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A direct method to obtain α-alkylidene β-oxo amides by the palladium-catalyzed carbonylation of α-chloro ketones in the presence of aromatic imines has been described. The methodology can be applied to a variety of C-aryl imines bearing N-aryl or N-alkyl substituents. The entire process is highly stereoselective and affords the α-alkylidene β-oxo amides only as (Z) isomers. A mechanistic hypothesis involving an acyl-β-lactam intermediate has also been proposed.

Introduction: Cannabidiol (CBD), the nonintoxicating constituent of cannabis, is largely employed for pharmaceutical and cosmetic purposes. CBD can be extracted from the plant or chemically synthesized. Impurities of psychotropic cannabinoids Δ9-tetrahydrocannabinol (Δ9-THC) and Δ8-THC have been found in extracted CBD, thus hypothesizing a possible contamination from the plant. Materials and Methods: In this study, synthetic and extracted CBD samples were analyzed by ultrahigh-performance liquid chromatography coupled to high-resolution mass spectrometry and the parameters that can be responsible of the conversion of CBD into THC were evaluated by an accelerated stability test. Results: In synthetic and extracted CBD no trace of THC species was detected. In contrast, CBD samples stored in the dark at room temperature on the benchtop for 3 months showed the presence of such impurities. Experiments carried out under inert atmosphere in the absence of humidity or carbon dioxide led to no trace of THC over time even at high temperature. Conclusions: The results suggested that the copresence of carbon dioxide and water from the air could be the key for creating the acidic environment responsible for the cyclization of CBD. These findings suggest that it might be appropriate to review the storage conditions indicated on the label of commercially available CBD.

The recent discovery of the novel heptyl phytocannabinoids cannabidiphorol (CBDP) and Δ9-tetrahydrocannabiphorol (Δ9-THCP) raised a series of questions relating to the presence and abundance of these new unorthodox compounds in cannabis inflorescence or derived products. As fresh inflorescence contains mainly their acid precursors, which are not commercially available, an ad hoc stereoselective synthesis was performed in order to obtain cannabidiphorolic acid (CBDPA) and Δ9-tetrahydrocannabiphorolic acid (THCPA) to be used as analytical standards for quantitative purposes. The present work reports an unprecedented targeted analysis of both pentyl (C5) and heptyl (C7) CBD- and THC-type compounds in forty-nine cannabis samples representing four different chemotypes. Moreover, the ultrahigh performance liquid chromatography coupled to high-resolution mass spectrometry-based method was applied for the putative identification of other heptyl homologs of the most common phytocannabinoid acids, including cannabigerophorolic acid (CBGPA), cannabichromephorolic acid (CBCPA), cannabinophorolic acid (CBNPA), cannabielsophorolic acid (CBEPA), cannabicyclophorolic acid (CBLPA), cannabitriophorolic acid (CBTPA), and cannabiripsophorolic acid (CBRPA).

L-Kynurenine (KYN) and kynurenic acid (KYNA) are products of the metabolism of L-tryptophan (TRP) in the central nervous system of animals, but they are not commonly found in plants. In particular, KYNA is known for its interesting pharmacological properties (anti-oxidative, anti-inflammatory, hypolipidemic, and neuroprotective), which suggest a potential functional food ingredient role. The three compounds were identified in samples of Cannabis sativa L. by means of high-performance liquid chromatography coupled to high-resolution mass spectrometry using an untargeted metabolomics approach. Their concentrations were evaluated using a targeted metabolomics method in three organs of the plant (roots, stem, and leaves) in soil at two different growth stages and in hydroponics conditions. The distribution of TRP, KYN and KYNA was found tendentially higher in leaves compared to stem and roots and changed over time. Moreover, the levels of KYNA found in this study are unprecedentedly high compared to those found so far in other plant species, suggesting that Cannabis sativa L. could be a promising alternative source of this metabolite.

Inorganic nanoparticles are widely investigated as drug delivery systems. In particular micro and nanoparticles of CaCO3 offer smart features for different biomaterials applications. In this work we exploit the phospholipids coating of nano-CaCO3. We prepare modified phospholipids through a chemo-enzymatic approach using Phospholipase D to efficiently transform the most abundant natural phospholipids in two products, snglycero-3-phosphocholine (GPC) and sn-glycero-3-phosphoserine (GPS). We have investigated and modified the Spray Drier process to obtain nano-crystals with specific phase, surface zeta-potential and morphology. The intracellular localization of coated nano-crystals was achieved by ultrastructural microscopy analysis. The synthetized phospholipids, GPC and GPS, improve nano-CaCO3 proprieties and promote a specific targeting as opposed to a widespread and nonspecific localization in the cell.

