Novel selective agents for the degradation of AR/AR-V7 to treat advanced prostate cancer.

The androgen receptor AR antagonists, such as enzalutamide and apalutamide, are efficient therapeutics for the treatment of prostate cancer (PCa). Even though they are effective at first, resistance to both drugs occurs frequently. Resistance is mainly driven by aberrations of the AR signaling pathway including AR gene amplification and the expression of AR splice variants (e.g. AR-V7). This highlights the urgent need for alternative therapeutic strategies. Here, a total of 24 compounds were synthesized and biologically evaluated to disclose compound 20i, exhibiting potent AR antagonistic activities (IC50 = 172.85 ± 21.33 nM), promising AR/AR-V7 protein degradation potency, and dual targeting site of probably AR (ligand-binding domain, LBD and N-terminal domain, NTD). It potently inhibits cell growth with IC50 values of 4.87 ± 0.52 and 2.07 ± 0.34 µM in the LNCaP and 22RV1 cell lines, respectively, and exhibited effective tumor growth inhibition (TGI = 50.9 %) in the 22RV1 xenograft study. These data suggest that 20i has the potential for development as an AR/AR-V7 inhibitor with degradation ability to treat advanced prostate cancer.

Discovery of N-(4-(Benzyloxy)-phenyl)-sulfonamide Derivatives as Novel Antagonists of the Human Androgen Receptor Targeting the Activation Function 2.

Androgen receptor (AR) antagonists have been widely used for the treatment of prostate cancer (PCa). As a link between the AR and its transcriptional function, the activation function 2 (AF2) region has recently been revealed as a novel targeting site for developing AR antagonists. Here, we reported a series of N-(4-(benzyloxy)-phenyl)-sulfonamide derivatives as new-scaffold AR antagonists targeting the AR AF2. Therein, compound T1-12 showed excellent AR antagonistic activity (IC50 = 0.47 µM) and peptide displacement activity (IC50 = 18.05 µM). Furthermore, the in vivo LNCaP xenograft study confirmed that T1-12 offered effective inhibition on tumor growth when administered intratumorally. The study represents the first successful attempt to identify a small molecule targeting the AR AF2 with submicromolar AR antagonistic activity by structure-based virtual screening and provides important clues for the development of novel therapeutics for PCa treatment.

Discovery of ARD-2585 as an Exceptionally Potent and Orally Active PROTAC Degrader of Androgen Receptor for the Treatment of Advanced Prostate Cancer.

We report herein the discovery of exceptionally potent and orally bioavailable PROTAC AR degraders with ARD-2585 being the most promising compound. ARD-2585 achieves DC50 values of ≤0.1 nM in the VCaP cell line with AR gene amplification and in the LNCaP cell line carrying an AR mutation. It potently inhibits cell growth with IC50 values of 1.5 and 16.2 nM in the VCaP and LNCaP cell lines, respectively, and achieves excellent pharmacokinetics and 51% of oral bioavailability in mice. It is more efficacious than enzalutamide in inhibition of VCaP tumor growth and does not cause any sign of toxicity in mice. ARD-2585 is a promising AR degrader for extensive investigations for the treatment of advanced prostate cancer.

Exploration and Biological Evaluation of Basic Heteromonocyclic Propanamide Derivatives as SARDs for the Treatment of Enzalutamide-Resistant Prostate Cancer.

A series of propanamide derivatives were designed, synthesized, and pharmacologically characterized as selective androgen receptor degraders (SARDs) and pan-antagonists that exert a broad-scope androgen receptor (AR) antagonism. Incorporating different basic heteromonocyclic B-ring structural elements in the common A-ring-linkage-B-ring nonsteroidal antiandrogen general pharmacophore contributed to a novel scaffold of small molecules with SARD and pan-antagonist activities even compared to our recently published AF-1 binding SARDs such as UT-69 (11), UT-155 (12), and UT-34 (13). Compound 26f exhibited inhibitory and degradation effects in vitro in a wide array of wtAR, point mutant, and truncation mutant-driven prostate cancers (PCs). Further, 26f inhibited tumor cell growth in a xenograft model composed of enzalutamide-resistant (EnzR) LNCaP cells. These results demonstrate an advancement toward the development of novel SARDs and pan-antagonists with efficacy against EnzR prostate cancers.

