Novel alkylaminoethyl derivatives of androstane 3-oximes as anticancer candidates: synthesis and evaluation of cytotoxic effects

Abstract

Steroid anticancer drugs are the focus of numerous scientific research efforts. Due to their high cytotoxic effects against tumor cells, some natural or synthetic steroid compounds seem to be promising for the treatment of different classes of cancer. In the present study, fourteen novel O-alkylated oxyimino androst-4-ene derivatives were synthesized from isomerically pure 3E-oximes, using different alkylaminoethyl chlorides. Their in vitro cytotoxic activity was evaluated against eight human cancer cell lines, as well as against normal fetal lung (MRC-5) and human foreskin (BJ) fibroblasts, to test the efficiency and selectivity of the compounds. Most derivatives displayed strong activity against malignant melanoma (G-361), lung adenocarcinoma (A549) and colon adenocarcinoma (HT-29) cell lines. Angiogenesis was assessed in vitro using migration scratch and tube formation assays on HUVEC cells, where partial inhibition of endothelial cell migration was observed for the 17α-(pyridin-2-yl)methyl 2-(morpholin-4-yl)ethyl derivative. Among the compounds that most impaired the growth of lung cancer A549 cells, the (17E)-(pyridin-2-yl)methylidene derivative bearing a 2-(pyrrolidin-1-yl)ethyl substituent induced significant apoptosis in these cells. In combination with low cytotoxicity toward normal MRC-5 cells, this molecule stands out as a good candidate for further anticancer studies. In addition, in vitro investigations against cytochrome P450 enzymes revealed that certain compounds can bind selectively in the active sites of human steroid hydroxylases CYP7, CYP17A1, CYP19A1 or CYP21A2, which could be important for the development of novel activity modulators of these enzymes and identification of possible side effects.

Fig. 1. Simplified scheme of biosynthesis of steroid hormones (a) and bile acids (b).

Scheme 1. Synthetic routes to compounds 2–10 and 12–20. Reagents and conditions: (a) NH₂OH·HCl, AcONa, 95% EtOH, reflux, 1.5 h; isomer separation by flash chromatography; (b) R–Cl·HCl, anh. KOH, anh. K₂CO₃, anh. butanone, reflux.

Fig. 2. Migration of HUVECs after 20 h of treatment with 8 (10 and 50 μM) compared with untreated control and positive control (2-methoxyestradiol). Experiments were repeated three times in triplicate.

Fig. 3. Results of apoptosis obtained by the double-fluorescent staining method. The chart shows the ratio of red and green channel density measured in ImageJ computer program for samples of untreated A549 cells (Ctrl) and samples treated 72 h with IC₅₀ concentrations of the selected compounds 12, 13 and 14. Images of stained cells are photographed from 6-well plates turned bottom up, using a fluorescence microscope (Olympus BX51) at 400× magnification.

Fig. 4. Fragment of active site of CYP17A1 in complex with compound 19 and abiraterone (PDB ID: 3RUK). In the stick and sphere representations, non-carbon atoms are indicated in blue (N) and red (O), Fe atom is indicated as an orange sphere. Molecules are colored by dark grey (heme), salmon (compound 19) and light blue (abiraterone). Surface and key residues, forming hydrophobic pocket are colored by forest green. Key residues, forming bonds with derivative 19, are colored cornflower blue.