BDBM50053408 Haloperidol Decanoate R-13672 Haldol Haldol Dec
CHEMBL545608 4-[4-(4-Chloro-phenyl)-4-hydroxy-piperidin-1-yl]-1-(4-fluoro-phenyl)-butan-1-one;propionate(HCl) 4-[4-(4-chlorophenyl)-4-hydroxypiperidin-1-yl]-1-(4-fluorophenyl)butan-1-one BDBM21398 Haloperidol CHEMBL54 Haloperidol, 1
- Sampson, D; Bricker, B; Zhu, XY; Peprah, K; Lamango, NS; Setola, V; Roth, BL; Ablordeppey, SY Further evaluation of the tropane analogs of haloperidol. Bioorg Med Chem Lett 24: 4294-7 (2014)
- Barbaraci, C; Giurdanella, G; Leotta, CG; Longo, A; Amata, E; Dichiara, M; Pasquinucci, L; Turnaturi, R; Prezzavento, O; Cacciatore, I; Zuccarello, E; Lupo, G; Pitari, GM; Anfuso, CD; Marrazzo, A Haloperidol Metabolite II Valproate Ester ( J Med Chem 64: 13622-13632 (2021)
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- De Voss, JJ; Sui, Z; DeCamp, DL; Salto, R; Babé, LM; Craik, CS; Ortiz de Montellano, PR Haloperidol-based irreversible inhibitors of the HIV-1 and HIV-2 proteases. J Med Chem 37: 665-73 (1994)
- Peprah, K; Zhu, XY; Eyunni, SV; Etukala, JR; Setola, V; Roth, BL; Ablordeppey, SY Structure-activity relationship studies of SYA 013, a homopiperazine analog of haloperidol. Bioorg Med Chem 20: 1671-8 (2012)
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- Sikazwe, DM; Li, S; Lyles-Eggleston, M; Ablordeppey, SY The acute EPS of haloperidol may be unrelated to its metabolic transformation to BCPP+. Bioorg Med Chem Lett 13: 3779-82 (2003)
- Kato, Y; Nakajima, M; Oda, S; Fukami, T; Yokoi, T Human UDP-glucuronosyltransferase isoforms involved in haloperidol glucuronidation and quantitative estimation of their contribution. Drug Metab Dispos 40: 240-8 (2012)
- Peprah, K; Zhu, XY; Eyunni, SV; Setola, V; Roth, BL; Ablordeppey, SY Multi-receptor drug design: Haloperidol as a scaffold for the design and synthesis of atypical antipsychotic agents. Bioorg Med Chem 20: 1291-7 (2012)
- Marrazzo, A; Fiorito, J; Zappalà, L; Prezzavento, O; Ronsisvalle, S; Pasquinucci, L; Scoto, GM; Bernardini, R; Ronsisvalle, G Antiproliferative activity of phenylbutyrate ester of haloperidol metabolite II [(±)-MRJF4] in prostate cancer cells. Eur J Med Chem 46: 433-8 (2010)
- Lyles-Eggleston, M; Altundas, R; Xia, J; Sikazwe, DM; Fan, P; Yang, Q; Li, S; Zhang, W; Zhu, X; Schmidt, AW; Vanase-Frawley, M; Shrihkande, A; Villalobos, A; Borne, RF; Ablordeppey, SY Design, synthesis, and evaluation of metabolism-based analogues of haloperidol incapable of forming MPP+-like species. J Med Chem 47: 497-508 (2004)
- Sozio, P; Fiorito, J; Di Giacomo, V; Di Stefano, A; Marinelli, L; Cacciatore, I; Cataldi, A; Pacella, S; Turkez, H; Parenti, C; Rescifina, A; Marrazzo, A Haloperidol metabolite II prodrug: asymmetric synthesis and biological evaluation on rat C6 glioma cells. Eur J Med Chem 90: 1-9 (2015)
- Sikazwe, DM; Li, S; Mardenborough, L; Cody, V; Roth, BL; Ablordeppey, SY Haloperidol: towards further understanding of the structural contributions of its pharmacophoric elements at D2-like receptors. Bioorg Med Chem Lett 14: 5739-42 (2004)
- Burt, DR; Creese, I; Snyder, SH Properties of [3H]haloperidol and [3H]dopamine binding associated with dopamine receptors in calf brain membranes. Mol Pharmacol 12: 800-12 (1976)
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- Salama, I; Löber, S; Hübner, H; Gmeiner, P Synthesis and binding profile of haloperidol-based bivalent ligands targeting dopamine D(2)-like receptors. Bioorg Med Chem Lett 24: 3753-6 (2014)
- Jaen, JC; Caprathe, BW; Pugsley, TA; Wise, LD; Akunne, H Evaluation of the effects of the enantiomers of reduced haloperidol, azaperol, and related 4-amino-1-arylbutanols on dopamine and sigma receptors. J Med Chem 36: 3929-36 (1994)
- Yamaguchi, K; Kazuta, Y; Hirano, K; Yamada, S; Matsuda, A; Shuto, S Synthesis of 1-arylpiperazyl-2-phenylcyclopropanes designed as antidopaminergic agents: cyclopropane-based conformationally restricted analogs of haloperidol. Bioorg Med Chem 16: 8875-81 (2008)
- Nakazono, M; Hasegawa, S; Yamamoto, T; Zaitsu, K Synthesis of 61-bis(1-adamantylcarbamoyl)-1,2-methano[60]fullerene and its antagonistic effect on haloperidol-induced catalepsy in mice. Bioorg Med Chem Lett 14: 5619-21 (2004)
