chloro, bromo, iodo, amino, amido, alkyl, hydroxy, alkoxy, alkylamido, alkenyl, alkynyl, alkoxycarbonyl, acyl, formyl, arylcarbonyl, aryloxycarbonyl, aryloxy, carboxy, haloalkyl, hydroxy, cyano, nitroso, nitro, azido, trifluoromethyl, trifluoromethoxy, thio, alkylthio, arylthiol, alkylsulfonyl, alkylsulfinyl, dialkylaminosulfonyl, sulfonic acid, carboxylic acid, dialkylamino, and dialkylamido. Where there are two or more adjacent substituents, the substituents can be linked to form a carbocyclic or heterocyclic ring. Such adjacent groups can have a vicinal or germinal relationship, or they can be adjacent on a ring in, for example, an orthoarrangement. Each instance of substituted is understood to be independent. For example, a substituted aryl can be substituted with bromo and a substituted heterocycle on the same compound can be substituted with alkyl. It is envisaged that a substituted group can be substituted with one or more non-fluoro groups. As another example, a substituted group can be substituted with one or more non- cyano groups. As another example, a substituted group can be substituted with one or more groups other than haloalkyl. As yet another example, a substituted group can be substituted with one or more groups other than tert-butyl. As yet a further example, a substituted group can be substituted with one or more groups other than trifluoromethyl. As yet even further examples, a substituted group can be substituted with one or more groups other than nitro, other than methyl, other than methoxymethyl, other than dialkylaminosulfonyl, other than bromo, other than chloro, other than amido, other than halo, other than benzodioxepinyl, other than polycyclic heterocyclyl, other than polycyclic substituted aryl, other than methoxycarbonyl, other than alkoxycarbonyl, other than thiophenyl, or other than nitrophenyl, or groups meeting a combination of such descriptions. Further, substituted is also understood to include fluoro, cyano, haloalkyl, tert-butyl, trifluoromethyl, nitro, methyl, methoxymethyl, dialkylaminosulfonyl, bromo, chloro, amido, halo, benzodioxepinyl, polycyclic heterocyclyl, polycyclic substituted aryl, methoxycarbonyl, alkoxycarbonyl, thiophenyl, and nitrophenyl groups.

[0082] In some instances, the compounds described herein (for example, compounds of the formulae (l)-(lll)) can contain chiral centers. All diastereomers of the compounds described herein are contemplated herein, as well as racemates.

[0083] Each embodiment described herein is envisaged to be applicable in each combination with other embodiments described herein. For example, embodiments corresponding to formula (I) are equally envisaged as being applicable to formulae (II) and (III). Likewise, embodiments corresponding to formula (II) are equally envisaged as being applicable to formulae (I) and (III), and so forth.

[0084] Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” As used herein in connection with a measured quantity, the term “about” refers to that variation in the measured quantity as would be expected by the skilled artisan making the measurement and exercising a level of care commensurate with the objective of the measurement and the precision of the measuring equipment used. Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0085] Values expressed in a range format should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range were explicitly recited. For example, a range of “0.1 % to 5%” or “0.1 % to 5%” should be interpreted to include not just as 0.1 % to 5%, but also the individual values (for example, 1%, 2%, 3%, and 4%) and the sub-ranges (for example, 0.1 % to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the indicated range. In addition, the term “in the range” or “within a range” (and similar statements) includes the endpoints of the stated range. Herein, for example, “up to” a number (for example, “up to 50”) includes the number (for example, 50).

[0086] In this document, the terms “a,” “an,” or “the” are used to include one or more than one unless the context clearly dictates otherwise. The term “or” is used to refer to a nonexclusive “or” unless otherwise indicated. Further, term “and/or” means one or all of the listed elements or a combination of any two or more of the listed elements.

[0087] In addition, it is to be understood that the phraseology or terminology employed herein, and not otherwise defined, is for the purpose of description only and not of limitation. Any use of section headings is intended to aid reading of the document and is not to be interpreted as limiting. Further, information that is relevant to a section heading may occur within or outside of that particular section. Furthermore, all publications, patents, and patent documents referred to in this document are incorporated by reference herein in their entirety, as though individually incorporated by reference. In the event of inconsistent usages between this document and those documents so incorporated by reference, the usage in the incorporated reference should be considered supplementary to that of this document; for irreconcilable inconsistencies, the usage in this document controls. [0088] In the methods described herein, the steps may be carried out in any order without departing from the principles of the invention, except when a temporal or operational sequence is explicitly recited. Furthermore, specified steps may be carried out concurrently unless explicit claim language recites that they be carried out separately. For example, a claimed step of doing X and a claimed step of doing Y may be conducted simultaneously within a single operation, and the resulting process will fall within the literal scope of the claimed process. [0089] The term “comprises” and variations thereof do not have a limiting meaning where these terms appear in the description and claims. Such terms will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.

