New 99mTc-Labeled Digitoxigenin Derivative for Cancer Cell Identification

In recent years, cardiac glycosides (CGs) have been investigated as potential antiviral and anticancer drugs. Digitoxigenin (DIG) and other CGs have been shown to bind and inhibit Na+/K+-adenosinetriphosphatase (ATPase). Tumor cells show a higher expression rate of the Na+/K+-ATPase protein or a stronger affinity towards the binding of CGs and are therefore more prone to CGs than non-tumor cells. Cancer imaging techniques using radiotracers targeted at specific receptors have yielded successful results. Technetium-99m (99mTc) is one of the radionuclides of choice to radiolabel pharmaceuticals because of its favorable physical and chemical properties along with reasonable costs. Herein, we describe a new Na+/K+-ATPase targeting radiotracer consisting of digitoxigenin and diethylenetriaminepentaacetic acid (DTPA), a bifunctional chelating ligand used to prepare 99mTc-labeled complexes, and its evaluation as an imaging probe. We report the synthesis and characterization of the radiolabeled compound including stability tests, blood clearance, and biodistribution in healthy mice. Additionally, we investigated the binding of the compound to A549 human non-small-cell lung cancer cells and the inhibition of the Na+/K+-ATPase by the labeled compound in vitro. The 99mTc-labeled DTPA–digitoxigenin (99mTc-DTPA–DIG) compound displayed high stability in vitro and in vivo, a fast renal excretion, and a specific binding towards A549 cancer cells in comparison to non-tumor cells. Therefore, 99mTc-DTPA–DIG could potentially be used for non-invasive visualization of tumor lesions by means of scintigraphic imaging

New 99mTc-labeled digitoxigenin derivative for cancer cell identification

In recent years, cardiac glycosides (CGs) have been investigated as potential antiviral and anticancer drugs. Digitoxigenin (DIG) and other CGs have been shown to bind and inhibit Na+ /K+ -adenosinetriphosphatase (ATPase). Tumor cells show a higher expression rate of the Na+ /K+ - ATPase protein or a stronger affinity towards the binding of CGs and are therefore more prone to CGs than non-tumor cells. Cancer imaging techniques using radiotracers targeted at specific receptors have yielded successful results. Technetium99m (99mTc) is one of the radionuclides of choice to radiolabel pharmaceuticals because of its favorable physical and chemical properties along with reasonable costs. Herein, we describe a new Na+ /K+ -ATPase targeting radiotracer consisting of digitoxigenin and diethylenetriaminepentaacetic acid (DTPA), a bifunctional chelating ligand used to prepare 99mTc-labeled complexes, and its evaluation as an imaging probe. We report the synthesis and characterization of the radiolabeled compound including stability tests, blood clearance, and biodistribution in healthy mice. Additionally, we investigated the binding of the compound to A549 human non-small-cell lung cancer cells and the inhibition of the Na+ /K+ - ATPase by the labeled compound in vitro. The 99mTc-labeled DTPA−digitoxigenin (99mTc-DTPA−DIG) compound displayed high stability in vitro and in vivo, a fast renal excretion, and a specific binding towards A549 cancer cells in comparison to nontumor cells. Therefore, 99mTc-DTPA−DIG could potentially be used for non-invasive visualization of tumor lesions by means of scintigraphic imaging

Butenolide ring closure via a malonic ester of 21-hydroxy-pregnane, substrate synthesis, uptake, biotransformation and ring closure in vitro

