Synthesis of Estrone Heterodimers and Evaluation of Their In Vitro Antiproliferative Activity.

Directed structural modifications of natural products offer excellent opportunities to develop selectively acting drug candidates. Natural product hybrids represent a particular compound group. The components of hybrids constructed from different molecular entities may result in synergic action with diminished side effects. Steroidal homo- or heterodimers deserve special attention owing to their potentially high anticancer effect. Inspired by our recently described antiproliferative core-modified estrone derivatives, here, we combined them into heterodimers via Cu(I)-catalyzed azide-alkyne cycloaddition reactions. The two trans-16-azido-3-(O-benzyl)-17-hydroxy-13α-estrone derivatives were reacted with 3-O-propargyl-D-secoestrone alcohol or oxime. The antiproliferative activities of the four newly synthesized dimers were evaluated against a panel of human adherent gynecological cancer cell lines (cervical: Hela, SiHa, C33A; breast: MCF-7, T47D, MDA-MB-231, MDA-MB-361; ovarian: A2780). One heterodimer (12) exerted substantial antiproliferative activity against all investigated cell lines in the submicromolar or low micromolar range. A pronounced proapoptotic effect was observed by fluorescent double staining and flow cytometry on three cervical cell lines. Additionally, cell cycle blockade in the G2/M phase was detected, which might be a consequence of the effect of the dimer on tubulin polymerization. Computational calculations on the taxoid binding site of tubulin revealed potential binding of both steroidal building blocks, mainly with hydrophobic interactions and water bridges.

Synthesis and Study of the Structure-Activity Relationship of Antiproliferative N-Substituted Isosteviol-Based 1,3-Aminoalcohols.

Starting from isosteviol, a series of diterpenoid 1,3-aminoalcohol derivatives were prepared via stereoselective transformations. The acid-catalysed hydrolysis and rearrangement of natural stevioside produced isosteviol, which was transformed into the key intermediate methyl ester. In the next step, an 1,3-aminoalcohol library was prepared by the reductive amination of the intermediate 3-hydroxyaldehyde obtained from isosteviol in a two-step synthesis. To study the effect of the carboxylate ester function at position 4, the free carboxylic acid, benzyl ester and acryloyl ester analogues were prepared as elongated derivatives in comparison with our earlier results in this field. The antiproliferative activity of compounds against human tumour cell lines (A2780, HeLa, MCF-7 and MDA-MB-231) was investigated. In our preliminary study, the 1,3-aminoalcohol function with N-benzyl or (1H-imidazol-1-yl)-propyl substitution and benzyl ester moiety seemed essential for the reliable antiproliferative activity. The results obtained could be a good starting point to further functionalisation towards more efficient antiproliferative diterpenes.

Isolation, Structure Determination of Sesquiterpenes from Neurolaena lobata and Their Antiproliferative, Cell Cycle Arrest-Inducing and Anti-Invasive Properties against Human Cervical Tumor Cells.

Seven new germacranolides (1-3, 5-8), among them a heterodimer (7), and known germacranolide (4), eudesmane (9) and isodaucane (10) sesquiterpenes were isolated from the aerial parts of Neurolaena lobata. Their structures were determined by using a combination of different spectroscopic methods, including HR-ESIMS and 1D and 2D NMR techniques supported by DFT-NMR calculations. The enantiomeric purity of the new compounds was investigated by chiral HPLC analysis, while their absolute configurations were determined by TDDFT-ECD and OR calculations. Due to the conformationally flexible macrocycles and difficulties in assigning the relative configuration, 13C and 1H NMR chemical shift and ECD and OR calculations were performed on several stereoisomers of two derivatives. The isolated compounds (1-10) were shown to have noteworthy antiproliferative activities against three human cervical tumor cell line with different HPV status (HeLa, SiHa and C33A). Additionally, lobatolide C (6) exhibited substantial antiproliferative properties, antimigratory effect, and it induced cell cycle disturbance in SiHa cells.

