Coumarin-modified androgen for treating prostate cancer

Abstract

To provide novel therapeutic methods for prostate cancer.SOLUTION: Provided are androstane and dihydrotestosterone compounds functionalized with carbocyclic groups or heterocyclic groups that may be saturated or unsaturated. The compounds may be used in methods of inhibiting cell growth of malignant cells and / or hyperplastic cells and / or treating individuals having diseases associated with malignant cell growth (e.g., cancer, such as, for example, prostate cancer) and / or hyperplastic cell growth and / or molecular imaging of malignant cells and / or hyperplastic cells and / or inducing degradation of a target protein. Also provided are compositions.SELECTED DRAWING: Figure 2

Description

Cross - Reference to Related Applications

[0001] This application claims priority to U.S. Provisional Application No. 62 / 839,676, filed on April 27, 2019; U.S. Provisional Application No. 62 / 844,062, filed on May 6, 2019; U.S. Provisional Application No. 62 / 844,073, filed on May 6, 2019; and U.S. Provisional Application No. 62 / 890,292, filed on 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 No. CA77739, P20 RR020171, R21 CA2051, and P30 GM110787 awarded by the National Cancer Institute, and Contract No. 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 Art]

[0003] The growth and progression of prostate cancer (CaP) depend on the interaction between the androgen receptor (AR), testosterone (T) (produced by the testes), which is the androgen in the circulating blood, and its metabolite, 5α-dihydrotestosterone (DHT) (produced by the 5α-reduction of T in prostate tissue). T and DHT have similar affinities for AR in the ligand-binding domain (LBD), but DHT is more potent because it has a slower off rate. The ligand-bound AR forms a dimer, translocates to the nucleus, binds to the androgen response element (ARE), and initiates the transcription of AR-regulated genes that promote the proliferation of CaP cells. Most men with metastatic disease or those who have failed treatment that could have been curative are treated with androgen deprivation therapy (ADT), which reduces circulating T levels, impairs AR transactivation, and causes CaP regression. However, ADT is symptomatic, and CaP always recurs as lethal castration-recurrent / resistant CaP (CRPC). One mechanism contributing to the transition of CaP to CRPC is intratumoral androgen metabolism. In the absence of circulating T during ADT, the androgen dehydroepiandrosterone or 4-androstene-3,17-dione (ASD) undergoes conversion to DHT via T through 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 backdoor pathway, 5α-androstan-3α-ol-17-one undergoes enzymatic reduction to 5α-androstan-3α,17β-diol (DIOL), which then undergoes enzymatic oxidation to yield DHT (Figure 1). In the secondary backdoor pathway, ASD is enzymatically reduced to 5α-androstan-3,17-dione (5α-DIONE), which is then enzymatically reduced to DHT (Figure 1). In summary, three enzyme-driven pathways provide access to DHT in CaP tissue, and each pathway has an enzyme that catalyzes an important step in the later stages of the biosynthetic pathway. Finasteride or dutasteride inhibits 5α-reductase (SRD5A) to reduce or inhibit the conversion of T to DHT (i.e., the front-door pathway in Figure 1).Although all drugs fail clinically, part of the reason is that the backdoor pathway produces sufficient DHT concentrations to activate the growth of AR and CaP. Other inhibitors such as abiraterone, which are cytochrome P450 17A1 (CYP17A1) inhibitors, inhibit the biosynthesis from pregnane (C. 21 compound) to androstane (C 19 compound) well before the end of the three pathways converging to DHT. Abiraterone (and other CYP17A1 inhibitors) only extend the survival period of CRPC by several months. This is because multiple mechanisms, including increased CYP17A1 expression levels and the accumulation of progesterone that can overcome abiraterone for CYP17A, 25, CYP11A1 or AKR1C3, enhance DHT production and CaP growth.

[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 different orders (Figure 1). ASD reaches DHT either through T or proceeds to 5α-DIONE, which can either proceed directly to DHT or shift to a primary back door pathway that goes 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 the "alternative" pathway of Sharifi, the "alternate" pathway of Penning, and the "5α-DIONE" pathway of Corcoran. The role of intratumoral metabolism of HSD17B3 via the front door and secondary back door pathways can be separated using ASD and 5α-DIONE as substrates, respectively. Preclinical studies using indomethacin, an AKR1C3 inhibitor, demonstrated proof of principle that AKR1C3 inhibition overcomes CaP resistance to abiraterone and enzalutamide. This study provided evidence regarding the need for the identification and development of AKR1C3 inhibitors. The first / second phase clinical trial that verified the efficacy of ASP9521, an AKR1C3 inhibitor, ended without evidence of a clinical response. The authors suggested that insufficient CaP cell expression of AKR1C3 causes treatment failure, which is probably a problem not shared by the four 3α-oxidoreductases discussed herein.

[0005] Among the methods to achieve ADT, anti-androgens occupy a central position in the chemical castration of men suffering from CaP. Bicalutamide or enzalutamide, which are anti-androgen drugs, compete with T or DHT for the AR-LBD. Due to the lower binding affinity for AR-LBD than DHT, the clinical performance of bicalutamide was poor. Furthermore, bicalutamide showed unwanted AR agonist (not antagonist) activity after long-term treatment. Enzalutamide binds to AR-LBD with a higher affinity than bicalutamide, inhibits AR nuclear translocation, and inhibits the interaction between the ARE of AR-regulated genes essential for CaP growth and AR. However, enzalutamide resulted in a modest response to CRPC and only extended the survival period by 4.8 months. One of the mechanisms contributing to abiraterone and anti-androgen resistance is the expression of the constitutively active AR splice variant 7 (AR-V7) lacking the LBD. The lack of the LBD avoids the need for T or DHT, eliminates the binding sites for anti-androgens, and thereby renders them ineffective. Small molecule inhibitors (such as EPI-506) targeting the N-terminal domain of AR rather than AR-LBD inactivated AR-V7 when examined in vitro. EPI-506 was found to be safe in a Phase I trial, but the Phase II clinical trial failed. The limited success of current treatment methods for progressive CaP and especially CRPC requires the pursuit of new treatment methods. SUMMARY OF THE INVENTION

[0006] The present disclosure provides androstane and dihydrotestosterone compounds functionalized with a carbocyclic or heterocyclic group that can be saturated or unsaturated (e.g., a coumarin-containing ring group (e.g., a coumarin group) and a coumarin isostar group). This compound can be used in a method of inhibiting the cell growth of malignant cells and / or hyperplastic cells, and / or a method of treating an individual having a disease associated with malignant cell growth and / or hyperplastic cell growth (e.g., cancer, e.g., prostate cancer, etc.), and / or molecular imaging of malignant cells and / or hyperplastic cells, and / or a method of inducing the degradation of a target protein.

[0007] The compound is 1) capable of inhibiting a redox enzyme that appears in the terminal stage of the primary backdoor pathway for DHT biosynthesis and / or 2) capable of functioning as an antiandrogen by competing with AR-LBD binding and by suppressing the cell proliferation of CaP cell lines that express high levels of the AR-V7 variant, and perhaps by impairing AR-AR-V7 dimerization and / or 3) capable of providing a tool for carrying a small molecule that disrupts AR function or even degrades AR. Compounds having one or more of these properties will show significant clinical progress.

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

[0010] The compounds of the present disclosure may have the following structure: TIFF2025098059000002.tif35169where R 1 is hydrogen or an alkyl group (e.g., a methyl group, an ethyl group, n- and isopropyl groups, etc.), In TIFF2025098059000003.tif14169, X is a hydroxyl group, Y is hydrogen or an alkyl group (e.g., a methyl group, an ethyl group, n- and isopropyl groups, etc.), or X and Y together are a spiro-condensed, substituted or unsubstituted coumarin group or coumarin isostar group, and L 3 is optional and is a linking group. In TIFF2025098059000004.tif30169, L 1 is a linking group, and L 2 is optional and is a linking group, Z is a terminal group containing a substituted or unsubstituted carbocyclic group, or a substituted or unsubstituted heterocyclic group, or a substituted or unsubstituted coumarin group, or a substituted or unsubstituted coumarin isostar group, and R 2 is an alkyl group (e.g., a methyl group, an ethyl group, n- and isopropyl groups, and the like), or hydrogen. R 5 is a photoactive group. Note that TIFF2025098059000005.tif34169

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

[0012] In one aspect, the present disclosure provides a method of using one or more compounds or compositions thereof. This compound is suitable in methods for treating various diseases. For example, one or more compounds of the present disclosure or the compositions of the present disclosure can be used to treat cancer, other diseases, or combinations thereof. The methods of the present disclosure can be used to inhibit the cell growth of malignant cells and / or proliferating cells, and / or to induce the selective degradation of target proteins, and / or for molecular imaging. The methods may be carried out in combination with one or more known treatments.

[0013] In one aspect, the present disclosure provides a kit. In various examples, the kit includes a pharmaceutical formulation containing any one or any combination of the compounds of the present disclosure. In one example, the present disclosure includes a sealed or airtight package containing the pharmaceutical formulation. In various examples, the package includes one or more sealed or airtight vials, bottles, blister (bubble) packs, or any other suitable package for the sale, distribution, or use of the pharmaceutical compounds and compositions containing them. The printed matter can include printed information. The printed information may be attached on a label, on an insert sheet, or printed on a packaging material. The printed information may include information identifying the compounds in the package, the amounts and types of other active and / or inactive ingredients in the composition, and instructions for taking the compounds and / or the composition. The instructions can include information such as the number of doses to be taken over a given period of time, and / or information directed to another healthcare 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 drug provided therein is for the treatment of a subject having prostate cancer (e.g., castration-resistant prostate cancer) and / or any disorder associated with other diseases and / or cancers and / or other diseases. In various examples, the kit includes a label that describes the contents of the kit and provides instructions and / or explanations regarding the use of the contents of the kit for treating a subject having any cancer and / or other disease.

