Click estradiol dimers with novel aromatic bridging units: synthesis and anticancer evaluation.

Estradiol dimers (EDs) possess significant anticancer activity by targeting tubulin dynamics. In this study, we synthesised 12 EDs variants via copper-catalysed azide-alkyne cycloaddition (CuAAC) reaction, focusing on structural modifications within the aromatic bridge connecting two estradiol moieties. In vitro testing of these EDs revealed a marked improvement in selectivity towards cancerous cells, particularly for ED1-8. The most active compounds, ED3 (IC50 = 0.38 µM in CCRF-CEM) and ED5 (IC50 = 0.71 µM in CCRF-CEM) demonstrated cytotoxic effects superior to 2-methoxyestradiol (IC50 = 1.61 µM in CCRF-CEM) and exhibited anti-angiogenic properties in an endothelial cell tube-formation model. Cell-based experiments and in vitro assays revealed that EDs interfere with mitotic spindle assembly. Additionally, we proposed an in silico model illustrating the probable binding modes of ED3 and ED5, suggesting that dimers with a simple linker and a single substituent on the aromatic central ring possess enhanced characteristics compared to more complex dimers.

A facile synthesis of precursor for the σ-1 receptor PET radioligand [18 F]FTC-146 and its radiofluorination.

The σ-1 receptor is a non-opioid transmembrane protein involved in various human pathologies including neurodegenerative diseases, inflammation, and cancer. The previously published ligand [18 F]FTC-146 is among the most promising tools for σ-1 molecular imaging by positron emission tomography (PET), with a potential for application in clinical diagnostics and research. However, the published six- or four-step synthesis of the tosyl ester precursor for its radiosynthesis is complicated and time-consuming. Herein, we present a simple one-step precursor synthesis followed by a one-step fluorine-18 labeling procedure that streamlines the preparation of [18 F]FTC-146. Instead of a tosyl-based precursor, we developed a one-step synthesis of the precursor analog AM-16 containing a chloride leaving group for the SN 2 reaction with 18 F-fluoride. 18 F-fluorination of AM-16 led to a moderate decay-corrected radiochemical yield (RCY = 7.5%) with molar activity (Am ) of 45.9 GBq/µmol. Further optimization of this procedure should enable routine radiopharmaceutical production of this promising PET tracer.

Triazole-based estradiol dimers prepared via CuAAC from 17α-ethinyl estradiol with five-atom linkers causing G2/M arrest and tubulin inhibition.

Microtubule dynamic is exceptionally sensitive to modulation by small-molecule ligands. Our previous work presented the preparation of microtubule-targeting estradiol dimer (ED) with anticancer activity. In the present study, we explore the effect of selected linkers on the biological activity of the dimer. The linkers were designed as five-atom chains with carbon, nitrogen or oxygen in their centre. In addition, the central nitrogen was modified by a benzyl group with hydroxy or methoxy substituents and one derivative possessed an extended linker length. Thirteen new dimers were subjected to cytotoxicity assay and cell cycle profiling. Dimers containing linker with benzyl moiety substituted with one or more methoxy groups and longer branched ones were found inactive, whereas other structures had comparable efficacy as the original ED (e.g. D1 with IC50 = 1.53 µM). Cell cycle analysis and immunofluorescence proved the interference of dimers with microtubule assembly and mitosis. The proposed in silico model and calculated binding free energy by the MM-PBSA method were closely correlated with in vitro tubulin assembly assay.

Synthesis and biological evaluation of cationic TopFluor cholesterol analogues.

Cholesterol is not only a major component of the cell membrane, but also plays an important role in a wide range of biological processes and pathologies. It is therefore crucial to develop appropriate tools for visualizing intracellular cholesterol transport. Here, we describe new cationic analogues of BODIPY-Cholesterol (TopFluor-Cholesterol, TF-Chol), which combine a positive charge on the sterol side chain and a BODIPY group connected via a C-4 linker. In contrast to TF-Chol, the new analogues TF-1 and TF-3 possessing acetyl groups on the A ring (C-3 position on steroid) internalized much faster and displayed slightly different levels of intracellular localization. Their applicability for cholesterol monitoring was indicated by the fact that they strongly label compartments with accumulated cholesterol in cells carrying a mutation of the Niemann-Pick disease-associated cholesterol transporter, NPC1.

