Microfluidic Fabricated Liposomes for Nutlin-3a Ocular Delivery as Potential Candidate for Proliferative Vitreoretinal Diseases Treatment.

Purpose: Proliferative vitreoretinal diseases (PVDs) represent a heterogeneous group of pathologies characterized by the presence of retinal proliferative membranes, in whose development retinal pigment epithelium (RPE) is deeply involved. As the only effective treatment for PVDs at present is surgery, we aimed to investigate the potential therapeutic activity of Nutlin-3a, a small non-genotoxic inhibitor of the MDM2/p53 interaction, on ARPE-19 cell line and on human RPE primary cells, as in vitro models of RPE and, more importantly, to formulate and evaluate Nutlin-3a loaded liposomes designed for ophthalmic administration. Methods: Liposomes were produced using an innovative approach by a microfluidic device under selection of different conditions. Liposome size distribution was evaluated by photon correlation spectroscopy and centrifugal field flow fractionation, while the liposome structure was studied by transmission electron microscopy and Fourier-transform infrared spectroscopy. The Nutlin-3a entrapment capacity was evaluated by ultrafiltration and HPLC. Nutlin-3a biological effectiveness as a solution or loaded in liposomes was evaluated by viability, proliferation, apoptosis and migration assays and by morphological analysis. Results: The microfluidic formulative study enabled the selection of liposomes composed of phosphatidylcholine (PC) 5.4 or 8.2 mg/mL and 10% ethanol, characterized by roundish vesicular structures with 150-250 nm mean diameters. Particularly, liposomes based on the lower PC concentration were characterized by higher stability. Nutlin-3a was effectively encapsulated in liposomes and was able to induce a significant reduction of viability and migration in RPE cell models. Conclusion: Our results lay the basis for a possible use of liposomes for the ocular delivery of Nutlin-3a.

Nutlin-3 Loaded Ethosomes and Transethosomes to Prevent UV-Associated Skin Damage.

The skin's protective mechanisms, in some cases, are not able to counteract the destructive effects induced by UV radiations, resulting in dermatological diseases, as well as skin aging. Nutlin-3, a potent drug with antiproliferative activity in keratinocytes, can block UV-induced apoptosis by activation of p53. In the present investigation, ethosomes and transethosomes were designed as delivery systems for nutlin-3, with the aim to protect the skin against UV damage. Vesicle size distribution was evaluated by photon correlation spectroscopy and morphology was investigated by cryogenic transmission electron microscopy, while nutlin-3 entrapment capacity was evaluated by ultrafiltration and HPLC. The in vitro diffusion kinetic of nutlin-3 from ethosomes and transethosomes was studied by Franz cell. Moreover, the efficiency of ethosomes and transethosomes in delivering nutlin-3 and its protective role were evaluated in ex vivo skin explants exposed to UV radiations. The results indicate that ethosomes and transethosomes efficaciously entrapped nutlin-3 (0.3% w/w). The ethosome vesicles were spherical and oligolamellar, with a 224 nm mean diameter, while in transethosome the presence of polysorbate 80 resulted in unilamellar vesicles with a 146 nm mean diameter. The fastest nutlin-3 kinetic was detected in the case of transethosomes, with permeability coefficients 7.4-fold higher, with respect to ethosomes and diffusion values 250-fold higher, with respect to the drug in solution. Ex vivo data suggest a better efficacy of transethosomes to promote nutlin-3 delivery within the skin, with respect to ethosomes. Indeed, nutlin-3 loaded transethosomes could prevent UV effect on cutaneous metalloproteinase activation and cell proliferative response.

Microwave-assisted enzymatic synthesis of geraniol esters in solvent-free systems: optimization of the reaction parameters, purification and characterization of the products, and biocatalyst reuse.

Various geraniol esters act as insect pheromones and display pharmacological activities, especially as neuroprotective agents. Therefore, the search for synthetic strategies alternative to traditional chemical synthesis could help designing ecofriendly routes for the preparation of such bioactive compounds. Hence, this work aims at the microwave-assisted enzymatic synthesis of geranyl esters in solvent-free systems. The process variables were optimized for the synthesis of geranyl acetoacetate, achieving 85% conversion after 60 min using a 1:5 substrates molar ratio (ester to geraniol), 80 °C and 8.4% of Lipozyme 435 lipase without removal of the co-produced methanol. On the other hand, a 95% conversion was reached after 30 min using 1:6 substrates molar ratio, 70 °C and 7% lipase in the presence of 5Å molecular sieves for the methanol capture. In addition, the lipase showed good reusability, maintaining the same activity for five reaction cycles. Finally, under the above optimized conditions, other geraniol esters were successfully synthetized such as the geranyl butyrate (98%), geranyl hexanoate (99%), geranyl octanoate (98%), and geranyl (R)-3-hydroxybutyrate (56%). These results demonstrate the microwave-assisted lipase-catalyzed transesterification in a solvent-free system as an excellent and sustainable catalytic methodology to produce geraniol esters.