Brief Description of the Drawings

[0014] To more fully understand the nature and objects of the present disclosure, reference is made to the following detailed description in conjunction with the accompanying drawings.

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

[0016] FIG. 2 shows the synthetic pathway of a fluorescent steroid 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-benzo[de]anthracen-10-one hydrochloride; d LiAlH(OtBu)3; e C6H5CO2H, DIAD

[0017] Figure 3 shows the synthetic route of a fluorescent steroid modified at the C-17 keto group. Explanation 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-benzo[de]anthracen-10-one hydrochloride; d Py-SO3

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

[0019] Figure 5 shows the 2D ROESY spectrum of DHT-3C (Compound Ig). The spectrum was recorded at 25 °C using an Agilent 400 MHz. The dotted line is NH2 + and the ring A protons (i.e., H-5 (H-1 overlapping), H-4, H-2, and CH2 adjacent to NH2 +) shows an extended region that displays the nuclear Overhauser effect (NOE) connectivity with

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

[0021] Figure 7 shows the results of computer modeling. 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: An enlarged view of the DHT-3C binding shows the interaction with specific residues in AR, indicating that the hairpin domain of AR (759 - 771) was "opened" approximately 4.6 Å from its initial position to accommodate DHT-3C.

[0022] Figure 8 shows that 3α-hydroxysteroid dehydrogenase is expressed in clinical specimens.

[0023] Figure 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-hydroxysteroid dehydrogenase variants reduced DHT concentration better than dutasteride alone. In each series, the columns from left to right are "empty vector", HSD17B6, RDH16, DHRS9, and RDH5.

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

[0027] Figure 13 shows that inhibition of front door and primary backdoor DHT synthesis decreased VCaP proliferation.

[0028] Figure 14 shows that DIOL-17C treatment decreased VCaP cell proliferation.

[0029] Figure 15 shows RDH16-YFP and DIOL-17C co-localization by ImageStream.

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

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

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

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

[0034] Figure 20 shows proliferation data demonstrating that DHT-17C decreases the proliferation of AR- and AR-V7-expressing VCaP and CWR-1 cell lines, but not that of PC-3 (AR-negative CaP cell line) or non-CaP, non-AR CV-1 or 293 cell lines.

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

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

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

[0038] Figure 24 shows ImageStream data demonstrating nuclear co-localization between DHT-3C and AR-GFP, but not between free coumarin and AR-GFP. The AR-GFP studies were confirmed using 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 results 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] Figure 27 shows a three-pronged attack on AR ligand binding, AR dimerization, and AR transactivation.

[0042] Figure 28 shows the utility of DIOL-17C (Compound Ia), DHT-17C (Compound Id), and DHT-3C (Compound Ig). DIOL-17C inhibits the intratumoral synthesis of DHT by redox enzymes, DHT-17C competes with DHT for the AR-LBD, prevents dimerization between AR and AR-V7, and DHT-3C provides a carrier for PROTACs or photo- or thermo-activated agents, disrupting AR transactivation or even degrading AR.

[0043] Figure 29 shows representative photoactive derivatives of DHT-3C or C-3O-(carboxymethyl)oxime of DHT.

[0044] Figure 30 shows the synthetic route for the synthesis of PROTACs for AR degradation.

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

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

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

[0048] Figure 34 shows the quadrant of DIOL-17C synthetic modification.

[0049] Figure 35 shows the synthetic route of DIOL-17C. Explanation: 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] Figure 36 shows the synthetic route of a DIOL-17C analog having a modification in quadrant 2. Explanation: 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 in which the amine substituent in coumarin is modified by substituent X. Explanation: 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 INVENTION

[0052] The subject matter is described with respect to specific examples, but other examples including those not providing all of the advantages and features described herein are also within the scope of the present disclosure. Without departing from the scope of the present disclosure, various structural, logical, and process steps can be performed.

[0053] The present disclosure provides androstane and dihydrotestosterone compounds functionalized with a carbocyclic or heterocyclic group that can be saturated or unsaturated (e.g., a coumarin-containing ring group (e.g., a coumarin group) and a coumarin isostar group). The compounds can be used in a method of inhibiting the cell proliferation of malignant and / or proliferating cells, and / or in a method of treating an individual having a disease associated with malignant cell proliferation and / or proliferating cell proliferation (e.g., cancer, e.g., prostate cancer, etc.), and / or in a method of molecular imaging of malignant and / or proliferating cells, and / or in a method of inducing the degradation of a target protein.

[0054] The compounds can 1) inhibit the redox enzyme that appears in the terminal stage of the primary backdoor pathway for DHT biosynthesis, and / or 2) function as an anti-androgen by competing with AR-LBD binding and by suppressing the cell proliferation of CaP cell lines that express high levels of the AR-V7 variant, presumably by impairing AR-AR-V7 dimerization, and / or 3) provide a tool for carrying a small molecule that disrupts AR function or even degrades AR. Compounds having one or more of these properties will show significant clinical progress.

[0055] Ranges of values are disclosed herein. The ranges set a lower limit value and an upper limit value. Unless otherwise specified, the ranges include all values up to the magnitude of the minimum value (either the lower limit value or the upper limit value) and the ranges between the values of the recited ranges.

[0056] As used herein, the term "group" refers to a chemical entity that is monovalent (i.e., having one terminus that can covalently bond to other chemical species, such as a methyl or phenyl group), divalent, or polyvalent (i.e., having two or more termini that can covalently bond to other chemical species, such as a methylene or phenylene group), unless otherwise indicated. 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 group, ethyl group, n- and isopropyl groups, n-, sec-, iso- and tert-butyl groups, etc. The alkyl group may be an alkyl group of C1-C 12 which may be an alkyl group of any integer value of all carbons in between, and a range of the number of carbons (e.g., C1, C2, C3, C4, C5, C6, C7, C8, C9, C 10 C 11 or C 12 ). The alkyl group may or may not be substituted, and may be substituted with one or more substituents. Examples of substituents include, for example, halogen (e.g., -F, -Cl, -Br, and -I), aliphatic groups (e.g., alkyl group, alkenyl group, and alkynyl group), aryl group, alkoxide group, carboxylate group, carboxylic acid, ether group, alcohol group, amine group, thiol group, thioether group, etc., and various substituents such as combinations thereof, but are not limited thereto.

[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 halogen. The heteroalkyl group may or may not be substituted, and may be substituted with one or more substituents. Examples of substituents include, for example, halogen (e.g., -F, -Cl, -Br, and -I), aliphatic groups (e.g., alkyl group, alkenyl group, and alkynyl group), aryl group, alkoxide group, carboxylate group, carboxylic acid, ether group, alcohol group, amine group, thiol group, thioether group, etc., and various substituents such as combinations thereof, but are not limited thereto.

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

[0060] As used herein, unless otherwise indicated, the terms "carbocyclic" or "heterocyclic" each mean a carbon-containing ring, or a carbon-containing ring in which one or more of the carbon atoms are substituted by heteroatoms. These groups may be non-aromatic or aromatic. A carbocyclic group or a 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, halogen (e.g., -F, -Cl, -Br, and -I), aliphatic groups (e.g., alkyl group, alkenyl group, and alkynyl group), aryl group, alkoxide group, carboxylate group, carboxylic acid, ether group, alcohol group, amine group, thiol group, thioether group, etc., and combinations thereof.

[0061] As used herein, unless otherwise indicated, the term "heterocyclic group" means a C3-C 20refers to a cyclic group, and the C3-C 20 includes all integers of the carbon atoms between them and the range of the number of carbon atoms (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 ). The heterocyclic group may or may not be substituted and / or may have additional unsaturations. Examples of substituents include, but are not limited to, halogen (e.g., -F, -Cl, -Br, and -I), aliphatic groups (e.g., alkyl group, alkenyl group, and alkynyl group), aryl group, alkoxide group, carboxylate group, carboxylic acid, ether group, alcohol group, amine group, thiol group, thioether group, etc., and combinations thereof. The heterocyclic group may be fused to a carbocyclic group or to each other. Non-limiting examples of heterocyclic groups include furanyl group, oxazolyl group, isothiazolyl group, thiazolyl group, tetrahydropyranyl group, piperazinyl group, dioxanyl group, pyrrolidinyl group, tetrahydrothiophenyl group, tetrahydrofuranyl group, quinuclidinyl group, azabicyclo[3.2.1]octanyl group, decahydroquinolinyl group, etc.

[0062] As used herein, unless otherwise indicated, the term "heteroaryl group" means a 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, where the 5 to 18 includes 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 means a polycyclic (e.g., bicyclic) heteroaromatic group where the heteroatom is selected from N, O, or S. The aromatic group may optionally be independently substituted with one or more substituents described herein. The substituents themselves may optionally be substituted. Examples of substituents include, but are not limited to, halogen (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, etc., 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, 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, indolyl, indolinyl, indolinonyl, dihydrobenzothienyl, 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]pyrazinyl, 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, benzoisoxazolyl, 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, including but not limited to these. Further, 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, the compounds of the present disclosure include 5α-androstane-3α,17β-diol (DIOL), 5α-androstane-3α-ol-17-one, 5α-androstane-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, the compounds may be modified (e.g., functionalized) with a coumarin-containing group or a coumarin isostere group at either the C-3 or C-17 position of these steroids.

