Beyond Pseudo-natural Products: Sequential Ugi/Pictet-Spengler Reactions Leading to Steroidal Pyrazinoisoquinolines That Trigger Caspase-Independent Death in HepG2 Cells.

In this work, we describe how stereochemically complex polycyclic compounds can be generated by applying a synthetic sequence comprising an intramolecular Ugi reaction followed by a Pictet-Spengler cyclization on steroid-derived scaffolds. The resulting compounds, which combine a fragment derived from a natural product and a scaffold not found in nature. are both structurally distinct and globally similar to natural products at the same time, and interrogate an alternative region of the chemical space. One of the new compounds showed significant antiproliferative activity on HepG2 cells through a caspase-independent cell-death mechanism, an appealing feature when new antitumor compounds are searched.

Design and Microwave-Assisted Synthesis of Aza-Resveratrol Analogs with Potent Cholinesterase Inhibition.

BACKGROUND: Currently approved Alzheimer's disease medications mainly comprise acetylcholinesterase inhibitors. Many of these inhibitors are either natural compounds or synthetic molecules inspired in natural compounds. Hybrid molecules that can interact with different target sites of the enzyme could lead to the discovery of effective multitarget drugs. OBJECTIVE: To design, synthesize, and evaluate a series of new aza-resveratrol analogs as in vitro acetyl- and butyrylcholinesterase inhibitors. METHODS: The synthesis is achieved by a simple and efficient microwave-assisted method, from commercially available starting materials. Compounds are designed as hybrids of an aza-stilbene nucleus (Schiff base) connected to a tertiary amine by a hydrocarbon chain of variable length, designed to interact both with the peripheric anionic site and the catalytic site of the enzyme. RESULTS: All the derivatives inhibit both enzymes in a concentration-dependent manner, acting as moderate to potent cholinesterase inhibitors. The most potent inhibitors are compounds 12b (IC50 = 0.43 µM) and 12a (IC50 = 0.31 µM) for acetyl- and butyrylcholinesterase, respectively. Compounds 12a and 12b also exhibit significant acetylcholinesterase inhibition in SH-SY5Y human neuroblastoma cells without cytotoxic properties. Enzyme kinetic studies and molecular modeling reveal that inhibitor 12b targets both the catalytic active site and the peripheral anionic site of acetylcholinesterase what makes it able to modulate the self-induced ß-amyloid aggregation. Furthermore, the molecular modeling analysis helps to assess the impact of the linker length in the inhibitory activity of this family of new cholinesterase inhibitors. CONCLUSION: These compounds have the potential to serve as a dual binding site inhibitor and might provide a useful template for the development of new anti-Alzheimer's disease agents.

Gas phase structure and reactivity of doubly charged microhydrated calcium(II)-catechol complexes probed by infrared spectroscopy.

Doubly charged microhydrated adducts formed from catechol and calcium(II) were produced in the gas phase using electrospray ionization (ESI) appearing as the most important ions in the mass spectra recorded. The gas phase structures of [Ca(catechol)2(H2O)](2+) and [Ca(catechol)2(H2O)2](2+) have been assayed by IR multiphoton dissociation (IRMPD) spectroscopy, recording their vibrational spectra in the 3450-3750 cm(-1) range (OH stretching region) and in the 900-1700 cm(-1) fingerprint spectral region. The agreement between experimental and calculated IR spectra of the selected cluster ions confirmed the suitability of the proposed geometries. In addition, quantum chemical calculations at the B3LYP/6-311+G(d,p) level of theory were performed for [Ca(catechol)2(H2O)](2+) to gain insight into the major routes of dissociation. The results suggest that loss of the water molecule is the lowest energy fragmentation channel followed by charge separation products and neutral loss of one catechol molecule, in agreement with the product ions observed upon collision-induced dissociation (CID).

The Caenorhabditis elegans DAF-12 nuclear receptor: structure, dynamics, and interaction with ligands.