Terpene Research Is Providing New Inspiration for Scientists.

This current Special Issue of Molecules gathers selected communications on terpenes and terpene derivatives, clearly demonstrating the sustained interest in and importance of natural products in this field; fields connected to secondary metabolites; and renewable resources of plant and animal compounds for medicinal, material, supramolecular, and general chemistry research [...].

Growing Importance of Natural Products Research.

Natural products and preparations based on them play a stable and ever-increasing role in human and veterinary medicine, agriculture, in food and the cosmetic industry, and in other increasing numbers of fields. [...].

BODIPY-based fluorescent liposomes with sesquiterpene lactone trilobolide.

Like thapsigargin, which is undergoing clinical trials, trilobolide is a natural product with promising anticancer and anti-inflammatory properties. Similar to thapsigargin, it has limited aqueous solubility that strongly reduces its potential medicinal applications. The targeted delivery of hydrophobic drugs can be achieved using liposome-based carriers. Therefore, we designed a traceable liposomal drug delivery system for trilobolide. The fluorescent green-emitting dye BODIPY, cholesterol and trilobolide were used to create construct 6. The liposomes were composed of dipalmitoyl-3-trimethylammoniumpropane and phosphatidylethanolamine. The whole system was characterized by atomic force microscopy, the average size of the liposomes was 150 nm in width and 30 nm in height. We evaluated the biological activity of construct 6 and its liposomal formulation, both of which showed immunomodulatory properties in primary rat macrophages. The uptake and intracellular distribution of construct 6 and its liposomal formulation was monitored by means of live-cell fluorescence microscopy in two cancer cell lines. The encapsulation of construct 6 into the liposomes improved the drug distribution in cancer cells and was followed by cell death. This new liposomal trilobolide derivative not only retains the biological properties of pure trilobolide, but also enhances the bioavailability, and thus has potential for the use in theranostic applications.

Plant Secondary Metabolites Used for the Treatment of Diseases and Drug Development.

The importance of natural products in medicine, and in particular, plant secondary metabolites used for the treatment of diseases and drug development, has been obvious for several thousands of years [...].

Betulinic Acid Decorated with Polar Groups and Blue Emitting BODIPY Dye: Synthesis, Cytotoxicity, Cell-Cycle Analysis and Anti-HIV Profiling.

Betulinic acid (BA) is a potent triterpene, which has shown promising potential in cancer and HIV-1 treatment. Here, we report a synthesis and biological evaluation of 17 new compounds, including BODIPY labelled analogues derived from BA. The analogues terminated by amino moiety showed increased cytotoxicity (e.g., BA had on CCRF-CEM IC50 > 50 µM, amine 3 IC50 0.21 and amine 14 IC50 0.29). The cell-cycle arrest was evaluated and did not show general features for all the tested compounds. A fluorescence microscopy study of six derivatives revealed that only 4 and 6 were detected in living cells. These compounds were colocalized with the endoplasmic reticulum and mitochondria, indicating possible targets in these organelles. The study of anti-HIV-1 activity showed that 8, 10, 16, 17 and 18 have had IC50i > 10 µM. Only completely processed p24 CA was identified in the viruses formed in the presence of compounds 4 and 12. In the cases of 2, 8, 9, 10, 16, 17 and 18, we identified not fully processed p24 CA and p25 CA-SP1 protein. This observation suggests a similar mechanism of inhibition as described for bevirimat.

Steroid Glycosides Hyrcanoside and Deglucohyrcanoside: On Isolation, Structural Identification, and Anticancer Activity.

Cardiac glycosides (CGs) represent a group of sundry compounds of natural origin. Most CGs are potent inhibitors of Na+/K+-ATPase, and some are routinely utilized in the treatment of various cardiac conditions. Biological activities of other lesser known CGs have not been fully explored yet. Interestingly, the anticancer potential of some CGs was revealed and thereby, some of these compounds are now being evaluated for drug repositioning. However, high systemic toxicity and low cancer cell selectivity of the clinically used CGs have severely limited their utilization in cancer treatment so far. Therefore, in this study, we have focused on two poorly described CGs: hyrcanoside and deglucohyrcanoside. We elaborated on their isolation, structural identification, and cytotoxicity evaluation in a panel of cancerous and noncancerous cell lines, and on their potential to induce cell cycle arrest in the G2/M phase. The activity of hyrcanoside and deglucohyrcanoside was compared to three other CGs: ouabain, digitoxin, and cymarin. Furthermore, by in silico modeling, interaction of these CGs with Na+/K+-ATPase was also studied. Hopefully, these compounds could serve not only as a research tool for Na+/K+-ATPase inhibition, but also as novel cancer therapeutics.