[0065] The compounds of the present disclosure may have the following structures: TIFF2025098059000006.tif35169Here, R 1 is hydrogen or an alkyl group (e.g., methyl group, ethyl group, n- and isopropyl groups, etc.), TIFF2025098059000007.tif14169wherein X is a hydroxyl group, Y is hydrogen or an alkyl group (e.g., methyl group, ethyl group, n- and isopropyl groups, etc.), or X and Y together are a spiro-fused, substituted or unsubstituted coumarin group or a coumarin isostar group, L 3 is optional and is a linking group. TIFF2025098059000008.tif30169wherein L 1 is a linking group, L 2 is optional and is a 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 containing a substituted or unsubstituted coumarin isostar group, and, R 2 is an alkyl group (e.g., methyl group, ethyl group, n- and isopropyl groups, and the like), or hydrogen. R 5 is a photoactive group. Here, TIFF2025098059000009.tif34169Z can be various terminal groups. In various examples, Z is a carbocyclic group or a heterocyclic group, which may be saturated or unsaturated and may have one or more substituents (e.g., hydroxy, alkoxy, thioalkoxy, halogen, etc., and combinations thereof). Non-limiting examples of the Z group include the following: TIFF2025098059000010.tif64169TIFF2025098059000011.tif95169TIFF2025098059000012.tif177169Wherein, X' is independently selected from hydrogen, an alkyl group, a cycloalkyl group, an alkoxy group, a halogen, and combinations thereof. The Z group may have various substituents. Non-limiting examples of 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, etc., and combinations thereof.

[0066] Various photoactive groups can be used. The photoactive group (R 5 ) Non-limiting examples include the following: TIFF2025098059000013.tif28169

[0067] The general formula of an androgen with a substituted coumarin bonded to C-3 or C-17. The compound may have the following structure: TIFF2025098059000014.tif46169Wherein, R 1 and R 2 are each selected from hydrogen and an alkyl group (e.g., methyl group, ethyl group, n- and isopropyl groups, etc.); X and Y are each a hydroxyl group or hydrogen, or X and Y contain a carbonyl group; Z' is a substituted coumarin ring or a coumarin isostar. In various examples, R 1 and R 2 are each selected from hydrogen and an alkyl group (e.g., methyl group, ethyl group, n- and isopropyl groups, etc.); X and Y are spiro-condensed, substituted coumarin rings or coumarin isostars; and Z' is hydrogen.

[0068] The compound may have the following structure: TIFF2025098059000015.tif44169Wherein, R1 are each selected from hydrogen and alkyl groups (such as methyl group, ethyl group, n- and isopropyl groups, etc.); X and Y are each a hydroxyl group or hydrogen; Z'' is O-(carboxymethyl)oxime linked to a substituted coumarin or coumarin isostar. In various examples, R 1 are each selected from hydrogen and alkyl groups (such as methyl group, ethyl group, n- and isopropyl groups, etc.); X and Y are spiro-condensed, substituted coumarin rings or coumarin isostars; Z'' is oxygen.

[0069] Representative examples of compound (I) with a substituted coumarin bonded at the C-17 position. Non-limiting examples of the compounds of the present disclosure include those in which the X group in compound (I) 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 isostar. Examples of such compounds include, but are not limited to, compounds (Ia), (Ib), and (Ic): TIFF2025098059000016.tif48169 wherein X' are each independently selected from hydrogen, an alkyl group (such as methyl, ethyl, n- and isopropyl, etc.), a cycloalkyl group, an alkoxy group (such as -O(CH2) n CH3, n is 0, 1, or 2), and / or a halogen (i.e., -F, -Cl, -Br, and -I), and L is a linker (such as a linker group). Examples of linkers include, but are not limited to, the following structures (n and m are independently 1, 2, 3, 4, 5, or 6): TIFF2025098059000017.tif29169

[0070] Non-limiting examples of the compounds of the present disclosure include those in which the X group and the Y group in compound (I) 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 isostar group. Examples of the compounds include, but are not limited to, compounds (Id), (Ie), and (If): TIFF2025098059000018.tif51169In the formula, each X' is independently selected from hydrogen, an alkyl group (e.g., methyl, ethyl, n- and isopropyl, etc.), a cycloalkyl group, an alkoxy group (e.g., -O(CH2) n CH3, where n is 0, 1, or 2), and / or a halogen (i.e., -F, -Cl, -Br, and -I), and L is a linker (e.g., a linker group). Examples of the linker include, but are not limited to, the following structures (n and m are independently 1, 2, 3, 4, 5, or 6): TIFF2025098059000019.tif28169

[0071] Representative examples of compound (II) with a substituted coumarin attached at the C-17 position. Non-limiting examples of the compounds of the present disclosure include those in which the X group and the Y group of compound (II) are a hydroxyl group and hydrogen, respectively, or a carbonyl group or an oxime. Examples of the groups include, but are not limited to, compounds (IIa) and (IIb): TIFF2025098059000020.tif39169

[0072] General representative examples of compound (I) with a substituted coumarin attached at the C-3 position. Non-limiting examples of the compounds of the present disclosure include those in which the X group and the Y group of compound (I) are a spiro-condensed substituted coumarin ring group or a coumarin isostar group, and Z' is hydrogen. Examples include, but are not limited to, compounds (Ig), (Ih), and (Ii): TIFF2025098059000021.tif68169

[0073] Non-limiting examples of the compounds of the present disclosure include those in which the R 1 groups of compound (II) are each independently selected from hydrogen and an alkyl group (e.g., methyl group, ethyl group, n- and isopropyl groups, etc.), the X group and the Y group are a spiro-condensed substituted coumarin ring or a coumarin isostar, and Z'' is oxygen. Examples include, but are not limited to, compounds (Ij), (Ik), and (Il): TIFF2025098059000022.tif72169

[0074] In various examples, the compounds of the present disclosure are PROTACs. A PROTAC may have the following structure: A-L-B, 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, the E3 ligase recruiter can recruit proteins such as, for example, VHL, CRBN, RNF114, MDM2, DCAF15, DCAF16, SCF, etc. (which can ubiquitinate target proteins such as AR). Non-limiting examples of E3 ligase recruiter groups include groups formed from: TIFF2025098059000023.tif70169TIFF2025098059000024.tif143147 or, a ligase recruiter group that binds to cereblon, von Hippel-Lindau (VHL), or Mouse-Double-Minute-2.

[0076] The compounds of the present disclosure (e.g., PROTAC compounds) can include various linkers (e.g., linking groups). The linker links 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 2-4 (e.g., 2, 3, or 4).

[0077] Non-limiting examples of target proteins include proteins related to cancer (e.g., leukemia, lung cancer (e.g., non-small cell lung cancer), dermatological cancer, pre-cancerous lesions of the upper digestive tract, prostate malignancy, brain malignancy, breast malignancy, 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 may contain an R 5 group, may contain a spiro-coumarin ring group, or may contain both. Non-limiting examples of compounds suitable for molecular imaging (e.g., imaging of proliferating cells and / or malignant cells) include the following: TIFF2025098059000025.tif173169TIFF2025098059000026.tif229169TIFF2025098059000027.tif85169

[0079] Specific examples of the compounds of the present disclosure include, but are not limited to, the following: TIFF2025098059000028.tif134169TIFF2025098059000029.tif215169TIFF2025098059000030.tif239169TIFF2025098059000031.tif204169TIFF2025098059000032.tif212169TIFF2025098059000033.tif233169TIFF2025098059000034.tif209169TIFF2025098059000035.tif209169TIFF2025098059000036.tif229169TIFF2025098059000037.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 the coumarin moiety is attached to the C-3 or C-17 position of the steroid. Non-limiting examples of the methods are provided herein. The synthetic routes for these compounds are shown in FIGS. 2, 3, and 4. The compounds were made by the methods of the present disclosure and characterized with respect to structure and purity as follows. Nuclear magnetic resonance spectra were measured on a Varian instrument ( 1 H, 400 or 500 MHz; 13 C, 100 Mz). 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 to 100,000 (400 m / z). Samples were introduced by direct injection at a flow rate of 3 μL / min using a syringe pump. The purity of the compounds was established using 13 C NMR and exact mass spectra. Unless otherwise specified, the compounds were chromatographed on preparative layer Merck silica gel F254.

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

[0082] The composition may further comprise additional components. For example, the composition may include 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 pharmaceutically acceptable carrier.

[0083] The composition may include one or more standard pharmaceutically 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 can 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, alcohols, and the like, and combinations thereof. Furthermore, the injectables may contain stabilizers, solubilizers, suspending agents, emulsifying agents, soothing agents, buffering agents, preservatives, and the like. The injection 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, used after sterilization, or used after being dissolved in sterile water for injection or other sterile diluents immediately before use. Non-limiting examples of pharmaceutically acceptable carriers are described in Remington: 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. This compound is suitable in methods for treating various diseases. For example, one or more compounds of the present disclosure or the compositions of the present disclosure can be used to treat cancer, other diseases, or combinations thereof. The methods of the present disclosure can be used to inhibit the cell growth of malignant cells and / or proliferating cells and / or to induce the selective degradation of target proteins and / or for molecular imaging. The methods can be carried out in combination with one or more known treatments.

[0085] Non-limiting examples of cancer include leukemia, lung cancer (e.g., non-small cell lung cancer), dermatological cancer, premalignant lesions of the upper digestive tract, prostate malignancy, brain malignancy, breast malignancy, 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, etc., and combinations thereof.

[0087] The methods of the present disclosure can be used, for example, to treat various types of cancer such as hormone cancer. Non-limiting examples of hormone cancer include breast cancer, ovarian cancer, uterine cancer or endometrial cancer, prostate cancer, etc.