In der vorliegenden Arbeit wurde die Hypothese der Bildung von Cardenoliden über den Malonsäureester eines 21-Hydroxypregnans geprüft. Im ersten Schritt galt es eine Methode zur chemischen Malonylierung zu entwickeln, welche die Synthese eines in Position 14ß hydroxylierten 21-O-Malonylpregnans erlaubt. Diese Verbindung sollte nämlich das ideale Substrat für eine hypothetische Butenolidring-Cyclase darstellen. Das für die Malonylierung verwendete Edukt, das 20,21-Ketol eines 14ß-Pregnans, wurde durch Hydrolyse von ß-Methyldigitoxin, anschließender 3ß-O-Acetylierung des entstandenen Digitoxigenins und Ozonisierung des Butenolidringes des 3ß-O-Acetyldigitoxigenins mit nachfolgender reduktiver Spaltung des Ozonids gewonnen. Die effiziente 21-O-Malonylierung gelang schließlich erstmals durch Umsetzung des 14ß-hydroxylierten 20,21-Ketols (3ß-O-Acetyl-21-O-malonyl-5ß-pregnan-14ß-ol-20-on) mit einem Überschuss von Malonyldichlorid bei tiefer Temperatur. Verschiedene spektroskopische Methoden wurden für die Strukturaufklärung der synthetisierten Verbindungen verwendet. Im nächsten Schritt wurde die Aufnahme und Metabolisierung des 21-O-Malonyl- und 21-O-Aceytlesters von Desoxycorticosteron durch Digitalis lanata (W.1.4) Zellsuspensionskulturen untersucht. Neben einer hohen Esterase-Aktivität konnte hierbei die Bildung einer Reihe weiterer Biotransformationsprodukte beobachtet werden. Die Motivation für die Isolierung und Charakterisierung dieser Verbindungen stellte die für den Butenolidring typische Kedde-positive Farbreaktion eines dieser Biotransformationsproduktes dar. Es wurden aus der D. lanata Zellsuspensionskultur (W.1.4) neun Biotransformationsprodukte des 21-O-Acetyl-Desoxycorticosterons isoliert. Ausgehend von dem durch Esterspaltung entstandenen Desoxycorticosteron wurden folgende Reaktionen beobachtet: 5-Alfa-Reduktion, 5ß-Reduktion, 3-Alfa-Reduktion, 3ß-Reduktion, 2ß-Hydroxylierung, 21-O-Glucosidierung und 3ß-O-Glucosidierung. Die 2ß-Hydroxylierung sowie die 21-O-Glucosidierung wurden erstmals in pflanzlichen Zellkulturen nachgewiesen. Das Auftreten von 5ß-Pregnan-3-Alfa,21-diol-20-on wurde als in Hinweis auf das Vorhandensein einer 5ß-Progesteron Reduktase interpretiert, deren Anwesenheit in Digitalis Zellsuspensionskulturen damit ebenfalls erstmals belegt wurde. Zwei bisher nicht beschriebene Verbindungen 4-Pregnen-2ß,3ß,21-triol-20-on und 4-Pregnen-2ß,3-Alfa,21-triol-20-on mit Ecdysteroid-ähnlicher Struktur konnten isoliert werden. Für die Strukturaufklärung der Biotransformationsprodukte kamen 1H- und 13C-NMR, HREI-MS und/oder MALDI-TOF zum Einsatz. Das Kedde-positive Biotransformationsprodukt konnte als 21-O-Glucosid des Desoxycorticosterons identifiziert werden. Dies zeigt, dass das Kedde-Reagenz in manchen Fällen falsch-positive Resultate liefern kann. Die Fütterung von 5ß-Pregnan-3ß-ol-20-on, 3ß-O-Acetyl-5ß-pregnan-14ß,21-diol-20-on (20,21-Ketol) und 3ß-O-Acetyl-21-O-malonyl-5ß-pregnan¬-14ß-ol-20-on (21-O-Malonyl-20,21-ketol) an Sprosskulturen von D. purpurea führte zu einer Erhöhung des Cardenolidgehalts. Die Biosynthese wurde durch das 21-O-Malonyl-20,21-Ketol geringer stimuliert als durch das 20,21-Ketol