Bioavailability and structural study of 20-hydroxyecdysone complexes with cyclodextrins.

20-Hydroxyecdysterone - (2ß,3ß,5ß,22R)-2,3,14,20,22,25-hexahydroxycholest-7-en-6-one was isolated in satisfactory yield using ethanol extraction from the aerial part of Silene wolgensis (Hornem.) Otth; sometimes Silene wolgensis (Willd.) Bess. ex Spreng. The complexation of the phytoecdysteroid with ß-cyclodextrin was studied by NMR spectroscopy. By studying the changes in chemical shifts of protons of substrates and receptors it was found that ecdysterone interacts with cyclodextrins to form supramolecular inclusion complexes of stoichiometric composition of 1:1 or 1:2. Ecdysterone-ß-cyclodextrin complexes exhibit 100 times higher solubility in water than the parent compound.

Estradiol dimer inhibits tubulin polymerization and microtubule dynamics.

Microtubule dynamics is one of the major targets for new chemotherapeutic agents. This communication presents the synthesis and biological profiling of steroidal dimers based on estradiol, testosterone and pregnenolone bridged by 2,6-bis(azidomethyl)pyridine between D rings. The biological profiling revealed unique properties of the estradiol dimer including cytotoxic activities on a panel of 11 human cell lines, ability to arrest in the G2/M phase of the cell cycle accompanied with the attenuation of DNA/RNA synthesis. Thorough investigation precluded a genomic mechanism of action and revealed that the estradiol dimer acts at the cytoskeletal level by inhibiting tubulin polymerization. Further studies showed that estradiol dimer, but none of the other structurally related dimeric steroids, inhibited assembly of purified tubulin (IC50, 3.6 µM). The estradiol dimer was more potent than 2-methoxyestradiol, an endogenous metabolite of 17ß-estradiol and well-studied microtubule polymerization inhibitor with antitumor effects that was evaluated in clinical trials. Further, it was equipotent to nocodazole (IC50, 1.5 µM), an antimitotic small molecule of natural origin. Both estradiol dimer and nocodazole completely and reversibly depolymerized microtubules in interphase U2OS cells at 2.5 µM concentration. At lower concentrations (50 nM), estradiol dimer decreased the microtubule dynamics and growth life-time and produced comparable effect to nocodazole on the microtubule dynamicity. In silico modeling predicted that estradiol dimer binds to the colchicine-binding site in the tubulin dimer. Finally, dimerization of the steroids abolished their ability to induce transactivation by estrogen receptor α and androgen receptors. Although other steroids were reported to interact with microtubules, the estradiol dimer represents a new structural type of steroid inhibitor of tubulin polymerization and microtubule dynamics, bearing antimitotic and cytotoxic activity in cancer cell lines.

Immunoassay for determination of trilobolide.

Trilobolide (Tb) is a pharmacologically interesting sesquiterpene lactone isolated from Laser trilobum (L.) Borkh. Structural relation to a sarco/endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin bring promising prospects for Tb to be used in the development of new anti-cancer drugs. As long as there are still unanswered questions regarding its investigation, a need for novel analytical tools emerge. Since immunoassays serve as one of powerful tools within the investigation of natural products, the development of indirect competitive enzyme-linked immunosorbent assay (ELISA) utilizing coating based on avidin-biotin technology is described. In our set-up of ELISA, newly synthesized biotinylated Tb served as immobilized competitor. Tb-carboxymethyloxime-bovine serum albumin (BSA) and Tb-succinoyl-BSA conjugates were used separately for immunization of rabbits. Two sets of polyclonal antibodies (RAbs) were obtained. Antibodies against Tb-succinoyl-BSA conjugate (RAb No. 206) were chosen as the best. Under optimized conditions, limit of detection and 50% intercept of our ELISA were 849pg/mL and 8.89ng/mL, respectively. The cross-reactivity (CR) was tested on 10 structurally related compounds and CR did not exceed 6.1%. The reproducibility of the system is expressed as intra- and inter-assay coefficients of variation (9.7% and 11.4%, respectively). Based on conducted experiments, we proposed the use of ELISA for quantification of Tb in complex biological matrices such as plant extracts. A method was applied to analyze three extracts obtained from different parts of L. trilobum. Data obtained were compared to those acquired by UHPLC-MS/MS. The concordance between the methods (103-87%) showed the ability of ELISA to quantify Tb.