Cotinus coggygria Scop. induces cell cycle arrest, apoptosis, genotoxic effects, thermodynamic and epigenetic events in MCF7 breast cancer cells.

Current plant-derived anticancer therapeutics aim to reach higher effectiveness, to potentiate chemosensitivity and minimize the toxic side effects compared to conventional chemotherapy. Cotinus coggygria Scop. is a herb with high pharmacological potential, widely applied in traditional phytotherapy. Our previous study revealed that leaf aqueous ethanolic extract from C. coggygria exerts in vitro anticancer activity on human breast, ovarian and cervical cancer cell lines. The objective of the present research was to investigate possible molecular mechanisms and targets of the antitumor activity of the extract in breast cancer MCF7 cells through analysis of cell cycle and apoptosis, clonogenic ability assessment, evaluation of the extract genotoxic capacity, characterization of cells thermodynamic properties, and analysis on the expression of genes involved in cellular epigenetic processes. The obtained results indicated that in MCF7 cells C. coggygria extract causes S phase cell cycle arrest and triggers apoptosis, reduces colony formation, induces DNA damage, affects cellular thermodynamic parameters, and tends to inhibit the relative expression of DNMT1, DNMT3a, MBD3, and p300. Further studies on the targeted molecules and the extract anti-breast cancer potential on animal experimental model system, need to be performed in the future.

Isolation and Structure Determination of Antiproliferative Secondary Metabolites from the Potato Earthball Mushroom, Scleroderma bovista (Agaricomycetes).

Mycochemical examination of a methanol extract of Scleroderma bovista Fr. (Agaricomycetes) led to the isolation of 7 compounds, which were, to our knowledge, identified for the first time in this species. The chemical structures of these compounds were determined through extensive spectroscopic methods (nuclear magnetic resonance and mass spectrometry). The fungal metabolites were identified as steroids based on ergostane (compounds 1-4) and lanostane (compounds 6 and 7) skeletons, whereas compound 5 was a ceramide derivative. We evaluated the antiproliferative activity of compounds 4-7 against human cancer cell lines (HeLa, A2780, MDA-MB-231, and MCF-7) using the MTT assay. The lanostane-type derivatives (compounds 6 and 7) and ergosterol peroxide 3-glucoside (compound 4) exerted significant antiproliferative property on 1 or more human cancer cell lines.

Abietane diterpenoids from Sideritis montana L. and their antiproliferative activity.

The present study aimed at the phytochemical and pharmacological investigation of Sideritis montana L. (Lamiaceae). Two new abietane diterpenes [sideritins A (1) and B (2)] were isolated from the methanol extract of the plant. Six known compounds [pomiferin E (3), 9α,13α-epi-dioxyabiet-8(14)-en-18-ol (4), paulownin (5), 6-methoxysakuranetin (6), 3-oxo-α-ionol (7) and 4-allyl-2,6-dimethoxyphenol glucoside (8)] were also obtained from the plant. The structures were determined by means of HREIMS and NMR experiments. The antiproliferative effect of the isolated compounds was investigated on human cancer cell lines (HeLa, SiHa and C33A) at 10 and 30µM concentrations, using the MTT assay. The results demonstrated that pomiferin E (3) and 6-methoxysakuranetin (6) displayed considerable activity [inhibition (%)±SEM: 46.93±2.35 on HeLa (pomiferin E), and 51.52±2.45 on C33A (6-methoxysakuranetin)] at 30µM concentration.

Mechanism of antiproliferative action of a new d-secoestrone-triazole derivative in cervical cancer cells and its effect on cancer cell motility.

