Syntheses and Cytotoxicities of Quinazolinone-Based Conjugates.

Two novel series of quinazolinone-based hybrids, including quinazolinone-1,3,4-oxadiazoles (10a-l) and quinazolinone-1,3,4-oxadiazole-benzimidazoles (8a-e), were designed and synthesized and their cytotoxic activities against three human cancer cell lines, lung cancer (A549), cervical cancer (HeLa), and breast cancer (MCF-7), were evaluated. The cytotoxic assays revealed that 10i with a lipophilic 4-fluoro-phenyl moiety at the C-2 position of the quinazolinone ring displayed good cytotoxicities against the A549 and MCF-7 cell lines, while 8b-d with the thioether-linked benzimidazole moiety incorporated on the right side of the oxadiazole ring induced comparable stronger activities toward the MCF-7 cell line, relative to the simple two-heterocycle-containing hybrid 10i. These novel quinazolinone-based hybrids could be considered as lead compounds that merit further optimization and development as anti-cancer agents.

3D screen printing - An innovative technology for large-scale manufacturing of pharmaceutical dosage forms.

Three-dimensional (3D) screen printing was used to fabricate oral dosage forms of different geometry and size. The paste required as starting material for the 3D screen printing process was designed for delayed release and contained the model drug paracetamol (acetaminophen). A prototype screen printing unit was used to fabricate different tablets in a single production process. The resulting tablets were produced with three different sizes and designed geometries (disk, donut, cuboid, oval and grid). Investigation of size and mass of the individual tablets demonstrated high uniformity within the various groups of tablets. Further characterization of their physical properties, such as breaking force and friability, yielded results comparing favorably to conventionally produced tablets. Finally, drug release tests in artificial gastric media showed paracetamol release to depend on the surface-area-to-volume ratio. In conclusion, the study shows the potential of 3D screen printing to fabricate more complex oral dosage forms in the setting of mass production with high reproducibility.

Case Report: Anesthetic management for Cesarean section in a parturient with unspecified inherited bleeding disorder.

Neuraxial anesthesia, as the standard of care for Cesarean deliveries, is associated with decreased blood loss. However, parturients with inherited bleeding disorders are at increased risk for epidural hematomas. A small retrospective study has shown that parturients with known factor deficiencies can safely undergo neuraxial anesthesia once the specific factors are replenished. We present a patient who had a considerably increased risk of peripartum bleeding from an unspecified inherited bleeding disorder and was provided a successful neuraxial anesthetic without complications. We discuss the multidisciplinary approach among the surgeons, anesthesiologists, hematologist, and nursing staff to maximize patient safety and comfort.

Role of the deposition temperature on the self-assembly of the non-planar molecule benzene-1,3,5-triphosphonic acid (BTP) at the liquid-solid interface.

Benzene-1,3,5-triphosphonic acid (BTP) contains three non-planar phosphonic acid groups which enable three-dimensional hydrogen bonding. Because of these versatile 3D functional groups, BTP is an interesting intermediate to design both 2D and 3D supramolecular hydrogen-bonded architectures and organic-inorganic hybrid frameworks. However, the adsorption of BTP has surprisingly not been the subject of scanning tunneling microscopy (STM) investigations so far. Here a STM study of the adsorption pattern of BTP as obtained from deposition out of a solution in undecanol on an interface to highly-oriented pyrolytic graphite (HOPG) is presented. Furthermore, the influence of the substrate temperature during the deposition from solution on the self-assembly is investigated. High-resolution STM images reveal that the BTB molecules usually form various structures by co-adsorption with undecanol and that the BTP molecules as parts of self-assembled aggregates adsorb with their benzene ring planes tilted with respect to the substrate plane. The specific supramolecular pattern and the 2D packing density of BTP can be precisely tuned by adjusting the initial substrate temperature during deposition. The experimental results are compared to corresponding model structures obtained from semi-empirical simulations and explained by the influence of temperature on the concentration at the solution-solid interface and the kinetics of the self-assembly process. Based on these results, the control of the deposition substrate temperature has been proven to be a versatile tool to control the polymorphism of molecular patterns deposited out of solutions.

An appraisal of oxoketene cycloaddition methodology for the synthesis of 2,6-dideoxysugars and fluorinated 2,6-dideoxysugars.

The cycloaddition reaction of acylketenes with vinyl ethers affords an extremely direct route to 2,6-dideoxysugars and their methyl ethers. The lithium enolate of commercial 2,6,6-trimethyldioxinone 3 was fluorinated in good yield to afford fluorinated dioxinone 8. An illustrative range of fluorinated 2,6-dideoxysugar derivatives was prepared via the acetyl ketene-vinyl ether cycloadduct. Electronic structure calculations were carried out to investigate the effect of the fluorine atom on ease of formation and subsequent reaction of the (fluoroacetyl)ketene reactive intermediate. A single fluorine atom lowers the barrier to fragmentation by ca. 7.5 kJ mol(-1), consistent with experimental findings, but has almost no effect on the barrier to rate determining vinyl ether addition, or to oxoketene dimerisation.

In vitro resistance study of AG-021541, a novel nonnucleoside inhibitor of the hepatitis C virus RNA-dependent RNA polymerase.