Benzothiadiazine type compounds (BTDs) have gained great attention for their potential therapeutic activity as nootropic and neuroprotective agents. BTDs, acting as AMPA positive allosteric modulators, potentiate the glutamatergic neurotransmission without the side effects typically associated with direct agonists. Studies regarding the binding mode of racemic BTDs into the receptor binding pocket demonstrated that one enantiomer establishes a more favourable interaction and possesses a higher biological activity with respect to the other one. The S enantiomer was proved to be the eutomer for both IDRA21 and S18986, two of the most studied BTD AMPA positive allosteric modulators. However, recent data highlighted an opposite stereoselectivity for some substituted BTDs (7-chloro-9-(furan-3-yl)-2,3,3a,4-tetrahydro-1H-benzo[e]pyrrolo[2,1-c][1,2,4]thiadiazine 5,5-dioxide and 7-chloro-2,3,4-trimethyl-3,4-dihydro-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide) showing unexpected structure–activity relationships. In this work, the synthesis and configuration assignment of the stereoisomers of 7-chloro-5-(3-furanyl)-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide, one of the most active BTDs, are reported. Electrophysiological tests demonstrated that the R form is the eutomer. Docking and molecular dynamics simulations on the AMPA GluA2 binding site revealed new insights into the stereodiscrimination process. Lastly, metabolic studies disclosed a stereoselective hepatic metabolization of this chiral BTD.

LY2157299 (LY), which is very small molecule bringing high cancer diffusion, is a pathway antagonist against TGFβ. LY dosage can be diluted by blood plasma, can be captured by immune system or it might be dissolved during digestion in gastrointestinal tract. The aim of our study is to optimize a “nano-elastic” carrier to avoid acidic pH of gastrointestinal tract, colon alkaline pH, and anti-immune recognition. Polygalacturonic acid (PgA) is not degradable in the gastrointestinal tract due to its insolubility at acidic pH. To avoid PgA solubility in the colon, we have designed its conjugation with Polyacrylic acid (PAA). PgA-PAA conjugation has enhanced their potential use for oral and injected dosage. Following these pre-requisites, novel polymeric nano-micelles derived from PgA-PAA conjugation and loading LY2157299 are developed and characterized. Efficacy, uptake and targeting against a hepatocellular carcinoma cell line (HLF) have also been demonstrated.

In the last few years, there has been a boost in the use of cannabis-based extracts for medicinal purposes, although their preparation procedure has not been standardized but rather decided by the individual pharmacists. The present work describes the development of a simple and rapid high performance liquid chromatography method with UV detection (HPLC-UV) for the qualitative and quantitative determination of the principal cannabinoids (CBD-A, CBD, CBN, THC and THC-A) that could be applied to all cannabis-based medicinal extracts (CMEs) and easily performed by a pharmacist. In order to evaluate the identity and purity of the analytes, a high-resolution mass spectrometry (HPLC-ESI-QTOF) analysis was also carried out. Full method validation has been performed in terms of specificity, selectivity, linearity, recovery, dilution integrity and thermal stability. Moreover, the influence of the solvent (ethyl alcohol and olive oil) was evaluated on cannabinoids degradation rate. An alternative extraction method has then been proposed in order to preserve cannabis monoterpene component in final CMEs.

Almost all Iridium(III) complexes employed both as dopants in PhOLEDs and as pharmaceuticals and fluorescence bioprobes are racemic mixtures. In this study the single enantiomers of the most stable diastereomeric form fac-trans-N–N , bis[2-(4,6-difluorophenyl)pyridinato-C 2 , N ](picolinato)iridium(III) (FIrpic) were separated and analysed. The data obtained showed that the complex can be separated into stable optically active Λ and Δ isomers employing cellulose based chiral stationary phase both in normal and polar phase mode. Their chirality was confirmed and their absolute configuration assigned employing several methods (DFT and TDDFT calculations, CD and VCD). The CPL spectroscopy of the isolated enantiomers of FIrpic was also recorded due to its possible value in the OLEDs field. The chromatographic method was applied for a semipreparative purpose demonstrating that polar organic solvent chromatography (POSC) could be used to avoid the low-solubility issues associated with these Iridium(III) complexes. Finally, the chemical and stereochemical stability of the single isomers was evaluated under thermal stress by liquid chromatography coupled to high-resolution mass spectrometry (LC-QTOF) on both chiral and achiral columns. No racemization and/or isomerization was observed; however, the dissociation of the ancillary ligand was demonstrated employing LC-QTOF.

