[18F]Fluspidine-A PET Tracer for Imaging of σ1 Receptors in the Central Nervous System.

σ1 receptors play a crucial role in various neurological and neurodegenerative diseases including pain, psychosis, Alzheimer's disease, and depression. Spirocyclic piperidines represent a promising class of potent σ1 receptor ligands. The relationship between structural modifications and σ1 receptor affinity and selectivity over σ2 receptors led to the 2-fluoroethyl derivative fluspidine (2, Ki = 0.59 nM). Enantiomerically pure (S)-configured fluspidine ((S)-2) was prepared by the enantioselective reduction of the α,ß-unsaturated ester 23 with NaBH4 and the enantiomerically pure co-catalyst (S,S)-24. The pharmacokinetic properties of both fluspidine enantiomers (R)-2 and (S)-2 were analyzed in vitro. Molecular dynamics simulations revealed very similar interactions of both fluspidine enantiomers with the σ1 receptor protein, with a strong ionic interaction between the protonated amino moiety of the piperidine ring and the COO- moiety of glutamate 172. The 18F-labeled radiotracers (S)-[18F]2 and (R)-[18F]2 were synthesized in automated syntheses using a TRACERlab FX FN synthesis module. High radiochemical yields and radiochemical purity were achieved. Radiometabolites were not found in the brains of mice, piglets, and rhesus monkeys. While both enantiomers revealed similar initial brain uptake, the slow washout of (R)-[18F]2 indicated a kind of irreversible binding. In the first clinical trial, (S)-[18F]2 was used to visualize σ1 receptors in the brains of patients with major depressive disorder (MDD). This study revealed an increased density of σ1 receptors in cortico-striato-(para)limbic brain regions of MDD patients. The increased density of σ1 receptors correlated with the severity of the depressive symptoms. In an occupancy study with the PET tracer (S)-[18F]2, the selective binding of pridopidine at σ1 receptors in the brain of healthy volunteers and HD patients was shown.

Negative allosteric modulators of NMDA receptors with GluN2B subunit: synthesis of ß-aminoalcohols by epoxide opening and subsequent rearrangement.

In order to obtain novel antagonists of GluN2B subunit containing NMDA receptors, aryloxiranes were opened with benzylpiperidines. Phenyloxiranes 6 and (indazolyl)oxirane 15 were opened regioselectively at the position bearing the aryl moiety. Reaction of the resulting ß-aminoalcohols 7 and 16 with carboxylic acids under Mitsunobu conditions (DIAD, PPh3) led to rearrangement and after ester hydrolysis to the regioisomeric ß-aminoalcohols 9 and 18. This strategy allows the synthesis of amino-ifenprodil 12 as well using phthalimide in the Mitsunobu reaction. Unexpectedly, the isomeric (indazolyl)oxirane 21 reacted with benzylpiperidines to afford both regioisomeric ß-aminoalcohols 22 and 23. In radioligand receptor binding studies, the indazolyl derivative 18a, which can be regarded as indazole bioisostere of ifenprodil, showed high GluN2B affinity (Ki = 31 nM). Replacement of the benzylic OH moiety of ifenprodil by the NH2 moiety in amino-ifenprodil 12 also resulted in low nanomolar GluN2B affinity (Ki = 72 nM). In TEVC experiments, 18a inhibited the ion flux to the same extent as ifenprodil proving that the phenol of ifenprodil can be replaced bioisosterically by an indazole ring maintaining affinity and inhibitory activity. Whereas 10-fold selectivity was found for the ifenprodil binding site over σ1 receptors, only low preference for the GluN2B receptor over σ2 receptors was detected. The log D7.4 value of 18a (log D7.4 = 2.08) indicates promising bioavailability.

Diastereoselective synthesis and structure-affinity relationships of σ1 receptor ligands with spirocyclic scaffold.

