A Photoactivatable Version of Ivabradine Enables Light-Induced Block of HCN Current In Vivo.

Therapeutic drugs, whose bioactivity is hindered by a photoremovable cage, offer the advantage of spatiotemporal confinement of their action to the target diseased tissue with improved bioavailability and efficacy. Here, we have applied such an approach to ivabradine (IVA), a bradycardic agent indicated for angina pectoris and heart failure, acting as a specific HCN channel blocker. To overcome the side effects due to its poor discrimination among HCN channel subtypes (HCN1-4), we prepared a caged version of IVA linked to a photocleavable bromoquinolinylmethyl group (BHQ-IVA). We show that upon illumination with blue light (440 nm), BHQ-IVA releases active IVA that blocks HCN channel currents in vitro and exerts a bradycardic effect in vivo. Both BHQ-IVA and the cage are inactive. Caging is stable in aqueous medium and in the dark, and it does not impair aqueous solubility and cell permeation, indispensable for IVA activity. This approach allows for bypassing the poor subtype-specificity of IVA, expanding its prescription to HCN-related diseases besides cardiac.

Selective Potentiation of the (α4)3(ß2)2 Nicotinic Acetylcholine Receptor Response by NS9283 Analogues.

NS9283, 3-(3-pyridyl)-5-(3-cyanophenyl)-1,2,4-oxadiazole, is a selective positive allosteric modulator of (α4)3(ß2)2 nicotinic acetylcholine receptors (nAChRs). It has good subtype selective therapeutic potential afforded by its specific binding to the unique α4-α4 subunit interface present in the (α4)3(ß2)2 nAChR. However, there is currently a lack of structure activity relationship (SAR) studies aimed at developing a class of congeners endowed with the same profile of activity that can help consolidate the druggability of the α4-α4 subunit interface. In this study, new NS9283 analogues were designed, synthesized, and characterized for their ability to selectively potentiate the ACh activity at heterologous (α4)3(ß2)2 nAChRs vs nAChR subtypes (α4)2(ß2)3, α5α4ß2, and α7. With few exceptions, all the NS9283 analogues exerted positive modulation of the (α4)3(ß2)2 nAChR ACh-evoked responses. Above all, those modified at the 3-cyanophenyl moiety by replacement with 3-nitrophenyl (4), 4-cyanophenyl (10), and N-formyl-4-piperidinyl (20) showed the same efficacy as NS9283, although with lower potency. Molecular dynamics simulations of NS9283 and some selected analogues highlighted consistency between potentiation activity and pose of the ligand inside the α4-α4 site with the main interaction being with the complementary (-) side and induction of a significant conformational change of the Trp156 residue in the principal (+) side.

Medicinal Cannabis: Extended Stability of Cannabis Extracts Produced Using a New Ethanol-based Extraction Method.

Cannabis as a therapeutic agent is increasing in popularity all around the globe, particularly in Western countries, and its potential is now well assessed. On the other hand, each country has its own regulation for the preparation of cannabis macerated oils; in Italy, there are only a few preparation methods allowed. With this work, we aim to perform a stability study of cannabis oils produced with a novel method for the extraction of cannabinoids from cannabis inflorescence. Three different varieties of cannabis were used, with and without the adding of tocopherol acetate as an antioxidant. Cannabinoids were extracted using ethanol at room temperature; then, the solvent was evaporated under reduced pressure and the preparations reconstituted with olive oil. In this work, we assessed the stability of both cannabinoids and terpenes in these formulas over 8 months. Cannabinoid stability was assessed by monitoring the concentrations of THC and CBD, while terpene stability was assessed by monitoring ß-Caryophyllene and α-Humulene concentrations. Stability of the extracts was not influenced by the presence of tocopherol acetate, though refrigeration seems to be detrimental for a long storage of products, especially regarding THC concentrations. The improvements offered by this method reside in the flexibility in controlling the concentration of the extract and the ability to produce highly concentrated oils, alongside the possibility to produce standardized oils despite the variability of the starting plant material.

