Supplementary Materials and Methods. Figure S1: MALDI-TOF spectrum of the single-stranded oligonucleotide products of primer extension experiment with human DNA polymerase γ, AB61-TP, primer Prim248 and template (bio)-OligoAterm.

The purpose of the paper is to draw readers' attention to the low stability of radioactively labelled compounds and its potential to distort experimental results. Different modes of decay of radioactively labelled compounds with the high specific activity are discussed. Attention is drawn to the decrease of the specific activity, compared to the freshly synthetized preparation, of the compounds multilabelled by tritium and repurified after several years of storage at -196 degrees C in liquid nitrogen. The possible pathways of decay are illustrated by the stability study of 9-([5'endo,6'endo-H-3(2)]norborn-2-yl)-6-chloro-purine. Full text English translation available in the on-line version.

Characterisation of G protein-coupled receptors (GPCR) relies on the availability of a toolbox of ligands that selectively modulate different functional states of the receptors. To uncover such molecules, we explored a unique strategy for ligand discovery that takes advantage of the evolutionary conservation of the 600-million-year-old oxytocin/vasopressin signalling system. We isolated the insect oxytocin/vasopressin orthologue inotocin from the black garden ant (Lasius niger), identified and cloned its cognate receptor and determined its pharmacological properties on the insect and human oxytocin/vasopressin receptors. Subsequently, we identified a functional dichotomy: inotocin activated the insect inotocin and the human vasopressin V-1b receptors, but inhibited the human V1aR. Replacement of Arg8 of inotocin by D-Arg8 led to a potent, stable and competitive V1aR-antagonist ([D-Arg8]-inotocin) with a 3,000-fold binding selectivity for the human V1aR over the other three subtypes, OTR, V1bR and V2R. The Arg8/D-Arg8 ligand-pair was further investigated to gain novel insights into the oxytocin/vasopressin peptide-receptor interaction, which led to the identification of key residues of the receptors that are important for ligand functionality and selectivity. These observations could play an important role for development of oxytocin/vasopressin receptor modulators that would enable clear distinction of the physiological and pathological responses of the individual receptor subtypes.

In our project, ghrelin analogs possessing enhanced stability and potential to significantly increase food intake were used. Three newly synthesized ghrelin analogs with fatty acid residues consisting of 8, 10, and 14 carbon atoms were studied. The main goal of this work was to develop a suitable analytical method for the determination of the stability of the novel ghrelin analogs in plasma. An appropriate liquid chromatography-mass spectrometry method was developed and optimized. The results obtained were compared with the data measured by using a commercial enzyme-linked immunosorbent assay kit, and a good correlation was found. A preparation strategy for plasma samples was optimized and consisted of simple dilution of the plasma samples followed by direct injection onto a very short monolithic column in combination with mass spectrometric detection. The developed analytical method was utilized for the determination of the stability of the prepared lipopeptides in plasma and for the quantification of the lipopeptides in a preliminary pharmacokinetic study. The feasibility of the developed separation method was clearly demonstrated. Accuracy and precision were within 80-120% and ±20% limits, respectively. Calibration curves were constructed in the range of 1-250 μg/mL.

Brassinosteroids (BRs) are plant-specific steroid hormones that play essential roles in the regulation of many important physiological processes in plant life. Their extremely low concentrations (~pmoles/g FW) in plant tissue and huge differences in polarity of individual members within the BR family hamper their detection and quantification. To address this problem, an immunoaffinity sorbent with broad specificity and high capacity for different BR metabolites containing a monoclonal antibody (mAb) against a BR spacer (20S)−2α,3α-dihydroxy-7-oxa-7α-homo-5α-pregnane-6-one-20 carboxylic acid (BR4812) was used for the rapid and highly selective isolation of endogenous BRs containing a 2α,3α-diol in ring A from minute plant samples. This enrichment procedure was successfully applied as a sample preparation method prior to quantitative analysis of BRs in real plant tissues by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Use of immunoaffinity chromatography (IAC) increased the sensitivity of the UHPLC-MS/MS analysis owing to improvements in the BR signal-to-noise ratio (S/N) and matrix factor (MF). Although MF values of BRs analyzed in classical samples ranged from 8.9% to 47.4%, MF values for the IAC purified samples reached 44.5–96.6%. Thus, the developed IAC-UHPLC-MS/MS approach was shown to be a simple, robust, effective and extremely fast procedure requiring minute amounts of plant samples suitable for the quantitative profiling of many BR metabolites, helping to overcome the major problems associated with their determination in very complex plant matrices.

Compound 1 is a selective and potent agonist of the G protein-coupled receptor GPR139 (EC50 = 39 nM).