[0090] By “consisting of” is meant including, and limited to, whatever follows the phrase “consisting of.” Thus, the phrase “consisting of” indicates that the listed elements are required or mandatory, and that no other elements may be present. By “consisting essentially of” is meant including any elements listed after the phrase and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase “consisting essentially of” indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they materially affect the activity or action of the listed elements.

[0091] Those skilled in the art will appreciate that many modifications to the embodiments described herein are possible without departing from the spirit and scope of the present disclosure. Thus, the description is not intended and should not be construed to be limited to the examples given but should be granted the full breadth of protection afforded by the appended claims and equivalents thereto. In addition, it is possible to use some of the features of the present disclosure without the corresponding use of other features. Accordingly, the foregoing description of or illustrative embodiments is provided for the purpose of illustrating the principles of the present disclosure and not in limitation thereof and may include modification thereto and permutations thereof.

EXAMPLES

[0092] The present disclosure can be better understood by reference to the following examples which are offered by way of illustration. The disclosure is not limited to the examples given herein.

Example 1

[0093] Progesterone derivatives of the disclosure can be synthesized as shown in Scheme 1 :

[0094] ¹H-NMR (CDC , 400MHz), 6 = 5.89 (s, 1 H), 5.04 (d, J = 3Hz, 1 H), 4.92 (d,

J = 4Hz, 1 H), 2.11 (s, 3H), 1.07 (s, 3H), 0.64 (s, 3H). MS: 327 (M⁺+1).

[0095] ¹H-NMR (CDCh, 400MHz), 5 = 5.77 (s, 1 H), 3.16-3.08 (m, 2H), 2.96-2.01 (m, 1 H), 2.11 (s, 3H), 1.46 (s, 9H), 1.17 (s, 3H), 0.66 (s, 3H). MS: 497 (M⁺+Na).

[0096] ¹H-NMR (CDCh, 400MHz), 5 = 5.96 (s, 1 H), 3.28 (s, 2H), 2.97-2.93 (m, 1 H), 2.71-2.68 (m, 1 H), 2.18 (s, 3H), 1.19 (s, 3H), 0.67 (s, 3H). MS: 417 (M⁺-1).

[0097] ¹H-NMR (CDCI3, 400MHz), 5 = 5.80 (s, 1 H), 3.46-3.41 (m, 2H), 3.05-3.01 (m, 1 H), 2.86 (br, 4H), 2.04 (s, 3H), 1.19 (s, 3H), 0.67 (s, 3H). MS: 516 (M⁺).

Example 2 [0098] Adrenosterone derivatives of the disclosure can be synthesized as shown in Scheme 2:

[0099] ¹H-NMR (CDCI3, 400MHz), 6 = 5.92 (s, 1 H), 5.09 (d, J = 4Hz, 1 H), 4.98 (d, J = 4Hz, 1 H), 1 .11 (s, 3H), 0.91 (s, 3H).

[00100] ¹H-NMR (CDCI3, 400MHz), 5 = 5.78 (s, 1 H), 3.20-3.14 (m, 2H), 3.09-2.96 (m, 1 H), 1.47 (s, 9H), 1.23 (s, 3H), 0.91 (s, 3H). MS: 485 (M⁺+K).

[00101] ¹H-NMR (CDCh, 400MHz), 6 = 5.99 (s, 1 H), 3.31-3.28 (m, 2H), 3.04-2.97 (m, 2H), 1.22 (s, 3H), 0.95 (s, 3H). MS: 389 (M⁺-1).

[00102] ¹H-NMR (CDCh, 400MHz), 5 = 5.82 (s, 1 H), 3.54-3.44 (m, 2H), 3.08-3.04 (m, 1 H), 2.86 (br, 4H), 1.25 (s, 3H), 0.92 (s, 3H). MS: 488 (M⁺+1).

Example 3

[00103] Derivatives of the disclosure also include the product shown in Scheme 3:

[00104] ¹H-NMR (CDCh, 400MHz), 6 = 5.87 (s, 1 H), 5.07 (d, J = 3Hz, 1 H), 4.95 (d, J = 3Hz, 1 H), 2.10 (s, 3H), 1.23 (s, 3H), 0.86 (s, 3H). MS: 341 (M⁺+1).

[00105] ¹H-NMR (CDCh, 400MHz), 6 = 5.75 (s, 1 H), 3.18-3.06 (m, 2H), 2.93 (m, 1 H), 2.04 (s, 3H), 1.46 (s, 9H), 1.40(s, 3H), 0.89 (s, 3H). MS: 489 (M⁺+1).

