Nonsteroidal Androgen Receptor Ligands: Versatile Syntheses and Biological Data

We report herein a stereoselective and straightforward methodology for the synthesis of new androgen receptor ligands with (anti)-agonistic activities. Oxygen–nitrogen replacement in bicalutamide-like structures paves the way to the disclosure of a new class of analogues, including cyclized/nitrogen-substituted derivatives, with promising antiandrogen (or anabolic) activity

A Small-Molecule Targeting the MicroRNA Binding Domain of Argonaute 2 improves the Retinoic Acid Differentiation Response of the Acute Promyelocytic Leukemia Cell Line NB4

Argonaute proteins are pivotal regulators of gene expression mediating miRNAs function. Modulating their activity would be extremely useful to elucidate the processes governing small-RNAs-guided gene silencing. We report the identification of a chemical compound able to compete with Argonaute 2 miRNAs binding, and we demonstrate that this functional inhibition determines effects similar to Argonaute 2 shRNA-mediated down-regulation, favoring granulocytic differentiation of the acute promyelocytic leukemia cell line NB4 in response to retinoic acid

Effect of Small Molecules Modulating Androgen Receptor (SARMs) in Human Prostate Cancer Models

<div><p>The management of hormone-refractory prostate cancer represents a major challenge in the therapy of this tumor, and identification of novel androgen receptor antagonists is needed to render treatment more effective. We analyzed the activity of two novel androgen receptor antagonists, (<i>S</i>)-<b>11</b> and (<i>R</i>)-<b>9</b>, in <i>in vitro</i> and <i>in vivo</i> experimental models of hormone-sensitive or castration-resistant prostate cancer (CRPC). <i>In vitro</i> experiments were performed on LNCaP, LNCaP-AR, LNCaP-Rbic and VCaP human prostate cancer cells. Cytotoxic activity was assessed by SRB and BrdU uptake, AR transactivation by luciferase reporter assay and PSA levels by Real Time RT-PCR and ELISA assays. Cell cycle progression-related markers were evaluated by western blot. <i>In vivo</i> experiments were performed on SCID mice xenografted with cells with different sensitivity to hormonal treatment. In hormone-sensitive LNCaP and LNCaP-AR cells, the latter expressing high androgen receptor levels, (<i>R</i>)-<b>9</b> and (<i>S</i>)-<b>11</b> exhibited a higher cytotoxic effect compared to that of the reference compound ((<i>R</i>)-bicalutamide), also in the presence of the synthetic androgen R1881. Furthermore, the cytotoxic effect produced by (<i>R</i>)-<b>9</b> was higher than that of (<i>S</i>)-<b>11</b> in the two hormone-resistant LNCaP-AR and VCaP cells. A significant reduction in PSA levels was observed after exposure to both molecules. Moreover, (<i>S</i>)-<b>11</b> and (<i>R</i>)-<b>9</b> inhibited DNA synthesis by blocking the androgen-induced increase in cyclin D1 protein levels. <i>In vivo</i> studies on the toxicological profile of (<i>R</i>)-<b>9</b> did not reveal the presence of adverse events. Furthermore, (<i>R</i>)-<b>9</b> inhibited tumor growth in various <i>in vivo</i> models, especially LNCaP-Rbic xenografts, representative of recurrent disease. Our <i>in vitro</i> results highlight the antitumor activity of the two novel molecules (<i>R</i>)-<b>9</b> and (<i>S</i>)-<b>11</b>, making them a potentially attractive option for the treatment of CRPC.</p></div

Structure compounds and their AR-specific binding properties.

<p>(<b>A</b>) Chemical structure and molecular weight (m.w.) of (<i>R</i>)-bicalutamide, (<i>S</i>)-11 and (<i>R</i>)-9. (<b>B</b>) AR ligand binding displacement analysis in LNCaP cells<b>.</b> Quiescent LNCaP cells were incubated with 10 nM [3H] R1881 in the absence or presence of the indicated excess (from 0.5 µM to 4 µM) of radio inert compounds. Intracellular radioactivity was assayed. Inset in panel B shows the AR binding sites assayed in 10³ cells incubated with 10 nM of [3H] R1881 (R1881*) in the absence or presence of the indicated excess of unlabeled (<i>R</i>)-<b>9</b>, (<i>S</i>)-<b>11</b> or (<i>R</i>)-bicalutamide (<i>R</i>)-bic. Data from three different experiments were collected and residual binding was calculated and expressed as % of total AR binding sites. <i>n</i> =  number of experiments. The statistical significance of results was also evaluated by the paired <i>t</i> test and P values <0.005 were considered significant. No significance was attributed to the difference in the residual binding between the cells incubated with 10 nM [3H] R1881 in the presence of (<i>S</i>)-11 or (<i>R</i>)-9 and those incubated with 10 nM [3H] R1881 in the presence of (<i>R</i>)-bicalutamide ((<i>R</i>)-bic). LNCaP cells were also incubated with 10 nM [3H] R1881 in the absence or presence of 100-fold excess (1 µM) of unlabeled R1881 or Casodex®. Residual binding was 13% and 14% for unlabeled R1881 or Casodex®, respectively.</p

Effect of (R)-bicalutamide, (<i>R</i>)-9 and (<i>S</i>)-11 on BrdU incorporation induced by R1881 in LNCaP cells.

