Coumarin-modified androgens for the treatment of prostate cancer

Abstract

Androstane and dihydrotestosterone compounds functionalized with carbocyclic or heterocyclic groups, which may be saturated or unsaturated, are provided. The compounds can be used in methods for inhibiting cell proliferation of malignant and / or hyperplastic cells, and / or for treating individuals with diseases associated with malignant and / or hyperplastic cell proliferation (e.g., cancer, e.g., prostate cancer) and / or diseases associated with hyperplastic cell proliferation, and / or for molecular imaging of malignant and / or hyperplastic cells, and / or for inducing target protein degradation. Compositions are also provided.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 62 / 839,676, filed April 27, 2019, U.S. Provisional Application No. 62 / 844,062, filed May 6, 2019, U.S. Provisional Application No. 62 / 844,073, filed May 6, 2019, and U.S. Provisional Application No. 62 / 890,292, filed August 22, 2019, the disclosures of which are incorporated herein by reference.

[0002] [Statement Regarding Federally Sponsored Research] This invention was made with Government support under Contract Nos. CA77739, P20 RR020171, R21 CA2051, and P30 GM110787 awarded by the National Cancer Institute, and Contract Nos. W81XWH-16-1-0635 and W81XWH-15-1-0409 awarded by the Department of Defense. The U.S. Government has certain rights in this invention. [Background technology]

[0003] Prostate cancer (CaP) growth and progression depend on the interaction of the androgen receptor (AR) with the circulating androgens testosterone (T) (produced by the testes) and its metabolite 5α-dihydrotestosterone (DHT) (produced by 5α-reduction of T in prostate tissue). T and DHT have similar affinities for the AR at the ligand-binding domain (LBD), but DHT is more potent due to its slower off-rate. Ligand-bound AR dimerizes and translocates to the nucleus, where it binds to androgen response elements (AREs) and initiates the transcription of AR-regulated genes that promote CaP cell proliferation. Most men who present with metastatic disease or who fail potentially curative treatments are treated with androgen deprivation therapy (ADT), which reduces circulating T levels, impairs AR transactivation, and induces CaP regression. However, ADT is symptomatic, and CaP invariably relapses as fatal castration-relapsed / resistant CaP (CRPC). One mechanism that contributes to the transition of CaP to CRPC is intratumoral androgen metabolism. In the absence of circulating T during ADT, the androgens dehydroepiandrosterone or 4-androstene-3,17-dione (ASD) undergo conversion to DHT via T by the front-door pathway (Figure 1). CaP cells utilize two other pathways that do not require T as an intermediate to synthesize DHT. In the primary back-door pathway, 5α-androstan-3α-ol-17-one undergoes enzymatic reduction to 5α-androstane-3α,17β-diol (DIOL) and then enzymatic oxidation to yield DHT (Figure 1). In the secondary backdoor pathway, ASD is enzymatically reduced to 5α-androstane-3,17-dione (5α-DIONE), which is then enzymatically reduced to DHT (Figure 1). In summary, three enzyme-driven pathways provide CaP tissues with access to DHT, with each pathway having an enzyme that catalyzes a key step late in the biosynthetic pathway. Finasteride or dutasteride inhibit 5α-reductase (SRD5A) to reduce or prevent the conversion of T to DHT (i.e., the frontdoor pathway in Figure 1).Both drugs fail clinically, in part because the backdoor pathway generates sufficient DHT concentrations to activate AR and CaP proliferation. Still other inhibitors, such as abiraterone, an inhibitor of cytochrome P450 17A1 (CYP17A1), inhibit pregnane (C17A1) well before the terminal ends of the three pathways converge on DHT. 21 Compound) to Androstane (C 19 Abiraterone (and other CYP17A1 inhibitors) only extend survival by a few months in CRPC because of multiple mechanisms including increased CYP17A1 expression levels and accumulation of progesterone that can outcompete abiraterone for CYP17A,25, CYP11A1, or AKR1C3, enhancing DHT production and CaP proliferation.

[0004] ASD is metabolized to DHT via a front door and two back door pathways that utilize the same metabolic steps at C-3 and C-17 in a different order (Figure 1). ASD can either go through T to reach DHT or proceed to 5α-DIONE, which can either proceed directly to DHT or to the primary back door pathway through androsterone, then DIOL, and finally DHT (Figure 1). The secondary back door pathway was discovered after the primary back door pathway and is synonymous with Sharifi's "alternative", Penning's "alternate", and Corcoran's "5α-DIONE" pathways. The role of HSD17B3 intratumoral metabolism via the front door and secondary back door pathways can be separated using ASD and 5α-DIONE as substrates, respectively. Preclinical studies with the AKR1C3 inhibitor indomethacin demonstrated proof of principle that AKR1C3 inhibition overcomes CaP resistance to abiraterone and enzalutamide. This study provided evidence for the need to identify and develop AKR1C3 inhibitors. A phase I / II clinical trial testing the efficacy of ASP9521, an AKR1C3 inhibitor, was terminated without evidence of clinical response. The authors suggested that insufficient CaP cell expression of AKR1C3 causes treatment failure, a problem that is likely not shared by the four 3α-oxidoreductases discussed herein.

[0005] Among the methods to achieve ADT, antiandrogens occupy a central position in chemical castration of men with CaP. Antiandrogen drugs bicalutamide or enzalutamide compete with T or DHT for the AR-LBD. Due to its lower binding affinity to the AR-LBD than DHT, bicalutamide has had poor clinical outcomes. In addition, bicalutamide showed undesirable AR agonist (but not antagonist) activity after long-term treatment. Enzalutamide binds to the AR-LBD with higher affinity than bicalutamide, impairs AR nuclear translocation, and inhibits the interaction between the ARE of AR-regulated genes essential for CaP proliferation and AR. However, enzalutamide provided a modest response in CRPC, prolonging survival by only 4.8 months. One of the mechanisms contributing to abiraterone and antiandrogen resistance is the expression of AR splice variant 7 (AR-V7), which is constitutively active and lacks the LBD. The lack of LBD avoids the need for T or DHT and eliminates the binding sites for antiandrogens, thereby rendering them ineffective. Small molecule inhibitors that target the N-terminal domain of AR rather than the AR-LBD (such as EPI-506) inactivated AR-V7 when examined in vitro. EPI-506 was found to be safe in a phase I trial, but failed a phase II clinical trial. The limited success of current therapies for advanced CaP and especially CRPC necessitates the pursuit of novel therapies. Summary of the Invention

[0006] The present disclosure provides androstane and dihydrotestosterone compounds functionalized with carbocyclic or heterocyclic groups, which may be saturated or unsaturated, such as coumarin-containing ring groups (e.g., coumarin groups and coumarin isostere groups). The compounds may be used in methods of inhibiting cell proliferation of malignant and / or hyperplastic cells, and / or in methods of treating individuals having diseases associated with malignant and / or hyperplastic cell proliferation (e.g., cancer, such as prostate cancer), and / or in molecular imaging of malignant and / or hyperplastic cells, and / or in methods of inducing degradation of target proteins.

[0007] The compound is 1) inhibiting an oxidoreductase that appears at the terminal step of the primary backdoor pathway for DHT biosynthesis; and / or 2) may function as an antiandrogen by competing with AR-LBD binding and inhibiting cell proliferation of CaP cell lines expressing high levels of AR-V7 variants, possibly by impairing AR-AR-V7 dimerization; and / or 3) It may provide a tool to deliver small molecules that disrupt AR function or even degrade AR. Compounds possessing one or more of these properties would represent a significant clinical advance.

[0008] In one aspect, the present disclosure provides functionalized androstane and dihydrotestosterone compounds. The compounds can be modified with a coumarin ring group or a coumarin isostere group.

[0009] In various examples, compounds of the present disclosure include 5α-androstan-3α,17β-diol (DIOL), 5α-androstan-3α-ol-17-one, 5α-androstan-3,17-dione (5α-DIONE), and 5α-dihydrotestosterone (DHT), which may be modified with various carbocyclic or heterocyclic groups that may be saturated or unsaturated. In various examples, compounds may be modified with coumarin-containing or coumarin isostere groups at either the C-3 or C-17 positions of these steroids.

[0010] The compounds of the present disclosure may have the structure: TIFF0007648538000001.tif35169, where R 1 is hydrogen or an alkyl group (e.g., methyl, ethyl, n- and isopropyl groups, etc.); TIFF0007648538000002.tif14169, wherein X is a hydroxyl group, Y is a hydrogen or an alkyl group (e.g., methyl, ethyl, n- and isopropyl groups, etc.), or X and Y together are spiro-fused, substituted or unsubstituted coumarin or coumarin isostere groups, and L 3 is optional and is a linking group. TIFF0007648538000003.tif30169, L 1 is a linking group, L 2 is an optional linking group; Z is a substituted or unsubstituted carbocyclic group, or a substituted or unsubstituted heterocyclic group, or a substituted or unsubstituted coumarin group, or a terminal group comprising a substituted or unsubstituted coumarin isostere group; and R 2 R is an alkyl group (e.g., methyl, ethyl, n- and isopropyl, and the like), or hydrogen. 5 is a photoactive group. TIFF0007648538000004.tif34169

[0011] In one aspect, the disclosure provides a composition comprising a compound of the disclosure. The composition further comprises one or more pharma- ceutically acceptable carriers.

[0012] In one aspect, the present disclosure provides a method of using one or more compounds or compositions thereof. The compounds are suitable in methods for treating various diseases. For example, one or more compounds of the present disclosure or compositions of the present disclosure can be used to treat cancer, other diseases, or combinations thereof. The method of the present disclosure can be used to inhibit cell proliferation of malignant cells and / or hyperplastic cells, and / or to induce selective degradation of target proteins, and / or for molecular imaging. The method can be performed in combination with one or more known therapies.

[0013] In one aspect, the disclosure provides a kit. In various examples, the kit includes a pharmaceutical formulation containing any one or any combination of the compounds of the disclosure. In one example, the disclosure includes a closed or sealed package containing the pharmaceutical formulation. In various examples, the package includes one or more closed or sealed vials, bottles, blister (bubble) packs, or any other suitable package for sale, distribution, or use of the pharmaceutical compounds and compositions containing them. The printed matter can include printed information. The printed information can be placed on a label, an insert, or printed on the packaging material. The printed information can include information identifying the amount and type of the compound in the package, other active and / or inactive ingredients in the composition, as well as instructions for taking the compound and / or composition. The instructions can include information such as, for example, the number of doses to take over a given period of time, and / or information directed to another health care provider, such as a pharmacist and / or a physician, or to the patient. The printed matter may include an indication that the pharmaceutical composition and / or any other agents provided therein are for the treatment of a subject having prostate cancer (e.g., castration-resistant prostate cancer) and / or other disease and / or any disorder associated with cancer and / or other disease. In various examples, the kit includes a label describing the contents of the kit and providing directions and / or instructions for using the contents of the kit to treat a subject having any cancer and / or other disease. [Brief description of the drawings]

[0014] For a fuller understanding of the nature and objects of the present disclosure, reference should be made to the following detailed description taken in conjunction with the accompanying drawings, in which:

[0015] Figure 1 shows the (A and B) biosynthetic pathways to DHT, including the front door pathway, the primary backdoor pathway, and the secondary backdoor pathway.

[0016] FIG. 2 shows the synthetic route to fluorescent steroids modified at the C-17β hydroxyl group. Description of the reagents used in the synthesis: a N2CH2CO2Et, Rh2(OAc)4, CH2Cl2; b NaOH, aqueous CH3OH; c N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC), 1-hydroxybenzotriazole hydrate (HOBt), and 8-(2-aminoethyl)-2,3,4,5-tetrahydro-1H,4H-11-oxa-3a-aza-benz[de]anthracen-10-one hydrochloride; d LiAlH(OtBu)3; e C6H5CO2H, DIAD

[0017] FIG. 3 shows the synthesis route of fluorescent steroids modified at the C-17 keto group. Description of the reagents used in the synthesis: a H2NOCH2CO2Et; b NaOH, aq CH3OH; c N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC), 1-hydroxybenzotriazole hydrate (HOBt), and 8-(2-aminoethyl)-2,3,4,5-tetrahydro-1H,4H-11-oxa-3a-aza-benz[de]anthracen-10-one hydrochloride; d Py-SO3

[0018] Figure 4 shows the Pictet-Spengler reaction of DHT with 8-(2-aminoethyl)-2,3,4,5-tetrahydro-1H,4H-11-oxa-3a-aza-benzo[de]anthracen-10-one hydrochloride. Description of the reagents used in the synthesis: a 1:10 (v / v) concentration HCl-abs. ethanol, reflux

[0019] FIG. 5 shows the 2D ROESY spectrum of DHT-3C (compound Ig). The spectrum was recorded at 25° C. on an Agilent 400 MHz. The dotted line indicates the NH + and ring A protons (i.e., H-5 (H-1 overlap), H-4, H-2, and CH2 adjacent NH2 +) is shown in an extended region displaying Nuclear Overhauser Effect (NOE) connectivity between

[0020] The results of computer modeling are shown in Figure 6. Panel A: 5α-DIONE (proximal NADP + Panel B: Compound Ig (proximal NADP + in the SP1-binding site of AKR1C3 (PDB:1XF0) containing

[0021] The results of computer modeling are shown in Figure 7. Panel A: Computer modeling of the binding of DHT-3C to AR showed that the BCD ring of DHT-3C was inserted into the AR-LBD binding pocket. Panel B: A close-up of DHT-3C binding shows interactions with specific residues in AR, indicating that the hairpin domain of AR (759-771) was "opened" by approximately 4.6 Å from its initial position to accommodate DHT-3C.

[0022] FIG. 8 shows that 3α oxidoreductase is expressed in clinical specimens.

[0023] FIG. 9 shows that RDH16 expression increased DHT production in LAPC-4.

[0024] Figure 10 shows the ARE-luciferase signals produced by DIOL and DHT treatment. In each series, the columns from left to right are SFM, DHT, DIOL, and androsterone (AND).

[0025] Figure 11 shows that the combination of dutasteride and 3α-oxidoreductase mutants reduced DHT levels better than dutasteride alone. In each series, the columns from left to right are "empty vector", HSD17B6, RDH16, DHRS9, and RDH5.

[0026] FIG. 12 shows that RDH16 knockdown reduced DHT production in LAPC-4 cells.

[0027] FIG. 13 shows that inhibition of frontdoor and primary backdoor DHT synthesis reduced VCaP proliferation.

[0028] FIG. 14 shows that DIOL-17C treatment reduced VCaP cell proliferation.

[0029] FIG. 15 shows RDH16-YFP and DIOL-17C colocalization by ImageStream.

[0030] FIG. 16 shows that DIOL-17C and DIOL compete for RDH16-YFP.

[0031] FIG. 17 shows that RDH16 metabolizes DIOL or DIOL-17C to DHT or DHT-17C, respectively.

