A novel, highly potent and selective phosphodiesterase-9 inhibitor for the treatment of sickle cell disease.

The most common treatment for patients with sickle cell disease (SCD) is the chemotherapeutic hydroxyurea, a therapy with pleiotropic effects, including increasing fetal hemoglobin (HbF) in red blood cells and reducing adhesion of white blood cells to the vascular endothelium. Hydroxyurea has been proposed to mediate these effects through a mechanism of increasing cellular cGMP levels. An alternative path to increasing cGMP levels in these cells is through the use of phosphodiesterase-9 inhibitors that selectively inhibit cGMP hydrolysis and increase cellular cGMP levels. We have developed a novel, potent and selective phosphodiesterase-9 inhibitor (IMR-687) specifically for the treatment of SCD. IMR-687 increased cGMP and HbF in erythroid K562 and UT-7 cells and increased the percentage of HbF positive erythroid cells generated in vitro using a two-phase liquid culture of CD34+ progenitors from sickle cell blood or bone marrow. Oral daily dosing of IMR-687 in the Townes transgenic mouse SCD model, increased HbF and reduced red blood cell sickling, immune cell activation and microvascular stasis. The IMR-687 reduction in red blood cell sickling and immune cell activation was greater than that seen with physiological doses of hydroxyurea. In contrast to other described phosphodiesterase-9 inhibitors, IMR-687 did not accumulate in the central nervous system, where it would inhibit phosphodiesterase-9 in neurons, or alter rodent behavior. IMR-687 was not genotoxic or myelotoxic and did not impact fertility or fetal development in rodents. These data suggest that IMR-687 may offer a safe and effective oral alternative for hydroxyurea in the treatment of SCD.

A small molecule activator of Nav 1.1 channels increases fast-spiking interneuron excitability and GABAergic transmission in vitro and has anti-convulsive effects in vivo.

Nav 1.1 (SCN1A) channels primarily located in gamma-aminobutyric acid (GABA)ergic fast-spiking interneurons are pivotal for action potential generation and propagation in these neurons. Inappropriate function of fast-spiking interneurons, leading to disinhibition of pyramidal cells and network desynchronization, correlates with decreased cognitive capability. Further, reduced functionality of Nav 1.1 channels is linked to various diseases in the central nervous system. There is, at present, however no subtype selective pharmacological activators of Nav 1.1 channels available for studying pharmacological modulation of interneuron function. In the current study, we identified a small molecule Nav 1.1 activator, 3-amino-5-(4-methoxyphenyl)thiophene-2-carboxamide, named AA43279, and provided an in vitro to in vivo characterization of the compound. In HEK-293 cells expressing human Nav 1.1 channels, AA43279 increased the Nav 1.1-mediated current in a concentration-dependent manner mainly by impairing the fast inactivation kinetics of the channels. In rat hippocampal brain slices, AA43279 increased the firing activity of parvalbumin-expressing, fast-spiking GABAergic interneurons and increased the spontaneous inhibitory post-synaptic currents (sIPSCs) recorded from pyramidal neurons. When tested in vivo, AA43279 had anti-convulsive properties in the maximal electroshock seizure threshold test. AA43279 was tested for off-target effects on 72 different proteins, including Nav 1.2, Nav 1.4, Nav 1.5, Nav 1.6 and Nav 1.7 and exhibited reasonable selectivity. Taken together, AA43279 might constitute a valuable tool compound for revealing biological functions of Nav 1.1 channels.

Subtype-Specific Agonists for NMDA Receptor Glycine Binding Sites.

A series of analogues based on serine as lead structure were designed, and their agonist activities were evaluated at recombinant NMDA receptor subtypes (GluN1/2A-D) using two-electrode voltage-clamp (TEVC) electrophysiology. Pronounced variation in subunit-selectivity, potency, and agonist efficacy was observed in a manner that was dependent on the GluN2 subunit in the NMDA receptor. In particular, compounds 15a and 16a are potent GluN2C-specific superagonists at the GluN1 subunit with agonist efficacies of 398% and 308% compared to glycine. This study demonstrates that subunit-selectivity among glycine site NMDA receptor agonists can be achieved and suggests that glycine-site agonists can be developed as pharmacological tool compounds to study GluN2C-specific effects in NMDA receptor-mediated neurotransmission.

Identification and electrophysiological evaluation of 2-methylbenzamide derivatives as Nav1.1 modulators.

Voltage-gated sodium channels (Nav) are crucial to the initiation and propagation of action potentials (APs) in electrically excitable cells, and during the past decades they have received considerable attention due to their therapeutic potential. Here, we report for the first time the synthesis and the electrophysiological evaluation of 16 ligands based on a 2-methylbenzamide scaffold that have been identified as Nav1.1 modulators. Among these compounds, N,N'-(1,3-phenylene)bis(2-methylbenzamide) (3a) has been selected and evaluated in ex-vivo experiments in order to estimate the activation impact of such a compound profile. It appears that 3a increases the Nav1.1 channel activity although its overall impact remains moderate. Altogether, our preliminary results provide new insights into the development of small molecule activators targeting specifically Nav1.1 channels to design potential drugs for treating CNS diseases.

The grasshopper: a novel model for assessing vertebrate brain uptake.

