Investigating the photosensitization activities of flavins irradiated by blue LEDs.

Due to their ability to easily absorb light and to generate highly reactive species, photosensitizers emerged as promising tools in a wide variety of physico-chemical and biological processes. Natural photosensitizers have the benefit of a life-compatible toxicological profile. Porphyrins and flavins are such examples that already proved their efficiency as photo-dynamic therapeutics. The present article describes a reliable, easy-to-implement, readily available and reproducible method that can be used to characterize the photosensitizing activity of flavins. Several key factors were investigated during this study, the optimum parameters were: (i) a blue LED light source (λ em = 455 nm) at 6.69 mW; (ii) a pH of 6 mimicking the tumoral environment; (iii) an air-saturated atmosphere reaction medium, (iv) a tetrazolium dye (MTT) was used to monitor the photosensitization efficacy via the generation of the colored MTT-formazan product. This method can be used to rank a series of flavins based on their photosensitizing activities. Such structure-photosensitization activity relationships are essential for the discovery of future potent photosensitizers for photodynamic therapy.

Enhancing the photosensitizing activity of natural flavins: Tuning the heavy-atom effect in the isoalloxazine series.

Structure-photosensitizing activity relationships for a series of flavin analogues were investigated with the final goal of identifying the most potent photosensitizer in these series. The main structural modifications involved the introduction of various halogen atoms in C7- and/or C8-positions on the isoalloxazine ring. These compounds were synthesized by reacting judiciously-functionalized anilines with alloxan. The SAR trends showed that the photosensitizing activity increased with the size of the halogen atoms, confirming the importance of the heavy-atom effect on the photosensitizer's activity. The halogens in C8 were more active than the di-substituted halogens, which in turn were more active than the C7-substituted equivalents. However, even if the photosensitizing activity is slightly less important for the 7- compared to the 8-substituted derivatives, the 7-haloisoalloxazines are promising photosensitizers, as they present a better cellular toxicity profile than the 8-substituted analoges. The photosensitizing activity perfectly correlated with the determined fluorescence for the same compounds. Except for the dihalogeno derivatives, all the compounds were not toxic up to a 50 µM range.

Flavin-Conjugated Nanobombs: Key Structural Requirements Governing Their Self-Assemblies' Morphologies.

In contrast to artificial molecules, natural photosensitizers have the benefit of excellent toxicity profiles and of life-compatible activating energy ranges. Flavins are such photosensitizers that were selected by nature in a plethora of light-triggered biochemical reactions. Flavin-rich nanoparticles could thus emerge as promising tools in photodynamic therapies and in active-targeting drug delivery. Self-assembled flavin-conjugated phospholipids improve the pharmacokinetics of natural flavins and, in the case of controlled morphologies, reduce photobleaching phenomena. The current article presents a proof of concept for the design of riboflavin-rich nanoparticles of tunable morphology from multilamellar patches to vesicular self-assemblies. Coarse-grained simulations of the self-assembling process revealed the key interactions governing the obtained nanomaterials and successfully guided the synthesis of new flavin-conjugates of predictable self-assembly. The obtained flavin-based liposomes had a 65 nm hydrodynamic diameter, were stable, and showed potential photosensitizer activity.

Productive Syntheses of Privileged Scaffolds Inspired by the Recognition of a Diels-Alder Pattern Common to Three Classes of Natural Products.

Identification of a common Diels-Alder pattern in three classes of bioactive natural products led us to study the synthesis and cycloaddition of a new class of cyclic dienes readily available from ß,γ-unsaturated lactams. A practical and readily scalable route to the parent p-methoxybenzyl-protected 6- and 7-membered ß,γ-unsaturated lactams was developed. These were readily transformed into the corresponding O-silylated dienes, which were reacted with dimethyl and diethyl fumarate to yield stereoselectively highly functionalized bicyclic adducts. These exhibited unexpected and versatile transformations upon acid hydrolysis depending on the nature of the dienophile substituents and the acid catalyst. All reactions have been performed on multigram quantities. These transformations provide a convenient, economical, and easily scalable pathway for the rapid construction of functionally and stereochemically dense privileged scaffolds for the construction of libraries of natural products-inspired molecules of pharmacological relevance.

Discovery of a subnanomolar and selective spirocyclic agonist of the glucocorticoid receptor.

Pure diastereomeric spirocyclic analogs of fluorocortivazol were conveniently prepared by a short and efficient synthetic sequence recently developed in our laboratory. The structures and conformations of several key products were confirmed by single crystal X-ray diffraction analysis. Conformational assignments were also supported by DFT calculations. Biological evaluation led to the identification of a highly potent hGR agonist with excellent anti-inflammatory effects in the subnanomolar range. All tested compounds from this series were also selective versus the progesterone receptor.

Development of Potent and Selective Tissue Transglutaminase Inhibitors: Their Effect on TG2 Function and Application in Pathological Conditions.

Potent-selective peptidomimetic inhibitors of tissue transglutaminase (TG2) were developed through a combination of protein-ligand docking and molecular dynamic techniques. Derivatives of these inhibitors were made with the aim of specific TG2 targeting to the intra- and extracellular space. A cell-permeable fluorescently labeled derivative enabled detection of in situ cellular TG2 activity in human umbilical cord endothelial cells and TG2-transduced NIH3T3 cells, which could be enhanced by treatment of cells with ionomycin. Reaction of TG2 with this fluorescent inhibitor in NIH3T3 cells resulted in loss of binding of TG2 to cell surface syndecan-4 and inhibition of translocation of the enzyme into the extracellular matrix, with a parallel reduction in fibronectin deposition. In human umbilical cord endothelial cells, this same fluorescent inhibitor also demonstrated a reduction in fibronectin deposition, cell motility, and cord formation in Matrigel. Use of the same inhibitor in a mouse model of hypertensive nephrosclerosis showed over a 40% reduction in collagen deposition.