[0032] Figure 18 shows ImageStream data demonstrating similar AR-GFP colocalization patterns between DHT-17C and DHT-C3. ImageStream also confirmed nuclear colocalization between DHT-17C and AR-V2-GFP.

[0033] FIG. 19 shows competition data demonstrating that DHT-17C and enzalutamide compete for AR-V2-GFP.

[0034] FIG. 20 shows proliferation data demonstrating that DHT-17C reduces proliferation of AR and AR-V7 expressing VCaP and CWR-1 cell lines, but does not reduce proliferation of PC-3 (an AR-free CaP cell line) or non-CaP, non-AR CV-1 or 293 cell lines.

[0035] FIG. 21 shows proliferation data demonstrating that DHT-17C (20 nM) inhibited proliferation of AR-V7 positive VCaP at a dose 1000-fold lower than enzalutamide (20 μM).

[0036] FIG. 22 shows proliferation data using the castration-relapse AR-V7 positive CRPC CWR-R1 cell line demonstrating that DHT-17C (50 nM) and enzalutamide (20 μM) exhibit similar proliferation impairment.

[0037] FIG. 23 shows qRT-PCR data demonstrating that DHT-17C treatment of CWR-R1 impairs AR-regulated gene kallikrein-related peptidase 2 (KLK2) transcript induction.

[0038] Figure 24 shows ImageStream data demonstrating nuclear colocalization between DHT-3C and AR-GFP, but not free coumarin and AR-GFP. The AR-GFP studies were confirmed with AR-V2-GFP.

[0039] Figure 25 shows proliferation data demonstrating similar proliferation patterns among AR-positive CaP cell lines treated with DHT-3C or DHT. The various cell lines are (A) LAPC-4; (B) VCaP; (C) LNCaP; (D) CRW-R1; (E) 22rv1; (F) PC-3; and (G) DU145.

[0040] Figure 26 shows that DHT-3C or DHT treatment of CaP cell lines resulted in similar AR-regulated gene, KLK2, protein expression levels. The various cell lines are (A) LAPC-4; (B) VCaP; (C) LNCaP; (D) CRW-R1; and (E) 22rv1.

[0041] FIG. 27 shows a three-pronged attack on AR ligand binding, AR dimerization, and AR transactivation.

[0042] Figure 28 shows the usefulness of DIOL-17C (compound Ia), DHT-17C (compound Id) and DHT-3C (compound Ig). DIOL-17C inhibits intratumoral synthesis of DHT by oxidoreductase, DHT-17C competes with DHT for AR-LBD and prevents dimerization between AR and AR-V7, and DHT-3C provides a carrier for PROTACS or light or heat activators, disrupts AR transactivation or even degrades AR.

[0043] FIG. 29 shows a representative photoactive derivative of DHT, DHT-3C or C-3O-(carboxymethyl)oxime.

[0044] FIG. 30 shows a synthetic route for the synthesis of a PROTAC for AR degradation.

[0045] FIG. 31 shows the synthesis and Pictet Spengler reaction with DHT or 17α-methyl-5α-dihydrotestosterone from various 4-(2-aminoethyl)coumarins.

[0046] Figure 32 shows the role of fluorescent 5α-dihydrotestosterone surrogate (DHT-C). A. Structure of DHT-C. B. Schematic diagram of DHT-C activation of AR and stimulation of AR-regulated gene transcript induction.

[0047] Figure 33 shows the synthetic route of DIOL-17C and several analogues. (A) Synthesis of DIOL-17C; (B) Structures of oxazole and benzoxazole targets; (C) Structures of imidazole and benzimidazole targets. Description: a N2CH2CO2Et, Rh2(OAc)4, CH2Cl2; b LiAlH(OtBu)3; c C6H5CO2H, DIAD; d NaOH, aq CH3OH; e EDC, HOBt, aminocoumarin

[0048] FIG. 34 shows a quadrant of DIOL-17C synthetic modifications.

[0049] FIG. 35 shows a synthetic route for DIOL-17C. Description: a C6H5CO2H, DEAD; b NaBH4; c NaH, BrCH2CH2Br; d 8-(2-aminoethyl)-2,3,4,5-tetrahydro-1H,4H-11-oxa-3a-aza-benzo[de]anthracen-10-one hydrochloride, Et3N; e NaOH, MeOH

[0050] FIG. 36 shows the synthetic route to DIOL-17C analogs with modifications in quadrant 2. Description: a N2CH2CO2Et, Rh2(OAc)4; b LiAlH(OtBu)3; c C6H5CO2H, DIAD; d NaOH, aq MeOH; e Various standard synthetic reactions.

[0051] Figure 37 shows analogs of DIOL-17C where the amine substituent in the coumarin is modified with a substituent X. Legend: DIOL-17C-2, X = -NH2; DIOL-17C-3, X = -N(CH3)2; DIOL-17C-4, X = -NHC(=O)OC2H5; DIOL-17C-5, X = -OCH3; DIOL-17C-6, X = -NHCH2CH2N(CH3)2; DIOL-17C-7, X = -N(CH2CH2)2NCH3. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0052] Although the subject matter has been described with respect to particular examples, other examples, including examples that do not provide all of the advantages and features described herein, are also within the scope of this disclosure.Various structural, logical, and process steps may be made without departing from the scope of the disclosure.

[0053] The present disclosure provides androstane and dihydrotestosterone compounds functionalized with carbocyclic or heterocyclic groups, which may be saturated or unsaturated (e.g., coumarin-containing ring groups (e.g., coumarin groups) and coumarin isostere groups). The compounds may be used in methods of inhibiting cell proliferation of malignant and / or hyperplastic cells, and / or in methods of treating individuals having diseases associated with malignant and / or hyperplastic cell proliferation (e.g., cancer, such as prostate cancer), and / or in molecular imaging of malignant and / or hyperplastic cells, and / or in methods of inducing degradation of target proteins.

[0054] The compound is 1) inhibiting an oxidoreductase that appears at the terminal step of the primary backdoor pathway for DHT biosynthesis; and / or 2) may function as an antiandrogen by competing with AR-LBD binding and inhibiting cell proliferation of CaP cell lines expressing high levels of AR-V7 variants, possibly by impairing AR-AR-V7 dimerization; and / or 3) It may provide a tool to deliver small molecules that disrupt AR function or even degrade AR. Compounds possessing one or more of these properties would represent a significant clinical advance.

[0055] A range of values ​​is disclosed herein. The ranges are set forth with lower and upper limits. Unless otherwise stated, the ranges include all values ​​up to the minimum value (either the lower or upper limit) and ranges between the values ​​in the stated ranges.

[0056] As used herein, the term "group," unless otherwise indicated, refers to a chemical entity that is monovalent (i.e., having one end that can be covalently bonded to other chemical species, such as a methyl or phenyl group), divalent, or polyvalent (i.e., having two or more ends that can be covalently bonded to other chemical species, such as a methylene or phenylene group). The term "group" also includes radicals (e.g., monovalent radicals and polyvalent radicals, e.g., divalent radicals, trivalent radicals, etc.).

[0057] As used herein, unless otherwise indicated, the term "alkyl group" refers to a branched or unbranched saturated hydrocarbon group. Examples of alkyl groups include, but are not limited to, methyl groups, ethyl groups, n- and isopropyl groups, n-, sec-, iso- and tert-butyl groups, and the like. An alkyl group is any of the C1 to C6 alkyl groups. 12 and any of the alkyl groups having a carbon number between them, including all integer values ​​of carbon and ranges of carbon numbers (e.g., C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, C24, C25, C26, C 10 , C 11 , or C 12 ). Alkyl groups can be unsubstituted or substituted with one or more substituents. Examples of substituents include, but are not limited to, various substituents such as, for example, halogens (e.g., -F, -Cl, -Br, and -I), aliphatic groups (e.g., alkyl, alkenyl, and alkynyl groups), aryl groups, alkoxide groups, carboxylate groups, carboxylic acids, ether groups, alcohol groups, amine groups, thiol groups, thioether groups, and the like, and combinations thereof.

[0058] As used herein, unless otherwise indicated, the term "heteroalkyl group" refers to a branched or unbranched, saturated or unsaturated hydrocarbon group containing at least one heteroatom. Examples of suitable heteroatoms include, but are not limited to, nitrogen, oxygen, sulfur, phosphorus, and halogens. Heteroalkyl groups can be unsubstituted or substituted with one or more substituents. Examples of substituents include, but are not limited to, various substituents such as, for example, halogens (e.g., -F, -Cl, -Br, and -I), aliphatic groups (e.g., alkyl, alkenyl, and alkynyl groups), aryl groups, alkoxide groups, carboxylate groups, carboxylic acids, ether groups, alcohol groups, amine groups, thiol groups, thioether groups, and the like, and combinations thereof.

[0059] As used herein, the term "aryl group" means, unless otherwise indicated, an aryl group having a C5 to C 12Aromatic or partially aromatic carbocyclic groups (including all integer carbon and carbon number ranges therebetween (e.g., C5, C6, C7, C8, C9, C 10 , C 11 Or C 12 ), which includes aryl groups. An aryl group can also be referred to as an aromatic group. An aryl group can include, for example, fused ring or polyaryl groups, such as biaryl groups. An aryl group can be unsubstituted or substituted with one or more substituents. Examples of substituents include, but are not limited to, halogens (e.g., -F, -Cl, -Br, and -I), aliphatic groups (e.g., alkyl, alkenyl, and alkynyl groups), aryl groups, alkoxide groups, carboxylate groups, carboxylic acids, ether groups, alcohol groups, amine groups, thiol groups, thioether groups, and the like, and combinations thereof. Examples of aryl groups include, but are not limited to, phenyl groups, biaryl groups (e.g., biphenyl groups, and the like), and fused ring groups (e.g., naphthyl groups, and the like).

[0060] As used herein, unless otherwise indicated, the term "carbocycle" or "heterocycle" refers to a carbon-containing ring or a carbon-containing ring in which one or more of the carbon atoms are replaced by a heteroatom, respectively. These groups may be non-aromatic or aromatic. A carbocyclic or heterocyclic group may be saturated or unsaturated and may have one or more substituents (e.g., hydroxy, alkoxy, thioalkoxy, halogen, etc.), and combinations thereof. Further examples of substituents include, but are not limited to, halogens (e.g., -F, -Cl, -Br, and -I), aliphatic groups (e.g., alkyl, alkenyl, and alkynyl groups), aryl groups, alkoxide groups, carboxylate groups, carboxylic acids, ether groups, alcohol groups, amine groups, thiol groups, thioether groups, and the like, and combinations thereof.

[0061] As used herein, unless otherwise indicated, the term "heterocyclic group" refers to a C3-C6 ring system that contains one or more heteroatoms (e.g., N, O, S, etc.) as part of the ring structure. 20The C3-C 20 includes all integers and ranges of numbers of carbons between them (C3, C4, C5, C6, C7, C8, C9, C 10 , C 11 , C 12 , C 13 , C 14 , C 15 , C 16 , C 17 , C 18 , C 19 , or C 20 ). Heterocyclic groups may be substituted or unsubstituted and / or may have additional degrees of unsaturation. Examples of substituents include, but are not limited to, halogens (e.g., -F, -Cl, -Br, and -I), aliphatic groups (e.g., alkyl, alkenyl, and alkynyl groups), aryl groups, alkoxide groups, carboxylate groups, carboxylic acids, ether groups, alcohol groups, amine groups, thiol groups, thioether groups, and the like, and combinations thereof. Heterocyclic groups may be fused to carbocyclic groups or to each other. Non-limiting examples of heterocyclic groups include furanyl, oxazolyl, isothiazolyl, thiazolyl, tetrahydropyranyl, piperazinyl, dioxanyl, pyrrolidinyl, tetrahydrothiophenyl, tetrahydrofuranyl, quinuclidinyl, azaadamantanyl, decahydroquinolinyl, and the like.

[0062] As used herein, unless otherwise indicated, the term "heteroaryl group" refers to a monovalent monocyclic or polycyclic aromatic group having 5 to 18 ring atoms, or a polycyclic aromatic group, containing one or more ring heteroatoms selected from N, O, or S, with the remaining ring atoms being C, including all integers and ranges of ring atoms therebetween (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18). Heteroaryl, as defined herein, also refers to a polycyclic (e.g., bicyclic) heteroaromatic group in which the heteroatoms are selected from N, O, or S. The aromatic group may be optionally substituted independently with one or more substituents described herein. The substituents themselves may be optionally substituted. Examples of substituents include, but are not limited to, halogens (e.g., -F, -Cl, -Br, and -I), aliphatic groups (e.g., alkyl, alkenyl, and alkynyl groups), aryl groups, alkoxide groups, carboxylate groups, carboxylic acids, ether groups, alcohol groups, amine groups, thiol groups, thioether groups, and the like, and combinations thereof.Examples of heteroaryl groups include benzothienyl, furyl, thienyl, pyrrolyl, pyridyl, pyrazinyl, pyrazolyl, pyridazinyl, pyrimidinyl, imidazolyl, isoxazolyl, oxazolyl, oxadiazolyl, pyrazinyl, indolyl, thiophen-2-yl, quinolyl, benzopyranyl, isothiazolyl, thiazolyl, thiadiazolyl, thieno[3,2-b]thiophene, and triazolyl. , triazinyl, imidazo[1,2-b]pyrazolyl, furo[2,3-c]pyridinyl, imidazo[1,2-a]pyridinyl, indazolyl, pyrrolo[2,3-c]pyridinyl, pyrrolo[3,2-c]pyridinyl, pyrazolo[3,4-c]pyridinyl, benzimidazolyl, thieno[3,2-c]pyridinyl, thieno[2,3-c]pyridinyl, thieno[2,3-b]pyridinyl, benzothiazolyl, isopropyl Indolyl, indolinyl, indolinonyl, dihydrobenzothiophenyl, dihydrobenzofuranyl, benzofuran, chromanyl, thiochromanyl, tetrahydroquinolinyl, dihydrobenzothiazine, dihydrobenzoxanyl, quinolinyl, isoquinolinyl, 1,6-naphthyridinyl, benzo[de]isoquinolinyl, pyrido[4,3-b][1,6]naphthyridinyl, thieno[2,3-b]pyrazine nyl, quinazolinyl, tetrazolo[1,5-a]pyridinyl, [1,2,4]triazolo[4,3-a]pyridinyl, isoindolyl, pyrrolo[2,3-b]pyridinyl, pyrrolo[3,4-b]pyridinyl, pyrrolo[3,2-b]pyridinyl, imidazo[5,4-b]pyridinyl, pyrrolo[1,2-a]pyrimidinyl, tetrahydropyrrolo[1,2-a]pyrimidinyl, 3,4-dihydro-2H-1λ. 2-pyrrolo[2,1-b]pyrimidine, dibenzo[b,d]thiophene, pyridin-2-one, furo[3,2-c]pyridinyl, furo[2,3-c]pyridinyl, 1H-pyrido[3,4-b][1,4]thiazinyl, benzoxazolyl, benzisoxazolyl, furo[2,3-b]pyridinyl, benzothiophenyl, 1,5-naphthyridinyl, furo[3,2-b]pyridine, [1,2,4]triazolo[1,5-a]pyridinyl, benzo[1,2,3]triazolyl, imidazo[1,2-a]pyrimidinyl, [1,2,4]triazolo[4, 3-b]pyridazinyl, benzo[c][1,2,5]thiadiazolyl, benzo[c][1,2,5]oxadiazole, 1,3-dihydro-2H-benzo[d]imidazol-2-one, 3,4-dihydro-2H-pyrazolo[1,5-b][1,2]oxazinyl, 4,5,6,7-tetrahydropyrazolo[1,5-a]pyridinyl, thiazolo[5,4-d]thiazolyl, imidazo[2,1-b][1,3,4]thiadiazolyl, thieno[2,3-b]pyrrolyl, 3H-indolyl, and derivatives thereof. Furthermore, when having two fused rings, the heteroaryl group as defined herein may have an unsaturated or partially saturated ring fused to a fully saturated ring.