- Seeman, P; Van Tol, HH Deriving the therapeutic concentrations for clozapine and haloperidol: the apparent dissociation constant of a neuroleptic at the dopamine D2 or D4 receptor varies with the affinity of the competing radioligand. Eur J Pharmacol 291: 59-66 (1995)
- Scherz, MW; Fialeix, M; Fischer, JB; Reddy, NL; Server, AC; Sonders, MS; Tester, BC; Weber, E; Wong, ST; Keana, JF Synthesis and structure-activity relationships of N,N'-di-o-tolylguanidine analogues, high-affinity ligands for the haloperidol-sensitive sigma receptor. J Med Chem 33: 2421-9 (1990)
- Ablordeppey, SY; Lyles-Eggleston, M; Bricker, B; Zhang, W; Zhu, X; Goodman, C; Roth, BL Evaluation of the eutomer of 4-{3-(4-chlorophenyl)-3-hydroxypyrrolidin-1-yl}-1-(4-fluorophenyl)butan-1-one, {(+)-SYA 09}, a pyrrolidine analog of haloperidol. Bioorg Med Chem Lett 16: 3219-23 (2006)
- ChEBML_58529 Displacement of [3H]haloperidol from Dopamine receptor D2 in rat brain
- ChEBML_61589 Inhibition of [3H]haloperidol binding for Dopamine receptor D2 in rat striatal membranes.
- ChEMBL_2197448 (CHEMBL5109964) Displacement of [3H]-haloperidol from rat striatal homogenate D2 receptor
- ChEMBL_59593 (CHEMBL672942) Inhibition of [3H]haloperidol binding to Dopamine receptor in calf caudate nuclei.
- ChEMBL_59869 (CHEMBL670610) Inhibition of [3H]haloperidol binding to dopamine receptors in rat striatal membranes.
- ChEMBL_62090 (CHEMBL674970) Affinity at dopamine D2 receptor, (For haloperidol Ki(nM)= 1.5+/-1.2)
- ChEBML_58225 Compound was evaluated for binding activity against [3H]haloperidol as radioligand for Dopamine receptor D2
- ChEMBL_59158 (CHEMBL671189) Inhibition of [3H]haloperidol binding for Dopamine receptor D2 in rat striatal membranes.
- ChEMBL_60173 (CHEMBL675873) Binding affinity against dopamine receptor using [3H]- haloperidol in rat brain
- ChEMBL_60175 (CHEMBL675875) Ability to inhibit [3H]haloperidol binding to dopamine receptor in rat striatal homogenate
- ChEMBL_61589 (CHEMBL675761) Inhibition of [3H]haloperidol binding for Dopamine receptor D2 in rat striatal membranes.
- ChEBML_58707 Compound was for its ability to displace [3H]haloperidol binding to rat striatal Dopamine receptor D2
- ChEMBL_58225 (CHEMBL858328) Compound was evaluated for binding activity against [3H]haloperidol as radioligand for Dopamine receptor D2
- ChEMBL_59872 (CHEMBL672998) Binding affinity against dopamine receptor in rat striatal membrane using [3H]haloperidol
- ChEMBL_60171 (CHEMBL675871) Binding affinity against dopamine receptor in rat striatal membrane using [3H]haloperidol
- ChEMBL_60172 (CHEMBL675872) Binding affinity for dopamine receptor in rat striatal membrane using [3H]haloperidol as radioligand
- ChEMBL_60174 (CHEMBL675874) Binding affinity against dopamine receptor in rat striatal membrane using [3H]haloperidol
- ChEBML_61425 Concentration inhibiting specific binding of [3H]haloperidol to Dopamine receptor D2 from rat striatal brain.
- ChEMBL_138661 (CHEMBL748804) Binding affinity against Muscarinic acetylcholine receptor in rat striatal membrane using [3H]haloperidol
- ChEMBL_226541 (CHEMBL846481) Binding affinity against sigma receptor in bovine cerebellum using 2.0 nM [3H]- haloperidol
- ChEMBL_58707 (CHEMBL672051) Compound was for its ability to displace [3H]haloperidol binding to rat striatal Dopamine receptor D2
- ChEMBL_60013 (CHEMBL675838) In vitro inhibition of [3H]haloperidol (HPD) binding to dopamine (DA) receptor of rat striatal membranes
- ChEBML_201295 Binding affinity towards sigma opioid receptor was determined in rat cerebral homogenate using [3H]haloperidol as radioligand
- ChEBML_61441 In vitro binding affinity against Dopamine receptor D2 by displacement of [3H]haloperidol from rat striatal membranes.
- ChEMBL_61425 (CHEMBL671399) Concentration inhibiting specific binding of [3H]haloperidol to Dopamine receptor D2 from rat striatal brain.
- ChEMBL_965431 (CHEMBL2395077) Displacement of [3H]Haloperidol from sigma 1 receptor in human jurkat cells after 4 hrs
- ChEBML_58715 Compound was tested for its inhibitory effect on the binding profile in Dopamine receptor D2 using [3H]haloperidol in rat brain.