Addressing Motor Dysfunction by a Selective α6-Containing Nicotinic Receptor Antagonist.

In the striatum, presynaptic α6-containig nicotinic receptors are crucially involved in the modulation of dopamine release. CVN417, a novel selective antagonist at this receptor subtype, attenuates motor dysfunction in a Parkinson's disease-relevant animal model, suggesting, for this pathology, a therapeutic strategy that could greatly profit from the restricted localization of α6* nicotinic receptors in the brain.

Green Oxidation of Heterocyclic Ketones with Oxone in Water.

The recently reported efficient conversion of cyclic ketones to lactones by Oxone in neutral buffered water is extended to heterocyclic ketones, namely, cyclic N-Boc azaketones and oxoethers with the aim of obtaining N-protected azalactones and their analogues with oxygen in place of nitrogen. N-Boc-4-piperidinone and all the cyclic oxoethers were successfully oxidized to lactones, while the azacyclic ketones with nitrogen α-positioned to carbonyl were univocally transformed into N-Boc-ω-amino acids and N-Boc-N-formyl-ω-amino acids operating in alkaline water and DMF, respectively.

Small molecule ligands for α9 * and α7 nicotinic receptors: A survey and an update, respectively.

The α9- and α7-containing nicotinic acetylcholine receptors (nAChRs) mediate numerous physiological and pathological processes by complex mechanisms that are currently the subject of intensive study and debate. In this regard, selective ligands serve as invaluable investigative tools and, in many cases, potential therapeutics for the treatment of various CNS disfunctions and diseases, neuropathic pain, inflammation, and cancer. However, the present scenario differs significantly between the two aforementioned nicotinic subtypes. Over the past few decades, a large number of selective α7-nAChR ligands, including full, partial and silent agonists, antagonists, and allosteric modulators, have been described and reviewed. Conversely, reports on selective α9-containing nAChR ligands are relatively scarce, also due to a more recent characterization of this receptor subtype, and hardly any focusing on small molecules. In this review, we focus on the latter, providing a comprehensive overview, while providing only an update over the last five years for α7-nAChR ligands.

Fractioning and Compared 1H NMR and GC-MS Analyses of Lanolin Acid Components.

The management of food and food-related wastes represents a growing global issue, as they are hard to recycle and dispose of. Foremost, waste can serve as an important source of biomasses. Particularly, fat-enriched biomasses are receiving more and more attention for their role in the manufacturing of biofuels. Nonetheless, many biomasses have been set aside over the years. Wool wax, also known as lanolin, has a huge potential for becoming a source of typical and atypical fatty acids. The main aim of this work was to evaluate and assess a protocol for the fractioning of fatty acids from lanolin, a natural by-product of the shearing of sheep, alongside the design of a new and rapid quantitative GC-MS method for the derivatization of free fatty acids in fat mixtures, using MethElute™. As the acid portion of lanolin is characterized by the presence of both aliphatic and hydroxylated fatty acids, we also evaluated a procedure for the parting of these two species, by using NMR spectroscopy, benefitting of the different solubilities of the components in organic solvents. At last, we evaluated and quantified the fatty acids and the α-hydroxy fatty acids present in each attained portion, employing both analytical and synthetic standards. The performed analyses, both qualitative and quantitative, showed a good performance in the parting of the different acid components, and GC-MS allowed to speculate that the majority of α-hydroxylated fatty acids is formed of linear saturated carbon chains, while the totality of properly said fatty acids has a much more complex profile.

Tannins-Based Extracts: Effects on Gut Chicken Spontaneous Contractility.