[0063] In one aspect, the present disclosure provides functionalized androstane and dihydrotestosterone compounds. The compounds may be functionalized (e.g., modified) with a coumarin ring group or a coumarin isostere group.

[0064] In various examples, compounds of the present disclosure include 5α-androstan-3α,17β-diol (DIOL), 5α-androstan-3α-ol-17-one, 5α-androstan-3,17-dione (5α-DIONE), and 5α-dihydrotestosterone (DHT), which may be modified with various carbocyclic or heterocyclic groups that may be saturated or unsaturated. In various examples, compounds may be modified (e.g., functionalized) with coumarin-containing or coumarin isostere groups at either the C-3 or C-17 positions of these steroids.

[0065] The compounds of the present disclosure may have the structure: TIFF0007648538000005.tif35169, where R 1 is hydrogen or an alkyl group (e.g., methyl, ethyl, n- and isopropyl groups, etc.); TIFF0007648538000006.tif14169, wherein X is a hydroxyl group, Y is a hydrogen or an alkyl group (e.g., methyl, ethyl, n- and isopropyl groups, etc.), or X and Y together are a spiro-fused, substituted or unsubstituted coumarin group or coumarin isostere group; L 3 is optional and is a linking group. TIFF0007648538000007.tif30169, L 1 is a linking group, L 2 is an optional linking group; Z is a substituted or unsubstituted carbocyclic group, or a substituted or unsubstituted heterocyclic group, or a substituted or unsubstituted coumarin group, or a terminal group comprising a substituted or unsubstituted coumarin isostere group; and R 2 R is an alkyl group (e.g., methyl, ethyl, n- and isopropyl, and the like), or hydrogen. 5 is a photoactive group, where TIFF0007648538000008.tif34169Z can be a variety of terminal groups. In various examples, Z is a carbocyclic or heterocyclic group, which may be saturated or unsaturated, and may have one or more substituents (e.g., hydroxy, alkoxy, thioalkoxy, halogen, and the like, and combinations thereof). Non-limiting examples of Z groups include the following: TIFF0007648538000009.tif64169TIFF0007648538000010.tif95169TIFF0007648538000011.tif177169In the formula, each X' is independently selected from hydrogen, alkyl groups, cycloalkyl groups, alkoxy groups, halogens, and combinations thereof. The Z group may have various substituents. Non-limiting examples of the substituents include halogens (e.g., -F, -Cl, -Br, and -I), aliphatic groups (e.g., alkyl groups, alkenyl groups, and alkynyl groups), aryl groups, alkoxide groups, carboxylate groups, carboxylic acids, ether groups, alcohol groups, amine groups, thiol groups, thioether groups, and the like, and combinations thereof.

[0066] A variety of photoactive groups can be used. 5 Non-limiting examples of ) include: TIFF0007648538000012.tif28169

[0067] A general formula for androgens with substituted coumarins attached at C-3 or C-17. The compounds may have the following structure: TIFF0007648538000013.tif46169In formula, R 1 and R 2 are each selected from hydrogen and an alkyl group (e.g., methyl, ethyl, n- and isopropyl, etc.); X and Y are each hydroxyl or hydrogen, or X and Y contain a carbonyl group; Z' is a substituted coumarin ring or a coumarin isostere. In various examples, R 1 and R 2 are each selected from hydrogen and an alkyl group (eg, methyl, ethyl, n- and isopropyl, etc.); X and Y are spiro-fused, substituted coumarin rings or coumarin isosteres; and Z' is hydrogen.

[0068] The compound may have the structure: TIFF0007648538000014.tif44169In formula, R1 are each selected from hydrogen and an alkyl group (e.g., methyl, ethyl, n- and isopropyl, etc.); X and Y are each hydroxyl or hydrogen; and Z″ is an O-(carboxymethyl)oxime linked to a substituted coumarin or coumarin isostere. In various embodiments, R 1 are each selected from hydrogen and an alkyl group (e.g., methyl, ethyl, n- and isopropyl, etc.); X and Y are spiro-fused, substituted coumarin rings or coumarin isosteres; and Z'' is oxygen.

[0069] Representative examples of compounds (I) with substituted coumarins attached at the C-17 position. Non-limiting examples of compounds of the present disclosure include compounds (I) in which the X group is a hydroxyl group; Y is hydrogen; and Z' is a substituted coumarin, where the substituent connects a linker L between the C-17β oxygen of the steroid and the C-4' position of the coumarin ring or coumarin isostere. Examples of such compounds include, but are not limited to, compounds (Ia), (Ib), and (Ic): TIFF0007648538000015.tif48169In the formula, each X' is independently hydrogen, an alkyl group (e.g., methyl, ethyl, n-, and isopropyl), a cycloalkyl group, an alkoxy group (e.g., -O(CH2) n wherein n is selected from CH3, n is 0, 1, or 2, and / or halogen (i.e., -F, -Cl, -Br, and -I), and L is a linker (e.g., a linker group). Exemplary linkers include, but are not limited to, the following structures (where n and m are independently 1, 2, 3, 4, 5, or 6): TIFF0007648538000016.tif29169

[0070] Non-limiting examples of compounds of the present disclosure include compounds (I) in which the X and Y groups are carbonyl groups and Z' is a substituted coumarin where the substituent connects a linker L between the C-17β oxygen of the steroid and the C-4' position of the coumarin ring group or coumarin isostere group. Exemplary compounds include, but are not limited to, compounds (Id), (Ie) and (If): TIFF0007648538000017.tif51169In the formula, each X' is independently hydrogen, an alkyl group (e.g., methyl, ethyl, n-, and isopropyl), a cycloalkyl group, an alkoxy group (e.g., -O(CH2) n wherein n is selected from CH3, n is 0, 1, or 2, and / or halogen (i.e., -F, -Cl, -Br, and -I), and L is a linker (e.g., a linker group). Exemplary linkers include, but are not limited to, the following structures (where n and m are independently 1, 2, 3, 4, 5, or 6): TIFF0007648538000018.tif28169

[0071] Representative examples of compound (II) with a substituted coumarin attached at the C-17 position. Non-limiting examples of compounds of the present disclosure include those in which the X and Y groups of compound (II) are hydroxyl and hydrogen, respectively, or carbonyl or oxime. Exemplary groups include, but are not limited to, compounds (IIa) and (IIb): TIFF0007648538000019.tif39169

[0072] General representative examples of compounds (I) with substituted coumarins attached at the C-3 position. Non-limiting examples of compounds of the present disclosure include those in which the X and Y groups of compound (I) are spiro-fused, substituted coumarin ring groups or coumarin isostere groups, and Z' is hydrogen. Examples include, but are not limited to, compounds (Ig), (Ih), and (Ii): TIFF0007648538000020.tif68169

[0073] Non-limiting examples of compounds of the present disclosure include R 1 wherein each of the X and Y groups is selected from hydrogen and an alkyl group (e.g., methyl, ethyl, n- and isopropyl groups, etc.), the X and Y groups are spiro-fused, substituted coumarin rings or coumarin isosteres, and Z″ is oxygen. Examples include, but are not limited to, compounds (Ij), (Ik) and (Il): TIFF0007648538000021.tif72169

[0074] In various examples, the compounds of the present disclosure are PROTACs. A PROTACs may have the following structure: ALB, where A is an E3 ligase recruiter, B is a ligand group for a target protein, and L is a linker that covalently links the E3 ligase recruiter and the ligand group.

[0075] In one example, an E3 ligase recruiter can recruit proteins such as, for example, VHL, CRBN, RNF114, MDM2, DCAF15, DCAF16, SCF, etc., which can ubiquitinate a target protein such as AR. Non-limiting examples of E3 ligase recruiter groups include groups formed from: TIFF0007648538000022.tif70169TIFF0007648538000023.tif143147or Ligase recruiter group that binds to cereblon, von Hippel-Lindau (VHL), or Mouse-Double-Minute-2.

[0076] The compounds (e.g., PROTAC compounds) of the present disclosure can include various linkers (e.g., linking groups). The linker connects the E3 ligase recruiter and the ligand group of the target protein via a covalent bond (e.g., two or more covalent bonds). Various linkers are known in the art. Non-limiting examples of linkers include: -NH(CH2) n NH-, -C(=O)(CH2) n NH-, -C(=O)(CH2) n C(=O)-, -NH(CH2CH2O)nCH2CH2NH-, -C(=O)(CH2CH2O)nCH2CH2NH-, -C(=O)(CH2CH2O)nCH2CH2C(=O)-, where n is 2 to 4 (e.g., 2, 3, or 4).

[0077] Non-limiting examples of target proteins include proteins associated with cancer (e.g., leukemia, lung cancer (e.g., non-small cell lung cancer), dermatological cancers, precancerous lesions of the upper gastrointestinal tract, prostate malignancies, brain malignancies, breast malignancies, solid tumors, etc.), infectious diseases, inflammatory diseases, immune disorders, sleep disorders, neurodegenerative disorders, etc., and combinations thereof.

[0078] The compounds of the present disclosure can be used for molecular imaging. Compounds suitable for molecular imaging include those having R 5 The compound may include a coumarin ring group, may include a spiro-coumarin ring group, or may include both. Non-limiting examples of compounds suitable for molecular imaging (e.g., imaging of hyperplastic and / or malignant cells) include the following: TIFF0007648538000024.tif173169TIFF0007648538000025.tif229169TIFF0007648538000026.tif85169

[0079] Specific examples of compounds of the present disclosure include, but are not limited to, the following: TIFF0007648538000027.tif134169TIFF0007648538000028.tif215169TIFF00076485380 00029.tif239169TIFF0007648538000030.tif204169TIFF0007648538000031.tif212169 TIFF0007648538000032.tif233169TIFF0007648538000033.tif209169TIFF00076485380 00034.tif209169TIFF0007648538000035.tif229169TIFF0007648538000036.tif116169

[0080] The synthesis of the compounds of the present disclosure is possible from commercially available materials. For example, the present disclosure provides methods for making compounds (I) and (II) from commercially available materials in which a coumarin moiety is attached to the C-3 or C-17 position of the steroid. Non-limiting examples of methods are provided herein. The synthetic routes of these compounds are shown in Figures 2, 3 and 4. The compounds were made by the methods of the present disclosure and characterized for structure and purity as follows. Nuclear magnetic resonance spectra were obtained using a Varian instrument ( 1 H, 400 or 500MHz; 13 C, 100 MHz). High-resolution electrospray ionization (ESI) mass spectra were recorded on a Q Exactive mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). The resolution was set at 100,000 (400 m / z). Samples were introduced by direct injection using a syringe pump at a flow rate of 3 μL / min. Compound purity was 13 C NMR and rigorous mass spectrometry were used to establish the identity of the compounds. Compounds were chromatographed on preparative Merck silica gel F254 unless otherwise stated.

[0081] In one aspect, the disclosure provides a composition comprising a compound of the disclosure. The composition further comprises one or more pharma- ceutically acceptable carriers.

[0082] The composition may include additional components. For example, the composition includes a buffer solution suitable for administration to an individual (e.g., a mammal, such as a human or non-human). The individual may be a subject. The buffer solution may be a pharma- ceutically acceptable carrier.

[0083] The composition may include one or more standard pharma- ceutically acceptable carriers. Non-limiting examples of the composition include solutions, suspensions, emulsions, solid injectable compositions that are dissolved or suspended in a solvent before use, and the like. Injectables may be prepared by dissolving, suspending, or emulsifying one or more active ingredients in a diluent. Non-limiting examples of diluents include distilled water for injection, physiological saline, vegetable oils, alcohol, and the like, and combinations thereof. In addition, the injectables may contain stabilizers, solubilizers, suspending agents, emulsifiers, soothing agents, buffers, preservatives, and the like. Injectables may be sterilized at the final formulation stage or prepared by a sterilization procedure. The composition may also be formulated into a sterile solid preparation, for example, by lyophilization, and used after sterilization, or may be used after dissolving in sterile water for injection or other sterile diluents immediately before use. Non-limiting examples of pharma- ceutically acceptable carriers are described in Remington: The Science and Practice of Pharmacy (2005) 21st Edition, Philadelphia, PA. Lippincott Williams & Wilkins.

[0084] In one aspect, the present disclosure provides a method of using one or more compounds or compositions thereof. The compounds are suitable in methods for treating various diseases. For example, one or more compounds of the present disclosure or compositions of the present disclosure can be used to treat cancer, other diseases, or combinations thereof. The method of the present disclosure can be used to inhibit cell proliferation of malignant cells and / or hyperplastic cells, and / or to induce selective degradation of target proteins, and / or for molecular imaging. The method can be performed in combination with one or more known therapies.

[0085] Non-limiting examples of cancer include leukemia, lung cancer (e.g., non-small cell lung cancer), dermatological cancer, precancerous lesions of the upper gastrointestinal tract, prostate malignancies, brain malignancies, breast malignancies, solid tumors, and the like, and combinations thereof.

[0086] Non-limiting examples of other diseases include infectious diseases, inflammatory diseases, immune disorders, sleep disorders, neurodegenerative disorders, and the like, and combinations thereof.

[0087] The methods of the present disclosure can be used to treat various types of cancer, such as, for example, hormonal cancers, non-limiting examples of which include breast cancer, ovarian cancer, uterine or endometrial cancer, prostate cancer, and the like.

[0088] The methods of the present disclosure can be used to treat CaP (e.g., androgen-stimulated CaP and CRPC) by inhibiting late oxidoreductases in androgen biosynthesis and / or impairing AR transactivation or AR dimerization. The methods described herein can be used to overcome AR-V7-mediated resistance in treating CRPC. The methods of the present disclosure can inhibit the proliferation of CaP cells better than methods involving treatment with enzalutamide alone.

[0089] The compounds of the present disclosure can be used in methods for treating diseases associated with malignant cells (e.g., CaP) and / or hyperplastic cells (e.g., benign prostatic hyperplasia or hypertrophy (BPH)).The compounds described either inhibit terminal substrate conversion to DHT or impair AR transactivation or AR dimerization.The compounds can be used together with or instead of current CaP therapy such as bicalutamide, enzalutamide, or abiraterone.