[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 by impairing AR transactivation or AR dimerization. The methods described herein can be used to overcome AR-V7-mediated resistance when treating CRPC. The methods of the present disclosure can inhibit the growth of CaP cells better than methods including 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 proliferating cells (e.g., benign prostatic hyperplasia or hypertrophy (BPH)). The compounds described either inhibit the terminal substrate conversion to DHT or impair AR transactivation or AR dimerization. This compound can be used with or instead of current CaP therapies such as bicalutamide, enzalutamide, or abiraterone.

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

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

[0092] A method of treating a disease (e.g., cancer, e.g., a hormone cancer such as prostate cancer, e.g., castration-resistant prostate cancer, e.g., castration-resistant prostate cancer expressing AR-V7, etc.) includes administering to a subject in need of treatment (e.g., an individual in need of treatment) a therapeutically effective amount of a compound or composition of the present disclosure (e.g., an amount of the compound or composition sufficient to treat the subject), whereby the disease of the subject is treated.

[0093] In various examples, the compounds of the present disclosure are used to inhibit the proliferation of cells (e.g., malignant cells such as cancer cells, e.g., hormone 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 proliferating cells (e.g., benign prostatic hyperplasia or hypertrophy (BPH) cells). 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 cancer cells with the compound 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 the proliferation of the cancer cells. The inhibition of proliferation can 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 the proliferation / regeneration of cells (e.g., the proliferation / regeneration of cancer cells).

[0094] A method of inhibiting cell proliferation includes contacting the cells with a compound of the present disclosure or a composition comprising a compound of the present disclosure.

[0095] The present disclosure provides a method for inducing the selective degradation of a target protein. The method for inducing the selective degradation of a target protein includes 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] A subject in need of treatment or an individual in need of treatment can 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 method and / or administration method of the present disclosure can be carried out in combination with contacting and / or administering with one or more additional drugs. The additional drugs can be selected from anti-androgen drugs, 5α-reductase drugs, androgen metabolism inhibitors, and combinations thereof. Non-limiting examples of anti-androgen drugs include flutamide, bicalutamide, enzalutamide, apalutamide, darolutamide, dilutamide, etc., and combinations thereof. Non-limiting examples of 5α-reductase drugs include finasteride, dutasteride, etc., and combinations thereof. Non-limiting examples of androgen metabolism drugs include abiraterone acetate, abiraterone acetate microparticles, ketoconazole, etc., and combinations thereof.

[0098] The compound of the present disclosure can be used as a tool for screening small molecule inhibitors of oxidoreductases or small molecule anti-androgens (which bind to AR).

[0099] In one example, the compounds and compositions are suitable in methods including fluorescence microscopy. Methods including 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] Methods including molecular tracking (e.g., intracellular tracking) in cells (e.g., prostate cells) include contacting the cells (e.g., prostate cells) with a compound of the disclosure and imaging the cells (e.g., prostate cells) at regular intervals (e.g., intervals known in the art) (e.g., imaging using a fluorescence microscope) to monitor activity. In one example, the activity is androgen metabolism.

[0101] In one aspect, the present disclosure provides a kit. In various examples, the kit includes a pharmaceutical formulation containing any one or any combination of the compounds of the present disclosure. In one example, the present disclosure includes a sealed or airtight package containing the pharmaceutical formulation. In various examples, the package includes one or more sealed or airtight vials, bottles, blister (bubble) packs, or any other suitable package for the sale, distribution, or use of the pharmaceutical compounds and compositions containing them. The printed matter can include printed information. The printed information may be attached on a label, on an insert sheet, or printed on a packaging material. The printed information may include information identifying the compounds in the package, the amounts and types of other active and / or inactive components in the composition, and instructions for taking the compounds and / or composition. The instructions can include, for example, information such as the number of doses to be taken over a given period, and / or information directed to another healthcare provider such as a pharmacist and / or a physician, or the patient. The printed matter may include an indication that the pharmaceutical composition and / or any other agent provided therein is for the treatment of a subject having CaP and / or any disorder associated with another disease and / or cancer and / or another disease. In various examples, the kit includes a label that describes the contents of the kit and provides instructions and / or explanations regarding the use of the contents of the kit for treating 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 steps of the methods described in the various embodiments and examples disclosed herein are sufficient to carry out the methods of the present disclosure. The methods described in the embodiments are combinations of the steps of the disclosed methods. In another embodiment, the method consists of such steps.

