σ(2) Receptor research is receiving increasing interest with regard to the potential of σ(2) proteins as targets for tumor therapy and diagnosis. Nevertheless, knowledge about the σ(2) receptor is far from conclusive. The paucity and modest affinity of known σ(2) antagonists represent one of the limitations to σ(2) receptor research. Previous studies of the high-affinity σ(2) agonist 1-cyclohexyl-4-[3-(5-methoxy-1,2,3,4-tetrahydronaphthalen-1-yl)-n-propyl]piperazine 4 (PB28) suggested that a decrease in lipophilicity might lead to σ(2) ligands devoid of antiproliferative activity (potential σ(2) antagonists). With the aim of producing σ(2) receptor antagonists, we replaced the tetralin nucleus of compound 4 with a 2-aminopyridine moiety. A series of compounds with high affinity for both σ subtypes and with no antiproliferative activity in various cells (mouse HT-22, human SK-N-SH, MCF-7wt, and MCF-7σ(1) ) were obtained. The effect on Ca(2+) mobilization was investigated for high-affinity compounds 18 and 4, which showed opposite effects. All of the data support the new 2-aminopyridines as high-affinity σ ligands with σ(2) antagonist and σ(1) agonist activity, and, despite the lack of significant σ(2) versus σ(1) selectivity, these novel compounds may be better tools for σ receptor research than the known low-affinity σ(2) antagonists.

IMPORTANCE OF THE FIELD: The 5-HT(7) receptors are discretely localized within the CNS (thalamus, hypothalamus, limbic and cortical regions). The 5-HT(7) receptors are also present in smooth muscle cells from blood vessels and have been reported in gastrointestinal tract as well as in rat lumbar dorsal root and sympathetic ganglia. The 5-HT(7) receptors have been implicated in depression, disorders related to circadian rhythms, pain and migraine. Thus, there is a great interest in developing potent and selective 5-HT(7) receptor modulators. AREAS COVERED IN THIS REVIEW: This review article highlights the research advances published in the patent literature between January 2004 and December 2009, giving emphasis to the medicinal chemist's standpoint. WHAT THE READER WILL GAIN: Readers will rapidly gain an overview of the various 5-HT(7) receptor modulators reported in the patent literature in the past 6 years. Furthermore, the readers will learn which structure type can interact with 5-HT(7) receptor and also the different companies that are the main players in the field. TAKE HOME MESSAGE: Although no 5-HT(7) modulator has entered clinical trials, the development and future use of different agonists and antagonists suitable for use in vivo seem very promising

Activatable fluorescent probes share the unique feature of being turned on only under specific conditions: they are "silent" when not interacting with a specific target protein, microenvironment, or reactive species. Several activatable fluorescence probes have demonstrated their potential in cell biology study, disease study and diagnosis, and even in the rapidly expanding field of image-guided surgery. In this review, we will summarize progress in the design of activatable probes and their application in studying cell biology or in optical imaging. Some of the most effective examples of activatablefluorescent probes will be resented and their application will be discussed.

A series of alkyloxyquinoline derivatives has been developed to evaluate the relationship between P-gp potency and lipophilicity. The results show a satisfactory lipophilicity-activity correlation although a series of derivatives showing higher P-gp potency is needed in order to confirm this hypothesis.

1-Cyclohexyl-4-[3-(5-methoxy-1,2,3,4-tetrahydronaphthalen-1-yl)propyl]piperazine 1 (PB28) and 2- Methoxy-5-methyl-N-[4-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)butyl]benzamide 2 (RHM-1) represent leads for tumor diagnosis, given their high affinity at sigma2 receptors. With the purpose of obtaining good candidates for s2 PET tracers development, hybrid structures between 1 and 2 were designed. Excellent sigma1/sigma2 selectivities were reached when 6,7-dimethoxytetrahydroisoquinolinewas linked to an omethoxy substituted arylamide (11a, 12a, 15a), and for these benzamides an intramolecular H-bond in the active conformation at the s sites, was hypothesized. However these excellent s2 ligands were accompanied by interaction with P-gp, which may limit their use as sigma2 receptor PET agents when tumors overexpress P-gp. Compound 15a whose P-gp interaction was just moderate represents an interesting tool for the development of sigma2 PET tracers useful in tumors overexpressing P-gp.

Although several pieces of information are still missing about sigma-2 receptor, the production of high affinity sigma-2 receptor ligands allowed important acquisitions. Morphans such as CB64D and CB184 were the first truly 2-selective ligands synthesized, and their use in cell cultures highlighted the relationship between sigma-2 receptors and cell proliferation, shedding light on important diagnostic and therapeutic potentials with which sigma-2 ligands are endowed. The most significant classes of compounds are herein discussed. The design and Structure-Affinity Relationship studies (SAfiR) of 2 receptor ligands belonging to the classes of morphans, indoles (siramesine analogues), granatanes, flexible benzamides and N-cyclohexylpiperazines are reported, together with the biological results which these compounds provided giving a crucial contribution to the sigma-2 receptor research. The pharmacophore models which were generated on the basis of different classes of the sigma-2 ligands and the attempts for 2 receptor purification are briefly described.