Spirocyclic scaffolds play an increasing role in drug discovery as they define a rigid three-dimensional space to increase specific interactions with protein binding sites. Herein, a spirocyclic center was introduced into the lead compound 1 to rigidify its flexible benzylaminoethyl side chain. The key step of the synthesis was the reaction of different α,ß-unsaturated amides 6 and 13-16 with methyl acrylate in the presence of TBDMSOTf. DFT calculations explain the mechanism of this transformation as concerted Diels-Alder reaction (functionals B3LYP and TPSS) or double (aza)-Michael addition (functionals PBE and wB97X-D). After separation of the diastereomeric spirocyclic products 8 and 17-20, LiAlH4 reduction provided the spirocyclic hydroxymethyl piperidines 21a,b-25a,b showing low nanomolar σ1 affinity (Ki < 100 nM). trans-Configured ligands (a-series) showed higher or equal σ1 affinity and higher selectivity over σ2 receptors and GluN2B-NMDA receptors than their cis-configured analogs (b-series). The additional hydroxymethyl moiety brings the log D7.4 value in a promising range. The high σ1 affinity (Ki = 3.6 nM) and the low lipophilicity result in the highest lipophilic ligand efficiency for the dispiro compound 23a (LLE = 6.0). The spirocyclic compounds reported herein and in particular the dispiro compound 23a demonstrate that ligands containing a large number of sp3 C-atoms possess favorable pharmacological (σ1 receptor affinity, receptor selectivity) and physicochemical properties (log D7.4 value) resulting in promising LLE.

Indazole as a Phenol Bioisostere: Structure-Affinity Relationships of GluN2B-Selective NMDA Receptor Antagonists.

Negative allosteric modulation of GluN2B subunit-containing NMDA receptors prevents overstimulation, resulting in neuroprotective effects. Since the phenol of prominent negative allosteric modulators is prone to rapid glucuronidation, its bioisosteric replacement by an indazole was envisaged. The key step in the synthesis was a Sonogashira reaction of non-protected iodoindazoles with propargylpiperidine derivatives. Modification of the alkynyl moiety allowed the introduction of several functional groups. The synthesized indazoles showed very high GluN2B affinity but limited selectivity over σ receptors. Molecular dynamics simulations revealed the same molecular interactions with the ifenprodil binding site as the analogous phenols. In two-electrode voltage-clamp experiments, enantiomeric 3-(4-benzylpiperidin-1-yl)-1-(1H-indazol-5-yl)propan-1-ols (S)-10a and (R)-10a displayed higher inhibitory activity than ifenprodil. In contrast to phenolic GluN2B antagonists, the indazoles were not conjugated with glucuronic acid. It can be concluded that the phenol of potent GluN2B antagonists can be replaced bioisosterically by an indazole, retaining the high GluN2B affinity and activity but inhibiting glucuronidation.

Conformationally Restricted σ1 Receptor Antagonists from (-)-Isopulegol.

Antagonists at σ1 receptors have great potential for the treatment of neuropathic pain. Starting from monoterpene (-)-isopulegol (1), aminodiols 8-11 were obtained and transformed into bicyclic 13-16 and tricyclic ligands 19-22. Aminodiols 8-11 showed higher σ1 affinity than the corresponding bicyclic 13-16 and tricyclic derivatives 19-22. (R)-configuration in the side chain of aminodiols (8 and 10) led to higher σ1 affinity than (S)-configuration (9 and 11). 4-Benzylpiperidines (b-series) revealed higher σ1 affinity than 4-phenylbutylamines (a-series). Aminodiol 8b showed very high σ1 affinity (Ki = 1.2 nM), excellent selectivity over σ2 receptors, and promising logD7.4 (3.05) and lipophilic ligand efficiency (5.87) values. Molecular dynamics simulations were conducted to analyze the σ1 affinity and selectivity on an atomistic level. In the capsaicin assay, 8b exhibited similar antiallodynic activity to the prototypical σ1 antagonist S1RA. The antiallodynic activity of 8b was removed by co-application of the σ1 agonist PRE-084, proving σ1 antagonism being involved in the antiallodynic effect.

