Streamlined Stereoselective Entry to (-)-Quinagolide and to 3-Substituted Octahydrobenzo[g]-Quinolines.

A very short stereoselective synthesis of enantiomerically pure (3S, 4aS, 10aR)-quinagolide has been developed. The key steps involved are a copper-catalyzed regioselective arylation of (S)-epichlorohydrin with 1,6-dimethoxynaphthalene and a diastereoselective trans-reduction of a cyclic enamine intermediate. The possibility to use both enantiomers of epichlorohydrin and the diastereodivergency found in the reduction process paves the way for a general preparation also in the nonracemic form of chiral trans-fused 3-substituted octahydrobenzo[g]quinolines that are privileged structures in medicinal chemistry.

Ring-Opening Reactions of Imidazolidines and Hexahydropyrimidines with Grignard Reagents.

We herein report an unprecedented ring-opening of unstrained cyclic aminals such as imidazolidines and hexahydropyrimidines by the use of Grignard and cuprate reagents to give secondary sulfonamides bearing diversely substituted tertiary amines in the ß- or γ-position. This synthetic procedure can be carried out in a one-pot fashion without collateral reactions that are commonly associated with sp3-organometallic multicomponent Mannich-type reactions, indicating the fundamental role of sulfonamide protection of the second nitrogen atom in the generation of the cyclic aminal and in the ring-opening process. Computational density functional theory (DFT) data point to the formation of a transient iminium ion intermediate, in which the Lewis acidity of the cationic component of the organometallic reagent triggers the ring-opening process by coordination. The presented method allows the nucleophilic decoration of diamines including those bearing an adjacent chiral center to the tertiary amine not easily achievable by means of alternative standard synthetic procedures.

Glycoconjugate coumarins exploiting metabolism-enhanced fluorescence and preferential uptake: New optical tools for tumor cell staining.

The possibility to visually discriminate cells based on their metabolism and capability to uptake exogenous molecules is an important topic with exciting fallback on translational and precision medicine. To this end, probes that combine several complementary features are necessary. The ideal probe is selectively uptaken and activated in tumor cells compared with control ones and is not fluorescent in the extracellular medium. Fluorogenic compounds that combine enzyme-activated pH sensitivity and good cell uptake can be an ideal solution, provided that the sensed enzymes are dysregulated in tumor cells. Here, we present synthesis and in vitro evaluation of a new class of glyco-coumarin based probes that merge all these features. These probes show uptake ratio in tumor vs. control cells up to 3:1, with a cell to background ratio upon administration of the probe up to 5:1. These features make this new family of fluorogenic targeted probes a promising tool in life science.

Development of an asymmetric formal synthesis of (-)-quinagolide via enzymatic resolution and stereoselective iminium ion reduction.

The stereoselective reduction of a diastereoisomeric mixture of benzo[g]octahydroquinolinium ion was examined in detail. A diastereoselective borohydride reduction in combination with an efficient deacylative enzymatic resolution of its ß-aminoester precursor are the key steps for a stereoselective installation of the three chiral centres present in the (3S,4aS,10aR)-eutomer of the medicinal drug quinagolide. The obtained data paves the way for an easy and practical attainment of chiral 3-substituted octahydrobenzo[g]quinolines that are privileged structures in medicinal chemistry.

New glycoconjugation strategies for Ruthenium(II) arene complexes via phosphane ligands and assessment of their antiproliferative activity.

