Design, Synthesis and Biological Evaluation of Novel N-Arylpiperazines Containing a 4,5-Dihydrothiazole Ring.

Arylpiperazines represent one of the most important classes of 5-HT1AR ligands and have attracted considerable interests for their versatile properties in chemistry and pharmacology, leading to the research of new derivatives that has been focused on the modification of one or more portions of such pharmacophore. An efficient protocol for the synthesis of novel thiazolinylphenyl-piperazines (2a-c) and the corresponding acetylated derivatives was used (3a-c). The new compounds were tested for their functional activity and affinity at 5-HT1A receptors, showing an interesting affinity profile with a Ki value of 412 nM for compound 2b. The cytotoxic activity of novel thiazolinylphenyl-piperazines (2a-c) and corresponding N-acetyl derivatives (3a-c) against human prostate and breast cancer cell lines (LNCAP, DU-145 and PC-3, MCF-7, SKBR-3 and MDA-MB231) was investigated according to the procedure described in the literature. The reported data showed a cytotoxic effect for 2a-c and 3a-c compounds (IC50 values ranging from 15 µM to 73 µM) on the investigated cancer cell lines, with no effect on noncancer cells. Future studies will be aimed to investigate the mechanism of action and therapeutic prospects of these new scaffolds.

Enhanced efficacy of formoterol-montelukast salt in relieving asthma features and in preserving ß2-agonists rescue therapy.

Adrenergic ß2-agonists represent a mainstay in asthma management. Their chronic use has been associated with decreased bronchoprotection and rebound hyperresponsiveness. Here we investigate on the possible therapeutic advantage of a pharmacological association of ß2-agonists with montelukast, a highly selective leukotriene receptor antagonist, in modulating bronchial reactivity and controlling asthma features. The study has been conducted in vitro and in vivo and also takes advantage of the synthesis of a salt that gave us the possibility to simultaneously administer in vivo formoterol and montelukast (MFS). In vitro studies demonstrate that montelukast (1) preserves ß2-agonist response in isolated bronchi by preventing homologous ß2-adrenoceptor desensitization; (2) reduces desensitization by modulating ß2-receptor translocation in bronchial epithelial cells. In vivo studies demonstrate that sensitized mice receiving formoterol or montelukast display a significant reduction in airway hyperresponsiveness, but the ß2-agonist relaxing response is still impaired. Allergen challenge causes ß2 heterologous desensitization that is further increased by treatment in vivo with formoterol. Conversely MFS not only inhibits airway hyperresponsiveness but it rescues the ß2-agonist response. Histological analysis confirms the functional data, demonstrating an enhanced therapeutic efficiency of MSF in controlling also pulmonary metaplasia and lung inflammation. MFS is efficacious also when sensitized mice received the drug by local administration. In conclusion, the data obtained evidenced a therapeutic advantage in the association of ß2-agonists with montelukast in the control of asthma-like features and a better rescue bronchodilation response to ß2-agonists.

Design, Synthesis and Evaluation of Novel Molecular Hybrids between Antiglaucoma Drugs and H2S Donors.

Glaucoma is a group of eye diseases consisting of optic nerve damage with corresponding loss of field vision and blindness. Hydrogen sulfide (H2S) is a gaseous neurotransmitter implicated in various pathophysiological processes. It is involved in the pathological mechanism of glaucomatous neuropathy and exerts promising effects in the treatment of this disease. In this work, we designed and synthetized new molecular hybrids between antiglaucoma drugs and H2S donors to combine the pharmacological effect of both moieties, providing a heightened therapy. Brinzolamide, betaxolol and brimonidine were linked to different H2S donors. The H2S-releasing properties of the new compounds were evaluated in a phosphate buffer solution by the amperometric approach, and evaluated in human primary corneal epithelial cells (HCEs) by spectrofluorometric measurements. Experimental data showed that compounds 1c, 1d and 3d were the hybrids with the best properties, characterized by a significant and long-lasting production of the gasotransmitter both in the aqueous solution (in the presence of L-cysteine) and in the intracellular environment. Because, to date, the donation of H2S by antiglaucoma H2S donor hybrids using non-immortalized corneal cells has never been reported, these results pave the way to further investigation of the potential efficacy of the newly synthesized compounds.

Serotoninergic receptor ligands improve Tamoxifen effectiveness on breast cancer cells.

