Synthesis, in vitro and in silico anticancer evaluation of novel pyridin-2-yl estra-1,3,5(10)-triene derivatives.

Aim: The aim of this study was the synthesis of steroid compounds with heterocyclic rings and good anticancer properties. Materials & methods: The synthesis, in silico and in vitro anticancer testing of novel pyridin-2-yl estra-1,3,5(10)-triene derivatives was performed. Results: All synthesized compounds have shown promising results for, antiproliferative activity, relative binding affinities for the ligand binding domains of estrogen receptors α, ß and androgen receptor, aromatase binding potential, and inhibition of AKR1C3 enzyme. Conclusion: 3-Benzyloxy (17E)-pycolinilidene derivative 9 showed the best antitumor potential against MDA-MB-231 cell line, an activity that can be explained by its moderate inhibition of AKR1C3. Molecular docking simulation indicates that it binds to AKR1C3 in a very similar orientation and geometry as steroidal inhibitor EM1404.

Synthesis and biological activity of thiophene bioisosteres of natural styryl lactone goniofufurone and related compounds.

Ten new thiophene derivatives related to goniofufurone have been obtained by multistep synthesis starting from d-glucose. The critical step of the synthesis was the Grignard reaction of 2-thienyl magnesium bromide with a protected dialdose, yielding the C-5 epimeric thiophene derivatives 9 and 10. The mixture was oxidized to the 5-keto derivative 11, which after deprotection was converted to the corresponding keto-lactone 14. Stereoselective reduction of 14 afforded the thiophene mimic of goniofufurone 3. Esterification of 3 with cinnamic or 4-fluorocinnamic acid gave hybrids 5-7. Synthesized analogues were evaluated for their in vitro cytotoxicity against several tumour cell lines. The vast majority of them showed better activity than lead 1. In the culture of K562 cells, compound 3 was more active than the commercial antitumour drug doxorubicin. Structural features of analogues important for their antiproliferative activities were identified by SAR analysis. Pro-apoptotic potential examination of compound 3 on the K562 cell line was performed using flow cytometry, double fluorescence staining and apoptotic morphology screening. Results show that this derivative induces cell membrane disruptions attributable to apoptosis and induces the apoptotic morphology, but decreasing simultaneously the population of cells in the subG1 phase of the cell cycle. The results further suggest that analogue 3 achieves strong cytotoxicity without causing DNA fragmentation. This is clearly indicated by the relatively low incidence of micronuclei, as well as the SAR analysis of all biological effects.

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.

Synthesis of new bile acid-fused tetrazoles using the Schmidt reaction.

A practical and high-yielding Schmidt reaction for the synthesis of fused tetrazoles from bile acid precursors was developed. Mild reaction conditions using TMSN3 instead of hydrazoic acid as an azide source produced good yields of the desired tetrazoles. These conditions could be applied to other steroidal precursors. Additionally, an improved methodology for the synthesis of different ketone and enone precursors from cholic acid, deoxycholic acid, and chenodeoxycholic acid was established. Newly obtained tetrazole derivatives were characterized by NMR and X-ray diffraction spectroscopy. In a number of cases, preliminary antiproliferative tests of new compounds showed strong and selective activity towards certain tumor cell lines.

1,2,3,4-Tetrahydroisoquinoline Derivatives as a Novel Deoxyribonuclease I Inhibitors.