[00106] ¹H-NMR (CDCh, 400MHz), 5 = 5.98(s, 1 H), 3.30-3.19(m, 2H), 2.96 (m, 1 H), 2.88 (m, 1 H), 2.17 (s, 3H), 1.41 (s, 3H), 0.90 (3, 3H).

[00107] ¹H-NMR (CDCh, 400MHz), 5 = 5.77 (s, 1 H), 3.55-3.40 (m, 2H), 3.05 (m, 1 H), 2.86 (br, 4H), 2.11 (s, 3H), 1.39 (s, 3H), 0.89 (s, 3H). MS: 530 (M⁺).

Example 4

[00108] 17-alpha-hydroxy progesterone derivatives of the disclosure can be synthesized as shown in Scheme 4:

[00109] ¹H-NMR (CDCh, 400MHz), 6 = 5.87 (s, 1 H), 5.03 (d, J = 3 Hz, 1 H), 4.91 (d, J =3Hz, 1 H), 3.05 (s, 1 H), 2.24 (s, 3H), 1.06 (s, 3H), 0.70 (s, 3H).

[00110] ¹H-NMR (CDCh, 400MHz), 5 = 5.77 (s, 1 H), 3.17-3.10 (m, 2H), 2.92 (m, 1 H), 2.28 (s, 3H), 1.47 (s, 9H), 1.18 (s, 3H), 0.76 (s, 3H).

[00111] ¹H-NMR (CDCh, 400MHz), 5 = 5.95 (s, 1 H), 3.30-3.20 (m, 2H), 3.01 (m, 1 H), 2.36 (s, 3H), 1.20 (s, 3H), 0.77 (s, 3H). MS: 433(M⁺-1).

[00112] ¹H-NMR (CDCh, 400MHz), 6 = 5.80 (s, 1 H), 3.49-3.40 (m, 2H), 3.05 (m, 1 H), 2.86 (br, 4H), 2.28 (s, 3H), 1.19 (s, 3H), 0.76 (s, 3H). MS: 532 (M⁺+1).

Example 5

[00113] This example shows the preparation of an NHS ester compound.

[00114] ¹H-NMR (CDCh, 400MHz), 5 = 5.82 (s, 1 H), 2.95-2.80 (m, 2H),

1.22 (s, 3H), 0.91 (s, 3H). MS: 403 (M⁺-1).

[00115] ¹H-NMR (CDCh, 400MHz), 5 = 5.77 (s, 1 H), 3.10-3.03 (m, 2H), 2.95-2.80 (m, 2H), 2.85 (, 4H), 1.22 (s, 3H), 0.76 (s, 3H). MS: 502 (M⁺+1).

Example 6

[00116] This example shows the preparation of an NHS ester compound that can, in turn, be conjugated to add the group G.

Scheme 6

[00117] ¹H-NMR (CDCh, 400MHz), 6 = 5.80 (s, 1 H), 2.90-2.60 (m, 8H), 2.12 (S, 3H), 1.15 (s, 3H), 0.66 (s, 3H). MS: 431 (M⁺-1).

[00118] ¹H-NMR (CDCh, 400MHz), 5 = 5.75 (s, 1 H), 3.10-3.0 (m, 1 H), 2.96-2.82 (m, 6H), 2.84 (s, 4H), 2.11 (S, 3H), 1.15 (s, 3H), 0.66 (s, 3H). MS: 530 (M⁺+1).

Example 7

[00119] This example shows the preparation of a compound comprising the group:

that can, in turn, be conjugated to add the group G.

[00122] ¹H-NMR (CDCh, 400MHz), 6 = 6.82 (s, 2H), 5.80 (br, 1 H), 5.78 (s, 1 H), 3.53-3.40 (m, 4H), 2.84-2.80 (m, 1 H), 2.68-2.60 (m, 2H), 1.09 (s, 3H), 0.92 (s, 3H). MS: 569 (M⁺+1).

Example 8

[00123] This example shows the preparation of a compound comprising the group:

that can, in turn, be conjugated to add the group G.

[00124] ¹H-NMR (CDCh, 400MHz), 5 = 5.77 (s, 1 H), 4.85 (br, 1 H), 3.30 (m, 2H), 2.86-2.78 (m, 1 H), 2.66-2.62 (m, 2H), 2.54-2.48 (m, 2H), 2.12 (s, 3H), 1.15 (s, 3H), 0.67 (s, 3H). MS: 504 (M⁺+1).

[00125] ¹H-NMR (CDCI3, 400MHz), 6 = 5.88 (s, 1 H), 3.30 (m, 2H), 2.96- 2.84 (m, 3H), 2.60-2.54 (m, 2H), 2.46-2.40 (m, 2H), 2.15 (s, 3H), 1.15 (s, 3H), 0.67 (s, 3H). MS: 405 (M⁺+1).