- ChEBML_201133 Inhibitory concentration against radioligand [3H](+)-NAN binding to haloperidol-sensitive sigma binding site in whole guinea pig brain
- ChEMBL_201271 (CHEMBL804960) Binding affinity towards sigma opioid receptor was determined in guinea pig cerebral homogenate using [3H]haloperidol as radioligand
- ChEMBL_832035 (CHEMBL2065331) Binding affinity to VAChT in Sprague-Dawley rat cerebral membrane after 1 hr by gamma counting in presence of haloperidol
- ChEBML_201714 Binding affinity against sigma receptor in bovine cerebellum using 2.0 nM [3H]haloperidol in the presence of 25 nM unlabeled spiperone
- ChEMBL_201133 (CHEMBL872418) Inhibitory concentration against radioligand [3H](+)-NAN binding to haloperidol-sensitive sigma binding site in whole guinea pig brain
- ChEMBL_201135 (CHEMBL803876) Inhibitory concentration against radioligand [3H]DTG binding to haloperidol-sensitive sigma binding site in whole guinea pig brain
- ChEMBL_216103 (CHEMBL817692) Ability to displace [3H]haloperidol from rat striatal membranes, in order to measure its intrinsic affinity for the dopamine (DA) receptor
- ChEMBL_2314573 Displacement of [3H]di-o-tolylguanidine from rat liver membrane sigma 2 receptor assessed as inhibition constant in presence of haloperidol
- ChEMBL_61590 (CHEMBL675762) Inhibition of [3H]haloperidol binding for Dopamine receptor D2 in rat striatal membranes. (15% inhibition at 10 e-6 M)
- ChEMBL_61591 (CHEMBL675763) Inhibition of [3H]haloperidol binding for Dopamine receptor D2 in rat striatal membranes. (2% inhibition at 10 e-6 M)
- ChEMBL_61592 (CHEMBL675764) Inhibition of [3H]haloperidol binding for Dopamine receptor D2 in rat striatal membranes. (25% inhibition at 10 e-6 M)
- ChEMBL_61593 (CHEMBL675765) Inhibition of [3H]haloperidol binding for Dopamine receptor D2 in rat striatal membranes. (36%inhibition at 10 e-6 M)
- ChEMBL_61594 (CHEMBL675766) Inhibition of [3H]haloperidol binding for Dopamine receptor D2 in rat striatal membranes. (41% inhibition at 10 e-6 M)
- ChEMBL_62089 (CHEMBL670518) Affinity at dopamine D2 receptor from rat striatum using [3H]spiroperidol as radioligand (For haloperidol Ki(nM)= 1.5+/-1.2)
- ChEMBL_201134 (CHEMBL803875) Inhibitory concentration against radioligand [3H]3-PPP binding to haloperidol-sensitive sigma binding site in whole guinea pig brain
- ChEMBL_201714 (CHEMBL803751) Binding affinity against sigma receptor in bovine cerebellum using 2.0 nM [3H]haloperidol in the presence of 25 nM unlabeled spiperone
- ChEMBL_226542 (CHEMBL846482) Binding affinity against sigma receptor in bovine cerebellum using 2.0 nM [3H]haloperidol in the presence of 25 nM unlabeled spiperone
- ChEMBL_2303194 Displacement of [3H]-(+)-pentazocine from sigma1 receptor in guinea pig brain membrane assessed as inhibition constant measured for 150 mins in presence of haloperidol
- ChEMBL_2303212 Displacement of [3H]DTG from sigma 2 receptor in rat liver membranes assessed as inhibition constant presence of haloperidol by scintillation counting analysis
- ChEMBL_62234 (CHEMBL675745) Binding affinity measured at the Dopamine receptor D2 by the inhibition of [3H]methylspiperone binding to rat striatum using unlabeled haloperidol for nonspecific binding.
- ChEMBL_154091 (CHEMBL873407) Compound was tested for its binding affinity towards PCP receptor using [3H](+)-NANM in the presence of 5 uM haloperidol from rat brain
- ChEMBL_154225 (CHEMBL760115) Compound was tested for its binding affinity towards PCP receptor using [3H](+)-NANM in the presence of 5 uM haloperidol from rat brain
- ChEMBL_2134926 (CHEMBL4844536) Displacement of [3H]-haloperidol from human D2L receptor expressed in HEK293 cell membranes measured after 1 hr by microbeta counting method
- ChEMBL_2314570 Displacement of [3H](+)-pentazocine from sigma 1 receptor in guinea pig brain membranes assessed as inhibition constant incubated for 120 mins in presence of haloperidol
- ChEMBL_2303207 Displacement of [3H]DTG from sigma 2 receptor (unknown origin) assessed as inhibition constant in presence of haloperidol measured for 120 mins by scintillation counting analysis
- ChEMBL_60671 (CHEMBL671707) Binding affinity measured at the Dopamine receptor D4 by the inhibition of [3H]spiperone binding to human recombinant CHO cells using unlabeled haloperidol for nonspecific binding.