Modulable Photocatalyzed Strategies for the Synthesis of α-C-Glycosyl Alanine Analogues via the Giese Reaction with Dehydroalanine Derivates.

Herein, we present the α-selective Giese reaction between pyranosyl/furanosyl bromides and dehydroalanine analogues, which provides access to a library of highly valuable α-C-glycosyl alanines. The key C-glycosyl radical is generated through photocatalysis by either the new generation copper(I) complex [(DPEPhos)(bcp)Cu]PF6 or [Ru(bpy)3](BF4)2. The reactions proceed smoothly, affording the desired α-C-glycosyl alanines in up to 99% yield when diethyl 1,4-dihydro-2,6-dimethyl-3,5-pyridinedicarboxylate [Hantzsch ester (HE)] is used as an additive. N,N-Diisopropylethylamine (DIPEA) has been selected as a reductant in both protocols. A mechanistic study by means of transient absorption spectroscopy unveils a halogen-atom transfer (XAT) process in C-glycosyl radical formation.

Humin Formation on SBA-15-pr-SO3H Catalysts during the Alcoholysis of Furfuryl Alcohol to Ethyl Levulinate: Effect of Pore Size on Catalyst Stability, Transport, and Adsorption.

Herein, the alcoholysis of furfuryl alcohol in a series of SBA-15-pr-SO3H catalysts with different pore sizes is reported. Elemental analysis and NMR relaxation/diffusion methods show that changes in pore size have a significant effect on catalyst activity and durability. In particular, the decrease in catalyst activity after catalyst reuse is mainly due to carbonaceous deposition, whereas leaching of sulfonic acid groups is not significant. This effect is more pronounced in the largest-pore-size catalyst C3, which rapidly deactivates after one reaction cycle, whereas catalysts with a relatively medium and small average pore size (named, respectively, C2 and C1) deactivate after two reaction cycles and to a lesser extent. CHNS elemental analysis showed that C1 and C3 experience a similar amount of carbonaceous deposition, suggesting that the increased reusability of the small-pore-size catalyst can be attributed to the presence of SO3H groups mostly present on the external surface, as corroborated by results on pore clogging obtained by NMR relaxation measurements. The increased reusability of the C2 catalyst is attributed to a lower amount of humin being formed and, at the same time, reduced pore clogging, which helps to maintain accessible the internal pore space.

Oxidative NHC Catalysis: An Unconventional Tool for Macrocyclic Oligoesters Synthesis.

The application of N-heterocyclic carbene (NHC) catalysis under highly diluted oxidative condition to the polycondensation of dialdehydes and diols is herein presented as an alternative, atom-economical synthetic route to macrocyclic oligoesters (MCOs). The disclosed protocol paves the way to the straightforward access to MCOs, starting from commercial dialdehydes, avoiding the use of toxic diacyl chlorides, commonly employed in traditional MCOs synthetic processes. The method is totally metal-free, takes place in the green Me-THF solvent and requires the use of a fully recyclable quinone oxidant. The protocol versatility is confirmed by the employment of fossil-based and bio-based monomers such as 2,5-diformylfuran (DFF), 2,5-bis(hydroxymethyl)furan (BHMF), and isomannide, synthesizing a series of novel and known synthetically relevant macrocyclic oligoesters, fully characterized by NMR and MALDI-TOF MS analysis, with product yields (51-86 %) comparable to those obtained by traditional synthetic routes. Finally, to emphasize the synthetic relevance of the target macrocycles, an entropically-driven ring opening polymerization (ED-ROP) key study has been performed, optimizing the organocatalyzed synthesis of poly(2,5-furan-dimethylene 2,5 furandicarboxylate) (PBHMF) with number-average molecular weight up to 8200 g mol-1 and 66 % isolated yield.

Oxidative N-Heterocyclic Carbene Catalysis.