The impossibility of using drugs for the health of farm animals leads to the search for alternative strategies with two purposes: to maintain animal health and safeguard human health. In this perspective, tannins have shown great promises. These phytocomplexes obtained from natural matrices with multiple health properties may be used as a feed supplement in chicken farms. In this work, we studied two tannin-based extracts (from Castanea sativa Mill. wood and from Schinopsis balansae Engl. Quebracho Colorado hardwood) with different chemical compositions on the spontaneous contractility on the isolated intestinal tissues of healthy chicken. The results showed that the chemical composition of the two phytocomplexes influenced the spontaneous intestinal contractility in different ways by regulating the tone and consequent progression of the food bolus. The chemical analysis of the two extracts revealed that Castanea sativa Mill. wood mainly contains hydrolysable tannins, while Schinopsis balansae Engl. hardwood mainly contains condensed tannins. The two phytocomplexes showed different effects towards gastrointestinal smooth muscle contractility, with Castanea sativa Mill. wood providing a better activity profile than Schinopsis balansae Engl. hardwood.

Subnanomolar Affinity and Selective Antagonism at α7 Nicotinic Receptor by Combined Modifications of 2-Triethylammonium Ethyl Ether of 4-Stilbenol (MG624).

Modifications of the cationic head and the ethylene linker of 2-(triethylammonium)ethyl ether of 4-stilbenol (MG624) have been proved to produce selective α9*-nAChR antagonism devoid of any effect on the α7-subtype. Here, single structural changes at the styryl portion of MG624 lead to prevailing α7-nAChR antagonism without abolishing α9*-nAChR antagonism. Nevertheless, rigidification of the styryl into an aromatic bicycle, better if including a H-bond donor NH, such as 5-indolyl (31), resulted in higher and more selective α7-nAChR affinity. Hybridization of this modification with the constraint of the 2-triethylammoniumethyloxy portion into (R)-N,N-dimethyl-3-pyrrolidiniumoxy substructure, previously reported as the best modification for the α7-nAChR affinity of MG624 (2), was a winning strategy. The resulting hybrid 33 had a subnanomolar α7-nAChR affinity and was a potent and selective α7-nAChR antagonist, producing at the α7-, but not at the α9*-nAChR, a profound loss of subsequent ACh function.

Design of a H2 O2 -generating P450SPα fusion protein for high yield fatty acid conversion.

Sphingomonas paucimobilis' P450SPα (CYP152B1) is a good candidate as industrial biocatalyst. This enzyme is able to use hydrogen peroxide as unique cofactor to catalyze the fatty acids conversion to α-hydroxy fatty acids, thus avoiding the use of expensive electron-donor(s) and redox partner(s). Nevertheless, the toxicity of exogenous H2 O2 toward proteins and cells often results in the failure of the reaction scale-up when it is directly added as co-substrate. In order to bypass this problem, we designed a H2 O2 self-producing enzyme by fusing the P450SPα to the monomeric sarcosine oxidase (MSOX), as H2 O2 donor system, in a unique polypeptide chain, obtaining the P450SPα -polyG-MSOX fusion protein. The purified P450SPα -polyG-MSOX protein displayed high purity (A417 /A280  = 0.6) and H2 O2 -tolerance (kdecay  = 0.0021 ± 0.000055 min-1 ; ΔA417  = 0.018 ± 0.001) as well as good thermal stability (Tm : 59.3 ± 0.3°C and 63.2 ± 0.02°C for P450SPα and MSOX domains, respectively). The data show how the catalytic interplay between the two domains can be finely regulated by using 500 mM sarcosine as sacrificial substrate to generate H2 O2 . Indeed, the fusion protein resulted in a high conversion yield toward fat waste biomass-representative fatty acids, that is, lauric acid (TON = 6,800 compared to the isolated P450SPα TON = 2,307); myristic acid (TON = 6,750); and palmitic acid (TON = 1,962).

The Antioxidant Role of Hemp Phytocomplex in Cannabis Oil-Based Extracts.