[0090] In various examples, one or more compositions comprising one or more compounds and / or one or more compounds described herein are administered to a subject in need of treatment using any known method and route, including oral, parenteral, subcutaneous, intraperitoneal, intrapulmonary, intranasal and intracranial injection. Parenteral injection includes intramuscular, intravenous, intraarterial, intraperitoneal and subcutaneous administration. Topical and / or transdermal administration are also included.

[0091] The method may be performed in a subject in need of treatment who has been diagnosed with or is suspected of having CaP (e.g., a male subject (e.g., individual) with CaP). The method may also be performed in a subject who relapses or has a high risk of relapse after undergoing treatment for CaP. A subject may also be referred to as an individual.

[0092] A method of treating a disease (e.g., cancer, such as a hormonal cancer, such as prostate cancer, such as castration-resistant prostate cancer, such as castration-resistant prostate cancer that expresses AR-V7) includes administering to a subject in need of treatment (e.g., an individual in need of treatment) a therapeutic amount of a compound or composition of the present disclosure (e.g., an amount of the compound or composition sufficient to treat the subject), thereby treating the disease in the subject.

[0093] In various examples, the compounds of the present disclosure are used to inhibit the proliferation of cells (e.g., malignant cells, e.g., cancer cells, e.g., hormonal cancer cells, e.g., prostate cancer cells, e.g., castration-resistant prostate cancer cells, e.g., castration-resistant prostate cancer cells expressing AR-V7), or hyperplastic cells (e.g., benign prostatic hyperplasia or hypertrophy (BPH) cells, etc.). For example, the proliferation of cancer cells (e.g., prostate cancer cells, e.g., castration-resistant prostate cancer cells, e.g., castration-resistant prostate cancer cells expressing AR-V7) is inhibited by contacting the compound with the cancer cells in an amount (e.g., 1 nM to 1 mM) and for a time sufficient to cause degradation of the androgen receptor (AR) in the cancer cells, where degradation of the AR results in inhibition of proliferation of the cancer cells. The inhibition of proliferation may be better than the inhibition of cell proliferation caused by other compounds / treatments known in the art (e.g., treatment with enzalutamide). Inhibition of cell proliferation refers to any decrease in cell growth / reproduction (eg, cancer cell growth / reproduction).

[0094] The method of inhibiting cell proliferation comprises contacting a cell with a compound of the present disclosure or a composition comprising a compound of the present disclosure.

[0095] The present disclosure provides a method of inducing selective degradation of a target protein, comprising: i) contacting a cell (e.g., a cell in a subject in need of treatment) with a compound and / or composition of the present disclosure, wherein the compound binds to an E3 ligase and to the target protein in the cell.

[0096] The subject or individual in need of treatment may be a human or a non-human mammal. Non-limiting examples of non-human mammals include cows, pigs, mice, rats, rabbits, cats, dogs, or other agricultural animals, pets, service animals, etc. In various examples, the subject or individual is male or has male reproductive organs. In various examples, the subject or individual has a prostate.

[0097] The contacting and / or administering methods of the present disclosure may be performed in combination with contacting and / or administering one or more additional drugs. The additional drugs may be selected from antiandrogens, 5α-reductase drugs, androgen metabolism inhibitors, and combinations thereof. Non-limiting examples of antiandrogens include flutamide, bicalutamide, enzalutamide, apalutamide, darolutamide, dilutamide, and the like, and combinations thereof. Non-limiting examples of 5α-reductase drugs include finasteride, dutasteride, and the like, and combinations thereof. Non-limiting examples of androgen metabolism drugs include abiraterone acetate, abiraterone acetate microparticles, ketoconazole, and the like, and combinations thereof.

[0098] The compounds of the present disclosure can be used as tools to screen for small molecule inhibitors of oxidoreductases or small molecule antiandrogens (that bind to the AR).

[0099] In one example, the compounds and compositions are suitable for the method comprising fluorescence microscopy.The method comprising fluorescence microscopy can be combined with other techniques such as flow cytometry.Techniques for fluorescence microscopy are known in the art.For example, such techniques can be used to monitor cell activity (e.g., androgen metabolism).

[0100] A method involving molecular tracking (e.g., intracellular tracking) in a cell (e.g., a prostate cell) includes contacting the cell (e.g., a prostate cell) with a compound of the present disclosure and imaging (e.g., imaging using a fluorescent microscope) the cell (e.g., a prostate cell) at regular intervals (e.g., intervals known in the art) to monitor activity. In one example, the activity is androgen metabolism.

[0101] In one aspect, the disclosure provides a kit. In various examples, the kit includes a pharmaceutical formulation containing any one or any combination of the compounds of the disclosure. In one example, the disclosure includes a closed or sealed package containing the pharmaceutical formulation. In various examples, the package includes one or more closed or sealed vials, bottles, blister (bubble) packs, or any other suitable package for sale, distribution, or use of the pharmaceutical compounds and compositions containing them. The printed matter can include printed information. The printed information can be placed on a label, an insert, or printed on the packaging material. The printed information can include information identifying the amount and type of the compound in the package, other active and / or inactive ingredients in the composition, as well as instructions for taking the compound and / or composition. The instructions can include information such as, for example, the number of doses to take over a given period of time, and / or information directed to another health care provider, such as a pharmacist and / or a physician, or to the patient. The printed matter may include an indication that the pharmaceutical composition and / or any other agents provided therein are for the treatment of a subject having CaP and / or other diseases and / or any disorder associated with cancer and / or other diseases. In various examples, the kit includes a label describing the contents of the kit and providing directions and / or instructions for using the contents of the kit to treat a subject having any cancer and / or other disease.

[0102] In various examples, the kit includes materials that can be used for administration to an individual in need of treatment for CaP. The kit can include, for example, one or more therapeutic agents, which may be in lyophilized form, optionally a reconstitution medium, and instructions for administration. The kit can include a single dose or multiple doses.

[0103] The method steps described in the various embodiments and examples disclosed herein are sufficient to carry out the disclosed method. The method described in the embodiments is a combination of the disclosed method steps. In another embodiment, the method consists of such steps.

[0104] The following statement provides examples of the disclosed compounds, methods of using the disclosed compounds, and uses of the disclosed compounds. Statement 1. A compound having the structure: TIFF0007648538000037.tif35169, where R 1 is hydrogen or an alkyl group (e.g., methyl, ethyl, n- and isopropyl groups, etc.); TIFF0007648538000038.tif14169, wherein X is a hydroxyl group, Y is a hydrogen or an alkyl group (e.g., methyl, ethyl, n- and isopropyl groups, etc.), or X and Y together are spiro-fused, substituted or unsubstituted coumarin or coumarin isostere groups, and L 3 is an optional linking group; TIFF0007648538000039.tif30169, L 1 is a linking group, L 2 is an optional linking group; Z is a substituted or unsubstituted carbocyclic group, or a substituted or unsubstituted heterocyclic group, or a substituted or unsubstituted coumarin group, or a terminal group comprising a substituted or unsubstituted coumarin isostere group; and R 2 is an alkyl group or hydrogen; R 5 is a photoactive group, where TIFF0007648538000040.tif34169 Statement 2. The compound according to statement 1, wherein L1 and / or L2 and / or L3 are independently selected from: TIFF0007648538000041.tif39169 Statement 3. The compound of Statement 1 or Statement 2, wherein the compound has the structure: TIFF0007648538000042.tif48169, where Z is selected from: TIFF0007648538000043.tif66169TIFF0007648538000044.tif219169TIFF0007648538000045.tif40169In the formula, each X' is independently selected from hydrogen, an alkyl group, a cycloalkyl group, an alkoxy group, a halogen, and combinations thereof. Statement 4. The compound of any one of the preceding statements, wherein the compound has the structure: TIFF0007648538000046.tif41169where n is 1, 2, or 3. Statement 5. The compound of any one of the preceding statements, wherein the compound has the structure: TIFF0007648538000047.tif86169TIFF0007648538000048.tif204169TIFF0007648538000049.tif58169 Statement 6. The compound of statement 1 above, wherein the compound has the structure: TIFF0007648538000050.tif32169In the formula, R is L 1 -A, R 5 , -OH, -NH2, -CO2Et, -CN, -CHO, -SO3H, and -CO2H, or three adjacent R groups are independently selected from a fused ring system (e.g., TIFF0007648538000051.tif28169, n is 1, 2, 3 or 4, L 1 is a linking group, A is an E3 ligase recruiter group, where only one R is L 1 -A. Statement 7. The compound of statement 1 or statement 6, wherein A is formed from: TIFF0007648538000052.tif40169TIFF0007648538000053.tif57169 Statement 8. 7. The compound of claim 6, wherein A is formed from a ligase recruiter group that binds to cereblon, von Hippel-Lindau (VHL), or Mouse-Double-Minute-2. Statement 9. The compound of Statement 1 or Statement 6, wherein the compound has the structure: TIFF0007648538000054.tif94169 Statement 10. The compound of Statement 1 or Statement 6, wherein the compound has the structure: TIFF0007648538000055.tif37169TIFF0007648538000056.tif176169 Statement 11. The compound of statement 1, wherein the compound has the structure: TIFF0007648538000057.tif49169, where Z is selected from: TIFF0007648538000058.tif218169TIFF0007648538000059.tif101169In the formula, each X' is independently selected from hydrogen, an alkyl group, a cycloalkyl group, an alkoxy group, a halogen, and combinations thereof. Statement 12. The compound of Statement 1 or Statement 11, wherein the compound has the structure: TIFF0007648538000060.tif42169 where n is 1, 2, or 3. Statement 13. The compound of Claim 1 or Claims 11-12, wherein the compound has the following structure: TIFF0007648538000061.tif46169TIFF0007648538000062.tif187169TIFF0007648538000063.tif115169 Statement 14. The compound of statement 1, wherein the compound has the structure: TIFF0007648538000064.tif56169, where Z is selected from: TIFF0007648538000065.tif30169TIFF0007648538000066.tif238169TIFF0007648538000067.tif56169In the formula, each X' is independently selected from hydrogen, an alkyl group, a cycloalkyl group, an alkoxy group, a halogen, and combinations thereof. Statement 15. The compound of statement 1 or statement 14, wherein the compound has the structure: TIFF0007648538000068.tif43169where n is 1, 2, or 3. Statement 16. The compound of Statement 1 or Statements 14-15, wherein the compound has the structure: TIFF0007648538000069.tif89169TIFF0007648538000070.tif203169TIFF0007648538000071.tif57169 Statement 17. The compound of statement 1, wherein the compound has the structure: TIFF0007648538000072.tif56169, where Z is selected from: TIFF0007648538000073.tif65169TIFF0007648538000074.tif219169TIFF0007648538000075.tif38169In the formula, each X' is independently selected from hydrogen, an alkyl group, a cycloalkyl group, an alkoxy group, and a halogen. Statement 18. The compound of Statement 1 or Statement 17, wherein the compound has the structure: TIFF0007648538000076.tif50169where n is 1, 2, or 3. Statement 19. The compound of Claim 1 or Claims 17-18, wherein the compound has the following structure: TIFF0007648538000077.tif90169TIFF0007648538000078.tif210169TIFF0007648538000079.tif113169 Statement 20. The compound of statement 1, wherein the compound has the structure: TIFF0007648538000080.tif34169In formula, R 5 is selected from: TIFF0007648538000081.tif30169 Statement 21. The compound of statement 1 or statement 20, wherein the compound has the structure: TIFF0007648538000082.tif39169TIFF0007648538000083.tif188169 Statement 22. A composition comprising a compound according to any one of the preceding statements and a pharma- ceutically acceptable carrier. Statement 23. 23. The composition of claim 22, further comprising one or more additional drugs. Statement 24. 24. The composition of claim 23, wherein the one or more additional drugs are selected from antiandrogens, 5α-reductase drugs, androgen metabolism inhibitors, and combinations thereof. Statement 25 and / or the antiandrogen is selected from flutamide, bicalutamide, enzalutamide, apalutamide, darolutamide, dilutamide, and combinations thereof; and / or the 5α-reductase drug is selected from finasteride, dutasteride, and combinations thereof; the androgen metabolism agent is selected from abiraterone acetate, abiraterone acetate microparticles, ketoconazole, and combinations thereof; 25. The composition according to any one of claims 22 to 24. Statement 26. 26. The compound of any one of claims 22-25, wherein the compound has the structure: TIFF0007648538000084.tif181169TIFF0007648538000085.tif233169TIFF0007648538000086.tif51169 Statement 27. A method for impairing androgen receptor (AR) transactivation and / or androgen receptor dimerization, comprising contacting a cell with a compound of any one of statements 1-21 or a composition of any one of statements 22-26 in an amount and for a time sufficient to impair androgen receptor transactivation and / or androgen receptor dimerization. Statement 28. 28. The method of statement 27, further comprising contacting the cell with flutamide, bicalutamide, enzalutamide, apalutamide, darolutamide, dilutamide, finasteride, dutasteride, or a combination thereof. Statement 29. The method of claim 27 or 28, wherein the compound has the structure: TIFF0007648538000087.tif91169TIFF0007648538000088.tif52169 Statement 30. A method of inhibiting cell proliferation, comprising contacting a cell with a compound of any one of statements 1-21 or a composition of any one of statements 22-26 in an amount and for a time sufficient to inhibit cell proliferation. Statement 31. 31. The method of statement 30, further comprising contacting the cells with flutamide, bicalutamide, enzalutamide, apalutamide, darolutamide, dilutamide, finasteride, dutasteride, abiraterone acetate, abiraterone acetate microparticles, ketoconazole, or a combination thereof. Statement 32. The method of claim 30 or 31, wherein the compound has the structure: TIFF0007648538000089.tif86169TIFF0007648538000090.tif49169Statement 33. 33. The method of claim 31 or 32, wherein the inhibited cell growth is malignant cell growth and / or hyperplastic cell growth. Statement 34. 34. The method of any one of claims 31 to 33, wherein the cell proliferation (e.g., malignant cell proliferation and / or hyperplastic cell proliferation) is cancer cell proliferation. Statement 35. 35. The method of any one of claims 31 to 34, wherein the cell proliferation (e.g., malignant cell proliferation and / or hyperplastic cell proliferation, which may be cancer cell proliferation) is hormonal cancer cell proliferation. Statement 36. 36. The method of claim 35, wherein the hormonal cancer is selected from breast cancer, ovarian cancer, uterine or endometrial cancer, prostate cancer, and combinations thereof. Statement 37. The method of any one of claims 31 to 35, wherein the cell proliferation (e.g., malignant cell proliferation and / or hyperplastic cell proliferation, which may be cancer cell proliferation, which may be hormonal cancer cell proliferation) is prostate cancer cell proliferation. Statement 38. The method of any one of claims 31 to 35 or claim 37, wherein the cell proliferation (e.g., malignant cell proliferation and / or hyperplastic cell proliferation, which may be cancer cell proliferation, which may be hormonal cancer cell proliferation, which may be prostate cancer cell proliferation) is castration-resistant prostate cancer cell proliferation. Statement 39. The method of any one of claims 31-35 or 37-38, wherein the cell (e.g., a malignant cell and / or a hyperplastic cell, which may be a cancer cell, which may be a hormonal cancer cell, which may be a prostate cancer cell, which may be a castration-resistant prostate cancer cell) expresses AR-V7. Statement 40. A method of treating a hyperplastic cell proliferative disease and / or a malignant cell proliferative disease in an individual, comprising administering to the individual a compound of any one of statements 1-21 or a composition of any one of statements 22-26, wherein the malignant cell proliferative disease and / or hyperplastic cell proliferative disease in the individual is treated. Statement 41. 41. The method of claim 40, wherein the malignant cell proliferation disease is cancer. Statement 42. 42. The method of claim 40 or 41, wherein the malignant cell proliferation disease (e.g., cancer) is a hormonal cancer. Statement 43. 43. The method of claim 42, wherein the hormonal cancer is selected from breast cancer, ovarian cancer, uterine or endometrial cancer, prostate cancer, and combinations thereof. Statement 44. 44. The method of any one of claims 40 to 43, wherein the malignant cell proliferation disease (eg, cancer, which may be a hormonal cancer) is prostate cancer. Statement 45. 45. The method of any one of claims 40 to 44, wherein the malignant cell proliferation disease (e.g., cancer, which may be a hormonal cancer, which may be prostate cancer) is castration-resistant prostate cancer. Statement 46. The method of any one of claims 40 to 45, wherein a cell of the malignant cell proliferative disease (e.g., cancer, which may be a hormonal cancer, which may be prostate cancer, which may be castration-resistant prostate cancer) expresses AR-V7. Statement 47. 47. The method of any one of claims 40-46, wherein the compound has the following structure: TIFF0007648538000091.tif84169TIFF0007648538000092.tif51169 Statement 48. 1. A method of imaging malignant and / or hyperproliferative cells in an individual, comprising administering to the individual one or more compounds of any one of statements 1 or 6 or statements 9-10 or statements 20-21, or a composition of any one of statements 22-26, exposing the individual, or a part thereof, to electromagnetic radiation to excite the one or more compounds, detecting the one or more excited compounds, and imaging the individual, or a part thereof, to obtain an image of the malignant and / or hyperproliferative cells. Statement 49. 49. The method of claim 48, wherein the malignant cells are cancer cells. Statement 50. 50. The method of claim 48 or claim 49, wherein the malignant cell (e.g., cancer cell) is a hormonal cancer cell. Statement 51. 51. The method of claims 48 to 50, wherein the malignant cells (e.g., cancer cells, which may be hormonal cancer cells) are selected from breast cancer cells, ovarian cancer cells, uterine or endometrial cancer cells, and prostate cancer cells. Statement 52. The method of any one of claims 48 to 51, wherein the malignant cell (eg, cancer cell, which may be a hormonal cancer cell) is a prostate cancer cell. Statement 53. The method of any one of claims 48 to 52, wherein the malignant cell (e.g., a cancer cell, which may be a hormonal cancer cell, which may be a prostate cancer cell) is a castration-resistant prostate cancer cell. Statement 54. The method of any one of claims 48 to 53, wherein the malignant cell (e.g., a cancer cell, which may be a hormonal cancer cell, which may be a prostate cancer cell, which may be a castration-resistant prostate cancer cell) expresses AR-V7. Statement 55. The method of any one of claims 48 to 54, wherein the imaging is repeated so that androgen metabolic activity can be measured. Statement 56. The method of any one of claims 48 to 55, wherein the compound has the structure: TIFF0007648538000093.tif213169TIFF0007648538000094.tif228169TIFF0007648538000095.tif47169