[00126] ¹H-NMR (CDCI3, 400MHz), 5 = 6.86 (s, 2H), 5.78 (s, 1 H), 4.69 (s, 1 H), 3.53-3.50 (m, 4H), 2.84-2.80 (m, 1 H), 2.68-2.40 (m, 4H), 2.12 (s, 3H), 1.09 (s, 3H), 0.67 (s, 3H). MS: 597 (M⁺+1).

Example 9

[00127] This example provides a synthetic method used for a hydrocortisol derivative according to the disclosure as described in Scheme 9:

[00129] ¹H-NMR (CDCI₃, 400MHz), 5 = 5.87 (s, 1H), 5.69 (s, 1H), 5.10 (d, J = 2Hz, 1H), 5.04 (d, J = 17.2Hz, 1H), 5.01 (d, J = 3Hz, 1H), 4.69 (d, J = 17.3Hz, 1H), 2.85-2.77 (m, 1H), 1.99 (s, 3H), 1.18 (s, 3H), 0.87 (s, 3H).

[00130] ¹H-NMR (CDCh, 400MHz), 6 = 5.74 (s, 1H), 5.68 (s, 1H), 5.05 (d, J = 17.2Hz, 1H), 4.68 (d, J = 17.2Hz, 1H), 3.10 (dd, Ji = 22Hz, J₂ = 7.2Hz, 2H), 2.95- 2.90 (m, 1H), 2.85-2.60 (m, 1H), 2.15 (s, 3H), 1.47 (s, 3H), 0.82 (s, 3H).

[00131] ¹H-NMR (CDCh, 400MHz), 5 = 5.71 (s, 1H), 4.65 (d, J = 17.2Hz, 1H), 4.47 (s, 1H), 4.30 (d, J = 17.2Hz, 1H), 3.11 (dd, Ji = 22Hz, J₂ = 7.2Hz, 2H), 2.93-2.89 (m, 1H), 1.25 (s, 3H), 0.87 (s, 3H). MS: 561 (M⁺ + K⁺)

[00132] ¹H-NMR (CD3OD, 400MHz), 5 = 5.70 (s, 1H), 4.62 (d, J = 17.2Hz, 1H), 4.39 (s, 1H), 4.26 (d, J = 17.2Hz, 1H), 3.25 (d, J = 2.4Hz, 2H), 2.96-2.91 (m, 1H), 2.85-2.66 (m, 2H), 2.65-2.60 (m, 1H), 1.42 (s, 3H), 0.88 (3, 3H). MS: 465 (M⁺- 1).

[00133] ¹H-NMR (CDCI3, 400MHz), 6 = 5.73 (s, 1 H), 4.65 (dd, Ji = 17.2Hz, J₂ = 4Hz, 1 H), 4.32 (s, 1 H), 4.30 (dd, Ji = 17.2Hz, J₂ = 4Hz, 1 H), 3.49 (dd, Ji = 23.2Hz, J₂ = 8.2Hz, 2H), 3.1-3.0 (m, 2H), 2.86 (s, 4H), 2.80-2.65 (m, 2H), 1.41 (s, 3H), 0.96 (s, 3H). MS: 564 (M⁺+1).

Example 10

[00134] This example shows the preparation of an ester compound that can, in turn, be conjugated to add the group G.

[00136] 0.1 mmol of 6-methyleme-steroid in 20mL of CH3CN, with stirring under Argon, 0.5 mmol of K₂COs added, continue to stir for 1 hour, the thiol compound (0.15 mmol) added, keep stirring at room temperature under Argon for 1-3 days, check TLC until the 6-methylene steroids disappear, then concentrate in vacuo to remove the solvent, the residue diluted with 50 mL of ethyl acetate, washed with brine (20 mL). The organic phase dried over Na₂SC>4 and concentrated in vacuo, the residue purified by preparative TLC plate (20cm x 20cm, 500pm), using ethyl acetate/hexane system to develop the TLC plate.

[00137] ¹H-NMR (CDCI₃, 500MHz), 6 = 5.72 (s, 1 H), 5.04 (d, J = 20Hz, 1 H), 4.86 (d, J = 20Hz, 1 H), 4.50 (d, J = 5 Hz, 1 H), 3.74 (s, 3H), 3.10-3.05 (m, 1 H), 2.95-2.90 (m, 2H), 2.85-2.60 (m, 4H), 2.20 (s, 3H), 1.48 (s, 3H), 0.90 (s, 3H). MS: 537 (M⁺ +1)

Example 11

[00138] This example shows the preparation of the steroid precursor used as a starting material in Examples 12, 13, and 14.