A structure for the ligand binding domain (LBD) of the DAF-12 receptor from Caenorhabditis elegans was obtained from the X-ray crystal structure of the receptor LBD from Strongyloides stercoralis bound to (25R)-Δ(7)-dafachronic acid (DA) (pdb:3GYU). The model was constructed in the presence of the ligand using a combination of Modeller, Autodock, and molecular dynamics (MD) programs, and then its dynamical behavior was studied by MD. A strong ligand binding mode (LBM) was found, with the three arginines in the ligand binding pocket (LBP) contacting the C-26 carboxylate group of the DA. The quality of the ceDAF-12 model was then evaluated by constructing several ligand systems for which the experimental activity is known. Thus, the dynamical behavior of the ceDAF-12 complex with the more active (25S)-Δ(7)-DA showed two distinct binding modes, one of them being energetically more favorable compared with the 25R isomer. Then the effect of the Arg564Cys and Arg598Met mutations on the (25R)-Δ(7)-DA binding was analyzed. The MD simulations showed that in the first case the complex was unstable, consistent with the lack of transactivation activity of (25R)-Δ(7)-DA in this mutant. Instead, in the case of the Arg598Met mutant, known to produce a partial loss of activity, our model predicted smaller effects on the LBM with a more stable MD trajectory. The model also showed that removal of the C-25 methyl does not impede the simultaneous strong interaction of the carboxylate with the three arginines, predicting that 27-nor-DAs are putative ceDAF-12 ligands.

Differentiation of isomeric hydroxypyridine N-oxides using metal complexation and electrospray ionization mass spectrometry.

Differentiation between two isomers of hydroxypyridine N-oxide according to the metal cation adducts generated by electrospray ionization (ESI) was investigated for different metal cations, namely Mg(II), Al(III), Ca(II), Sc(III), Fe(III), Co(II), Ni(II), Cu(II), Zn(II), Ga(III), besides the diatomic cation VO(IV). Protonated molecules of the isomeric hydroxypyridine N-oxides as well as the singly/doubly charged adducts formed from neutral or deprotonated ligands and a doubly/triply charged cation were produced in the gas phase using ESI, recording mass spectra with different metal ions for each isomer. While complex formation was successful for 2-hydroxypyridine N-oxide with trivalent ions, in the case of 3-hydroxypyridine N-oxide, only peaks related to the protonated molecule were present. On the other hand, divalent cations formed specific species for each isomer, giving characteristic spectra in every case. Hence, differentiation was possible irrespective of the metal cation utilized. In addition, quantum chemical calculations at the B3LYP/6-31+G(d,p) level of theory were performed in order to gain insight into the different complexation of calcium(II) with the isomers of hydroxypyridine N-oxide. The relative stability in the gas phase of the neutral complexes of calcium made up of two ligands, as well as the singly charged and doubly charged complexes, was investigated. The results of these calculations improved the understanding of the differences observed in the mass spectra obtained for each isomer.

Unraveling the molecular basis for ligand binding in truncated hemoglobins: the trHbO Bacillus subtilis case.

Truncated hemoglobins (trHbs) are heme proteins present in bacteria, unicellular eukaryotes, and higher plants. Their tertiary structure consists in a 2-over-2 helical sandwich, which display typically an inner tunnel/cavity system for ligand migration and/or storage. The microorganism Bacillus subtilis contains a peculiar trHb, which does not show an evident tunnel/cavity system connecting the protein active site with the solvent, and exhibits anyway a very high oxygen association rate. Moreover, resonant Raman results of CO bound protein, showed that a complex hydrogen bond network exists in the distal cavity, making it difficult to assign unambiguously the residues involved in the stabilization of the bound ligand. To understand these experimental results with atomistic detail, we performed classical molecular dynamics simulations of the oxy, carboxy, and deoxy proteins. The free energy profiles for ligand migration suggest that there is a key residue, GlnE11, that presents an alternate conformation, in which a wide ligand migration tunnel is formed, consistently with the kinetic data. This tunnel is topologically related to the one found in group I trHbs. On the other hand, the results for the CO and O(2) bound protein show that GlnE11 is directly involved in the stabilization of the cordinated ligand, playing a similar role as TyrB10 and TrpG8 in other trHbs. Our results not only reconcile the structural data with the kinetic information, but also provide additional insight into the general behaviour of trHbs. Proteins 2010. (c) 2009 Wiley-Liss, Inc.