selbst. Eine mögliche Erklärung dafür ist eine von Esterasen katalysierte Hydrolyse des 21-O-Malonylrests während oder nach der Aufnahme. Außerdem wurde der chemische Ringschluss des Butenolidrings, ausgehend von Malonylhemiestern (mit freier Carboxylgruppe), erforscht. Die Ergebnisse deuten darauf hin, dass die chemische Bildung des Ringschlusses in Gegenwart von Puffer über eine intramolekulare Knoevenagel-Reaktion möglich ist. Jedoch kann ein chemischer Ringschluss über einen Krapcho-Mechanismus oder über einen intermediär auftretenden sechsgliedrigen Ring noch nicht vollständig ausgeschlossen werden. Abschließend wurde ein möglicher enzymatischer Ringschluss untersucht. Mit dem Ziel eine enzymatische Reaktion zu erfassen, die durch Decarboxylierung bzw. Erhöhung der Azidität der Methylengruppe des Malonylrests durch Diesterbildung zum Ringschluss führt wurden getestet: der Einfluss zweiwertiger Metallionen, das Vorhandensein eines Malat-Decarboxylase ähnlichen Enzyms, sowie der Einfluss der Cofaktoren Mg+2/ATP und Coenzym A. Jedoch waren immer nur die Aktivitäten der 21-O-Malonylesterase und der 3ß-O-Acetylesterase zu sehen. Daher konnte die Hypothese eines Wegs der Cardenolidbildung über 21-O-malonylierte Pregnane experimentell weder in vivo noch biochemisch bestätigt werden. Dennoch kann nicht ausgeschlossen werden, dass dieser Weg in der Pflanze beschritten werden kann und ein entsprechendes Enzym diese Reaktion katalysiert. Die Option eines nicht-enzymatischen Ringschlusses in planta bleibt weiterhin bestehen. Dieser müsste jedoch in einem Kompartiment stattfinden, wo keine 21-O-Malonylesterase vorhanden ist.The hypothesis of the cardenolides formation via a malonic ester of 21-hydroxy-pregnane was analysed in the present work. The first step comprised the development of a chemical malonylation method to obtain hydroxylated 21-O-Malonyl-pregnanes at position 14ß, considered to be adequate substrates for a putative butenolide ring cyclase. The substrate used in the malonylation was obtained through acid hydrolysis of ß-metyldigitoxin followed by 3ß-O-acetylation and ozonolysis with reductive cleavage of ozonides and subsequent isolation of the 20,21-ketol of a 14ß-hydroxypregnane. For the first time, the effective malonylation of 14ß-hydroxylated 20,21-Ketol (3ß-O-acetyl-21-O-malonyl-5ß-pregnan-14ß-ol-20-on) was achieved by using an excess of malonyl dichloride at low temperature. The spectrometric methods 1H and 13C NMR, CG-MS and/or HPLC-MS were performed for structure elucidation. In the next step, the uptake and metabolization of 21-O malonyl and acetyl esters of desoxycorticosteron by cell suspension cultures of Digitalis lanata (W.1.4) were evaluated. After application, an elevated esterase activity was found accompanied by a series of biotransformation products. These products were isolated because of the presence of a Kedde-positive compound indicating the presence of a butenolide ring. Altogether nine biotransformation products of 21-O-acetyl-desoxycorticosterone were isolated from cell suspension cultures of D. lanata (W.1.4). After ester hydrolysis of the substrate the following reactions were observed: 5-Alfa-reduction