Sigma 2 receptor expression levels in blood and bladder from healthy and bladder cancer cattle.

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.

Radiopharmaceuticals for assessing ABC transporters at the blood-brain barrier.

ABC transporters protect the brain by transporting neurotoxic compounds from the brain back into the blood. P-glycoprotein (P-gp) is the most investigated ABC (efflux) transporter, as it is implicated in neurodegenerative diseases such as Alzheimer's disease. Altered function of P-gp can be studied in vivo, using Positron Emission Tomography (PET). To date, several radiopharmaceuticals have been developed to image P-gp function in vivo. So far, attempts to image expression levels of P-gp using radiolabeled P-gp inhibitors have not been successful. Improved knowledge of compound behavior toward P-gp from in vitro studies should increase predictability of in vivo outcome.

Activatable fluorescent probes: a new concept in optical molecular imaging.

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 activatable fluorescent probes will be presented and their application will be discussed.

Sigma-2 receptor expression in bovine papillomavirus-associated urinary bladder tumours.

The expression of sigma-2 receptors was investigated in nine urothelial tumours of the urinary bladder of cattle. Each tumour was associated with the presence of DNA of bovine papillomavirus type-2 (BPV-2) and expression of the E5 viral oncoprotein. Five tumours were classified as low-grade carcinoma on the basis of morphological criteria and calculation of mean nuclear area (MNA) and mean nuclear perimeter (MNP). Four tumours were classified as high-grade carcinoma. Sigma-2 receptors were overexpressed in both types of carcinoma. In control normal bovine bladder tissue the density of receptors (expressed as the B(max)) was 0.37 pmol/mg of protein. Low-grade carcinomas had a mean B(max) of 1.37+/-0.32 pmol/mg of protein (range 1.03-1.86) and in high-grade carcinomas the mean B(max) was 10.9+/-2.8 pmol/mg of protein (range 8.2-14). The difference in B(max) between low- and high-grade carcinomas was statistically significant (P=0.0001).

trans-4-[4-(Methoxyphenyl)cyclohexyl]-1-arylpiperazines: a new class of potent and selective 5-HT(1A) receptor ligands as conformationally constrained analogues of 4-[3-(5-methoxy-1,2,3,4-tetrahydronaphthalen-1-yl)propyl]-1-arylpiperazines.

The present paper concerns the influence of conformational parameters on the recognition by rat 5-HT1A receptors of derivatives 4-[3-(5-methoxy-1,2,3,4-tetrahydronaphthalen-1-yl)propyl]-1-(2-pyridinyl)piperazine (1a) and 3-(5-methoxy-1,2,3,4-tetrahydronaphthalen-1-yl)-N-[2-(2-pyridyloxy)ethyl]propanamine (3b), two highly potent and selective 5-HT1A receptor ligands. Fifteen corresponding flexible and rigid analogues were prepared following several synthetic routes and were tested in binding assays with radioligands at 5-HT1A, D2, and alpha1 receptors from rat brain membranes. Among the new derivatives emerged trans-4-[4-(3-methoxyphenyl)cyclohexyl]-1-(2-pyridinyl)piperazine (trans-8a) and trans-N-[4-(3-methoxyphenyl)cyclohexyl]-2-(2-pyridyloxy)ethylamine (trans-8b). These compounds can be considered as conformationally constrained analogues of compounds 1a and 3a, respectively. In fact, compounds trans-8a and trans-8b showed a marked enhancement in 5-HT1A receptor affinity when compared to the corresponding cis isomers. Because compound trans-8a was a potent and selective 5-HT1A ligand (K(i), nM: 5-HT1A = 0.028, D2 = 2194, alpha1 = 767), it was chosen as a lead to prepare other analogues that were tested at 5-HT1A, D2, and alpha1 receptors from rat brain membranes, showing high affinity at the 5-HT1A and selectivity vs D2 and alpha1 receptors. Selected compounds were tested for their affinity at the human cloned 5-HT1A, alpha1a, alpha1b, alpha1d receptor subtypes. They were also submitted to the [35S]GTPgammaS binding assay stimulating the 5-HT1A receptor-mediated G-protein activation, therefore behaving as full or as partial agonists. Finally, the ability of iv administration of trans-8a to induce fore-paw treading in rats was evaluated in comparison with 8-OH-DPAT. Although the affinity (K(i)) and in vitro activity (pD'2) of trans-8a at the 5-HT1A receptor were higher than those of 8-OH-DPAT, the compound was less potent than the reference standard in inducing the symptom.

Determination of dopamine D(4) receptor density in rat striatum using PB12 as a probe.

