Synthesis, characterization and biological evaluation of some novel P-heterocyclic androst-4-ene derivatives.

The reactions of 21-hydroxyprogesterone with Lawesson's reagent in toluene or [Formula: see text] gave four P-heterocyclic androst-4-ene derivatives (two tautomeric pairs): 4-(3-thioxoandrost-4-en-17[Formula: see text]-yl)-1,3,2-oxathiaphosphole-2- sulfide (2), 4-(3-thioxoandrost-4-en-17[Formula: see text]-ylidene)-1,3,2-oxathiaphospholane-2-sulfide (3), 4-(3-oxoandrost-4-en-17[Formula: see text]-yl)-1,3,2-oxathiaphosphole-2-sulfide (4), and 4-(3-oxoandrost-4-en-17[Formula: see text]-ylidene)-1,3,2- oxathiaphospholane-2-sulfide (5). The structures of all novel 17-substituted steroids were elucidated from their analytic and spectral data (HRMS, IR, 1D NMR and 2D NMR-HSQC, HMBC, NOESY, COSY). The detailed NMR analysis for all compounds revealed the presence of two pairs of signals in approx. 8:2 ratio indicating the existence of two diastereoisomers (a and b) with different configurations at the phosphorus atom. A parallel analysis of heteronuclear 2D [Formula: see text]-[Formula: see text] spectra (HSQC and HMBC) and homonuclear 2D spectra (NOESY and COSY) enabled complete [Formula: see text] and [Formula: see text] assignments of each isomer and provided evidence for the preferred configuration on phosphorus atom. Cytotoxic activity in vitro was tested against four tumor cell lines (human cervix carcinoma HeLa cells, chronic myelogenous leukemia K-562 and two human breast carcinoma MDA-MB-361 and MDA-MB-453 cells). Compounds 3a,b and 4a,b showed a poor activity against HeLa and MDA-MB-453 cell lines, while against MDA-MB-361 cell line, all tested compounds exerted very weak cytotoxic effect. All compounds exerted moderate activity against K562 cells. Antimicrobial activity against Gram-positive, Gram-negative bacteria and fungal cells, and toxicity to brine shrimp Artemia salina were evaluated. All tested compounds showed strong antifungal activity.

Synthesis and preliminary screening for the biological activity of some steroidal Δ4-unsaturated semicarbazone derivatives.

Eleven new steroidal mono- and bis(semicarbazones) 2a-e, 4d and 3a-e have been prepared starting from various 3-oxo-α,ß-unsaturated steroids. Mono-semicarbazones 2a-e were further subjected to ethyl chloroacetate in boiling absolute ethanol but, instead of expected intramolecular cyclocondensation reaction products, the new carbazate esters 5a-e were obtained. The structures of all synthesized compounds and identification of each E/Z isomer were deduced by elemental analysis, HRMS, NMR, and IR spectroscopy. Preliminary screening for the cytotoxic activity in vitro of the new compounds has been conducted against three cancer cell lines, K562, Jurkat and HeLa cells. HeLa cells were the most sensitive while K562 cells were the least sensitive to the cytotoxic action of the novel steroid derivatives. Compounds 2e, 3c and 5e were found to have the best but still moderate cytotoxic effects. All tested compounds showed very weak antimicrobial activities. These results demonstrate that the replacement of thioxo group with carbonyl group in steroidal hydrazone derivatives resulted in decrease in their biological activity.

Synthesis and biological evaluation of some 17-picolyl and 17-picolinylidene androst-5-ene derivatives.

Starting from dehydroepiandrosterone (1) 17-picolyl (2), 17-picolinylidene (7), 17-picolinylidene-16-one (10 and 11), and 17-picolyl-16-one (15) derivatives of androst-5-ene were synthesized in one, two, four and five steps respectively. By the Oppenauer oxidation or dehydration of 2, 7, 10, and 11 with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ), the corresponding A and B ring modified derivatives 3, 5, 6, 8, 9, and 12-14 were obtained. The structure of 2 was unambiguously proved by the appropriate X-ray structural analysis. Compounds 3, 5, 9, 12-14 showed inhibitory activity against the enzyme aromatase. Antibacterial activity, toxicity to brine shrimp Artemia salina, antitumor activity against three tumor cell lines (human cervix carcinoma HeLa cells, human melanoma FemX cells, and human myelogenous leukemia K562 cells) and toxicity against peripheral blood mononuclear cells were evaluated. Three tested compounds, namely 11, 13, and 15, showed strong activity against all three cell lines, the IC(50) values being in the range of 4-10 microM.

Anticancer potential of new steroidal thiazolidin-4-one derivatives. Mechanisms of cytotoxic action and effects on angiogenesis in vitro.