Structure-property and structure-activity relationships of phenylferrocene derivatives as androgen receptor antagonists.

Ferrocene is a representative organometallic compound having a sandwich structure with high stability and hydrophobicity. In this study, we determined the physicochemical properties of a series of nitro- and cyanophenylferrocenes, and evaluated their biological activity as androgen receptor (AR) antagonists. Ferrocene derivatives exhibited hydrophobicity parameter π values in the range between 2.54 and 3.23, depending on the substituents, indicating that the hydrophobicity of ferrocene is suitable for its application as a hydrophobic core structure of nuclear receptor ligands. The synthesized ferrocene derivatives showed AR-antagonistic activity, and among them, 3-nitrophenylferrocene 14 exhibited the most potent activity with an IC50 value of 0.28 µM. The developed compounds may be candidates for further structural development as AR antagonists. These findings also support the utility of organometallic species as structural options for drug discovery.

Synthesis of dihydrotestosterone derivatives modified in the A-ring with (hetero)arylidene, pyrazolo[1,5-a]pyrimidine and triazolo[1,5-a]pyrimidine moieties and their targeting of the androgen receptor in prostate cancer.

One of the main directions of steroid research is the preparation of modified derivatives in which, in addition to changes in physicochemical properties, receptor binding is significantly altered, thus a bioactivity different from that of the parent compound predominates. In the frame of this work, 2-arylidene derivatives were first synthesized by regioselective modification of the A-ring of natural sex hormone, 5α-dihydrotestosterone (DHT). After Claisen-Schmidt condensations of DHT with (hetero)aromatic aldehydes in alkaline EtOH, heterocyclizations of the α,ß-enones were performed with 3-amino-1,2,4-triazole, 3-aminopyrazole and 3-amino-5-methylpyrazole in the presence of t-BuOK in DMF to afford 7'-epimeric mixtures of A-ring-fused azolo-dihydropyrimidines, respectively. Depending on the electronic demand of the substituents of the arylidene moiety, spontaneous or 2,3-dichloro-5,6-dicyanobenzoquinone (DDQ)-induced oxidation of the heteroring led to triazolo[1,5-a]pyrimidines and pyrazolo[1,5-a]pyrimidines in good yields, while, using the Jones reagent as a strong oxidant, 17-oxidation also occurred. The crystal structures of an arylidene and a triazolopyrimidine product have been determined by single crystal X-ray diffraction and both were found to crystallize in the monoclinic crystal system at P21 space group. Most derivatives were found to diminish the transcriptional activity of androgen receptor (AR) in reporter cell line. The candidate compound (17ß-hydroxy-2-(4-chloro)benzylidene-5α-androstan-3-one, 2f) showed to suppress androgen-mediated AR transactivation in a dose-dependent manner. We confirmed the cellular interaction of 2f with AR, described the binding in AR-binding cavity by the flexible docking and showed the ability of the compound to suppress the expression of AR-regulated genes in two prostate cancer cell lines.

Identification of novel androgen receptor degrading agents to treat advanced prostate cancer.

Prostate cancer (PCa) is one of the most common malignancies affecting men worldwide. Androgen receptor (AR) has been a target of PCa treatment for nearly six decades. AR antagonists/degraders can effectively treat PCa caused by increased AR overexpression. However, all approved AR antagonists have similar chemical structures and exhibit the same mode of action on the protein. Although initially effective, resistance to these AR antagonists usually develops. Therefore, this calls for the identification of novel chemical structures of AR antagonists to overcome the resistance. Herein, we employed the synergetic combination of virtual and experimental screening to identify a flavonoid compound which not only effectively inhibits AR transcriptional activity, but also induces the degradation of the protein. Based on this compound, we designed and synthesized a series of derivatives. We discovered that the most potent compound 10e could effectively inhibit AR transcriptional activity, and possessed a profound ability to cause degradation of both full length- and ARv7 truncated forms of human AR. Notably, 10e efficiently inhibited the growth of ARv7 dependent prostate cancer cell-lines, which are completely resistant to all current anti-androgens. Compound 10e also showed strong antitumor activity in the LNCaP (androgen dependent prostate cancer cell line) in vivo xenograft model. These results provide a foundation for the development of a new class of AR antagonists.

