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Carbapenems are vital antibiotics, but their efficacy is increasingly compromised by metallo-beta-lactamases (MBLs). Here we report the discovery and optimization of potent broad-spectrum MBL inhibitors. A high-throughput screen for NDM-1 inhibitors identified indole-2-carboxylates (InCs) as potential beta-lactamase stable beta-lactam mimics. Subsequent structure-activity relationship studies revealed InCs as a new class of potent MBL inhibitor, active against all MBL classes of major clinical relevance. Crystallographic studies revealed a binding mode of the InCs to MBLs that, in some regards, mimics that predicted for intact carbapenems, including with respect to maintenance of the Zn(II)-bound hydroxyl, and in other regards mimics binding observed in MBL-carbapenem product complexes. InCs restore carbapenem activity against multiple drug-resistant Gram-negative bacteria and have a low frequency of resistance. InCs also have a good in vivo safety profile, and when combined with meropenem show a strong in vivo efficacy in peritonitis and thigh mouse infection models.

Subanesthetic rather than anesthetic doses are thought to bring the rapid antidepressant effects of the NMDAR (N-methyl-D-aspartate receptor) antagonist ketamine. Among molecular mechanisms, activation of BDNF receptor TrkB along with the inhibition of GSK3 beta (glycogen synthase kinase 3 beta) are considered as critical molecular level determinants for ketamine's antidepressant effects. Hydroxynorketamines (2R,6R)-HNK and (2S,6S) HNK), non-anesthetic metabolites of ketamine, have been proposed to govern the therapeutic effects of ketamine through a mechanism not involving NMDARs. However, we have shown that nitrous oxide, another NMDAR blocking anesthetic and a putative rapid-acting antidepressant, evokes TrkB-GSK3 beta signaling alterations during rebound slow EEG (electroencephalogram) oscillations. We investigated here the acute effects of ketamine, 6,6-d(2)-ketamine (a ketamine analogue resistant to metabolism) and cis-HNK that contains (2R,6R) and (2S,6S) enantiomers in 1:1 ratio, on TrkB-GSK3 beta signaling and concomitant electroencephalographic (EEG) alterations in the adult mouse cortex. Ketamine dose-dependently increased slow oscillations and phosphorylations of TrkB(Y816) and GSK3 beta(59) in crude brain homogenates (i.e. sedative/anesthetic doses ( > 50 mg/kg, i.p.) produced more prominent effects than a subanesthetic dose (10 mg/kg, i.p.)). Similar, albeit less obvious, effects were seen in crude synaptosomes. A sedative dose of 6,6-d(2)-ketamine (100 mg/kg, i.p.) recapitulated the effects of ketamine on TrkB and GSK3 beta phosphorylation while cis-HNK at a dose of 20 mg/kg produced negligible acute effects on TrkB-GSK3 beta signaling or slow oscillations. These findings suggest that the acute effects of ketamine on TrkB-GSK3 beta signaling are by no means restricted to subanesthetic (i.e. antidepressant) doses and that cis-HNK is not responsible for these effects.

Membrane-bound pyrophosphatases are homodimeric integral membrane proteins that hydrolyze pyrophosphate into orthophosphates, coupled to the active transport of protons or sodium ions across membranes. They are important in the life cycle of bacteria, archaea, plants, and parasitic protists, but no homologous proteins exist in vertebrates, making them a promising drug target. Here, we report the first nonphosphorus allosteric inhibitor of the thermophilic bacterium Thermotoga maritima membrane-bound pyrophosphatase and its bound structure together with the substrate analog imidodiphosphate. The unit cell contains two protein homodimers, each binding a single inhibitor dimer near the exit channel, creating a hydrophobic clamp that inhibits the movement of beta-strand 1-2 during pumping, and thus prevents the hydrophobic gate from opening. This asymmetry of inhibitor binding with respect to each homodimer provides the first clear structural demonstration of asymmetry in the catalytic cycle of membrane-bound pyrophosphatases.