- ChEMBL_62095 (CHEMBL674975) Binding affinity (Ki) at Dopamine receptor D2 using rat caudate cells with nemonapride (0.075 nM) as a radioligand and 10 M haloperidol as a blank agent
- ChEMBL_940867 (CHEMBL2330486) Displacement of [3H]-(+)-pentazocine from sigma1 receptor in human jurkat cell membrane after 2 hrs by liquid scintillation counting analysis in presence of haloperidol
- ChEMBL_2260983 (CHEMBL5215994) Displacement of [3H] LSD from 5-HT6R in HEK293 cells assessed as inhibition constant incubated for 1 hr in presence of haloperidol by microbeta plate reader analysis
- Pharmacological Activity Assay Procedures employed by the PDSP as described in the NIMH-PDSP Assay Protocol Book, Version II. The standard drug used in both Sigma subtype assays is haloperidol.
- ChEMBL_2314577 Displacement of [3H]-(+)-pentazocine from human sigma1 receptor transfected in HEK293 cell membranes assessed as inhibition constant incubated for 120 mins in presence of haloperidol by microbeta scintillation counter analysis
- Receptor Binding Assay Binding assays were carried out on rat recombinant D3 receptors (Perkin-Elmer, Cat. No. 6110139) expressed in Sf9 cells using [3H]spiperone (0.44-1.49 nM) as ligand and haloperidol (10 μM) for determination of non-specific binding. The assay was performed according to the supplier's assay protocol (Cat. No.: 3110139).
- Radiolabel Binding Assay A stock concentration of 5 nM 3H-1,3-di-(2-tolyl)guanidine (3H-DTG) is prepared in 50 mM Tris-HCl, 10 mM MgCl2, 1 mM EDTA, pH 7.4 (Assay Buffer). Aliquots (50 μl) of radioligand are dispensed into the wells of a 96-well plate containing 100 μl of Assay Buffer. Duplicate 50-μl aliquots of the compound of the disclosure test and Haloperidol positive control reference compound serial dilutions are added.
- Competitive Radioligand Binding Assay The affinity of candidate sigma-2 ligand compounds at sigma-1 and sigma-2 receptors was also determined by displacement of different known labeled sigma-2 or sigma-1 ligands. Filtration assays were conducted according the previously published procedure (Xu, et al., 2005). Test compounds were dissolved in N,N-Dimethylformamide (DMF), dimethyl sulfoxide (DMSO) or ethanol and then diluted in 50 mM Tris-HCl pH 7.4 buffer containing 150 mM NaCl and 100 mM EDTA. Membrane homogenates were made from guinea pig brain for sigma-1 binding assay and rat liver for sigma-2 binding assay. Membrane homogenates were diluted with 50 mM Tris-HCl buffer, pH 8.0 and incubated at 25° C. in a total volume of 150 uL in 96 well plates with the radioligand and test compounds with concentrations ranging from 0.1 nM to 10 uM. After incubation was completed, the reactions were terminated by the addition of 150 uL of ice-cold wash buffer (10 mM Tris HCl, 150 mM NaCl, pH 7.4) using a 96 channel transfer pipette (Fisher Scientific, Pittsburgh, Pa.) and the samples harvested and filtered rapidly through 96 well fiber glass filter plate (Millipore, Billerica, Mass.) that had been presoaked with 100 uL of 50 mM Tris-HCl buffer. Each filter was washed four times with 200 uL of ice-cold wash buffer (10 mM Tris-HCl, 150 mM NaCl, pH 7.4). A Wallac 1450 MicroBeta liquid scintillation counter (Perkin Elmer, Boston, Mass.) was used to quantitate the bound radioactivity. The sigma-1 receptor binding assays were conducted using guinea pig brain membrane homogenates ( 300 ug protein) and 5 nM [3H](+)-pentazocine (34.9 Ci/mmol, Perkin Elmer, Boston, Mass.), incubation time was 90 min at room temperature. Nonspecific binding was determined from samples that contained 10 μM of cold haloperidol. The sigma-2 receptor binding assays were conducted using rat liver membrane homogenates ( 300 ug protein) and 2 nM sigma-2 highly selective radioligand [3H]RHM-1 only (no other blockers) (America Radiolabeled Chemicals Inc. St. Louis, Mo.), 10 nM [3H]DTG (58.1 Ci/mmol, Perkin Elmer, Boston, Mass.) or 10 nM [3H]Haloperidol (America Radiolabeled Chemicals Inc., St. Louis, Mo.) in the presence of 1 uM (+)-pentazocine to block sigma-1 sites, incubation times were 6 minutes for [3H]RHM-1, 120 min for [3H]DTG and [3H]haloperidol at room temperature. Nonspecific binding was determined from samples that contained 10 uM of cold haloperidol.