and 5ß-reduction, 3-Alfa-reduction and 3ß-reduction of the carbonyl group at C-3, hydroxylation of steroidal nucleus at position 2ß, as well as 21-O- and 3ß-O-glucosylation. Hydroxylation at position 2ß of a steroidal nucleus and glycosylation of the position 21 were observed in plant cell cultures for the first time. The occurrence of 5ß-Pregnan-3-Alfa,21-diol-20-one evidenced for the first time the presence of 5ß-Progesteron reductase in the cell suspension cultures of Digitalis ssp.. Two new compounds, namely 4-Pregnen-2ß,3ß,21-triol-20-one and 4-Pregnen-2ß,3-Alfa,21-triol-20-one showing ecdysteroid-like structures were isolated. For structure elucidation of all isolated compounds 1H and 13C NMR, CG-MS, HREI-MS and/or MALDI-TOF were employed. The Kedde-positive biotransformation product was identified as desoxycorticosterone 21-O-ß-glycoside, a compound without butenolide ring, demonstrating that the use of Kedde reagent can, in some cases, lead to false-positive results. The feeding of 5ß-pregnan-3ß-ol-20-on, 3ß-O-acetyl-5ß-pregnane-14ß,21-diol-20-on (20,21-ketol) and 3ß-O-acetyl-21-O-malonyl-5ß-pregnan¬-14ß-ol-20-on (21-O-malonyl-ketol) in shoot cultures of D. purpurea resulted in an increase of cardenolide biosynthesis. For the compound 21-O-malonyl-ketol, this increase was lower than that found for 20,21-ketol. A probable explanation for this observation is the existence of a malonylesterase hydrolysing the 21-O-malonyl-ketol during or after uptake. Moreover, the chemical butenolide ring formation using malonic acid hemiesters (with a free carboxyl group) as the educt was studied. The results indicate that butenolide ring closure via intramolecular Knoevenagel reaction is possible in the presence of buffer. However, the chemical formation of butenolide ring closure by the Krapcho mechanism or via a six-membered ring intermediate cannot be ruled out completely. Finally, the enzymatic butenolide ring closure was investigated. In these experiments several different extraction methods e.g. the effects of antioxidants, protease inhibitors and surfactants were tested. Different tissues (roots and leaves) as well as different plants (D. lanata and D. purpurea) were investigated. With a view to finding conditions leading to an enzymatic ring closure with concomitant decarboxylation and increase of acidity of the malonyl acid moiety’s methylene group via diester formation respectively the following factors were tested: The influence of bivalent metal 2+ ions, the presence of an enzyme having a reaction mechanism similar to malate decarboxylase as well as the influcence of the cofactors Mg+2/ATP and Coenzyme A. However, only the activities of 21-O-malonylesterase and 3ß-O-acetylesterase were found. Thus, the hypothesis of a pathway for the formation of the butenolide ring via a 21-O-malonyl-pregnane could be confirmed neither in vivo nor biochemically. Despite of this, it cannot be rueld out that this pathway is operative and that an enzyme catalyzing the reaction exists. Moreover, non-enzymatic ring closure in planta remains an alternative, however this would require a compartment, where 21-O-Malonylesterase is not present