N-[2-[4-(4-Chlorophenyl)piperazin-1-yl]ethyl]-3-methoxybenzamide (PB12), a potent and selective dopamine D(4) receptor ligand, was used as a probe for the direct determination of the dopamine D(4) receptor density in rat striatum as an alternative to the subtraction method. The experiment was performed using [(3)H]spiroperidol to label D(2), D(3) and D(4) receptors and PB12 to determine directly dopamine D(4) receptor specific binding. The determined B(max) value was 82 fmol/mg protein. The contribution of the dopamine D(4) receptor to the overall population of D(2)-like receptors was 63%; however, this value cannot be considered reliable because of the observed difference in the kinetic profiles of D(2), D(3) and D(4) receptors.

A multireceptorial binding reinvestigation on an extended class of sigma ligands: N-[omega-(indan-1-yl and tetralin-1-yl)alkyl] derivatives of 3,3-dimethylpiperidine reveal high affinities towards sigma1 and EBP sites.

New 1-[omega-(2,3-dihydro-1H-inden-1-yl)- and (2,3-dihydro-5-methoxy-1H-inden-1-yl)alkyl]- and 1-[omega-(1,2,3,4-tetrahydronaphthalen-1-yl)- and (6-methoxy- or 6-fluoro-1,2,3,4-tetrahydronaphthalen-1-yl)alkyl] derivatives of 3,3-dimethylpiperidine were synthesized, as homologous compounds of an existing series of sigma ligands, in order to carry out sigma receptor subtypes structure-affinity relationships. The new compounds and some of their related analogues, already reported, were tested in new multireceptorial radioligand binding assays. As reference compounds, the known sigma(1) ligands SA 4503, BD 1008 and NE 100 were also prepared and tested. All reported compounds showed high sigma(1) affinity assayed by (+)-[(3)H]-pentazocine on guinea-pig brain (apparent K(i)=1.75-72.2 nM) and moderate or low sigma(2) affinity by [(3)H]-DTG on rat liver, in contrast with previous results. One tertiary amine function spaced by a five-membered chain from a phenyl group is the structural feature shared by the most active compounds 26 and 43 and some reference sigma(1) ligands. The reported sigma(1) ligands, including reference compounds, also demonstrated a high affinity towards EBP (Delta(8)-Delta(7) sterol isomerase) site (apparent K(i)=0.48-14.8 nM) and some of them (37 and 44) were good ligands at L-type Ca(++) channel. 1-[4-(2,3-Dihydro-1H-inden-1-yl)butyl]-3,3-dimethylpiperidine (26) was the best mixed sigma(1) and EBP ligand (apparent K(i)=1.75 and 1.54 nM, respectively) with a good selectivity versus sigma(2) receptor (138- and 157-fold, respectively).

1-Substituted-4-[3-(1,2,3,4-tetrahydro-5- or 7-methoxynaphthalen-1-yl)propyl]piperazines: influence of the N-1 piperazine substituent on 5-HT1A receptor affinity and selectivity versus D2 and alpha1 receptors. Part 6.

In the present paper, we report the synthesis and the binding profiles on 5-HT1A, D2, and alpha1 receptors of 1-substituted-4-[3-(5- or 7-methoxy-1,2,3,4-tetrahydronaphthalen-1-yl)propyl]piperazine derivatives 19-32 and some related heteroalkyl derivatives 33-35. The results obtained are compared to those previously reported for the 1-phenyl, 1-(2-methoxyphenyl), 1-(2-pyridyl) analogues 2-9. The results pointed out the critical role of the group linked in the N-1 position of the piperazine in terms of 5-HT1A binding affinity. In fact, 1-cyclohexyl, 1-(3-benzisoxazolyl), 1-(benzothiazole-2-carbonyl), 1-(2-benzothiazolyl), 1-(2-quinolyl) substituted piperazines 21-30 displayed moderate or low 5-HT1A receptor affinity; on the contrary, 1-(3-benzisothiazolyl) and 1-(1-naphthalenyl) substituted piperazines 19, 20 and 32 displayed high 5-HT1A receptor affinity, the Ki values being in the subnanomolar range. Furthermore, compounds 19, 20 and 32 demonstrated better selectivity over alpha1 receptors than the reference compounds 2-9.

A structure-affinity relationship study on derivatives of N-[2-[4-(4-Chlorophenyl)piperazin-1-yl]ethyl]-3-methoxybenzamide, a high-affinity and selective D(4) receptor ligand.