BACKGROUND: Serotonin (or 5-Hydroxytryptamine, 5-HT) signals in mammary gland becomes dysregulated in cancer, also contributing to proliferation, metastasis, and angiogenesis. Thus, the discovery of novel compounds targeting serotonin signaling may contribute to tailor new therapeutic strategies usable in combination with endocrine therapies. We have previously synthesized serotoninergic receptor ligands (SER) with high affinity and selectivity towards 5-HT2A and 5-HT2C receptors, the main mediators of mitogenic effect of serotonin in breast cancer (BC). Here, we investigated the effect of 10 SER on viability of MCF7, SKBR3 and MDA-MB231 BC cells and focused on their potential ability to affect Tamoxifen responsiveness in ER+ cells. METHODS: Cell viability has been assessed by sulforhodamine B assay. Cell cycle has been analyzed by flow cytometry. Gene expression of 5-HT receptors and Connective Tissue Growth Factor (CTGF) has been checked by RT-PCR; mRNA levels of CTGF and ABC transporters have been further measured by qPCR. Protein levels of 5-HT2C receptors have been analyzed by Western blot. All data were statistically analyzed using GraphPad Prism 7. RESULTS: We found that treatment with SER for 72 h reduced viability of BC cells. SER were more effective on MCF7 ER+ cells (IC50 range 10.2 µM - 99.2 µM) compared to SKBR3 (IC50 range 43.3 µM - 260 µM) and MDA-MB231 BC cells (IC50 range 91.3 µM - 306 µM). This was paralleled by accumulation of cells in G0/G1 phase of cell cycle. Next, we provided evidence that two ligands, SER79 and SER68, improved the effectiveness of Tamoxifen treatment in MCF7 cells and modulated the expression of CTGF, without affecting viability of MCF10A non-cancer breast epithelial cells. In a cell model of Tamoxifen resistance, SER68 also restored drug effect independently of CTGF. CONCLUSIONS: These results identified serotoninergic receptor ligands potentially usable in combination with Tamoxifen to improve its effectiveness on ER+ BC patients.

N-Acylethanolamine acid amidase (NAAA) is dysregulated in colorectal cancer patients and its inhibition reduces experimental cancer growth.

BACKGROUND AND PURPOSE: N-Acylethanolamine acid amidase (NAAA) is a lysosomal enzyme accountable for the breakdown of N-acylethanolamines (NAEs) and its pharmacological inhibition has beneficial effects in inflammatory conditions. The knowledge of NAAA in cancer is fragmentary with an unclarified mechanism, whereas its contribution to colorectal cancer (CRC) is unknown to date. EXPERIMENTAL APPROACH: CRC xenograft and azoxymethane models were used to assess the in vivo effect of NAAA inhibition. Further, the tumour secretome was evaluated by an oncogenic array, CRC cell lines were used for in vitro studies, cell cycle was analysed by cytofluorimetry, NAAA was knocked down with siRNA, human biopsies were obtained from surgically resected CRC patients, gene expression was measured by RT-PCR and NAEs were measured by LC-MS. KEY RESULTS: The NAAA inhibitor AM9053 reduced CRC xenograft tumour growth and counteracted tumour development in the azoxymethane model. NAAA inhibition affected the composition of the tumour secretome inhibiting the expression of EGF family members. In CRC cells, AM9053 reduced proliferation with a mechanism mediated by PPAR-α and TRPV1. AM9053 induced cell cycle arrest in the S phase associated with cyclin A2/CDK2 down-regulation. NAAA knock-down mirrored the effects of NAAA inhibition with AM9053. NAAA expression was down-regulated in human CRC tissues, with a consequential augmentation of NAE levels and dysregulation of some of their targets. CONCLUSION AND IMPLICATIONS: Our results show novel data on the functional importance of NAAA in CRC progression and the mechanism involved. We propose that this enzyme is a valid drug target for the treatment of CRC growth and development.

Trends in H2S-Donors Chemistry and Their Effects in Cardiovascular Diseases.