A Computational Approach Using Bioinformatics to Screening Drug Targets for Leishmania infantum

Background. The development of new therapeutic strategies to treat patients for leishmaniasis has become a priority. The antileishmanial activity of the strychnobiflavone flavonoid was recently demonstrated against Leishmania amazonensis and Leishmania infantum amastigotes and promastigotes. The biological effect of this molecule was identified due to its capacity to interfere in the parasite mitochondrial membrane; however, the underlying molecular mechanism remains unclear. Methods and Results. In this study, a computational approach using bioinformatics was performed to screen biological targets of strychnobiflavone in L. infantum. Computational programs, such as the target fishing approach and molecular docking assays, were used. Results showed that the putative pathway targeted by strychnobiflavone in L. infantum is the methylglyoxal degradation superpathway, and one hydrolase-like protein was predicted to be the molecular target of this flavonoid in the parasites. Conclusion. In this context, this study provides the basis for understanding the mechanism of action of strychnobiflavone in L. infantum and presents a strategy based on bioinformatics programs to screen targets of other molecules with biological action against distinct pathogens

Semisynthetic Cardenolides Acting as Antiviral Inhibitors of Influenza A Virus Replication by Preventing Polymerase Complex Formation

Influenza virus infections represent a major public health issue by causing annual epidemics and occasional pandemics that affect thousands of people worldwide. Vaccination is the main prophylaxis to prevent these epidemics/pandemics, although the effectiveness of licensed vaccines is rather limited due to the constant mutations of influenza virus antigenic characteristics. The available anti-influenza drugs are still restricted and there is an increasing viral resistance to these compounds, thus highlighting the need for research and development of new antiviral drugs. In this work, two semisynthetic derivatives of digitoxigenin, namely C10 (3β-((N-(2-hydroxyethyl)aminoacetyl)amino-3-deoxydigitoxigenin) and C11 (3β-(hydroxyacetyl)amino-3-deoxydigitoxigenin), showed anti-influenza A virus activity by affecting the expression of viral proteins at the early and late stages of replication cycle, and altering the transcription and synthesis of new viral proteins, thereby inhibiting the formation of new virions. Such antiviral action occurred due to the interference in the assembly of viral polymerase, resulting in an impaired polymerase activity and, therefore, reducing viral replication. Confirming the in vitro results, a clinically relevant ex vivo model of influenza virus infection of human tumor-free lung tissues corroborated the potential of these compounds, especially C10, to completely abrogate influenza A virus replication at the highest concentration tested (2.0 µM). Taken together, these promising results demonstrated that C10 and C11 can be considered as potential new anti-influenza drug candidates

Strychnos pseudoquina

The development of new and cost-effective alternative therapeutic strategies to treat leishmaniasis has become a high priority. In the present study, the antileishmanial activity of Strychnos pseudoquina St. Hil. was investigated and pure compounds that presented this biological effect were isolated. An ethyl acetate extract was prepared, and it proved to be effective against Leishmania amazonensis. A bioactivity-guided fractionation was performed, and two flavonoids were identified, quercetin 3-O-methyl ether and strychnobiflavone, which presented an effective antileishmanial activity against L. amazonensis, and studies were extended to establish their minimum inhibitory concentrations (IC50), their leishmanicidal effects on the intra-macrophage Leishmania stage, as well as their cytotoxic effects on murine macrophages (CC50), and in O+ human red blood cells. The data presented in this study showed the potential of an ethyl acetate extract of S. pseudoquina, as well as two flavonoids purified from it, which can be used as a therapeutic alternative on its own, or in association with other drugs, to treat disease evoked by L. amazonensis

Uma quinta portuguesa no interior do Brasil ou A saga do ilustrado dom frei Cipriano e o jardim do antigo palácio episcopal no final do século XVIII A Portuguese manor in rural Brazil or The saga of the enlightened Dom Frei Cipriano and the garden of the former episcopal palace in the late eighteenth century

Reconstrói a trajetória do bispo dom frei Cipriano de São José e do jardim histórico que construiu em seu palácio, na cidade de Mariana (MG), no final do século XVIII e início do seguinte. A área, com pomar e outros tipos vegetais, como a maioria dos quintais das casas mineiras do período, sofreu significativa transformação e tornou-se um clássico jardim europeu. Ordenado com preocupação estética, no final do período colonial era um espaço admirável, representante do processo de encantamento da ilustrada elite metropolitana pela história natural e pelos conhecimentos de botânica. Variada documentação foi utilizada para reconstruir a trajetória dessa área verde bem como a de seu idealizador, o ilustrado dom frei Cipriano de São José.<br>This is a reconstruction of the career of bishop Cipriano de São José and the historical garden he built for his palace in Mariana, Minas Gerais, in the late eighteenth and early nineteenth centuries. The grounds, which, like most of their counterparts in Minas Gerais, contained an orchard and kitchen garden, underwent a major reform to fit the mold of the classical European garden. Orderly and showing aesthetic concern, they were an admirable space at the end of the colonial period, representing as they did the enlightened metropolitan elite's growing interest in natural history and botany. A variety of documental sources were used to reconstruct the history of the grounds and the career of the person behind their design, the enlightened Dom Frei Cipriano de São José