N-Heterocyclic carbene (NHC) catalysis is a by now consolidated organocatalytic platform for a number of synthetic (asymmetric) transformations via diverse reaction modes/intermediates. In addition to the typical umpolung processes involving acyl anion/homoenolate equivalent species, implementation of protocols under oxidative conditions greatly expands the possibilities of this methodology. Oxidative NHC-catalysis allows for oxidative and oxygenative transformations through specific manipulations of Breslow-type species depending upon the oxidant used (external oxidant or O2 /air), the derived NHC-bound intermediates paving the way to non-umpolung processes through activation of carbon atoms and heteroatoms. This review is intended to update the state of the art in oxidative NHC-catalyzed reactions that appeared in the literature from 2014 to present, with a strong focus to crucial intermediates and their mechanistic implications.

Photoredox Cross-Dehydrogenative Coupling of N-Aryl Glycines Mediated by Mesoporous Graphitic Carbon Nitride: An Environmentally Friendly Approach to the Synthesis of Non-Proteinogenic α-Amino Acids (NPAAs) Decorated with Indoles.

Indole-decorated glycine derivatives are prepared through an environmentally benign cross-dehydrogenative coupling between N-aryl glycine analogues and indoles (yield of ≤81%). Merging heterogeneous organocatalysis and photocatalysis, C-H functionalization has been achieved by selective C-2 oxidation of N-aryl glycines to afford the electrophilic imine followed by Friedel-Crafts alkylation with indole. The sustainability of the process has been taken into account in the reaction design through the implementation of a metal-free recyclable heterogeneous photocatalyst and a green reaction medium. Scale-up of the benchmark reaction (gram scale, yield of 69%) and recycling experiments (over seven runs without a loss of efficiency) have been performed to prove the robustness of the protocol. Finally, mechanistic studies were conducted employing electron paramagnetic resonance spectroscopy to unveil the roles of the photocatalyst and oxygen in the formation of odd-electron species.

Preparations of Poly(lactic acid) Dispersions in Water for Coating Applications.

A green, effective methodology for the preparation of water-based dispersions of poly(lactic acid) (PLA) for coating purposes is herein presented. The procedure consists of two steps: in the first one, an oil-in-water emulsion is obtained by mixing a solution of PLA in ethyl acetate with a water phase containing surfactant and stabilizer. Different homogenization methods as well as oil/water phase ratio, surfactant and stabilizer combinations were screened. In the second step, the quantitative evaporation of the organic provides water dispersions of PLA that are stable, at least, over several weeks at room temperature or at 4 °C. Particle size was in the 200-500 nm range, depending on the preparation conditions, as confirmed by scanning electron microscope (SEM) analysis. PLA was found not to suffer significant molecular weight degradation by gel permeation chromatography (GPC) analysis. Furthermore, two selected formulations with glass transition temperature (Tg) of 51 °C and 34 °C were tested for the preparation of PLA films by drying in PTFE capsules. In both cases, continuous films that are homogeneous by Fourier-transform infrared spectroscopy (FT-IR) and SEM observation were obtained only when drying was performed above 60 °C. The formulation with lower Tg results in films which are more flexible and transparent.

Exploring Oxidative NHC-Catalysis as Organocatalytic Polymerization Strategy towards Polyamide Oligomers.

The polycondensation of diamines and dialdehydes promoted by an N-heterocyclic carbene (NHC) catalyst in the presence of a quinone oxidant and hexafluoro-2-propanol (HFIP) is herein presented for the synthesis of oligomeric polyamides (PAs), which are obtained with a number-average molecular weight (Mn ) in the range of 1.7-3.6 kg mol-1 as determined by NMR analysis. In particular, the utilization of furanic dialdehyde monomers (2,5-diformylfuran, DFF; 5,5'-[oxybis(methylene)]bis[2-furaldehyde], OBFA) to access known and previously unreported biobased PAs is illustrated. The synthesis of higher molecular weight PAs (poly(decamethylene terephthalamide, PA10T, Mn = 62.8 kg mol-1 ; poly(decamethylene 2,5-furandicarboxylamide, PA10F, Mn = 6.5 kg mol-1 ) by a two-step polycondensation approach is also described. The thermal properties (TGA and DSC analyses) of the synthesized PAs are reported.

Exploring the Synergy Between HPTLC and HPLC-DAD for the Investigation of Wine-Making By-Products.