Hydrogen sulfide (H2S) is an endogenous gasotransmitter recently emerged as an important regulatory mediator of numerous human cell functions in health and in disease. In fact, much evidence has suggested that hydrogen sulfide plays a significant role in many physio-pathological processes, such as inflammation, oxidation, neurophysiology, ion channels regulation, cardiovascular protection, endocrine regulation, and tumor progression. Considering the plethora of physiological effects of this gasotransmitter, the protective role of H2S donors in different disease models has been extensively studied. Based on the growing interest in H2S-releasing compounds and their importance as tools for biological and pharmacological studies, this review is an exploration of currently available H2S donors, classifying them by the H2S-releasing-triggered mechanism and highlighting those potentially useful as promising drugs in the treatment of cardiovascular diseases.

ROS-Mediated Apoptotic Cell Death of Human Colon Cancer LoVo Cells by Milk δ-Valerobetaine.

δ-Valerobetaine (δVB) is a constitutive milk metabolite with antioxidant and anti-inflammatory activities. Here, we tested the antineoplastic properties of milk δVB on human colorectal cancer cells. CCD 841 CoN (non-tumorigenic), HT-29 (p53 mutant adenocarcinoma) and LoVo (APC/RAS mutant adenocarcinoma) cells were exposed to 3 kDa milk extract, δVB (2 mM) or milk+δVB up to 72 h. Results showed a time- and dose-dependent capability of δVB to inhibit cancer cell viability, with higher potency in LoVo cells. Treatment with milk+δVB arrested cell cycle in G2/M and SubG1 phases by upregulating p21, cyclin A, cyclin B1 and p53 protein expressions. Noteworthy, δVB also increased necrosis (P < 0.01) and when used in combination with milk it improved its activity on live cell reduction (P < 0.05) and necrosis (P < 0.05). δVB-enriched milk activated caspase 3, caspase 9, Bax/Bcl-2 apoptotic pathway and reactive oxygen species (ROS) production, whereas no effects on ROS generation were observed in CCD 841 CoN cells. The altered redox homeostasis induced by milk+δVB was accompanied by upregulation of sirtuin 6 (SIRT6). SIRT6 silencing by small interfering RNA blocked autophagy and apoptosis activated by milk+δVB, unveiling the role of this sirtuin in the ROS-mediated apoptotic LoVo cell death.

Anticancer activities of fatty acids and their heterocyclic derivatives.

Traditional chemotherapy relies on the premise that rapidly proliferating cancer cells are more likely to be killed by a cytotoxic agent, but in reality, the long-standing problem of chemotherapy is the lack of tumor-specific treatments. Apart from the impact on tumor cells, the drugs' major limitation is their severe adverse side effects on normal cells and tissues. Nutritional and epidemiological studies have indicated that cancer progression is correlated with the consumption of fatty acids, but the exact mechanisms still remain unknown. In the first part of our review, we discussed the beneficial effects of free fatty acids (saturated and unsaturated) on the progress of carcinogenesis in different tumor cell lines. We presented various mechanisms proposed in the literature, which explain the possible impact on the cells metabolism. The second part describes modifications of different fatty acids with existing anticancer drugs and heterocyclic moieties by condensation reactions. Such conjugations increased the tissue selectivity and made chemotherapy potentially more effective and less toxic in in vivo and in vitro studies. This fatty acid modifications, which change the activity of compounds, their uptake selectivity and alter drug delivery methods, may be the key to unlocking true medical potential of fatty acids.

PCB levels in adipose tissue of dogs from illegal dumping sites in Campania region (Italy).

The aim of the study is to investigate the potential relationship between exposure to PCBs and cancer. In doing so we relied on a sample of dogs coming from a peculiar area of the Campania region (Italy), that has been suffering for illegal waste dumping and open air burning of plastic waste for many years. The latter determined the release of organic and inorganic pollutants, such as the PCBs. By comparing dogs with cancer and healthy dogs, we found much higher PCB concentrations in the former, with a significant difference (p < 0.05) for the non-indicator ∑10NDL-PCB and the DL-PCBs. A regression analysis, controlling for three potentially confounding factors, that are sex, age and weight, confirmed the higher ∑10NDL-PCB concentration in dogs with cancer. Hence, our evidence suggests a potential health hazard for animals and likewise people living in a risky area due to the presence of environmental organic pollutants.

Design of Sphingosine Kinases Inhibitors: Challenges and Recent Developments.