A “heart-cut” two-dimensional achiral-chiral liquid chromatography triple-quadrupole mass spectrometry method (LC–LC–MS/MS) was developed and coupled to in vivo cerebral microdialysis to evaluate the brain response to the chiral compound (±)-7-chloro-5-(3-furanyl)-3-methyl-3,4-dihydro-2 H -1,2,4-benzothiadiazine-1,1-dioxide ((±)- 1 ), a potent positive allosteric modulator (PAM) of AMPA receptor. The method was successfully employed to evaluate also its stereoselective metabolism and in vitro biological activity. In particular, the LC achiral method developed, employs a pentafluorinated silica based column (Discovery HS-F5) to separate dopamine, acetylcholine, serotonin, (±)- 1 and its two hepatic metabolites. In the “heart-cut” two-dimension achiral-chiral configuration, (±)- 1 and (±)- 1 - d 4 eluted from the achiral column (1st dimension), were transferred to a polysaccharide-based chiral column (2nd dimension, Chiralcel OD-RH) by using an automatic six-port valve. Single enantiomers of (±)- 1 were separated and detected using electrospray positive ionization mode and quantified in selected reaction monitoring mode. The method was validated and showed good performance in terms of linearity, accuracy and precision. The new method employed showed several possible applications in the evaluation of: (a) brain response to neuroactive compounds by measuring variations in the brain extracellular levels of selected neurotransmitters and other biomarkers; (b) blood brain barrier penetration of drug candidates by measuring the free concentration of the drug in selected brain areas; (c) the presence of drug metabolites in the brain extracellular fluid that could prove very useful during drug discovery; (d) a possible stereoselective metabolization or blood brain barrier stereoselective crossing of chiral drugs. Finally, compared to the methods reported in the literature, this technique avoids the necessity of euthanizing an animal at each time point to measure drug concentration in whole brain tissue and provides continuous monitoring of extracellular concentrations of single chiral drug enantiomers along with its metabolites in specific brain regions at each selected time point for a desired period by using a single animal.

5-Arylbenzothiadiazine type compounds acting as positive allosteric modulators of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPA-PAMs) have received particular attention in the past decade for their nootropic activity and lack of the excitotoxic side effects of direct agonists. Recently, our research group has published the synthesis and biological activity of 7-chloro-5-(3-furanyl)-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide (1), one of the most active benzothiadiazine-derived AMPA-PAMs in vitro to date. However, 1 exists as two stereolabile enantiomers, which rapidly racemize in physiological conditions, and only one isomer is responsible for the pharmacological activity. In the present work, experiments carried out with rat liver microsomes show that 1 is converted by hepatic cytochrome P450 to the corresponding unsaturated derivative 2 and to the corresponding pharmacologically inactive benzenesulfonamide 3. Surprisingly, patch-clamp experiments reveal that 2 displays an activity comparable to that of the parent compound. Molecular modeling studies were performed to rationalize these results. Furthermore, mice cerebral microdialysis studies suggest that 2 is able to cross the blood-brain barrier and increases acetylcholine and serotonin levels in the hippocampus. The experimental data disclose that the achiral hepatic metabolite 2 possesses the same pharmacological activity of its parent compound 1 but with an enhanced chemical and stereochemical stability, as well as an improved pharmacokinetic profile compared with 1.

Owing to their nano-sized porous structure, CaCO3 nanocrystals (CaCO3NCs) hold the promise to be utilized as desired materials for encapsulating molecules which demonstrate wide promise in drug delivery. We evaluate the possibility to encapsulate and release NVP-BEZ235, a novel and potent dual PI3K/mTOR inhibitor that is currently in phase I/II clinical trials for advanced solid tumors, from the CaCO3NCs. Its chemical nature shows some intrinsic limitations which induce to administer high doses leading to toxicity; to overcome these problems, here we proposed a strategy to enhance its intracellular penetration and its biological activity. Pristine CaCO3 NCs biocompatibility, cell interactions and internalization in in vitro experiments on T-cell lymphoma line, were studied. Confocal microscopy was used to monitor NCs-cell interactions and cellular uptake. We have further investigated the interaction nature and release mechanism of drug loaded/released within/from the NCs using an alternative approach based on liquid chromatography coupled to mass spectrometry. Our approach provides a good loading efficiency, therefore this drug delivery system was validated for biological activity in T-cell lymphoma: the anti-proliferative test and western blot results are very interesting because the proposed nano-formulation has an efficiency higher than free drug at the same nominal concentration.

