Many new chemotherapeutic agents are under preclinical investigation and, despite efforts to more selectively target cancer cells, limitations such as toxicity and inherent resistance are often encountered. Therefore, alternative strategies are needed to treat cancer and overcome such limitations. We describe novel cyclohexylpiperazine derivatives, designed as mixed affinity ligands for sigma (s) receptors and human D8– D7 sterol isomerase (HSI) ligands, which also exhibit P-glycoprotein (P-gp) inhibitory activity, with the aim of exploiting the antiproliferative effects mediated by s and HSI sites while overcoming P-gp-mediated resistance. All of the compounds displayed high affinities for s receptors and HSI sites, P-gp inhibitory activity, and s2 receptor agonist antiproliferative activity. Antiproliferative activity was also tested in PC-3 cells to establish s1 and HSI contribution. Compound cis-11, which displayed the best antiproliferative and P-gp inhibitory activities, was coadministered with 0.1 mm doxorubicin in MDCK-MDR1 cells. Compound cis-11 caused 70% and 90% cell death when coadministered at 30 mm and 50 mm, respectively. When administered alone, cis-11 resulted in 50% cell death, demonstrating its single agent antitumor properties in a tumor cell line overexpressing P-gp.

New fluorescent derivatives for s receptors were designed and synthesized. To achieve this purpose, a 4-nitro-2,1,3-benzoxadiazole fluorescent tag was connected through a piperazine linker to a modified skeleton derived from selected s receptor agonists or antagonists. Compounds 5g, 7b, 7e and 7g displayed high s1 affinity and low s1/s2 selectivity (Kis1 ranging from 31.6 nM to 48.5 nM, Kis1/s2 ¼ 5–18), while compound 5d exhibited high s2 affinity and selectivity (Kis2 ¼ 56.8 nM, Kis1 > 5000 nM). Binding affinity studies revealed that compounds 5d, 5g, 7b, 7e and 7g showed no affinity towards several receptors including opioid, dopaminergic, serotonergic, adrenergic, muscarinic, histaminergic, N-methyl-D-aspartate (NMDA), NMDA receptor channel, or dopamine and serotonine transporters. The fluorescent properties, cellular uptake and confocal microscopy studies on 5d suggest a potential use of this probe to further clarify the molecular role of s2 receptor subtypes in normal and cancer cells.

CSCs are responsible for the high rate of recurrence and chemoresistance of different types of cancer. The current antineoplastic agents able to inhibit bulk replicating cancer cells and radiation treatment are not efficacious toward CSCs since this subpopulation has several intrinsic mechanisms of resistance. Among these mechanisms, the expression of ATP-Binding Cassette (ABC) transporters family and the activation of different signaling pathways (such as Wnt/β-catenin signaling, Hedgehog, Notch, Akt/PKB) are reported. Therefore, considering ABC transporters expression on CSCs membranes, compounds able to modulate MDR could induce cytotoxicity in these cells disclosing an exciting and alternative strategy for targeting CSCs in tumor therapy. The next challenge in the cure of cancer relapse may be a multimodal strategy, an approach where specific CSCs targeting drugs exert simultaneously the ability to circumvent tumor drug resistance (ABC transporters modulation) and cytotoxic activity toward CSCs and the corresponding differentiated tumor cells. The efficacy of suggested multimodal strategy could be probed by using several scaffolds active toward MDR pumps on CSCs isolated by tumor specimens.

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.

A set of basic aryl-group-containing compounds was synthesized with the aim of developing potent and selective P-glycoprotein (P-gp) modulators that are able to reverse multidrug resistance (MDR). The natures of the spacer (dicyclohexylamine or dialkylamine) and the aryl moieties were modified to investigate selectivity and the mechanism of P-gp interaction. The inhibitory activities of the compounds toward P-gp, multidrug resistance-associated protein 1 (MRP1), and breast cancer resistance protein (BCRP), the most relevant ATP binding cassette (ABC) transporters for MDR, were evaluated. The mechanism of P-gp interaction for each compound was investigated with three biological assays: apparent permeability (Papp ) determination (B→A/A→B) in Caco-2 cell monolayers, ATP cell depletion, and inhibition of Calcein-AM transport in MDCK-MDR1 cells. These assays allowed us to estimate the selectivity of the compounds for the three efflux pumps and to identify the structural requirements that define the P-gp-interaction profile. All dicyclohexylamine derivatives were found to be P-gp substrates, whereas one dialkylamine derivative was shown to be a P-gp inhibitor. The good MRP1 activity of one cis/cis isomer highlighted this as a lead candidate for the development of MRP1 ligands.