The synthesis and cytotoxic activities determination of new steroidal mono- and bis(thiazolidin-4-ones) 4a-f and 5a-f have been performed. Their anticancer action was also evaluated in comparison to previously synthesized and reported corresponding steroidal thiosemicarbazones. All compounds were obtained as stereoisomeric mixtures with different configuration (E or Z) in the hydrazone moiety at the C-3 position. After several consecutive crystallizations diastereomerically pure major (E)-isomers of mono-thiazolidin-4-ones were isolated. The structure and stereochemistry of 2,4-thiazolidinedione,2-[(17-oxoandrost-4-en-3-ylidene)hydrazone] were confirmed by X-ray analysis. A pathway for the formation of thiazolidin-4-one ring was proposed. The steroid thiazolidinone derivatives examined in this study exerted selective concentration-dependent cytotoxic activities on six tested malignant cell lines. Ten out of twelve examined compounds exhibited strong cytotoxic effects on K562 cells (IC50 values from 8.5µM to 14.9µM), eight on HeLa cells (IC50 values ranging from 8.9µM to 15.1µM) while against MDA-MB-361 cells six compouds exerted similar or even higher cytotoxic action (IC50 values from 12.7µM to 25.6µM) than cisplatin (21.5µM) which served as a positive control. Eight of these ten compounds showed high selectivity in the cytotoxic action against HeLa and K562 cancer cell lines when compared with normal human fibroblasts MRC-5 and normal human PBMC. The study of mechanisms of the anticancer activity of the two selected compounds, mono- and bis(thiazolidin-4-one) derivatives of 19-norandrost-4-ene-3,17-dione 4a and 5a, revealed that both of these compounds induced apoptosis in HeLa cells through extrinsic and intrinsic signalling pathways. Treatment of EA.hy926 cells with sub-toxic concentrations of these compounds led to the inhibition of cell connecting and sprouting, and tube formation. The synthesized compounds exhibited poor antioxidant activity.

Steroid dimers-in vitro cytotoxic and antimicrobial activities.

The in vitro cytotoxic activity of previously synthesized steroid dimers with different spacer group (sulfide, trithiolane ring or phosphorotrithioate) and the substituent at C-17 position was tested for their possible effects against following human tumor cell lines: cervical adenocarcinoma (HeLa), chronic myelogenous leukemia (K562) and two human breast cancer cell lines (MDA-MB-361 and MDA-MB-453). These compounds, applied at micromolar concentrations, exhibited cytotoxic activity of different intensity (compared with cisplatin as a control), modality and selectivity in these malignant cell lines. The best activity against all four cell cancer lines was exhibited by dimer-sulfides. All screened compounds exerted concentration-dependent cytotoxic activity against leukemia K562 cells. The compounds which exerted the most pronounced cytotoxic action exhibited notably higher cytotoxic activities against K562, HeLa and MDA-MB-453 cells in comparison to resting and PHA-stimulated PBMC, pointing to a significant selectivity in their antitumor actions. Examination of the mechanisms of cytotoxicity on leukemia K562 cells revealed pro-apoptotic action of each of the investigated compounds applied at concentrations 2IC50. The most prominent pro-apoptotic action was exhibited by dimer-sulfide of cholest-4-en-3-one. Furthermore, almost all of the tested compounds at IC50 concentrations induced G1 phase cell cycle arrest in K562 cells. Antimicrobial activity against Gram-positive, Gram-negative bacteria and fungal cells, and toxicity to brine shrimp Artemia salina, were evaluated. There was no antibacterial activity. The best antifungal activity was exhibited against Saccharomyces cerevisiae by dimers linked with trithiolane ring, indicating a selective activity of investigated compounds.

New androst-4-en-17-spiro-1,3,2-oxathiaphospholanes. Synthesis, assignment of absolute configuration and in vitro cytotoxic and antimicrobial activities.

The reactions of 17α-hydroxyprogesterone with Lawesson's reagent (LR) in toluene, CH(2)Cl(2) and/or CCl(4) gave, depending on the duration of the reaction, two diastereoisomeric androst-4-en-17-spiro-1,3,2-oxathiaphospholane-2-sulfide pairs 2a,b and 3a,b in approximately 7:3 ratio, differing in configuration at the phosphorus atom. A parallel analysis of heteronuclear 2D (1)H-(13)C spectra (HSQC and HMBC) and homonuclear 2D spectra (NOESY) enabled complete (1)H and (13)C assignments of each isomer. Also, analysis of NOESY correlations provided evidence for the preferred conformation. X-ray analysis of 3a confirmed the structure and absolute configuration on phosphorus. A pathway for the formation of 1,3,2-oxathiaphospholane ring was proposed. Cytotoxic activity in vitro was tested against three tumor cell lines (human cervix carcinoma HeLa cells and two human breast carcinoma MDA-MB-361 and MDA-MB-453 cells). Compound 3a and mixture 3a,b showed a moderate activity against HeLa and MDA-MB-453 cell lines while against MDA-MB-361 cell line all tested compounds exerted very weak cytotoxic effect. Antimicrobial activity against Gram-positive, Gram-negative bacteria and fungal cells, toxicity to brine shrimp Artemia salina, were evaluated. All tested compounds showed strong antifungal activity.