- Radioligand Binding Assay Dopamine, D2s: Radioligand: [3H]Spiperone (20-60 Ci/mmol) or [3H]-7-hydroxy DPAT, 1.0 nMControl Compound: Haloperidol or ChlorpromazineIncubation Conditions: The reactions were carried out in 50 mM TRIS-HCl (pH 7.4) containing 120 mM NaCl, 5 mM KCl, 5 mM MgCl2, 1 mM EDTA for 60 minutes at 25 C. The reaction was terminated by rapid vacuum filtration onto glass fiber filters. Radioactivity trapped onto the filters was determined and compared to control values in order to ascertain any interactions of test compounds with the cloned dopamine D2 short binding site
- Binding Assay Dopamine, D2S: Materials and Methods: Receptor Source: Human recombinant expressed in CHO cells Radioligand: [3H]Spiperone (20-60 Ci/mmol) Control Compound: Haloperidol Incubation Conditions: The reactions were carried out in 50 mM TRIS-HCl (pH 7.4) containing 120 mM NaCl, 5 mM KCl, 5 mM MgCl2, 1 mM EDTA for 60 minutes at 25 C. The reaction was terminated by rapid vacuum filtration onto glass fiber filters. Radioactivity trapped onto the filters was determined and compared to control values in order to ascertain any interactions of test compounds with the cloned dopamine D2 short binding site
- Radioligand Binding Assay, Dopamine, D2S Receptor Source: Human recombinant D2S expressed mammalian cellsRadioligand: [3H]Spiperone (20-60 Ci/mmol) or [3H]-7-hydroxy DPAT, 1.0 nMControl Compound: Haloperidol or ChlorpromazineIncubation Conditions: The reactions were carried out in 50 mM TRIS-HCl (pH 7.4) containing 120 mM NaCl, 5 mM KCl, 5 mM MgCl2, 1 mM EDTA for 60 minutes at 25 C. The reaction was terminated by rapid vacuum filtration onto glass fiber filters. Radioactivity trapped onto the filters was determined and compared to control values in order to ascertain any interactions of test compounds with the cloned dopamine-D2 short binding site.
- Receptor Assay Compounds were evaluated for σ-1 and σ-2 binding in rat brain homogenates. Twelve concentrations of each test ligand (0.001-1,000 nM) were incubated for 120 min at 25° C. in 50 mM Tris-HCl, pH 8.0 with 500 μg membrane protein, and 5 nM [3H](+)-pentazocine (for σ1 assays) or 3 nM [3H]DTG plus 300 nM (+)-pentazocine (for σ2 assays); non-specific binding was determined in the presence of 10 μM haloperidol. The assays were terminated with ice-cold 10 mM Tris-HCl, pH 8.0, followed by two washes through glass fiber filters that were pre-soaked for at least 30 min in 0.5% polyethyleneimine.
- Radioligand Binding Assay Radioligand binding assays for Sigma-1 receptors and Sigma-2 receptors were carried out by a commercial contract research organization. For Sigma-1 binding, various concentrations of test compounds from 100 μM to 1 nM were used to displace 8 nM [3H](+)pentazocine from endogenous receptors on Jurkat cell membranes (Ganapathy M E et al. 1991, J Pharmacol. Exp. Ther. 289:251-260). 10 μM Haloperidol was used to define non-specific binding. For Sigma-2 receptors various concentrations of test compounds from 100 μM to 1 nM were used to displace 5 nM [3H] 1,3-Di-(2-tolyl)guanidine from endogenous receptors on membranes from rat cerebral cortex in the presence of 300 nM (+)pentazocine to mask Sigma-1 receptors. (Bowen W D, et al. 1993, Mol. Neuropharmcol 3:117-126). 10 μM Haloperidol was used to define non-specific binding. Reactions were terminated by rapid filtration through Whatman GF/C filters using a Brandel 12R cell harvester followed by two washes with ice-cold buffer. Radioactivity on the dried filter discs was measured using a liquid scintillation analyzer (Tri-Carb 2900TR; PerkinElmer Life and Analytical Sciences). The displacement curves were plotted and the Ki values of the test ligands for the receptor subtypes were determined using GraphPad Prism (GraphPad Software Inc., San Diego, Calif.). The percentage specific binding was determined by dividing the difference between total bound (disintegrations per minute) and nonspecific bound (disintegrations per minute) by the total bound (disintegrations per minute).
- Binding Assay Receptor Source: Human recombinant expressed in CHO cells Radioligand: [3H]Spiperone (20-60 Ci/mmol) Control Compound: Haloperidol Incubation Conditions: The reactions were carried out in 50 mM TRIS-HCl (pH 7.4) containing 120 mM NaCl, 5 mM KCl, 5 mM MgCl2, 1 mM EDTA for 60 minutes at 25 C. The reaction was terminated by rapid vacuum filtration onto glass fiber filters. Radioactivity trapped onto the filters was determined and compared to control values in order to ascertain any interactions of test compounds with the cloned dopamine—D2 short binding site (Literature Reference: Jarvis, K. R. et al. Journal of Receptor Research 1993, 13(1-4), 573-590; Gundlach, A. L. et al. Life Sciences 1984, 35, 1981-1988.)
- Human Sigma-1 Receptor Radioligand Assay To investigate binding properties of test compounds to human Sigma-1 receptor, transfected HEK-293 membranes and [3H](+)-pentazocine (Perkin Elmer, NET-1056), as the radioligand, were used. The assay was carried out with 7 μg of membrane suspension, 5 nM of [3H](+)-pentazocine in either absence or presence of either buffer or 10 μM Haloperidol for total and non-specific binding, respectively. Binding buffer contained Tris-HCl 50 mM at pH 8. Plates were incubated at 37° C. for 120 minutes. After the incubation period, the reaction mix was then transferred to Multiscreen HTS, FC plates (Millipore), filtered and plates were washed 3 times with ice-cold 10 mM Tris-HCL (pH7.4). Filters were dried and counted at approximately 40% efficiency in a MicroBeta scintillation counter (Perkin-Elmer) using EcoScint liquid scintillation cocktail.