N-[2-[4-(4-Chlorophenyl)piperazin-1-yl]ethyl]-3-methoxybenzamide (1), a high-affinity and selective dopamine D(4) receptor ligand, was chosen as a lead, and structural modifications were done on its amide bond and on its alkyl chain linking the benzamide moiety to the piperazine ring and by preparing some semirigid analogues. The binding profile at dopamine D(4) and dopamine D(2), serotonin 5-HT(1A), and adrenergic alpha(1) receptors of 16 new compounds was determined. From the results emerged that the modification of the amide bond and the elongation of the intermediate alkyl chain caused a decrease in dopamine D(4) receptor affinity. All prepared semirigid analogues displayed D(4) receptor affinity values in the same range of the opened counterparts.

1-aryl-4-[(5-methoxy-1,2,3, 4-tetrahydronaphthalen-1-yl)alkyl]piperazines and their analogues: influence of the stereochemistry of the tetrahydronaphthalen-1-yl nucleus on 5-HT1A receptor affinity and selectivity versus alpha1 and D2 receptors. 5.

Some 1-aryl-4-[(5-methoxy-1,2,3, 4-tetrahydronaphthalen-1-yl)-n-propyl]piperazines and their alkylamino and alkylamido analogues, previously studied as 5-HT1A ligands, were prepared in enantiomerically pure form, and their absolute configuration was determined by chemical correlation or by chiroptical properties. They were evaluated for in vitro 5-HT1A, D2, and alpha1 receptor affinity by radioligand binding assays, to study the influence of the chiral carbon atom of the tetrahydronaphthalene nucleus on the 5-HT1A affinity and selectivity. Results indicated that, as regarding the 5-HT1A receptor affinity, there was no difference in affinity between (-)- and (+)-enantiomers as well as the racemate of each compound. The stereochemistry, instead, influenced the selectivity: all (-)-enantiomers displayed affinity values higher than those of (+)-isomers at D2 receptors, and conversely, all (+)-enantiomers displayed affinity values higher than those of (-)-isomers at alpha1 receptors. As a result of this trend, it is not possible to predict the isomer with a better selectivity profile. However, compounds (S)-(+)-2, (S)-(+)-4, and (R)-(+)-6 displayed high affinity for the 5-HT1A receptor (IC50 values ranging between 7.0 and 2.3 nM) and good selectivity (>/=250-fold) versus both D2 and alpha1 receptors. Furthermore, compounds (S)-(+)-4 and (R)-(-)-4 were submitted to the [35S]GTPgammaS binding assay for a preliminary evaluation of their intrinsic activity on the 5-HT1A receptor.

N-[2-[4-(4-Chlorophenyl)piperazin-1-yl]ethyl]-3-methoxybenzamide: a potent and selective dopamine D4 ligand.

A series of new 1-aryl-4-alkylpiperazines containing a terminal benzamide fragment or a tetralin-1-yl nucleus on the alkyl chain were synthesized and tested for binding at cloned human dopamine D4 and D2 receptor subtypes. A SAFIR (structure-affinity relationship) study on this series is herein discussed. The most relevant D4 receptor affinities were displayed by N-[omega-[4-arylpiperazin-1-yl]alkyl]-methoxybenzamides (compounds 5, 16-20), their IC50 values ranging between 0.057 and 7.8 nM. Among these, N-[2-[4-(4-chlorophenyl)piperazin-1-yl]ethyl]-3-methoxybenzamide (17) emerged since it exhibited very high affinity for dopamine D4 receptor (IC50 = 0.057 nM) with selectivity of >10 000 for the D4 versus the D2 receptor; compound 17 was also selective versus serotonin 5-HT1A and adrenergic alpha1 receptors.

Structure-activity relationship studies on the 5-HT(1A) receptor affinity of 1-phenyl-4-[omega-(alpha- or beta-tetralinyl)alkyl]piperazines. 4.

The synthesis of 1-phenylpiperazines, linked in the alpha or beta position of the tetralin moiety on the terminal part of the N-4 alkyl chain, and their radioligand binding affinities for 5-HT(1A), 5-HT(2A), D-1, D-2, alpha1, and alpha2 receptors along with SAR studies on the 5-HT(1A) receptor are reported. Several changes have been carried out on previous structures of type 2, by inserting the alkyl chain with variable length in the alpha or beta position of the tetralin moiety and by changing the position of the methoxy group on the aromatic ring of the tetralin nucleus. The highest affinity (IC50 = 0.50 nM) and selectivity for the 5-HT(1A) receptor were showed by 1-phenylpiperazine 2a with a three-membered alkyl chain bearing a 5-methoxytetralin-1-yl ring in the omega position.

New sigma and 5-HT1A receptor ligands: omega-(tetralin-1-yl)-n-alkylamine derivatives.

