A novel prodrug-based strategy to increase effects of bumetanide in epilepsy.

OBJECTIVE: There is considerable interest in using bumetanide, a chloride importer Na-K-Cl cotransporter antagonist, for treatment of neurological diseases, such as epilepsy or ischemic and traumatic brain injury, that may involve deranged cellular chloride homeostasis. However, bumetanide is heavily bound to plasma proteins (~98%) and highly ionized at physiological pH, so that it only poorly penetrates into the brain, and chronic treatment with bumetanide is compromised by its potent diuretic effect. METHODS: To overcome these problems, we designed lipophilic and uncharged prodrugs of bumetanide that should penetrate the blood-brain barrier more easily than the parent drug and are converted into bumetanide in the brain. The feasibility of this strategy was evaluated in mice and rats. RESULTS: Analysis of bumetanide levels in plasma and brain showed that administration of 2 ester prodrugs of bumetanide, the pivaloyloxymethyl (BUM1) and N,N-dimethylaminoethylester (BUM5), resulted in significantly higher brain levels of bumetanide than administration of the parent drug. BUM5, but not BUM1, was less diuretic than bumetanide, so that BUM5 was further evaluated in chronic models of epilepsy in mice and rats. In the pilocarpine model in mice, BUM5, but not bumetanide, counteracted the alteration in seizure threshold during the latent period. In the kindling model in rats, BUM5 was more efficacious than bumetanide in potentiating the anticonvulsant effect of phenobarbital. INTERPRETATION: Our data demonstrate that the goal of designing bumetanide prodrugs that specifically target the brain is feasible and that such drugs may resolve the problems associated with using bumetanide for treatment of neurological disorders.

Synthesis, biological evaluation and 3D-QSAR studies of new chalcone derivatives as inhibitors of human P-glycoprotein.

P-glycoprotein (P-gp) is an ATP-dependent multidrug resistance efflux transporter that plays an important role in anticancer drug resistance and in pharmacokinetics of medicines. Despite a large number of structurally and functionally diverse compounds, also flavonoids and chalcones have been reported as inhibitors of P-gp. The latter share some similarity with the well studied class of propafenones, but do not contain a basic nitrogen atom. Furthermore, due to their rigidity, they are suitable candidates for 3D-QSAR studies. In this study, a set of 22 new chalcone derivatives were synthesized and evaluated in a daunomycin efflux inhibition assay using the CCRF.CEM.VCR1000 cell line. The compound 10 showed the highest activity (IC50=42nM), which is one order of magnitude higher than the activity for an equilipohillic propafenone analogue. 2D- and 3D-QSAR studies indicate the importance of H-bond acceptors, methoxy groups, hydrophobic groups as well as the number of rotatable bonds as pharmacophoric features influencing P-gp inhibitory activity.

Exploration of natural compounds as sources of new bifunctional scaffolds targeting cholinesterases and beta amyloid aggregation: the case of chelerythrine.

The presented project started by screening a library consisting of natural and natural based compounds for their acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activity. Active compounds were chemically clustered into groups and further tested on the human cholinesterases isoforms. The aim of the presented study was to identify compounds that could be used as leads to target two key mechanisms associated with the AD's pathogenesis simultaneously: cholinergic depletion and beta amyloid (Aß) aggregation. Berberin, palmatine and chelerythrine, chemically clustered in the so-called isoquinoline group, showed promising cholinesterase inhibitory activity and were therefore further investigated. Moreover, the compounds demonstrated moderate to good inhibition of Aß aggregation as well as the ability to disaggregate already preformed Aß aggregates in an experimental set-up using HFIP as promotor of Aß aggregates. Analysis of the kinetic mechanism of the AChE inhibition revealed chelerythrine as a mixed inhibitor. Using molecular docking studies, it was further proven that chelerythrine binds on both the catalytic site and the peripheral anionic site (PAS) of the AChE. In view of this, we went on to investigate its effect on inhibiting Aß aggregation stimulated by AChE. Chelerythrine showed inhibition of fibril formation in the same range as propidium iodide. This approach enabled for the first time to identify a cholinesterase inhibitor of natural origin-chelerythrine-acting on AChE and BChE with a dual ability to inhibit Aß aggregation as well as to disaggregate preformed Aß aggregates. This compound could be an excellent starting point paving the way to develop more successful anti-AD drugs.

The exploration of thienothiazines as selective butyrylcholinesterase inhibitors.

The role of butyrylcholinesterase (BChE) in the progression of Alzheimer's disease (AD) has recently become more crucial. In the AD brain, selective BChE inhibitors have been demonstrated to have a beneficial effect in vivo, probably by recovering cholinergic activity and/or by restoring AChE:BChE activity ratios to the levels observed in the healthy brain. Thienothiazines are compounds sharing some structural features with phenothiazines, which are known to be potent BChE inhibitors. Thus, in this contribution 45 thienothiazines were investigated for their BChE inhibitory activity. Six of them were proven to be potent and selective inhibitors of equine BChE's hydrolase activity. Structure-activity relationships were laid out, and a tentative pharmacophore model for BChE inhibitors of the thienothiazine type was proposed. The most active compound, 3f, displayed a mixed type of inhibition and was also active against the human BChE (huBChE) with an IC(50) huBChE of 0.51 ± 0.07 µM. Computational studies suggested that 3f likely binds to the catalytic site and nearby to the peripheral site of the huBChE in an extended form. In addition, the chemical space occupied by the active thienothiazines, as opposed to phenothiazines and other representative chemical classes of BChE inhibitors, was explored with the aid of ChemGPS-NP, and the relevant chemical space regions were identified. This study shows for the first time that thienothiazines represent a new group of BChE inhibitors that can be used as molecular probes for studying the role of BChE in the brain or for developing newer drug leads for AD therapy.

