Halogen-substituted anthranilic acid derivatives provide a novel chemical platform for androgen receptor antagonists.

Androgen receptor (AR) antagonists are used for hormone therapy of prostate cancer (PCa). However resistance to the treatment occurs eventually. One possible reason is the occurrence of AR mutations that prevent inhibition of AR-mediated transactivation by antagonists. To offer in future more options to inhibit AR signaling, novel chemical lead structures for new AR antagonists would be beneficial. Here we analyzed structure-activity relationships of a battery of 36 non-steroidal structural variants of methyl anthranilate including 23 synthesized compounds. We identified structural requirements that lead to more potent AR antagonists. Specific compounds inhibit the transactivation of wild-type AR as well as AR mutants that render treatment resistance to hydroxyflutamide, bicalutamide and the second-generation AR antagonist enzalutamide. This suggests a distinct mode of inhibiting the AR compared to the clinically used compounds. Competition assays suggest binding of these compounds to the AR ligand binding domain and inhibit PCa cell proliferation. Moreover, active compounds induce cellular senescence despite inhibition of AR-mediated transactivation indicating a transactivation-independent AR-pathway. In line with this, fluorescence resonance after photobleaching (FRAP) - assays reveal higher mobility of the AR in the cell nuclei. Mechanistically, fluorescence resonance energy transfer (FRET) - assays indicate that the amino-carboxy (N/C)-interaction of the AR is not affected, which is in contrast to known AR-antagonists. This suggests a mechanistically novel mode of AR-antagonism. Together, these findings indicate the identification of a novel chemical platform as a new lead structure that extends the diversity of known AR antagonists and possesses a distinct mode of antagonizing AR-function.

Antiandrogenic activity of anthranilic acid ester derivatives as novel lead structures to inhibit prostate cancer cell proliferation.

A plant extract from the fruits of saw palmetto, which is currently used to treat the androgen-dependent benign prostatic hyperplasia and PCa, served as source for new structure variants. We investigated the antiandrogenic potential of an ethanolic total extract and one of its main aromatic components anthranilic acid. An androgen receptor-antagonistic (antiandrogenic) effect of the extract was evident, and although anthranilic acid itself revealed no remarkable effect, its methyl ester, methyl anthranilate, exhibited antiandrogenic potential. Based on this chemical structure, we synthesized and investigated the antiandrogenic activity of four AnA ester derivatives, which were either novel or only little described in literature. These AnA esters inhibit the androgen-dependent transactivation of both the wild-type (wt) androgen receptor and the androgen receptor point mutant T877A, which often occurs in refractory PCa. Moreover, they inhibit the androgen receptor-induced expression of the endogenous prostate-specific antigen. Importantly, AnA esters repress the growth of human PCa cells. Deletion analyses of androgen receptor propose that the antiandrogenic effect of anthranilic acid esters is mediated by the ligand-binding domain, most likely through direct binding, without affecting androgen receptor protein levels. Taken together, the data suggest antiandrogenic potential of anthranilic acid ester derivatives, which can serve as lead structures for novel antiandrogens.

NBBS isolated from Pygeum africanum bark exhibits androgen antagonistic activity, inhibits AR nuclear translocation and prostate cancer cell growth.

Extracts from Pygeum africanum are used in the treatment of prostatitis, benign prostatic hyperplasia (BPH) and prostate cancer (PCa). The ligand-activated human androgen receptor (AR) is known to control the growth of the prostate gland. Inhibition of human AR is therefore a major goal in treatment of patients. Here, we characterize the compound N-butylbenzene-sulfonamide (NBBS) isolated from P. africanum as a specific AR antagonist. This antihormonal activity inhibits AR- and progesterone receptor- (PR) mediated transactivation, but not the related human glucocorticoid receptor (GR) or the estrogen receptors (ERα or ERß). Importantly, NBBS inhibits both endogenous PSA expression and growth of human PCa cells. Mechanistically, NBBS binds to AR and inhibits its translocation to the cell nucleus. Furthermore, using a battery of chemically synthesized derivatives of NBBS we revealed important structural aspects for androgen antagonism and have identified more potent AR antagonistic compounds. Our data suggest that NBBS is one of the active compounds of P. africanum bark and may serve as a naturally occurring, novel therapeutic agent for treatment of prostatic diseases. Thus, NBBS and its derivatives may serve as novel chemical platform for treatment prostatitis, BPH and PCa.

Analysis of the hydrodistillate from the fruits of Serenoa repens.

The chemical composition of the hydrodistillate won by steam distillation from the fruits of Serenoa repens (W. Bartram), a well known phytomedicine against benign prostatic hyperplasia (BPH), was analyzed by GC-MS. It resulted in the identification of 144 steam-volatile components including about 100 structures which have not been described as constituents of the fruits from S. repens so far. The main component detected was lauric acid (40.4 %).