[00139] A suspension of sodium acetate (1.0g) in Chloroform (30mL) containing formaldehyde dimethyl acetal (20 mL), then phosphoryl chloride (3mL) added. The mixture was stirred at reflux for 1 hour. Then steroid starting material (3 mmol) was added, continue to stir at reflux 4~6 hours. The suspension was allowed to cool at room temperature, with vigorous stirring, sodium carbonate is added dropwise until pH = 10, the organic layer separated, the aqueous layer extracted with dichloromethane (25 mL x 3), combined with organic phase, dried with sodium sulfate. Concentrated in vacuo, purified the mixture via silica column, elution with ethyl acetate- hexane system.

[00140] The foregoing synthetic method was adapted from Annen et al.,

5.95 (s, 1 H), 5.14 (d, Ji = 20Hz, J₂ = 5Hz, 1 H), 5.02 (s, 1 H), 4.70 (d, J = 15Hz, 1 H), 4.25-4.20 (m, 2H), 3.74 (s, 3H), 3.10-3.05 (m, 1 H), 2.95-2.90 (m, 3H), 2.85-2.60 (m, 4H), 2.19 (s, 3H), 1.42 (s, 3H), 0.70 (s, 3H).

Example 12

[00142] This example shows the preparation of an ester compound that can, in turn, be conjugated to add the group G.

(dd, Ji = 15Hz, J₂ = 5Hz, 1 H), 4.68 (d, J = 15Hz, 1 H), 3.77 (s, 0.6H), 3.73 (S, 2.4H), 2.95-2.90 (m, 1 H), 2.85-2.60 (m, 4H), 2.19 (s, 3H), 1.48 (s, 3H), 0.71 (s, 3H). MS: 535 (M⁺ +1).

Example 13

[00144] This example shows the preparation of an ester compound that can, in turn, be conjugated to add the group G.

(dd, Ji = 20Hz, J₂ = 5Hz, 1 H), 4.68 (d, J = 15Hz, 1 H), 3.73 (s, 1.2H), 3.71 (S, 1.8H), 2.95-2.90 (m, 1 H), 2.85-2.60 (m, 1 H), 2.19 (s, 3H), 1.48 (s, 3H), 0.72 (s,

3H). MS: 549 (M⁺ +1). Example 14

[00146] This example shows the preparation of a chlorophenyl compound that can, in turn, be conjugated to add the group G.

[00149] To a solution of the substrate (I) in anhydrous EtOH, a stock solution of triflic acid in EtOH (1 M, 1 eq), and a stock solution of 30% hydrogen peroxide (1 M, 2eq) were added. The reaction was stirred at room temperature, with control by ESI-MS. The starting material disappeared after 30 min. The reaction was quenched with the addition of water and extracted with EtOAc. The combined extract was passed through a short plug of silica mixed with dry Na2COs. Volatiles were removed in a vacuum. Yield 81 %.

[00150] ¹H-NMR (CDCh, 500MHz), 5 = 5.80 (s, 1 H), 3.74 (s, 3H), 2.88 (m, 1 H), 2.80 (t, J = 7.3Hz, 2H), 2.65 (t, J = 7.3Hz, 2H), 2.57-2.35 (m, 1 H), 2.95-2.35 (m, 5H), 2.30 (dt, Ji = 12.3Hz, J₂ =3.7Hz, 1 H), 2.16-1.99 (m, 4H), 1.89 (m, 1 H), 1.62 (s, 3H), 1.47 (m, 1 H), 1.35-1.28 (m, 2H), 1.23 (s, 3H), 1.05 (m, 1 H), 0.95 (s, 3H). MS: 435 (M⁺+1).

Example 16

[00151] Steroids are immunogenic haptens and, when conjugated to protein carriers, can produce specific anti-steroid antibody responses. The resulting antibodies may be used in immunoassays, examples of which are shown in FIGS. 1-7. The resulting polyclonal antibodies may be used; or, in some cases, a monoclonal antibody (for example, a monoclonal antibody further selected from the resulting polyclonal antibodies) may be used.

[00152] This Example describes exemplary competitive immunoassays. The competitive immunoassays may include the conjugates of the formula (I), (II) or (III), described herein, or may include antibodies generated using the conjugates of the formula (I), (II) or (III), described herein.