The therapeutic use of Cannabis oil extracts is constantly increasing. However, in Italy, they are allowed to be prepared with only a few methods and matrices. With this work, we aimed to assess how the different processes might affect the chemical composition of two different matrices (olive oils and medium chain triglycerides oils - MCT), accounting as variables for both the presence of Cannabis dried apices of the female flower and the adding of tocopherol acetate as an antioxidant. The macerated oils were prepared with four of the methods allowed according to the Italian legislation (Romano-Hazekamp, Cannazza-Citti, SIFAP and Calvi) and analyzed for normal and oxidized tocopherols, oxidized and conjugated fatty acids and volatile carbonyl compounds (VCCs), all using liquid chromatography coupled to UV or PDA detectors. According to our results, neither normal nor oxidized tocopherols are affected by the addition of antioxidants or Cannabis, while the oxidation state (according to the levels of oxidized and conjugated fatty acids) is often altered in either case. The VCCs concentrations, on the other hand, are never notably altered. These results suggest a worthless use of antioxidants in Cannabis macerated oils preparations, while the dried apices of female flowers might have a protective role in maintaining the oil oxidation state.

From 2-Triethylammonium Ethyl Ether of 4-Stilbenol (MG624) to Selective Small-Molecule Antagonists of Human α9α10 Nicotinic Receptor by Modifications at the Ammonium Ethyl Residue.

Nicotinic acetylcholine receptors containing α9 subunits (α9*-nAChRs) are potential druggable targets arousing great interest for pain treatment alternative to opioids. Nonpeptidic small molecules selectively acting as α9*-nAChRs antagonists still remain an unattained goal. Here, through modifications of the cationic head and the ethylene linker, we have converted the 2-triethylammonium ethyl ether of 4-stilbenol (MG624), a well-known α7- and α9*-nAChRs antagonist, into some selective antagonists of human α9*-nAChR. Among these, the compound with cyclohexyldimethylammonium head (7) stands out for having no α7-nAChR agonist or antagonist effect along with very low affinity at both α7- and α3ß4-nAChRs. At supra-micromolar concentrations, 7 and the other selective α9* antagonists behaved as partial agonists at α9*-nAChRs with a very brief response, followed by rebound current once the application is stopped and the channel is disengaged. The small or null postapplication activity of ACh seems to be related to the slow recovery of the rebound current.

Pyrrolidinyl benzofurans and benzodioxanes: Selective α4ß2 nicotinic acetylcholine receptor ligands with different activity profiles at the two receptor stoichiometries.

A series of racemic benzofurans bearing N-methyl-2-pyrrolidinyl residue at C(2) or C(3) has been synthesized and tested for affinity at the α4ß2 and α3ß4 nicotine acetylcholine receptors (nAChRs). As previously reported for the benzodioxane based analogues, hydroxylation at proper position of benzene ring results in high α4ß2 nAChR affinity and α4ß2 vs. α3ß4 nAChR selectivity. 7-Hydroxy-N-methyl-2-pyrrolidinyl-1,4-benzodioxane (2) and its 7- and 5-amino benzodioxane analogues 3 and 4, which are all α4ß2 nAChR partial agonists, and 2-(N-methyl-2-pyrrolidinyl)-6-hydroxybenzofuran (12) were selected for functional characterization at the two α4ß2 stoichiometries, the high sensitivity (α4)2(ß2)3 and the low sensitivity (α4)3(ß2)2. The benzene pattern substitution, which had previously been found to control α4ß2 partial agonist activity and α4ß2 vs. α3ß4 selectivity, proved to be also involved in stoichiometry-selectivity. The 7-hydroxybenzodioxane derivative 2 selectively activates (α4)2(ß2)3 nAChR, which cannot be activated by its 5-amino analogue 4. A marginal structural modification, not altering the base pyrrolidinyl benzodioxane scaffold, resulted in opposite activity profiles at the two α4ß2 nAChR isoforms providing an interesting novel case study.

Evidence of a dual mechanism of action underlying the anti-proliferative and cytotoxic effects of ammonium-alkyloxy-stilbene-based α7- and α9-nicotinic ligands on glioblastoma cells.