Supporting Information 1 2 Development of a human vasopressin V1a-receptor antagonist from an 3 evolutionary-related insect neuropeptide 4 5 6 Maria Giulia Di Giglio, Markus Muttenthaler, Kasper Harpsøe, Zita Liutkeviciute, Peter Keov, 7 Thomas Eder, Thomas Rattei, Sarah Arrowsmith, Susan Wray, Ales Marek, Tomas Elbert, 8 Paul F. Alewood, David E. Gloriam and Christian W. Gruber 9 10 Deleted: From ants to man:

The radioactively labelled 6-amino-5-nitroso-uracil (1) and 5-acetyl-6-amino-1,3-dimethyl-uracil (2) were required for metabolic studies to assess their suitability as drug candidates. A common precursor for both compounds was [cyano-14 C]cyanoacetic acid (6), readily prepared from potassium [14 C]cyanide. ACS reagents, namely, diethyl ether, acetic acid and acetic anhydride, had to be rigorously repurified to achieve a successful synthesis of 14 C-labelled compounds on a tenth-of-a-milligramme scale. 6-Amino-5-nitroso-[6-14 C]uracil (1-14 C) (0.55 mCi) was prepared with radiochemical purity > 98% and specific activity (SA) = 55.6 mCi/mmol. 5-Acetyl-6-amino-1,3-dimethyl-[6-14 C]uracil (2-14 C) (8 mCi) was prepared with radiochemical purity > 97% and SA = 55.6 mCi/mmol. It has been shown that a SA assay can be made from standard 13 C NMR spectra, thus avoiding the need to perform lengthier inverse-gated 13 C NMR experiments.

3-Hydroxycyclopent-1-ene-1-carboxylic acid (HOCPCA (1)) is a potent ligand for high-affinity γ-hydroxybutyric acid binding sites in the central nervous system. Various approaches to the introduction of a hydrogen label onto the HOCPCA skeleton are reported. The outcomes of the feasible C─H activation of olefin carbon (C-2) by iridium catalyst are compared with the reduction of the carbonyl group (C-3) by freshly prepared borodeuterides. The most efficient iridium catalysts proved to be Kerr bulky phosphine N-heterocyclic species providing outstanding deuterium enrichment (up to 91%) in a short period of time. The highest deuterium enrichment (>99%) was achieved through the reduction of ketone precursor 2 by lithium trimethoxyborodeuteride. Hence, analogical conditions were used for the tritiation experiment. [3 H]-HOCPCA selectively labeled on the position C-3 was synthetized with radiochemical purity >99%, an isolated yield of 637 mCi and specific activity = 28.9 Ci/mmol.

A regiospecific and enantiospecific synthesis of tritium-labeled 28-homocastasterone is reported. Appropriate chlorocarbonate, efficiently synthesized from the starting 28-homocastasterone in an overall yield of 46%, undergoes catalytic tritium dechlorination by the T2/Pd[0]/Et3N system, providing 28-[3 beta-H-3] homocastasterone, in a good yield, radiochemical purity(>97%), and with a high specific activity (5.8 Ci/mmol).

Abstract7-(2-Thienyl)-7-deazaadenosine (AB61) showed nanomolar cytotoxic activities against various cancer cell lines but only mild (micromolar) activities against normal fibroblasts. The selectivity of AB61 was found to be due to inefficient phosphorylation of AB61 in normal fibroblasts. The phosphorylation of AB61 in the leukemic CCRF-CEM cell line proceeds well and it was shown that AB61 is incorporated into both DNA and RNA, preferentially as a ribonucleotide. It was further confirmed that a triphosphate of AB61 is a substrate for both RNA and DNA polymerases in enzymatic assays. Gene expression analysis suggests that AB61 affects DNA damage pathways and protein translation/folding machinery. Indeed, formation of large 53BP1 foci was observed in nuclei of AB61-treated U2OS-GFP-53BP1 cells indicating DNA damage. Random incorporation of AB61 into RNA blocked its translation in an in vitro assay and reduction of reporter protein expression was also observed in mice after 4-hour treatment with AB61. AB61 also significantly reduced tumor volume in mice bearing SK-OV-3, BT-549, and HT-29 xenografts. The results indicate that AB61 is a promising compound with unique mechanism of action and deserves further development as an anticancer agent. Mol Cancer Ther; 15(5); 922–37. ©2016 AACR.

The brassinosteroids (BRs) are a class of native plant growth regulating substances with high biological activity even at very low concentration. These compounds have been rigorously explored and it has been found that they are not only growth regulators in plants but also promising antiviral agents. Recently, it has been reported that natural BRs exhibit relatively interesting anticancer activities. Up to now, the basic anticancer potential of BRs against several normal and human cancer cell lines has been determined. Natural BRs, at micromolar concentrations, impart cell growth-inhibitory responses in several human cancer cell lines without affecting the normal cells. To study the mechanism of action of BRs at the molecular level, the corresponding isotopically labelled compounds are essential. The latter BRs are essential for the investigation of biosynthesis, metabolism, transport and distribution in plants. This venture ultimately led us to explore the labeling of BRs by isotopes of hydrogen and carbon and the related technique to do this. The present review will shed light on the synthetic avenues in this field from the time of the discovery of labelled BRs up until their most recent advances.