[00153] FIGS. 1 and 2 are a schematic of an exemplary competitive binding immunoenzymatic assay to detect an analyte, which in this example is cortisol. Making reference to FIG. 1 , a sample was added to a reaction vessel (not shown, but can be any suitable vial) with rabbit antibody (first binding member 106) specific to cortisol 116, cortisol-alkaline phosphatase conjugate (cortisol-reporter conjugate 108), which can include cortisol 112 conjugated to alkaline phosphatase (reporter 110) either directly or, optionally, via a suitable linker 114, and a plurality of paramagnetic particles (particles 100) coated with goat anti-rabbit capture antibody (second binding member 102), optionally linked to the paramagnetic particles (particles 100) via a suitable linker 104. Cortisol 116 in the sample competes with the cortisol-alkaline phosphatase conjugate (cortisol-reporter conjugate 108) for binding sites on a limited amount of specific anti-cortisol antibody (first binding member 106). Making reference to FIG. 2, resulting antigemantibody complexes 118 (including cortisol 116) and 120 (including cortisol-alkaline phosphatase conjugate (cortisol-reporter conjugate 108)) bound to the capture antibody (second binding member 102). A paramagnetic particle (particle 100), goat anti-rabbit capture antibody (second binding member 102), with the second binding member 102 optionally linked to the paramagnetic particles 100 via a suitable linker 104, is an example of “the solid phase.”

[00154] Making reference to FIG. 2, after incubation (for example, at 36- 37°C for ten minutes) in a reaction vessel, the bound phase 300 including materials (for example, cortisol 116, cortisol-alkaline phosphatase (ALP) conjugate (cortisol- reporter conjugate 108), and rabbit antibody specific to cortisol (first binding member 106)) bound to the solid phase 200 were held in a magnetic field while unbound materials (not shown) were washed away (for example, with a suitable buffer). Making reference to FIG. 3, a chemiluminescent substrate 302 was added to the vessel (not shown) and light 304 generated by the reaction between the chemiluminescent substrate 302 and the ALP from the cortisol-ALP conjugate (cortisol-reporter conjugate 108) was measured with a luminometer 306. The light production 304 was inversely proportional to the concentration of analyte (in this example, cortisol) in the sample. The amount of analyte in the sample was determined from a stored, multi-point calibration curve.

[00155] Suitable chemiluminescent substrates include chemiluminescent dioxetanes such as those described in Published PCT Appl. No. WO 2021/086977. [00156] FIG. 4 shows an alternative competitive binding assay to detect cortisol. A sample is added to a reaction vessel with a complex 120 including an antibody specific to cortisol (binding member 106 specific for the steroid) conjugated to ALP (reporter 110) either directly or optionally via a suitable linker 121 ; and a plurality of paramagnetic particles (particle 100) coated with cortisol 112, optionally linked to the paramagnetic particles (particle 100) via a suitable linker 204. Cortisol 116 in the sample competes with the cortisol 112 bound to paramagnetic particles (particle 100) for binding sites on a limited amount of ALP conjugated-antibody to cortisol 120, as shown in FIG. 5. As shown in FIG. 5, a first complex 500 is formed between the steroid 112 coated on the particle 100 and the binding member 106 conjugated to a ALP, and a second complex 502 is formed between the steroid 116 in the sample and the binding member 106 conjugated to ALP. A particle 100, associated with cortisol 112 and, optionally, with an antibody specific to cortisol conjugated to ALP, is an example of “the solid phase.”

[00157] Making reference to FIG. 6, after incubation (for example, at 36- 37°C for ten minutes) in a reaction vessel, materials bound to the solid phase 400 are held in a magnetic field while unbound materials are washed away. Then, as shown in FIG. 7, a chemiluminescent substrate 302 is added to the vessel (not shown) and light 304 generated by the reaction is measured with a luminometer

306. The light production 304 is inversely proportional to the concentration of cortisol 116 in the sample. The amount of analyte (in this example, cortisol) in the sample may be determined from a stored, multi-point calibration curve.

Example 17

[00158] A rabbit was immunized by injecting the rabbit with an adjuvant and the following conjugate:

(referred to herein as the “cortisol-7-BSA conjugate”).

[00159] Antiserum comprising antibodies to the cortisol-7-BSA conjugate was isolated from blood of the immunized rabbit using methods known in the art. The antiserum was diluted in BSA buffer (HEPES) at 1 :5,000 serum to buffer.

[00160] Cortisol-7-ALP and cortisol-6-ALP were diluted in BSA buffer at 150 ng/mL and 400 ng/m, respectively:

Cortisol-7-ALP conjugate Cortisol-6-ALP conjugate

Samples comprising cortisol were assayed using methods known in the art that employ paramagnetic particles coated with goat anti-rabbit capture antibody.

Those coated paramagnetic particles are mixed with rabbit antibodies raised against cortisol-7-BSA, and either cortisol-7-ALP or cortisol-6-ALP. After incubation and washing to remove excess reactants, the bound conjugate was detected with LUMI-PHOS 530 (Lumigen, Inc., Southfield, Ml), a chemiluminescent substrate.