BACKGROUND: Sphingosine kinases (SphKs) catalyze the phosphorylation of sphingosine to form the bioactive sphingolipid metabolite sphingosine-1-phosphate (S1P). S1P is an important lipid mediator with a wide range of biological functions; it is also involved in a variety of diseases such as inflammatory diseases, Alzheimer's disease and cancer. METHODS: This review reports the recent advancement in the research of SphKs inhibitors. Our purpose is also to provide a complete overview useful for underlining the features needed to select a specific pharmacological profile. DISCUSSION: Two distinct mammalian SphK isoforms have been identified, SphK1 and SphK2. These isoforms are encoded by different genes and exhibit distinct subcellular localizations, biochemical properties and functions. SphK1 and SphK2 inhibition can be useful in different pathological conditions. CONCLUSION: SphK1 and SphK2 have many common features but different and even opposite biological functions. For this reason, several research groups are interested in understanding the therapeutic usefulness of a selective or non-selective inhibitor of SphKs. Moreover, a compensatory mechanism for the two isoforms has been demonstrated, thus leading to the development of dual inhibitors.

Anti-metastatic Properties of Naproxen-HBTA in a Murine Model of Cutaneous Melanoma.

The beneficial effects of H2S-release and of COXs-inhibition have been exploited in the design of novel anti-inflammatory drugs, the H2S-releasing non-steroidal anti-inflammatory drugs (H2S-NSAIDs), showing promising potential for chemoprevention in cancers. Here, we evaluated the efficacy of a new H2S-releasing derivative of naproxen, named naproxen-4-hydroxybenzodithioate (naproxen-HBTA), in reducing metastatic melanoma features, both in vitro and in vivo. The novel H2S donor has been prepared following a synthetic scheme that provided high yields and purity. In particular, we investigated the effect of naproxen-HBTA in vitro on several metastatic features of human melanoma cells such as proliferation, migration, invasion, and colonies formation and in vivo in a model of cutaneous melanoma. Cell culture studies demonstrated that naproxen-HBTA induced caspase 3-mediated apoptosis and inhibited motility, invasiveness, and focus formation. Finally, daily oral treatment with naproxen-HBTA significantly suppressed melanoma growth and progression in mice. In conclusion, by using this dual approach we propose that the COX-2 and H2S pathways could be regarded as novel therapeutic targets/tools to generate new treatment options based on "combination therapy" for melanoma.

Anxiolytic-like effects of the new arylpiperazine derivatives containing isonicotinic and picolinic nuclei: behavioral and biochemical studies.

Anxiety disorder is a great challenge for modern psychopharmacology. Although a variety of single drugs are used in the treatment of anxiety, it is important to search for new therapeutics with faster onset of action, fewer side effects, and higher efficacy. In this work, we studied the possible anxiolytic action mechanism of two new arylpiperazine derivatives: compounds 4p N-(3-(4-(piperonyl)piperazin-1-yl)propyl)isonicotinamide and 3o N-(2-(4-(pyrimidin-2-yl)piperazin-1-yl)ethyl)picolinamide, focusing on their effects on the GABAergic and 5-HT systems. The elevated plus-maze test (EPM) was used for measuring anxiety. Additionally, in order to elucidate whether the new compounds have impact on the central redox balance, we conducted biochemical studies. In doing so, the relative activity of the enzymes responsible for glutathione metabolism - glutathione peroxidase and reductase (GPx and GR) - was measured. The results of the presented studies confirmed the anxiolytic effects of the new compounds 4p (60 mg/kg) and 3o (7.5 mg/kg), and suggested in the mechanism of their action, direct 5-HT1A receptors' participation and indirect involvement of the GABAergic system. Furthermore, the compounds exerted significant agonistic effect with buspirone (BUS, the 5-HT1A partial agonist, 1 mg/kg i.p.) and diazepam (DZ, the classic benzodiazepine anxiolytic, 0.25 mg/kg s.c.), while WAY 100635 (N-{2-[4-(2-methoxyphenyl)-1-piperazinyl] ethyl}-N-(2-pyridyl) cyclohexanecarboxamide, a selective 5-HT1A antagonist, 0.1 mg/kg s.c.), but not flumazenil (a GABAA -BDZ receptor complex antagonist, 10 mg/kg i.p.) was able to reverse their anxiolytic effects in EPM. A concomitant decrease in GPx by the compound 4p (and to a lesser degree, by compound 3o) further seemed to confirm their anxiolytic and antioxidant activity.

