New Insights into Bitopic Orthosteric/Allosteric Ligands of Cannabinoid Receptor Type 2.

Very recently, we have developed a new generation of ligands targeting the cannabinoid receptor type 2 (CB2R), namely JR compounds, which combine the pharmacophoric portion of the CB2R positive allosteric modulator (PAM), EC21a, with that of the CB2R selective orthosteric agonist LV62, both synthesized in our laboratories. The functional examination enabled us to identify JR14a, JR22a, and JR64a as the most promising compounds of the series. In the current study, we focused on the assessment of the bitopic (dualsteric) nature of these three compounds. Experiments in cAMP assays highlighted that only JR22a behaves as a CB2R bitopic (dualsteric) ligand. In parallel, computational studies helped us to clarify the binding mode of these three compounds at CB2R, confirming the bitopic (dualsteric) nature of JR22a. Finally, the potential of JR22a to prevent neuroinflammation was investigated on a human microglial cell inflammatory model.

Design, synthesis and biological evaluation of novel orthosteric-allosteric ligands of the cannabinoid receptor type 2 (CB2R).

It is well known that G protein-coupled receptors (GPCRs) assume multiple active states. Orthosteric ligands and/or allosteric modulators can preferentially stabilize specific conformations, giving rise to pathway-biased signaling. One of the most promising strategies to expand the repertoire of signaling-selective GPCR activators consists of dualsteric agents, which are hybrid compounds consisting of orthosteric and allosteric pharmacophoric units. This approach proved to be very promising showing several advantages over monovalent targeting strategies, including an increased affinity or selectivity, a bias in signaling pathway activation, reduced off-target activity and therapeutic resistance. Our study focused on the cannabinoid receptor type 2 (CB2R), considered a clinically promising target for the control of brain damage in neurodegenerative disorders. Indeed, CB2R was found highly expressed in microglial cells, astrocytes, and even in some neuron subpopulations. Here, we describe the design, synthesis, and biological evaluation of two new classes of potential dualsteric (bitopic) CB2R ligands. The new compounds were obtained by connecting, through different linkers, the pharmacophoric portion of the CB2R positive allosteric modulator (PAM), EC21a, with that of the CB2R selective orthosteric agonist LV62, both developed in our laboratories. A preliminary screening enabled us to identify compound JR64a as the most promising of the series. Indeed, functional examination highlighted a signaling 'bias' in favor of G protein activation over ßarrestin2 recruitment, combined with high affinity for CB2R and the ability to efficiently prevent inflammation in human microglial cells (HMC3) exposed to LPS/TNFα stimulation, thus demonstrating great promise for the treatment of neurodegenerative diseases.

Design, Synthesis, and Biological Activity of New CB2 Receptor Ligands: from Orthosteric and Allosteric Modulators to Dualsteric/Bitopic Ligands.

The design of dualsteric/bitopic agents as single chemical entities able to simultaneously interact with both the orthosteric and an allosteric binding site represents a novel approach in medicinal chemistry. Biased dualsteric/bitopic agents could enhance certain signaling pathways while diminishing the others that cause unwanted side effects. We have designed, synthesized, and functionally characterized the first CB2R heterobivalent bitopic ligands. In contrast to the parent orthosteric compound, our bitopic ligands selectively target CB2R versus CB1R and show a functional selectivity for the cAMP signaling pathway versus ßarrestin2 recruitment. Moreover, the most promising bitopic ligand FD-22a displayed anti-inflammatory activity in a human microglial cell inflammatory model and antinociceptive activity in vivo in an experimental mouse model of neuropathic pain. Finally, computational studies clarified the binding mode of these compounds inside the CB2R, further confirming their bitopic nature.

Design, Synthesis, and In Vitro Evaluation of Novel 8-Amino-Quinoline Combined with Natural Antioxidant Acids.

Overproduction of reactive oxygen species (ROS) and alterations in metallostasis are common and related hallmarks in several neurodegenerative diseases (NDDs). Nature-based derivatives always represent an attractive tool in MTDL drug design, especially against ROS in NDDs. On this notion, we designed a new series of 8-quinoline-N-substituted derivatives with a natural antioxidant portion (i.e., lipoic, caffeic, and ferulic acids). These compounds were shown to chelate copper, a metal involved in ROS-induced degeneration, and scavenger oxygen radicals in DPPH assay. Then, selected compounds 4 and 5 were evaluated in an in vitro model of oxidative stress and shown to possess cytoprotective effects in 661W photoreceptor-like cells. The obtained results may represent a starting point for the application of the proposed class of compounds in retinal neurodegenerative diseases such as retinitis pigmentosa (RP), comprising a group of hereditary rod-cone dystrophies that represent a major cause of blindness in patients of working age, where the progression of the disease is a multifactorial event, with oxidative stress contributing predominantly.