Introduction: P-glycoprotein (P-gp) plays a crucial role in beta-amyloid efflux from the blood–brain barrier thus becoming a promising pharmacological target in the treatment of Alzheimer's disease (AD). The increase of P-glycoprotein expression and activity by a P-gp inducer could be an effective pharmacological strategy in slowing or halting the progression of AD. Commonly used in vitro methods to classify a P-gp interacting molecule as substrate, inhibitor, modulator or inducer are not always confirmed by in vivo experiments. Here we validate the new dye-probe beta-amyloid (1–40) HiLyte Fluor™ TR-labeled (Ab-HiLyte) (Anaspec) P-gp mediated transport in the ex vivo rat everted gut sac assay by using MC18 or MC266, a fully characterized P-gp inhibitor and substrate, respectively, and compare it with the commonly used dye rhodamine. Methods: Male Wistar rats' everted intestines were divided into sacs, each sac was filled with 10 μM Ab-HiLyte with or without 50 μM of MC18 or MC266. Ab-HiLyte concentrations in mucosal fluid were measured spectrophotometrically at 594 nm at each appropriate time. Results: The Ab-HiLyte P-gp mediated efflux had a K=1.00×10−2 min−1 and t1/2=68.74 min, while in the presence of MC18, the Ab-HiLyte efflux turned out to be reduced by an order of magnitude (K=1.65×10−3 min−1) and the half life is extremely increased (t1/2=419 min). A P-gp substrate, likeMC266, determines no change in the efflux of Ab: the kinetic constant and the half life turned out to be unmodified (K=1.81×10−2 min−1 and t1/2=38.28 min). Discussion: The results demonstrate that the new dye probe, Ab-HiLyte, could be a probe of choice to unequivocally distinguish between a P-gp substrate and an inhibitor. This is particularly important as different groups obtain a controversial classification of the same compound.

We report the synthesis of compounds structurally related to the high-affinity dopamine D4 receptor ligand N-{2-[4-(3-cyanopyridin-2-yl)piperazin-1-yl]ethyl}-3-methoxybenzamide (1e). All compounds were specifically designed as potential PET radioligands for brain D4 receptor visualization, having lipophilicity within a range for brain uptake and weak non-specific binding (0.75<cLogP<3.15) and bearing a substituent for easy access to labeling with the positron emitter isotope (11) C or (18) F. The best compound of the series, N-{2-[4-(4-chlorophenyl)piperazin-1-yl]ethyl}-6-fluoropyridine-3-carboxamide (7a), displayed excellent selectivity over D2 and D3 receptors (>100-fold), but its D4 receptor affinity was suboptimal for imaging of brain D4 receptors (Ki =30 nM).

Here we describe the design, synthesis, and evaluation of physicochemical and pharmacological properties of D(4) dopamine receptor ligands related to N-[2-[4-(4-chlorophenyl)piperazin-1-yl]ethyl]-3-methoxybenzamide (2). Structural features were incorporated to increase affinity for the target receptor, to improve selectivity over D(2) and σ(1) receptors, to enable labeling with carbon-11 or fluorine-18, and to adjust lipophilicity within the range considered optimal for brain penetration and low nonspecific binding. Compounds 7 and 13 showed the overall best characteristics: nanomolar affinity for the D(4) receptor, &gt;100-fold selectivity over D(2) and D(3) dopamine receptors, 5-HT(1A), 5-HT(2A), and 5-HT(2C) serotonin receptors and σ(1) receptors, and log P = 2.37-2.55. Following intraperitoneal administration in mice, both compounds rapidly entered the central nervous system. The methoxy of N-[2-[4-(3-cyanopyridin-2-yl)piperazin-1-yl]ethyl]-3-methoxybenzamide (7) was radiolabeled with carbon-11 and subjected to PET analysis in non-human primate. [(11)C]7 time-dependently accumulated to saturation in the posterior eye in the region of the retina, a tissue containing a high density of D(4) receptors.

Sigma2 Receptors are promising biomarkers for cancer diagnosis given the relationship between the proliferative status of tumors and their density. With the aim of contributing to the research of sigma2 receptor Positron Emission Tomography (PET) probes, we developed 2-[3-[6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl]propyl]-3,4-dihydroisoquinolin-1(2H)-one (3), with optimal 2 pharmacological properties and appropriate lipophilicity. Hence, 3 served as the lead compound for the development of a series of dihydroisoquinolinones amenable to radiolabeling. Radiosynthesis for compound 26, which displayed the most appropriate 2 profile, was developed and 2 specific binding for the corresponding [18F]-26 was confirmed by in vitro autoradiography on rat brain slices. Despite the excellent in vitro properties, [18F]-26 could not successfully image 2 receptors in the rat brain in vivo, maybe because of its interaction with P-gp. Nevertheless, [18F]-26 may still be worthy of further investigation for the imaging of sigma2 receptors in peripheral tumors devoid of P-gp overexpression.

Despite their controversial physiology, sigma-1 (σ1) receptors are intriguing targets for the development of therapeutic agents for central nervous system diseases. With the aim of providing versatile pharmacological tools to study σ1 receptors, we developed three σ1 fluorescent tracers by functionalizing three well characterized σ1 ligands with a fluorescent tag. A good compromise between σ1 binding affinity and fluorescent properties was reached, and the σ1 specific targeting of the novel tracers was demonstrated by confocal microscopy and flow cytometry. These novel ligands were also successfully used in competition binding studies by flow cytometry, showing their utility in nonradioactive binding assays as an alternative strategy to the more classical radioligand binding assays. To the best of our knowledge these are the first σ1 fluorescent ligands to be developed and successfully employed in living cells, representing promising tools to strengthen σ1 receptors related studies

σ2 Receptorsubtypeisoverexpressedinavarietyofhumantumors,with σ2 agonists showing antiproliferativeeffectstowardstumorcellsthroughmultiplepathwaysthatdependbothonthetumor cell typeandonthemoleculetype.Therefore, σ2 receptorisanintriguingtargetfortumordiagnosisand treatment despitethefactthatthatithasnotyetbeencloned.Oneofthelastattemptstocharacterize σ2 receptorsledtoidentifyitastheprogesteronereceptormembranecomponent1(PGRMC1).Although still controversial,suchidentityappearstohavebeenaccepted.Wetheaimofcontributingtosolvethis controversy,inthisworkwestablysilencedoroverexpressedPGRMC1proteininhumanMCF7 adenocarcinoma cells.WesternblottinganalyseswereperformedtoquantifythepresenceofPGRMC1 proteinoneachofthethreeMCF7celllinesvariants,whilescatchardanalyseswithradioligandwere performed inordertodeterminetheexpressionofthe σ2 receptors.Inordertocorrelatethe antiproliferativeeffectof σ2 receptoragonistwithPGRMC1density,some σ2 ligands wereadministered to eachofthethreeMCF7cellsvariants.TheresultssuggestedthatPGRMC1and σ2 receptorsaretwo different molecularentities.