Herein we report an assessment of 24 1,2,3,4-tetrahydroisoquinoline derivatives for potential DNase I (deoxyribonuclease I) inhibitory properties in vitro. Four of them inhibited DNase I with IC50 values below 200 µM. The most potent was 1-(6,7-dimethoxy-1,2,3,4-tetrahydroisoquinolin-1-yl)propan-2-one (2) (IC50 =134.35±11.38 µM) exhibiting slightly better IC50 value compared to three other active compounds, 2-[2-(4-fluorophenyl)-1,2,3,4-tetrahydroisoquinolin-1-yl]-1-phenylethan-1-one (15) (IC50 =147.51±14.87 µM), 2-[2-(4-fluorophenyl)-1,2,3,4-tetrahydroisoquinolin-1-yl]cyclohexan-1-one (18) (IC50 =149.07±2.98 µM) and 2-[6,7-dimethoxy-2-(p-tolyl)-1,2,3,4-tetrahydroisoquinolin-1-yl]cyclohexan-1-one (22) (IC50 =148.31±2.96 µM). Cytotoxicity assessment of the active DNase I inhibitors revealed a lack of toxic effects on the healthy cell lines MRC-5. Molecular docking and molecular dynamics simulations suggest that interactions with Glu 39, His 134, Asn 170, Tyr 211, Asp 251 and His 252 are an important factor for inhibitors affinity toward the DNase I. Observed interactions would be beneficial for the discovery of new active 1,2,3,4-tetrahydroisoquinoline-based inhibitors of DNase I, but might also encourage researchers to further explore and utilize potential therapeutic application of DNase I inhibitors, based on a versatile role of DNase I during apoptotic cell death.

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

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.

Synthesis and anticancer potential of novel 5,6-oxygenated and/or halogenated steroidal d-homo lactones.

A series of 5,6-modified steroidal d-homo lactones, comprising of halogenated and/or oxygenated derivatives, was synthesized and evaluated for potential anticancer properties. Preparation of many of these compounds involved investigating alternative synthetic pathways. In silico ADME testing was performed for both novel and some previously synthesized compounds. Calculated physicochemical properties were in accordance with the Lipinski, Veber, Egan, Ghose and Muegge criteria, suggesting the potential of these molecules as orally active agents. Cytotoxicity of the synthesized steroid derivatives was tested on six tumor and one normal human cell line. None of the investigated derivatives was toxic to non-cancerous MRC-5 control cells. Most of the compounds showed significant cytotoxicity against the treated cancer cell lines. Most notably, the 3ß,5α,6ß-trihydroxy derivative exhibited strong cytotoxicity against multiple cell lines (MCF-7, MDA-MB-231 and HT-29), with the highest effect observed for lung adenocarcinoma (A549) cells, for which this steroid was more cytotoxic than all of the three commercial chemotherapeutic agents used as reference compounds. Molecular docking suggests the 3ß,5α,6ß-trihydroxy derivative could bind the EGFR tyrosine kinase domain with high affinity, providing a potential mechanism for its cytotoxicity via inhibition of EGFR signaling. The most active compounds were further studied for their potential to induce apoptosis by the double-staining fluorescence method; where the 5α,6ß-dibromide, 5α,6ß-dichloride and 3ß,5α,6ß-triol induced apoptotic changes in all three treated cell lines: MDA-MB-231, HT-29 and A549. To predict interactions with nuclear steroidal receptors, affinity for the ligand binding domains of ERα, ERß and AR was measured using a yeast-based fluorescence assay. The 5ß,6ß-epoxide, dibromide and 5α-hydroxy-3,6-dioxo derivatives showed affinity for ERα, while the 5α-fluoro-6ß-hydroxy and 3ß-acetoxy-5α,6ß-dihydroxy derivatives were identified as ERß ligands. None of the tested compounds showed affinity for AR. Structure-activity relationships of selected compounds were also examined.

Synthesis, characterization, HSA/DNA interactions and antitumor activity of new [Ru(η6-p-cymene)Cl2(L)] complexes.