Design and synthesis of isothiocyanate-containing hybrid androgen receptor (AR) antagonist to downregulate AR and induce ferroptosis in GSH-Deficient prostate cancer cells.

Sustained androgen receptor (AR) signaling and apoptosis evasion are among the main hurdles of castration-resistant prostate cancer (CRPC) treatment. We designed and synthesized isothiocyanate (ITC)-containing hybrid AR antagonist (ITC-ARi) and rationally combined ITC-ARi with GSH synthesis inhibitor buthionine sulfoximine (BSO) to efficiently downregulate AR/AR splice variant and induce ferroptosis in CRPC cells. The representative ITC-ARi 13 is an AR ligand that contains an N-acetyl cysteine-masked ITC moiety and gradually releases parental unconjugated ITC 12b in aqueous solution. The in vitro anti-PCa activities of 13, such as growth inhibition and AR downregulation, are significantly enhanced when combined with BSO. The drug combination caused notable lipid peroxidation and the cell viability was effectively rescued by iron chelator, antioxidants or the inhibitor of heme oxygenase-1, supporting the induction of ferroptosis. 13 and BSO cooperatively downregulate AR and induce ferroptosis likely through increasing the accessibility of 13/12b to cellular targets, escalating free intracellular ferrous iron and attenuating GSH-centered cellular defense and adaptation. Further studies on the combination of ITC-ARi and GSH synthesis inhibitor could result in a new modality against CRPC.

Design, synthesis and biological evaluation of novel thiohydantoin derivatives as potent androgen receptor antagonists for the treatment of prostate cancer.

Prostate cancer (PC) is the most common malignancy in men worldwide. Here, two series of novel thiohydantoin derivatives of enzalutamide as potent androgen receptor (AR) antagonists were designed and synthesized. Among them, compound 31c was identified as an AR antagonist which is 2.3-fold more potent than enzalutamide. Molecular docking studies were performed to explain the improved potency of 31c at AR. In cell proliferation assays, 31c exhibited similar anti-proliferative activities with enzalutamide against hormone sensitive LNCaP cells and AR-overexpressing LNCaP/AR cells. These data indicate that 31c can be a good lead compound for further structure optimization for the treatment of prostate cancer.

Regulation and targeting of androgen receptor nuclear localization in castration-resistant prostate cancer.

Nuclear localization of the androgen receptor (AR) is necessary for its activation as a transcription factor. Defining the mechanisms regulating AR nuclear localization in androgen-sensitive cells and how these mechanisms are dysregulated in castration-resistant prostate cancer (CRPC) cells is fundamentally important and clinically relevant. According to the classical model of AR intracellular trafficking, androgens induce AR nuclear import and androgen withdrawal causes AR nuclear export. The present study has led to an updated model that AR could be imported in the absence of androgens, ubiquitinated, and degraded in the nucleus. Androgen withdrawal caused nuclear AR degradation, but not export. In comparison with their parental androgen-sensitive LNCaP prostate cancer cells, castration-resistant C4-2 cells exhibited reduced nuclear AR polyubiquitination and increased nuclear AR level. We previously identified 3-(4-chlorophenyl)-6,7-dihydro-5H-pyrrolo[1,2-a]imidazole (CPPI) in a high-throughput screen for its inhibition of androgen-independent AR nuclear localization in CRPC cells. The current study shows that CPPI is a competitive AR antagonist capable of enhancing AR interaction with its E3 ligase MDM2 and degradation of AR in the nuclei of CRPC cells. Also, CPPI blocked androgen-independent AR nuclear import. Overall, these findings suggest the feasibility of targeting androgen-independent AR nuclear import and stabilization, two necessary steps leading to AR nuclear localization and activation in CRPC cells, with small molecule inhibitors.