[0105] The following examples are presented to illustrate the present disclosure and are not intended to be limiting in any respect.

[0106] [Example 1] This example provides an illustration of the compounds of the present disclosure.

[0107] Synthesis of compound (Ia; also known as DIOL-17C), where R1 and R2 are hydrogen, as shown below and in FIG. TIFF0007648538000096.tif43169Ethyl (3-keto-5α-androstan-17β-yl) glycolate. To a suspension of 1 g (3.44 mmol) androstan-17β-ol-3-one and 95 mg (0.22 mmol, 0.064 equiv) dirhodium tetraacetate in 10 mL of CHCl was added dropwise at 0° C. with vigorous stirring 393 mg (3.44 mmol, 1 equiv) of ethyl diazoacetate. After 2 h at 25° C., another 393 mg (3.44 mmol, 1 equiv) of ethyl diazoacetate was added dropwise and the mixture was stirred for another 2 h. The mixture was diluted with CHCl, washed with water and brine, dried over anhydrous MgSO4, and concentrated. The product was purified by column chromatography using 1:3 EtOAc-hexanes to give 687 mg (53%) of ethyl (3-keto-5α-androstan-17β-yl)glycolate as a semi-solid. 1H NMR (CDCl3, 500 MHz): δ 4.20 (q, 2H, J=7.2Hz), 4.09 (s, 2H), 3.40(t, 1H, J=8.5Hz), 2.42-2.35 (m, 1H), 2.32-2.24 (m, 2H), 2.11-2.00 (m, 3H), 1.96-1.92(m, 1H), 1.72-1.68 (m, 1H), 1.58-1.26 (m, 13H), 1.21-1.15 (m, 1H), 1.02 (s, 3H), 1.00-0.83 (m, 2H), 0.82 (s, 3H), 0.75-0.69 (m, 1H). 13 C NMR (CDCl3, 100 MHz): δ 212.22, 171.03, 90.02, 67.80, 60.94, 54.09, 51.12, 46.91, 44.89, 43.28, 38.77, 38.36, 37.90, 35.93, 35.38, 31.43, 28.99, 27.76, 23.48, 21.27, 14.42, 11.86, 11.68.C 23 H 37 HRMS (ESI) calcd for O4[MH+]: 377.2686. Found: 377.2687.

[0108] Ethyl (3β-hydroxy-5α-androstan-17β-yl) glycolate. To a solution of 300 mg (0.80 mmol) of ethyl (3-keto-5α-androstan-17β-yl) glycolate in 4 mL of THF was added 2.1 mL (1.04 mmol, 1.3 equiv) of lithium tri-tert-butoxyaluminum hydride (0.5 M in bis(2-methoxyethyl) ether) dropwise at −20° C. The mixture was stirred for 3 h, quenched with water, extracted with CHCl, dried over anhydrous MgSO, and concentrated. The product was purified by column chromatography using 1:2 EtOAc-hexane to give 280 mg (93%) of ethyl (3β-hydroxy-5α-androstan-17β-yl) glycolate as a white solid: mp 72-74° C. 1H NMR (CDCl3, 500 MHz): δ 4.20 (q, 2H, J=7.0Hz), 4.10, 4.06 (ABq, 2H, J AB =16.2Hz), 3.61-3.55 (m, 1H), 3.39 (t, 1H, J=8.2Hz), 2.05-1.98 (m, 1H), 1.93-1.90 (m, 1H), 1.81-1.77 (m, 1H), 1.72-1.64 (m, 3H), 1.59-1.53 ​​(m, 4H), 1.43-1.36 (m, 2H), 1.32-1.21 (m, 8H), 1.17-1.07 (m, 2H), 0.99-0.82 (m, 3H), 0.81 (s, 3H), 0.79 (s, 3H), 0.65-0.59 (m, 1H). 13 C NMR (CDCl3, 100 MHz): δ 170.89, 89.94, 71.31, 67.60, 60.72, 54.45, 51.12, 44.87, 43.09, 38.18, 37.86, 37.02, 35.55, 35.29, 31.59, 31.50, 28.56, 27.59, 23.27, 20.88, 14.22, 12.34, 11.66.C 23 H 39 HRMS (ESI) calcd for O4[MH+]: 379.2843. Found: 379.2843.

[0109] Ethyl (3α-benzoyloxy-5α-androstan-17β-yl) glycolate. To a solution containing 200 mg (0.53 mmol) of ethyl (3β-hydroxy-5α-androstan-17β-yl) glycolate and 129 mg (1.06 mmol, 2 equiv.) of benzoic acid in 3 mL of anhydrous THF was added 214 mg (1.06 mmol, 2 equiv.) of diisopropyl azodicarboxylate. The solution was stirred at 25° C. for 12 h, quenched with water, extracted with CHCl, washed successively with 10% aqueous KCO and water, dried over anhydrous MgSO, and concentrated. The product was purified by elution with 1:5 EtOAc-hexane (R f=0.53) to afford 181 mg (71%) of ethyl (3α-benzoyloxy-5α-androstan-17β-yl)glycolate as a white solid: 1 H NMR (CDCl3, 400 MHz): δ 8.07-8.05 (m, 2H), 7.58-7.54 (m, 1H), 7.47-7.44 (m, 2H), 5.28 (br s, 1H), 4.20 (q, 2H, J=7.2Hz), 4.12, 4.07 (ABq, 2H, J AB =16.4Hz), 3.40 (t, 1H, J=8.4Hz), 2.05-1.98 (m, 1H), 1.95-1.85 (m, 2H), 1.80-1.52 (m, 7H), 1.45-1.14 (m, 12H), 1.03-0.87 (m, 2H), 0.85 (s, 3H), 0.81 (s, 3H), 0.79-0.75 (m, 1H). 13 C NMR (CDCl3, 100 MHz): δ 170.85, 165.87, 132.69, 131.18, 129.53 (two C), 128.32 (two C), 89.97, 70.68, 67.63, 60.70, 54.46, 51.16, C 30 H 43 HRMS (ESI) calculated for O5[MH+]: 483.3105. Found: 483.3108.

[0110] N-2'-(2,3,4,5-tetrahydro-1H,4H-10-keto-11-oxa-3a-azabenzo[de]anthracenyl)ethyl(3α-hydroxy-5α-androstan-17β-yl)glycolamide (Ia: alias DIOL-17C). To a solution containing 160 mg (0.33 mmol) of ethyl(3α-benzoyloxy-5α-androstan-17β-yl)glycolate in 3 mL of methanol, 0.66 mL (1.33 mmol, 4 equiv.) of 2N aqueous NaOH was added. The suspension was stirred at 50°C for 12 h, diluted with water and acidified to pH 2 with 3N HCl solution. The precipitate was collected and dried in vacuum to give 103 mg (89%) of (3α-hydroxy-5α-androstan-17β-yl)glycolic acid, which was pure enough to be used in the next reaction without further purification. To a mixture of 80 mg (0.23 mmol) of this acid and 90 mg (0.25 mmol, 1.1 equiv) of 8-(2-aminoethyl)-2,3,4,5-tetrahydro-1H,4H-11-oxa-3a-aza-benzo[de]anthracen-10-one hydrochloride in 2 mL of MeOH-CHCl (1:1), 66 mg (0.34 mmol, 1.5 equiv) of EDC, 46 mg (0.34 mmol, 1.5 equiv) of HOBt, and 81 mg (0.80 mmol, 3.5 equiv) of triethylamine were added successively. The mixture was stirred at 25° C. for 12 h, diluted with water, extracted with CHCl, dried over anhydrous MgSO, and concentrated. The product was obtained by eluting the 1:10 mixture of CHOH-CHCl (R f =0.53) to give 76 mg (54%) of Ia (also known as DIOL-17C) as a yellow foam: 1 H NMR (CDCl3, 400 MHz): δ 7.12 (s, 1H), 6.74 (t, 1H, J=5.8Hz), 5.90 (s, 1H), 4.04 (br s, 1H), 3.95, 3.90 (ABq, 2H, J AB=15.4Hz), 3.60 (q, 2H, J=7.2Hz), 3.28-3.23 (m, 5H), 2.92-2.87 (m, 4H), 2.79 (t, 2H, J=6.2Hz), 2.00-1.88 (m, 5H), 1.78-1.71 (m, 1H), 1.69-1.16 (m, 17H), 1.08-1.01 (m, 1H), 0.95-0.80 (m, 2H), 0.78 (s, 3H), 0.75-0.70 (m, 1H), 0.68 (s, 3H). 13 C NMR (CDCl3, 100 MHz): δ 170.61, 162.19, 153.65, 151.44, 145.97, 121.67, 118.18, 107.86, 107.71, 106.92, 90.00, 69.21, 66.49, 54.33, 51.06, 49.96, 49.51, 43.00, 39.12, 38.15, 37.77, 36.15, 35.84, 35.21, 32.19, 31.64, 31.49, 29.02, 28.36, 27.79, 27.66, 23.25, 21.54, 20.65, 20.50, 20.36, 11.77, 11.21.C 38 H 53 HRMS (ESI) calcd for N2O5[MH+]: 617.3949. Found: 617.3940.

[0111] [Example 2] This example provides an illustration of the compounds of the present disclosure. Synthesis of compound Id (also known as DHT-17C) where R1 and R2 are hydrogen, as shown in Figure 2: TIFF0007648538000097.tif43169N-2'-(2,3,4,5-tetrahydro-1H,4H-10-keto-11-oxa-3a-azabenzo[de]anthracenyl)ethyl(3-keto-5α-androstan-17β-yl)glycolamide. To a solution of 100 mg (0.27 mmol) of ethyl(3-keto-5α-androstan-17β-yl)glycolate in 4 mL of THF was added 0.28 mL (0.54 mmol, 2 equiv.) of 2N aqueous NaOH. The mixture was stirred at 25° C. for 5 h and concentrated. The residue was diluted with water and acidified to pH 2 with 3N HCl solution to produce a white precipitate which was collected to give 83 mg (90%) of (3-keto-5α-androstan-17β-yl)glycolic acid which was used in the next step without further purification. To a mixture of 37 mg (0.11 mmol) of this acid and 42 mg (0.12 mmol, 1.1 equiv) of 8-(2-aminoethyl)-2,3,4,5-tetrahydro-1H,4H-11-oxa-3a-aza-benzo[de]anthracen-10-one hydrochloride in 1 mL of CHCl was added 30 mg (0.16 mmol, 1.5 equiv) of EDC, 24 mg (0.16 mmol, 1.5 equiv) of HOBt, and 38 mg (0.37 mmol, 3.5 equiv) of triethylamine. The mixture was stirred at 25° C. for 12 h, diluted with water, extracted with CH2Cl2, dried over anhydrous MgSO4, and concentrated. The product was purified by elution with 5:1 EtOAc-hexane (after two runs, R f =0.53) to afford 33 mg (51%) of Id (aka DHT-17C) as a yellow foam. 1 H NMR (CDCl3, 400 MHz): δ 7.10 (s, 1H), 6.72 (t, 1H, J=6.0Hz), 5.87 (s, 1H), 3.93, 3.89 (ABq, 2H, J AB=15.2Hz), 3.59 (q, 2H, J=6.8Hz), 3.27-3.22 (m, 5H), 2.91-2.85 (m, 4H), 2.78 (t, 2H, J=6.4Hz), 2.42-2.21 (m, 3H), 2.09-1.88 (m, 7H), 1.78-1.74 (m, 1H), 1.70-1.64 (m, 1H), 1.59-1.20 (m, 10H), 1.10-1.04 (m, 1H), 0.99 (s, 3H), 0.95-0.80 (m, 2H), 0.73-0.66 (m, 4H). 13 C NMR (CDCl3, 100 MHz): δ 211.90, 170.49, 162.12, 153.61, 151.43, 145.97, 121.65, 118.19, 107.80, 107.72, 106.89, 89.83, 69.19, 53.77, 50.79, 49.95, 49.50, 46.66, 44.66, 42.99, 38.52, 38.13, 38.07, 37.61, 35.70, 35.11, 31.65, 31.17, 28.73, 27.78, 27.65, 23.28, 21.52, 21.01, 20.64, 20.51, 11.76, 11.48.C 38 H 51 HRMS (ESI) calcd for N2O5[MH+]: 615.3792. Found: 615.3793.