Both environmental and economic issues are increasingly pushing for the revalorization of agri-food by-products, including those arising from wine industry. Wastes produced from wine-making processes are important sources of biologically active compounds, mainly phenolic acids and flavonoids, which could be re-used for several applications, for example as additive surrogates or new ingredients in foodstuffs and/or pharmaceuticals. Therefore, the development of methods aimed at isolating, characterizing and quantifying molecules present in winery by-products acquires considerable importance in view of their re-utilization on a large scale. In this connection, this study demonstrated that high-performance thin-layer chromatography (HPTLC) and high-performance liquid chromatography with diode array detection (HPLC-DAD) can operate in synergy for the investigation of pomace and seed materials arising from both white and red cultivars of Vitis Vinifera. By virtue of fingerprint profiling, mass spectrometry (MS) interfacing and band comparison method, HPTLC enabled detection and identification of phenolic acids, non-anthocyanic flavonoids and anthocyanins. On the contrary, only anthocyanins could be identified by HPLC-DAD, and their subsequent quantification showed that malvidin-3-O-glucoside (oenin) was the most abundant one. In parallel, HPTLC has allowed to detect and quantify proanthocyanidins (PAC), showing that only catechin was present in the test samples. Both quantitative analytical methods were validated in terms of linearity, detection and quantification limits and precision.

Oxidative NHC-Catalysis as Organocatalytic Platform for the Synthesis of Polyester Oligomers by Step-Growth Polymerization.

The application of N-heterocyclic carbene (NHC) catalysis to the polycondensation of diols and dialdehydes under oxidative conditions is herein presented for the synthesis of polyesters using fossil-based (ethylene glycol, phthalaldehydes) and bio-based (furan derivatives, glycerol, isosorbide) monomers. The catalytic dimethyl triazolium/1,8-diazabicyclo[5.4.0]undec-7-ene couple and stoichiometric quinone oxidant afforded polyester oligomers with a number-average molecular weight (Mn ) in the range of 1.5-7.8 kg mol-1 as determined by NMR analysis. The synthesis of a higher molecular weight polyester (polyethylene terephthalate, PET) by an NHC-promoted two-step procedure via oligoester intermediates is also illustrated together with the catalyst-controlled preparation of cross-linked or linear polyesters derived from the trifunctional glycerol. The thermal properties (TGA and DSC analyses) of the synthesized oligoesters are also reported.

Enantioselective Desymmetrization of 1,4-Dihydropyridines by Oxidative NHC Catalysis.

The unprecedented desymmetrization of prochiral dialdehydes catalyzed by N-heterocyclic carbenes under oxidative conditions was applied to the highly enantioselective synthesis of 1,4-dihydropyridines (DHPs) starting from 3,5-dicarbaldehyde substrates. Synthetic elaboration of the resulting 5-formyl-1,4-DHP-3-carboxylates allowed for access to the class of pharmaceutically relevant 1,4-DHP-3,5-dicarboxylates (Hantzsch esters). DFT calculations suggested that the enantioselectivity of the process is determined by the transition state involving the oxidation of the Breslow intermediate by the external quinone oxidant.

Enzymatic synthesis of biobased aliphatic-aromatic oligoesters using 5,5'-bis(hydroxymethyl)furoin as a building block.

5,5'-Dihydroxymethyl furoin (DHMF) is a novel biobased difuranic polyol scaffold, achievable from the benzoin condensation of 5-hydroxymethylfurfural (HMF), which has recently been employed as a monomer for the preparation of cross-linked polyesters and polyurethane. Its upgrading by means of enzymatic reactions has not yet been reported. Here we demonstrated that Candida antarctica lipase B (CALB) is a suitable biocatalyst for the selective esterification of the primary hydroxyl groups of DHMF. Exploiting this enzymatic activity, DHMF has been reacted with the diethyl esters of succinic and sebacic acids obtaining fully biobased linear oligoesters with number-average molecular weight around 1000 g mol-1 and free hydroxyl groups on the polymer backbone. The structures of the DHMF-diacid ethyl ester dimers and of the oligomers were elucidated by NMR and MS analyses.

Native Quercetin as a Chloride Receptor in an Organic Solvent.

The binding properties of quercetin toward chloride anions were investigated by means of ¹H-NMR, 13C-NMR, and electrospray ionization mass spectrometry (ESI-MS) measurements, as well as computational calculations. The results indicate that quercetin behaves primarily as a ditopic receptor with the binding site of the B ring that exhibits stronger chloride affinity compared to the A ring. However, these sites are stronger receptors than those of catechol and resorcinol because of their conjugation with the carbonyl group located on the C ring. The 1:1 and 1:2 complexation of this flavonoid with Cl- was also supported by ESI mass spectrometry.

Aerobic oxidation of 5-hydroxymethylfurfural to 5-hydroxymethyl-2-furancarboxylic acid and its derivatives by heterogeneous NHC-catalysis.