The one-pot synthesis of butyl-1H-indol-3-alkylcarboxylic acid derivatives in ionic liquid as potent dual-acting agent for management of BPH.

Based on the SAR of both α1-AR antagonists and 5α-reductase (5AR) inhibitors, the dual-acting agent 4-(1-(4-(4-(2-methoxyphenyl)piperazin-1-yl)butyl)-1H-indol-3-yl)butanoic acid 4aaa was designed against BPH and synthesized by two steps of N-alkylation. One-pot protocol towards 4aaa was newly developed. With IL [C6min]Br as solvent, the yield of 4aaa was increased to 75.1% from 16.0% and the reaction time was shortened in 1.5 h from 48 h. 25 derivatives structurally based on arylpiperazine and indolyl butyric acid with alkyl linker were prepared. The protocol was futher extended to get another 14 derivatives wherein O-alkylation was involved, and applied to the synthesis of biologically efficient molecules DPQ and Aripiprazole. Expectedly, compound 4aaa exhibited dual inhibition of α1-AR and 5α-reductase, and exhibited no obvious cytotoxicity against human cells. The pharmacokinetic properties of 4aaa was also determined.

Activity-Directed Synthesis: A Flexible Approach for Lead Generation.

Activity-directed synthesis (ADS) is a structure-blind, functional-driven molecular discovery approach. In this Concept, four case studies highlight the general applicability of ADS and showcase its flexibility to support different medicinal chemistry strategies. ADS deliberately harnesses reactions with multiple possible outcomes, and allows many chemotypes to be evaluated in parallel. Resources are focused on bioactive molecules, which emerge in tandem with associated synthetic routes. Some of the future challenges for ADS are highlighted, including the realisation of an autonomous molecular discovery platform. The prospects for ADS to become a mainstream lead generation approach are discussed.

Metabolically stable diphenylamine derivatives suppress androgen receptor and BET protein in prostate cancer.

Androgen receptor (AR) is a crucial driver of prostate cancer (PC). AR-relevant resistance remains a major challenge in castration-resistant prostate cancer (CRPC). Bromodomain and extra-terminal domain (BET) family are critical AR coregulators. Here, we developed several diphenylamine derivatives and identified compound 7d that disrupted the functions of AR and BET family in prostate cancer and exhibited favorable metabolic stability in vitro and high drug exposure in vivo. We showed 7d not only bound to AR, suppressed transactivation of wild-type AR (wt-AR) and the mutant that mediates Enzalutamide resistance, but also reduced c-Myc protein expression through BET inhibition. In addition, 7d inhibited the proliferation of AR-positive PC cells with favorable selectivity and suppressed AR-V7-expressing VCaP and 22Rv1 xenografts growth in vivo. Collectively, these results indicate the potential of lead compound 7d as an orally available AR and BET inhibitor to treat CRPC and overcome antiandrogen resistance.

Discovery of pyridine tetrahydroisoquinoline thiohydantoin derivatives with low blood-brain barrier penetration as the androgen receptor antagonists.

Prostate cancer (PC) is the most diagnosed type of malignancy in men and the major frequently cause of cancer-related death worldwide. The androgen receptor (AR) has become a promising drug target for the treatment of PC. Here, we reported the design, optimization and evaluation of pyridine tetrahydroisoquinoline thiohydantoin derivatives with improved activity and safety as potent AR antagonists. The most promising compound 42f exhibited potent inhibitory activity on AR and strongly blocked AR nuclear translocation. Moreover, 42f displayed promising in vitro antitumor activity toward AR-dependent prostate cancer cell lines (LNCaP) and also demonstrated therapeutic effects in LNCaP xenograft tumor model in mice (TGI: 79%) with no apparent toxicity observed in vivo. More importantly, 42f showed negligible penetration of the brain-blood barrier (BBB) compared with enzalutamide. These results provide a foundation for the development of a new class of androgen receptor antagonists for potential therapeutics against PC with lower seizurogenic risk for patients.