A novel class of nonnucleoside hepatitis C virus (HCV) polymerase inhibitors characterized by a dihydropyrone core was identified by high-throughput screening. Crystallographic studies of these compounds in complex with the polymerase identified an allosteric binding site close to the junction of the thumb and finger domains, approximately 30 A away from the catalytic center. AG-021541, a representative compound from this series, displayed measurable in vitro antiviral activity against the HCV genotype 1b subgenomic replicon with a mean 50% effective concentration of 2.9 muM. To identify mutations conferring in vitro resistance to AG-021541, resistance selection was carried out using HCV replicon cells either by serial passages in increasing concentrations of AG-021541 or by direct colony formation at fixed concentrations of the compound. We identified several amino acid substitutions in the AG-021541-binding region of the polymerase, including M423(T/V/I), M426T, I482(S/T), and V494A, with M423T as the predominant change observed. These mutants conferred various levels of resistance to AG-021541 and structurally related compounds but remained sensitive to interferon and HCV polymerase inhibitors known to interact with the active site or other allosteric sites of the protein. In addition, dihydropyrone polymerase inhibitors retained activity against replicons that contain signature resistance changes to other polymerase inhibitors, including S282T, C316N, M414T, and P495(S/L), indicating their potential to be used in combination therapies with these polymerase inhibitors. AG-021541-resistant replicon cell lines provide a valuable tool for mechanism-of-action studies of dihydropyrone polymerase inhibitors. The clinical relevance of in vitro resistance to HCV polymerase inhibitors remains to be investigated.

Apolipoprotein AII is a regulator of very low density lipoprotein metabolism and insulin resistance.

Apolipoprotein AII (apoAII) transgenic (apoAIItg) mice exhibit several traits associated with the insulin resistance (IR) syndrome, including IR, obesity, and a marked hypertriglyceridemia. Because treatment of the apoAIItg mice with rosiglitazone ameliorated the IR and hypertriglyceridemia, we hypothesized that the hypertriglyceridemia was due largely to overproduction of very low density lipoprotein (VLDL) by the liver, a normal response to chronically elevated insulin and glucose. We now report in vivo and in vitro studies that indicate that hepatic fatty acid oxidation was reduced and lipogenesis increased, resulting in a 25% increase in triglyceride secretion in the apoAIItg mice. In addition, we observed that hydrolysis of triglycerides from both chylomicrons and VLDL was significantly reduced in the apoAIItg mice, further contributing to the hypertriglyceridemia. This is a direct, acute effect, because when mouse apoAII was injected into mice, plasma triglyceride concentrations were significantly increased within 4 h. VLDL from both control and apoAIItg mice contained significant amounts of apoAII, with approximately 4 times more apoAII on apoAIItg VLDL. ApoAII was shown to transfer spontaneously from high density lipoprotein (HDL) to VLDL in vitro, resulting in VLDL that was a poorer substrate for hydrolysis by lipoprotein lipase. These results indicate that one function of apoAII is to regulate the metabolism of triglyceride-rich lipoproteins, with HDL serving as a plasma reservoir of apoAII that is transferred to the triglyceride-rich lipoproteins in much the same way as VLDL and chylomicrons acquire most of their apoCs from HDL.

Development of a novel dicistronic reporter-selectable hepatitis C virus replicon suitable for high-throughput inhibitor screening.

Hepatitis C virus (HCV) research and drug discovery have been facilitated by the introduction of cell lines with self-replicating subgenomic HCV replicons. Early attempts to carry out robust, high-throughput screens (HTS) using HCV replicons have met with limited success. Specifically, selectable replicons have required laborious reverse transcription-PCR quantitation, and reporter replicons have generated low signal-to-noise ratios. In this study, we constructed a dicistronic single reporter (DSR)-selectable HCV replicon that contained a humanized Renilla luciferase (hRLuc) gene separated from the selectable Neo(r) marker by a short peptide cleavage site. The mutations E1202G, T1280I, and S2197P were introduced to enhance replicative capability. A dicistronic dual-reporter HCV replicon cell line (DDR) was subsequently created by transfection of Huh-7 cells with the DSR replicon to monitor antiviral activity and by the introduction of the firefly luciferase (FLuc) reporter gene into the host cell genome to monitor cytotoxicity. The DDR cell line demonstrated low signal variation within the HTS format, with a calculated Z' value of 0.8. A pilot HTS consisting of 20 96-well plates with a single concentration (10 microM) of 1,760 different compounds was executed. Hits were defined as compounds that reduced hRLuc and FLuc signals > or =50 and < or =40%, respectively, relative to those in a compound-free control. Good reproducibility was demonstrated, with a calculated confirmation rate of >75%. The development of a robust, high-throughput HCV replicon assay where the effects of inhibitors can be monitored for antiviral activity and cytotoxicity should greatly facilitate HCV drug discovery.

Identification and structure-based optimization of novel dihydropyrones as potent HCV RNA polymerase inhibitors.

A novel class of non-nucleoside HCV NS5B polymerase inhibitors has been identified from screening. A co-crystal structure revealed an allosteric binding site in the protein that required a unique conformational change to accommodate inhibitor binding. Herein we report the structure-activity relationships (SARs) of this novel class of dihydropyrone-containing compounds that show potent inhibitory activities against the HCV RNA polymerase in biochemical assays.