The first total synthesis of the marine bromotyrosine purpurealidin I (1) using trifluoroacetoxy protection group and its dimethylated analog (29) is reported along with 16 simplified bromotyrosine derivatives lacking the tyramine moiety. Their cytotoxicity was evaluated against the human malignant melanoma cell line (A-375) and normal skin fibroblast cells (Hs27) together with 33 purpurealidin-inspired simplified amides, and the structure&ndash, activity relationships were investigated. The synthesized simplified analogs without the tyramine part retained the cytotoxic activity. Purpurealidin I (1) showed no selectivity but its simplified pyridin-2-yl derivative (36) had the best improvement in selectivity (Selectivity index 4.1). This shows that the marine bromotyrosines are promising scaffolds for developing cytotoxic agents and the full understanding of the elements of their SAR and improving the selectivity requires further optimization of simplified bromotyrosine derivatives.

Membrane-bound pyrophosphatases are homodimeric integral membrane proteins that hydrolyse pyrophosphate into orthophosphates, coupled to the active transport of protons or sodium ions across membranes. They are important in the life cycle of bacteria, archaea, plants, and protist parasites, but no homologous proteins exist in vertebrates, making them a promising drug target. Here, we report the first non-phosphorous allosteric inhibitor (Ki of 1.8 ± 0.3 μM) of the thermophilic bacterium Thermotoga maritima membrane-bound pyrophosphatase and its bound structure at 3.7 Å resolution together with the substrate analogue imidodiphosphate. The unit cell contains two protein homodimers, each binding a single inhibitor dimer near the exit channel, creating a hydrophobic clamp that inhibits the movement of β-strand 1–2 during pumping, and thus preventing the hydrophobic gate from opening. This asymmetry of inhibitor binding with respect to each homodimer provide the first clear demonstration of asymmetry in the catalytic cycle of membrane-bound pyrophosphatases.

Electroconvulsive therapy (ECT) remains among the most efficient antidepressants but it seldom brings immediate remedy. However, a subanesthetic dose of NMDA-R (N-methyl-D-aspartate receptor) blocker ketamine ameliorates symptoms of depression already within hours. Glutamatergic excitability and regulation of TrkB neurotrophin receptor and GSK3β (glycogen synthase kinase 3β) signaling are considered as molecular-level determinants for ketamine’s antidepressant effects. Recent clinical observations suggests that nitrous oxide (N2O, “laughing gas”), another NMDA-R blocking dissociative anesthestic, also produces rapid antidepressant effects but the underlying mechanisms remain essentially unstudied. In this animal study we show that N2O, with a clinically relevant dosing regimen, evokes an emergence of rebound slow EEG (electroencephalogram) oscillations, a phenomenon considered to predict the efficacy and onset-of-action ECT. Very similar rebound slow oscillations are induced by subanesthetic ketamine and flurothyl (a treatment analogous to ECT). These responses become best evident upon drug withdrawal, i.e. after the peak of acute pharmacological actions, when their most prominent effects on cortical excitability have subsided. Most importantly, TrkB and GSK3β signaling remain unchanged during N2O administration (ongoing NMDA-R blockade) but emerge gradually upon gas withdrawal along with increased slow EEG oscillations. Collectively these findings reveal that rapid-acting antidepressants produce cortical excitability that triggers “a brain state” dominated by ongoing slow oscillations, sedation and drowsiness during which TrkB and GSK3β signaling alterations are induced.

A series of 2-aminobenzothiazole and benzimidazole analogs based on the clathrodin scaffold was synthesized and investigated for their antimicrobial and antiproliferative activities as well as for their effects in hepatitis C virus (HCV) replicon model. Compound 7, derived from 2-aminobenzothiazole, exhibited moderate antimicrobial activity only against the Gram-positive bacterium, Enterococcus faecalis. In the antiviral assay, compounds 4d and 7 were found to suppress the HCV replicon by >70%, but also to exhibit cytotoxicity against the host cells (35 and 44%, respectively). Compounds 4a and 7 demonstrated good activity in the antiproliferative assays on the human melanoma cell line A-375. To assess the selectivity of the effects between cancerous and noncancerous cells, a mouse fibroblast cell line was used. The IC50 values for compound 7 against the melanoma cell line A-375 and the fibroblast cell line BALB/c 3T3 were 16 and 71 µM, respectively, yielding fourfold selectivity toward the cancer cell line. These results suggest that compound 7 should be studied further in order to fully explore its potential for drug development.