Novel androgen receptor antagonist identified by structure-based virtual screening, structural optimization, and biological evaluation.

Androgen receptor (AR) plays important roles in the development of prostate cancer (PCa), and therefore it has been regarded as the most important therapeutic target for both hormone-sensitive prostate cancer (HSPC) and advanced PCa. In this study, a novel hit (C18) with IC50 of 2.4 µM against AR transcriptional activity in LNCaP cell was identified through structure-based virtual screening based on molecular docking and free energy calculations. The structure-activity relationship analysis and structural optimization of C18 resulted in the discovery of a structural analogue (AT2), a more potent AR antagonist with 16-fold improved anti-AR potency. Further assays indicated that AT2 was capable of effectively inhibiting the transcriptional function of AR and blocking the nuclear translocation of AR like the second-generation AR antagonists. The antagonists discovered in this study may be served as the promising lead compounds for the development of AR-driven PCa therapeutics.

Design, Synthesis and Evaluation of Novel Substituted (5-methyl-1H-pyrazol-3-yl)- 1,3,4-oxadiazole as Potent Androgen Receptor Antagonist.

BACKGROUND: Androgen Receptor (AR) is one of the highly explored targets for the treatment of prostate cancer. The emergence of point mutation in the Ligand Binding Domain (LBD) of AR has resulted in the development of resistance against AR antagonist. The point mutation T877A, W741L and F876L confer resistance to flutamide, bicalutamide and enzalutamide respectively. There is no AR antagonist in the present clinical set up without resistance. Hence, our aim in this study is to design a novel molecule to overcome the resistance caused by point mutation. METHODS: Here, we developed novel AR antagonist bearing (5-methyl-1H-pyrazol-3-yl)-1, 3,4-oxadiazole core by rational drug design. The test molecules 8a-h were synthesized from the corresponding dihydrazide compounds 7a-h on treatment with phosphorous oxychloride on reflux conditions. The structure of the molecules was confirmed from spectral data such as IR, 1H-NMR, HRMS and 13C-NMR. The synthesized compounds were screened for cytotoxicity in prostate cancer cell lines LNCaP-FGC and PC3. The confirmation of AR mediated activity of the test compounds was confirmed by gene expression study. The interaction of the best active ligands with mutant AR was predicted and drug design was rationalized through docking studies. RESULTS: The test compounds 8a-h were synthesized and the structures were conformed using suitable techniques like IR, 1H-NMR, HRMS and 13C-NMR. Among the tested compounds, 8b and 8d showed potent antiproliferative activity against mutant AR cell lines. Further, these compounds significantly decreased the gene expression of prostate cancer biomarkers. CONCLUSION: In this study, we have identified a potential hit molecule for AR antagonism that could be further developed to obtain a potent clinical candidate.

Synthesis, biological evaluation and molecular docking of 4-Amino-2H-benzo[h]chromen-2-one (ABO) analogs containing the piperazine moiety.

Prostate cancer (PCa) is a major cause of cancer-related male death in worldwide. To develop of potential anti-prostate cancer agents, 22 kinds of 4-Amino-2H-benzo[h]chromen-2-one analogs were designed and synthesized as potent androgen receptor (AR) antagonist through rational drug modification leading to the discovery of a series of novel antiproliferative compounds. Analogs (3, 4, 5, 7, 8, 10, 11, 12, 16, 18, 21, 23, and 24) exhibited potent antagonistic potency against AR (inhibition >50%), and exhibited potent AR binding affinities as well as displayed the higher activities than finasteride toward LNCaP cells (AR-rich) versus PC-3 cells (AR-deficient). Moreover, the docking study suggested that the most potent antagonist 23 mainly bind to AR ligand binding pocket (LBP) site through Van der Waals' force interactions. The structure-activity relationship (SAR) of these designed 4-Amino-2H-benzo[h]chromen-2-one analogs was rationally explored and discussed. Collectively, this work provides a potential lead compound for anticancer agent development related to prostate cancer therapy, and took a step forward towards the development of novel and improved AR antagonists.