The Role of 5-HT1A Receptor in Cancer as a New Opportunity in Medicinal Chemistry.

The 5-HT1A receptor is a pharmacologically well characterized serotonin receptor subtype and it has long been investigated because of its involvement in several physiopathological mechanisms and treatment of neurological diseases like ansia and depression. Serotonin (5-HT) also shows many non-neural functions such as essential hypertension, embryogenesis, follicle maturation and behavior. Moreover, it exerts a growth factor function on different types of non-tumoral cells, and it was also found to be related to oncogenes. In fact, growth-stimulatory activity of serotonin in different human tumor cells has been reported. Recently, new chemical molecules binding the 5-HT1A receptor have been described as novel therapeutic entities useful in neuroprotection, cognitive impairment, Parkinson's Disease, pain treatment, malignant carcinoid syndrome and cancer. It was widely demonstrated that 5-HT1A receptor is involved in the carcinogenesis and consequently in many human tumor types, such as prostate, bladder, small cell lung, colonrectal and cholangiocarcinoma. Furthermore, depending on the tumor type, 5-HT1A receptor antagonists were shown to be capable of blocking the 5HT-induced increase in tumor growth. In this review, we have focused our attention on each tumor type where the 5-HT1A receptor is involved, investigating the role of this molecular target and the different classes of compounds that have shown the capability to modulate it. The analyzed aspects could represent a hint for the medical chemists to develop novel molecules as selective 5-HT1A agents are useful in further elucidating the role of this therapeutic target.

1,2,4-Thiadiazolidin-3,5-diones as novel hydrogen sulfide donors.

Hydrogen sulfide (H2S) is an endogenous modulator that plays significant physio-pathological roles in several biological systems. In this research field there is a large interest in developing selective CBS and CSE inhibitors and H2S releasing moieties, that could be either used as therapeutic agents or linked to known drugs. One of the major problem is the limited availability of chemicals that ensure a controlled release of H2S in vitro as well in vivo. Aiming to obtain novel H2S donors, whose release properties could be appropriately modulated, we have synthesized a series of 1,2,4-thiadiazolidine-3,5-diones (THIA 1-10) as innovative donors that could release H2S in controlled manner. All the synthesized compounds were evaluated for their H2S releasing properties by an amperometric approach and for their vasorelaxant ability on aorta rings. In order to rationalize the obtained results, a detailed study on the release mechanism has been performed using the most efficient H2S donor, THIA 3 (Cmax 65.4 µM and EC50 1.7 µM).

Development of 1,2,3-Triazole-Based Sphingosine Kinase Inhibitors and Their Evaluation as Antiproliferative Agents.

Two series of N-(aryl)-1-(hydroxyalkyl)pyrrolidine-2-carboxamides (2a-2g and 3a-3g) and 1,4-disubstituted 1,2,3-triazoles (5a-5h and 8a-8h) were synthesized. All the compounds, containing a lipophilic tail and a polar headgroup, were evaluated as sphingosine kinase (SphK) inhibitors by assessing their ability to interfere with the acetylcholine (Ach) induced relaxation of aortic rings pre-contracted with phenylephrine. Moreover, their antiproliferative activity was tested on several cell lines expressing both SphK1 and SphK2. Compounds 5h and 8f, identified as the most efficient antiproliferative agents, showed a different selectivity profile, with 8f being selective for SphK1.

Synthesis of Arylpiperazine Derivatives as Protease Activated Receptor 1 Antagonists and Their Evaluation as Antiproliferative Agents.

BACKGROUND: Protease activated receptor-1 (PAR1) is a G-coupled receptor activated by α-thrombin and other proteases. Several reports have demonstrated the PAR1 involvement in tumorigenesis and tumor progression. In order to investigate on potential use of PAR1 antagonists as antiproliferative agents. AIMS: We have identified a series of arylpiperazine derivatives acting as PAR1 antagonists; the selected molecules have been evaluated for their antiproliferative properties. METHOD: All the compounds inhibited the growth of a panel of cell lines expressing PAR1; two of them, compounds 13 and 15, were able to inhibit, in a dose dependent manner, the growth of the selected cell lines with the lowest IC50 values, and were further characterized to define the mechanism responsible for the observed antiproliferative effect. RESULT: This study directed us to the identification of two interesting leads that may help to further validate PAR1 as an important therapeutic target for cancer treatment.