Fluorescent derivatives of σ2 high affinity ligand 1-cyclohexyl-4-[3-(5-methoxy-1,2,3,4-tetrahydronaphthalen-1- yl)propyl]piperazine 1 (PB28) were synthesized. NBD or dansyl fluorescent tags were connected through a 5- or 6-atom linker in two diverse positions of 1 structure. Good σ2 affinities were obtained when the fluorescent tag was linked to 5-methoxytetralin nucleus replacing the methyl function. NBD-bearing compound 16 displayed high σ2 affinity (Ki = 10.8 nM) and optimal fluorescent properties. Its uptake in pancreatic tumor cells was evaluated by flow cytometry, showing that it partially occurs through endocytosis. In proliferating cells, the uptake was higher supporting that σ2 receptors are markers of cell proliferation and that the higher the proliferation is, the stronger the antiproliferative effect of σ2 agonists is. Colocalization of 16 with subcellular organelles was studied by confocal microscopy: the greatest was in endoplasmic reticulum and lysosomes. Fluorescent σ2 ligands show their potential in clarifying the mechanisms of action of σ2 receptors.

Generations of modulators of the efflux pump P-glycoprotein (P-gp) have been produced as tools to counteract the Multidrug Resistance (MDR) phenomenon in tumor therapy, but clinical trials were not successful so far. With the aim of contributing to the development of novel P-gp modulators, we started from recently studied high-affinity sigma-2 (σ2) receptor ligands that showed also potent interaction with P-gp. For σ2 receptors high-affinity binding, a basic N-atom is a strict requirement. Therefore, we reduced the basic character of the N-atom present in these ligands, and we obtained potent P-gp modulators with poor or null σ2 receptor affinity. We also evaluated whether modulation of P-gp by these novel compounds involved consumption of ATP (as P-gp substrates do), as a source of energy to support the efflux. Surprisingly, even small structural changes resulted in opposite behavior, with amide 13 depleting ATP, in contrast to its isomer 18. Two compounds, 15 and 25, emerged for their potent activity at P-gp, and deserve further investigations as tools for P-gp modulation.

The 5-HT(1A) receptor subtype is the most thoroughly studied serotonin receptor subtype. We report here the design, synthesis and characterization of two new fluorescent ligands for the 5-HT(1A) receptor. The new 1-arylpiperazine-based red-emitting fluorescent compound 6 displayed good binding affinity at the 5-HT(1A) receptor (K(i)=35 nM) and was able to label specifically the human 5-HT(1A) receptor stably expressed in CHO cells visualized using confocal laser scanning microscopy.

Sigma-2 (s2) binding sites are an emerging target for anti-neoplastic agents due to the strong apoptotic effect exhibited by s2 agonists in vitro and the overexpression of these sites in tumor cells. Nonetheless, no s2 receptor protein has been identified. Affinity chromatography using the high-affinity s2 ligand PB28 and human SK-N-SH neuroblastoma cells was previously utilized to identify s2 ligand binding proteins, specifically histones H1, H2A, H2B, and H3.3a. To rationalize this finding, homology modeling and automated docking studies were employed to probe intermolecular interactions between PB28 and human nucleosomal proteins. These studies predicted interaction of PB28 with the H2A/H2B dimer at a series of sites previously found to be implicated in chromatin compaction and nucleosomal assembly. To experimentally verify this prediction, a competitive binding assay was performed on the reconstituted H2A/H2B dimer using [3H]PB28 as radioligand, and an IC50 value of 0.50 nm was determined for PB28 binding. In addition, [3H]PB28 was found to accumulate with up to a fivefold excess in nuclear fractions over cytosolic fractions of SK-N-SH and MCF7 cells, indicating that PB28 is capable of entering the nucleus to interact with histone proteins. In conjunction with computational results, these data suggest that PB28 may exert its cytotoxic effect through direct interaction with nuclear material

A series of polymethyl-substituted piperidines linked to either a 6-methoxy-1,2,3,4-tetrahydronaphthalen-1-yl or a 6-methoxynaphthalen-1-yl moiety was generated with the aim of verifying a previously generated hypothesis: tetralin and naphthalene nuclei confer opposite activity at the 1 receptor. Compounds 6, 9 and 10 displayed appreciable affinity at both  subtypes, but none of the novel compounds displayed significant antiproliferative activity in MCF7wt and MCF71 cell lines. The effect on bradikynin-triggered Ca2+ mobilization was studied as a methodology to suggest sigma receptors mediated activity.

Microglial cells are responsible for clearing and maintaining the central nervous system (CNS) microenvironment. Upon brain damage, they move toward injuries to clear the area by engulfing dying neurons. However, in the context of many neurological disorders chronic microglial responses are responsible for neurodegeneration. Therefore, it is important to understand how these cells can be “switched-off” and regain their ramified state. Current research suggests that microglial inflammatory responses can be inhibited by sigma () receptor activation. Here, we take advantage of the optical transparency of the zebrafish embryo to study the role of 1 receptor in microglia in an intact living brain. By combining chemical approaches with real time imaging we found that treatment with PB190, a 1 agonist, blocks microglial migration toward injuries leaving cellular baseline motility and the engulfment of apoptotic neurons unaffected. Most importantly, by taking a reverse genetic approach, we discovered that the role of 1 in vivo is to “switch-off” microglia after they responded to an injury allowing for these cells to leave the site of damage. This indicates that pharmacological manipulation of 1 receptor modulates microglial responses providing new approaches to reduce the devastating impact that microglia have in neurodegenerative diseases.