[0112] [Example 3] This example provides an illustration of the compounds of the present disclosure.

[0113] Synthesis of compound (IIa) where R1 is hydrogen, as shown in FIG. TIFF0007648538000098.tif4216917-(O-Carboxymethyloximino)-5α-androstan-3α-ol. A mixture of 537 mg (1.85 mmol) of 5α-androstan-3α-ol-17-one and 404 mg (3.7 mmol) of carboxymethoxyamine hemiclolide in 20 ml of pyridine was stirred at 80° C. for 15 h. The product was cooled and concentrated. The product was diluted with water and extracted with CHCl. ​​The organic layer was washed with brine and dried over anhydrous MgSO. The solvent was evaporated and the residue was recrystallized from acetone to give 652 mg (97%) of a white solid: mp 171-173℃. mp 173-175℃. 1 H NMR (CDCl3, 400 MHz): δ 4.57 (s, 2H), 4.05 (br s, 1H), 2.54-2.48 (m, 2H), 1.94-0.82 (m, 20H), 0.91 (s, 3H), 0.79 (s, 3H). 13 C NMR (CDCl3, 100 MHz): δ 174.3, 172.9, 69.8, 66.4, 54.3, 53.8, 44.5, 39.0, 36.1, 35.7, 34.8, 33.9, 32.0, 31.4, 30.9, 28.9, 28.2, 26.1, 23.0, 20.2, 17.1, 11.1.

[0114] 8-Hydroxy-2,3,6,7-tetrahydro-1H,5H-benzo[ij]quinolizine. A mixture of 30 g (0.244 mol) m-anisidine and 91.7 mL (0.927 mol, 3.8 equiv.) 1-bromo-3-chloropropane was heated at 95 °C for 1-2 h, at which point an exothermic reaction occurred, so heating was discontinued and the temperature of the mixture peaked at 140-145 °C. The temperature began to decrease, so heating was resumed. The mixture was heated at 140-145 °C for 48 h and then at 175-180 °C for 21 h. The mixture was cooled, diluted with 370 mL concentrated HCl, and quenched by the slow addition of 120 mL water. After all solids had dissolved, the two phases were separated. The organic layer was washed with 100 mL 10% HCl and combined with the aqueous solution. The aqueous solution was extracted with ether to remove unreacted 1-bromo-3-chloropropane. To this acidic aqueous solution was added 200 mL of 25 M aqueous NaOH. The solution was extracted with toluene until the organic phase was no longer colored. The organic phase was dried over anhydrous MgSO4 and the solvent was removed under reduced pressure to give 18 g (39%) of a white solid: mp 126-128℃. mp 126-130℃). 1 H NMR (400 MHz, DMSO-d6): δ 8.62 (s, 1H), 6.47 (d, 1H, J=8.0 Hz), 5.99 (d, 1H, J=8.0 Hz), 3.01-2.96 (m, 4H), 2.57-2.48 (m, 4H), 1.85-1.79 (m, 4H). 13 C NMR (100 MHz, DMSO-d6): δ 153.0, 143.6, 126.1, 111.9, 107.7, 103.0, 49.6, 49.1, 26.7, 22.1, 21.4, 21.0.

[0115] Methyl-5-(benzyloxycarbonylamino)-3-oxopentanoate. To a solution of 35 mL (30 mmol) of dimethyl malonate in 40 mL of MeOH, 17.1 g (30 mmol) of KOH in 70 mL of MeOH was added dropwise over 1 h at 5-10 °C. The mixture was stirred at 25 °C for about 12 h. The precipitated potassium methyl malonate was collected and washed with cold MeOH. A suspension of 7.42 g (78 mmol, 1.04 equiv.) of MgCl2 and 17.6 g (113 mmol, 1.5 equiv.) of potassium methyl malonate in 110 mL of anhydrous THF was stirred at 50 °C for 4 h. In a separate flask, 14.6 g (90 mmol, 1.2 equiv.) of 1,1'-carbonyldiimidazole was added to a solution of 16.7 g (75 mmol, 1 equiv.) of N-benzyloxycarbonyl β-alanine in 50 mL of THF (5° C.). The imidazolide solution was added dropwise to a suspension of methylmagnesium malonate at 25° C. The mixture was stirred for about 12 h, concentrated under reduced pressure, and then diluted with ethyl acetate. The ethyl acetate solution was washed successively with aqueous KHSO4, aqueous NaHCO3, and brine. The organic layer was dried over anhydrous Na2SO4 and concentrated to give 18 g (98%) of a yellow oil that was used without further purification. 1 H NMR (400 MHz, CDCl3): δ 7.34 (br s, 5H), 5.24 (br s, 1H, NH), 5.07 (s, 2H), 3.72 (s, 3 H), 3.45 (s, 4 H), 2.80 (t, 2 H, J=5.2 Hz).

[0116] 8-(2-aminoethyl)-2,3,4,5-tetrahydro-1H,4H-11-oxa-3a-aza-benzo[de]anthracen-10-one hydrochloride. To a suspension of 8.33 g (44 mmol) of 8-hydroxy-2,3,6,7-tetrahydro-1H,5H-benzo[ij]quinolizine and 10.8 g (44 mmol) of methyl-5-(benzyloxycarbonylamino)-3-oxopentanoate in 132 mL of toluene was added 88 mL (88 mmol, 2 equiv.) of a 1 M solution of triisopropoxytitanium chloride in hexane. The mixture was heated to reflux for approximately 12 hours. The mixture was diluted with 500 mL of CHCl and poured into 500 mL of stirred saturated potassium sodium tartrate solution. The aqueous layer was extracted with CHCl. ​​The combined organic layers were dried over anhydrous NaSO. The filtered solution was concentrated and the residue was dissolved in a mixture of EtOH and hexane. This mixture was stored at 5° C. for 3 days. The resulting precipitate was collected and recrystallized from EtOH to give 13 g (77%) of a 1:9 mixture of benzyl and isopropyl [2-(10-oxo-2,3,5,6-tetrahydro-1H,4H,10H-11-oxa-3a-aza-benzo[de]anthracen-8-yl)-ethyl]carbamate as a yellow solid. To 5.05 g (13.6 mmol) of this ester mixture was added 12 mL of concentrated HCl. The solution was heated at 95° C. for 9 hours, cooled, and concentrated in vacuo. The residue was suspended in a mixture of MeOH-acetone and filtered to give 4.3 g (98%) of (7) as a yellow hydrochloride salt: mp 238-239℃. mp for free amine 114-118℃ 1 H NMR (400 MHz, CD3OD): δ 7.25 (s, 1 H), 6.00 (s, 1 H), 3.36-3.24 (m, 6 H), 3.09 (t, 2 H, J=7.2 Hz), 2.86 (m, 4 H), 2.01 (m, 4 H). 13C NMR (100 MHz, CD3OD): δ 164.2, 154.0, 152.6, 146.7, 123.0, 122.9, 121.5, 109.7, 109.4, 109.1, 109.0, 51.4, 50.9, 39.8, 30.5, 28.6, 22.4, 21.6, 21.5.

[0117] N-2'-(2,3,4,5-tetrahydro-1H,4H-10-keto-11-oxa-3a-azabenzo[de]anthracenyl)ethyl 17-(O-carbamidomethyloximino)-5α-androstan-3α-ol (IIa). To a solution (0 °C) of 122 mg (0.38 mmol) of 8-(2-aminoethyl)-2,3,4,5-tetrahydro-1H,4H-11-oxa-3a-aza-benzo[de]anthracen-10-one hydrochloride (7) in 4 mL of 1:3 MeOH-CHCl3, 75 μL (0.42 mmol, 1.1 equiv.) of N,N-diisopropylethylamine, 139 mg (0.38 mmol) of (6), 206 mg (1.5 mmol, 4 equiv.) of 1-hydroxybenzotriazole hydrate (HOBt), and 205 mg (1.07 mmol, 2.8 equiv.) of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) were added in succession in two equal portions, 30 min apart. The mixture was stirred at 0° C. for 1 h, concentrated, and diluted with 1:10 MeOH-CH2Cl2(R f =0.39) to give 134 mg (56%) of IIa as a yellow foam. 1H NMR (CDCl3, 400 MHz): δ 7.13 (s, 1H), 6.6 (t, 1H, J=6.4 Hz), 5.87 (s, 1H), 4.46 (s, 2H), 4.03 (br s, 1H), 3.65-3.59 (m, 2H), 3.28-3.23 (m, 4H), 2.92-2.85 (m, 4H), 2.79 (t, 2H), 2.46-2.33 (m, 2H), 1.99-1.94 (m, 4H), 1.82-0.73 (m, 20H), 0.84 (s, 3H), 0.79 (s, 3H). 13 C NMR (CDCl3, 100 MHz): δ 173.3, 170.8, 162.3, 153.9, 151.4, 146.1, 121.8, 118.4, 107.8, 107.4, 106.9, 72.6, 66.3, 54.4, 53.9, 50.0, 49.5, 44.5, 39.1, 38.1, 36.2, 35.9, 34.9, 34.1, 32.1, 31.9, 31.5, 29.0, 28.4, 27.8, 26.1, 23.2, 21.6, 20.7, 20.6, 20.3, 17.3, 11.3.C 38 H 52 HRMS (ESI) calcd for N3O5[MH+]: 630.3901. Found: 630.3899.

[0118] [Example 4] This example provides an illustration of the compounds of the present disclosure.

[0119] Synthesis of compound (IIb) where R1 is hydrogen as shown in FIG. 3: TIFF0007648538000099.tif41169N-2'-(2,3,4,5-tetrahydro-1H,4H-10-keto-11-oxa-3a-azabenzo[de]anthracenyl)ethyl 17-(O-carbamidomethyloximino)-5α-androstan-3-one (IIb) To a solution of 30 mg (0.048 mmol) of IIa in 1 mL of DMSO:CH2Cl2 (1:1) were successively added 24 mg (0.24 mmol, 5 equiv.) of triethylamine and 23 mg (0.14 mmol, 3 equiv.) of SO3-pyridine complex. The mixture was stirred at 25 °C for 5 h, diluted with CH2Cl2, washed with brine and water, dried over anhydrous MgSO4, and concentrated. The product was obtained by elution with a 1:10 mixture of CH3OH-CH2Cl2 (R f =0.56) to afford 16 mg (54%) of IIb as a yellow foam. 1 H NMR (CDCl3, 400 MHz): δ 7.10 (s, 1H), 6.46 (t, 1H, J=5.8Hz), 5.87 (s, 1H), 4.46 (s, 2H), 3.69-3.57 (m, 2H), 3.28-3.23 (m, 4H), 2.91-2.86 (m, 4H), 2.79 (t, 2H, J=6.2Hz), 2.50-2.24 (m, 5H), 2.11-1.94 (m, 5H), 1.87-1.73 (m, 3H), 1.65-1.60 (m, 1H), 1.57-1.26 (m, 8H), 1.18-1.12 (m, 1H), 1.02-0.92 (m, 4H), 0.88 (s, 3H), 0.83-0.72 (m, 1H). 13 C NMR (CDCl3, 100 MHz): δ 211.69, 172.96, 170.60, 162.09, 153.62, 151.42, 145.99, 121.64, 118.25, 107.67, 107.33, 106.82, 72.55, 53.81, 53.61, 49.94, 49.49, 46.58, 44.63, 44.41, 38.46, 38.11, 37.71, 35.76, 34.74, 33.97, 31.81, 31.11, 28.69, 27.78, 26.09, 23.20, 21.51, 20.92, 20.62, 20.51, 17.22, 11.45.C 38 H 50HRMS (ESI) calcd for N3O5[MH+]: 628.3745. Found: 628.3741.

[0120] [Example 5] This example provides an illustration of the compounds of the present disclosure.

