Martin Vlk

Cytotoxic heterocyclic triterpenoids derived from betulin and betulinic acid

The aim of this work was to synthesize a set of heterocyclic derivatives of lupane, lup-20(29)-ene, and 18α-oleanane, and to investigate their cytotoxic activities. Some of those heterocycles were previously known in the oleanane (allobetulin) group; however, to our knowledge the syntheses and biological activities of lupane heterocycles have not been reported before. Starting from betulin (1) and betulinic acid (2), we prepared 3-oxo compounds and 2-bromo-3-oxo compounds 3-10, 2-hydroxymethylene-3-oxo compounds 11-13 and β-oxo esters 14-16. Condensation of these intermediates with hydrazine, phenylhydrazine, hydroxylamine, or thiourea yielded the pyrazole and phenylpyrazole derivatives 17-22, pyrazolones 23-25, isoxazoles 26 and 27, and thiazoles 28-31. Fifteen compounds (14-16, 18-25, and 29-32) have not been reported before. The cytotoxicity was measured using panel of seven cancer cell lines with/without MDR phenotype and non tumor MRC-5 and BJ fibroblasts. The preferential cytotoxicity to cancer cell lines, particularly to hematological tumors was observed, the bromo acids 5, 6 showed highest activity and selectivity against tumor cells.

Synthesis of cytotoxic 2,2-difluoroderivatives of dihydrobetulinic acid and allobetulin and study of their impact on cancer cells.

In this article, we describe the preparation and cytotoxic properties of a small focused library of lupane and 18α-oleanane triterpenoids that contain a combination of two structural motifs known to enhance the biological activities. First, we introduced two fluorine atoms to position 2 of the skeleton. Second, we synthesized a set of hemiester prodrugs, which were intended to increase the solubility and activity. Starting from betulin, we obtained two hydroxyketones (derivatives of dihydrobetulinic acid and allobetulin) and their fluorination using DAST provided 2,2-difluoro-3-oxo-compounds as the main products. Then the 3-oxo group in each derivative was reduced by NaBH4 to obtain 3β-hydroxy compounds suitable for modifying by various hemiesters. We prepared 21 compounds, 11 of them new, their cytotoxicity was tested on T lymphoblastic leukemia CCRF-CEM cells first and the most active derivatives were selected for screening on another six tumor and two non-tumor cell lines. All of them showed selectivity against cancer lines with therapeutic index between 2 and 8. All hemiesters had activity in the same range as the free hydroxyl derivatives and they would be suitable prodrugs for future in vivo experiments. Interestingly, all hemiesters of 2,2-difluorodihydrobetulonic acid had higher activity against p53 knock-out p53-/- cancer cell line than against the non-mutated analog. In active derivatives, the cell cycle was analyzed by flow cytometry and several compounds slowed down cell cycle progression through G0/G1 or S-phase.

Determination of selected bisphenols, parabens and estrogens in human plasma using LC-MS/MS

In this study, a novel liquid chromatography - tandem mass spectrometry method for the simultaneous determination of bisphenols (BPA, BPS, BPF, BPAF), parabens (methyl-, ethyl-, propyl-, butyl-, benzyl-paraben) and estrogens (estrone, estradiol, estriol) in human plasma is presented. Since all analytes possess the phenolic group, dansyl chloride derivatization was applied in order to gain high sensitivity. The method was validated according to FDA guidelines, and all validation requirements were satisfactory. The lower limits of quantifications were 41.6, 54.9, 43.5 and 150.8pg/mL for BPA, BPS, BPF and BPAF; 172, 149, 171, 134 and 202pg/mL for methyl-, ethyl-, propyl-, butyl- and benzyl-paraben; 10.5, 6.7 and 9.4pg/mL for estrone, estradiol and estriol, respectively. This is the first method allowing the determination of plasma bisphenols, parabens and estrogens in one run, and also the first determination of BPF levels in human plasma. The method was used to examine the plasma levels of healthy normospermic men, where three times higher plasma levels of BPF than BPA were found.

15N-labelled pyrazines of triterpenic acids

Triterpenoid pyrazines from our research group were found selectively cytotoxic on several cancer cell lines with IC50 in low micromolar range. This sparked our interest in preparing their labeled analogs for metabolic studies. In this work, we prepared a set of non-labeled pyrazines from seven triterpenoid skeletal types along with their 15N labelled analogs. In this work, we present the synthesis and characterization of the target 15N labelled pyrazines. Currently, these compounds are being studied in complex metabolic studies.