Glioblastomas (GBMs), the most frequent brain tumours, are highly invasive and their prognosis is still poor despite the use of combination treatment. MG624 is a 4-oxystilbene derivative that is active on α7- and α9-containing neuronal nicotinic acetylcholine receptor (nAChR) subtypes. Hybridisation of MG624 with a non-nicotinic resveratrol-derived pro-oxidant mitocan has led to two novel compounds (StN-4 and StN-8) that are more potent than MG624 in reducing the viability of GBM cells, but less potent in reducing the viability of mouse astrocytes. Functional analysis of their activity on α7 receptors showed that StN-4 is a silent agonist, whereas StN-8 is a full antagonist, and neither alters intracellular [Ca2+] levels when acutely applied to U87MG cells. After 72 h of exposure, both compounds decreased U87MG cell proliferation, and pAKT and oxphos ATP levels, but only StN-4 led to a significant accumulation of cells in phase G1/G0 and increased apoptosis. One hour of exposure to either compound also decreased the mitochondrial and cytoplasmic ATP production of U87MG cells, and this was not paralleled by any increase in the production of reactive oxygen species. Knocking down the α9 subunit (which is expressed at relatively high levels in U87MG cells) decreased the potency of the effects of both compounds on cell viability, but cell proliferation, ATP production, pAKT levels were unaffected by the presence of the noncell-permeable α7/α9-selective antagonist αBungarotoxin. These last findings suggest that the anti-tumoral effects of StN-4 and StN-8 on GBM cells are not only due to their action on nAChRs, but also to other non-nicotinic mechanisms.

Synthesis of α-Hydroxy Fatty Acids from Fatty Acids by Intermediate α-Chlorination with TCCA under Solvent-Free Conditions: A Way to Valorization of Waste Fat Biomasses.

Within food wastes, including edible and inedible parts, fat biomasses represent a significant portion, often uneconomically used or improperly disposed causing pollution issues. Interesting perspectives for their management and valorization could be opened by conversion of fatty acids (FAs), which are their main constituents, into α-hydroxy FAs (α-HFAs), fine chemicals of great, but largely untapped potential, possibly due to current poor availability. Here, a simple and efficient procedure is reported to α-chlorinate FAs with trichloroisocyanuric acid (TCCA), a green halogenating agent, under solvent-free conditions and to directly convert the resultant α-chloro FAs, without previous purification, into α-HFAs. The procedure was applied to stearic, palmitic, and myristic acid and, with analogous success, to their mixture, ad hoc created to simulate a FAs mixture obtainable from a fat biomass.

Green Oxidation of Ketones to Lactones with Oxone in Water.

Cyclic ketones were quickly and quantitatively converted to 5-, 6-, and 7-membered lactones, very important synthons, by treatment with Oxone, a cheap, stable, and nonpollutant oxidizing reagent, in 1 M NaH2PO4/Na2HPO4 water solution (pH 7). Under such simple and green conditions, no hydroxyacid was formed, thus making the adoption of more complex and non-eco-friendly procedures previously developed to avoid lactone hydrolysis unnecessary. With some changes, the method was successfully applied also to water-insoluble ketones such as adamantanone, acetophenone, 2-indanone, and the challenging cycloheptanone.

Determinants for α4ß2 vs. α3ß4 Subtype Selectivity of Pyrrolidine-Based nAChRs Ligands: A Computational Perspective with Focus on Recent cryo-EM Receptor Structures.

The selectivity of α4ß2 nAChR agonists over the α3ß4 nicotinic receptor subtype, predominant in ganglia, primarily conditions their therapeutic range and it is still a complex and challenging issue for medicinal chemists and pharmacologists. Here, we investigate the determinants for such subtype selectivity in a series of more than forty α4ß2 ligands we have previously reported, docking them into the structures of the two human subtypes, recently determined by cryo-electron microscopy. They are all pyrrolidine based analogues of the well-known α4ß2 agonist N-methylprolinol pyridyl ether A-84543 and differ in the flexibility and pattern substitution of their aromatic portion. Indeed, the direct or water mediated interaction with hydrophilic residues of the relatively narrower ß2 minus side through the elements decorating the aromatic ring and the stabilization of the latter by facing to the not conserved ß2-Phe119 result as key distinctive features for the α4ß2 affinity. Consistently, these compounds show, despite the structural similarity, very different α4ß2 vs. α3ß4 selectivities, from modest to very high, which relate to rigidity/extensibility degree of the portion containing the aromatic ring and to substitutions at the latter. Furthermore, the structural rationalization of the rat vs. human differences of α4ß2 vs. α3ß4 selectivity ratios is here proposed.