[0121] Synthesis of a compound in which R1 and R2 are hydrogen (Ig: also known as DHT-3C) as shown in Figure 4. TIFF0007648538000100.tif44169(3R,5S,10S,13S,17S)-17-Hydroxy-10,13-dimethyl-1,2,2',3',4,5,6,7,8,8',9,9',10,11,12,12',13,13',14,15,16,17-docosahydro-7'H,11'H-spiro[cyclopenta[a]phenanthrene-3,4'-pyrido[3,2,1-ij]pyrido[4',3':4,5]pyrano[2,3-f]quinoline]-5'(1'H)-one hydrochloride (Ig) To a suspension of 66 mg (0.21 mmol, 1 equiv.) of 9-(2-aminoethyl)-2,3,6,7-tetrahydro-1H,5H,11H-pyrano[2,3-f]pyrido[3,2,1-ij]quinolin-11-one hydrochloride in 2 mL of absolute ethanol was added 50 mg (0.17 mmol, 1 equiv.) of DHT. The suspension was placed in a sealed tube, 0.2 mL of concentrated HCl was added, and the mixture was stirred under reflux for 48 h. The suspension became a clear solution within the first hour of heating, and then a precipitate of the desired product appeared. The reaction was quenched by the addition of approximately 3 mL of water, and the precipitate was collected by filtration to give 73 mg (92%) of Ig. Further purification was achieved by recrystallization from methanol: 1H NMR (400 MHz, DMSO-d6) δ9.41-9.13 (m, 2H), 7.14 (s, 1H), 4.43 (br s, 1H), 3.45 (t, 2H), 3.29-3.22 (m, 4H), 3.12-3 (m, 2H), 2.82-2.64 (m, 4H), 2.57 (t, J=14.2 Hz, 1H), 1.96-1.8 (m, 4H), 1.8-1.7 (m, 2H), 1.7-1.55 (m, 4H), 1.56- 1.41 (m, 4H), 1.42-1.3 (m, 3H), 1.28-1.06 (m, 5H), 1.05-0.77 (m, 7H), 0.65 (s, 3H). 13 C NMR (101 MHz, DMSO-d6) δ158.61, 149.23, 147.42, 145.69, 121.59, 118.47, 114.52, 106.22, 104.7, 80.04, 58.69, 52.91, 50.82, 49.16, 48.65, 42.59 (two C), 36.7, 35.26, 35.05, 34.83, 32.41, 31.64, 31.17, 29.84, 27.47, 27.09, 25.4, 23.05, 22.54, 20.88, 20.12, 19.96, 19.69, 11.42, 11.37.C 36 H 49 HRMS (ESI) calcd for N2O3[MH+]: 557.3738. Found: 557.3744. The hydrochloride salt of the desired product was suspended in dichloromethane and washed with a saturated aqueous solution of NaHCO. The dichloromethane phase was dried over anhydrous NaSO, filtered, concentrated and purified with 1:10 methanol-dichloromethane (R f =0.55) to give Ig as the free base: 1H NMR (400 MHz, DMSO-d6) δ7.03 (s, 1H), 4.4 (d, J=4.8 Hz, 1H), 3.48-3.37 (m, 1H), 3.2 (q, J=5.6 Hz, 4H), 2.84 (t, J=5.7 Hz, 2H), 2.7 (q, J=6 Hz, 4H), 2.58 (t, J=5.6 Hz, 2H), 2.52-2.43 (m, 1H), 2.32 (t, J=13 Hz, 1H), 1.95-1.76 (m, 6H), 1.75-1.67 (m, 1H), 1.66-1.54 (m, 2H), 1.54-1.43 (m, 2H), 1.39-1.26 (m, 4H), 1.25-1.03 (m, 5H), 1-0.75 (m, 7H), 0.74-0.64 (m, 1H), 0.62 (s, 3H). 13 C NMR (101 MHz, DMSO-d6) δ159.5, 149.38, 149.08, 144.58, 122.52, 121.11, 117.68, 108.18, 104.87, 80.11, 53.89, 53.82, 50.79, 49.16, 48.67, 42.59, 39.94, 36.76, 36.13, 35.69, 35.28, 35.25, 33.32, 31.48, 29.88, 28.18, 27.84, 27.13, 26.3, 23.11, 21.16, 20.28, 20.22, 19.88, 11.61, 11.39. C 36 H 49 N2O3[MH+]HRMS (ESI) calculated value: 557.3738. Measured value: 557.3738. C 36 H 48 Analytical calculated values ​​for N2O3: C, 77.66; H, 8.69; N, 5.03. Measured values: C, 77.53; H, 8.62, N, 4.95. Confirmation of the stereochemistry of the cyclic DHT adduct IgにおけるC-3R and the second dimension 1 H- 13C heteronuclear single quantum coherence (HSQC), gradient correlation spectroscopy (gCOSY), and 2D rotating frame NOESY (2D ROESY) experiments were relied upon. The resonance of the protonated amine in the spirocyclic ring system appears at δ 9.24 ppm in the 2D ROESY spectrum and was the starting point for this stereochemical assignment at C-3. The ammonium group (NH2 + ) was identified by DO exchange experiments in DMSO-d6. Correlation of the ROESY spectrum of this ammonium group with the individual C-1α, C-2α, C-4α, and C-5α protons (Figure 5) confirmed the 3α-orientation of the ammonium group in Ig.

[0122] [Example 6] This example provides an illustration of the compounds of the present disclosure.

[0123] Synthesis of compound (Ij) where R1 is hydrogen as shown in FIG. TIFF0007648538000101.tif42169(3R,5S,10S,13S)-10,13-Dimethyl-1,2',3',4,5,6,7,8,8',9,9',10,11,12,12',13,13',14,15,16-icosahydro-7'H,11'H-spiro[cyclopenta[a]phenanthrene-3,4'-pyrido[3,2,1-ij]pyrido[4',3':4,5]pyrano[2,3-f]quinoline]-5',17(1'H,2H)-dione (Ij) The procedure described for the preparation of Ig was repeated using 5α-androstane-3,17-dione to give Ij (74%), which was purified by trituration in hot methanol. 1H NMR (400 MHz, DMSO-d6) δ 7.04 (s, 1H), 3.21 (q, J=5.5 Hz, 4H), 2.85 (t, J=5.8 Hz, 2H), 2.71 (q, J=6.1 Hz, 4H), 2.58 (t, J=5.7 Hz, 2H), 2.46-2.30 (m, 3H), 2.08-1.95 (m, 1H), 1.93-1.78 (m, 5H), 1.74 (dd, J=12.7, 3.3 Hz, 1H), 1.7-1.59 (m, 2H), 1.59-1.42 (m, 3H), 1.4-1.32 (m, 2H), 1.31-1.18 (m, 4H), 1.18-1.08 (m, 3H), 1.02-0.96 (m, 1H), 0.95 (s, 3H), 0.79 (s, 3H), 0.79-0.7 (m, 1H). 13 C NMR (101 MHz, DMSO-d6) δ 219.89, 159.52, 149.46, 149.08, 144.61, 122.46, 121.13, 117.71, 108.15, 104.87, 53.87, 53.64, 50.83, 49.16, 48.66, 47.14, 36.12, 35.77, 35.32, 34.63, 33.23, 31.45, 30.65, 28.01, 27.8, 27.12, 26.28, 21.38, 21.15, 20.27, 19.86, 13.49, 11.57.

[0124] [Example 7] This example provides a description of the biological activity of 2-(((3R,5S,10S,13S,17S)-3-hydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)oxy)-N-(2-(11-oxo-2,3,6,7-tetrahydro-1H,5H,11H-pyrano[2,3-f]pyrido[3,2,1-ij]quinolin-9-yl)ethyl)acetamide, described in the present disclosure as DIOL-17C (also known as compound Ia). TIFF0007648538000102.tif47169

[0125] CaP cells survive and proliferate under ADT due to intratumoral synthesis of DHT and AR sensitization. Our group and others have previously demonstrated that AR is transactivated by picomolar levels of DHT, that CRPC produces sufficient DHT for AR transactivation, and that CRPC efficiently metabolizes DIOL to DHT. Identification of sufficient tissue levels of DHT for AR transactivation in CRPC led to the repurposing and FDA approval of abiraterone for the treatment of advanced CaP. The confluence of the front- and backdoor pathways to DHT was only recently evaluated. DIOL is the immediate precursor of DHT in the primary backdoor pathway (Figure 1), and preclinical studies have clearly demonstrated the conversion of DIOL to DHT using castration-recurrent CWR-R1 human CaP xenografts. Four 3α-oxidoreductase enzymes that metabolize DIOL to DHT were immunostained in tissue microarrays (TMAs) constructed from androgen-stimulated benign prostate (AS-BP; n=36), CaP (AS-CaP; n=36), and CRPC (n=36) (Figure 8). CaP cell pellets analyzed using LC-MS / MS showed that 3α-oxidoreductase expression increased DHT production (Figure 9).

[0126] Inhibition of DIOL conversion to DHT by 3α-oxidoreductase: DIOL was converted to DHT in CRPC xenografts, and four 3α-oxidoreductases were present in clinical specimens, indicating that DIOL is likely metabolized to DHT in advanced CaP or CRPC patients. In vitro administration results in AR-luciferase (Figure 10) transactivation, providing an opportunity to test inhibitors. Constraint-based Multiple Protein Alignment Tool (COBALT) showed that the four 3α-oxidoreductases share active-site, catalytic amino acid residues. LC-MS / MS confirmed that the four 3α-oxidoreductases convert DIOL to DHT and androsterone to 5α-DIONE. The 3α-oxidoreductase catalytic amino acid residues were mutated using site-directed mutagenesis. LC-MS / MS showed that single, double, or complete catalytic deletion of the common catalytic amino acids impaired enzyme activity. Furthermore, the impaired catalytic mutant combined with dutasteride reduced DHT levels in CaP cell lines more than dutasteride alone (Figure 11). The experiment was repeated with LAPC-4 cells transfected with RDH16-targeting siRNA. DHT ELISA revealed that knockdown of RDH16 expression reduced DHT levels in LAPC-4 (Figure 12). Growth of VCaP (Figure 13) and LAPC-4 cells (data not shown) was assessed following expression of the catalytically impaired RDH16 Y176F, K180R mutant and treatment with dutasteride.

[0127] Identification of 3α-oxidoreductase inhibitors: Various nonsteroidal compounds were tested for antitumor activity by screening against PC-3 CaP cells. Coumarin-labeled androgens were developed to follow changes in the androgen metabolic pathway in response to castration or androgen metabolic inhibition using in vitro and in vivo CaP cell models. (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was used to assess cell viability and AR-regulated gene transcription. Data were collected on days 0 and 6 and expressed as relative proliferation percentage (mean day 6-mean day 0 / mean day 0). CaP cell lines were cultured in SFM alone or SFM containing 20 nM DIOL or 20 nM DIOL-17C for 6 days. MTT showed that DIOL-17C treatment impaired CaP cell proliferation (Figure 14).

[0128] ImageStream(ImageStream XA RDH16-MKII Flow Cytometer (Amnis, Seattle, WA), a hybrid technology combining the statistical power of flow cytometry with the imaging capabilities of fluorescence microscopy, was adapted to simultaneously study the compartmental localization and activity of DIOL-17C. ImageStream data were normalized to single staining controls and analyzed using Image Data Exploration and Analysis Software (IDEAS; Amnis). Similarity scores were measured by pixel-by-pixel comparison of two images of the same cell. If the similarity score was >0, the two objects (RDH16-YFP and DIOL-17C) were colocalized. DIOL-17C (blue) colocalizes with RDH16-YFP (recolored green in IDEA for comparison) and nuclear DRAQ5 (white punctate signal) in PC-3-RDH16-YFP cells (Figure 15A.1 single and merged fluorescent images; 15A.2 high similarity in upper right quadrant), but not in SFM-treated PC-3 RDH16-YFP cells (Figure 15B.1 no punctate signal in merged image; 15B.2 high similarity in upper right quadrant). Competition studies using ImageStream demonstrated that DIOL and DIOL-17C compete for RDH16-YFP in PC-3 cells stably expressing RDH16-YFP (Figure 16).

[0129] To ensure that cells were not producing free DIOL and orphan coumarin, LC-MS / MS was performed with CaP cells treated with DIOL-17C. No orphan fluorophores were detected. Addition of coumarin did not impair 3α-oxidoreductase metabolism of DIOL-17C to DHT-17C (Figure 17).

[0130] [Example 8] This example provides a description of the biological activity of (((5S,10S,13S,17S)-10,13-dimethyl-3-oxohexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)oxy)-N-(2-(11-oxo-2,3,6,7-tetrahydro-1H,5H,11H-pyrano[2,3-f]pyrido[3,2,1-ij]quinolin-9-yl)ethyl)acetamide, described in the present disclosure as DHT-17C (also known as compound Id). TIFF0007648538000103.tif49169

[0131] Androgen-sensitive AR-positive LAPC-4 and VCaP, castration-relapse and AR-positive CWR-R1 and CWR-22rv1 (22rv1), and AR-negative PC-3 and DU145 cells were transfected with either an empty pCMV expression plasmid (control) or a pCMV plasmid encoding wild-type AR tagged with a C-terminal green fluorescent protein (GFP) (AR-GFP). AR-positive CaP cell lines were transfected with AR-GFP to assess whether DHT-17C or DHT-3C colocalize with AR and translocate to the nucleus. To test the effect of coumarin on antiandrogenic activity, the fluorescent coumarin moiety was moved from the C-17 position to the C-3 position (DHT-3C, also known as compound Ig). The data demonstrated that the position of the coumarin moiety on DHT is essential to distinguish DHT-like from antiandrogenic activity. TIFF0007648538000104.tif38169

[0132] AR-positive CaP cell lines were treated with androgen-free serum-free complete medium (SFM) alone or with SFM containing 20 nM or 50 nM of fluorescent androgen or C alone for 16 hours. These time points were chosen to measure differences in fluorescent androgen uptake and nuclear or cytosolic localization of fluorescent androgen and / or AR-GFP. ImageStream analysis showed that DHT-17C and DHT-3C were uptaken into VCaP, LAPC-4, CWR-R1, 22Rv1, PC-3, and DU145 cells (data reported for VCaP and PC-3-AR-GFP, representative of all cell lines studied). DHT-17C (blue) or DHT-3C colocalized with AR-GFP (green) and the nuclear marker DRAQ5 (red) in the nuclei of VCaP-AR-GFP and PC-3-AR-GFP cells (white punctate signals) (Figure 18).

[0133] DHT-3C (also known as compound Ig) colocalized with AR-GFP and DRAQ5 (FIG. 18). DHT-3C served as a negative control for DHT-17C (also known as compound Id), which was shown to be an AR-LBD inhibitor in contrast to DHT-3C, which retains DHT activity (as described above).

[0134] No nuclear localization of DHT-17C or DHT-3C was observed in AR- or AR-GFP-negative PC-3 cells, suggesting that nuclear localization of DHT-17C or DHT-3C is AR-dependent. ImageStream studies were repeated using VCaP and PC-3 cells stably expressing AR-V2 (a splice variant lacking the N-terminus) with C-terminal GFP (AR-V2-GFP). ImageStream analysis revealed DHT-17C and DHT-3C colocalizing with DRAQ5 and AR-V2-GFP (Figure 18). ImageStream data was confirmed using immunoprecipitation (IP) studies performed with a DHT-targeting antibody.

[0135] To establish that DHT-17C targets the AR-LBD, we performed a competition study between DHT-17C and enzalutamide for AR-GFP or AR-V2 using ImageStream. ImageStream data confirmed that DHT-17C and enzalutamide compete for the AR-LBD of wild-type AR and AR-V2 (Figure 19).

[0136] VCaP, CWR-R1 and PC-3 cells were treated with complete culture medium (CM), serum-free culture medium (SFM), 10 μM bicalutamide (Bic), 20 μM coumarin C, 10 nM DHT, 20 nM DHT-17C and 20 nM DHT-3C for 6 days (Figure 20). MTT assay analysis revealed that 6 days of treatment with DHT-3C promoted VCaP, LAPC-4 and LNCaP cell proliferation, as expected for a DHT surrogate (data not shown) and CWR-R1 cell proliferation (compared to DHT) (Figure 20). In contrast, DHT-17C treatment impaired VCaP and CWR-R1 cell proliferation and enhanced the effect of bicalutamide (Figure 20; p-value < 0.05). MTT experiments with LAPC-4, LNCaP and 22Rv1 produced similar responses (data not shown; p value < 0.05). DHT-17C did not impair cell proliferation in AR-negative PC-3 (Figure 20), DU145 and non-CaP cell lines (CV-1 monkey kidney and 293 human kidney cell lines). MTT analysis was performed to compare cell proliferation impairment of VCaP cells treated with DHT-17C to enzalutamide. Cells were treated with DHT17C at 1 nM, 12 nM, 20 nM, 50 nM or 100 nM concentrations or enzalutamide at 1 μM, 12 μM, 20 μM, 50 μM or 100 μM concentrations. DHT-17C at nM concentrations impaired VCaP cell proliferation to the same extent as enzalutamide at μM concentrations (Figure 21). MTT performed with mutant AR and AR-V7 positive CWR-R1 cells treated with ezulutamide (20 uM) or DHT-17C (50 nM) supported the VCaP MTT data (Figure 22).