Glycoconjugation is a powerful tool to improve the anticancer activity of metal complexes. Herein, we modified commercial arylphosphanes with carbohydrate-derived fragments for the preparation of novel glycoconjugated ruthenium(II) p-cymene complexes. Specifically, d-galactal and d-allal-derived vinyl epoxides (VEß and VEα) were coupled with (2-hydroxyphenyl)diphenylphosphane, affording the 2,3-unsaturated glycophosphanes 1ß and 1α. Ligand exchange with [Ru(C2O4)(η6-p-cymene)(H2O)] gave the glycoconjugated complexes Ru1ß and Ru1α which were subsequently dihydroxylated with OsO4/N-methylmorpholine N-oxide to Ru2ß and Ru2α containing O-benzyl d-mannose and d-gulose units respectively. Besides, aminoethyl tetra-O-acetyl-ß-d-glucopyranoside was condensed with borane-protected (4-diphenylphosphanyl)benzoic acid by HATU/DIPEA under MW heating, to afford the amide 3∙BH3. Zemplén deacylation with MeONa/MeOH gave the deprotected d-glucopyranoside derivative 4∙BH3. The glycoconjugated phosphane complexes Ru3 and Ru4 were obtained by reaction of the phosphane-boranes 3∙BH3 and 4∙BH3 with [Ru(C2O4)(η6-p-cymene)(H2O)]. The employed synthetic strategies were devised to circumvent unwanted phosphine oxidation. The compounds were purified by silica chromatography, isolated in high yield and purity and characterized by analytical and spectroscopic (IR and multinuclear NMR) techniques. The behaviour of the six glycoconjugated Ru complexes in aqueous solutions was assessed by NMR and MS measurements. All compounds were screened for their in vitro cytotoxicity against A2780/A2780R human ovarian and MCF7 breast cancer cell lines, revealing a significant cytotoxicity for complexes containing the 2,3-unsaturated glycosyl unit (Ru1ß, Ru1α). Additional studies on five other human cancer cells, as well as time-dependent toxicity and cell-uptake analyses on ovarian cancer cells, confirmed the prominent activity of these two compounds - higher than cisplatin - and the better performance of the ß anomer. However, Ru1ß, Ru1α did not show preferential activity against cancer cells with respect to fetal lung fibroblast and human embryonic kidney cells as models of normal cells. The effects of the two ruthenium glycoconjugated compounds in A2780 ovarian cancer cells were further investigated by cell cycle analysis, induction of apoptosis, intracellular ROS production, activation of caspases 3/7 and disruption of mitochondrial membrane potential. The latter is a relevant factor in the mechanism of action of the highly cytotoxic Ru1ß, inducing cell death by apoptosis.

Influence of Divalent Cations in the Protein Crystallization Process Assisted by Lanthanide-Based Additives.

The use of lanthanide complexes as powerful auxiliaries for biocrystallography prompted us to systematically analyze the influence of the commercial crystallization kit composition on the efficiency of two lanthanide additives: [Eu(DPA)3]3- and Tb-Xo4. This study reveals that the tris(dipicolinate) complex presents a lower chemical stability and a strong tendency toward false positives, which are detrimental for its use in a high-throughput robotized crystallization platform. In particular, the crystal structures of (Mg(H2O)6)3[Eu(DPA)3]2·7H2O (1), {(Ca(H2O)4)3[Eu(DPA)3]2}n·10nH2O (2), and {Cu(DPA)(H2O)2}n (3), resulting from spontaneous crystallization in the presence of a divalent alkaline-earth cation and transmetalation, are reported. On the other hand, Tb-Xo4 is perfectly soluble in the crystallization media, stable in the presence of alkaline-earth dications, and slowly decomposes (within days) by transmetalation with transition metals. The original structure of [Tb4L4(H2O)4]Cl4·15H2O (4) is also described, where L represents a bis(pinacolato)triazacyclononane ligand. This paper also highlights a potential synergy of interactions between Tb-Xo4 and components of the crystallization mixtures, leading to the formation of complex adducts like {AdkA/Tb-Xo4/Mg2+/glycerol} in the protein binding sites. The observation of such multicomponent adducts illustrated the complexity and versatility of the supramolecular chemistry occurring at the surface of the proteins.

Stereoselective synthesis of new pyran-dioxane based polycycles from glycal derived vinyl epoxide.