Trilobolide-steroid hybrids: Synthesis, cytotoxic and antimycobacterial activity.

Sesquiterpene lactone trilobolide is a sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitor, thus depleting the Ins(1,4,5)P3-sensitive intracellular calcium stores. Here, we describe a synthesis of a series of 6 trilobolide-steroids conjugates (estradiol, pregnene, dehydroepiandrosterone, and testosterone). We found that the newly synthesized Tb-based compounds possess different remarkable biological activities. Cancer cell cytotoxicity and preferential selectivity is represented in our study by a Tb-pregnene derivative. The most cytotoxic clickates of estradiol and pregnene were studied by FACS where impact on cell cycle and RNA synthesis was observed; live-cell microscopy revealed the impact on cell organelle morphology particularly endoplasmic reticulum, mitochondria and nucleus. Further, we have studied the estrogenic and androgenic properties of the clickate molecules using cell-based luciferase assays. Finally, antimycobacterial tests revealed that testosterone and estradiol derivatives potentiated the antimycobacterial activity up to IC50 of 10.6µM.

Synthesis and biological evaluation of nandrolone-bodipy conjugates.

Here, we report synthesis and biological evaluation of fluorescent nandrolone-3-carboxymethyloxime derivatives conjugated with green-emitting bodipy dye via PEG linkers. All the newly-synthesized compounds were evaluated for their effect on cell proliferation in vitro in MCF-7, LNCaP, PC-3 and HEK 293T model cell lines using WST-1 assay. By means of live-cell fluorescence microscopy, the intracellular localization of nandrolone-bodipy conjugates was revealed in endoplasmic reticulum. Moreover, we performed competitive localization study with nonfluorescent nandrolone, metandrolone, boldenone, trenbolone, and testosterone.

Trilobolide-porphyrin conjugates: on synthesis and biological effects evaluation.

Trilobolide (Tb), a potent natural counterpart of thapsigargin, is a sesquiterpene lactone of guaianolide type isolated from horse caraway (Laser trilobum, L. Borkh). Tb exerts remarkable pharmacological properties based on irreversible inhibition of sarco/endoplasmic reticulum calcium ATPase (SERCA), thus being of increasing interest for cancer cure. Additionally, another pharmacological activity of Tb, as well as of thapsigargin, was reported in several studies, Tb as being an effective inductor of nitric oxide and cytokine production. These extraordinary biological properties move these molecules in further pre-clinical evaluation. Because of ubiquitous character of SERCA expression, development of specifically targeted bioactive molecules is inevitable. Since it is well known that porphyrins are preferentially taken up by cancer cells, we have designed and synthesized novel Tb-porphyrin conjugates. Copper-catalyzed azide-alkyne cycloaddition was used to link Tb with porphyrin at once. Two model conjugates of Tb and porphyrin were synthesized and properly characterized. Employing naturally occurring fluorescence properties of porphyrins, we investigated the intracellular localization of the conjugates employing fluorescence microscopy in living cells. Intriguingly, the prepared conjugates localized both in mitochondria and lysosomes of HeLa and LNCaP cells. Furthermore, the cytotoxicity of Tb-porphyrin conjugates was assessed in a number of human cancer cell lines and rat peritoneal cells. Likewise in cancer cell lines, viability of rat peritoneal cells was not affected by the tested conjugates. Interestingly, we observed dose-dependent nitric oxide (iNOS) production induced by the tested conjugates. The effect was related to the type of a linker used and the overall size of the molecule. Another potent immunobiological effects are under evaluation. In summary, the results presented here indicate notable immunobiological potential of the prepared Tb conjugates. Moreover, they could help to decipher the molecular mechanism of Tb for its possible biomedical applications.