Three new ruthenium(II) complexes were synthesized from different substituted isothiazole ligands 5-(methylamino)-3-pyrrolidine-1-ylisothiazole-4-carbonitrile (1), 5-(methylamino)-3-(4-methylpiperazine-1-yl)isothiazole-4-carbonitrile (2) and 5-(methylamino)-3-morpholine-4-ylisothiazole-4-carbonitrile (3): [Ru(η6-p-cymene)Cl2(L1)]·H2O (4), [Ru(η6-p-cymene)Cl2(L2)] (5) and [Ru(η6-p-cymene)Cl2(L3)] (6). All complexes were characterized by IR, UV-Vis, NMR spectroscopy, and elemental analysis. The molecular structures of all ligands and complexes 4 and 6 were determined by an X-ray. The results of the interactions of CT-DNA (calf thymus deoxyribonucleic acid) and HSA (human serum albumin) with ruthenium (II) complexes reveal that complex 4 binds well to CT-DNA and HSA. Kinetic and thermodynamic parameters for the reaction between complex and HSA confirmed the associative mode of interaction. The results of Quantum mechanics (QM) modelling and docking experiments toward DNA dodecamer and HSA support the strongest binding of the complex 4 to DNA major groove, as well as its binding to IIa domain of HSA with the lowest ΔG energy, which agrees with the solution studies. The modified GOLD docking results are indicative for Ru(p-cymene)LCl··(HSA··GLU292) binding and GOLD/MOPAC(QM) docking/modelling of DNA/Ligand (Ru(II)-N(7)dG7) covalent binding. The cytotoxic activity of compounds was evaluated by MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) assay. Neither of the tested compounds shows activity against a healthy MRC-5 cell line while the MCF-7 cell line is the most sensitive to all. Compounds 3, 4 and 5 were about two times more active than cisplatin, while the antiproliferative activity of 6 was almost the same as with cisplatin. Flow cytometry analysis showed the apoptotic death of the cells with a cell cycle arrest in the subG1 phase.

New A-homo lactam D-homo lactone androstane derivative: Synthesis and evaluation of cytotoxic and anti-inflammatory activities in vitro.

This paper describes the synthesis of a new A-homo lactam D-homo lactone androstane derivative from dehydroepiandrosterone. To evaluate the impact of the introduction of nitrogen in the parental scaffold on biological activity, a new androstane enamide-type lactam derivative was prepared and characterized. The new compound as well as starting compounds were screened for cytotoxic, anti-angiogenic and anti-inflammatory activities using several human cancer cell lines (MCF-7, MDA-MB-231, PC3, CEM, G-361, HeLa), endothelial (HUVEC) and non-tumour (MRC-5 and BJ) cell lines. Strong cytotoxic and anti-inflammatory activity with a broad therapeutical window was demonstrated by the A-homo lactam D-homo lactone androstane derivative. The induction of apoptosis in treated PC3 cultures was confirmed using apoptotic morphology screening and a fluorescent double-staining method. New A-homo lactam D-homo lactone androstane derivative induced apoptosis more than the tested reference compounds, Formestane and Doxorubicin. An in silico ADME analysis showed that the compounds possess drug-like properties.

Evaluation of A-ring fused pyridine d-modified androstane derivatives for antiproliferative and aldo-keto reductase 1C3 inhibitory activity.

New A-ring pyridine fused androstanes in 17a-homo-17-oxa (d-homo lactone), 17α-picolyl or 17(E)-picolinylidene series were synthesized and validated by X-ray crystallography, HRMS, IR and NMR spectroscopy. Novel compounds 3, 5, 8 and 12 were prepared by treatment of 4-en-3-one or 4-ene-3,6-dione d-modified androstane derivatives with propargylamine catalyzed by Cu(ii), and evaluated for potential anticancer activity in vitro using human cancer cell lines and recombinant targets of steroidal anti-cancer drugs. Pyridine fusion to position 3,4 of the A-ring may dramatically enhance affinity of 17α-picolyl compounds for CYP17 while conferring selective antiproliferative activity against PC-3 cells. Similarly, pyridine fusion to the A-ring of steroidal d-homo lactones led to identification of new inhibitors of aldo-keto reductase 1C3, an enzyme targeted in acute myeloid leukemia, breast and prostate cancers. One A-pyridine d-lactone steroid 5 also has selective submicromolar antiproliferative activity against HT-29 colon cancer cells. None of the new derivatives have affinity for estrogen or androgen receptors in a yeast screen, suggesting negligible estrogenicity and androgenicity. Combined, our results suggest that A-ring pyridine fusions have potential in modulating the anticancer activity of steroidal compounds.

Anticancer activity of novel steroidal 6-substituted 4-en-3-one D-seco dinitriles.