[0137] Quantitative real-time polymerase chain reaction (qRT-PCR) analysis showed that treatment of non-transfected VCaP, LAPC-4, LNCaP, CWR-R1 and 22rv1 cells with DHT or DHT-3C for 24 hours similarly induced expression of the AR-regulated gene, KLK2 (Figure 23; representative example CWR-R1). In contrast, DHT-17C treatment did not induce KLK2 transcript formation in any of the five AR-positive CaP cell lines (Figure 23; representative example CWR-R1), suggesting that the site of fluorophore placement on the DHT molecule (and not the presence of C) was responsible for the lack of KLK2 induction. Completion of these studies revealed that DHT-17C has antiandrogenic properties, but produced another unexpected finding.

[0138] [Example 9] DHT-3C is a surrogate for DHT that is used as a carrier for molecules that disrupt AR transactivation or even degrade the AR.

[0139] The cellular uptake of DHT-3C (also known as Compound Ig) by AR-negative PC-3 cells carrying an empty plasmid, PC-3 cells stably expressing AR-GFP (PC-3-AR-GFP), VCaP cells carrying an empty plasmid, VCaP cells stably expressing AR-GFP (VCaP-AR-GFP), or VCaP cells stably expressing the AR170 LBD with only the splice variant V2 (AR-V2-GFP) was assessed using ImageStream (Figure 4) and confirmed the DHT-like activity of DHT-3C. 27Using SFM, we obtained androgen-deprived cells. PC-3 cells containing an empty plasmid were treated with SFM alone, SFM with C, or SFM with DHT-3C. The cells showed minimal autofluorescence and did not produce nuclear fluorescence consistent with C or DHT-3C. SFM-treated PC-3 AR-GFP cells showed no autofluorescence. SFM with C-treated PC-3 AR-GFP cells showed no nuclear colocalization between C and AR-GFP (Figure 24A.1 and A.2). DHT-3C-treated PC-3-AR-GFP showed nuclear colocalization between DHT-3C and AR-GFP (Figure 24B.1 and B.2). C-treated VCaP cells stably expressing AR-GFP did not produce nuclear colocalization between C and AR-GFP (Figure 24C.1 and C.2). DHT-3C-treated VCaP stably expressing AR-GFP showed colocalization between DHT-3C and AR-GFP (Figure 24D.1 and D.2). DHT-3C-treated VCaP stably expressing AR-V2-GFP showed DHT-3C colocalized with the AR LBD (Figure 24E.1 and E.2).

[0140] ImageStream data revealed that DHT-3C was incorporated into CaP cell lines and colocalized with AR in the nucleus. MTT analysis was used to evaluate whether treatment with DHT-3C promoted CaP cell proliferation during androgen deprivation. AR-positive androgen-sensitive VCaP, LAPC-4, and LNCaP; AR-positive castration relapse CWR-R1 and CWR22rv1; and AR-negative PC-3 and DU145 CaP cell lines were treated for 6 days under five sets of conditions: complete cell culture medium (CM) to establish baseline CaP cell proliferation; SFM to simulate androgen deprivation; SFM with C alone as a negative control to confirm that C alone does not affect CaP cell proliferation; SFM with DHT as a positive control; and SFM with DHT-3C. SFM with DHT-3C stimulated proliferation in three of the five cell lines (Figure 25).

[0141] In summary, these data suggested that increased CaP cell proliferation was AR-dependent and stimulated by either DHT or DHT-3C, which functions as a DHT substitute.

[0142] Additional data showed that DHT-3C, like DHT itself, affected AR-regulated gene transcript induction. This finding was consistent with computer modeling, in which C alone did not affect AR-regulated transcript induction and the coumarin moiety in DHT-3C was primarily outside the AR-LBD binding pocket. Western blot analysis confirmed that DHT-3C and DHT treatments resulted in similar AR-regulated protein expression levels (Figure 26).

[0143] In summary, DHT-3C (aka compound Ig) in FIG. 4 serves as a true surrogate, and its replacement with other ligands provides a means for the evaluation of antiandrogens or for delivering small molecules to disrupt AR transactivation or even degrade the AR.

[0144] The use of genetic "knockdown" techniques to downregulate AR expression has been clinically challenging due to low cellular uptake of oligonucleotides and technical difficulties in delivering them to the desired target. Moreover, the recently reported development of SNIPER agents, as well as the hydrophobic tagging approach (HyT) for AR degradation, in their current indications, represented experimental tools for discovery biology rather than viable therapeutic strategies. There were questions about their reported micromolar potency, far above the current level of anti-cancer drugs; and questions about undesirable off-target effects. On the other hand, targeted AR proteolysis induced by targeted proteolysis chimeras (PROTACs) via the ubiquitin proteasome system (UPS) represented an attractive alternative for which DHT-3C was well designed.

[0145] Targeted protein degradation chimera (PROTAC) agents have three components: a ligand that binds to the protein targeted for destruction, an E3 ligase "recruiter" that promotes ubiquitination of the target protein, and a spacer that links the ligand and recruiter. Several studies have described the development of PROTACs to induce AR protein degradation by linking various ligands for AR to other ligands that recruit E3 ubiquitin ligases. For example, one of these studies reported the chemical conjugation of a selective AR modulator (SARM) (with nanomolar affinity for AR) to nutlin, a known ligand for MDM2 E3 ligase. This approach generated a SARM-nutlin PROTAC that reduced AR levels in HeLa cells in a proteasome-dependent manner (but only at 10 μM concentration). Two other studies described the development of DHT-based PROTACs by linking DHT to a ligand for the recruitment of SCFβ-TRCP as well as a ligand for the von Hippel-Lindau (VHL) E3 ligase. The resulting PROTACs showed proteasomal degradation of AR protein, but only at relatively high 10-25 μM concentrations. Furthermore, SCFβ-TRCP-based PROTACs were not cell-permeable and required microinjection.

[0146] In summary, the shortcomings in reported DHT-based PROTACs in terms of efficacy and cell permeability reside in the nature of the chemistry used to target the AR, and thus there is an ongoing, unmet need for improved PROTAC therapies. DHT-3C provides a platform for developing new PROTAC agents as a potent alternative to DHT. For example, modification of the aniline group in compound Ii with a linker and appropriate E3 ligase recruiter provides a new family of potent PROTAC agents. Modification of either the aniline group in compound Ii, or the C-3O-(carboxymethyl)oxime derivative of DHT itself, with photoactive groups (including diazirine, benzophenone, or perfluorinated aryl azide) (Figure 29) could provide a means to specifically crosslink these agents to the AR, or to late enzymes in the pathway leading to DHT, and provide a new photodynamic / thermal therapy for patients with advanced CaP or CRPC.

[0147] [Example 10] The following examples describe the use of compounds Ij and Ig.

[0148] Computer modeling provides information regarding the binding of coumarin-modified androgens to the active sites of late enzymes in the biosynthetic pathways that converge on DHT or to the AR-LBD.

[0149] In the former case, computer modeling of the binding of compound Ij to AKR1C3 (aka 17β-hydroxysteroid dehydrogenase-5) showed that the compact nature of this fluorescent androgen Ij does not interfere with its binding to the active site (Figure 6). The X-ray structure of the ligand-binding domain in human AKR1C3 (PDB: 1XF0) with 5α-DIONE (Figure 6A) was selected as a template to model the binding of compound Ij (aka 5α-DIONE-F) (Figure 6B). The initial enzyme structure was downloaded from the RCSB Protein Data Bank and prepared for docking using Autodock Tools. Compound Ij was docked into the position occupied by 5α-DIONE in AKR1C3 using Autodock Vina. The binding pose of Ij obtained from Vina contained a high overlap with that of 5α-DIONE. The binding pose was further refined by performing a series of energy minimization procedures. Briefly, the AMBER14SB force field and the second generation of the general AMBER force field (gaff2) were used for proteins and ligands, respectively. 9 The program generated the partial charges for Ij using the AM1-BCC model. It used a hybrid protocol with 2500 steps of steepest descent minimization, followed by a convergence criterion of 2500 iteration steps or a convergence criterion of 1×10 -4 A bicoastal minimization was performed using conjugate gradient minimization until a minimum of 100 kcal / mol Å was met. During the first stage of minimization, 2A force constant of 0.01 was applied to the protein atoms. The second minimization step consisted of 1,000 steps of steepest descent minimization followed by 1,500 steps of conjugate gradient minimization, with no constraints on either the ligand or protein atoms. In summary, compound j adopted the same state as the naturally occurring ligand 5α-DIONE. The BCD ring of compound Ij inserted into the SP1 binding pocket in the same manner as seen for 5α-DIONE, and the C-18 and C-19 internuclear methyl groups of 5α-DIONE and Ij were linked to Y55, H117 and NADP + The compound Ij protruded into the oxyanion hole of 17β-HSD5 bound by S129. Hydrogen bonds with S129 further stabilize the observed binding mode with Ij. These binding characteristics indicated that compound Ij has a binding mode consistent with that of 5α-DIONE itself. Similarly, compound Ij occupied the same binding pocket as seen for the previously described inhibitor, 3-carboxamido-1,3,5-(10)-estratriene-17R-spiro-2-(5,5-dimethyl-6-oxo)tetrahydropyran (EM1404) (bound to AKR1C3 (PDB:1ZQ5)).

[0150] In the latter case, computer modeling of the binding of compound Ig (also known as DHT-3C) and AR showed that the BCD ring of compound Ig was inserted into the AR-LBD binding pocket (Figure 7). The X-ray structure of human AR-LBD in complex with DHT (PDB: 2ama) was selected as a template to model AR. An initial structure of AR(694-919) was generated using SWISS-MODEL and the missing atoms were added. Compound Ig was superimposed on the position of DHT to obtain its complex with AR. This complex was carefully refined by performing a series of energy minimization procedures and constrained MD simulations. Briefly, the AMBER14SB force field and the second generation of the general AMBER force field (gaff2) were used for the protein and the ligand, respectively. TIP3P water molecules were added as solvent, and the solute atoms were at least 10 Å away from the boundaries of the rectangular box (using AmberTools18). Counterions, i.e., chlorine atoms, were added to neutralize the net charge of each system. Long-range electrostatic interactions were treated with a particle mesh Ewald (PME) algorithm, with a nonbonded cutoff for real-space interactions set at 10 Å. A hybrid protocol of 8000 steps of steepest descent minimization was used, followed by a convergence criterion of up to 2000 iterative steps or a convergence criterion (root mean square of the energy gradient is between 1 Å and 10 -4 A two-stage minimization was performed using conjugate gradient minimization until a minimum of 100 kcal / mol Å was met. During the first stage minimization, a minimum of 100 kcal / mol Å was met. 2 A force constant of 100 kcal / mol Å was applied to the ligand and protein atoms except for residues 711, 752, and 759–771; for the second-stage minimization, 100 kcal / mol Å 2 A force constant of 0.2 kcal / mol·Å was applied to the protein backbone atoms except for residues 711, 752, and 759–771. The system was heated linearly from 0 to 303.15 K over a period of 50 ps with restraints on all heavy atoms in the NVT ensemble (force constant of 10 kcal / mol·Å2), followed by a force constant of 0.2 kcal / mol·Å2. 2The compound was equilibrated for 325 ps on a Langevin thermostat in the NPT (P = 1 atm and T = 303.15 K) ensemble by gradually decreasing the temperature to 0.5°C. Finally, restraints (2 kcal / mol Å) were placed on the backbone of residues 694-759 and 771-919. 2 A 40 ns production step was performed with Amber12 in the NPT (P = 1 atm and T = 303.15 K) ensemble with a force constant of 1.5 Å. The SHAKE algorithm was used to suppress covalent bonds between heavy and hydrogen atoms, the time step was set to 2 fs, and snapshots were saved every 8 ps. The RMSD converged well in the second half of the simulation, with RMSD fluctuations of less than 1 Å in the last 20 ns. Using Chimera, the conformations of the last 20 ns trajectory were clustered, and the top conformation was selected and subjected to the minimization process described above (step 2). The final conformation was used for further analysis. Modeling revealed that the hairpin domain (759-771) of AR was "opened" by about 4.6 Å from its initial position to accommodate compound Ig. Compound Ig bound to the AR-LBD in a binding mode similar to that of compound Ig. Compound Ig formed two hydrogen bond interactions with N705 and T877, and favorable van der Waals interactions with residues L701, F876, M780, L873, L704, M742, W741, and L707 (Figure 7B). Besides these features, the coumarin moiety of compound Ig formed hydrophobic interactions with F764, M749, Y763, and R752, and hydrogen bond interactions with Q711. These binding features indicated that compound Ig has a more favorable binding mode for AR than DHT.

[0151] In summary, computer modeling suggested that successful binding of compounds Ij and Ig occurred to the enzyme binding pockets of AKR1C3 or AR-LBD, respectively, despite the bulky nature of the fluorescent labels attached to either 5α-DIONE or DHT. Thus, these fluorescent compounds Ij and Ig are useful tools for screening small molecule inhibitors of oxidoreductases or small molecule antiandrogens (binding to AR).

[0152] Although the present disclosure has been described with reference to one or more specific examples, it will be understood that other examples of the present disclosure are possible without departing from the scope of the present disclosure.

Claims

1. A compound having the structure: 【Chemistry 1】 (where: R 1 is hydrogen or an alkyl group; L 1 and L 2 are linking groups having the following structure: 【Chemistry 2】 (wherein n is 1, 2, 3, 4, 5 or 6) Z is the following structure: 【Chemistry 3】 R 2 is an alkyl group or hydrogen).

2. The compound has the following structure: 【Chemistry 4】 (wherein n is 1, 2 or 3) The compound of claim 1.

3. The compound has the following structure: 【Chemistry 5】 The compound according to claim 2.

4. 10. A composition comprising the compound of claim 1 and a pharma- ceutically acceptable carrier.

5. The composition of claim 4 further comprising one or more additional drugs.

6. 6. The composition of claim 5, wherein the one or more additional drugs are selected from antiandrogens, 5α-reductase drugs, androgen metabolism inhibitors, and combinations thereof.

7. 7. The composition of claim 6, wherein the antiandrogen drug is selected from flutamide, bicalutamide, enzalutamide, apalutamide, darolutamide, dilutamide, and combinations thereof, and / or the 5α-reductase drug is selected from finasteride, dutasteride, and combinations thereof, and / or the androgen metabolism inhibitor is selected from abiraterone acetate, abiraterone acetate microparticles, ketoconazole, and combinations thereof.

8. The compound has the following structure: 【Chemistry 6】 The composition of claim 4.

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