Androstane derivatives induce apoptotic death in MDA-MB-231 breast cancer cells.

Biological investigation was conducted to study in vitro antiproliferative and pro-apoptotic potential of selected 17α-picolyl and 17(E)-picolinylidene androstane derivatives. The antiproliferative impact was examined on six human tumor cell lines, including two types of breast (MCF-7 and MDA-MB-231), prostate (PC3), cervical (HeLa), colon (HT 29) and lung cancer (A549), as well as one normal fetal lung fibroblasts cell line (MRC-5). All derivatives selectively decreased proliferation of estrogen receptor negative MDA-MB-231 breast cancer cells after 48 h and 72 h treatment and compounds showed time-dependent activity. We used this cell line to investigate cell cycle modulation and apoptotic cell death induction by flow cytometry, expression of apoptotic proteins by Western blot and apoptotic morphology by visual observation. Tested androstane derivatives affected the cell cycle distribution and induced apoptosis and necrosis. Compounds had different and specific mode of action, depending on derivative type and exposure time. Some compounds induced significant apoptosis measured by Annexin V test compared to reference compound formestane. Higher expression of pro-apoptotic BAX, downregulation of anti-apoptotic Bcl-2 and cleavage of PARP protein were confirmed in almost all treated samples, but the lack of caspase-3 activation suggested the induction of apoptosis in caspase-independent manner. More cells with apoptotic morphology were observed in samples after prolonged treatment. Structure-activity relationship analysis was performed to find correlations between the structure variations of investigated derivatives and observed biological effects. Results of this study showed that some of the investigated androstane derivatives have good biomedical potential and could be candidates for anticancer drug development.

Synthesis, structural analysis and antitumor activity of novel 17α-picolyl and 17(E)-picolinylidene A-modified androstane derivatives.

The heterocyclic ring at C-17 position of the androstane compounds plays an important role in biological activity. The aim of the present study was to synthesize and evaluate potential antitumor activity of different A-modified 17α-picolyl and 17(E)-picolinylidene androstane derivatives. In several synthetic steps, novel derivatives bearing the hydroximino, nitrile or lactame functions in A-ring were synthesized and characterized according to the spectral data, by mass analysis as well as XRD analysis (compounds 6, 13 and 15). The structurally most promising compounds 6, 11-17 were investigated as antitumor agents. The in vitro antiproliferative activity was evaluated against six human cancer cell lines: estrogen receptor negative (ER-) breast adenocarcinoma (MDA-MB-231); estrogen receptor positive (ER+) breast adenocarcinoma (MCF-7); prostate cancer (PC-3); human cervical carcinoma (HeLa); lung adenocarcinoma (A549) and colon adenocarcinoma (HT-29) using MTT assay. The results of the 48h incubation time in vitro tests showed that compound 15 was the most effective against PC-3 (IC50 6.6µM), compound 17 against MCF-7 (IC50 7.9µM) cells, while compound 16 exhibited strong antiproliferative effect against both, MCF-7 (IC50 1.7µM) and PC-3 (IC50 8.7µM) cancer cells. It was also found that compounds 16 and 17 induced apoptosis in MCF-7 cells (dicyano derivative 17 stronger then dioxime 16 and reference formestane), with no distinct changes in the cell cycle of MCF-7 cells.

In silico ADMET analysis of the A-, B- and D-modified androstane derivatives with potential anticancer effects.

The major challenge in the fight against cancer is to design new drugs that will be more selective for cancer cells, with fewer side effects. Synthetic steroids such as cyproterone, fulvestrant, exemestane and abiraterone are approved powerful drugs for the treatment of hormone-dependent diseases such as breast and prostate cancers. Therefore, androstane derivatives in 17-substituted, 17a-homo lactone and 16,17-seco series, with potent anticancer activity, were selected for pharmacokinetic and druglike predictions from the absorption, distribution, metabolism and excretion (ADME) models. In silico determination of physico-chemical and ADMET properties was performed using SwissADME and ProTox-II web tools. The possibility of gastrointestinal absorption and brain penetration was analyzed using the BOILED-Egg model, while the in silico evaluation of the similarities between selected steroid derivatives and FDA-approved drugs was carried out using the SwissSimilarity tool. Of all tested, two compounds that showed good in silico ADMET results, in addition to promising cytotoxicity and molecular docking results, could potentially be evaluated in in vivo tests.

Antitumor potential of novel 5α,6ß-dibromo steroidal D-homo lactone.

New steroidal D-homo androstane derivative, 5α,6ß-dibromo-3ß-hydroxy-17-oxa-17a-homoandrostan-16-one was synthesized and its structure was confirmed by NMR spectroscopy. In silico ADME properties of this compound were assessed using the SwissADME online prediction tool. Six human cancer cell lines (MDA-MB-231, MCF-7, PC3, HT-29, HeLa, and A549) and one human noncancerous cell line (MRC-5) were used for in vitro cytotoxicity testing. Novel steroidal dibromide was also tested for relative binding affinity for the ligand binding domain of estrogen receptor α and ß or the androgen receptor using a published assay in yeast cells. Ligand binding domains of each steroid receptor were expressed in-frame with yellow fluorescent protein in yeast and the fluorescence intensity changes upon addition of test compound was measured. The new compound showed selective cytotoxic activity against HT-29 (colon adenocarcinoma) and A549 (lung adenocarcinoma) cell lines, as well as the potential to induce apoptosis in HT-29 cells, while results obtained from ligand binding assay in yeast suggested a lack of significant estrogenic or androgenic properties.