Chiral heteropolycyclic structures are widespread in compounds of high pharmaceutical relevance. In particular, linear fused pyran-dioxane based polycycles can be found in several naturally occurring molecules, and among them, cardiac glycosides and antibiotic spectinomycin are characterized by a cis-cisoid-trans geometry. Then, the stereocontrol in the synthesis of this type of polycyclic scaffold is of primary importance. Herein, we present two novel linear fused pyran-dioxane based bi- and tricycles, synthesized with total stereoselectivity from a glycal derived vinyl epoxide. The straightforward methodology described involves a substrate-dependent stereospecific glycosylation step followed by an intramolecular SN2' conjugate addition process, leading to a pyran-dioxane-cyclohexane tricycle with a cis-cisoid-trans stereochemistry, in agreement with the geometry of many natural products. The stereochemical analysis of these compounds, which was realized by a combined NMR/computational approach, is also reported.

New lipophilic glycomimetic DC-SIGN ligands: Stereoselective synthesis and SPR-based binding inhibition assays.

The design and synthesis of efficient ligands for DC-SIGN is a topic of high interest, because this C-type lectin has been implicated in the early stages of many infection processes. DC-SIGN membrane-protein presents four carbohydrate-binding domains (CRD) that specifically recognize mannose and fucose. Therefore, antagonists of minimal disaccharide epitope Manα(1,2)Man, represent potentially interesting antibacterial and antiviral agents. In the recent past, we were able to develop efficient antagonists, mimics of the natural moiety, characterized by the presence of a real d-carbamannose unit which confers greater stability to enzymatic breakdown than the corresponding natural disaccharide ligand. Herein, we present the challenging stereoselective synthesis of four new amino or azide glycomimetic DC-SIGN antagonists with attractive orthogonal lipophilic substituents in C(3), C(4) or C(6) positions of the real carba unit, which were expected to establish crucial interactions with lipophilic areas of DC-SIGN CRD. The activity of the new ligands was evaluated by SPR binding inhibition assays. The interesting results obtained, allow to acquire important information about the influence of the lipophilic substituents present in specific positions of the carba scaffold. Furthermore, C(6) benzyl C(4) tosylamide pseudodisaccharide displayed a good affinity for DC-SIGN with a more favorable IC50 value than those of the previously described real carba-analogues. This study provides valuable knowledge for the implementation of further structural modifications towards improved inhibitors.

New Coumarin Dipicolinate Europium Complexes with a Rich Chemical Speciation and Tunable Luminescence.

Europium (III) luminescent chelates possess intrinsic photophysical properties that are extremely useful in a wide range of applications. The lack of examples of coumarin-based lanthanide complexes is mainly due to poor photo-sensitization attempts. However, with the appeal of using such a versatile scaffold as antenna, especially in the development of responsive molecular probes, it is worth the effort to research new structural motifs. In this work, we present a series of two new tris coumarin-dipicolinate europium (III) complexes, specifically tailored to be either a mono or a dual emitter, tuning their properties with a simple chemical modification. We also encountered a rich chemical speciation in solution, studied in detail by means of paramagnetic NMR and emission spectroscopy.

Copper-Catalyzed Ring-Opening Reactions of Alkyl Aziridines with B2pin2: Experimental and Computational Studies.

The possibility to form new C-B bonds with aziridines using diboron derivatives continues to be a particularly challenging field in view of the direct preparation of functionalized ß-aminoboronates, which are important compounds in drug discovery, being a bioisostere of ß-aminoacids. We now report experimental and computational data that allows the individuation of the structural requisites and of reaction conditions necessary to open alkyl aziridines using bis(pinacolate)diboron (B2pin2) in a regioselective nucleophilic addition reaction under copper catalysis.

Coumarin-based fluorescent biosensor with large linear range for ratiometric measurement of intracellular pH.

Intracellular pH is a critical parameter involved in cell machinery, and its dysregulation can either cause or signal pathologic states. Currently described fluorescent pH probes are based on single acid-base equilibria, and for this reason are intrinsically unable to capture the wide range of cell pH, usually spanning over more than four units. Here we describe a fluorescent pH biosensor based on a conjugated coumarin-triazine scaffold that is excitable in the visible range, shows pseudo-linear ratiometric response over more than 6 pH units with a single fluorogenic unit, and allows imaging of the whole endo-lysosomal pH landscape of living cells with a single acquisition. The probe can discriminate, on the basis of intracellular acidity, between physiologic and tumor cells, being potentially suitable in perspective as diagnostic tool.