- Human Sigma1 Receptor Radioligand Assay To investigate binding properties of test compounds to human σ1 receptor, transfected HEK-293 membranes and [3H](+)-pentazocine (Perkin Elmer, NET-1056), as the radioligand, were used. The assay was carried out with 7 μg of membrane suspension, 5 nM of [3H](+)-pentazocine in either absence or presence of either buffer or 10 μM Haloperidol for total and non-specific binding, respectively. Binding buffer contained Tris-HCl 50 mM at pH 8. Plates were incubated at 37° C. for 120 minutes. After the incubation period, the reaction mix was then transferred to MultiScreen HTS, FC plates (Millipore), filtered and plates were washed 3 times with ice-cold 10 mM Tris-HCL (pH 7.4). Filters were dried and counted at approximately 40% efficiency in a MicroBeta scintillation counter (Perkin-Elmer) using EcoScint liquid scintillation cocktail.
- Radioligand Assay To investigate binding properties of test compounds to human σ1 receptor, transfected HEK-293 membranes and [3H](+)-pentazocine (Perkin Elmer, NET-1056), as the radioligand, were used. The assay was carried out with 7 μg of membrane suspension, 5 nM of [3H](+)-pentazocine in either absence or presence of either buffer or 10 μM Haloperidol for total and non-specific binding, respectively. Binding buffer contained Tris-HCl 50 mM at pH 8. Plates were incubated at 37° C. for 120 minutes. After the incubation period, the reaction mix was then transferred to MultiScreen HTS, FC plates (Millipore), filtered and plates were washed 3 times with ice-cold 10 mM Tris-HCL (pH7.4). Filters were dried and counted at approximately 40% efficiency in a MicroBeta scintillation counter (Perkin-Elmer) using EcoScint liquid scintillation cocktail.
- Receptor Radioligand Assay To investigate binding properties of sigma 1 receptor ligands to human sigma 1 receptor, transfected HEK-293 membranes and [3H](+)-pentazocine (Perkin Elmer, NET-1056), as the radioligand, were used. The assay was carried out with 7 μg of membrane suspension, 5 nM of [3H](+)-pentazocine in either absence or presence of either buffer or 10 μM Haloperidol for total and non-specific binding, respectively. Binding buffer contained Tris-HCl 50 mM at pH 8. Plates were incubated at 37° C. for 120 minutes. After the incubation period, the reaction mix was then transferred to MultiScreen HTS, FC plates (Millipore), filtered and plates were washed 3 times with ice-cold 10 mM TrisHCL (pH7.4). Filters were dried and counted at approximately 40% efficiency in a MicroBeta scintillation counter (Perkin-Elmer) using EcoScint liquid scintillation cocktail.
- Receptor Radioligand Assay To investigate binding properties of test compounds to human σ1 receptor, transfected HEK-293 membranes and [3H](+)-pentazocine (Perkin Elmer, NET-1056), as the radioligand, were used. The assay was carried out with 7 μg of membrane suspension, 5 nM of [3H](+)-pentazocine in either absence or presence of either buffer or 10 μM Haloperidol for total and non-specific binding, respectively. Binding buffer contained Tris-HCl 50 mM at pH 8. Plates were incubated at 37° C. for 120 minutes. After the incubation period, the reaction mix was then transferred to MultiScreen HTS, FC plates (Millipore), filtered and plates were washed 3 times with ice-cold 10 mM Tris-HCL (pH7.4). Filters were dried and counted at approximately 40% efficiency in a MicroBeta scintillation counter (Perkin-Elmer) using EcoScint liquid scintillation cocktail
- Competitive Radioligand Binding Assay Radioligand binding assays for Sigma-1 receptors and Sigma-2 receptors were carried out by a commercial contract research organization. For Sigma-1 binding, various concentrations of test compounds from 100 μM to 1 nM were used to displace 8 nM [3H](+)pentazocine from endogenous receptors on Jurkat cell membranes (Ganapathy M E et al. 1991, J Pharmacol. Exp. Ther. 289:251-260). 10 μM Haloperidol was used to define non-specific binding. For Sigma-2 receptors various concentrations of test compounds from 100 μM to 1 nM were used to displace 5 nM [3H] 1,3-Di-(2-tolyl)guanidine from endogenous receptors on membranes from rat cerebral cortex in the presence of 300 nM (+)pentazocine to mask Sigma-1 receptors. (Bowen W D, et al. 1993, Mol. Neuropharmcol 3:117-126). 10 μM Haloperidol was used to define non-specific binding. Reactions were terminated by rapid filtration through Whatman GF/C filters using a Brandel 12R cell harvester followed by two washes with ice-cold buffer. Radioactivity on the dried filter discs was measured using a liquid scintillation analyzer (Tri-Carb 2900TR; PerkinElmer Life and Analytical Sciences). The displacement curves were plotted and the Ki values of the test ligands for the receptor subtypes were determined using GraphPad Prism (GraphPad Software Inc., San Diego, Calif.). The percentage specific binding was determined by dividing the difference between total bound (disintegrations per minute) and nonspecific bound (disintegrations per minute) by the total bound (disintegrations per minute).Affinities for Sigma-1 and Sigma-2 receptors are typically obtained from published studies using cerebral tissue homogenates with [3H](+)pentazocine to measure displacement from Sigma-1 receptors and [3H] 1,3-Di-(2-tolyl)guanidine in the presence of 300 nM (+)pentazocine to measure displacement from Sigma-2 receptors.