We have determined the pharmacological profile of the new serotonin 5-HT7 receptor agonist N- (4-cyanophenylmethyl)-4-(2-diphenyl)-1-piperazinehexanamide (LP-211). Radioligand binding assays were performed on a panel of 5-HT receptor subtypes. The compound was also evaluated in vivo by examining its effect on body temperature regulation in mice lacking the 5-HT7 receptor (5-HT7−/−) and their 5-HT7 +/+ sibling controls. Disposition studies were performed in mice of both genotypes. It was found that LP-211 was brain penetrant and underwent metabolic degradation to 1-(2-diphenyl)piperazine (RA- 7). In vitro binding assays revealed that RA-7 possessed higher 5-HT7 receptor affinity than LP-211 and a better selectivity profile over a panel of 5-HT receptor subtypes. In vivo it was demonstrated that LP- 211, and to a lesser degree RA-7, induced hypothermia in 5-HT7 +/+ but not in 5-HT7−/− mice. Our results suggest that LP-211 can be used as a 5-HT7 receptor agonist in vivo.

BACKGROUND: Sigma-2 receptor ligands have been studied for treatment of pancreatic cancer because they are preferentially internalized by proliferating cells and induce apoptosis. This mechanism of apoptosis is poorly understood, with varying reports of caspase-3 dependence. We evaluated multiple sigma-2 receptor ligands in this study, each shown to decrease tumor burden in preclinical models of human pancreatic cancer. RESULTS: Fluorescently labeled sigma-2 receptor ligands of two classes (derivatives of SW43 and PB282) localize to cell membrane components in Bxpc3 and Aspc1 pancreatic cancer cells and accumulate in lysosomes. We found that interactions in the lysosome are critical for cell death following sigma-2 ligand treatment because selective inhibition of a protective lysosomal membrane glycoprotein, LAMP1, with shRNA greatly reduced the viability of cells following treatment. Sigma-2 ligands induced lysosomal membrane permeabilization (LMP) and protease translocation triggering downstream effectors of apoptosis. Subsequently, cellular oxidative stress was greatly increased following treatment with SW43, and the hydrophilic antioxidant N-acetylcysteine (NAC) gave greater protection against this than a lipophilic antioxidant, alpha-tocopherol (alpha-toco). Conversely, PB282-mediated cytotoxicity relied less on cellular oxidation, even though alpha-toco did provide protection from this ligand. In addition, we found that caspase-3 induction was not as significantly inhibited by cathepsin inhibitors as by antioxidants. Both NAC and alpha-toco protected against caspase-3 induction following PB282 treatment, while only NAC offered protection following SW43 treatment. The caspase-3 inhibitor DEVD-FMK offered significant protection from PB282, but not SW43. CONCLUSIONS: Sigma-2 ligand SW43 commits pancreatic cancer cells to death by a caspase-independent process involving LMP and oxidative stress which is protected from by NAC. PB282 however undergoes a caspase-dependent death following LMP protected by DEVD-FMK and alpha-toco, which is also known to stabilize the mitochondrial membrane during apoptotic stimuli. These differences in mechanism are likely dependent on the structural class of the compounds versus the inherent sigma-2 binding affinity. As resistance of pancreatic cancers to specific apoptotic stimuli from chemotherapy is better appreciated, and patient-tailored treatments become more available, ligands with high sigma-2 receptor affinity should be chosen based on sensitivities to apoptotic pathways.

P-glycoprotein (P-gp) is involved in MDR and in neurodegenerative disorders such as Parkinson disease (PD), Alzheimer disease (AD) and epilepsy. Cytochrome P450 enzymes (CYP450(s)) catalyze the metabolism of a wide variety of endogenous and exogenous compounds including xenobiotics, drugs, environmental toxins, steroids, and fatty acids. P-gp substrates, inhibitors and inducers should be designed and developed studying interacting mechanism with both P-gp an CYP450 enzymes before they could be employed in MDR and/or in neurodegenerative disorders. Here, the ex vivo rat everted gut sac assay has been proposed as an immediate approach to simultaneously study metabolism and transport of drugs. Elacridar, verapamil and cyclosporine A (CsA), P-gp inhibitor, substrate and modulator respectively, have been tested to validate this ex vivo approach. The new model have been used yet to develop our ligands MC18, MC266 and MC80, both as potential drugs for MDR and radiotracers for diagnosis of neurodegenerative disorders. Herein, a comparative evaluation of transport and metabolic results, by using in vitro, ex vivo and in vivo assays, is reported.

Although sigma-2 (s2) receptors are still enigmatic proteins, they are promising targets for tumor treatment and diagnosis. With the aim of clarifying their role in oncology, we developed a s2-selective fluorescent tracer compound 5) as a specific tool to study s2 receptors. By using flow cytometry with 5, we performed competition binding studies on three different cell lines where we also detected the content of the s2 receptors, avoiding the inconvenient use of radioligands. Comparison with a previously developed mixed s1/s2 fluorescent tracer (1) also allowed for the detection of s1 receptors within these cells. Results obtained by flow cytometry with tracers 1 and 5 were confirmed by standard methods (western blot for s1, and Scatchard analysis for s2 receptors). Thus, we have produced powerful new tools for research on the s whose reliability and adaptability to a number of fluorescence techniques will be useful to elucidate the roles of s receptors in oncology.

Despite the promising potentials of σ2 receptors in cancer therapy and diagnosis, there are still ambiguities related to the nature and physiological role of the σ2 protein. With the aim of providing potent and reliable tools to be used in σ2 receptor research, we developed a novel series of fluorescent σ2 ligands on the basis of our previous work, where high-affinity σ2 ligand 1-cyclohexyl-4-[3-(5-methoxy-1,2,3,4-tetrahydronaphthalen-1-yl)-n-propyl]piperazine (1, PB28) was used as the pharmacophore. Compared to the previous compounds, these novel ligands displayed improved fluorescence and σ2 binding properties, were σ2-specifically taken up by breast tumor cells, and were successfully employed in confocal microscopy. Compound 14, which was the best compromise between pharmacological and fluorescent properties, was successfully employed in flow cytometry, demonstrating its potential to be used as a tool in nonradioactive binding assays for studying the affinity of putative σ2 receptor ligands.