A series of chiral 2,3-dichlorophenoxy and 1-naphthyloxy alkylamines were synthesized, and their binding affinities towards 5-HT1D and h5-HT1B receptors were evaluated. In the naphthyloxy series, the (R)-prolinol derivative was the most selective 5-HT1D ligand, while (S)-N-methyl-2-(1-naphthyloxy)propan-1-amine showed the highest selectivity for h5-HT1B. Both compounds performed as 5-HT1D agonists in the isolated guinea pig assay and showed higher analgesic activity than both sumatriptan and the achiral analogue 20b in the mouse hotplate test. Neither ligand displayed any affinity for nicotinic ACh receptors present in mouse brain membranes, thus indicating that their analgesic activity does not arise through interaction with these receptors.

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).

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.

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

Here we report the design, synthesis, and 5-HT7 receptor affinity of a set of 1-(3-biphenyl)- and 1-(2-biphenyl)piperazines. The effect on 5-HT7 affinity of various substituents on the second (distal) phenyl ring was analyzed. Several compounds showed 5-HT7 affinities in the nanomolar range and >100-fold selectivity over 5-HT1A and adrenergic α1 receptors. 1-[2-(4-Methoxyphenyl)phenyl]piperazine (9a) showed 5-HT7 agonist properties in a guinea pig ileum assay but blocked 5-HT-mediated cAMP accumulation in 5-HT7-expressing HeLa cells.

A major limitation of cancer treatment is the ability of cancer cells to develop resistance to chemotherapeutic drugs, by the establishment of multidrug resistance. Here, we characterize MC70 as ABC transporters inhibitor and anticancer agent, alone or with chemotherapy. MC70 was analyzed for its interaction with ABCB1, ABCG2 and ABCC1 by specific transport assays. In breast and colon cancer cell lines, cell growth and apoptosis were measured by MTT assay and DNA laddering Elisa kit, respectively. Cell cycle perturbation and cellular targets modulation were analyzed by Flow-cytometry and Western blotting, respectively. MC70 interacted with ABC transporters. In breast cancer cells, MC70 slightly inhibited cell proliferation strongly enhancing doxorubicin effectiveness. By contrast, MC70 was found to inhibit cell growth in colon cancer cells without affecting doxorubicin efficacy and in combination with topoisomerase I inhibitors it could be a promising therapeutic approach. What is more, it was also observed that MC70 induced apoptosis, canceled in favor of necrosis when given in combination with high doses of doxorubicin. MC70 inhibited cell migration probably through its interaction with sigma-1 receptor. Modulations of i) cell cycle, ii) pAkt and the phosphorylation of the three MAPKs were highlighted, while any activity was excluded at transcription level, thus accounting for the phenotypic effects observed. MC70 might be considered as a new potential anticancer agent capable to i) enhance chemotherapy effectiveness and ii) to play a contributory role in the treatment of chemotherapy resistant tumors.

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.

The aim of the present work was to compare the mucoadhesive and efflux pump P-glycoprotein (P-gp) interacting properties of chitosan (CS)- and glycolchitosan (GCS)-based thiomers and corresponding unmodified parent polymers. For this purpose, the glycol chitosan-N-acetyl-cysteine (GCS-NAC) and glycol chitosan-glutathione (GCS-GSH) thiomers were prepared under simple and mild conditions. Their mucoadhesive characteristics were studied by turbidimetric and zeta potential measurements. The P-gp interacting properties were evaluated measuring the effects of thiolated- and unmodified-polymers on the bidirectional transport (BA/AB) of rhodamine-123 across Caco-2 cells as well as in the calcein-AM and ATPase activity assays. Although all the thiomers and unmodified polymers showed optimal-excellent mucoadhesive properties, the best mucoadhesive performances have been obtained by CS and CS-based thiomers. Moreover, it was found that the pretreatment of Caco-2 cell monolayer with GCS-NAC or GCS restores Rho-123 cell entrance by inhibiting P-gp activity. Hence, GCS-NAC and GCS may constitute new biomaterials useful for improving the bioavailability of P-gp substrates.

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.

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 multifactorial nature of chemotherapy failure in controlling cancer is often associated with the occurrence of multidrug resistance (MDR), a phenomenon likely related to the increased expression of members of the ATP binding cassette (ABC) transporter superfamily. In this respect, the most extensively characterized MDR transporters include ABCB1 (also known as MDR1 or P-glycoprotein) and ABCC1 (also known as MRP1) whose inhibition remains a priority to circumvent drug resistance. Herein, we report how the simple galloyl benzamide scaffold can be easily and properly decorated for the preparation of either MRP1 or P-gp selective inhibitors. In particular, some gallamides and pyrogallol-1-monomethyl ethers congeners showed remarkable affinity and selectivity toward MRP1 (e.g. 15g: IC50 = 9.50 μM, > 100 μM). On the other hand, trimethyl ether galloyl anilides, with few exceptions, exhibited moderate to very high and selective P-gp inhibition (e.g., 11g: IC50 = 0.2 μM, > 100μM).