Cervical cancer is the fourth most frequently diagnosed tumor and the fourth leading cause of cancer death in females worldwide. Cervical cancer is predominantly related with human papilloma virus (HPV) infection, with the most oncogenic types being HPV-18 and -16. Our previous studies demonstrated that some d-secoestrone derivatives exert pronounced antiproliferative activity. The aim of the current investigation was to characterize the mechanism of action of d-secoestrone-triazole (D-SET) on three cervical cancer cell lines with different pathological backgrounds. The growth-inhibitory effects of D-SET were determined by a standard MTT assay. We have found that D-SET exerts a pronounced growth-inhibitory effect on HPV 18-positive HeLa and HPV-negative C-33 A cells, but it has no substantial inhibitory activity on HPV 16-positive SiHa or on intact fibroblast MRC-5 cell lines. After 24h incubation, cells showed the morphological and biochemical signs of apoptosis determined by fluorescent double staining, flow cytometry and caspase-3 activity assay. Besides the elevation of the ratio of cells in the subG1 phase, flow cytometric analysis revealed a cell cycle arrest at G2/M in both HeLa and C-33 A cell lines. To distinguish the G2/M cell population immunocytochemical flow cytometric analysis was performed on HeLa cells. The results show that D-SET significantly increases the ratio of phosphorylated histone H3, indicating cell accumulation in the M phase. Additionally, D-SET significantly increased the maximum rate of microtube formation measured by an in vitro tubulin polymerization assay. Besides its direct antiproliferative activity, the antimigratory property of D-SET has been investigated. Our results demonstrate that D-SET significantly inhibits the migration and invasion of HeLa cells after 24h incubation. These results suggests that D-SET is a potent antiproliferative agent against HPV 16+ and HPV-negative cervical cancer cell lines, with an efficacious motility-inhibiting activity against HPV 16+ cells. Accordingly D-SET can be regarded as a potential drug candidate with a promising new mechanism of action among the antiproliferative steroids, potentially allowing for the design of novel anticancer agents.

Antiproliferative Effects of Various Furanoacridones Isolated from Ruta graveolens on Human Breast Cancer Cell Lines.

BACKGROUND/AIM: Thanks to its biologically active constituents, Ruta graveolens L. (Rutaceae) is a widely used medicinal plant. In our study, six furanoacridone alkaloids isolated from Ruta graveolens were investigated for their antiproliferative and pro-apoptotic effects on human breast cancer cell lines (MCF-7, MDA-MB-361, MDA-MB-231 and T47D). MATERIALS AND METHODS: The cell lines were pretreated with alkaloid components (rutacridone, isogravacridone chlorine (IGC), gravacridonediol monomethyl ether, gravacridonediol, gravacridonetriol, a 1:1 mixture of gravacridonetriol and - diol monoglucosides) and their antiproliferative effects were determined by the MTT assay. RESULTS: IGC had the most marked effect on cell proliferation of MDA-MB-231 (half maximal inhibitory concentration (IC50)=2.27 µM). Cell-cycle analysis was applied to quantify the effect of IGC on subpopulations of MDA-MB-231 and MCF-7 cells. It caused a cell-cycle disturbance by decreasing the G2/M and G0/G1 and increasing the S phase and the appearance of the subdiploid (sub-G1) population. Hoechst 33258-propidium iodide staining was used to evaluate the morphological changes in IGC-pretreated MDA-MB-231 and MCF-7 cells, revealing the appearance of apoptotic features. IGC was found to cause a modest activation of caspase-3 and -9, but not caspase-8, indicating the activation of an intrinsic apoptotic pathway in MDA-MB-231 cells. CONCLUSIONS: These in vitro findings indicate that furanoacridones are suitable candidates for anticancer drug development.

Synthesis and biological evaluation of 13α-estrone derivatives as potential antiproliferative agents.

13α-Estrone derivatives containing various substituents on C-3 and C-17 were synthesized, and evaluated by means of MTT assays for in vitro antiproliferative activity against a panel of human adherent cancer cell lines (HeLa, MCF-7, A2780 and A431). Compounds with N-benzyltriazolylmethoxy moieties on C-3 proved to be more potent than their 3-hydroxy or 3-ether counterparts. Some triazoles exerted substantial cytostatic effects against particular tumor cell lines, with submicromolar IC50 values.

Synthesis and in Vitro Antiproliferative Evaluation of C-13 Epimers of Triazolyl-d-Secoestrone Alcohols: The First Potent 13α-d-Secoestrone Derivative.