Development and Early Identification of Cannabis Chemotypes during the Plant Growth: Current Analytical and Chemometric Approaches.

The identification of cannabis chemotypes at an early stage of a plant's growth, which is long before anthesis, has been intensively pursued in order to control the on-target selection of the cultivar type at the beginning of cultivation, so as to avoid economic and legal drawbacks. However, this issue has been systematically addressed by only few and relatively recent studies of analytical chemistry, possibly because result validations require long-term monitoring of the content and ratio of cannabinoids and terpenes in a great number of plant specimens suitably selected and grown. Here, we review the procedures, the chromatographic techniques and the statistics used in topical investigations during the past thirteen years. Through heterogeneous and not easily comparable approaches, they prove the feasibility of chemotypes safe determination within the first month of a plant's life.

Characterization of chemotype-dependent terpenoids profile in cannabis by headspace gas-chromatography coupled to time-of-flight mass spectrometry.

A headspace method called full evaporation technique (FET) coupled to capillary gas chromatography with a mass detector operating in time-of-flight mode (HS-GC/MS-TOF) was developed to characterize the volatile components, especially the terpene fraction, in Cannabis sativa L. inflorescences. This analytical approach allows to reach a high equilibration temperature, that was able to obtain a complete quantification of the volatile components, providing simple sample preparation, specific qualitative detection, high sensitivity, a precise and accurate quantitative determination. The method was applied to 20 cannabis THC-dominant (I) and 13 CBD-dominant (III) chemotypes. The obtained results were then compared with a series of standard solutions consisting of 35 terpenoids and the mass spectra present in a computer library (NIST). The method has an accuracy of more than 90 % and a limit of detection of 5 ppm for all analytes. The main terpenoids in cannabis are namely (% Chemotypes III vs. I of the total terpene content): ß-Caryophyllene (25 vs. 19.3), ß-Mircene (18.2 vs. 20.0), Terpinolene (12.1 vs. 7.0), α-Humulene (6.5 vs. 8.5), D-Limonene (6.2 vs. 7.2), α-Pinene (5.8 vs. 4.9), ß-Pinene (5.0 vs. 5.8) and cis-ß-Ocimene (4.3 vs. 5.2), whose presence is confirmed in both plant chemotypes and account for more than 80 % of the total terpenoids amount. The terpenoids which can clearly distinguish the chemotype are α-Terpineol, Linalool, DL-Menthol, α-Cedrene, and Borneol. This application provides important data on the secondary volatile components of the plant, which may be useful for a better understanding of the therapeutic properties of the cannabis phyto-complex. It gives the possibility of establishing the aroma profile of different Cannabis batches, allowing possible similarities between samples and identifying any artificial adulteration such as hexyl butyrate ester and it provides the opportunity to highlight some target compounds characteristic of the different chemotypes.

Efficient conversion of d-mannitol into 1,2:5,6-diacetonide with Aquivion-H as a recyclable catalyst.

Heterogeneous solid catalysis by the commercially available perfluorosulfonic ionomer Aquivion-H allowed 1,2:5,6-diacetonide of d-mannitol (1), immediate precursor of important unichiral C3-synthons, to be efficiently obtained from d-mannitol and 2,2-dimethoxypropane in DMF at room temperature. The 1,2-monoacetonide, whose intermediate formation is the rate-limiting step, could be almost completely converted into 1 with limited concurrent transformation of 1 into triacetonides. In line with recent literature reports, these results indicate that heterogeneous catalysis by Aquivion-H surpasses the performances of homogeneous acidic catalysis assuring, presumably for its peculiar morphology, a higher product selectivity. Easy recovery at the end of the reaction and recyclability are additional advantages of this solid acid catalyst.