Starting from the previously developed P-gp ligands 1a and 1b (EC50 ¼ 0.25 mM and 0.65 mM, respectively), new arylmethyloxyphenyl derivatives have been synthesized as P-gp modulators in order to investigate: (i) the effect of small electron-donor groups (OMe) (5e11), (ii) the effect of the replacement of methoxy groups with an electron-withdrawal substituent (Cl) on C-ring (13) (iii) the effect induced by the replacement of C-ring with heteroaromatic cycles such as thiophene and pyrimidine (13, 15, 16), (iv) the effect induced by molecular constriction on C ring (14, 17, 18) on P-gp modulating activity. The results demonstrated that P-gp inhibition potency is strongly correlated to the number of methoxy groups in the A-ring whereas the methoxylation of C-ring seems to poorly affect P-gp activity. The best result was found for compound 10 that displays a nanomolar affinity (EC50 ¼ 7.1 nM) towards P-gp pump and, in the meantime lacks of activity against MRP1 pump.

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

Background: Nor-BNI, GNTI and JDTic induce selective k opioid antagonism that is delayed and extremely prolonged, but some other effects are of rapid onset and brief duration. The transient effects of these compounds differ, suggesting that some of them may be mediated by other targets. Results: In binding assays, the three antagonists showed no detectable affinity (Ki$10 mM) for most non-opioid receptors and transporters (26 of 43 tested). There was no non-opioid target for which all three compounds shared detectable affinity, or for which any two shared sub-micromolar affinity. All three compounds showed low nanomolar affinity for k opioid receptors, with moderate selectivity over m and d (3 to 44-fold). Nor-BNI bound weakly to the a2C-adrenoceptor (Ki = 630 nM). GNTI enhanced calcium mobilization by noradrenaline at the a1A-adrenoceptor (EC50 = 41 nM), but did not activate the receptor, displace radioligands, or enhance PI hydrolysis. This suggests that it is a functionally-selective allosteric enhancer. GNTI was also a weak M1 receptor antagonist (KB = 3.7 mM). JDTic bound to the noradrenaline transporter (Ki = 54 nM), but only weakly inhibited transport (IC50 = 1.1 mM). JDTic also bound to the opioid-like receptor NOP (Ki = 12 nM), but gave little antagonism even at 30 mM. All three compounds exhibited rapid permeation and active efflux across Caco-2 cell monolayers. Conclusions: Across 43 non-opioid CNS targets, only GNTI exhibited a potent functional effect (allosteric enhancement of a1A-adrenoceptors). This may contribute to GNTI’s severe transient effects. Plasma concentrations of nor-BNI and GNTI may be high enough to affect some peripheral non-opioid targets. Nonetheless, k opioid antagonism persists for weeks or months after these transient effects dissipate. With an adequate pre-administration interval, our results therefore strengthen the evidence that nor-BNI, GNTI and JDTic are highly selective k opioid antagonists.

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.

Inhibition of drug efflux pumps such as P-glycoprotein (P-gp) is an approach toward combating multidrug resistance, which is a significant hurdle in current cancer treatments. To address this, N-substituted aryloxymethyl pyrrolidines were designed and synthesized in their homochiral forms in order to investigate the stereochemical requirements for the binding site of Pgp. Our study provides evidence that the chiral property of molecules could be a strategy for improving the capacity for interacting with P-gp, as the most active compounds of the series stereoselectively modulated this efflux pump. The naphthalene- 1-yl analogue (R)-2-[(2,3-dichlorophenoxy)methyl]-1- (naphthalen-1-ylmethyl)pyrrolidine) [(R)-7a] emerged foremost for its potency and stereoselectivity toward P-gp, with the S enantiomer being nearly inactive. The modulation of P-gp by (R)-7a involved consumption of ATP, thus demonstrating that the compound behaves as a P-gp substrate.

Gastrin-releasing peptide receptors (GRP-Rs, also known as bombesin 2 receptors) are overexpressed in a variety of human cancers, including prostate cancer, and therefore they represent a promising target for in vivo imaging of tumors using positron emission tomography (PET). Structural modifications of the non-peptidic GRP-R antagonist PD-176252 ((S)-1a) led to the identification of the fluorinated analog (S)-3-(1H-indol-3-yl)-N-[1-[5-(2-fluoroethoxy)pyridin-2-yl]cyclohexylmethyl]-2-methyl-2-[3-(4-nitrophenyl)ureido]propionamide ((S)-1m) that showed high affinity and antagonistic properties for GRP-R. This antagonist was stable in rat plasma and towards microsomal oxidative metabolism in vitro. (S)-1m was successfully radiolabeled with fluorine-18 through a conventional radiochemistry procedure. [18F](S)-1m showed high affinity and displaceable interaction for GRP-Rs in PC3 cells in vitro.

