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.

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.

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.

1-Cyclohexyl-4-[3-(5-methoxy-1,2,3,4-tetrahydronaphthalen-1-yl)propyl]piperazine 1 (PB28) represents an excellent lead candidate for therapeutic and/or diagnostic applications in oncology. However, because its utility is limited by its relatively high degree of lipophilicity, novel analogues of 1 with reduced lipophilic character were designed by substituting methylene groups with more polar functional groups in the propylene linker and at the tetralin C4 position. For the chiral analogues, separate enantiomers exhibited substantial and roughly equal affinities within a given receptor subtype, with the greatest difference observed for compound 9 at σ1 (7.5-fold; (-)-(S)-9 Ki=94.6 nM, (þ)-(R)-9 Ki=12.6 nM). Compound (-)-(S)-9 was also found to be the most σ2-selective agent (σ2 Ki=5.92 nM), to possess a lipophilicity consistent with entry into tumor cells (log D7.4 = 2.38), and to show minimal antiproliferative activity. However, (-)-(S)-9 exhibited moderate activity (EC50= 8.1 μM) at the P-gp efflux pump.

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).

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.

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.

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.

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.

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 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).

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

The mammalian bombesin receptor family comprises three G protein-coupled receptors: the neuromedin B receptor, the gastrin-releasing peptide receptor (BB2), and the bombesin receptor subtype 3. BB2 receptor plays a role in gastrointestinal functions; however, at present the role of this subtype in physiological and pathological conditions is unknown due to the lack of specific binders for all subclasses of bombesin receptors. Here, we present a study focused on the properties of the peptoid bombesin antagonist called PD176252, and other structural analogues with the aim to elucidate causes of their different affinity towards the BB2 receptor. By means of computational techniques, based on QSAR, docking and homology building, supported by experimental data (X-ray diffraction and NMR spectroscopy) fresh insights on binding modes of this class of biological targets were achieved.

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.

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.

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.

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.