The application of the oxidative system composed of a heterogeneous triazolium pre-catalyst, iron(ii) phthalocyanine and air is described for the selective conversion of 5-hydroxymethylfurfural (HMF) into the added-value 5-hydroxymethyl-2-furancarboxylic acid (HMFCA). The disclosed one-pot two-step procedure involved sequential oxidative esterifications of HMF to afford a polyester oligomer having hydroxyl and carboxyl terminal groups (Mw = 389-1258), which in turn was hydrolyzed by a supported base (Ambersep 900 OH) to yield HMFCA in 87% overall yield. The same strategy was adopted for the effective synthesis of ester and amide derivatives of HMFCA by nucleophilic depolymerization of the oligomeric intermediate with methanol and butylamine, respectively. The utilization of the disclosed oxidative system for the direct conversion of HMF and furfural into their corresponding ester, amide, and thioester derivatives is also reported.

Enantioselective Dearomatization of Alkylpyridiniums by N-Heterocyclic Carbene-Catalyzed Nucleophilic Acylation.

A chiral NHC-catalyzed dearomatizing reaction of activated N-alkylpyridinium salts with aliphatic aldehydes is described. The resulting acylated 1,4-dihydropyridines have been obtained with complete C4 regioselectivity and enantioselectivities in the range 52-78% ee. The (4R)-absolute configuration of the synthesized compounds has been determined by the TD-DFT simulation of the electronic circular dichroism spectra.

Synthesis of functionalized imidazolidine-2-thiones via NHC/base-promoted aza-benzoin/aza-acetalization domino reactions.

A strategy for the synthesis of biologically relevant 5-hydroxy-imidazolidine-2-thione derivatives is presented. A novel class of α-sulfonylamines have been suitably prepared (46-81% yield) as precursors of formal benzylidenethiourea acceptors; these are generated in situ and intercepted by N-heterocyclic carbene (NHC)-activated aldehydes affording open-chain aza-benzoin-type adducts, which in turn undergo an intramolecular aza-acetalization reaction in a one-pot fashion. A thiazolium salt/triethylamine couple proved to be the more effective system to trigger the domino sequence giving the target heterocycles in good yields (45-97%) and diastereoselectivities (up to 99 : 1 dr). The multigram scale synthesis and elaboration of a selected 5-hydroxy-imidazolidine-2-thione compound is also described.

KuQuinones Equilibria Assessment for Biomedical Applications.

A small library of pentacyclic quinoid compounds, called KuQuinones (KuQs), has been prepared through a one-pot reaction. KuQuinones complex structure is made up by two naphthoquinone units connected by a five-membered ring. Due to KuQs structural features, keto-enol tautomerization in solution likely occurs, leading to the generation of four different species, i.e., the enol, the enolate, the external enol and the diquinoid species. The interchange among KuQ tautomers leads to substantial spectral variations of the dye depending on the experimental conditions used. The comprehension of tautomeric equilibria of this new class of quinoid compounds is strongly required in order to explain their behavior in solution and in biological environment. UV-vis, 1H NMR spectroscopies, and DFT calculations resulted appropriate tools to understand the nature of the prevalent KuQuinone species in solution. Moreover, due to the structural similarity of KuQuinones with camptothecin (CPT), a largely used anticancer agent, KuQs have been tested against Cisplatin-resistant SKOV3 and SW480 cancer cell lines. Results highlighted that KuQs are highly active toward the analyzed cell lines and almost nontoxic for healthy cell, indicating a high specific activity.

Research Progress in the Modification of Quercetin Leading to Anticancer Agents.

The flavonoid quercetin (3,3',4',5,7-pentahydroxyflavone) is widely distributed in plants, foods, and beverages. This polyphenol compound exhibits varied biological actions such as antioxidant, radical-scavenging, anti-inflammatory, antibacterial, antiviral, gastroprotective, immune-modulator, and finds also application in the treatment of obesity, cardiovascular diseases and diabetes. Besides, quercetin can prevent neurological disorders and exerts protection against mitochondrial damages. Various in vitro studies have assessed the anticancer effects of quercetin, although there are no conclusive data regarding its mode of action. However, low bioavailability, poor aqueous solubility as well as rapid body clearance, fast metabolism and enzymatic degradation hamper the use of quercetin as therapeutic agent, so intense research efforts have been focused on the modification of the quercetin scaffold to obtain analogs with potentially improved properties for clinical applications. This review gives an overview of the developments in the synthesis and anticancer-related activities of quercetin derivatives reported from 2012 to 2016.