The aim of the present study was to develop a blood-brain barrier (BBB) permeability model that is applicable in the drug discovery phase. The BBB ensures proper neural function, but it restricts many drugs from entering the brain, and this complicates the development of new drugs against central nervous system diseases. Many in vitro models have been developed to predict BBB permeability, but the permeability characteristics of the human BBB are notoriously complex and hard to predict. Consequently, one single suitable BBB permeability screening model, which is generally applicable in the early drug discovery phase, does not yet exist. A new refined ex vivo insect-based BBB screening model that uses an intact, viable whole brain under controlled in vitro-like exposure conditions is presented. This model uses intact brains from desert locusts, which are placed in a well containing the compound solubilized in an insect buffer. After a limited time, the brain is removed and the compound concentration in the brain is measured by conventional liquid chromatography-mass spectrometry. The data presented here include 25 known drugs, and the data show that the ex vivo insect model can be used to measure the brain uptake over the hemolymph-brain barrier of drugs and that the brain uptake shows linear correlation with in situ perfusion data obtained in vertebrates. Moreover, this study shows that the insect ex vivo model is able to identify P-glycoprotein (Pgp) substrates, and the model allows differentiation between low-permeability compounds and compounds that are Pgp substrates.

New DNA polymerase IIIC inhibitors: 3-subtituted anilinouracils with potent antibacterial activity in vitro and in vivo.

The development of resistance has rendered several antibiotics clinically ineffective, and there is an urgent medical need for potent and safe antibacterials with a novel and valid mode of action. To avoid cross-resistance, they should preferably inhibit targets that are not addressed by established antibiotics. In this respect, 6-anilinouracils represent a promising lead structure. They target the Gram-positive DNA polymerase IIIC, a target that is associated with a bactericidal mode of action. Moreover, they have no cross-resistance to marketed antibiotics. This paper describes the synthesis and biological characterization of structurally novel anilinouracils, some of which display potent in vivo efficacy in murine models of bacterial septicemia.

Improved synthesis of antibacterial 3-substituted 6-anilinouracils.

3-Substituted 6-anilinouracils, presently the most promising class of inhibitors of the bacterial DNA polymerase in Gram-positive bacteria, have been prepared by a general and straightforward three-step procedure starting from a readily available 1-benzyloxymethyl-protected derivative of 6-chlorouracil.

Pyrrolidinedione derivatives as antibacterial agents with a novel mode of action.

The pseudopeptide pyrrolidinedione natural products moiramide B and andrimid represent a new class of antibiotics that target bacterial fatty acid biosynthesis. Structure-activity relationship (SAR) studies revealed a high degree of variability for the fatty acid side chain, allowing optimization of physicochemical parameters, and a restricted SAR for the pyrrolidinedione group, indicating major relevance of this subunit for efficient target binding.

Biological characterization of novel inhibitors of the gram-positive DNA polymerase IIIC enzyme.

Novel N-3-alkylated 6-anilinouracils have been identified as potent and selective inhibitors of bacterial DNA polymerase IIIC, the enzyme essential for the replication of chromosomal DNA in gram-positive bacteria. A nonradioactive assay measuring the enzymatic activity of the DNA polymerase IIIC in gram-positive bacteria has been assembled. The 6-anilinouracils described inhibited the polymerase IIIC enzyme at concentrations in the nanomolar range in this assay and displayed good in vitro activity (according to their MICs) against staphylococci, streptococci, and enterococci. The MICs of the most potent derivatives were about 4 microg/ml for this panel of bacteria. The 50% effective dose of the best compound (6-[(3-ethyl-4-methylphenyl)amino]-3-{[1-(isoxazol-5-ylcarbonyl)piperidin-4-yl]methyl}uracil) was 10 mg/kg of body weight after intravenous application in a staphylococcal sepsis model in mice, from which in vivo pharmacokinetic data were also acquired.

A Pyrazole to Furan Rearrangement. Thermolysis of 5-Azido-4-formylpyrazoles.

5-Azido-3-benzyl-4-formyl-1-phenylpyrazoles 1a-c extrude dinitrogen upon heating in toluene to give the corresponding nitrenes, which immediately rearrange via a new ring-opening ring-closure reaction to produce an equimolar mixture of 4-cyano-2-phenyl-3-phenylazofurans 2a-c and 3-benzyl-4-cyano-1-phenylpyrazoles 3a-c. The formation of the 4-cyano-2-phenyl-3-phenylazofurans 2a-c is the first example in the pyrazole series of a nitrene rearrangement, in which the parent heterocyclic system of the product differs from that of the starting material. The isolation of equimolar amounts of the two products points to the fact that their formation occurs by two mechanistically interconnected pathways, between which the exchange of a redox equivalent takes place. Evidence for the existence of two mechanistically interlinked pathways is presented, and the insight into the stoechiometry of the reaction is taken advantage of to optimize the reaction with respect to either of the two products 2 or 3. Thus, it is demonstrated how one can bias the two pathways using external reagents, thereby changing the product distribution ratio 2:3 from 1:1 in the unbiased case, to 1:4 in one direction, and to better than 20:1 in the other direction.

Tetrathiafulvalene Belts with Large Cavities.

The latest member of the cyclophane family, the macrotricyclic tetrathiafulvalene (TTF) "belt", is now available. A general synthetic strategy for the construction of tetraconnected belt-type TTFs (shown schematically) has been developed, made possible by the use of a TTF with two different protecting groups. In the solid-state structure of one of the three TTF-belts prepared two chloroform molecules reside inside the spacious cavity.