In vitro ADME characterization of a very potent 3-acylamino-2-aminopropionic acid-derived GluN2C-NMDA receptor agonist and its ester prodrugs.

The GluN2C subunit exists predominantly, but not exclusively in NMDA receptors within the cerebellum. Antagonists such as UBP1700 and positive allosteric modulators including PYD-106 and 3-acylamino-2-aminopropionic acid derivatives such as UA3-10 ((R)-2-amino-3-{[5-(2-bromophenyl)thiophen-2-yl]carboxamido}propionic acid) represent promising tool compounds to investigate the role of GluN2C-containing NMDA receptors in the signal transduction in the brain. However, due to its high polarity the bioavailability and CNS penetration of the amino acid UA3-10 are expected to be rather low. Herein, three ester prodrugs 12a-c of the NMDA receptor glycine site agonist UA3-10 were prepared and pharmacokinetically characterized. The esters 12a-c showed higher lipophilicity (higher logD 7.4 values) than the acid UA3-10 but almost the same binding at human serum albumin. The acid UA3-10 was rather stable upon incubation with mouse liver microsomes and NADPH, but the esters 12a-c were fast hydrolyzed to afford the acid UA3-10. Incubation with pig liver esterase and mouse serum led to rapid hydrolysis of the esters 12a-c. The isopropyl ester 12c showed a promising logD 7.4 value of 3.57 and the highest stability in the presence of pig liver esterase and mouse serum. These results demonstrate that ester prodrugs of UA3-10 can potentially afford improved bioavailability and CNS penetration.

Multifunctional cubic liquid crystalline nanoparticles for chemo- and photodynamic synergistic cancer therapy.

With the aim of engineering multifunctional nanoparticles useful for cancer therapy, a diketopyrrolopyrrole-porphyrin based photosensitizer was here conjugated to a block copolymer (Pluronic F108), and used to stabilize in water lipidic cubic liquid crystalline nanoparticles (cubosomes), also loaded with the antineoplastic agent docetaxel. The physicochemical characterization by SAXS, DLS, and cryo-TEM demonstrated that the formulation consisted of cubosomes, about 150 nm in size, possessing a bicontinuous cubic structure (space group Pn3m). The cellular imaging experiments proved that these nanoparticles localized in lysosomes and mitochondria, while cytotoxicity tests evidenced a slight but significant synergistic effect which, after irradiation, increased the toxicity induced by docetaxel alone, allowing further reduction of cell viability.

A degradable fluorinated surfactant for emulsion polymerization of vinylidene fluoride.

An original degradable fluorinated surfactant, 3-hydroxy-2-(trifluoromethyl)propanoic acid (MAF-OH), was applied for the emulsion polymerization of vinylidene fluoride (VDF) to yield a latex of PVDF, consisting of typically 100 nm particle diameters. This surfactant was shown to be easily decomposed in subcritical water, releasing fluoride anions. The % reversed head-to head (H-H) addition is greatly minimized (2.4%) in the PVDF, prepared in the presence of MAF-OH surfactant.

Polymer-free cubosomes for simultaneous bioimaging and photodynamic action of photosensitizers in melanoma skin cancer cells.

We designed novel polymer-free cubic bicontinuous liquid crystalline dispersions (cubosomes) using monoolein as molecular building block, phospholipids as stabilizers, propylene glycol as hydrotrope. Their kinetic stability was evaluated by analysing the backscattering profiles upon ageing, and the most stable formulation was chosen as potential photosensitizers delivery vehicle for photodynamic therapy (PDT) of human skin melanoma cells. Morphological and topological features of such formulation alternatively loaded with Chlorin e6 or meso-Tetraphenylporphine-Mn(III) chloride photosensitizing dyes were investigated by cryo-TEM, DLS, and SAXS. Bioimaging studies demonstrated that Me45 and MeWo cell lines effectively internalized these cubosomes formulations. Particularly, photodynamic activity experiments proved both the very low cytotoxicity of the cubosomes formulation loaded with Chlorin e6 dye in the "dark" condition, and its significant cytotoxic effect after photoirradiation. The toxic effect recorded when the photosensitizer was encapsulated within the cubosomes was shown to be one order of magnitude higher than that caused by the free photosensitizer. This is the first report of biocompatible polymer-free cubosomes for potential application in both PDT and bioimaging of skin malignant melanoma.

