Petr Konečný

Synthesis and Significant Cytostatic Activity of 7-Hetaryl-7-deazaadenosines

A series of 7-aryl- and 7-hetaryl-7-deazaadenosines was prepared by the cross-coupling reactions of unprotected or protected 7-iodo-7-deazaadenosines with (het)arylboronic acids, stannanes, or zinc halides. Nucleosides bearing 5-membered heterocycles at the position 7 exerted potent in vitro antiproliferative effects against a broad panel of hematological and solid tumor cell lines. Cell cycle analysis indicated profound inhibition of RNA synthesis and induction of apoptosis in treated cells. Intracellular conversion to triphosphates has been detected with active compounds. The triphosphate metabolites showed only a weak inhibitory effect on human RNA polymerase II, suggesting potentially other mechanisms for the inhibition of RNA synthesis and quick onset of apoptosis. Initial in vivo evaluation demonstrated an effect of 7-(2-thienyl)-7-deazaadenine ribonucleoside on the survival rate in syngeneic P388D1 mouse leukemia model.

Synthesis, Cytostatic, Antimicrobial, and Anti-HCV Activity of 6-Substituted 7-(Het)aryl-7-deazapurine Ribonucleosides

A series of 80 7-(het)aryl- and 7-ethynyl-7-deazapurine ribonucleosides bearing a methoxy, methylsulfanyl, methylamino, dimethylamino, methyl, or oxo group at position 6, or 2,6-disubstituted derivatives bearing a methyl or amino group at position 2, were prepared, and the biological activity of the compounds was studied and compared with that of the parent 7-(het)aryl-7-deazaadenosine series. Several of the compounds, in particular 6-substituted 7-deazapurine derivatives bearing a furyl or ethynyl group at position 7, were significantly cytotoxic at low nanomolar concentrations whereas most were much less potent or inactive. Promising activity was observed with some compounds against Mycobacterium bovis and also against hepatitis C virus in a replicon assay.

Synthesis and biological activity evaluation of hydrazone derivatives based on a Tröger’s base skeleton

We report the design and synthesis of novel anticancer agents based on bis-hydrazones separated by a rigid Trögers base skeleton. This novel approach combines a biologically active moiety (hydrazone) with this scaffold (Trögers base) to construct DNA intercalators. Evaluation of the anticancer activity of these agents using seven cancer cell lines and two healthy cell lines found that several derivatives had potent anticancer activity and excellent selectivity indexes toward cancer cells. The antimicrobial activities were tested on a set of thirteen bacterial stains, but the prepared compounds were not active. Complexation studies using biologically important metal ions demonstrated that these compounds are able to bind Cu(2+), Fe(3+), Co(2+), Ni(2+) and Zn(2+). DNA intercalation studies showed that the compounds themselves do not interact with DNA, but their metallocomplexes do interact, most likely via intercalation into DNA.

Structural Basis for Inhibition of Mycobacterial and Human Adenosine Kinase by 7-Substituted 7-(Het)aryl-7-deazaadenine Ribonucleosides

Adenosine kinase (ADK) from Mycobacterium tuberculosis (Mtb) was selected as a target for design of antimycobacterial nucleosides. Screening of 7-(het)aryl-7-deazaadenine ribonucleosides with Mtb and human (h) ADKs and testing with wild-type and drug-resistant Mtb strains identified specific inhibitors of Mtb ADK with micromolar antimycobacterial activity and low cytotoxicity. X-ray structures of complexes of Mtb and hADKs with 7-ethynyl-7-deazaadenosine showed differences in inhibitor interactions in the adenosine binding sites. 1D (1)H STD NMR experiments revealed that these inhibitors are readily accommodated into the ATP and adenosine binding sites of Mtb ADK, whereas they bind preferentially into the adenosine site of hADK. Occupation of the Mtb ADK ATP site with inhibitors and formation of catalytically less competent semiopen conformation of MtbADK after inhibitor binding in the adenosine site explain the lack of phosphorylation of 7-substituted-7-deazaadenosines. Semiempirical quantum mechanical analysis confirmed different affinity of nucleosides for the Mtb ADK adenosine and ATP sites.

Pathophysiologically relevant in vitro tumor models for drug screening.

The alarming rate of failure of clinical trials is a major hurdle in cancer therapy that partly results from the inadequate use of in vitro tumor models for the screening of promising hits and leads in preclinical studies. 2D cultures of cancer cell lines that are primarily used for drug screening do not adequately recapitulate tumor microenvironment (TME) complexities compared with 3D cancer cell cultures and tumor-derived primary cell cultures. In this review, we focus on the potential use of in vitro tumor models that reproduce in vivo tumor complexities for effective drug selection in the preclinical stages of drug development.

6-Alkyl-, 6-aryl- or 6-hetaryl-7-deazapurine ribonucleosides as inhibitors of human or MTB adenosine kinase and potential antimycobacterial agents

Title 6-alkyl-, 6-aryl- and 6-hetaryl-7-deazapurine ribonucleosides previously known as nanomolar cytostatics were found to be potent inhibitors of either human or mycobacterial (MTB) adenosine kinase (ADK). Several new derivatives bearing bulky substituents at position 6 were non-cytotoxic but selectively inhibited MTB ADK. However, most of the nucleosides (ADK inhibitors) as well as their octadecylphosphate prodrugs were inactive in the whole cell assay of inhibition of Mycobacterium bovis growth. 6-Methyl-7-deazapurine ribonucleoside was found to be a potent antimycobacterial agent.