Identification and characterization of diarylimidazoles as hybrid inhibitors of butyrylcholinesterase and amyloid beta fibril formation.

In this contribution, a chemical collection of aromatic compounds was screened for inhibition on butyrylcholinesterase (BChE)'s hydrolase activity using Ellman's reaction. A set of diarylimidazoles was identified as highly selective inhibitors of BChE hydrolase activity and amyloid ß (Aß) fibril formation. New derivatives were synthesized resulting in several additional hits, from which the most active was 6c, 4-(3-ethylthiophenyl)-2-(3-thienyl)-1H-imidazole, an uncompetitive inhibitor of BChE hydrolase activity (IC50 BChE=0.10 µM; K(i)=0.073 ± 0.011 µM) acting also on Aß fibril formation (IC50=5.8 µM). With the aid of structure-activity relationship (SAR) studies, chemical motifs influencing the BChE inhibitory activity of these imidazoles were proposed. These bifunctional inhibitors represent good tools in basic studies of BChE and/or promising lead molecules for AD therapy.

Synthesis, spasmolytic activity and structure-activity relationship study of a series of polypharmacological thiobenzanilides.

Recently we presented a series of benzanilide derivatives with a selective spasmolytic effect on terminal ileum preparations of the guinea pig. In this report we demonstrate a further development of these compounds. The exchange of the amide oxygen against a sulfur atom resulted in an up to 325 fold increase of the antispasmodic activity of the thiobenzanilide (IC(50) of 0.1 µM) compared to its benzanilide derivative. Considering their mode of action the compounds interacted with several molecular targets, suggesting that we identified a chemical identity able to modulate multiple targets simultaneously. Furthermore, based on this data set, we present a structure-activity relationship study supporting the important role of the sulfur atom.

A journey from benzanilides to dithiobenzanilides: Synthesis of selective spasmolytic compounds.

A series of dithiobenzanilide derivatives was synthesized and each compound was evaluated for its ability to reduce KCl-induced contractions of smooth muscle preparations of the guinea pig. Starting from a recent publication describing benzanilide derivatives as antispasmodic agents, structure-activity guided synthesis was performed to obtain compounds with improved spasmolytic activity. First, compounds with two amide bonds were designed and second, both amide oxygens were replaced by two sp² sulfur atoms resulting in dithiobenzanilide derivatives. The most potent antispasmodic dithiobenzanilide 19 showed improved activity with an IC50 value of 0.4 µM. Moreover, the study also demonstrated that these active compounds were able to antagonize the effect of spasmogens like acetylcholine and phenylephrine and that the activity is not mediated by activation of ATP-dependent potassium channels (K(ATP)-channels) or inhibition of endothelial nitric oxide synthase (eNOS).

Pros and cons of using resazurin staining for quantification of viable Staphylococcus aureus biofilms in a screening assay.

Staining of Staphylococcus aureus biofilms with 20 microM resazurin during 20 min was shown to provide a good screening assay in 96-well micro titer plates. However, data quality was found to be dependent on the staining duration and biofilm concentration. Also, the inadequacy of using resazurin calibration curves with planktonic cells to estimate S. aureus biofilm concentrations was demonstrated.

Benzanilides with spasmolytic activity: chemistry, pharmacology, and SAR.

The following study describes the synthesis of new benzanilide derivatives and their pharmacological investigation on smooth muscle preparations of guinea pigs. All compounds were synthesized in good yields and showed a spasmolytic activity without significant effect on vascular smooth muscles and heart muscle preparations. Moreover, further pharmacological investigations as well as in silico studies were performed to elucidate the mechanism of action. Compound 3 showed the most potent spasmolytic activity with an IC(50) of 3.25microM.

'Bridged' stilbene derivatives as selective cyclooxygenase-1 inhibitors.

Resveratrol ((E)-3,4',5-trihydroxy-stilbene), a phytoalexin found in various plants, shows non-selective cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) inhibition. In order to find more selective COX inhibitors a series of bridged stilbene derivatives was synthesized and evaluated for their ability to inhibit both COX-1 and COX-2 in vitro. The compounds showed a high rate of COX-1 inhibition with the most potent compounds exhibiting submicromolar IC(50) values and high selectivity indices. A prediction model for COX-inhibiting activity was also developed using the classical LIE approach resulting in consistent docking data for our molecule sample. Phenyl substituted 1,2-dihydronaphthalene derivatives and 1H-indene derivatives therefore represent a novel class of highly selective COX-1 inhibitors and land promising candidates for in vivo studies.