Here we report the synthesis, pharmacological and pharmacokinetic evaluation of a pilot set of compounds structurally related to the potent and selective 5-HT7 ligand LP-211. Among the studied compounds, N-pyridin-3-ylmethyl-3-[4-[2-(4-methoxyphenyl)phenyl]piperazin-1-yl]ethoxy]propanamide (4b) showed high affinity for 5-HT7 receptors (Ki=23.8nM), selectivity over 5-HT1A receptors (>50-fold), in vitro metabolic stability (82%) and weak interaction with P-glycoprotein (BA/AB=3.3). Compound 4b was injected ip in mice to preliminarily evaluate its distribution between blood and brain.

To combat the emergence of drug-resistance in tumors novel strategies are urgently needed. With this in mind we designed a novel class of thiosemicarbazones able to target simultaneously 2 receptors and P-glycoprotein efflux pump while chelating metals such as Iron. The combined effect of these targets would lead to the activation of multiple pathways to which resistant tumors are sensitive. Indeed, most of the novel thiosemicarbazones displayed antiproliferative activity in both parent (MCF7 breast adenocarcinoma and A549 lung carcinoma) and corresponding doxorubicine-resistant cells (MCF7dx and A549dx). A few compounds emerged for their potent antiproliferative activity or for their more potent effect in doxorubicine-resistant cells than in the parent ones, while other compounds emerged for their remarkable P-gp modulatory activity. These results pave the way for further studies on these targets in the oncology field, while the availability of promising molecules for resistant tumors treatment that warrant deeper investigations was increased.

The invention relates to a new class of compounds able to bind with high affinity and selectivity the 5-HT7 receptor. The invention also relates to the utilization of such compounds as medicaments useful in the treatment and prevention of 5-HT7 receptor relating disorders of the central nervous system. The invention also relates to the isotopically labeled compounds for use in vivo diagnosis or imaging of a 5-HT7 condition.

Compounds 8a–d have been designed as bioisosters of tariquidar for imaging P-gp expression and density by PET. The results displayed that compounds 8b and 8d could be considered potential P-gp/BCRP ligands suitable as 11C and 18F radiotracers, respectively.

The development of P-glycoprotein (P-gp) ligands remains of considerable interest mostly for investigating protein structure and transport mechanism. In the last years many ligands, belonging to different generations, have been tested for modulating P-gp activity. Aim of the present work is to perform SAR studies on tetrahydroisoquinoline derivatives in order to design potent and selective P-gp ligands. For this purpose the effect of bioisosteric replacement and the role of flexibility have been investigated and four series of tetrahydroisoquinoline ligands have been developed: a) 2-aryoxazole bioisosteres; b) elongated analogues; c) 2H-Chromene and d) 2-biphenyl derivatives. Obtained results showed that both 2-biphenyl derivative 20b and elongated derivative 6g behaved as strong P-gp substrates. In conclusion important items have been highlighted for developing potent and selective P-gp ligands providing a solid starting point for further optimization.

The serotonin 7 (5-HT7) receptor was the last serotonin receptor subtype to be discovered in 1993. This receptor system has been implicated in several central nervous system (CNS) functions, including circadian rhythm, rapid eye movement sleep, thermoregulation, nociception, memory and neuropsychiatric symptoms and pathologies, such as anxiety, depression and schizophrenia. In 1999, medicinal chemistry efforts led to the identification of SB-269970, which became the gold standard selective 5-HT7 receptor antagonist, and later of various selective agonists such as AS-19, LP-44, LP-12, LP-211 and E-55888. In this review, we summarize the preclinical pharmacological studies performed using these agonists, highlighting their strengths and weaknesses. The data indicate that 5-HT7 receptor agonists can have neuroprotective effects against N-methyl-d-aspartate-induced toxicity, modulate neuronal plasticity in rats, enhance morphine-induced antinociception and alleviate hyperalgesia consecutive to nerve lesion in neuropathic animals

Since its discovery in the 1940s in serum, the mammalian intestinal mucosa, and in the central nervous system, serotonin (5-HT) has been shown to be involved in virtually all cognitive and behavioral human functions, and alterations in its neurochemistry have been implicated in the etiology of a plethora of neuropsychiatric disorders. The cloning of 5-HT receptor subtypes has been of importance in enabling them to be classified as specific protein molecules encoded by specific genes. The 5-HT7 receptor is the most recently classified member of the serotonin receptor family. Since its identification, it has been the subject of intense research efforts driven by its presence in functionally relevant regions of the brain. The availability of some selective antagonists and agonists, in combination with genetically modified mice lacking the 5-HT7 receptor, has allowed for a better understanding of the pathophysiological role of this receptor. This paper reviews data on localization and pharmacological properties of the 5-HT7 receptor, and summarizes the results of structure-activity relationship studies aimed at the discovery of selective 5-HT7 receptor ligands. Additionally, an overview of the potential therapeutic applications of 5-HT7 receptor agonists and antagonists in central nervous system disorders is presented.

The expression of sigma-2 receptor (S2R) was assayed in blood and bladder samples from healthy cattle and in blood and bladder of cattle with deltapapillomavirus-associated urothelial tumors. Samples of bladder from cattle with neoplasia had significantly higher S2R than samples of bladder from healthy cattle (95% CI 0.31-0.82, P < 0.05). In addition, significantly higher S2R was detected in the blood of cattle with bladder cancer than blood from healthy cattle (95% CI 0.22-0.41, P < 0.05). The results provide evidence that increased expression of SR2 in blood could be useful as circulating biomarker for bladder cancer in cattle. PGRMC1 protein levels were also found to be increased in blood and bladder from cattle with cancer and increased expression of PGRMC1 transcripts was detected by quantitative real time PCR in samples from cattle neoplasia. Furthermore, electron microscopy revealed phagophores and numerous autophagosomes, ultrastructural hallmark of autophagy.