Complex, Dynamic Behavior of Extremely Asymmetric Di-n-Alkylphosphate-Anion Aggregates, the Long-Chain Effect and the Role of a Limiting Size: Cryo-TEM, SANS, and X-Ray Diffraction Studies.

For extremely asymmetric n-hexyl(n-decyl)phosphate (HDeP), n-hexyl(n-dodecyl)phosphate (HDoP), and n-hexyl(n-cetyl)phosphate (HCeP), the effect of the long-chains on the dynamic behavior of their aggregate structures in water was examined by cryo-TEM imaging, SANS, and X-ray diffraction techniques. The cryo-TEM images demonstrated the complex and dynamic behavior of the aggregates, and its dependence on the length of the long-chain. Application of the one-dimensional aggregate theory to the SANS results led to the conclusion that the existence of a limiting size also depended on the length of the main long-hydrocarbon chain and affected strongly the dynamic behavior of the aggregates, causing breakage of thread-like micelles or ribbon-like aggregates. The X-ray diffraction patterns of the lyotropic liquid crystalline samples of HDeP and HCeP were used to estimate the aggregate structures of this limited size.

Cubosomes for in vivo fluorescence lifetime imaging.

Herein we provided the first proof of principle for in vivo fluorescence optical imaging application using monoolein-based cubosomes in a healthy mouse animal model. This formulation, administered at a non-cytotoxic concentration, was capable of providing both exogenous contrast for NIR fluorescence imaging with very high efficiency and chemospecific information upon lifetime analysis. Time-resolved measurements of fluorescence after the intravenous injection of cubosomes revealed that the dye rapidly accumulated mainly in the liver, while lifetimes profiles obtained in vivo allowed for discriminating between free dye or dye embedded within the cubosome nanostructure after injection.

Dual-Responsive Lipid Nanotubes: Two-Way Morphology Control by pH and Redox Effects.

Lipid nanotubes are the preferred structures for many applications, especially biological ones, and thus have attracted much interest recently. However, there is still a significant need for developing more lipid nanotubes that are reversibly controllable to improve their functionality and usability. Here, we presented a two-way reversible morphology control of the nanotubes formed by the recently designed molecule AQUA (C25H29NO4). Because of its special design, the AQUA has both pH-sensitive and redox-active characters provided by the carboxylic acid and anthraquinone groups. Upon chemical reduction, the nanotubes turned into thinner ribbons, and this structural transformation was significantly reversible. The reduction of the AQUA nanotubes also switched the nanotubes from electrically conductive to insulative. Nanotube morphology can additionally be altered by decreasing the pH below the pKa value of the AQUA, at ∼4.9. Decreasing the pH caused the gradual unfolding of the nanotubes, and the interlayer distance in the nanotube's walls increased. This morphological change was fast and reversible at a wide pH range, including the physiological pH. Thus, the molecular design of the AQUA allowed for an unprecedented two-way and reversible morphology control with both redox and pH effects. These unique features make AQUA a very promising candidate for many applications, ranging from electronics to controlled drug delivery.

Spontaneous structural transition and crystal formation in minimal supramolecular polymer model.