Experimental and Computational Studies Unraveling the Peculiarity of Enolizable Oxoesters in the Organocatalyzed Mannich-Type Addition to Cyclic N-Acyl Iminium Ions.

γ- and δ-Oxoesters are easily available starting materials that have been sparingly used in some organocatalyzed reactions proceeding with a high enantioselectivity. In our experimentation we found that the use of these compounds as the enolizable (nucleophilic) component in organocatalyzed Mannich-type reactions using in situ-generated cyclic N-acyl iminium ions gave low diastereoselectivity and low to moderate values of enantioselectivity. This significant drop of facial selectivity with respect to simple aliphatic aldehydes has been rationalized by means of density functional theory (DFT) calculations.

New 1,3-Disubstituted Benzo[h]Isoquinoline Cyclen-Based Ligand Platform: Synthesis, Eu3+ Multiphoton Sensitization and Imaging Applications.

The development of lanthanide-based luminescent probes with a long emission lifetime has the potential to revolutionize imaging-based diagnostic techniques. By a rational design strategy taking advantage of computational predictions, a novel, water-soluble Eu3+ complex from a cyclen-based ligand bearing 1,3-disubstituted benzo[h]isoquinoline arms was realized. The ligand has been obtained overcoming the lack of reactivity of position 3 of the isoquinoline moiety. Notably, steric hindrance of the heteroaromatic chromophore allowed selective and stoichiometry-controlled insertion of two or three antennas on the cyclen platform without any protection strategy. The complex bears a fourth heptanoic arm for easy conjugation to biomolecules. This new chromophore allowed the sensitization of the metal center either with one or two photons excitation. The suitability as a luminescent bioprobe was validated by imaging BMI1 oncomarker in lung carcinoma cells following an established immunofluorescence approach. The use of a conventional epifluorescence microscope equipped with a linear structured illumination module disclosed a simple and inexpensive way to image confocally Ln-bioprobes by single photon excitation in the 350-400 nm window, where ordinary confocal systems have no excitation sources.

First Examples of H2S-Releasing Glycoconjugates: Stereoselective Synthesis and Anticancer Activities.

H2S donors are currently emerging as promising therapeutic agents in a wide variety of pathologies, including tumors. Cancer cells are characterized by an enhanced uptake of sugars, such as glucose. Therefore, novel glycoconjugated H2S donors were synthesized so that high concentrations of H2S can be selectively achieved therein. Dithiolethione portions or isothiocyanate portions were selected for their well-known H2S-releasing properties in the presence of biological substrates. A synthetic procedure employing trichloroacetimidate glycosyl donors was applied to produce, in a stereoselective fashion, C1-glycoconjugates, whereas C6-glycoconjugates were obtained by a Mitsunobu-based transformation. The resulting molecules were then tested for their anticancer effects on human pancreas adenocarcinoma ascites metastasis cell line AsPC-1. The most potent inhibitors of cell viability (6aß and 7b) proved to release H2S inside the AsPC-1 cells and to alter the basal cell cycle.

Organocatalytic alkylation of carbohydrate-containing aldehydes with dihydroquinoline N,O-acetals: Absolute configuration of 1,2-dihydroquinolines.

The direct catalytic α-amidoalkylation of dihydroquinolines with aldehydes bearing oxygen functionalities at different positions in a Mannich-type reaction has been studied. ß-Alkoxy-aldehyde 1d gave high enantioselectivity, albeit with an inherently poor diastereoselectivity, while the use of α-alkoxy aldehydes 1c was detrimental also to enantioselectivity. Mannich-type reactions have been studied for the first time using new chiral carbohydrate-derived aldehydes 1a,b showing a reactivity markedly influenced by the presence of water. The chiral glycidic backbone showed a slight but significant influence on the overall stereochemical outcome only when present in α-position of the aldehyde. The absolute stereochemistry of the products was studied by electronic circular dichroism (ECD) spectra and compared with theoretical calculations. ECD analysis easily provides the absolute configuration of 1,2-dihydroquinoline derivatives such as quinoline-1(2H)-carboxylates.