Fragment-based de novo design of a cystathionine γ-lyase selective inhibitor blocking hydrogen sulfide production.

Hydrogen sulfide is an essential catabolite that intervenes in the pathophysiology of several diseases from hypertension to stroke, diabetes and pancreatitis. It is endogenously synthesized mainly by two pyridoxal-5'-phosphate-dependent enzymes involved in L-cysteine metabolism: cystathionine-ß-synthase (CBS) and cystathionine-γ-lyase (CSE). Research in this field is currently impaired by the lack of pharmacological tools such as selective enzymatic inhibitors that could target specifically only one of these pathways. We used a novel approach based on a hybrid method that includes drug design, synthetic biology, metabolomics and pharmacological assays to rationally design a new inhibitor selective for the CSE enzyme. The identification of this compound opens new frontiers towards a better understanding of the role of CSE over CBS in the pathophysiology of diseases where a role for the H2S pathway has been proposed and the development of new lead compounds that could target the CSE enzyme.

5-HT(1A) receptor: an old target as a new attractive tool in drug discovery from central nervous system to cancer.

The serotonin receptor subtype 5-HT(1A) was one of the first serotonin receptor subtypes pharmacologically characterized. This receptor subtype has long been object of intense research and is implicated in the pathogenesis and treatment of anxiety and depressive disorders. In recent years, new chemical entities targeting the 5-HT(1A) receptor (alone or in combination with other molecular targets) have been proposed for novel therapeutic uses in neuroprotection, cognitive impairment, Parkinson's disease, pain treatment, malignant carcinoid syndrome, and prostate cancer. This Perspective compares existing data on expression and signaling activity of the 5-HT(1A) receptor to a ligand with an intrinsic agonist or antagonist profile. Our purpose is also to make a complete overview, useful for underlining the features needed to select a specific pharmacological profile rather than another one. This aspect could be really interesting to consider and justify the 5-HT(1A) receptor as a new attractive target for drug discovery.

Identification of a pepducin acting as S1P3 receptor antagonist.

Sphingosine-1-phosphate (S1P) is a bioactive lipid with key functions in the immune, inflammatory, and cardiovascular systems. S1P exerts its action through the interaction with a family of five known G protein-coupled receptors, named S1P(1-5). Among them, S1P(3) has been implicated in the pathological processes of a number of diseases, including sepsis and cancer. KRX-725 (compound 1) is a pepducin that mimics the effects of S1P by triggering specifically S1P(3). Here, aiming to identify novel S1P(3) antagonists, we carried out an alanine scanning analysis to address the contribution of the side chains of each amino acid residue to the peptide function. Then, deleted peptides from both the C- and N-terminus were prepared in order to determine the minimal sequence for activity and to identify the structural requirements for agonistic and, possibly, antagonistic behaviors. The pharmacological results of the Ala-scan derived compounds (2-10) suggested a high tolerance of the pepducin 1 to amino acid substitutions. Importantly, the deleted peptide 16 has the ability to inhibit, in a dose-dependent manner, both pepducin 1-induced vasorelaxation and fibroblast proliferation. Finally, a computational analysis was performed on the prepared compounds, showing that the supposed antagonists 16 and 17 appeared to be aligned with each other but not with the others. These results suggested a correlation between specific conformations and activities.

Synthesis of benzamide derivatives and their evaluation as antiprion agents.

A new set of 5-(2-(pyrrolidin-1-yl)acetamido)-N-butyl-2-(substituted)benzamide and 5-(2-(piperidin-1-yl)acetamido)-N-butyl-2-(substituted) benzamide derivatives were synthesized in which as structural features the 2-(1-pyrrolidinyl)- or 2-(1-piperidyl)acetylamino group or a diphenylether moiety are associated to a benzamide scaffold. Their binding affinity for human PrP(C) and inhibition of its conversion into PrP(Sc) were determined in vitro; moreover, the antiprion activity was assayed by inhibition of PrP(Sc) accumulation in scrapie-infected mouse neuroblastoma cells (ScN2a) and scrapie mouse brain (SMB) cells. The results clearly indicate the benzamide derivatives as attractive lead compounds for the development of potential therapeutic agents against prion disease.