Malaria continues to be a major global health problem, being particularly devastating in the African population under the age of five. Artemisinin-based combination therapies (ACTs) are the first-line treatment recommended by the WHO to treat Plasmodium falciparum malaria, but clinical resistance against them has already been reported. As a consequence, novel chemotypes are urgently needed. Herein we report a novel, in vivo active, fast-acting antimalarial chemotype based on a benzimidazole core. This discovery is the result of a medicinal chemistry plan focused on improving the developability profile of an antichlamydial chemical class previously reported by our group.

A set of 56 2-arylbenzimidazoles was designed, synthesized and tested against Leishmania donovani amastigotes. The left- and right-hand side rings of the molecule, as well as the amide linker were modified. Structurally different derivatives were screened on L. donovani axenic amastigotes at concentrations of 5, 15 and 50 μM, and the ten most active derivatives were selected for further testing. 2-Arylbenzimidazole derivative 24 was active against L. donovani -infected THP-1 cells showing 46% parasite inhibition at 5 μM.

Chlamydia pneumoniae is an intracellular bacterium that responds poorly to antibiotic treatment. Insufficient antibiotic usage leads to chronic infection, which is linked to disease processes of asthma, atherosclerosis, and Alzheimer's disease. The Chlamydia research lacks genetic tools exploited by other antimicrobial research, and thus other approaches to drug discovery must be applied. A set of 2-arylbenzimidazoles was designed based on our earlier findings, and 33 derivatives were synthesized. Derivatives were assayed against C. pneumoniae strain CWL-029 in an acute infection model using TR-FIA method at a concentration of 10 μM, and the effects of the derivatives on the host cell viability were evaluated at the same concentration. Fourteen compounds showed at least 80% inhibition, with only minor changes in host cell viability. Nine most potential compounds were evaluated using immunofluorescence microscopy on two different strains of C. pneumoniae CWL-029 and CV-6. The N-[3-(1H-benzimidazol-2-yl)phenyl]-3-methylbenzamide (42) had minimal inhibitory concentration (MIC) of 10 μM against CWL-029 and 6.3 μM against the clinical strain CV-6. This study shows the high antichlamydial potential of 2-arylbenzimidazoles, which also seem to have good characteristics for lead compounds.

I. E. Marko [Universite catholique de Louvain, Belgium]—Novel Orthoesters in Organic Synthesis. S. J. Harwood [GlaxoSmithKline, UK]—The Synthesis of Stable Labelled Ketamine. Th. Moenius, R. Salter, B. Wietfeld, P. Burtscher, C. Zueger [Novartis, Switzerland]—C-13, C-14, N-15 Labelling of the 4-Cyanobenzyl bromide – A Versatile Labelling Building Block. J. Clayden [University of Manchester, UK]—Synthesis and Stereocontrol with Lithiated Amides. R. Salter, K. Bordeaux, P. Burtscher, Y. Metz, Th. Moenius, I. Rodriguez, R. Ruetsch, R. Voges, C. Zueger [Novartis, Switzerland]—Isotopic Labelling of STI571 and its Metabolites in the Development of Gleevec®. J. M. Gardiner, W. Stimpson, N. Panchal [University of Manchester, UK], J. Herbert, G. J. Ellames [Sanofi-Aventis, UK]—Synthesis of Labelled Carbohydrates. M. Beller [Rostock University, Germany]—Homogeneous Catalysis – A Powerful Tool for the Synthesis of Pharmaceuticals. J. Kozempel, J. Kadeřavek, L. Lesetický, O. Lebeda [University of Prague, Czech]—Preparation of Ortho-radiohalogenated Phenylazides and Triazenes, Versatile Building Blocks for Indirect Labelling. K. Wahala, P. Kiuru, E. Leppala, M. Pohjoispaa, K. Parikka, B. Raffaelli [University of Helsinki, Finland]—Controlled Polydeuteration of Estrogenic Compounds. J. M. Herbert [Sanofi-Aventis, UK]—Catalyst Stability in Iridium-mediated Isotope Exchange. C. G. M. Janssen, W.L.M. Verluyten, M. Vliegen [Johnson & Johnson Pharmaceutical, Belgium]—Pd/C Catalysed Hydrogenolytic H/T Exchange in Solvents, Effects on Solvents and Product in a Bromine-tritium Exchange Reaction. P. Mading, F. Fuchtner, R. Bergmann, J. Pietzsch, C. Hultsch, F. Wust [Forschungszentrum Rossendorf, Germany]—A New Module-Assisted Synthesis of the Versatile, Bifunctional Labelling Agent [18F]SFB: From Radiochemistry to Applications. M. Scheunemann, J. Vercouillie, St. Fischer, J. Steinbach [Institut fur Interdisziplinare Isotopenforschung Leipzig, Germany], D. Sorger, U. Grosmann, O. Sabri, R. Schliebs [University Leipzig, Germany]—Syntheses and First Biological Evaluation of 18F Labelled Bicyclic Analogues of Vesamicol as Potential VAChT Imaging Agents.