[0104] The following statements provide compounds of the present disclosure, methods of using the compounds of the present disclosure, and examples of uses of the compounds of the present disclosure. Statement 1. A compound having the following structure: TIFF2025098059000038.tif35169 Here, R 1 is hydrogen or an alkyl group (e.g., methyl group, ethyl group, n- and isopropyl groups, etc.), TIFF2025098059000039.tif14169 wherein X is a hydroxyl group, Y is hydrogen or an alkyl group (e.g., methyl group, ethyl group, n- and isopropyl groups, etc.), or X and Y together are a spiro-condensed, substituted or unsubstituted coumarin group or coumarin isostar group, L 3 is optional and is a linking group; TIFF2025098059000040.tif30169 wherein L 1 is a linking group, L 2 is optional and is a 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 substituted or unsubstituted coumarin isostar group-containing terminal group, and, R 2 is an alkyl group or hydrogen; R 5 is a photoactive group, where, TIFF2025098059000041.tif34169 Statement 2. The compound according to statement 1, wherein L1 and / or L2 and / or L3 are independently selected from the following: TIFF2025098059000042.tif39169 Statement 3. The compound according to statement 1 or statement 2, wherein the compound has the following structure: TIFF2025098059000043.tif48169 Here, Z is selected from the following: TIFF2025098059000044.tif66169TIFF2025098059000045.tif219169TIFF2025098059000046.tif40169Wherein, X' is independently selected from hydrogen, an alkyl group, a cycloalkyl group, an alkoxy group, a halogen, and combinations thereof. Statement 4. The compound according to any one of the preceding statements, wherein the compound has the following structure: TIFF2025098059000047.tif41169Wherein, n is 1, 2, or 3. Statement 5. The compound according to any one of the preceding statements, wherein the compound has the following structure: TIFF2025098059000048.tif86169TIFF2025098059000049.tif204169TIFF2025098059000050.tif58169 Statement 6. The compound according to statement 1 above, wherein the compound has the following structure: TIFF2025098059000051.tif32169Wherein, R is L 1 -A, R 5 , -OH, -NH2, -CO2Et, -CN, -CHO, -SO3H, and -CO2H, or three adjacent R groups form a fused ring system (e.g., TIFF2025098059000052.tif28169forming, and n is 1, 2, 3, or 4, and L 1 is a linking group, and A is an E3 ligase recruiter group, where only one R is L 1 -A. Statement 7. The compound according to statement 1 or statement 6 above, wherein A is formed from the following: TIFF2025098059000053.tif40169TIFF2025098059000054.tif57169 Statement 8. The compound according to statement 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 according to statement 1 or statement 6, wherein the compound has the following structure: TIFF2025098059000055.tif94169 Statement 10. The compound according to statement 1 or statement 6, wherein the compound has the following structure: TIFF2025098059000056.tif37169TIFF2025098059000057.tif176169 Statement 11. The compound according to statement 1, wherein the compound has the following structure: TIFF2025098059000058.tif49169Here, Z is selected from the following: TIFF2025098059000059.tif218169TIFF2025098059000060.tif101169Wherein X' is independently selected from hydrogen, an alkyl group, a cycloalkyl group, an alkoxy group, a halogen, and combinations thereof. Statement 12. The compound according to statement 1 or statement 11, wherein the compound has the following structure: TIFF2025098059000061.tif42169Wherein n is 1, 2, or 3. Statement 13. The compound according to statement 1 or statements 11 - 12, wherein the compound has the following structure: TIFF2025098059000062.tif46169TIFF2025098059000063.tif187169TIFF2025098059000064.tif115169 Statement 14. The compound according to statement 1, wherein the compound has the following structure: TIFF2025098059000065.tif56169Here, Z is selected from the following: TIFF2025098059000066.tif30169TIFF2025098059000067.tif238169TIFF2025098059000068.tif56169Wherein, X' is independently selected from hydrogen, an alkyl group, a cycloalkyl group, an alkoxy group, a halogen, and combinations thereof. Statement 15. The compound according to statement 1 or statement 14, wherein the compound has the following structure: TIFF2025098059000069.tif43169Wherein, n is 1, 2, or 3. Statement 16. The compound according to statement 1 or statements 14 to 15, wherein the compound has the following structure: TIFF2025098059000070.tif89169TIFF2025098059000071.tif203169TIFF2025098059000072.tif57169 Statement 17. The compound according to statement 1, wherein the compound has the following structure: TIFF2025098059000073.tif56169Here, Z is selected from the following: TIFF2025098059000074.tif65169TIFF2025098059000075.tif219169TIFF2025098059000076.tif38169Wherein, X' is independently selected from hydrogen, an alkyl group, a cycloalkyl group, an alkoxy group, and a halogen. Statement 18. The compound according to statement 1 or statement 17, wherein the compound has the following structure: TIFF2025098059000077.tif50169Wherein, n is 1, 2, or 3. Statement 19. The compound according to statement 1 or statements 17 to 18, wherein the compound has the following structure: TIFF2025098059000078.tif90169TIFF2025098059000079.tif210169TIFF2025098059000080.tif113169 Statement 20. The compound according to statement 1, wherein the compound has the following structure: TIFF2025098059000081.tif34169wherein R 5 is selected from the following: TIFF2025098059000082.tif30169 Statement 21. The compound according to statement 1 or statement 20, wherein the compound has the following structure: TIFF2025098059000083.tif39169TIFF2025098059000084.tif188169 Statement 22. A composition comprising the compound according to any one of the preceding statements and a pharmaceutically acceptable carrier. Statement 23. The composition according to statement 22, further comprising one or more additional drugs. Statement 24. The composition according to statement 23, wherein the one or more additional drugs are selected from antiandrogen drugs, 5α-reductase drugs, androgen metabolism inhibitors, and combinations thereof. Statement 25 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 drug is selected from abiraterone acetate, abiraterone acetate microparticles, ketoconazole, and combinations thereof, The composition according to any one of statements 22 to 24. Statement 26. The compound according to any one of statements 22 to 25, wherein the compound has the following structure: TIFF2025098059000085.tif181169TIFF2025098059000086.tif233169TIFF2025098059000087.tif51169 Statement 27. A method for impairing androgen receptor (AR) transactivation and / or androgen receptor dimerization, the method comprising contacting a cell with a compound according to any one of Statements 1 to 21 or a composition according to any one of Statements 22 to 26 in an amount and for a time sufficient to impair androgen receptor transactivation and / or androgen receptor dimerization. Statement 28. The method according to Statement 27, further comprising contacting the cell with flutamide, bicalutamide, enzalutamide, apalutamide, darolutamide, dilutamide, finasteride, dutasteride, or a combination thereof. Statement 29. The method according to Statement 27 or Statement 28, wherein the compound has the following structure: TIFF2025098059000088.tif91169TIFF2025098059000089.tif52169 Statement 30. A method for inhibiting cell proliferation, the method comprising contacting a cell with a compound according to any one of Statements 1 to 21 or a composition according to any one of Statements 22 to 26 in an amount and for a time sufficient to inhibit cell proliferation. Statement 31. The method according to Statement 30, further comprising contacting the cell with flutamide, bicalutamide, enzalutamide, apalutamide, darolutamide, dilutamide, finasteride, dutasteride, abiraterone acetate, abiraterone acetate microparticles, ketoconazole, or a combination thereof. Statement 32. The method according to Statement 30 or Statement 31, wherein the compound has the following structure: TIFF2025098059000090.tif86169TIFF2025098059000091.tif49169Statement 33. The method according to statement 31 or 32, wherein the inhibited cell proliferation is malignant cell proliferation and / or proliferative cell proliferation. Statement 34. The method according to any one of statements 31 to 33, wherein the cell proliferation (e.g., malignant cell proliferation and / or proliferative cell proliferation) is cancer cell proliferation. Statement 35. The method according to any one of statements 31 to 34, wherein the cell proliferation (e.g., malignant cell proliferation and / or proliferative cell proliferation, which may be cancer cell proliferation) is hormone cancer cell proliferation. Statement 36. The method according to statement 35, wherein the hormone cancer is selected from breast cancer, ovarian cancer, uterine cancer or endometrial cancer, prostate cancer, and combinations thereof. Statement 37. The method according to any one of statements 31 to 35, wherein the cell proliferation (e.g., malignant cell proliferation and / or proliferative cell proliferation, which may be cancer cell proliferation, which may be hormone cancer cell proliferation) is prostate cancer cell proliferation. Statement 38. The method according to any one of statements 31 to 35 or statement 37, wherein the cell proliferation (e.g., malignant cell proliferation and / or proliferative cell proliferation, which may be cancer cell proliferation, which may be hormone cancer cell proliferation, which may be prostate cancer cell proliferation) is castration-resistant prostate cancer cell proliferation. Statement 39. The method according to any one of statements 31 to 35 or statements 37 to 38, wherein the cell (e.g., malignant cell and / or proliferative cell, which may be cancer cell, which may be hormone cancer cell, which may be prostate cancer cell, which may be castration-resistant prostate cancer cell) expresses AR-V7. Statement 40. A method for treating a proliferative cell growth disorder and / or a malignant cell growth disorder in an individual, comprising administering to the individual a compound as described in any one of Statements 1 to 21 or a composition as described in any one of Statements 22 to 26, wherein the malignant cell growth disorder and / or the proliferative cell growth disorder of the individual is treated. Statement 41. The method according to Statement 40, wherein the malignant cell growth disorder is cancer. Statement 42. The method according to Statement 40 or Statement 41, wherein the malignant cell growth disorder (e.g., cancer) is a hormone cancer. Statement 43. The method according to Statement 42, wherein the hormone cancer is selected from breast cancer, ovarian cancer, uterine cancer or endometrial cancer, prostate cancer, and combinations thereof. Statement 44. The method according to any one of Statements 40 to 43, wherein the malignant cell growth disorder (e.g., cancer, which may be a hormone cancer) is prostate cancer. Statement 45. The method according to any one of Statements 40 to 44, wherein the malignant cell growth disorder (e.g., cancer, which may be a hormone cancer and which may be prostate cancer) is castration-resistant prostate cancer. Statement 46. The method according to any one of Statements 40 to 45, wherein cells of the malignant cell growth disorder (e.g., cancer, which may be a hormone cancer, which may be prostate cancer, and which may be castration-resistant prostate cancer) express AR-V7. Statement 47. The method according to any one of Statements 40 to 46, wherein the compound has the following structure: TIFF2025098059000092.tif84169TIFF2025098059000093.tif51169 Statement 48. A method for imaging malignant cells and / or proliferating cells of an individual, comprising administering to the individual one or more compounds described in any one of Statement 1 or Statement 6 or Statement 9 to 10 or Statement 20 to 21, or a composition described in any one of Statement 22 to 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 malignant cells and / or hyperproliferating cells. Statement 49. The method according to Statement 48, wherein the malignant cells are cancer cells. Statement 50. The method according to Statement 48 or Statement 49, wherein the malignant cells (e.g., cancer cells) are hormone cancer cells. Statement 51. The method according to Statements 48 to 50, wherein the malignant cells (e.g., cancer cells, which may be hormone cancer cells) are selected from breast cancer cells, ovarian cancer cells, uterine cancer cells or endometrial cancer cells, and prostate cancer cells. Statement 52. The method according to Statements 48 to 51, wherein the malignant cells (e.g., cancer cells, which may be hormone cancer cells) are prostate cancer cells. Statement 53. The method according to Statements 48 to 52, wherein the malignant cells (e.g., cancer cells, which may be hormone cancer cells and which may be prostate cancer cells) are castration-resistant prostate cancer cells. Statement 54. The method according to Statements 48 to 53, wherein the malignant cells (e.g., cancer cells, which may be hormone cancer cells, which may be prostate cancer cells, and which may be castration-resistant prostate cancer cells) express AR-V7. Statement 55. The method according to Statements 48 to 54, wherein imaging is repeated so that the activity of androgen metabolism can be measured. Statement 56. The method according to Statements 48 to 55, wherein the compound has the following structure: TIFF2025098059000094.tif213169TIFF2025098059000095.tif228169TIFF2025098059000096.tif47169

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

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

[0107] Synthesis of compound (Ia; alias DIOL-17C) in which R1 and R2 are hydrogen as shown below and in Figure 2: TIFF2025098059000097.tif43169Ethyl (3-keto-5α-androstan-17β-yl) glycolate. To a 10 mL CH2Cl2 suspension containing 1 g (3.44 mmol) of androstan-17β-ol-3-one and 95 mg (0.22 mmol, 0.064 equiv) of dirhodium tetraacetate, 393 mg (3.44 mmol, 1 equiv) of ethyl diazoacetate was added dropwise at 0 °C with vigorous stirring. After 2 hours at 25 °C, an additional 393 mg (3.44 mmol, 1 equiv) of ethyl diazoacetate was added dropwise and the mixture was stirred for an additional 2 hours. The mixture was diluted with CH2Cl2, washed with water and brine, dried over anhydrous MgSO4, and concentrated. The product was purified by column chromatography using 1:3 EtOAc-hexane to give 687 mg (53%) of ethyl (3-keto-5α-androstan-17β-yl) glycolate as a semi-solid. 11H NMR (CDCl3, 500 MHz): δ 4.20 (q, 2H, J = 7.2 Hz), 4.09 (s, 2H), 3.40 (t, 1H, J = 8.5 Hz), 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 13C 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 Calculated HRMS (ESI) for C22H37O4[MH+]: 377.2686. Found: 377.2687.

[0108] Ethyl (3β - hydroxy - 5α - androstan - 17β - yl) glycolate. To a solution containing 300 mg (0.80 mmol) of ethyl (3 - keto - 5α - androstan - 17β - yl) glycolate in 4 mL of THF, 2.1 mL (1.04 mmol, 1.3 equiv) of lithium tri - tert - butoxyaluminum hydride (0.5 M in bis(2 - methoxyethyl) ether) was added dropwise at - 20 °C. The mixture was stirred for 3 h, quenched with water, extracted with CH2Cl2, dried over anhydrous MgSO4, 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. 11H NMR (CDCl3, 500 MHz): δ 4.20 (q, 2H, J = 7.0 Hz), 4.10, 4.06 (ABq, 2H, J AB = 16.2 Hz), 3.61 - 3.55 (m, 1H), 3.39 (t, 1H, J = 8.2 Hz), 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 13C 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) calculated value for C24H36O4[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 CH2Cl2, washed successively with 10% aqueous K2CO3 solution and water, dried over anhydrous MgSO4, and concentrated. The product was purified by column chromatography on silica gel eluting with a 1:5 EtOAc - hexane (R fPurified by chromatography using =0.53) to obtain 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, 43.10, 40.47, 37.84, 35.97, 35.26, 33.24, 33.00, 31.46, 28.24, 27.59, 26.30, 23.23, 20.47, 14.23, 11.68, 11.43. C 30 H 43 HRMS (ESI) calculated value for C29H43O5[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 was added 0.66 mL (1.33 mmol, 4 equivalents) of 2N aqueous NaOH solution. 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 vacuo to give 103 mg (89%) of (3α-hydroxy-5α-androstan-17β-yl) glycolic acid. This was pure enough to be used in the next reaction without further purification. To a mixture containing 80 mg (0.23 mmol) of this acid and 90 mg (0.25 mmol, 1.1 equivalents) 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-CH2Cl2 (1:1) were successively added 66 mg (0.34 mmol, 1.5 equivalents) of EDC, 46 mg (0.34 mmol, 1.5 equivalents) of HOBt, and 81 mg (0.80 mmol, 3.5 equivalents) 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 chromatography using 1:10 CH3OH-CH2Cl2 (R f = 0.53) to give 76 mg (54%) of Ia (alias 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= 15.4 Hz), 3.60 (q, 2H, J = 7.2 Hz), 3.28 - 3.23 (m, 5H), 2.92 - 2.87 (m, 4H), 2.79 (t, 2H, J = 6.2 Hz), 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 13C 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 Calculated HRMS (ESI) for [MH+] of N2O5: 617.3949. Found: 617.3940.