The syntheses of C-13 epimeric 3-[(1-benzyl-1,2,3-triazol-4-yl)methoxy]-d-secoestrones are reported. Triazoles were prepared from 3-(prop-2-inyloxy)-d-secoalcohols and p-substituted benzyl azides via Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC). The antiproliferative activities of the products and their precursors were determined in vitro against a panel of human adherent cervical (HeLa, SiHa and C33A), breast (MCF-7, MDA-MB-231, MDA-MB-361 and T47D) and ovarian (A2780) cell lines by means of MTT assays. The orientation of the angular methyl group and the substitution pattern of the benzyl group of the azide greatly influenced the cell growth-inhibitory potential of the compounds. The 13ß derivatives generally proved to be more potent than their 13α counterparts. Introduction of a benzyltriazolylmethyl group onto the 3-OH position seemed to be advantageous. One 13α compound containing an unsubstituted benzyltriazolyl function displayed outstanding antiproliferative activities against three cell lines.

Stereocontrolled synthesis of the four 16-hydroxymethyl-19-nortestosterone isomers and their antiproliferative activities.

Novel 16-hydroxymethyl-19-nortestosterone diastereomers were prepared by Birch reduction from the corresponding 3-methoxy-16-hydroxymethylestra-1,3,5(10)-trien-17-ol isomers with known configurations. The synthesized compounds are 16α- and 16ß-hydroxymethyl-substituted 19-nortestosterone and their 17α-epimers. To prepare 17α-19-nortestosterone, the Mitsunobu inversion reaction of 19-nortestosterone with different alkyl and aryl carboxylic acids was chosen. Deacylation of the new compounds by the Zemplén method yielded the required 17α-19-nortestosterone. The antiproliferative activities of the structurally related compounds were determined in vitro through microculture tetrazolium assays on a panel of human adherent cervical (HeLa, SiHa and C33A), breast (MCF-7, MDA-MB-231, MDA-MB-361 and T47D) and ovarian (A2780) cell lines. The 17α epimer of 19-nortestosterone demonstrated considerable activity, selectively for HeLa cells, with a calculated IC50 of 0.65 µM. The reference compound, cisplatin, displayed an order of magnitude higher IC50 (12.4 µM). The cancer selectivity of 17α-19-nortestosterone was tested by MTT assay performed with noncancerous human fibroblast cell line MRC-5. The results indicated that 17α-19-nortestosterone selectively disturbs the viability of HeLa cells without greatly affecting other cancer cell types and intact fibroblasts.

A molecular understanding of D-homoestrone-induced G2/M cell cycle arrest in HeLa human cervical carcinoma cells.

2-Methoxyestradiol (ME), one of the most widely investigated A-ring-modified metabolites of estrone, exerts significant anticancer activity on numerous cancer cell lines. Its pharmacological actions, including cell cycle arrest, microtubule disruption and pro-apoptotic activity, have already been described in detail. The currently tested D-ring-modified analogue of estrone, D-homoestrone, selectively inhibits cervical cancer cell proliferation and induces a G2/M phase cell cycle blockade, resulting in the development of apoptosis. The question arose of whether the difference in the chemical structures of these analogues can influence the mechanism of anticancer action. The aim of the present study was therefore to elucidate the molecular contributors of intracellular processes induced by D-homoestrone in HeLa cells. Apoptosis triggered by D-homoestrone develops through activation of the intrinsic pathway, as demonstrated by determination of the activities of caspase-8 and -9. It was revealed that D-homoestrone-treated HeLa cells are not able to enter mitosis because the cyclin-dependent kinase 1-cyclin B complex loses its activity, resulting in the decreased inactivation of stathmin and a concomitant disturbance of microtubule formation. However, unlike 2-ME, D-homoestrone does not exert a direct effect on tubulin polymerization. These results led to the conclusion that the D-homoestrone-triggered intracellular processes resulting in a cell cycle arrest and apoptosis in HeLa cells differ from those in the case of 2-ME. This may be regarded as an alternative mechanism of action among steroidal anticancer compounds.