- D1R Functional Assay The [35S]GTPγS binding assay was performed at 30 °C for 30 min containing 10 μgof membrane protein in a final volume of 100 μL with various concentration of the compound. The antagonism effects of the compounds were tested in the existence of10 μm haloperidol for the D1R. The binding buffer contains 50 mm Tris, pH 7.5, 5 mm MgCl2, 1 mm ethylenediamine tetraacetic acid (EDTA), 100 mm NaCl, 1 mm DL-dithiothreitol, and 40 μm guanosine triphosphate. The reaction was initiated by the addition of [35S]GTPγS (final concentration of 0.1 nm). Non-specificbinding was measured in the presence of 100 μm 50-guanylimidodiphosphate (Gpp(NH)p). The reaction was terminated by the addition of 1 mL of ice-coldwashing buffer (50 mm Tris, pH 7.5, 5 mm MgCl2, 1 mm EDTA, and 100 mm NaCl) and was rapidly filtered with GF/C glass fiber filters (Whatman) and washed three times. Radioactivity was determined by liquid scintillation counting. Again, l-SPDwas used as the control
- Binding Affinity Assay Human-cloned dopamine D2L receptors stably expressed in C6 rat glioma cells (kindly donated by Professor Roberto Maggio, Università di L'Aquila, Italy) were radiolabeled with [3H]spiroperidol according to Scarselli et al. with minor modifications [Scarselli et al., J. Biol. Chem., 276:30308-14]. The incubation buffer (120mM NaCl, 5.0mM KCl, 5.0mM MgCl2, 1mM EDTA, 50mM Tris-HCl, pH 7.4) contained 100 μg of dopamine D2L receptor membranes, 0.30-0.50nM [3H]spiroperidol (Kd = 0.093 nM) and six to nine concentrations of drug solution in a final volume of 500 μL. The samples were incubated for 120 min at 25 °C, then the incubation was stopped by rapid filtration through Whatman GF/C glass fiber filters (presoaked in 0.5% polyethylenimine for 60 min). The filters were washed three times in 1mL of ice-cold 50mM Tris, 0.9% NaCl, pH 7.4. Nonspecific binding was determined in the presence of 10 μM haloperidol. The radioactivity bound to the filters was measured by liquid scintillation using a LS6500 Multi-Purpose scintillation Counter, Beckman.
- Radioligand Assay To investigate binding properties of test compounds to human σ1 receptor, transfected HEK-293 membranes and [3H](+) -pentazocine (Perkin Elmer, NET-1056), as the radioligand, were used. The assay was carried out with 7 μg of membrane suspension, 5 nM of [3H](+)-pentazocine in either absence or presence of either buffer or 10 μM Haloperidol for total and non-specific binding, respectively. Binding buffer contained Tris-HCl 50 mM at pH 8. Plates were incubated at 37° C. for 120 minutes. After the incubation period, the reaction mix was then transferred to Multiscreen HTS, FC plates (Millipore), filtered and plates were washed 3 times with ice-cold 10 mM Tris-HCL (pH 7.4). Filters were dried and counted at approximately 40% efficiency in a MicroBeta scintillation counter (Perkin-Elmer) using EcoScint liquid scintillation cocktail Results: As this invention is aimed at providing a compound or a chemically related series of compounds which act as ligands of the σ1 receptor it is a very preferred embodiment in which the compounds are selected which act as ligands of the σ1 receptor and especially compounds which have a binding expressed as Ki which is preferably <1000 nM, more preferably <500 nM, even more preferably <100 nM.
- Receptor Binding Assays Receptor binding assays were performed by the Psychoactive Drug Screening Program (PDSP) at Chapel Hill, N.C. The assay protocol book can be accessed free of charge at: website pdsp.med.unc.edu/PDSP %20Protocols %20II %202013-03-28.pdf, which is incorporated by reference in it's entirety for all purposes. Briefly, Sig1Rs and Sig2Rs were sourced from homogenates of Guinea pig brains and rat livers, respectively. Assessment of Sig1R binding affinity was determined via competition binding assays with the radioligand [3H]-(+)-pentazocine. Sig2R binding affinity was determined through competition binding assays using the radioligand [3H]-ditolylguanidine in the presence of (+)-pentazocine to block Sig1R binding sites. For primary binding results, non-specific binding in the presence of 10 mM is set as 100% inhibition; total binding in the absence of haloperidol is set to 0% inhibition. The radioactivity in the presence of the test compound is calculated with the following equation and expressed as a percent inhibition: % inhibition=(sample CPM non-specific CPM)/Total CPM−non-specific CPM)×100. The normalization process is carried out in Prism or Excel. To determine secondary binding results, CPM/well are pooled and fitted to a three parameter logistic function for competition binding in Prism v 5.0 to determine IC50 values, which are converted to Ki according to the Cheng-Prusoff equation.