New steroidal oxazolines, benzoxazoles and benzimidazoles related to abiraterone and galeterone.

Seven new oxazoline, benzoxazole and benzimidazole derivatives were synthesized from 3ß-acetoxyandrosta-5,16-dien-17-carboxylic, 3ß-acetoxyandrost-5-en-17ß-carboxylic and 3ß-acetoxypregn-5-en-21-oic acids. Docking to active site of human 17α-hydroxylase/17,20-lyase revealed that all oxazolines, as well as benzoxazoles and benzimidazoles comprising Δ16 could form stable complexes with enzyme, in which steroid moiety is positioned similarly to that of abiraterone and galeterone, and nitrogen atom coordinates heme iron, while 16,17-saturated benzoxazoles and benzimidazoles could only bind in a position where heterocycle is located nearly parallel to heme plane. Modeling of the interaction of new benzoxazole and benzimidazole derivatives with androgen receptor revealed the destabilization of helix 12, constituting activation function 2 (AF2) site, by mentioned compounds, similar to one induced by known antagonist galeterone. The synthesized compounds inhibited growth of prostate carcinoma LNCaP and PC-3 cells at 96 h incubation; the potency of 2'-(3ß-hydroxyandrosta-5,16-dien-17-yl)-4',5'-dihydro-1',3'-oxazole and 2'-(3ß-hydroxyandrosta-5,16-dien-17-yl)-benzimidazole was superior and could inspire further investigations of these compounds as potential anti-cancer agents.

Comprehensive QSRR modeling as a starting point in characterization and further development of anticancer drugs based on 17α-picolyl and 17(E)-picolinylidene androstane structures.

The selection of the most promising anticancer compounds from the pool of the huge number of synthesized molecules is a quite complex task. There are many compounds characterization approaches which can suggest the best structural features of a molecule with the highest antiproliferative effect on the certain type of cancer cell lines. One of these approaches is the lipophilicity determination of compounds and the analysis of its correlation with the anticancer activity. Since the importance of the lipophilicity is underlined in many earlier studies, this study is focused on determination of lipophilicity of previously synthesized 17α-picolyl and 17(E)-picolinylidene androstane derivatives by using reversed-phase high performance liquid chromatography (RP-HPLC) as a very fast, effective and relatively cheap method. Determination of the chromatographic lipophilicity of the studied androstanes can be considered as the part of their physicochemical characterization, which is a very important step in their further selection as drug candidates. The present study does not neglect the in silico approach. The determined chromatographic lipophilicity was analyzed by quantitative structure-retention relationship (QSRR) approach in order to reveal which molecular characteristics contribute mostly to the typical behavior of the androstanes in the applied chromatographic system, and thus to their lipophilicity. Classical statistical approach and Sum of Ranking Differences method were used for selection of the best QSRR models which should be used in prediction of chromatographic lipophilicity of studied androstane derivatives.

Preselection of A- and B- modified d-homo lactone and d-seco androstane derivatives as potent compounds with antiproliferative activity against breast and prostate cancer cells - QSAR approach and molecular docking analysis.

The problem with trial-and-error approach in organic synthesis of targeted anticancer compounds can be successfully avoided by computational modeling of molecules, docking studies and chemometric tools. It has been proven that A- and B- modified d-homo lactone and d-seco androstane derivatives are compounds with significant antiproliferative activity against estrogen-independent breast adenocarcinoma (ER-, MDA-MB-231) and androgen-independent prostate cancer cells (AR-, PC-3). This paper presents the quantitative structure-activity relationship (QSAR) models based on artificial neural networks (ANNs) which are able to predict whether d-homo lactone and/or d-seco androstane-based compounds will express antiproliferative activity against breast cancer cells (MDA-MB-231) or not. Also, the present paper describes the molecular docking study of 3ß-acetoxy-5α,6α-epoxy- (3) and 6α,7α-epoxy-1,4-dien-3-one (24) d-homo lactone androstane derivatives, as well as 4-en-3-one (15) d-seco androstane derivative, which are compounds with strong or moderate antiproliferative activity against prostate cancer cells (PC-3), and compares them with commercially available medicament for prostate cancer - abiraterone. The obtained promising results can be used as guidelines in further syntheses of novel d-homo lactone and d-seco androstane derivatives with antiproliferative activity against breast and prostate cancer cells.

Non-linear assessment of anticancer activity of 17-picolyl and 17-picolinylidene androstane derivatives--chemometric guidelines for further syntheses.

The present paper deals with prediction of cytotoxic activity of 17-picolyl and 17-picolinylidene androstane derivatives toward androgen receptor negative prostate cancer cell line (PC-3). The prediction was achieved applying artificial neural networks (ANNs) method on the basis of molecular descriptors. The most important descriptors (skin permeability (SP), Madin-Darby canine kidney cell permeability (MDCK) and universal salt solubility factor (S+SF)) were selected by using stepwise selection coupled with partial least squares method. The ANN modelling was carried out in order to obtain reliable models which can facilitate further synthesis of androstane derivatives with high antiproliferative activity toward PC-3 cell line. The modelling procedure resulted in three ANN models with the best statistical performance. The obtained results show that the established ANN models can be applied for required purpose.