Starting from two carbocyclic analogs, a series of 3,3-dimethylpiperidine derivatives was prepared and tested in radioligand binding assays at r1 and r2 receptors, and at D8–D7 sterol isomerase (SI) site. The novel compounds mostly bear heterocyclic rings or bicyclic nucleus of differing lipophilicities. Compounds 18a and 19a,b demonstrated the highest r1 affinity (Ki = 0.14–0.38 nM) with a good selectivity versus r2 binding. Among them, 18a had the lowest ClogD value (3.01) and only 19b was selective versus SI too. Generally, it was observed that more planar and hydrophilic heteronuclei conferred a decrease in affinity for both r receptor subtypes.

Introduction: Both subtypes of sigma (σ) receptors, σ1 and σ2, are over-expressed in many cancers with σ2 proposed as a biomarker of tumor proliferation. We are interested in developing a high affinity selective σ2 radioligand for in vivo monitoring of proliferative status of solid tumors and response to anti-cancer therapies. 1-Cyclohexyl-4-[3-(5-methoxy-1,2,3,4-tetrahydronaphthalen-1-yl)propyl]piperazine (PB28) represents one of the lead candidates in the development of σ receptor ligands for therapeutic and diagnostic applications. However, the utility of PB28 is limited due to its relatively high lipophilicity. Methods: A more hydrophilic analogue (–)-(S)-1was radiolabeledwith 11C via standard O-alkylation. In vitro autoradiography with [11C](–)-(S)-1 was done using rat brain slices. PET imaging was performed in mice bearing EMT6, C6 or PC-3 tumors after i.v. injection of [11C](–)-(S)-1. Results: [11C](–)-(S)-1 was produced in 53% ± 7% isolated decay-corrected yield with radiochemical and chemical purity over 99% and specific activity greater than 100 GBq/μmol. In vitro autoradiography with [11C(–)-(S)-1 resulted in a heterogeneous binding of the tracer in the rat brain with the highest radioactivity signals in the cortex region followed by cerebellum. This binding was successfully blocked by 10 μM of either haloperidol, (+)- (R)-1 or PB28. For C6 xenografts low target-to-nontarget ratio and high non-specific binding did not allow clear tumor visualization. No accumulation was visible in EMT6 tumor or in PC-3 tumor. Rat and mouse brain uptake was low and homogeneous while stronger signal was detected in the spinal cord. High accumulation of radioactivity was observed in liver and intestine suggesting hepatobiliary clearance. Conclusions: Despite excellent in vitro properties, [11C](–)-(S)-1 did not provide high enough specific binding in vivo and is, therefore, not a useful PET tracer for imaging σ2 expression in tumors

Both subtypes of sigma receptors, s1 and s2, are overexpressed in many cancers with s2 proposed as a biomarker of tumor proliferation. Sigma receptors are also abundant and are involved in various pathological processes in central nervous system. We are interested in developing a high affinity selective s2 radioligand, in particular for in vivo monitoring of proliferative status of solid tumors and response to anti‐cancer therapies. We report here the synthesis and evaluation of two piperazine analogues. 1‐cyclohexyl‐4‐[3‐(5‐methoxy‐1,2,3,4‐tetrahydronaphthalen‐1‐yl)propyl]piperazine (PB28) represents one of the lead candidates in the development of sigma receptor ligands for therapeutic and diagnostic applications. However, its utility is limited due to relatively high lipophilicity. We synthesized a more hydrophilic analogue 1 (Figure 1, logD7.4 = 2.38). Binding affinity (Ki) of its S‐enantiomer, (–)‐(S)‐1, to 2 was 5.92 ± 0.52 nM with ~15‐fold selectivity over 1 (Ki = 94.6 ± 12.6 nM). (–)‐(S)‐1 was radiolabeled with 11C via standard O‐alkylation with 11C‐methyliodide in an automated module. 11C‐(–)‐(S)‐1 was produced in 53 ± 7 % isolated decay‐corrected yield with radiochemical and chemical purity over 99% and specific activity greater than 100 GBq/micromol. Average synthesis time including semi‐preparative HPLC purification was 30 min. Fluoroethylation of desmethyl precursor 3 negatively affected binding to both sigma receptor subtypes (1 Ki = 316 ± 21 nM, 2 60% of 3H‐(+)‐DTG binding inhibition at 10‐11 M) and as a result the fluorinated compound was not pursued further. PET imaging was performed in mice bearing EMT6, C6 or PC3 tumors after i.v. injection of 11C‐(–)‐(S)‐ 1. For C6 xenografts low target‐to‐nontarget ratio did not allow clear tumor visualization. No accumulation was visible in EMT6 tumor, which is commonly used to evaluate s2 ligands, or PC3 tumor with SUV 0 suggesting poorly perfused tissue. Brain uptake was low and homogeneous (SUV 0.9‐1.3). High accumulation of radioactivity was observed in liver and intestine suggesting hepatobiliary clearance. In vitro autoradiography with 11C‐(–)‐(S)‐1 using rat brain slices resulted in a heterogeneous binding of the tracer with the highest accumulation in olfactory bulb, cerebellum, and cortex. This binding was successfully blocked by 10 μM of either, haloperidol, (+)‐(R)‐1, or PB28. In conclusion, despite excellent in vitro properties, 11C‐(–)‐(S)‐1 was not successful in imaging s2 receptors in vivo. Other analogues are currently being evaluated.