Direct enantioselective vinylogous Mannich-type reactions of acyclic enals: New experimental insights into the E/Z-dilemma.

The direct heterofunctionalization of acyclic α,ß-unsaturated aldehydes with N-acylquinolinium ions contemplating the formation of two stereocentres is studied using dienamine catalysis. This work gives some new experimental insights on the remote stereocontrol in dienamine catalysis using unbiased aliphatic systems and large electrophiles, pointing to a (Z)-preference of the reactive configuration of the second double bond.

Remarkable Effect of [Li(G4)]TFSI Solvate Ionic Liquid (SIL) on the Regio- and Stereoselective Ring Opening of α-Gluco Carbasugar 1,2-Epoxides.

Carba analogues of biologically relevant natural carbohydrates are promising structures for the development of future drugs endowed with enhanced hydrolytic stability. An open synthetic challenge in this field is the optimization of new methodologies for the stereo- and regioselective opening of α-gluco carbasugar 1,2-epoxides that allow for the preparation of pseudo mono- and disaccharides of great interest. Therefore, we investigated the effect of Lewis acids and solvate ionic liquids (SILs) on the epoxide ring opening of a model substrate. Of particular interest was the complete stereo- and regioselectivity, albeit limited to simple nucleophiles, toward the desired C(1) isomer that was observed using LiClO4. The results obtained with SILs were also remarkable. In particular, Li[NTf2]/tetraglyme ([Li(G4)]TFSI) was able to function as a Lewis acid and to direct the attack of the nucleophile preferentially at the pseudo anomeric position, even with a more complex and synthetically interesting nucleophile. The regioselectivity observed for LiClO4 and [Li(G4)]TFSI was tentatively ascribed to the formation of a bidentate chelating system, which changed the conformational equilibrium and ultimately permitted a trans-diaxial attack on C(1). To the best of our knowledge, we report here the first case in which SILs were successfully employed in a ring-opening process of epoxides.

Unveiling the Binding Modes of the Crystallophore, a Terbium-based Nucleating and Phasing Molecular Agent for Protein Crystallography.

Crystallophores are lanthanide complexes that act as powerful auxiliary for protein crystallography due to their strong nucleating and phasing effects. To get first insights on the mechanisms behind nucleation induced by Crystallophore, we systematically identified various elaborated networks of supramolecular interactions between Tb-Xo4 and subset of 6 protein structures determined by X-ray diffraction in complex with terbium-Crystallophore (Tb-Xo4). Such interaction mapping analyses demonstrate the versatile binding behavior of the Crystallophore and pave the way to a better understanding of its unique properties.

"Breathing" Motion of a Modulable Molecular Cavity.

A class of hemicryptophane cages that adopt imploded conformations in solution and in the solid state has been described and studied by NMR spectroscopy and X-ray crystallography. It is reported that the degree of collapse of the molecular cavity can be controlled by changing the stereochemistry of the chiral elements of the hemicryptophanes, leading to a modulation of their physical and chemical properties. Upon the binding of an oxidovanadium unit, the collapsed molecular cavity can inflate to give an expanded conformation. Removal of the vanadium core by an ancillary complexing ligand restores the initial folded structure. Thus, coordination/de-coordination of the metal ion controls the dynamic motions of the cage, leading to a reversible nanomechanical process. This controlled motion between a collapsed and expanded cavity can be seen as that of a breathable molecular cage.

Self-aggregation propensity of the Tat peptide revealed by UV-Vis, NMR and MD analyses.

By a combination of UV-Vis analyses, NMR-based diffusion measurements and MD simulations we have demonstrated for the first time that the HIV-1 Tat arginine-rich peptide (Tat11) is able to self-aggregate in both its fluorescently labeled and unlabeled variants. We propose Tat11 dimerization as the dominant aggregation process and show that the associated equilibrium constant increases ten-fold by labeling with the standard TAMRA dye. Also, we extend similar conclusions to other cationic cell penetrating peptides (CPPs), such as Antennapedia (Ant) and nona-arginine (R9).