[00161] Light generated by the reaction between the chemiluminescent substrate from the cortisol-ALP conjugate in relative luminescence units (RLU) was measured. The relative light units (RLU) data from the assay is shown in Table 1 , below. The data from Table 1 were fitted using a four-parameter logistic to generate calibration curves that were, in turn, used to generate the CV conversion factors shown in Table 2.

Table 1

¹Average RLU across two replicates.

²Access cortisol assay available from Beckman Coulter, Inc.

³Animal immunization and ALP conjugate use a homologous cortisol derivative.

Table 2

[00162] The cortisol concentration range of 1-60 ng/mL was used to generate the data in Tables 1 and 2 because those ranges may be considered medically relevant. See, for example, ACCESS Immunoassay Systems, Access Cortisol Ref. 33600 (available online at www. beckmancoulter. com/download/phxC78424B-EN_US?type=pdf).

[00163] The data in Table 2 show increased precision (reduced error) when 6-ALP-cortisol conjugate is used with antibodies from an animal immunized with the 7-BSA-cortisol conjugate (compared with the results when 7-ALP-cortisol conjugate is used with antibodies from an animal immunized with the 7-BSA- cortisol conjugate). The increased precision is evidenced by a decrease in the CV conversion factor when the cortisol-6-ALP conjugate is used relative to when the cortisol-7-ALP conjugate is used. The CV conversion factors at the cortisol concentrations used herein are 25% smaller when one uses the cortisol-6-ALP conjugate (versus a cortisol-6-ALP conjugate) when the animal has been immunized with the cortisol-7-BSA conjugate. One can therefore expect up to a 25% decrease in error and an average of 12% decrease in error by using the cortisol-6-ALP conjugate at medically relevant cortisol concentrations.

[00164] The data in Table 2, as evidenced by the lower S/SO particularly at the higher antigen concentrations, indicate that antigen displacement is better with the cortisol-6-ALP conjugate. This result demonstrates that one obtains better displacement of the cortisol-ALP conjugate by cortisol when heterologous (different) cortisol derivatives are used to make the immunogen and the ALP conjugate. While not wishing to be bound by any specific theory, it is believed that one observes better displacement because there is a population of antibodies in the antiserum that recognize the conjugation chemistry as opposed to the cortisol portion of the cortisol-ALP conjugate. For example, if one uses cortisol-7-BSA conjugate to immunize and cortisol-7-ALP conjugate in an immunoassay to detect cortisol, there is a population of antibodies generated by the host that recognize the conjugation chemistry, not the cortisol portion of the cortisol-7-ALP conjugate. If that population of antibodies is used to detect a cortisol-7-ALP conjugate in an immunoassay, the portion of antibodies that are recognizing the conjugation chemistry as opposed to cortisol result in increased error in the immunoassay. In contrast, if one uses cortisol-7-BSA conjugate to immunize and cortisol-6-ALP conjugate in the assay, the portion of antibodies that recognize the conjugation chemistry do not recognize the cortisol-6-ALP conjugate, resulting in increased precision (reduced error). Similarly, it is expected that if an animal were immunized with a cortisol-6-BSA conjugate, that one would obtain better immunoassay precision by using a cortisol-7-ALP conjugate in a competition assay to detect cortisol.

Claims

What is claimed is:

1 . A conjugate of the formula (I):

wherein:

R¹ is hydrogen, acyl, or alkoxy;

G is or includes a magnetic particle, a reporter, a carrier protein, or an immunogen.

2. The conjugate of claim 1 , wherein R² is:

R⁵ is methyl or ethyl;

R¹³ is oxygen, ethylene dioxy, or =C-COOR¹⁰;

R¹⁴ is alkyl or haloalkyl; and R¹⁵ is hydroxy, acyloxy, nitrooxy, alkoxy, or an acetal or hemithioacetal group of the formula -OCH(R⁹)(ZR¹⁰); and

R¹⁶ is alkyl, alkenyl or alkynyl, each of which can optionally be substituted by fluorine, chlorine or bromine. A conjugate of the formula (II):

wherein:

R¹⁷ and R¹⁸ are each, independently, H, alklyl, alkenyl, alkynyl, acyl, or OR²² (wherein R²² is H or alkyl), or R¹⁷ and R¹⁸, together with the carbon atom to which they are attached, form a carbonyl;

R¹⁹ is H, alkyl, or acyl;

R²¹ is H or alkyl;

(CH₂)_PX¹ , wherein: t is 0, 1 or 2; n and p are each, independently, an integer from 1 to 10; and

4. The conjugate of claim 3, wherein R¹⁷ is acyl.

5. The conjugate of claim 3, wherein R¹⁷ is alkylacyl.

6. The conjugate of claim 3 or 4, wherein R¹⁷ is -C(O)-R²³-OR^24A or -C(O)R²³, wherein R²³ is alkyl and R^24A is H or alkyl.