Progress in Targeted Alpha-Particle Therapy. What We Learned about Recoils Release from In Vivo Generators

This review summarizes recent progress and developments as well as the most important pitfalls in targeted alpha-particle therapy, covering single alpha-particle emitters as well as in vivo alpha-particle generators. It discusses the production of radionuclides like 211At, 223Ra, 225Ac/213Bi, labelling and delivery employing various targeting vectors (small molecules, chelators for alpha-emitting nuclides and their biomolecular targets as well as nanocarriers), general radiopharmaceutical issues, preclinical studies, and clinical trials including the possibilities of therapy prognosis and follow-up imaging. Special attention is given to the nuclear recoil effect and its impacts on the possible use of alpha emitters for cancer treatment, proper dose estimation, and labelling chemistry. The most recent and important achievements in the development of alpha emitters carrying vectors for preclinical and clinical use are highlighted along with an outlook for future developments.

Separation of curium from americium using composite sorbents and complexing agent solutions

The EXAm and the AmSel liquid–liquid extraction processes have been used as bases for the development of chromatographic systems for separation of curium(III) from americium(III). The liquid organic phases were replaced by composite sorbents with PAN binding matrix and complexing agent in nitric acid solutions were employed as aqueous phases. The influence of complexing agent and nitric acid concentrations on weight distribution coefficients and separation factor and the kinetics of the actinide uptake were determined in batch experiments with trace amounts of 241Am and 244Cm. The efficiency of Cm(III) separation from Am(III) was evaluated in column experiment.

Environmental Aspects of Radiopharmaceuticals Extraction and Translocation of Ra 223 in Plants

Radiopharmaceuticals represent an attractive and efficient treatment of oncological diseases. Medical radionuclide use might bring a particular safety issue with penetration of a radioactive material into environment via urinal and colonal excretion. Therefore, the waste water cleaning and decontamination of food chain ought to be studied. Radium-223 is FDA and EMA approved therapeutic radionuclide for the treatment of bone metastases originating from castration resistant prostate cancer. Its introduction to clinical praxis opened the possibility of Radium retention and translocation into roots and shoot plant parts in the ecosystem. Though Ra uptake was investigated in vitro on cultivated plants Avena sativa and Zea mays using electronic autoradiography. Stimulators (Atonik, Racine, Rexan, Sunagreen, Stimulator Z) increasing the water transport, the plant stress management additives (Vermaktiv Stimul and Vermaktiv RP), together with the chelating agent ethylenediaminetetraacetic acid (EDTA) were added to the cultivars. Results of plants growth without any regulators indicate over 90% uptake of Ra in root system with minimal translocation into other parts. An addition of growth regulators decreased the overall uptake, however significantly increased Radium translocation into shoot parts. Surprisingly, an addition of EDTA decreases the overall retention in oat under the lowest detectable limit, nevertheless an increased translocation to shoot parts was observed. Experiments reveal potential of phytoextraction technologies for waste water cleaning in hospitals, on the other hand, indicates possibility of food chain contamination particularly when growth regulators were used. Keywords—uptake, radium-223, radiopharmaceuticals. environment contamination

Abstract 2641: Diversity of anticancer mechanisms of action of tritepenoids derived from betulinic acid

Betulinic acid and its derivatives are cytotoxic compounds with the specific effect to the tumor cells. In our betulinic acid derivative chemical library we have identified several potent compounds which were highly active in the wide spectrum of the cancer cell lines and the primary tumour cells as well as. Cell cycle modulating effects of selected compounds were monitored and surprisingly the compounds with different effect on the cell cycle profile were observed. Some derivatives were inhibiting the progression through the early S or G2-M phases and some were rapidly inducing the apoptosis. To identify the mechanisms of the action we have used genomic expressional (Affymetrix) and proteomic profiling (LC-MS/MS, SILAC). Derivatives inducing the rapid induction of the apoptosis were identified to regulate and affect the mRNA expression of the genes and proteins closely related to the oxidative stress and to the detoxification enzymes. Glutathione detoxification pathway and the oxidative phosphorylation cascade (upregulation of glutathion peroxidase, peroxiredoxine, thioredoxin, sulfiredoxine, cytochrome c oxidase, down regulation of glutathion S transferase) were affected and direct interaction of derivatives with cytochrome c was observed. All previously mentioned mechanisms are closely related to the mitochondrion damage. However, derivatives inducing the G2/M block were also increasing the expression of the tubulin chaperone cofactor A (TBCA) and the underexpression of other proteins like chaperonin containing TCP1, subunit 3 (CCT3) related to tubulin polymerization pathway and cell cycle regulation. Tubulin chaperone cofactors were recently suggested to be potential targets for the cancer therapy and current works are showing the upregulation of tubulin chaperone cofactor C correlated to the suppression of the tumor growth and increased chemosensitivity in resistant cancer phenotypes. This work was supported by the GACR 301/09/P433, GACR 305/09/1216 and CZ.1.05/2.1.00/01.0030). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2641. doi:10.1158/1538-7445.AM2011-2641