Deuterated analogs of estrogen fatty acid esters are needed as internal standards for isotope dilution GC/MS analyses. We have developed a rapid and efficient synthesis for 2,4,16,16-D 4 -estrone palmitate, stearate, oleate, linoleate, and linolenate and the corresponding 2,4,16,16,17α-D 5 -estradiol fatty acid 17-mono and 3,17-diesters using analogous fatty acid chlorides or fatty acid anhydrides and 4-(dimethylamino)pyridine under microwave irradiation. Chemoselective hydrolysis of fatty acid diesters was carried out by KOH in t -BuOH.

The metabolism of endogenous estrogens is complicated and certain metabolic patterns may reflect an individual risk of estrogen-dependent diseases such as breast cancer. Since the 1960s we have been constantly involved in developing estrogen profiling methods, in the beginning using gas chromatography and later gas chromatography-mass spectrometry (GC-MS) in the selected ion monitoring mode (SIM) and finally utilizing isotope dilution (ID-GC-MS-SIM). The addition of the dietary phytoestrogens to the profile rendered the method even more complicated. The present work presents the final estrogen profile method for 15 endogenous estrogens, four lignans, seven isoflavonoids and coumestrol in one small urine sample (1/150th of a 24 h human urine sample, minimum 2.5-5 ml) with complete validation including investigations as to the precision, sensitivity, accuracy and specificity. The method does not include the minimal amounts of unconjugated estrogens in urine. It may also be used for animal (e.g. rat and mouse) urine using a minimum of 2 ml of usually pooled sample. Despite its complexity it was found to fulfill the reliability criteria, resulting in highly specific and accurate results. (C) 2004 Elsevier Ltd. All rights reserved. Journal of Steroid Biochemistry and Molecular Biology

In the search for novel anticancer drugs, the potassium channel KV10.1 has emerged as an interesting cancer target. Here, we report a new group of KV10.1 inhibitors, namely the purpurealidin analogs. These alkaloids are produced by the Verongida sponges and are known for their wide variety of bioactivities. In this study, we describe the synthesis and characterization of 27 purpurealidin analogs. Structurally, bromine substituents at the central phenyl ring and a methoxy group at the distal phenyl ring seem to enhance the activity on KV10.1. The mechanism of action of the most potent analog 5 was investigated. A shift of the activation curve to more negative potentials and an apparent inactivation was observed. Since KV10.1 inhibitors can be interesting anticancer drug lead compounds, the effect of 5 was evaluated on cancerous and non-cancerous cell lines. Compound 5 showed to be cytotoxic and appeared to induce apoptosis in all the evaluated cell lines. ispartof: PLoS One vol:12 issue:12 ispartof: location:United States status: published

The estrogen metabolite 2-methoxyestradiol was synthesized from estradiol bis-THP-ether which was 2-hydroxylated using the superbase LIDAKOR, trimethyl borate, and H 2 O 2 , then methylated and deprotected to obtain 2-methoxyestradiol in three steps and 61% yield. 2-Hydroxyestradiol was obtained by deprotecting the 2-hydroxyestradiol bis-THP-ether from the first step.