[0111] [Example 2] This example provides an illustration of the compound of the present disclosure. As shown in Figure 2, synthesis of the compound where R1 and R2 are hydrogen (Id: alias DHT - 17C): TIFF2025098059000098.tif 43169N - 2'-(2,3,4,5 - Tetrahydro - 1H,4H - 10 - keto - 11 - oxa - 3a - azabenzo[de]anthracenyl)ethyl (3 - keto - 5α - androstan - 17β - yl)glycolamide. A solution containing 100 mg (0.27 mmol) of ethyl (3 - keto - 5α - androstan - 17β - yl) glycolate in 4 mL of THF was added to 0.28 mL (0.54 mmol, 2 equivalents) of 2N aqueous NaOH solution. The mixture was stirred at 25 °C for 5 hours and concentrated. The residue was diluted with water and acidified to pH 2 with 3N HCl solution to form a white precipitate, which was collected to obtain 83 mg (90%) of (3 - keto - 5α - androstan - 17β - yl) glycolic acid, which was used in the next step without further purification. In 1 mL of CH2Cl2, to a mixture containing 37 mg (0.11 mmol) of this acid and 42 mg (0.12 mmol, 1.1 equivalents) of 8 - (2 - aminoethyl) - 2,3,4,5 - tetrahydro - 1H,4H - 11 - oxa - 3a - azabenzo[de]anthracen - 10 - one hydrochloride, 30 mg (0.16 mmol, 1.5 equivalents) of EDC, 24 mg (0.16 mmol, 1.5 equivalents) of HOBt, and 38 mg (0.37 mmol, 3.5 equivalents) of triethylamine were sequentially added. The mixture was stirred at 25 °C for 12 hours, diluted with water, extracted with CH2Cl2, dried over anhydrous MgSO4, and concentrated. The product was purified by chromatography using 5:1 EtOAc - hexane (after two developments, R f = 0.53) to obtain 33 mg (51%) of Id (alias 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= 15.2 Hz), 3.59 (q, 2H, J = 6.8 Hz), 3.27 - 3.22 (m, 5H), 2.91 - 2.85 (m, 4H), 2.78 (t, 2H, J = 6.4 Hz), 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 Calculated HRMS (ESI) for N2O5[MH+]: 615.3792. Found: 615.3793.

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

[0113] As shown in Figure 3, synthesis of compound (IIa) where R1 is hydrogen: TIFF2025098059000099.tif4216917-(O-Carboxymethyloximino)-5α-androstan-3α-ol. A mixture containing 537 mg (1.85 mmol) of 5α-androstan-3α-ol-17-one and 404 mg (3.7 mmol) of carboxymethoxyamine hemichloride 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 CH2Cl2. The organic layer was washed with brine and dried over anhydrous MgSO4. The solvent was evaporated and the residue was recrystallized from acetone to give 652 mg (97%) of a white solid: mp 171-173 °C. mp 173-175 °C. 1 1H 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 13C 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) of m-anisidine and 91.7 mL (0.927 mol, 3.8 equiv) of 1-bromo-3-chloropropane was heated at 95 °C for 1 - 2 h, at which point an exothermic reaction occurred, so heating was stopped and the temperature of the mixture reached a peak at 140 - 145 °C. Since the temperature began to drop, 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 of concentrated HCl, and quenched by slowly adding 120 mL of water. After all the solids had dissolved, the two phases were separated. The organic layer was washed with 100 mL of 10% HCl and combined with the aqueous solution. The aqueous solution was extracted with ether to remove unreacted 1-bromo-3-chloropropane. 200 mL of 25 M aqueous NaOH was added to this acidic aqueous solution. The solution was extracted with toluene until the organic phase no longer became 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 °C. mp 126 - 130 °C). 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. A solution of 35 mL (30 mmol) of dimethyl malonate in 40 mL of MeOH was added dropwise to a solution of 17.1 g (30 mmol) of KOH in 70 mL of MeOH at 5 - 10 °C over 1 hour. The mixture was stirred at 25 °C for about 12 hours. The precipitated potassium methyl malonate was collected and washed with cold MeOH. A suspension containing 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 hours. In a separate flask, 14.6 g (90 mmol, 1.2 equiv) of 1,1'-carbonyldiimidazole was added to a 50 mL THF solution (5 °C) containing 16.7 g (75 mmol, 1 equiv) of N-benzyloxycarbonyl-β-alanine. This imidazolide solution was added dropwise to the suspension of methyl magnesium malonate at 25 °C. The mixture was stirred for about 12 hours, concentrated under reduced pressure, and then diluted with ethyl acetate. This ethyl acetate solution was washed successively with aqueous KHSO4, aqueous NaHCO3, and brine. The organic layer was dried over anhydrous Na2SO4, concentrated to give 18 g (98%) of a yellow oil, which was used without further purification. 1 1H 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 containing 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 eq) of a 1 M solution of triisopropoxytitanium chloride in hexane. The mixture was heated to reflux for about 12 h. The mixture was diluted with 500 mL of CH2Cl2 and poured into 500 mL of stirred saturated potassium sodium tartrate solution. The aqueous layer was extracted with CH2Cl2. The combined organic layers were dried over anhydrous Na2SO4. The filtered solution was concentrated and the residue was dissolved in a mixture of EtOH and hexane. The 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 h, cooled and concentrated in vacuo. The residue was suspended in a mixture of MeOH-acetone, filtered to give 4.3 g (98%) of (7) as the yellow hydrochloride: mp 238-239 °C. mp 114-118 °C for the free amine 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). 1313C 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) containing 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 1:3 MeOH-CHCl3 (4 mL) were successively added 75 μL (0.42 mmol, 1.1 eq) of N,N-diisopropylethylamine, 139 mg (0.38 mmol) of (6), 206 mg (1.5 mmol, 4 eq) of 1-hydroxybenzotriazole hydrate (HOBt) and 205 mg (1.07 mmol, 2.8 eq) of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC). The EDC was divided into two equal portions and added at 30-minute intervals. The mixture was stirred at 0 °C for 1 h, concentrated and chromatographed using 1:10 MeOH-CH2Cl2 (R f = 0.39) to give 134 mg (56%) of IIa as a yellow foam. 11H 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 13C 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) calculated value for [MH+] of N3O5: 630.3901. Measured value: 630.3899.

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

[0119] Synthesis of compound (IIb) where R1 is hydrogen as shown in Figure 3: TIFF2025098059000100.tif41169N - 2'-(2,3,4,5 - Tetrahydro - 1H,4H - 10 - keto - 11 - oxa - 3a - azabenzo[de]anthracenyl)ethyl 17-(O - carbamidomethyloxymino)-5α - androstan - 3 - one (IIb) To a solution containing 30 mg (0.048 mmol) of IIa in 1 mL of DMSO:CH2Cl2 (1:1), 24 mg (0.24 mmol, 5 equiv) of triethylamine and 23 mg (0.14 mmol, 3 equiv) of the SO3-pyridine complex were successively added. 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 purified by silica gel chromatography using 1:10 CH3OH-CH2Cl2 (R f = 0.56) to give 16 mg (54%) of IIb as a yellow foam. 1 1H NMR (CDCl3, 400 MHz): δ 7.10 (s, 1H), 6.46 (t, 1H, J = 5.8 Hz), 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 = 6.2 Hz), 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 13C 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. 13C 38 1H 50Calculated HRMS (ESI) value of N3O5[MH+]: 628.3745. Measured value: 628.3741.

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

[0121] As shown in Figure 4, synthesis of the compound (Ig: alias DHT-3C) where R1 and R2 are hydrogen. TIFF2025098059000101.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 containing 66 mg (0.21 mmol, 1 equivalent) 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, 50 mg (0.17 mmol, 1 equivalent) of DHT was added. This suspension was placed in a sealed tube, 0.2 mL of concentrated HCl was added, and the mixture was stirred under reflux for 48 hours. This suspension became a clear solution within the first hour of heating, and then precipitation of the desired product appeared. The reaction was stopped by the addition of approximately 3 mL of water, and the precipitate was collected by filtration to obtain 73 mg (92%) of Ig. Further purification was achieved by recrystallization from methanol: 11H 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 13C 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) calculated 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 NaHCO3. The dichloromethane phase was dried over anhydrous Na2SO4, filtered, concentrated, and purified by silica gel chromatography using 1:10 methanol - dichloromethane (R f = 0.55) to give Ig as the free base: 11H 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 13C 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 HRMS (ESI) calculated value for [M+H]+ of N2O3: 557.3738. Found: 557.3738. C 36 H 48 Analytical calculated values for N2O3: C, 77.66; H, 8.69; N, 5.03. Found: C, 77.53; H, 8.62, N, 4.95. Confirmation of the C-3R stereochemical assignment in the spirocyclic DHT adduct Ig was by two-dimensional 1 1H- 13It relied on C-heteronuclear single quantum coherence (HSQC), gradient correlation spectroscopy (gCOSY), and 2D rotating frame NOESY (2D ROESY) experiments. The resonance of the protonated amine in the spirocyclic ring system appeared at δ 9.24 ppm in the 2D ROESY spectrum, which was the starting point for this stereochemical assignment at C-3. The ammonium group (NH2 + ) at C-3 was identified by D2O exchange experiments in DMSO-d6. From the ROESY spectrum correlations (Figure 5) between this ammonium group and the individual C-1α, C-2α, C-4α, and C-5α protons, the 3α-orientation of the ammonium group in Ig was confirmed.