Synthesis and In Vitro Characterization of Selective Cannabinoid CB2 Receptor Agonists: Biological Evaluation against Neuroblastoma Cancer Cells.

1,8-naphthyridine-3-carboxamide structures were previously identified as a promising scaffold from which to obtain CB2R agonists with anticancer and anti-inflammatory activity. This work describes the synthesis and functional characterization of new 1,8-naphthyridin-2(1H)-one-3-carboxamides with high affinity and selectivity for CB2R. The new compounds were able to pharmacologically modulate the cAMP response without modulating CB2R-dependent ß-arrestin2 recruitment. These structures were also evaluated for their anti-cancer activity against SH-SY5Y and SK-N-BE cells. They were able to reduce the cell viability of both neuroblastoma cancer cell lines with micromolar potency (IC50 of FG158a = 11.8 µM and FG160a = 13.2 µM in SH-SY5Y cells) by a CB2R-mediated mechanism. Finally, in SH-SY5Y cells one of the newly synthesized compounds, FG158a, was able to modulate ERK1/2 expression by a CB2R-mediated effect, thus suggesting that this signaling pathway might be involved in its potential anti-cancer effect.

Tyrosol-Enriched Tomatoes by Diffusion across the Fruit Peel from a Chitosan Coating: A Proposal of Functional Food.

Chitosan is receiving increasing attention from the food industry for being a biodegradable, non-toxic, antimicrobial biopolymer able to extend the shelf life of, and preserve the quality of, fresh food. However, few studies have investigated the ability of chitosan-based coatings to allow the diffusion of bioactive compounds into the food matrix to improve its nutraceutical quality. This research is aimed at testing whether a hydrophilic molecule (tyrosol) could diffuse from the chitosan-tyrosol coating and cross the tomato peel. To this end, in vitro permeation tests using excised tomato peel and an in vivo application of chitosan-tyrosol coating on tomato fruit, followed by tyrosol quantification in intact fruit, peel and flesh during a seven-day storage at room temperature, were performed. Both approaches demonstrated the ability of tyrosol to permeate across the fruit peel. Along with a decreased tyrosol content in the peel, its concentration within the flesh was increased, indicating an active transfer of tyrosol into this tissue. This finding, together with the maintenance of constant tyrosol levels during the seven-day storage period, is very promising for the use of chitosan formulations to produce functional tomato fruit.

Design and synthesis of H2S-donor hybrids: A new treatment for Alzheimer's disease?

Hydrogen sulphide (H2S) is an endogenous gasotransmitter, largely known as a pleiotropic mediator endowed with antioxidant, anti-inflammatory, pro-autophagic, and neuroprotective properties. Moreover, a strong relationship between H2S and aging has been recently identified and consistently, a significant decline of H2S levels has been observed in patients affected by Alzheimer's disease (AD). On this basis, the use of H2S-donors could represent an exciting and intriguing strategy to be pursued for the treatment of neurodegenerative diseases (NDDs). In this work, we designed a small series of multitarget molecules combining the rivastigmine-scaffold, a well-established drug already approved for AD, with sulforaphane (SFN) and erucin (ERN), two natural products deriving from the enzymatic hydrolysis of glucosinolates contained in broccoli and rocket, respectively, endowed both with antioxidant and neuroprotective effects. Notably, all new synthetized hybrids exhibit a H2S-donor profile in vitro and elicit protective effects in a model of LPS-induced microglia inflammation. Moreover, a decrease in NO production has been observed in LPS-stimulated cells pre-treated with the compounds. Finally, the compounds showed neuroprotective and antioxidant activities in human neuronal cells. The most interesting compounds have been further investigated to elucidate the possible mechanism of action.

Anti-Proliferative Properties and Proapoptotic Function of New CB2 Selective Cannabinoid Receptor Agonist in Jurkat Leukemia Cells.

