Novel Approach to the Construction of Fused Indolizine Scaffolds: Synthesis of Rosettacin and the Aromathecin Family of Compounds.

Camptothecin-like compounds are actively employed as anticancer drugs in clinical treatments. The aromathecin family of compounds, which contains the same indazolidine core structure as the camptothecin family of compounds, is also expected to display promising anticancer activity. Therefore, the development of a suitable and scalable synthetic method of aromathecin synthesis is of great research interest. In this study, we report the development of a new synthetic approach for constructing the pentacyclic scaffold of the aromathecin family by forming the indolizidine moiety after synthesizing the isoquinolone moiety. Thermal cyclization of 2-alkynylbenzaldehyde oxime to the isoquinoline N-oxide, followed by a Reissert-Henze-type reaction, forms the key strategy in this isoquinolone synthesis. Under the optimum reaction conditions for the Reissert-Henze-type reaction step, microwave irradiation-assisted heating of the purified N-oxide in acetic anhydride at 50 °C reduced the formation of the 4-acetoxyisoquinoline byproduct to deliver the desired isoquinolone at a 73% yield after just 3.5 h. The eight-step sequence employed afforded rosettacin (simplest member of the aromathecin family) at a 23.8% overall yield. The synthesis of rosettacin analogs was achieved by applying the developed strategy and may be generally applicable to the production of other fused indolizidine compounds.

Involvement of Ca2+/calmodulin-dependent protein kinase II in heparin-stimulated release of hepatic lipase activity from rat hepatocytes.

The release of hepatic lipase (HTGL), which is responsible for the hydrolysis of lipoprotein triacylglyceride, produced by heparin from the isolated rat hepatocytes in primary culture has been examined. Tyrosine kinase (TK) inhibitors (ST-638 and biochanin A) inhibited the heparin-stimulated release of HTGL activity. The activity of partially purified TK preparation from the hepatocytes was found to be increased following incubation with heparin in a manner which was both time- and dose-dependent. An intracellular Ca(2+)-chelator (Quin2/AM), a calmodulin inhibitor (W-7) and a Ca(2+)/calmodulin-dependent protein kinase II (CaMK-II) inhibitor (KN-93) suppressed the release of HTGL activity by heparin. In addition, CaMK-II activity in the hepatocytes incubated with heparin was recognized to elevate in a time- and dose-dependent manner. The increase in CaMK-II activity by heparin was markedly reduced in the presence of the inhibitors of TK. These results suggest that the release of HTGL activity from the hepatocytes by heparin is, in part, caused through a pathway involving an activation of CaMK-II associated with an increase in membrane TK activity.

Orthovanadate stimulates cAMP phosphodiesterase 3 activity in isolated rat hepatocytes through mitogen-activated protein kinase activation dependent on cAMP-dependent protein kinase.

Orthovanadate (vanadate) as well as insulin stimulated phosphodiesterase 3 (PDE3) in the particulate fraction of rat hepatocytes. The vanadate-induced activations of PDE3 and mitogen-activated protein kinase (MAPK) were inhibited by H-89 and PD98059, suggesting that the MAPK activation via cAMP-dependent protein kinase (PKA) and MAPK kinase is involved in the vanadate action. On the other hand, the insulin-induced activations of PDE3 and Akt were inhibited by wortmannin, suggesting involvement of the Akt activation via phosphatidylinositol 3-kinase (PI3K) in the insulin action. The vanadate-induced activations of PKA and PDE3 were inhibited in part by propranolol or genistein, suggesting that vanadate may exert its actions via dual signaling pathways of beta-adrenergic receptors and receptor tyrosine kinases of growth factors. Vanadate, in contrast to insulin, did not promote the phosphorylation of insulin receptor substrate-1. The vanadate-induced increase in the phosphorylation of a main isoform of MAPKs, p44 protein, was detected by immunoblotting migration patterns of SDS-PAGE. A partially purified PDE3 activity was increased by addition of MAPK or Akt to the reaction mixture, suggesting that MAPK as well as Akt acts upstream of PDE3. The activation of PDE3 by insulin was independent of a transient increase in the MAPK activity, probably due to the dephosphorylated inactivation mediated by the induced activation of MAPK phosphatases (MKPs). Vanadate did not affect the MKP activity. These results indicate that vanadate stimulates the particulate PDE3 activity by activating mainly p44 MAPK via a PKA-dependent process, and that it differs from insulin with regard to a phosphorylation cascade of PDE3 activation.