The association of building blocks into supramolecular polymers allows the fabrication of diverse functional architectures at the nanoscale. The use of minimal assembly units to explore polymer dynamics and phase transitions significantly contributes to the application of polymer physicochemical paradigms in the field of supramolecular polymers. We present a minimal model that displays spontaneous coordinated structural transitions between micro- and nanostructures, hydrogels with nanoscale order, and single crystals. The simple amphiphilic 9-fluorenylmethoxycarbonyl-3,4-dihydroxyphenylalanine (Fmoc-DOPA) modified amino acid undergoes a noninduced transition from spherical assemblies into nanofibrils followed by sol-gel transition, nanotube formation via intermediate assembly, and crystallization within the gel. Notably, the transition kinetics is slow enough to allow both multistage and multiscale characterization of the supramolecular arrangement using electron microscopy, vibrational and circular dichroism spectroscopies, nuclear magnetic resonance, and x-ray crystallography. This minimalistic system is the first comprehensive model for a complete spontaneous structural transition between diverse states governed by distinct molecular interactions.

Docetaxel-Loaded Fluorescent Liquid-Crystalline Nanoparticles for Cancer Theranostics.

Here, we describe a novel monoolein-based cubosome formulation engineered for possible theranostic applications in oncology. The Docetaxel-loaded nanoparticles were stabilized in water by a mixture of commercial Pluronic (poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) triblock copolymer) F108 (PF108) and rhodamine- and folate-conjugated PF108 so that the nanoparticles possess targeting, therapeutic, and imaging properties. Nanoparticles were investigated by DLS, cryo-TEM, and SAXS to confirm their structural features. The fluorescent emission characterization of the proposed formulation indicated that the rhodamine conjugated to the PF108 experiences an environment less polar than water (similar to chloroform), suggesting that the fluorescent fragment is buried within the poly(ethylene oxide) corona surrounding the nanoparticle. Furthermore, these nanoparticles were successfully used to image living HeLa cells and demonstrated a significant short-term (4 h incubation) cytotoxicity effect against these cancer cells. Furthermore, given their analogy as nanocarriers for molecules of pharmaceutical interest and to better stress the singularities of these bicontinuous cubic nanoparticles, we also quantitatively evaluated the differences between cubosomes and multilamellar liposomes in terms of surface area and hydrophobic volume.

Cubosome formulations stabilized by a dansyl-conjugated block copolymer for possible nanomedicine applications.

We present here an innovative, fluorescent, monoolein-based cubosome dispersion. Rather than embedded within the monoolein palisade, the fluorescent imaging agent, namely dansyl, was conjugated to the terminal ethylene oxide moieties of the block copolymer Pluronic F108. We discuss the physicochemical and photophysical properties of this fluorescent Pluronic and of a cubosome formulation stabilized by a mixture of dansyl-conjugated and non-conjugated Pluronic, also including an anticancer drug (quercetin). Furthermore, we performed biocompatibility tests against HeLa cells to assess internalization and cytotoxicity features of this nanoparticles aqueous dispersion. Cryo-TEM, SAXS, and DLS analysis, proved the bicontinuous cubic inner nanostructure and the morphology of this fluorescent cubosome dispersion, while photophysical measurements and biocompatibility results basically validate their potential use for theranostic nanomedicine applications.

Capacious and programmable multi-liposomal carriers.

Spherical polycationic brushes (SPBs) were synthesized by grafting polycationic chains onto 100 nm polystyrene particles. These particles were exposed to unilamellar egg-lecithin (EL) liposomes with a mean diameter of 40 nm that had been rendered anionic via the presence of 10 molar% of phosphatidylserine (PS(1-)). The liposomes also contained 30 mole% of a morpholinocyclohexanol-based lipid (MOCH) that undergoes a conformational flip when the pH is decreased from 7.0 to 5.0. Mixtures of SPBs and liposomes at pH 7 gave an electrostatically-driven complex possessing, on average, about 40 liposomes for each SPB particle. It was found that the bound liposomes rapidly release much of their contents when the pH is reduced from 7.0 to 5.0 owing mostly to a MOCH conformational change that creates defects in the bilayer membrane. The drop in pH does not, however, induce a separation of the liposomes from the SPBs. Around 50-60% of the liposome contents escape before, it is reasoned, lateral and transmembrane motion of the membrane components heals the defects and prevents further release. Remarkably, the liposomes complexed with SPB release their cargo much faster than the identical but non-complexed liposomes.