In this investigation, differently shaped and surface functionalized TiO2 anatase nanoparticles and human serum albumin (HSA) were selected to study protein-nanoparticles interaction both in a solution and on flat surfaces, thereby mimicking a medical device. Anatase nanocrystals were characterized by transmission electron microscopy (TEM), Brunauer-Emmett-Teller (BET) surface analysis and dynamic light scattering (DLS). The protein-nanoparticles' interactions and their eventual reversibility were studied by pH dependent ζ- potential measurements in different media: ultra-pure water, a phosphate buffer simulating physiological conditions and in a culture medium supplemented with foetal bovine serum. The protein corona masking effect was evidenced and the interaction HSA-nanocrystals resulted irreversible. The interaction HSA-silicon supported TiO2 nanocrystals films was studied by atomic force microscopy (AFM), and resulted driven by the substrate hydrophilicity degree plus was different for the diverse range of nanocrystals tested. Surface roughness measurements showed that on some of the nanocrystals, HSA were arranged in a more globular manner. A lower protein affinity was found for nanocrystals that had a smaller primary particle size, which may correspond to their higher biocompatibility. This nano-bio interface research aimed to study the HSA protein-TiO2 anatase nanocrystals under conditions similar to those for in vitro and in vivo toxicity analyses.

Nanotechnology is a promising branch of the medical field, directed to improve diagnostic and therapeutics strategies, applying nanovectors as drug delivery systems. Efficient encapsulation of anticancer drugs in nanocolloids and microcapsules was recently developed by G. Ciccarella research group (1). Based on our collaboration with the Nantional Nanotechnology Laboratory of the University of Salento and our previous experience with target therapies, we encapsulated BEZ235, a phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin inhibitor (mTOR). BEZ235 efficiently blocks the dysfunctional activation of the PI3K/mTOR pathway in cellular and in vivo settings, thus inhibiting the growth and proliferation of various cancer cells, and phase I/II clinical trials were open in solid cancer. However the scarse solubility limited further development of this promising compound. In order to overcome the solubility issue BEZ235-loaded nanocapsules were generated by the stepwise adsorption of oppositely charged polyelectrolytes into biocompatible CaCO3 cores. First nanocapsules were tested for biocompatibility. The exposition of lymphoma cell lines to empty nanocapsules up to 48 hours, did not induce any cititoxicity, confirming their biocompatibility. Second, encapsulated BEZ235 was compared with free-drug to test the cytotoxicity in a T lymphoma cell line (HUT78) by MTT assay (Fig. 1). The results suggested that nanoencapsulated-BEZ235 was extremely efficient compared with free-BEZ235, reaching IC50 just after 5 hours of exposure compared with an IC65% at 48 hours with the free drug. A validated LC-MS/MS method was developed in order to quantify intracellular concentration of BEZ235 over time. Intracellular concentration of BEZ235 in the lymphoma cell line was consistent with biological results since the internalization kinetic and efficiency was increased by the coating. In order to confirm that the encapsuled-BEZ235 was still effective on cell apoptosis, we tested free BEZ and encapsulated BEZ235 at a concentration of 1µM in T cell lymphoma cell lines. Encapsulated-BEZ235 induced apoptosis evidenced by the cleavage of caspase 8, 9 and 3 at an earlier time point compared with free BEZ235 and at significantly lower concentration. We also confirmed that the encapsulated-BEZ235 maintained its effect on the target mTOR/AKT pathway: p-AKT was dephosphorylated at 5h while the free BEZ235 operates at least after 24 hours at concentrations 100 times higher, as previously demonstrated (2). Keeping in mind a future clinical application of these polymeric particles/capsules, our data can be regarded as a promising new nanotechnology-based strategy to improve the efficacy and bioavailability of old and new drugs. Functional biological studies of BEZ235-encapsulated carrier and its mechanism of internalization are already under way, and animal in vivo studies to evaluated toxicity and distribution of the nanocapsuled compound are ongoing. 1 F. Baldassare et al., Macromolecular Bioscience, Volume 12, Issue 5, pages 656-665, 2012 2 Civallero M, Cosenza M, Marcheselli L, Pozzi S, Sacchi S. Expert Opin Investig Drugs. 2012 Nov;21(11):1597-606. Disclosures No relevant conflicts of interest to declare.