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

[0123] Synthesis of compound (Ij) in which R1 is hydrogen as shown in Figure 4 TIFF2025098059000102.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 hot methanol trituration. 11H 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 13C 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 an explanation 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, which is described as DIOL-17C (also known as Compound Ia) in the present disclosure. TIFF2025098059000103.tif47169

[0125] CaP cells survive and proliferate under ADT due to intratumoral synthesis of DHT and AR hypersensitization. Our group and other researchers 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 convergence of the front and backdoor pathways to DHT has only recently been evaluated. DIOL is the immediate precursor to DHT in the primary backdoor pathway (Figure 1), and preclinical studies have clearly shown the conversion of DIOL to DHT using castration-recurrent CWR-R1 human CaP xenografts. Four 3α-hydroxysteroid dehydrogenases that metabolize DIOL to DHT were immunostained in a tissue microarray (TMA) 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-hydroxysteroid dehydrogenase expression increased DHT production (Figure 9).

[0126] Inhibition of DIOL conversion to DHT by 3α-hydroxysteroid dehydrogenases: DIOL is converted to DHT in CRPC xenografts, and since four 3α-oxidoreductases were present in clinical specimens, it was shown that DIOL is likely to be metabolized to DHT in patients with progressive CaP or CRPC. In vitro administration results in AR-luciferase (Figure 10) transactivation, providing an opportunity to test inhibitors. The constraint-based multiple protein alignment tool (COBALT) showed that the four 3α-oxidoreductases share active sites and 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 deletions of the common catalytic amino acids impair enzyme activity. Furthermore, the mutant catalytically impaired in combination with dutasteride decreased DHT levels in CaP cell lines more than dutasteride alone (Figure 11). Experiments were repeated using LAPC-4 cells transfected with RDH16-targeted siRNA. DHT ELISA revealed that knockdown of RDH16 expression decreased DHT levels in LAPC-4 (Figure 12). The growth of VCaP (Figure 13) and LAPC-4 cells (data not shown) was evaluated after expression of the catalytically impaired RDH16 Y176F, K180R mutants and treatment with dutasteride.

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

[0128] ImageStream(ImageStream X-MKII Flow Cytometer (Amnis, Seattle, WA) is a hybrid technology that combines the statistical output of flow cytometry and the imaging capabilities of fluorescence microscopy and is adapted to simultaneously study the compartmental localization and activity of DIOL-17C. ImageStream data were normalized against single-stained controls and analyzed using Image Data Exploration and Analysis Software (IDEAS; Amnis). Similarity scores were measured by comparing two images of the same cell on a pixel-by-pixel basis. If the similarity score was >0, the two targets (RDH16-YFP and DIOL-17C) were co-localized. DIOL-17C (blue) co-localizes with RDH16-YFP (color-changed to green in IDEA for comparison) and nuclear DRAQ5 (white punctate signal) in PC-3-RDH16-YFP cells (Figure 15A.1 single and merged fluorescence images; 15A.2 high similarity in the upper right quadrant), but not in SFM-treated PC-3 RDH16-YFP cells (Figure 15B.1 no punctate signal in the merged image; 15B.2 high similarity in the 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 do not produce free DIOL and orphan coumarin, LC-MS / MS was performed using CaP cells treated with DIOL-17C. No orphan fluorophore was detected. The addition of coumarin did not impair the 3α-oxidoreductase metabolism of DIOL-17C to DHT-17C (Figure 17).

[0130] [Example 8] This example provides an explanation 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 as DHT-17C (also known as Compound Id) in the present disclosure. TIFF2025098059000104.tif49169

[0131] Either an empty pCMV expression plasmid (control) or a pCMV plasmid encoding wild-type AR (AR-GFP) labeled with a C-terminal green fluorescent protein (GFP) was transfected into androgen-sensitive AR-positive LAPC-4 and VCaP, castration-recurrent and AR-positive CWR-R1 and CWR-22rv1 (22rv1), and AR-negative PC-3 and DU145 cells. AR-positive CaP cell lines were transfected with AR-GFP to evaluate whether DHT-17C or DHT-3C co-localizes with AR and translocates to the nucleus. To test the effect of coumarin on anti-androgenic 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 activity from anti-androgenic-like activity. TIFF2025098059000105.tif38169

[0132] AR-positive CaP cell lines were treated for 16 hours with serum-free complete medium (SFM) alone without androgen, or SFM containing 20 nM or 50 nM of fluorescent androgen or C alone. These time points were selected to measure the uptake of fluorescent androgen and the differences in the nuclear or cytoplasmic localization of fluorescent androgen and / or AR-GFP. ImageStream analysis showed that DHT-17C and DHT-3C were taken up by VCaP, LAPC-4, CWR-R1, 22Rv1, PC-3, and DU145 cells (data reported for VCaP and PC-3-AR-GFP, which are representative of all cell lines studied). DHT-17C (blue) or DHT-3C co-localized with AR-GFP (green) and 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) co-localized with AR-GFP and DRAQ5 (Figure 18). DHT-3C served as a negative control for DHT-17C (also known as Compound Id), and DHT-17C was shown to be an AR-LBD inhibitor, in contrast to DHT-3C, which retains DHT activity (as described above).

[0134] Nuclear localization of DHT-17C or DHT-3C was not observed in AR- or AR-GFP-negative PC-3 cells, suggesting that the 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 co-localizing with DRAQ5 and AR-V2-GFP (Figure 18). ImageStream data were confirmed using immunoprecipitation (IP) studies performed with DHT-targeting antibodies.

[0135] Using ImageStream, a competition study between DHT-17C and enzalutamide was performed for AR-GFP or AR-V2 to establish that DHT-17C targets AR-LBD. 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 medium (CM), serum-free 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-day treatment with DHT-3C promoted VCaP, LAPC-4, and LNCaP cell proliferation, as expected for DHT surrogates (data not shown) and CWR-R1 cell growth (compared to DHT) (Figure 20). In contrast, DHT-17C treatment impaired VCaP and CWR-R1 cell growth and enhanced the effect of bicalutamide (Figure 20; p-value < 0.05). MTT experiments using LAPC-4, LNCaP, and 22Rv1 yielded similar responses (data not shown; p-value < 0.05). DHT-17C did not impair cell growth 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 the cell growth impairment of VCaP cells treated with DHT-17C to enzalutamide. Cells were treated with DHT17C at concentrations of 1 nM, 12 nM, 20 nM, 50 nM, or 100 nM, or enzalutamide at concentrations of 1 μM, 12 μM, 20 μM, 50 μM, or 100 μM. DHT-17C at nM concentrations impaired VCaP cell growth to the same extent as enzalutamide at μM concentrations (Figure 21). MTT performed using mutant AR- and AR-V7-positive CWR-R1 cells treated with enzaltamide (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 the expression of KLK2, an AR-regulated gene (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 (rather than the presence of C) was responsible for the lack of KLK2 induction. Completion of these studies revealed that DHT-17C has anti-androgen-like properties but produces another unexpected finding.

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

[0139] Cell uptake of DHT-3C (also known as Compound Ig) by AR-negative PC-3 cells with an empty plasmid, PC-3 cells stably expressing AR-GFP (PC-3-AR-GFP), VCaP cells with an empty plasmid, VCaP cells stably expressing AR-GFP (VCaP-AR-GFP), or VCaP stably expressing only the splice variant V2 of AR170 LBD (AR-V2-GFP) was evaluated using ImageStream (Figure 4), confirming the DHT-like activity exhibited by DHT-3C. Serum-free complete medium (SFM) 27Androgen-depleted cells were obtained using []. PC-3 cells containing empty plasmid were treated with SFM alone, SFM and C, or SFM and DHT-3C. The cells showed minimal autofluorescence and did not generate nuclear fluorescence consistent with C or DHT-3C. SFM-treated PC-3 AR-GFP cells did not show autofluorescence. In SFM using C-treated PC-3 AR-GFP cells, no nuclear co-localization was observed between C and AR-GFP (Figs. 24A.1 and A.2). DHT-3C-treated PC-3-AR-GFP showed nuclear co-localization between DHT-3C and AR-GFP (Figs. 24B.1 and B.2). C-treated VCaP stably expressing AR-GFP did not result in nuclear co-localization between C and AR-GFP (Figs. 24C.1 and C.2). DHT-3C-treated VCaP stably expressing AR-GFP showed co-localization between DHT-3C and AR-GFP (Figs. 24D.1 and D.2). DHT-3C-treated VCaP stably expressing AR-V2-GFP showed DHT-3C co-localized with the AR LBD (Figs. 24E.1 and E.2).