The natural compound atraric acid is an antagonist of the human androgen receptor inhibiting cellular invasiveness and prostate cancer cell growth.

Extracts from Pygeum africanum are used in the treatment of prostatitis, benign prostatic hyperplasia and prostate cancer (Pca), major health problems of men in Western countries. The ligand-activated human androgen receptor (AR) supports the growth of the prostate gland. Inhibition of human AR by androgen ablation therapy and by applying synthetic anti-androgens is therefore the primary goal in treatment of patients. Here, we show that atraric acid (AA) isolated from bark material of Pygeum africanum has anti-androgenic activity, inhibiting the transactivation mediated by the ligand-activated human AR. This androgen antagonistic activity is receptor specific and does not inhibit the closely related glucocorticoid or progesterone receptors. Mechanistically, AA inhibits nuclear transport of AR. Importantly, AA is able to efficiently repress the growth of both the androgen-dependent LNCaP and also the androgen-independent C4-2 Pca cells but not that of PC3 or CV1 cells lacking AR. In line with this, AA inhibits the expression of the endogenous prostate specific antigen gene in both LNCaP und C4-2 cells. Analyses of cell invasion revealed that AA inhibits the invasiveness of LNCaP cells through extracellular matrix. Thus, this study provides a molecular insight for AA as a natural anti-androgenic compound and may serve as a basis for AA derivatives as a new chemical lead structure for novel therapeutic compounds as AR antagonists, that can be used for prophylaxis or treatment of prostatic diseases.

Activity-guided isolation of an antiandrogenic compound of Pygeum africanum.

Inactivation of the androgen receptor (AR) through androgen ablation and treatment with antiandrogens is a major goal in the therapy for prostate hyperplasia and prostate cancer. Bioactivity-directed fractionation of a selective dichloromethane extract from the stem bark of Pygeum africanum led to the isolation of the antiandrogenic compound atraric acid. Its activity was examined by an androgen receptor responsive reporter gene assay. For lead structure optimization we transformed the natural occurring compound atraric acid into its ethyl, N-propyl and N-butyl esters and their antiandrogenic activities were examined as well. In addition, benzoic acid was isolated. The structures of all compounds were determined and characterized by means of 1H- and 13C-NMR, HR-EI-mass, IR and UV spectroscopy.

Extracts from Pygeum africanum and other ethnobotanical species with antiandrogenic activity.

Extracts from Pygeum africanum, Serenoa repens and Cucurbita pepo are used in the treatment of benign prostatic hyperplasia (BPH) and prostate cancer (PCa). The activity of the androgen receptor (AR) is known to control growth of the prostate. Here, we examined extracts of these plants for their antiandrogenic activity using an AR responsive reporter gene assay for drug discovery. A selective dichloromethane extract from the stem barks of Pygeum africanum revealed the highest antiandrogenic effect. Bioactivity-directed fractionation of this extract led to the isolation of N-butylbenzenesulfonamide (NBBS) indicating that extracts of the stem bark of P. africanum harbour androgen antagonistic activity. This compound may provide a novel approach for the prevention and treatment of BPH and human PCa.

Biotransformation of desoxypeganine by microsomal enzymes of the rabbit liver.

The biotransformation of the anticholinergic quinazoline alkaloid Desoxypeganine is studied by means of aerobic incubation with the non-induced supernatant obtained at 9000g from rabbit liver homogenates as enzyme source followed by an admixture of NADPH. The metabolites were identified by high-performance liquid chromatography, chemical ionisation mass spectrometry (LC-CI MS) and electron impact mass spectrometry (LC-EI MS) in comparison with synthetic reference compounds and typical ion fragments taken from literature data. C-oxidation of Desoxypeganine to the major metabolite Pegenone was observed as well as the hydroxylation of the alicyclic ring. The incubation mixture followed Michaelis-Menten kinetics characterised by K(m) = 5.8 x 10(-5) mol L(-1) and V(max) = 4.32 nmol Pegenone/min per mg protein or 3.37 nmol Pegenone/min per nmol CYP 450, respectively. These in vitro results demonstrate that the bioactive substance Desoxypeganine is easily oxidised to its ineffective metabolite Pegenone. This provokes a problem for correct dosage finding in formulations for the treatment of Alzheimer's disease and in the therapy of alcoholism and nicotine dependence.

Anhydronium bases from Rauvolfia serpentina.

Five anhydronium bases were isolated by preparative HPLC from a methanolic extract of Rauvolfia serpentina roots. For the first time 3,4,5,6-tetradehydroyohimbine, 3,4,5,6-tetradehydro-(Z)-geissoschizol, 3,4,5,6-tetradehydrogeissoschizol and 3,4,5,6-tetradehydrogeissoschizine-17-O-beta-D-glucopyranoside were isolated from a natural source. In addition, the well-known anhydronium base serpentine was isolated. The structures of the compounds were determined by 1H and 13C NMR, MS and UV.