Antiproliferative Activity of Some Higher Mushrooms from Hungary against Human Cancer Cell Lines.

In the present work, aqueous and organic extracts of 16 Basidiomycetes mushrooms and 1 Ascomycetes mushroom were investigated in vitro for their antiproliferative activity against HeLa (cervix epithelial adenocarcinoma), A431 (skin epidermoid carcinoma), A2780 (ovarian carcinoma), and MCF7 (breast epithelial adenocarcinoma) cells, using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. A total of 68 n-hexane, chloroform, 50% methanol, and water extracts of selected species were screened for their potential cell growth inhibitory activity. Our experiments revealed that 7 of 17 species demonstrated notable antiproliferative activity (at least 50% inhibition of cell proliferation) against one or more cell lines. Kuehneromyces mutabilis, Lactarius quietus, and Lentinellus cochleatus, which exerted the highest activity on cancer cells, are considered valuable species in the perspective of further mycochemical studies.

Syntheses and antiproliferative effects of D-homo- and D-secoestrones.

Substituted and/or heterocyclic d-homoestrone derivatives were synthetized via the intramolecular cyclization of a δ-alkenyl-d-secoaldehyde, -d-secoalcohol or -d-secocarboxylic acid of estrone 3-benzyl ether. The d-secoalcohol was modified at three sites in the molecule. The in vitro antiproliferative activities of the new d-homo- and d-secoestrone derivatives were determined on HeLa, MCF-7, A431 and A2780 cells through use of MTT assay. d-Homoalcohols 3 and 5 displayed cell line-selective cytostatic effects against ovarian and cervical cell lines, respectively. Two d-secoestrones (6 and 12c) proved to be effective, with IC50 values comparable with those of the reference agent cisplatin. A selected compound (6) was tested by tubulin polymerization assay and its cancer specificity was additionally determined by using noncancerous human fibroblast cells.

Antiproliferative effect of normal and 13-epi-D-homoestrone and their 3-methyl ethers on human reproductive cancer cell lines.

The possibility of the therapeutic use of estrogens emerged following the recognition that certain estradiol analogs, and particularly metabolites (e.g. the A-ring metabolite 2-hydroxyestrone, etc.) inhibit the differentiation of diverse tumor cell lines. Until recently, despite the investigation of numerous synthetic d-ring-substituted estrone derivatives, no analysis had been published on the effects of D-ring expansion of estrone on its tumor-suppressing activity. The aim of the present study was to characterize the antiproliferative effects of normal and 13-epi-D-homoestrone and their 3-methyl ethers (1-4) on human reproductive cancer cell lines. The antitumor activities of the two epimer pairs on HeLa, MCF-7 and Ishikawa cells were determined. Normal D-homoestrone exerted the greatest cytostatic effect on HeLa cells (IC(50)=5.5 µM) and was subjected to further investigations to elucidate its mechanism of action on apoptosis induction. Morphological changes detected by Hoechst 33258-propidium iodide double staining, the cell cycle arrest at phase G2/M and the subsequent increase in the proportion of the subG1 fraction determined by flow cytometric analysis and the significant increase in the activity of caspase-3 confirmed the induction of apoptosis in HeLa cells treated with D-homoestrone. D-Homoestrone was also tested on a non-cancerous human lung fibroblast cell line (MRC-5) to determine its selective toxicity. The concentration in which it inhibited cell proliferation by 50% was at least six times higher for the fibroblast cells than for cervical cancer cells. No significant in vivo estrogenic activity was observed as concerns the uterus weight of gonadectomized rats after a 7-day treatment with normal D-homoestrone. These results led to the conclusion that normal D-homoestrone is a novel antitumor compound with a similar activity on HeLa cells as that of the reference agent cisplatin, but its selectivity toward non-cancerous cells is significantly higher than that of cisplatin. It may be considered to be a basic lead molecule for the preclinical development of potential anticancer agents.