Several studies demonstrated that cannabinoids reduce tumor growth, inhibit angiogenesis, and decrease cancer cell migration. As these molecules are well tolerated, it would be interesting to investigate the potential benefit of newly synthesized compounds, binding cannabinoid receptors (CBRs). In this study, we describe the synthesis and biological effect of 2-oxo-1,8-naphthyridine-3-carboxamide derivative LV50, a new compound with high CB2 receptor (CB2R) affinity. We demonstrated that it decreases viability of Jurkat leukemia cells, evaluated by Trypan Blue and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), but mainly induces a proapoptotic effect. We observed an increase of a hypodiploid peak by propidium iodide staining and changes in nuclear morphology by Hoechst 33258. These data were confirmed by a significant increase of Annexin V staining, cleavage of the nuclear enzyme poly(ADP-ribose)-polymerase (PARP), and caspases activation. In addition, in order to exclude that LV50 non-specifically triggers death of all normal leukocytes, we tested the new compound on normal peripheral blood lymphocytes, excluding the idea of general cytotoxicity. To characterize the involvement of CB2R in the anti-proliferative and proapoptotic effect of LV50, cells were pretreated with a specific CB2R antagonist and the obtained data showed reverse results. Thus, we suggest a link between inhibition of cell survival and proapoptotic activity of the new compound that elicits this effect as selective CB2R agonist.

Oleocanthal and oleacein contribute to the in vitro therapeutic potential of extra virgin oil-derived extracts in non-melanoma skin cancer.

Although the anticancer properties of extra virgin olive oil (EVOO) extracts have been recognized, the role of single compounds in non-melanoma skin cancer is still unknown. The in vitro chemopreventive and anticancer action of EVOO extracts and oil-derived compounds in non-melanoma skin cancer models were evaluated on cutaneous squamous cell carcinoma cells and on immortalized human keratinocytes stimulated with epidermal growth factor. Preparation of EVOO extracts and isolation of single compounds was carried out by chromatographic methods. Antitumor activity was assessed by cell-based assays (cell viability, migration, clonogenicity, and spheroid formation) and apoptosis documented by internucleosomal DNA fragmentation. Finally, inhibition of key oncogenic signaling nodes involved in the progression from actinic keratosis to cutaneous squamous cell carcinoma was studied by western blot. EVOO extracts reduced non-melanoma skin cancer cell viability and migration, prevented colony and spheroid formation, and inhibited proliferation of atypical keratinocytes stimulated with epidermal growth factor. Such a pharmacological activity was promoted by oleocanthal and oleacein through the inhibition of Erk and Akt phosphorylation and the suppression of B-Raf expression, whereas tyrosol and hydroxytyrosol did not have effect. The current study provides in vitro evidence for new potential clinical applications of EVOO extracts and/or single oil-derived compounds in the prevention and treatment of non-melanoma skin cancers.

Novel analogs of PSNCBAM-1 as allosteric modulators of cannabinoid CB1 receptor.

In this work, we explored the molecular framework of the known CB1R allosteric modulator PSNCBAM-1 with the aim to generate new bioactive analogs and to deepen the structure-activity relationships of this type of compounds. In particular, the introduction of a NH group between the pyridine ring and the phenyl nucleus generated the amino-phenyl-urea derivative SN15b that behaved as a positive allosteric modulator (PAM), increasing the CB1R binding affinity of the orthosteric ligand CP55,940. The functional activity was evaluated using serum response element (SRE) assay, which assesses the CB1R-dependent activation of the MAPK/ERK signaling pathway. SN15b and the biphenyl-urea analog SC4a significantly inhibited the response produced by CP55,940 in the low µM range, thus behaving as negative allosteric modulators (NAMs). The new derivatives presented here provide further insights about the modulation of CB1R binding and functional activity by allosteric ligands.

Sulfonamido-derivatives of unsubstituted carbazoles as BACE1 inhibitors.

A novel series of variously substituted N-[3-(9H-carbazol-9-yl)-2-hydroxypropyl]-arylsulfonamides has been synthesized and assayed for ß-Secretase (BACE1) inhibitory activity. BACE1 is a widely recognized drug target for the prevention and treatment of Alzheimer's Disease (AD). The introduction of benzyl substituents on the nitrogen atom of the arylsulfonamide moiety has so far led to the best results, with three derivatives showing IC50 values ranging from 1.6 to 1.9 µM. Therefore, a significant improvement over the previously reported series of N-carboxamides (displaying IC50's ≥ 2.5 µM) has been achieved, thus suggesting an active role of the sulfonamido-portion in the inhibition process. Preliminary molecular modeling studies have been carried out to rationalize the observed structure-activity relationships.

Application of An Improved HPLC-FL Method to Screen Serine Palmitoyl Transferase Inhibitors.

