Alexandros Kiriazis

Synthesis and anti-leishmanial activity of heterocyclic betulin derivatives

Betulin, a naturally occurring abundant triterpene is converted in four steps to 3,28-di-O-acetyllupa-12,18-diene. When various 4-substituted urazoles were oxidized to the corresponding urazines with iodobenzene diacetate in the presence of 3,28-di-O-acetyllupa-12,18-diene, new heterocyclic betulin derivatives were produced. These betulin derivatives were examined in a microplate assay at 50 microM for their ability to inhibit the growth of Leishmania donovani axenic amastigotes, a species that causes the fatal visceral leishmaniasis. GI(50) (concentration for 50% growth inhibition) values of the most effective compounds were determined and their cytotoxicity on the human macrophage cell line THP-1 evaluated. The anti-leishmanial activity on L. donovani amastigotes growing in macrophages was also examined. The heterocycloadduct between 3,28-di-O-acetyllupa-12,18-diene and 4-methylurazine was the most effective derivative with an GI(50)=8.9 microM against L. donovani amastigotes.

Enzyme-assisted synthesis and characterization of glucuronide conjugates of neuroactive steroids.

Synthesis of reference standards is needed to determine the presence and function of steroid glucuronides in the brain or other tissues, because commercial sources of steroid glucuronide standards are limited or unavailable. In the present study porcine, rat, and bovine liver microsomes were tested to evaluate their ability to glucuronidate eight neurosteroids and neuroactive steroids of various types: dehydroepiandrosterone, pregnenolone, isopregnanolone, 5alpha-tetrahydrodeoxycorticosterone, corticosterone, cortisol, beta-estradiol, and testosterone. In general, the glucuronidation efficiency of rat liver was rather poor compared with that of bovine and porcine liver microsomes. Since porcine liver apparently has a relatively large amount of dehydrogenase, its microsomes also produced dehydrogenated steroids and their glucuronides, as well as various regioisomers in which the site of glucuronidation varied. In contrast, bovine liver microsomes produced mainly a single major glucuronidation product and few dehydrogenation products and gave the best overall yield for two-third of the steroids tested. The enzymatic synthesis of five glucuronides of four steroids was carried out and the conditions, purification, and analytical methods for the glucuronidation products were optimized. The steroid glucuronides synthesized were characterized by nuclear magnetic resonance spectroscopy (NMR) and liquid chromatography-mass spectrometry (LC-MS). The stereochemically pure steroid glucuronide conjugates were recovered in milligram amounts (yield 10-78%) and good purity (>85-90%), which is sufficient for LC-MS/MS method development and analyses of steroid glucuronides in biological matrices such as brain, urine, or plasma.

Enzyme-assisted synthesis and structure characterization of glucuronic acid conjugates of losartan, candesartan, and zolarsartan

Three angiotensin II receptor antagonists--losartan, candesartan, and zolarsartan--were investigated. All the compounds, which are structural analogues, are metabolized via conjugation to glucuronic acid. Interestingly, both O- and N-glucuronidation take place, so that regioisomers are formed. One ether O-glucuronide, two acyl O-glucuronides, and five tetrazole-N-glucuronides were biosynthesized, in milligram scale, from the three sartan aglycones. Liver microsomes from bovine, moose, rat, and pig and recombinant human UDP-glucuronosyltransferases were used as catalysts. The synthesized compounds were identified as sartan glucuronides by mass spectrometry, while the sites of glucuronidation were determined by nuclear magnetic resonance spectroscopy. Drug metabolites are needed as standards for pharmaceutical research and, as the present study shows, they can easily be produced with enzymes as catalyst.

Tricyclic Benzo[cd]azulenes Selectively Inhibit Activities of Pim Kinases and Restrict Growth of Epstein-Barr Virus-Transformed Cells

Oncogenic Pim family kinases are often overexpressed in human hematopoietic malignancies as well as in solid tumours. These kinases contribute to tumorigenesis by promoting cell survival and by enhancing resistance against chemotherapy and radiation therapy. Furthermore, we have recently shown that they increase the metastatic potential of adherent cancer cells. Here we describe identification of tricyclic benzo[cd]azulenes and their derivatives as effective and selective inhibitors of Pim kinases. These compounds inhibit Pim autophosphorylation and abrogate the anti-apoptotic effects of Pim kinases. They also reduce cancer cell motility and suppress proliferation of lymphoblastoid cell lines infected and immortalized by the Epstein-Barr virus. Thus, these novel Pim-selective inhibitors provide promising compounds for both research and therapeutic purposes.

Nucleophilic Substitution of Hydrogen Facilitated by Quinone Methide Moieties in Benzo[cd]azulen-3-ones

The built-in o- and p-QM (QM = quinone methide) moieties in benzo[cd]azulen-3-ones account for an easy switch between the bridged 10π- and 6π-aromatic systems in organic synthesis. We report conjugate additions, oxidative nucleophilic substitutions of hydrogen, and reversible Michael additions under very mild conditions. In the presence of thiol nucleophiles, the protonated σH-adducts could be isolated and characterized. The typical preference for either the o- or p-QM moiety led to high regioselectivity. Furthermore, the inhibitory potency of the novel benzo[cd]azulenes against the human Pim-1 kinase was evaluated.

Asymmetry in catalysis by Thermotoga maritima membrane-bound pyrophosphatase demonstrated by a non-phosphorous allosteric inhibitor

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.