Sigma-2 receptors are intriguing targets under study for their involvement in tumors and neurodegenerative diseases. However, the ‘sigma enigma’ is not over yet, with controversies about sigma-2 receptor identity. In one of the last attempts for the characterization, the identification of the sigma-2 protein with the progesterone receptor membrane component 1 (PGRMC1) was proposed and generally accepted. Recently, the so-called PGRMC1/sigma-2 protein was identified as a neuronal receptor for Abeta oligomers binding, with Abeta oligomers behaving as ‘regular’ ligands at such proteins. Very importantly, a few sigma-2 receptor antagonists were able to displace in a dose-dependent manner synthetic Abeta oligomers from synaptic puncta, as well as human Abeta oligomers from brain sections of Alzheimer’s disease (AD) patients. In vivo, these compounds showed to reverse cognitive deficits restoring memory and sustaining long-term improvement in AD mice models. The PGRMC1/sigma-2 protein involvement in these activities was demonstrated by radioactive sigma-2 ligand binding together with PGRMC1 silencing, starting from the consideration that PGRMC1 and sigma-2 are the same molecular entity. Nevertheless, we feel that the clear identification of the proteins involved in the described effects is crucial for future development of AD disease-modifying therapies. With this perspective, we verified how the expression of sigma-2 receptor is independent of PGRMC1 expression by western blotting and scatchard analyses on cell lines where sigma-2 receptors were constitutively present, and where PGRMC1 was alternatively silenced or overexpressed (human breast adencarcinoma MCF7 cells). In addition, the sigma-2 mediated activity, which was studied through sigma-2 agonists, was independent of the presence and amount of PGRMC1. In order to further investigate the connection between sigma-2 and PGRMC1, the profiling of these proteins in a number of cell lines is in progress. Confocal microscopy and flow-cytometry studies employing sigma-2 fluorescent tracers are undergoing in cell lines where PGRMC1 is silenced, overexpressed and/or constitutively present. Results from this work will contribute to clarify the controversial relationship between sigma-2 and PGRMC1, so that unbiased research focused on these targets for the development of AD-modifying agents may be conducted.

Background A number of σ receptor ligands have been demonstrated to possess antidepressant-like effect in some experimental paradigms (e.g. forced swim test, tail suspension test, olfactory bulbectomy model, conditioned fear stress). The objective of the present study was to find out whether PB190 and PB212, new σ1 receptor ligands, show the effects in some models predictive of antidepressant activity. Methods The impact of PB190 and PB212 on the immobility time in the forced swim test (FST) and tail suspension test (TST) was assessed in C57BL/6J male mice. Extracellular bradykinin triggers a transient increase in intracellular calcium concentration by activating the phospholipase C/IP3 pathway. The intracellular calcium concentration was estimated with the dual wavelength ratiometric probe Fura-2. Results In the FST model, PB190 showed a moderate antidepressant-like effect (only in the dose of 3 mg/kg) which was enhanced by joint treatment with amantadine (AMA), 10 mg/kg (inactive per se). The decrease in the immobility time induced by the combined treatment with PB190 and AMA was counteracted by PB212 and by BD1047, a σ1-receptor antagonist. The in vitro studies indicated that Ca2+-response was increased by 1 μM PB190, like by the σ1-agonist (+)-pentazocine, while 1 μM PB212 behaved line σ1-antagonist, BD1063. On the other hand, 100 μM PB190 negatively affected the Ca2+-response after bradykinin. Conclusions The obtained results: 1/indicated that in the in vivo conditions PB190 behaved as a σ1-receptor agonist while PB212 counteracted its effect, confirming the in vitro data; 2/gave support to the hypothesis that σ1-receptors might be one of possible mechanisms by which drugs induce antidepressant-like activity; 3/revealed that this effect may be potentiated by NMDA receptor antagonists, e.g. AMA.

Sigma-1 receptors are specifically located at the endoplasmic reticulum–mitochondrion interface, but upon stimulation by ligands or under prolonged cellular stress, they translocate to other areas of the cell. Sigma-1 receptors are involved in the regulation of intracellular [Ca2+] by affecting the Ca2+-influx or the release from intracellular stores. In SK-N-SH cells, we measured the affinity of 4-methyl-1-[4-(6-methoxynaphthalen-1-yl)butyl]piperidine (PB212) at sigma-1 receptor by using a competition binding assay with specific radioligand; we obtained a Ki value = 316 ± 19 nM. PB212 also showed an antiproliferative effect in SK-N-SH cells (EC50 = 32 ± 4 μM) but had no effect in MCF7 cells, which only express sigma-2 receptor; these findings suggest that PB212 behaves as a sigma-1 receptor antagonist. We have studied the effect of PB212 on Ca2+ homeostasis of the SK-N-SH cell line with the fluorescent probe Fura-2. 100 μM PB212 induced a Ca2+-efflux from the endoplasmic reticulum through the inositol (1, 4, 5)-trisphosphate (IP3) receptor. Moreover, [PB212] ranging from 1 to 100 μM reduced the Ca2+-response, triggered by carbachol or bradykinin that engage the phospholipase C/IP3 pathway; such a response is generally increased by sigma-1 receptor agonists. On the other hand, PB212 did not reduce the Ca2+-response mediated by IP3 in LoVo cells, which do not express neither sigma-1 nor sigma-2 receptors, and in MCF7 cells. The fact that the activity of the sigma-1 receptor can be experimentally modulated by agonists and antagonists supports the intriguing hypothesis that some endogenous molecules, unknown at the moment, modulate the sigma-1 receptor and its cellular targets.

Introduction: The 5-HT1A receptors are implicated in mood disorders (anxiety, depression), in cognition, and in modulation of pain. Nearly 30 years of research in this field, there is still interest in developing new chemical entities capable of 5-HT1A receptor activation or blockade. 10 Areas covered: This review article will highlight and review the research advances published in the patent literature between January 2007 and December 2011, giving emphasis to the medicinal chemist’s standpoint. Literature search methodology included search in Scifinder and PubMed Databases and browsing clinicaltrials.gov website. 15 Expert opinion: Almost no new therapeutic applications have been proposed for molecules targeting the 5-HT1A receptor, during the years covered by the present review. The discovery that stimulation of 5-HT1A receptor can promote neurogenesis will likely renew the interest for selective 5-HT1A receptor agonists as therapeutics to replace neural populations damaged by disease 20 or injury.