In this work, we reported the application and validation of an improved high-performance liquid chromatography method coupled with a fluorimetric detector (HPLC-FL) to screen the activity of two heterocyclic derivatives reported as serine palmitoyl transferase (SPT) inhibitors. The analytical conditions were optimized in terms of the derivatization procedure, chromatographic condition, extraction procedure, and method validation according to EMEA guidelines. Once fully optimized, the method was applied to assess the SPT-inhibitory activity of the above-mentioned derivatives and of the reference inhibitor myriocin. The obtained results, expressed as a percentage of residual SPT activity, were compared to those obtained with the reference radio immune assay (RIA). The good correlation between the two types of assay demonstrated that the improved HPLC-FL method is suitable for a preliminary and rapid screening of potential SPT-inhibitors.

Structural Optimization of 4-Chlorobenzoylpiperidine Derivatives for the Development of Potent, Reversible, and Selective Monoacylglycerol Lipase (MAGL) Inhibitors.

Monoacylglycerol lipase (MAGL) inhibitors are considered potential therapeutic agents for a variety of pathological conditions, including several types of cancer. Many MAGL inhibitors are reported in literature; however, most of them showed an irreversible mechanism of action, which caused important side effects. The use of reversible MAGL inhibitors has been only partially investigated so far, mainly because of the lack of compounds with good MAGL reversible inhibition properties. In this study, starting from the (4-(4-chlorobenzoyl)piperidin-1-yl)(4-methoxyphenyl)methanone (CL6a) lead compound that showed a reversible mechanism of MAGL inhibition (Ki = 8.6 µM), we started its structural optimization and we developed a new potent and selective MAGL inhibitor (17b, Ki = 0.65 µM). Furthermore, modeling studies suggested that the binding interactions of this compound replace a structural water molecule reproducing its H-bonds in the MAGL binding site, thus identifying a new key anchoring point for the development of new MAGL inhibitors.

Cytotoxic Activity of Oleocanthal Isolated from Virgin Olive Oil on Human Melanoma Cells.

Oleocanthal is one of the phenolic compounds of extra virgin olive oil with important anti-inflammatory properties. Although its potential anticancer activity has been reported, only limited evidence has been provided in cutaneous malignant melanoma. The present study is aimed at investigating the selective in vitro antiproliferative activity of oleocanthal against human malignant melanoma cells. Since oleocanthal is not commercially available, it was obtained as a pure standard by direct extraction and purification from extra virgin olive oil. Cell viability experiments carried out by WST-1 assay demonstrated that oleocanthal had a remarkable and selective activity for human melanoma cells versus normal dermal fibroblasts with IC50s in the low micromolar range of concentrations. Such an effect was paralleled by a significant inhibition of ERK1/2 and AKT phosphorylation and downregulation of Bcl-2 expression. These findings may suggest that extra virgin olive oil phenolic extract enriched in oleocanthal deserves further investigation in skin cancer.

Synthesis and pharmacological evaluation of new biphenylic derivatives as CB2 receptor ligands.

Targeting type-2 cannabinoid receptor (CB2) is considered a feasible strategy to develop new drugs for the treatment of diseases like neuropathic pain, chronic inflammation, neurodegenerative disorders and cancer. Such drugs are devoid of the undesired central side effects that are typically mediated by the CB1 receptor. In this work we synthesized 18 biphenylic carboxamides as new CB2-selective ligands and evaluated their pharmacological profiles. The functional activity of these compounds is strongly influenced by the nature of the substituent at position 4' and 5 of the biphenyl scaffold. Position 5 seems to be responsible for the agonist or inverse agonist behaviour independently of the substituent in position 4', with the exception of the methoxyl group which transforms both full agonists and inverse agonists into neutral antagonists. This study provides a novel complete toolbox of CB2 functional modulators that derive from the same chemical scaffold. Such probes may be useful to investigate the biological role of CB2 receptors in cellular assays.

Design, synthesis and preliminary evaluation of (18)F-labelled 1,8-naphthyridin- and quinolin-2-one-3-carboxamide derivatives for PET imaging of CB2 cannabinoid receptor.

In the present work, we report the synthesis of new aryliodonium salts used as precursors of single-stage nucleophilic (18)F radiofluorination. The corresponding unlabelled fluorinated derivatives showed to be CB2 cannabinoid receptor specific ligands, with Ki values in the low nanomolar range and high CB2/CB1 selectivity. The radiolabelled compound [(18)F]CB91, was successfully formulated for in vivo administration, and its preliminary biodistribution was assessed with microPET/CT. This tracer presented a reasonable in vivo stability and a preferential extraction in the tissues that constitutionally express CB2 cannabinoid receptor. The results obtained indicate [(18)F]CB91 as a possible candidate marker of CB2 cannabinoid receptor distribution. This study would open the way to further validation of this tracer for assessing pathologies for which the expression of this receptor is modified.