Design, synthesis, and biological evaluation of novel androst-17ß-amide structurally related compounds as dual 5α-reductase inhibitors and androgen receptor antagonists.

Prostate cancer (PCa) is the second leading cause of death in men. Apart from androgen receptor, 5α-reductase has also been recognized as a potential target. In this study, a series of androst-17ß-amide compounds have been designed and synthesized targeting both AR and 5α-reductase. Their anti-proliferation activities were evaluated in AR + cell line 22RV1 and AR - cell line PC-3. The results indicated that most of the synthesized compounds inhibited the testosterone-stimulated cell proliferation with good selectivity and safety. Among all the compounds, androst[3,2-c]pyrazole derivatives (9a-9d) displayed the best inhibition activity comparable with flutamide. Moreover, most of the synthesized compounds displayed good 5α-reductase inhibitory activities with IC50 lower than 1 µM. The docking result of 9d-AR indicated that AR was forced to expands its binding cavity and maintain an antagonistic conformation since the steric hindrance of 9d impeded H12 transposition. Overall, compound 9d can be identified as a potential dual 5α-reductase inhibitor and AR antagonist, which might be of therapeutic importance for PCa treatment.

Discovery and biological evaluation of darolutamide derivatives as inhibitors and down-regulators of wild-type AR and the mutants.

Androgen receptor (AR) has been a target of prostate cancer (PC) for nearly six decades. Recently, downregulating or degrading AR and the mutants especially the splice variant 7 (AR-V7) lacking ligand binding domain (LBD) emerged as an advantageous therapeutic approach to overcome drug resistance. Here, the structural modification of darolutamide resulted in the discovery of dual-action AR inhibitors and down-regulators. Unlike other traditional AR antagonists targeting the AR-LBD, compounds 4k and 4b not only inhibit the activities of wt-AR and AR-F876L mutant but also downregulate the protein expression of full-length (AR-full) and AR variant 7 (AR-V7) at mRNA level. In cell proliferation assays, compounds 4k and 4b exhibited better antiproliferative activities than darolutamide and enzalutamide against AR-V7-positive 22Rv1 cells and VCaP cells. In addition, 4k demonstrated better antitumor activity than clinically used enzalutamide in castration-resistant VCaP xenograft model. Collectively, combining the activities of AR inhibition and downregulation, compound 4k is proposed as an advantageous lead compound to disrupt AR signaling and overcome resistance.

Synthesis of novel tanshinone derivatives for treatment of castration-resistant prostate cancer.

Prostate cancer is the second leading cause of death in USA and has developed serious resistance to current drugs. Thus, development of new drugs for the treatment of castration-resistant prostate cancer (CRPC) is urgently needed. In this study, 25 novel derivatives of tanshinone IIA as potential androgen receptor (AR) suppression reagents for the treatment of CRPC have been synthesized. The inhibition on DHT-mediated AR transactivation and cell viability assay were performed to test the synthesized compounds. The results showed that among 25 new compounds, seven methoxy-substituted tanshinone IIA derivatives showed significant inhibition effect on DHT-mediated AR transactivation. In particular, TAN-24 that contains three methoxy groups showed the strongest inhibition effect on DHT-mediated AR transactivation. In addition, TAN-24 also suppressed DHT-AR transactivation more effectively than those of tanshinoneIIA and enzalutamide. In cytotoxicity against human prostate cancer cell lines assay, TAN-24 exhibited the most potent in vitro cytotoxicity against LNCaP and CWR22Rv1 cells, with IC50 values 20- and 19-times lower than those of tanshinone IIA and comparable to enzalutamide. TAN-24 reported in the present work represents a novel and effective AR suppression drug, showing great potential for the treatment of CRPC.