Isolation of anticoagulant from the venom of tick, Boophilus calcaratus, from Uzbekistan.

Boophilus calcaratus is a tick found in Central Asia and a common parasite to domestic animals. Venom from this tick was fractionated by two-step column chromatography, Sephadex G-75, and DEAE-Sephadex A-25. The homogeneity of the anticoagulant was examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified component is named calcaratin and has a molecular weight of 14,500. The effect of the purified anticoagulant component (calcaratin) on various sites of the blood coagulation cascade scheme was examined and compared with crude venom. The chromogenic substrates S-2238 (H-D-Phe-Pip-Arg-pNA 2HCl) for thrombin and S-2765 (N-alpha-Z-D-Arg-Gly-Arg-pNA 2HCl) for factor Xa were also investigated. Activated partial clotting times were all prolonged, suggesting the anticoagulation nature of the purified component and crude venom. Prolongation of fibrinogen clotting time (FCT) is highly suggestive of the antithrombin property of the purified component and its original venom.

Okadaic acid decreases the leptin content in isolated mouse fat pads.

Okadaic acid (OA) decreased the leptin content in isolated mouse fat pads in a time and dose-dependent manner. MG-132, a membrane-permeable proteasome inhibitor, prevented the decrease by OA, suggesting the involvement of proteasome in the OA action. No significant decrease in the incorporation of [(3)H]leucine into leptin was observed with a 4-h incubation, although the amino acid incorporation was stimulated by insulin and decreased by cycloheximide. These results suggest that the OA action is independent of the decrease in protein synthesis. The proteasome fraction, which had been separated from the fat pads pretreated with OA, enhanced the proteolytic degradation of exogenous [(125)I]leptin in the presence of an ATP-regenerating system together with an ubiquitination system. No enhancement of hydrolytic activity against Suc-Leu-Leu-Val-Tyr-AMC was detected in the OA-treated proteasome fraction, suggesting that the activation of proteasome is not involved in the OA action. The OA-treated proteasome fraction had decreased phosphatase activity against p-nitrophenyl phosphate, suggesting that OA entering the cells may exert its action by preventing dephosphorylation of key molecules. OA may reduce the intracellular leptin content through the increased ubiquitination and proteolytic turnover of leptin by the proteasome, based on the decreased phosphatase activity.

Orthovanadate decreases the leptin content in isolated mouse fat pads via proteasome activation.

When isolated mouse fat pads were incubated with insulin or sodium orthovanadate (vanadate) for up to 4h, the intracellular leptin content was increased by insulin, while it was decreased by vanadate. Bupranolol, a beta3-adrenergic receptor antagonist, prevented both effects of vanadate, i.e., the decrease in intracellular leptin and increase in cellular cAMP content, while BRL 37344, a beta3-adrenergic receptor antagonist mimicked the action of vanadate. H-89 prevented the vanadate-induced decrease in intracellular leptin, suggesting the involvement of a cAMP-dependent protein kinase (PKA). No detectable difference in the incorporation of [3H]leucine into leptin was observed between incubations of the fat pads with and without vanadate, suggesting that the action of vanadate is independent of decreasing synthesis. Similar concentrations of MG-132, a membrane-permeable proteasome inhibitor, prevented the vanadate-induced decrease in both intracellular leptin content and leptin secretion, suggesting the involvement of the proteasome in the vanadate action. The proteasome fraction separated from the vanadate-treated fat pads increased the degradation of exogenous [125I]leptin in the presence of an ATP-regenerating system together with an ubiqutination system. The endopeptidase activity against Cbz-Leu-Leu-Glu-beta-naphthylamine also was increased by the proteasome fraction. MG-132 prevented both increased effects. The 8-Br-cAMP-treated proteasome fraction increased the degradation of the exogenous leptin. H-89 prevented the effect of 8-Br-cAMP. These results indicate that vanadate decreases the intracellular leptin content by increased degradation via a cAMP/PKA-dependent process involving proteasome activation.