Scanning-tunneling-spectroscopy-directed design of tailored deep-blue emitters.

Frontier molecular orbitals can be visualized and selectively set to achieve blue phosphorescent metal complexes. For this purpose, the HOMOs and LUMOs of tridentate Pt(II) complexes were measured using scanning tunneling microscopy and spectroscopy. The introduction of electron-accepting or -donating moieties enables independent tuning of the frontier orbital energies, and the measured HOMO-LUMO gaps are reproduced by DFT calculations. The energy gaps correlate with the measured and the calculated energies of the emissive triplet states and the experimental luminescence wavelengths. This synergetic interplay between synthesis, microscopy, and spectroscopy enabled the design and realization of a deep-blue triplet emitter. Finding and tuning the electronic "set screws" at molecular level constitutes a useful experimental method towards an in-depth understanding and rational design of optoelectronic materials with tailored excited state energies and defined frontier-orbital properties.

Cancer-cell-targeted theranostic cubosomes.

This work was devoted to the development of a new type of lipid-based (cubosome) theranostic nanoparticle able to simultaneously host camptothecin, a potent anticancer drug, and a squarain-based NIR-emitting fluorescent probe. Furthermore, to confer targeting abilities on these nanoparticles, they were dispersed using mixtures of Pluronic F108 and folate-conjugated Pluronic F108 in appropriate ratios. The physicochemical characterization, performed via SAXS, DLS, and cryo-TEM techniques, proved that aqueous dispersions of such cubosomes can be effectively prepared, while the photophysical characterization demonstrated that these nanoparticles may be used for in vivo imaging purposes. The superior ability of these innovative nanoparticles in targeting cancer cells was emphasized by investigating the lipid droplet alterations induced in HeLa cells upon exposure to targeted and nontargeted cubosomes.

Ravens notice dominance reversals among conspecifics within and outside their social group.

A core feature of social intelligence is the understanding of third-party relations, which has been experimentally demonstrated in primates. Whether other social animals also have this capacity, and whether they can use this capacity flexibly to, for example, also assess the relations of neighbouring conspecifics, remains unknown. Here we show that ravens react differently to playbacks of dominance interactions that either confirm or violate the current rank hierarchy of members in their own social group and of ravens in a neighbouring group. Therefore, ravens understand third-party relations and may deduce those not only via physical interactions but also by observation.

Statistical length measurement method by direct imaging of carbon nanotubes.

The influence of carbon nanotube (CNT) length on their macroscopic properties requires an accurate methodology for CNT length measurement. So far, existing techniques are limited to short (less than a few micrometers) CNTs and sample preparation methods that bias the measured values. Here, we show that the average length of carbon nanotubes (CNTs) can be measured by cryogenic transmission electron microscopy (cryo-TEM) of CNTs in chlorosulfonic acid. The method consists of dissolving at low concentration CNTs in chlorosulfonic acid (a true solvent), imaging the individual CNTs by cryo-TEM, and processing and analyzing the images to determine CNT length. By measuring the total CNT contour length and number of CNT ends in each image, and by applying statistical analysis, we extend the method to cases where each CNT is long enough to span many cryo-TEM images, making the direct length measurement of an entire CNT impractical. Hence, this new technique can be used effectively to estimate samples in a wide range of CNT lengths, although we find that cryo-TEM imaging may bias the measurement towards longer CNTs, which are easier to detect. Our statistical method is also applied to AFM images of CNTs to show that, by using only a few AFM images, it yields estimates that are consistent with literature techniques, based on individually measuring a higher number of CNTs.