New quinolone- and 1,8-naphthyridine-3-carboxamides as selective CB2 receptor agonists with anticancer and immuno-modulatory activity.

Several recent studies suggest that selective CB2 receptor agonists may represent a valid pharmacological approach in the treatment of various diseases due to the absence of relevant psychoactive side effect. In this study, we synthesized and tested a series of new quinoline-2(1H)-one- and 4-hydroxy-2-oxo-1,2-dihydro-1,8-naphthyridine derivatives characterized by a 4-methylcyclohexylamido substituent in position 3 of the heterocyclic nucleus with high CB2 receptor affinity and selectivity. Two compounds showing the best binding and selectivity profile behaved as a full agonist and a partial agonist at the CB2 receptor and induced a concentration-dependent decrease of cell viability on LNCaP, a prostatic cancer cell line expressing CB2 receptor. Moreover considering that the CB2 receptor is mainly expressed in cells and organs of the immune system, the same compounds were studied for their potential immune-modulatory and anti-inflammatory effects in activated lymphocytes isolated from healthy controls and multiple sclerosis (MS) patients.

Synthesis, biological activity and molecular modeling of new biphenylic carboxamides as potent and selective CB2 receptor ligands.

The CB2 receptor is a therapeutic target of increasing importance for several diseases, including pain, inflammation, neurodegeneration, cancer and osteoporosis. While several compounds showing CB2-selective agonist or inverse agonist properties have been developed, only few CB2 receptor selective neutral antagonists are actually known. Such type of compounds could be useful to study more in depth the role of the CB2 receptor, because they lack the ability to counteract its "constitutive" activity. Here we describe the synthesis and biological activity of a series of biphenylic carboxamides as a new class of CB2 receptor selective ligands. In binding assays, one of these compounds showed good CB2 receptor affinity and selectivity (Ki = 11.48 nM; Selectivity Index = 130). Furthermore, in functional assays, the same compound showed a very interesting pharmacological profile as CB2 receptor selective neutral antagonist. These results pave the way to further developments, including structural optimization, with the aim to obtain more potent CB2 receptor ligands with this peculiar feature.

CB2-selective cannabinoid receptor ligands: synthesis, pharmacological evaluation, and molecular modeling investigation of 1,8-Naphthyridin-2(1H)-one-3-carboxamides.

We have recently identified 1,8-naphthyridin-2(1H)-one-3-carboxamide as a new scaffold very suitable for the development of new CB2 receptor potent and selective ligands. In this paper we describe a number of additional derivatives in which the same central scaffold has been variously functionalized in position 1 or 6. All new compounds showed high selectivity and affinity in the nanomolar range for the CB2 receptor. Furthermore, we found that their functional activity is controlled by the presence of the substituents at position C-6 of the naphthyridine scaffold. In fact, the introduction of substituents in this position determined a functionality switch from agonist to antagonists/inverse agonists. Finally, docking studies showed that the difference between the pharmacology of these ligands may be in the ability/inability to block the Toggle Switch W6.48(258) (χ1 g+ → trans) transition.

1,2-Dihydro-2-oxopyridine-3-carboxamides: the C-5 substituent is responsible for functionality switch at CB2 cannabinoid receptor.

The relevance of CB2R-mediated therapeutic effects is well-known for the treatment of inflammatory and neuropathic pain and neurodegenerative disorders. In our search for new cannabinoid receptor modulators, we report the optimization of a series of 1,2-dihydro-2-oxopyridine-3-carboxamide derivatives as CB2R ligands. In particular, N-cycloheptyl-5-(4-methoxyphenyl)-1-(4-fluorobenzyl)-pyridin-2(1H)-on-3-carboxamide (17) showed high CB2R affinity (K(i) = 1.0 nM), accompanied by interesting K(i)(CB1R)/K(i)(CB2R) selectivity ratio (SI = 43.4). Compound 17 was also identified as a potent CB2R neutral antagonist/weak partial inverse agonist. Finally we found that the functionality activity of the series of 1,2-dihydro-2-oxopyridine is controlled by the presence of a substituent in position 5 of the heterocyclic nucleus. In fact when the hydrogen atom in position 5 of the unsubstituted compound 1 was replaced with a phenyl group (compound 18) the CB2R activity was shifted from agonism to inverse agonism whereas the introduction in the same position of p-methoxyphenyl group lead to compound 17 which showed a behavior as CB2R neutral antagonist/weak partial inverse agonist.