Trilobolide-steroid hybrids: Synthesis, cytotoxic and antimycobacterial activity

Graphical abstract Figure. No Caption available. HighlightsFive trilobolide‐steroid hybrids were synthesized using CuAAC approach.Cytotoxicity was tested on a 12 cancer and 3 non‐cancerous cell lines. The most cytotoxic compounds were tested for cell‐cycle analysis on CCRF‐CEM line.The potency on androgen (AR) and estrogen (&agr;,&bgr;‐ER) receptors was examined.Impact on cell morphology was studied by live‐cell microscopy.Compounds were tested against 8 sensitive and multiresistant bacterial and Candida strains. Abstract 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 10.6 &mgr;M.

Cell cycle profiling by image and flow cytometry: The optimised protocol for the detection of replicational activity using 5-Bromo-2′-deoxyuridine, low concentration of hydrochloric acid and exonuclease III

The approach for the detection of replicational activity in cells using 5-bromo-2′-deoxyuridine, a low concentration of hydrochloric acid and exonuclease III is presented in the study. The described method was optimised with the aim to provide a fast and robust tool for the detection of DNA synthesis with minimal impact on the cellular structures using image and flow cytometry. The approach is based on the introduction of breaks into the DNA by the low concentration of hydrochloric acid followed by the subsequent enzymatic extension of these breaks using exonuclease III. Our data showed that the method has only a minimal effect on the tested protein localisations and is applicable both for formaldehyde- and ethanol-fixed cells. The approach partially also preserves the fluorescence of the fluorescent proteins in the HeLa cells expressing Fluorescent Ubiquitin Cell Cycle Indicator. In the case of the short labelling pulses that disabled the use of 5-ethynyl-2′-deoxyuridine because of the low specific signal, the described method provided a bright signal enabling reliable recognition of replicating cells. The optimized protocol was also successfully tested for the detection of trifluridine, the nucleoside used as an antiviral drug and in combination with tipiracil also for the treatment of some types of cancer.

Abstract 4492: Novel carborane based inhibitors of carbonic anhydrase IX

Carborane-based compounds have emerged as promising lead structures for the development of carbonic anhydrase (CA) inhibitors. The aim of this study is to evaluate the effect of new carboranes with functional sulfonamide residues on CAIX function. CAIX is a transmembrane isoform of carbonic anhydrase with an extracellular-facing catalytic site and therefore is well positioned to act in the control of tumor pH, and is overexpressed in various solid tumors. The function of CAIX can be inhibited by CAIX selective sulphonamides, and the inhibition perturbs the in vitro survival, under hypoxia conditions. To investigate the potency of carboranes to inhibit CAIX, at cellular level, carboranes were chosen based on their enzymatic activity against CAIX and CAII, in vitro from a library of 28 new carboranes with sulphonamide residues (IOCB, ASCR, v.v.i., Czech Republic). 3 carboranes, viz., CB-30, CB-31, and CB-33, showed high binding constant (10, 16, and 64 nM, respectively) and high selectivity for CAIX, and were studied further. Extracellular pH in cell cultures was measured, in parallel with measurements of cellular cytotoxicity, in both 2D and 3D culture systems. At cellular level, the activity of CB-30 and CB-31 was significantly lower than CB-33, due to increased binding of CB-30 (99.7%) and CB-31 (99.3%) to the plasma proteins, compared with 93.3% binding for CB-33. This indicated that the activity at cellular level was preserved by the free fraction of CB-33. Moreover, CB-33 comparatively showed the highest change of extracellular pH under hypoxia conditions, and decreased migration of cells in anti-metastatic assay. Further, we developed a new method of Raman spectroscopy, to locate the distribution of carboranes in cells, and determined the distribution pattern of CB-30 and CB-31 in HT-29 cells, under hypoxia conditions. The highest Raman signal was detected on the cell membrane. Furthermore, we investigated the pharmacodynamics and pharmacokinetics profiles of carboranes. All tested carboranes were distributed using ADME methods, according to their plasma and microsomal stability, plasma protein binding, and presence of passive and active transport in cells. The rapidity of drug metabolism, suitability of carboranes for per-oral administration, and the ability of carboranes to penetrate through the blood brain barrier were then evaluated. Pharmacokinetic analysis is currently ongoing process, based on which anti-tumor studies will be performed, followed by the measurement of pH for intra-tumoral alteration. Our results show the ability of herein tested carboranes to inhibit CAIX at enzymatic and cellular level. In future, novel carboranes with functional sulfonamide residues may serve as selective inhibitors of CAIX, and potential drugs in anticancer treatment. Acknowledgement: This work was supported by ProMedChem (CZ.1.07/2.3.00/30.0060), and BIOMEDREG (CZ.1.05/2.1.00/01.0030). Citation Format: Jana Stěpankova, Pavlina Rezacova, Jiři Brynda, Monika Harvanova, Vlastimil Masek, Alice Nova, Michal Siller, Viswanath Das, Dalibor Doležal, Bohumir Gruner, Vaclav Sicha, Petr Konecný, Pawel Znojek, Petr Džubak, Marian Hajduch. Novel carborane based inhibitors of carbonic anhydrase IX. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4492. doi:10.1158/1538-7445.AM2015-4492