Chemical modifications of the N-methyl-laudanosine scaffold point to new directions for SK channels exploration.

An asparagine or a histidine are present in a similar position in the outer pore region of SK2 and SK3 channels, respectively. Therefore, this structural difference was targeted in order to develop selective blockers of SK channel subtypes. Following docking investigations, based on theoretical models of truncated SK2 and SK3 channels, the benzyl side chain of N-methyl-laudanosine (NML) was functionalized in order to target this specific amino-acid residues. Chiral butanamide and benzyloxy analogues were prepared, resolved and tested for their affinity for SK2 and SK3 channels. Isoquinolinium (NMIQ) derivatives have a higher affinity for both SK channel subtypes than the corresponding derivative with no functionalized side chain. This trend was observed also for the 1,2,3,4-tetrahydroisoquinoline (THIQ) analogues. A benzyloxy functionalized NML enantiomer has a higher affinity than NML stereoisomers. Otherwise, the conserved affinity of these analogues led to the opportunity to further investigate in terms of possible labeling for in vivo investigations of the role of SK channels.

Imidazolium-based warheads strongly influence activity of water-soluble peptidic transglutaminase inhibitors.

New peptidic water-soluble inhibitors are reported. In addition to the carboxylate moiety, a new polar warhead was explored. Depending on the size of its substituents, the newly appended imidazolium scaffold designed to enhance the hydrophilic character of the inhibitors could induce a good inhibition for tissue transglutaminase (TG2) and blood coagulation factor XIIIa (FXIIIa). Correlated with the narrow tunnel that hosts the target catalytic cysteine residue, the various modulations suggest a bent conformation of the ligands as the binding pattern mode. Analogues in the dialkylsulfonium series were also tested and showed specificity for TG2 over FXIIIa.

Implementation of a design space approach for enantiomeric separations in polar organic solvent chromatography.

This paper focuses on implementing a design space approach and on the critical process parameters (CPPs) to consider when applying the Quality by Design (QbD) concepts outlined in ICH Q8(R2), Q9 and Q10 to analytical method development and optimization for three chiral compounds developed as modulators of small conductance calcium-activated potassium (SK) channels. In this sense, an HPLC method using a polysaccharide-based stationary phase containing a cellulose tris (4-chloro-3-methylphenylcarbamate) chiral selector in polar organic solvent chromatography mode was considered. The effects of trifluoroacetic acid (TFA) and n-hexane concentration in an acetonitrile (MeCN) mobile phase were investigated under a wide range of column temperatures. Good correlations were found between the observed data obtained after using a central composite design and the expected chromatographic behaviours predicted by applying the design of experiments-design space (DoE-DS) methodology. The critical quality attribute represented here by the separation criterion (S(crit)) allowed assessing the quality of the enantioseparation. Baseline separation for the compounds of interest in an analysis time of less than 20 min was possible due to the original and powerful tools applied which facilitated an enhanced method comprehension. Finally, the advantage of the DoE-DS approach resides in granting the possibility to concurrently assess robustness and identify the optimal conditions which are compound dependent.

Synthesis and radioligand binding studies of bis-(8-isopropyl-isoquinolinium) derivatives as ligands for apamin-sensitive sites on cloned SK2 and SK3 channels.

A structure-activity relationship study of N-methyl-laudanosine, a SK channel blocker, has indicated that the 6,7-dimethoxy group could be successfully replaced by a hydrophobic moiety such as an isopropyl substituent in position 8 of the isoquinoline ring. In the present study, bis-(8-isopropyl-isoquinolinium) derivatives (2a-e) were synthesized and tested for their affinity for cloned SK2 and SK3 channels in comparison with their 6,7-dimethoxy analogues (4a-f). Several ligands were investigated, both in flexible (propyl, butyl and pentyl) and rigid (m- or p-xylyl) series, the m-xylyl derivative (2d) having the best profile in terms of affinity and selectivity for SK3/SK2 channels. Molecular studies showed that the optimal conformation of compound 2d fits well with our SK pharmacophore model.

SAR studies on new bis-aryls 5-HT7 ligands: Synthesis and molecular modeling.

Structure-activity relationships of a series of bis-arylic compounds, investigated as 5-HT(7)R ligands, are reported. The main structural modifications involved a central aryl moiety (phenyl, pyridine, diazine, triazine) and the nature and position of an amine-containing aliphatic chain. The affinity of the synthesized compounds (26 nM-10 microM) was systematically correlated with other previously reported series of bis-arylic ligands and rationalized by a ligand-based pharmacophore approach.

New insights into homopiperazine-based 5-HT1A/5-HT7R ligands: synthesis and biological evaluation.

The synthesis of new N-homopiperazinyl-based ligands is reported. Various structural modifications along with the corresponding biological activities on 5-HT(1A)/5-HT(7) receptors give further insights into this class of serotoninergic ligands. Among the tested central heterocyles, the 7-azaindole gave the best results on the above-mentioned receptors.

Benzimidazolone-based serotonin 5-HT1A or 5-HT7R ligands: synthesis and biological evaluation.

A new group of serotoninergic 5-HT(1A) or 5-HT(7) receptor ligands was identified. These compounds were designed and synthesized on a benzimidazolone scaffold and they enrich the well-known arylpiperazine class of 5-HT ligands. Diverse pharmacomodulations induced a shift in the affinity and selectivity profile with final identification of new potent hits.