A detailed study of regiospecific silylation, oxidation, and reduction of sterols is reported. The different reactivity of 2α and 3α hydroxy groups is demonstrated on a 2α,3α-dihydroxy-5α-pregnane-6,20-dione as a model substrate. A regiospecific silylation of the 2α-alcohol occurred when silyl chloride was used under mild reaction conditions. The selective oxidation of one of the 2α,3α-diols by dimethyldioxirane or the Dess–Martin periodinane led to a mixture of α-hydroxyketones with high prevalence of 3,6,20-trione over 2,6,20-trione in the ratio 10:1. A regiospecific reduction of a model substrate by lithium tri-tert-butoxyaluminium hydride gives the C6 alcohol.

A convenient method for the synthesis of tritium-labeled brassinosteroids with very high specific activity is reported.

A new catalytic enantiospecific approach to the synthesis of epibrassinosteroids (and other polyhydroxylated steroids) regiospecifically labeled by heavy isotopes of hydrogen is reported. Chlorocarbonate, efficiently synthesized from a-hydroxy ketone by a reaction with triphosgene, undergoes reductive tritium dechlorination catalyzed by the [Pd-0]/Et3N system, providing 24-[3 beta-H-3]epicastasterone and 24-[3 beta-H-3]epibrassinolide, respectively, in good yield and with high specific activity (5.8 Ci/mmol; 20% tritium enrichment per molecule). (C) 2015 Elsevier Ltd. All rights reserved.

Department of Drug Design and Pharma Sciences, University of Copenhagen, Denma Amsterdam Institute for Molecules, Medic Medicinal Chemistry, Faculty of Science, V 1083, 1081 HV Amsterdam, The Netherland Institute of Organic Chemistry and Biochem Republic, 16610, Prague 6, Czech Republic † Electronic supplementary information reductive debromination reactions characterisation of all compounds. Detai assays, clog P and log S calculations. See ‡ These authors contributed equally. Cite this: RSC Adv., 2016, 6, 947

6-Chloropurines substituted at the position 9 with variously modified bicyclic skeletons represent promising antiviral and anticancer agents. This work aimed to investigate the transport mechanisms of 9-[(1R*,2R*,4S*)-bicyclo[2.2.1]hept-2-yl]-6-chloro-9H-purine (9-norbornyl-6-chloropurine, NCP) and their relationship to the metabolism and biological activity of the compound. Transport experiments were conducted in CCRF-CEM cells using radiolabeled compound ([(3)H]NCP). The pattern of the intracellular uptake of [(3)H]NCP in CCRF-CEM cells pointed to a combination of passive and facilitated diffusion as prevailing transport mechanisms. NCP intracellular metabolism was found to enhance its uptake by modifying NCP concentration gradient. The transport kinetics reached steady state under the conditions of MRP and MDR proteins blockade, indicating that NCP is a substrate for these efflux pumps. Their inhibition also increased the cytotoxicity of NCP. Our findings suggest that the novel nucleoside analog NCP has potential to become a new orally available antileukemic agent due to its rapid membrane permeation.

The aim of this work was to synthesize deuterated and tritiated analogues of highly oxidized lupane derivatives known from our group. We selected compounds that previously showed very high cytotoxic activity on multiple cancer cell lines in order to further investigate the mechanism of their action. From starting material (compounds 1–4), we obtained benzyl platanate (5) and its reaction with deuteromethyltriphenylphosphonium iodide gave deuterated compound 6. Following benzyl deprotection gave free acid 7 and oxidation with SeO2 gave 30-oxo-[29-2H2]lup-20(29)-en-28-oic acid (8), which is one of the most active compounds synthesized in our group to date (IC50 6 μmol/L on CEM cell line). The alkylation of benzyl 2-hydroxy-3-oxolupa-1,20(29)-dien-28-oate (9) with methyliodide or deuteromethyliodide followed by a series of deprotection and hydrogenation steps gave compounds 10–14, where 2β-[31-2H3]methoxy-3-oxolupan-20(29)-en-28-oic acid (13) is especially interesting, it showed lower activity on CEM cell line (IC50 10 μmol/L) however, it was very active against Ph1—positive human leukemia BV-173 (IC50 0.91 μmol/L) and against human myelogenous leukemia K562 (IC50 0.52 μmol/L). Selectively labelled [3α-2H] and [3α-3H] methyl 3β-acetoxy-21,22-dioxolup-18-en-28-oates 24, 25 were prepared in three steps by reduction of corresponding 3-oxo derivatives and they showed moderate activity on CEM cell line (IC50 10 μmol/L). In total, 11 labelled compounds (6–8, 11, 14, 18, 19, 21, 22, 24 and 25) have not been reported before.