Serotonin 7 (5-hydroxytryptamine7 or 5-HT7) is the most recently identified serotonin receptor. It is involved in mood disorders and is studied as a target for antidepressants. Here, we report on the structural manipulation of the 5-HT7 receptor ligand 4-[2-(3-methoxyphenyl)ethyl]-1-(2-methoxyphenyl)piperazine (1a) aimed at obtaining 5-HT7 receptor ligands endowed with good in vitro metabolic stability. A set of N-[3-methoxyphenyl)ethyl-substituted] 1-arylpiperazine, 4-arylpiperidine and 1-aryl-4-aminopiperidine was synthesized and tested in radioligand binding assays at human cloned 5-HT7 and 5-HT1A receptors. In vitro metabolic stability of the target compounds was assessed after incubation with rat hepatic S9 microsomal fraction. Among the new compounds, 1-(2-biphenyl)-4-[2-(3-methoxyphenyl)ethyl]piperazine (1d) and 4-(2-biphenyl)-1-[2-(3-methoxyphenyl)ethyl]piperidine (2d) showed a good compromise between affinity at 5-HT7 receptor (K i = 7.5 nM and 13 nM, respectively) and in vitro metabolic stability (26 and 65 % recovery of parent compound, respectively) but were poorly selective over 5-HT1A receptor.

L'invenzione riguarda una nuova classe di composti con elevata affinità e selettività verso la glicoproteina P. L'invenzione riguarda anche l'utilizzo di tali composti nella diagnosi in vivo di malattie neurodegenerative e come medicamenti per la prevenzione e il trattamento di malattie neurodegenerative che coinvolgono la glicoproteina P.

L'invenzione riguarda una nuova classe di composti in grado di inibire con alta affinità e selettività il recettore 5-HT7. L'invenzione riguarda anche l'utilizzo di tali composti come medicamenti utili nel trattamento e nella prevenzione dei disordini relativi al recettore 5-HT7 del sistema nervoso centrale. L'invenzione riguarda anche i composti marcati isotopicamente per l'uso in diagnostica o imaging in vivo di una condizione 5-HT7.

The present invention relates to a new method for the determination of 'free' copper concentration in serum, i.e. the portion of serum copper not structurally bound to ceruloplasmin. The present invention also refers to a method with a high degree of sensitivity and precision for the determination of free copper in serum samples of patients with Alzheimer's disease.

The present invention relates to cyclohexyl-substituted piperazine, piperidine and cyclohexane compounds, composition comprising the same and process for their synthesis. The invention also relates to said compounds for use as medicament, in particular for the treatment and/or prevention of early stage of Alzheimer's disease.

The present invention refers to a method and a kit of screening for therapeutic compounds useful in detoxifying central nervous system from ß-amyloid peptide. The method and kit involve the use of the beta amyloid peptide in the everted gut sac assay and allow assessing the inducing activity of the test compounds on the P-gp-mediated ß-amyloid peptide transport

A series of N-(1,2,3,4-tetrahydronaphthalen-1-yl)-4-aryl-1-piperazinealkylamides was prepared and their affinity for serotonin 5-HT7, 5-HT1A, and 5-HT2A receptors was measured using in vitro binding assays. In relation to 5-HT7 receptor affinity, receptor binding studies indicated that: (i) the optimal alkyl chain length was five methylenes; (ii) an unsubstituted 1,2,3,4-tetrahydronaphthalenyl nucleus was selected for further substitutions; and (iii) the substitution pattern of the aryl ring linked to the piperazine ring played a significant role. Several compound with high affinity for 5-HT7 receptors were identified.; Among them, 4-(2-methoxyphenyl)-N-(1,2,3,4-tetrahydronaphthalen-1-yl)-1-piperazinehexanamide (28), 4-(2-acetylphenyl)-N-(1,2,3,4-tetrahydronaphthalen-1-yl)-1-piperazinehexanamide (34), 4-(2-methylthiophenyl)-N-(1,2,3,4-tetrahydronaphthalen-1-yl)-1-piperazinehexanamide (44), 4-(2-hydroxyphenyl)-N-(1,2,3,4-tetrahydronaphthalen-1-yl)-1-piperazinehexanamide (46), 4-(2-methylphenyl)-N-(1,2,3,4-tetrahydronaphthalen-1-yl)-1-piperazinehexanamide (49) were assayed for the 5-HT7 receptor mediated relaxation of substance P-induced guinea-pig ileum contraction. Compounds 28, 44, and 49 behaved as full agonists, compound 34 as a partial agonist, whereas derivative 46 acted as an antagonist.

The invention relates to a new class of compounds able to inhibit with high affinity and selectivity the 5-HT7 receptor. The invention also relates to the utilization of such compounds as medicaments useful in the treatment and prevention of 5-HT7 receptor relating disorders of the central nervous system. The invention also relates to the isotopically labeled compounds for use in vivo diagnosis or imaging of a 5-HT7 condition.

The invention relates to a new class of compounds with high affinity and selectivity towards P-glycoprotein. The invention also relates to the utilization of such compounds in the in vivo diagnosis of neurodegenerative diseases and as medicaments for use in the prevention and treatment of neurodegenerative disease involving P-glycoprotein.

The present invention relates to the identification, isolation and characterization of the proteins forming the sigma-2 receptor. The invention further relates to the utilization of said proteins for preparing a screening for the sigma-2 receptor, as well as assay of specific candidate ligands to the use of the ligands for setting up diagnostic assays for tumour tissues and for preparing antitumour drugs. Lastly, the isolated proteins are utilized for producing anti-receptor antibodies, which likewise find employ in the diagnosis and therapeutic treatment of neoplasias.