Two series of compounds that are structurally related to benzomorphans, derived by structural modification of arylpiperazines with high 5-HT1A affinity and moderate sigma affinity, were prepared in order to increase sigma affinity and selectivity. All new compounds are N-substituted-omega-(1,2,3,4-tetrahydronaphthalen-1-yl)- or -omega-(1,2-dihydronaphthalen-4-yl)-n-alkylamines with, in some cases, a methoxy group on the tetralin moiety. They were tested in radioligand binding assays on sigma ([3H]DTG and [3H]-(+)-pentazocine), D-2 dopaminergic, 5-HT1A and 5-HT2 serotonergic, and PCP (phencyclidine) receptors. A first set of compounds bearing a 4-(1-substituted)piperazine moiety as terminal fragment on the alkyl chain showed moderate to high sigma affinity (Ki = 5.3-139 nM), the most active and selective being 1-cyclohexyl-4-[3-(5-methoxy-1,2,3,4-tetrahydronaphthalen-1-yl)-n- propyl ]piperazine (14), with probable pronounced sigma 2 affinity (Ki = 5.3 nM on [3H]DTG and Ki = 71 nM on [3H]-(+)-pentazocine). Moreover, compound 13, a 1-benzylpiperazine analogue of 14, preserved a dual high 5-HT1A and sigma affinity (Ki = 3.6 nM on [3H]-5-HT and Ki = 7.0 nM on [3H]DTG). The second set of compounds includes some N-phenylalkyl derivatives of 3-(5-methoxy-1,2,3,4-tetrahydronaphthalen-1-yl)- n-propylamine that can be considered to be open-chain derivatives of 4-substituted-1-arylpiperazines. Among these compounds that had a lower activity toward sigma binding sites, a high 5-HT1A affinity was found for the N-(3-phenylpropyl) derivative 21 (Ki = 4.4 nM) which demonstrated very good selectivity.

High affinity and selectivity on 5-HT1A receptor of 1-aryl-4-[1-tetralin)alkyl]piperazines. 2.

Several 4-alkyl-1-arylpiperazines that present a tetralin moiety on the terminal part of the side chain were synthesized in order to increase the selectivity on the 5-HT1A versus D-2, alpha 1, sigma, and other 5-HT receptors. Many changes have been effected on previous structures of type 3(1-aryl-4-[3-(1,2-dihydronaphthalen-4-yl)-n-propyl]piperazines). Several synthetic procedures were followed to obtain the final products, depending on the presence or absence of a double bond, as well as of a heteroatom on the side chain. In the first case versatile use of Grignard reaction was made, whereas in the second one usual synthetic ways were applied. Final compounds were evaluated for in vitro activity on dopamine D-1 and D-2, serotonin 5-HT1A, 5-HT1B, 5-HT1C, and 5-HT2, alpha 1 adrenergic, and sigma receptors by radioreceptor binding assay. For the 2-MeO-Ph, 2-pyridyl, and unsubstituted phenyl N-piperazine derivatives, low IC50 values (0.3 nM) on 5-HT1A receptors and high selectivity values were observed.

Mixed 5-HT1A/D-2 activity of a new model of arylpiperazines: 1-aryl-4-[3-(1,2-dihydronaphthalen-4-yl)-n-propyl]piperazines. 1. Synthesis and structure-activity relationships.

A new model of 4-alkyl-1-arylpiperazines containing a terminal dihydronaphthalene fragment on the alkyl chain was synthesized in order to have mixed serotonergic and dopaminergic activity and to pursue the recent alternative approaches to the discovery of novel antipsychotic and anxiolytic agents. Title compounds were evaluated for in vitro activity on dopamine D-2 and serotonin 5-HT1A and 5-HT2 receptors by radioreceptor binding assays. They show high nanomolar affinity for 5-HT1A, moderate affinity for D-2, and low affinity for 5-HT2 receptors, and in particular, two compounds, 4-[3-(1,2-dihydro-6-methoxynaphthalen-4-yl)-n-propyl]-1-(2- methoxyphenyl)piperazine (8) and 4-[3-(1,2-dihydro-8-methoxynaphthalen-4-yl)-n-propyl]-1-(2- pyridyl)piperazine (15), show values (nM) of IC50 = 2.0 and 1.4 for 5-HT1A and IC50 = 90.6 and 119.3 for D-2, respectively. Some in vivo behavioral studies show compound 8 to be an antagonist on 5-HT1A receptors. These first findings place the new arylpiperazines on the same level as that of the azaspirone class, e.g., 1-(2-methoxyphenyl)-4-[4-(2-phthalimido)-n-butyl]piperazine (NAN-190) and buspirone.