Chlamydia pneumoniae is a worldwide cause of various respiratory track diseases ranging from asymptomatic pharyngeal infection to severe, sometimes fatal pneumonia. We have previously identified 2-arylbenzimidazoles as highly active antichlamydial compounds. In this work the importance of conformational effects on the structure–activity relationship of these compounds was studied. To simplify calculations, properly substituted N -phenylbenzamides, or the corresponding heterocyclic compounds, and 2-arylbenzimidazoles were used as model compounds. They were energy minimized and the energy differences between certain conformations were calculated. The main finding was that the compounds which can more easily adopt a non-planar conformation show higher bioactivity. This finding can be utilized in designing new derivatives or in constructing a pharmacophore model.

The search for novel compounds of relevance to the treatment of diseases caused by trypanosomatid protozoan parasites continues. Screening of a large library of known bioactive compounds has led to several drug-like starting points for further optimisation. In this study, novel analogues of the monoamine uptake inhibitor indatraline were prepared and assessed both as inhibitors of trypanothione reductase (TryR) and against the parasite Trypanosoma brucei. Although it proved difficult to significantly increase the potency of the original compound as an inhibitor of TryR, some insight into the preferred substituent on the amine group and in the two aromatic rings of the parent indatraline was deduced. In addition, detailed mode of action studies indicated that two of the inhibitors exhibit a mixed mode of inhibition.

The aza-Diels-Alder reaction of αβ-unsaturated hydrazones is a general methodology that has been applied both to the synthesis of natural products and in the development of multicomponent reactions. Trends have emerged as to the effect of substituents on the efficiency of this reaction with substituents at the C2 and C4-positions of the aza-diene in general suppressing the reaction. Here we report that 4,5-dihydropyrazoles can function as substrates in this process despite the presence of substituents at both of these positions. A one pot, four chemical step sequence carried out under standard thermal or microwave conditions results in the formation of the corresponding pyridine-containing compounds. The scope of the reaction is explored and additional insights into the proposed mechanism of this reaction are provided.

A rapid and efficient deuteration procedure was developed for estrone. Irradiation of estrone in CF 3 COOD in a microwave oven gave [2,4,16,16- 2 H 4 ]-estrone in 95% yield. Ultrasound and CF 3 COOD reflux deuterations of estrone were also studied.

Background: Lipophilic estradiol derivatives carried by lipoprotein particles in blood may mediate antioxidant or endocrine effects. We developed a new quantitative method to determine the concentration of circulating lipophilic estradiol fatty acid esters in human early- and late-pregnancy serum and in ovarian follicular fluid.Methods: After extraction from serum or follicular fluid, estradiol fatty acid esters were separated from nonesterified estradiol by Sephadex LH-20 column chromatography. The estradiol ester fraction was hydrolyzed by saponification and further purified by several chromatographic steps. The hydrolyzed estradiol esters were measured by time-resolved fluoroimmunoassay.Results: The average estradiol fatty acid ester concentration in serum increased 10-fold during pregnancy, from 40.4 pmol/L (expressed as pmol/L estradiol; range, 25.0–64.2 pmol/L) in early pregnancy (n = 8) to 404 pmol/L (196–731 pmol/L) in late pregnancy (n = 10). The ratio of estradiol ester to nonesterified estradiol remained relatively constant during pregnancy, at 0.4–0.6%. In 10 follicular fluid samples, the mean estradiol ester concentration was 106 nmol/L (56.9–262 nmol/L). Compared with serum, a greater proportion of estradiol in follicular fluid (3.0–10%) was in the esterified form.Conclusion: The new method provides a means to measure circulating estradiol fatty acid ester concentrations in human pregnancy serum.