<p>Quiescent LNCaP cells on coverslips were left untreated (control) or treated for 18 hours with the synthetic androgen R1881 (10 nM), in the absence or presence of the indicated antagonists (used at 10 µM or 20 µM). After <i>in vivo</i> pulsing with 100 µM BrdU, BrdU incorporation was analyzed by immunofluorescence and expressed as % of total nuclei. The numbers represent the mean of three independent experiments, with standard deviation <1.4. The statistical significance of results was also evaluated by the paired <i>t</i> test. No significance was attributed to the difference in BrdU incorporation between the control cells and cells stimulated with 10 nM R1881 in the presence of (<i>R)</i>-bicalutamide (<i>R</i>)-bic or (<i>S</i>)-<b>11</b> or (<i>R</i>)-<b>9</b>.</p

Cytotoxic activity <i>in vitro</i>.

<p>Cytotoxic activity of (<i>R</i>)-bicalutamide, (<i>S</i>)-<b>11</b>and (<i>R</i>)-<b>9</b> in human prostate cancer cell lines LNCaP, LNCaP-Rbic, LNCaP-AR and VCaP after a 144-hour exposure, measured by SRB assay (average of three independent experiments). The concentrations (µM) of (<i>R</i>)-bicalutamide, (<i>S</i>)-<b>11</b> and (<i>R</i>)-<b>9</b> causing 50% decrease in cell survival (IC₅₀) are shown to the right of the curves.</p

Interference of the anti-androgens on the effect of R1881 in naïve LNCaP and derivative LNCaP-AR lines.

<p>(<b>A</b>) Evaluation by SRB assay of the antitumor acitivity of scalar concentrations of (<i>R</i>)-bicalutamide, (<i>S</i>)-<b>11</b> or (<i>R</i>)-<b>9</b> in hormone-responsive LNCaP and AR-overexpressing LNCaP-AR cells, in the presence or not of the synthetic androgen, R1881 (10 nM). Bars represent the mean of two independent experiments<b>.</b> (<b>B</b>)<b>,</b> (<b>C</b>) Evaluation of mRNA levels of PSA gene after a 24-hour exposure to different concentration of (<i>S</i>)-<b>11</b> (C) or (<i>R</i>)-<b><i>9</i></b> (D) in the presence or absence of R1881 (points are mean of two independent experiments, *<i>P</i>>0.05).</p

(<i>S</i>)-11- and (<i>R</i>)-9-induced inhibition of G1/S progression in androgen-treated LNCaP cells.

<p>In <b>A and B</b>, quiescent LNCaP cells on coverslips were left untreated (control) or treated for 18 hours with the synthetic androgen R1881 (10 nM) in the absence or presence of the indicated antagonists (used at 10 or 20 µM). After <i>in vivo</i> pulsing with 100 µM BrdU, BrdU incorporation was analyzed by immunofluorescence and expressed as % of total nuclei. In <b>A</b> and <b>B</b>, the numbers at the top of each bar represent the mean of three independent experiments (<i>n = 3</i>), with standard deviation (SD) <1. The statistical significance of results in <b>A</b> and <b>B</b> was assessed with the paired <i>t</i> test. <i>P</i> values were <0.005 for cells stimulated with 10 nM R1881. No significance was attributed to the difference in BrdU incorporation between control cells and cells stimulated with 10 nM R1881 in the presence of Casodex®, (<i>S</i>)-<b>11</b> (<b>A</b>) or (<i>R</i>)-<b>9</b> (<b>B</b>). In <b>C</b> and <b>D,</b> quiescent LNCaP cells were left untreated or were treated for the indicated times with 10 nM R1881, in the absence or presence of 10 µM of the indicated antagonists (Casodex®, Cx; (<i>S</i>)-<b>11</b> or (<i>R</i>)-<b>9</b>). Lysate proteins were analyzed by Western blot using antibodies directed against cyclin D1 (Cyc D1) in <b>C,</b> or p27 in <b>D</b>. Filters were stripped and re-probed using the rabbit polyclonal anti CDK-4 antibody as a loading control. Western blots in <b>C</b> and <b>D</b> are representative of two different experiments. In <b>C</b>, a 3.3-fold increase in the levels of 4-hr hormone-induced Cyc D1 expression was detected using the NIH ImageJ program. Casodex, (<i>S</i>)-<b>11</b> and (<i>R</i>)-<b>9</b> inhibited this increase (16% 80% and 66% for Casodex®, for S-11 and R-9, respectively) to varying degrees.</p

A New Avenue toward Androgen Receptor Pan-antagonists: C2 Sterically Hindered Substitution of Hydroxy-propanamides

The androgen receptor (AR) represents the primary target for prostate cancer (PC) treatment even when the disease progresses toward androgen-independent (AIPC) or castration-resistant (CRPC) forms. Because small chemical changes in the structure of nonsteroidal AR ligands determine the pharmacological responses of AR, we developed a novel stereoselective synthetic strategy that allows sterically hindered C2-substituted bicalutamide analogues to be obtained. Biological and theoretical evaluations demonstrate that C2-substitution with benzyl and phenyl moieties is a new, valuable option toward improving pan-antagonist behavior. Among the synthesized compounds, (<i>R</i>)-<b>16m</b>, when compared to casodex, (<i>R</i>)-bicalutamide, and enzalutamide, displayed very promising in vitro activity toward five different prostate cancer cell lines, all representative of CPRC and AIPC typical mutations. Despite being less active than (<i>R</i>)-bicalutamide, (<i>R</i>)-<b>16m</b> also displayed marked in vivo antitumor activity on VCaP xenografts and thus it may serve as starting point for developing novel AR pan-antagonists