Steroidal 16,17-seco-16,17a-dinitriles possessing 4-ene-3,6-dione (3), 6-methylene-4-en-3-one (5), (6E)-hydroxyimino-4-en-3ß-ol (9) or (6E)-hydroxyimino-4-en-3-one (10) moiety were synthesized starting from 3ß-acetoxy-16,17-secoandrost-4-ene-16,17a-dinitrile (1). Antiproliferative activity of the newly synthesized compounds, as well as previously synthesized 3-oxo-16,17-secoandrosta-1,4-diene-16,17a-dinitrile (VII), was tested in vitro. Compound 9 displayed submicromolar antiproliferative activity against human cervical carcinoma (HeLa) cells (IC50 0.48 µM), and compounds 3 and 10 expressed strong inhibitory potential against HeLa cells (IC50 4.31 µM and 2.64 µM, respectively). Also, compound 10 was effective in inhibiting estrogen hormone-independent (MDA-MB-231) cells (IC50 2.78 µM). All tested compounds had no influence on the proliferation of healthy cells (MRC-5). Since MDA-MB-231 breast cancer cells and HeLa cervical cancer cells were most sensitive to treatment by 16,17-seco-16,17a-dinitriles, apoptosis induction after treatment by compounds 3, VII, 9 and 10 was studied in these cells, to reveal the mechanism underlying cell growth inhibition. All tested compounds significantly induced apoptosis in both treated cell lines, which was evident from results obtained by a double AO-EB staining test and quantified by counting cells with apoptotic morphology after staining with Giemsa dye. Among all tested substances, (6E)-hydroxyimino-4-en-3-one derivative 10 expressed the most proapoptotic activity.

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.

Synthesis and anticancer cell potential of steroidal 16,17-seco-16,17a-dinitriles: identification of a selective inhibitor of hormone-independent breast cancer cells.

We report the synthesis of steroidal 16,17-seco-16,17a-dinitriles and investigate their antitumor cell properties. Compounds were evaluated for anticancer potential by in vitro antiproliferation studies, molecular docking and virtual screening. Several compounds inhibit the growth of breast and prostate cancer cell lines (MCF-7, MDA-MB-231 and PC3), and/or cervical cancer cells (HeLa). Supporting this, molecular docking predicts that steroidal 16,17-seco-16,17a-dinitriles could bind with high affinity to multiple molecular targets of breast and prostate cancer treatment (aromatase, estrogen receptor α, androgen receptor and 17α-hydroxylase) facilitated by D-seco flexibility and nitrile-mediated contacts. Thus, 16,17-seco-16,17a-dinitriles may be useful for the design of inhibitors of multiple steroidogenesis pathways. Strikingly, 10, a 1,4-dien-3-on derivative, displayed selective submicromolar antiproliferative activity against hormone-dependent (MCF-7) and -independent (MDA-MB-231) breast cancer cells (IC50 0.52, 0.11µM, respectively). Ligand-based 3D similarity searches suggest AKR1C, 17ß-HSD and/or 3ß-HSD subfamilies as responsible for this antiproliferative activity, while fast molecular docking identified AKR1C and ERß as potential binders-both targets in the treatment of hormone-independent breast cancers.

17(E)-picolinylidene androstane derivatives as potential inhibitors of prostate cancer cell growth: antiproliferative activity and molecular docking studies.

We report a rapid and efficient synthesis of A-ring modified 17α-picolyl and 17(E)-picolinylidene androstane derivatives from dehydroepiandrosterone. Compounds were validated spectroscopically and structurally characterized by X-ray crystallography. Virtual screening by molecular docking against clinical targets of steroidal anticancer drugs (ERα, AR, Aromatase and CYP17A1) suggests that 17(E)-picolinylidene, but not 17α-picolyl androstanes could specifically interact with CYP17A1 (17α-hydroxylase) with similar geometry and affinity as Abiraterone, a 17-pyridinyl androstane drug clinically used in the treatment of prostate cancer. In addition, several 17(E)-picolinylidene androstanes demonstrated selective antiproliferative activity against PC3 prostate cancer cells, which correlates with Abiraterone antiproliferative activity and predicted CYP17A1 binding affinities. Based on these preliminary results, 17(E)-picolinylidene androstane derivatives could be a promising starting point for the development of new compounds for the treatment of prostate cancer.