7. The conjugate of claim 3, wherein R¹⁷ and R¹⁸, together with the carbon atom to which they are attached, form a carbonyl.

8. The conjugate of any of claims 3-7, wherein R¹⁹ is Ci-Ce-alkyl.

9. The conjugate of claim 3 or 5-7, wherein R¹⁹ is acyl.

10. The conjugate of claim 9, wherein R¹⁹ is -C(O)R^24B, wherein R^24B is H, alkyl, or OR²².

11 . The conjugate of any preceding claim, wherein R²⁰ is H.

12. The conjugate of claims 3-10, wherein R²⁰ is OR²².

13. The conjugate of claim 12, wherein R²² is H.

14. The conjugate of any claims 3-13, wherein R²¹ is Ci-Ce-alkyl.

15. The conjugate of claim 3, wherein the conjugate is a conjugate of the formula:

A conjugate of the formula (III):

wherein:

R²⁵ is a divalent linker of the formula -R²⁶-X²-R²⁷-X¹ , wherein R²⁶ is alkyl and

R²⁷ is alkyl or aryl; X² is O or S(O)t , wherein t is 0, 1 or 2;

X¹ is R^5a, -C(O)-, -C(O)NH-R⁴-, -NHC(O)-R⁴-R^5b or -C(O)NH-R⁴-R^5b wherein R⁴ is alkyl,

R^5a is:

17. The conjugate of claim 16, wherein R²⁶ is alkyl, R²⁷ is alkyl, and X² is S.

18. The conjugate of claim 16 or 17, wherein R²⁵ is:

-(CH₂)n-S-(CH₂)pX¹ or

-(CH₂)n-O-(CH₂)pX¹, wherein n and p are each, independently, an integer from 1 to 10.

19. The conjugate of claims 16-18, wherein R²⁵ comprises or is:

20. The conjugate of claims 16-19, wherein R²⁵ comprises or is:

21. The conjugate of any preceding claim, wherein the carrier protein comprises bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), or ovalbumin (OVA).

22. The conjugate of any of claims 1-21 , wherein the reporter comprises or is an enzyme, a chromogenic reporter, a chemiluminescent reporter, or a fluorescent reporter.

23. The conjugate of claim 22, wherein the enzyme is an alkaline phosphatase (ALP) or a horseradish peroxidase (HRP).

24. A method of determining one or both of the presence and the amount of a steroid in a sample, the method comprising: obtaining or providing in combination, in a medium,

(i) a sample suspected of containing a steroid, (ii) a first binding member specific for the steroid,

(iii) a particle comprising a second binding member specific for the first binding member, and

(iv) a conjugate of any of claims 1-20 or 22-23, the conjugate comprising a reporter; subjecting the combination to conditions for forming a first complex including the first binding member and the steroid, and a second complex including the first binding member and the conjugate, wherein the first complex and the second complex bind to the second binding member; washing, wherein the washing step removes any conjugate not bound to the second binding member via the first binding member and produces a washed composition, and measuring the amount of the reporter present in the washed composition. The method of claim 24, wherein the steroid is cortisol and/or the conjugate comprises a cortisol radical. The method of claim 24 or 25, wherein the reporter is an enzyme reporter. The method of claim 26, wherein the enzyme reporter comprises or is alkaline phosphatase (ALP). The method of any of claims 24-27, wherein the first binding member, the second binding member, or both the first binding member and the second binding member comprise an antibody. A method of determining one or both of the presence and the amount of a steroid in a sample, the method comprising: obtaining or providing in combination, in a medium,

(i) a sample suspected of containing a steroid,

(ii) a binding member specific for the steroid, the binding member comprising a reporter, and

(iii) a conjugate of any of claims 1-20 or 22-23, the conjugate comprising a paramagnetic particle; subjecting the combination to conditions for forming a first complex between a steroid on the conjugate and the binding member and a second complex between the steroid in the sample and the binding member; washing, wherein the washing step removes the second complex and produces a washed composition; and measuring the amount of reporter in the washed composition. The method of claim 29, wherein the steroid is cortisol and/or the conjugate comprises a cortisol radical. The method of claim 29 or 30, wherein the reporter comprises an enzyme reporter. The method of claim 31 , wherein the enzyme reporter comprises or is alkaline phosphatase (ALP). The method of claims 29-32, wherein binding member comprises an antibody. A kit comprising the conjugate of any of claims 1 to 23.