- Binding Assay Brain membrane preparation and binding assays for the σ1-receptor were performed as described (DeHaven-Hudkins, D. L., L. C. Fleissner, and F. Y. Ford-Rice, 1992, Characterization of the binding of [3H](+)pentazocine to a recognition sites in guinea pig brain, Eur. J. Pharmacol. 227, 371-378) with some modifications. Guinea pig brains were homogenized in 10 vols. (w/v) of Tris-HCl 50 mM 0.32 M sucrose, pH 7.4, with a Kinematica Polytron PT 3000 at 15000 r.p.m. for 30 s. The homogenate was centrifuged at 1000 g for 10 min at 4° C. and the supernatants collected and centrifuged again at 48000 g for 15 min at 4° C. The pellet was resuspended in 10 volumes of Tris-HCl buffer (50 mM, pH 7.4), incubated at 37° C. for 30 min, and centrifuged at 48000 g for 20 min at 4° C. Following this, the pellet was re-suspended in fresh Tris-HCl buffer (50 mM, pH 7.4) and stored on ice until use.The radioligand used was [3H]-(+)-pentazocine at 5.0 nM and the final volume was 200 μl. The incubation was initiated with the addition of 100 μl of membrane at a final tissue concentration of approximately 5 mg tissue net weight/mL and the incubation time was 150 m. at 37° C. After incubation, the membranes were collected onto pretreated glass fiber filterplate (MultiScreen-FC, Millipore), with polyethylenimine 0.1%. The filters were washed two times with 200 μl of washing buffer (50 mM Tris Cl, pH=7.4) and then 25 μl of Ecoscint H liquid scintillation cocktail were added. Microplates were allowed to set for several hours and then quantified by liquid scintillation spectrophotometry (1450 Microbeta, Wallac). Nonspecific binding was determined with 1 μM haloperidol.
- Binding Assay Brain membrane preparation and binding assays for the 61-receptor were performed as described (DeHaven-Hudkins, D. L., L. C. Fleissner, and F. Y. Ford-Rice, 1992, Characterization of the binding of [3H](+)pentazocine to a recognition sites in guinea pig brain, Eur. J. Pharmacol. 227, 371-378) with some modifications. Guinea pig brains were homogenized in 10 vols. (w/v) of Tris-HCl 50 mM 0.32 M sucrose, pH 7.4, with a Kinematica Polytron PT 3000 at 15000 r.p.m. for 30 s. The homogenate was centrifuged at 1000 g for 10 min at 4° C. and the supernatants collected and centrifuged again at 48000 g for 15 min at 4° C. The pellet was resuspended in 10 volumes of Tris-HCl buffer (50 mM, pH 7.4), incubated at 37° C. for 30 min, and centrifuged at 48000 g for 20 min at 4° C. Following this, the pellet was re-suspended in fresh Tris-HCl buffer (50 mM, pH 7.4) and stored on ice until use.The radioligand used was [3H]-(+)-pentazocine at 5.0 nM and the final volume was 200 μl. The incubation was initiated with the addition of 100 μl of membrane at a final tissue concentration of approximately 5 mg tissue net weight/mL and the incubation time was 150 m. at 37° C. After incubation, the membranes were collected onto pretreated glass fiber filterplate (MultiScreen-FC, Millipore), with polyethylenimine 0.1%. The filters were washed two times with 200 μl of washing buffer (50 mM Tris Cl, pH=7.4) and then 25 μl of Ecoscint H liquid scintillation cocktail were added. Microplates were allowed to set for several hours and then quantified by liquid scintillation spectrophotometry (1450 Microbeta, Wallac). Nonspecific binding was determined with 1 μM haloperidol.
- Binding Assay to Sigma Receptors The σ binding assays were performed according to Ganapathy et al. (Ganapathy, M. E.; Prasad, P. D.; Huang, W.; Seth, P.; Leibach, F. H.; Ganapathy, V. Molecular and ligand-binding characterization of the sigma-receptor in the Jurkat human T lymphocyte cell line. J. Pharmacol. Exp. Ther. 1999, 289, 251-260). The σ1 binding assay was carried out by incubating Jurkat cell membranes (10-20 mg protein per tube) with [3H](+)-pentazocine (15 nM) and a range of concentrations of tested compounds, at 37° C. for 2 hours, in 5 mM Tris/HCl buffer (pH=7.4). The σ2 binding assay was performed by incubating Jurkat cell membranes (10-20 mg protein per tube) with [3H]-DTG (25 nM) in presence of (+)-pentazocine (1 μM) to saturate σ1 receptors, and a range of concentrations of tested compounds, at room temperature for 1 hour in 5 mM Tris/HCl buffer (pH=7.4). The final assay volume was 0.5 mL. Binding was terminated by rapid filtration through Wathman GF/B filters, which were then washed with 5×1 mL ice-cold NaCl solution and allowed to dry before bound radioactivity was measured using liquid scintillation counting. Nonspecific binding was determined, in both assays, under similar conditions, but in presence of 10 μM unlabeled haloperidol. Inhibition constants (Ki) were calculated from the IC50 values according to the method of Cheng and Prusoff (Cheng, Y.; Prusoff, W. H. Relationship between the inhibition constant (Ki) and the concentration of inhibitor which causes 50 percent inhibition (IC50) of an enzymatic reaction. Biochem Pharmacol. 1973, 22 (23), 3099-108):K i = IC 50 1 + L K dWhere IC50=Inhibitory concentration at 50% L=Concentration of radioligand Kd=Affinity constant of radioligandThe σ1 binding assay was carried out with [3H](+)-pentazocine (L=15 nM, Kd=16 nM) as radioligand and the a binding assay with [3H]-DTG (L=25 nM, Kd=80.84 nM).