[0140] ImageStream data revealed that DHT-3C was taken up by the CaP cell line and co-localized with AR in the nucleus. MTT analysis was used to evaluate whether treatment with DHT-3C promoted CaP cell growth during androgen ablation. AR-positive androgen-sensitive VCaP, LAPC-4, and LNCaP; AR-positive castration-recurrent CWR-R1 and CWR22rv1; and AR-negative PC-3 and DU145 CaP cell lines were treated for 6 days under 5 sets of conditions: complete cell culture medium (CM) to establish baseline CaP cell growth; SFM to simulate androgen ablation; SFM containing C alone as a negative control to confirm that C alone did not affect CaP cell growth; SFM containing DHT as a positive control; and SFM containing DHT-3C. SFM containing DHT-3C stimulated growth in 3 out of 5 cell lines (Fig. 25).

[0141] In summary, these data suggested that the increased CaP cell growth was AR-dependent and was stimulated by either DHT or DHT-3C that 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 indicating that C alone did not affect AR-regulated transcript induction and that the coumarin moiety in DHT-3C was mainly outside the AR-LBD binding pocket. Western blot analysis confirmed that similar levels of AR-regulated protein expression were obtained with DHT-3C treatment and DHT treatment (Figure 26).

[0143] In summary, DHT-3C (alias Compound Ig) in Figure 4 functions as a true substitute, and its replacement by other ligands provides a means for anti-androgen evaluation 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 difficult due to the low cellular uptake of oligonucleotides and the technical difficulties associated with delivery to the desired target. Furthermore, the recently reported SNIPER agents, as well as the development of the hydrophobic tagging approach (HyT) for AR degradation, have shown to be experimental tools for discovery biology rather than feasible therapeutic strategies in their current form. There are questions about their reported micromolar potencies, which far exceed current levels of anti-cancer agents; and questions about unwanted off-target effects. On the other hand, target AR proteolysis induced by target protein degradation chimeras (PROTACs) via the ubiquitin proteasome system (UPS) represents an attractive alternative for which DHT-3C is well designed.

[0145] Targeted protein degradation chimeras (PROTACs) have three components: a ligand that binds to the protein to be degraded, an E3 ligase "recruiter" that promotes ubiquitination of the target protein, and a spacer that links the ligand and the recruiter. Some 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 androgen receptor modulator (SARM), which has nanomolar affinity for AR, to nutlin, a known ligand for the MDM2 E3 ligase. This approach generated a SARM-nutlin PROTAC that decreased AR levels in a proteasome-dependent manner in HeLa cells (however, only at a concentration of 10 μM). Two other studies described the development of DHT-based PROTACs by linking DHT to a ligand for 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 concentrations of 10 - 25 μM. Furthermore, the SCFβ-TRCP-based PROTACs were not cell permeable and required microinjection.

[0146] In summary, the reported drawbacks in DHT-based PROTACs regarding potency and cell permeability lie in the nature of the chemicals used to target 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 an 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 a photoactive group (including diazirine, benzophenone or perfluorinated aryl azide) (Figure 29) could provide a means to specifically crosslink these agents to AR or to late-stage enzymes in the pathway leading to DHT, and a new photodynamic / hyperthermia therapy for patients with progressive 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 site of late-stage enzymes in the biosynthetic pathway converging to DHT, or to the AR-LBD.

[0149] In the former case, computer modeling of the binding of compound Ij to AKR1C3 (alias 17β-hydroxysteroid dehydrogenase-5) showed that the compact nature of this fluorescent androgen Ij did not interfere with 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, and the binding of compound Ij (alias 5α-DIONE-F) was modeled (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 using Autodock Vina to the position occupied by 5α-DIONE in AKR1C3. The binding pose of Ij obtained from Vina contained high overlap with that of 5α-DIONE. The binding pose was further refined by performing a series of energy minimization processes. Briefly, the AMBER14SB force field and the second generation of the general AMBER force field (gaff2) were used for the protein and ligand, respectively. The Antechamber of AMBER 18 9 program was used to generate the partial charges of Ij using the AM1-BCC model. A hybrid protocol of 2500 steps of steepest descent minimization was used, followed by two courses of minimization using conjugate gradient minimization until a maximum of 2500 iterative steps was reached or the convergence criterion (root mean square of the energy gradient was less than 1×10 -4 kcal / mol·Å) was met. During the first stage of minimization, 100 kcal / mol·Å 2The force constant was applied to the protein atoms. The second minimization stage consisted of 1,000 steps of steepest descent minimization followed by 1,500 steps of conjugate gradient minimization, and this minimization process had no restrictions on either ligand atoms or protein atoms. In summary, compound j adopted the same conformation as the naturally occurring ligand 5α-DIONE. The BCD ring of compound Ij was 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 protruded into the oxyanion hole of 17β-HSD5 bound by Y55, H117, and NADP + and S129. The hydrogen bond with S129 further stabilized the observed binding mode with Ij. These binding characteristics indicated that compound Ij had 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) (which binds to AKR1C3 (PDB:1ZQ5)).

[0150] In the latter case, computer modeling of the binding of compound Ig (alias 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 for modeling AR. The initial structure of AR (694-919) was generated using SWISS-MODEL, and 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 processes and restrained MD simulations. Briefly, the AMBER14SB force field and the second generation of the general AMBER force field (gaff2) were used for the protein and ligand, respectively. TIP3P water molecules were added as the solvent, and solute atoms were at least 10 Å away from the boundary 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 the particle mesh Ewald (PME) algorithm, and the non-bonded cutoff for real-space interactions was set to 10 Å. A hybrid protocol of 8000 steps of steepest descent minimization was used, followed by conjugate gradient minimization for two-stage minimization until a maximum of 2000 iterative steps was reached or the convergence criterion (root mean square of the energy gradient was less than 1 Å~10 -4 kcal / mol·Å) was satisfied. During the first stage of minimization, a force constant of 100 kcal / mol·Å 2 was applied to the ligand and protein atoms except for residues 711, 752, and 759-771; for the second stage of minimization, a force constant of 100 kcal / mol·Å 2 was applied to the protein backbone atoms except for residues 711, 752, and 759-771. The system was linearly heated from 0 to 303.15 K over a period of 50 ps with constraints (force constant of 10 kcal / mol·Å2) on all heavy atoms in the NVT ensemble, and then the force constant was changed from 10 to 0.2 kcal / mol·Å 2By gradually decreasing it, equilibration was carried out for 325 ps with the Langevin thermostat13 in the NPT (P = 1 atm and T = 303.15 K) ensemble. Finally, a 40 ns production process was carried out using Amber12 in the NPT (P = 1 atm and T = 303.15 K) ensemble with restraints (force constant of 2 kcal / mol·Å 2 on the backbones of residues 694 - 759 and 771 - 919). The SHAKE algorithm was used to suppress the covalent bonds between heavy atoms 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, and the RMSD fluctuations in the last 20 ns were less than 1 Å. Using Chimera, the conformations of the last 20 ns trajectory were clustered, and the top conformations were selected and subjected to the above minimization process (step 2). The final conformation was used for further analysis. Modeling revealed that the hairpin domain (759 - 771) of AR was "opened" by approximately 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). In addition to these features, the coumarin moiety of compound Ig formed hydrophobic interactions with F764, M749, Y763, and R752 and a hydrogen bond interaction with Q711. These binding characteristics indicated that compound Ig had a more favorable binding mode to AR than DHT.

[0151] In summary, computer modeling suggested that the successful binding of compounds Ij and Ig occurs 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 anti-androgens (which bind 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 following structure: where: R 1 is hydrogen or an alkyl group; wherein X is a hydroxyl group and Y is hydrogen or an alkyl group, or X and Y together are a spiro-fused, substituted or unsubstituted coumarin group or coumarin isostere group; m is 1, 2, 3, 4, 5 or 6; n is 1, 2, 3, 4, 5 or 6; Z is a terminal group comprising a substituted or unsubstituted carbocyclic group, or a substituted or unsubstituted heterocyclic group, or a substituted or unsubstituted coumarin group, or a substituted or unsubstituted coumarin isostere group; R 2 is an alkyl group or hydrogen; however,

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

3. A compound described in claim 1.

4. L 2 The compound of claim 1 , wherein

5. 2. The compound of claim 1, wherein n is 1 or 2.

6. 2. The compound of claim 1, wherein m is 1, 2, or 3.

7. R 2 The compound of claim 1 , wherein is H.

8. 2. The compound of claim 1, wherein Z is selected from the group consisting of: (wherein each X' is independently selected from hydrogen, an alkyl group, a cycloalkyl group, an alkoxy group, a halogen, and combinations thereof).

9. The compound of claim 8, wherein X' is an alkyl group or an alkoxy group.

10. R 3 The compound of claim 1 , wherein is carbonyl.

11. A compound described in claim 1.

12. The compound of claim 1 selected from the group consisting of:

13. A compound having the following structure: where: R 1 is hydrogen or an alkyl group; R 3 is carbonyl; L 1 is a linking group; L 2 is an optional linking group, each X' is independently selected from hydrogen, an alkyl group, a cycloalkyl group, an alkoxy group, a halogen, and combinations thereof; R 2 is an alkyl group or hydrogen.

14. R 1 14. The compound of claim 13, wherein is hydrogen.

15. R 2 The compound of claim 13, wherein is H.

16. 14. The compound of claim 13, wherein X' is an alkyl group or an alkoxy group.

17. 14. A composition comprising a compound of claim 1 or 13 and a pharmaceutically acceptable carrier.

18. 17. The composition of claim 16, further comprising one or more additional drugs.

19. 18. The composition of claim 17, wherein the one or more additional drugs are selected from antiandrogens, 5α-reductase agents, androgen metabolism inhibitors, and combinations thereof.

20. 19. The composition of claim 18, wherein the antiandrogen is selected from flutamide, bicalutamide, enzalutamide, apalutamide, darolutamide, dilutamide, and combinations thereof, and / or the 5α-reductase inhibitor 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.

21. 18. The composition of claim 17, wherein the compound is selected from the group consisting of:

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