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.

The first synthesis of m-hydroxymexiletine (MHM) has been accomplished. MHM displayed hNav1.5 sodium channel blocking activity, and tests indicate it to be ∼2-fold more potent than the parent mexiletine and to have more favorable toxicological properties than mexiletine. Thus, MHM and possible related prodrugs might be studied as agents for the treatment of arrhythmias, neuropathic pain, and myotonias in substitution of mexiletine (metabolite switch), which has turned out to be tainted with common toxicity.

Four mexiletine analogues have been tested for their antiarrhythmic, inotropic, and chronotropic effects on isolated guinea pig heart tissues and to assess calcium antagonist activity, in comparison with the parent compound mexiletine. All analogues showed from moderate to high antiarrhythmic activity. In particular, three of them (1b,c,e) were more active and potent than the reference drug, while exhibiting only modest or no negative inotropic and chronotropic effects and vasorelaxant activity, thus showing high selectivity of action. All compounds showed no cytotoxicity and 1b,c,d did not impair motor coordination. All in, these new analogues exhibit an interesting cardiovascular profile and deserve further investigation.

In the search for a novel serotonin 7 (5-HT7) receptor PET radioligand we synthesized and evaluated a new series of biphenylpiperazine derivatives in vitro. Among the studied compounds, (R)-1-[4-[2-(4-methoxyphenyl)phenyl]piperazin-1-yl]-3-(2-pyrazinyloxy)-2-propanol ((R)-16), showed the best combination of affinity, selectivity, and lipophilicity, and was thus chosen for carbon-11 labelling and evaluation in pigs. After intravenous injection, [(11)C](R)-16 entered the pig brain and displayed reversible tracer kinetics. Pretreatment with the 5-HT7 receptor selective antagonist SB-269970 (1) resulted in limited decrease in the binding of [(11)C](R)-16, suggesting that this radioligand is not optimal for imaging the brain 5-HT7 receptor in vivo but it may serve as a lead compound for the design of novel 5-HT7 receptor PET radioligands.

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.

Copper is an essential trace element for the human body since it is a cofactor of several enzymes and proteins and plays a pivotal role in several biological functions (e.g., respiration, protection from oxidative damage, iron metabolism, etc.), also including the central nervous system development and functioning (e.g., synthesis of neurotransmitters, myelination, activation of neuropeptides, etc.). Therefore, copper dysmetabolism is associated with different toxic effects, mainly represented by oxidative stress, and it has been reported in many neurodegenerative disorders, such as Wilson's disease, Menkes disease, Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. This paper shows a detailed report of how copper is involved in the pathophysiology of these diseases. Moreover, a hint on novel therapeutic approaches based on restoring copper homeostasis through metal chelators will be pointed out.

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.

The P-glicoprotein (P-gp) inhibitor MC18 has been recently proposed as a valuable PET tracer to measure P-gp expression in vivo. The aim of this study was to evaluate the toxic hazard towards the vasculature of MC18 along with the structurally related and more potent P-gp inhibitor MC70. Their effects on A7r5 and EA.hy926 cells viability, on the mechanical activity of fresh and cultured rat aorta rings as well as on Cav 1.2 channel current (ICa1.2 ) of A7r5 cells were studied. At concentrations >10 μM, MC18 and MC70 decreased cell viability causing evident morphological changes. In fresh rat aorta rings, both compounds (0.1-100 μM) antagonized phenylephrine-induced contraction in a concentration-dependent manner, with IC50 values in the range of 1.67-14.49 μM, whereas only MC18 caused a concentration-dependent decrease of the 60 mM K(+) (K60)-induced responses. In rings cultured for 7 days with both compounds (1-10 μM), 10 μM MC70 significantly reduced, while 10 μM MC18 completely prevented the contractile response to both phenylephrine and K60. MC18 and MC70 (0.1-100 μM) inhibited ICa1.2 in a concentration-dependent manner with IC50 values of 16.81 and 32.13 μM, respectively. These findings demonstrate that MC18-induced vascular effects took place at concentrations that are at least three orders of magnitude higher than those (≤10 nM) allowing in vivo measurement of P-gp expression. Thus, MC18, and possibly MC70, can be considered promising PET tools for in vivo P-gp quantification.

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.

produzione di prodotti chimici organici di base per uso industriale; -attivita' di ricerca biomolecolare in ambito industriale;