DESIPRAMINE BDBM50010869
DMI [3-(10,11-Dihydro-dibenzo[b,f]azepin-5-yl)-propyl]-methyl-amine; hydrochloride Desipramine JB-8181 G-35020 desipramine HCl Norpramin EX-4355 CHEMBL1696 DESIPRAMINE HYDROCHLORIDE BDBM50028289 Pertofrane RMI-9384A
Desipramin DMI monodemethylimipramine Desipramine US9944618, Compound ID No. 174 demethylimipramine norimipramine 3-(10,11-dihydro-5H-dibenzo[b,f]azepin-5-yl)-N-methylpropan-1-amine 5-(gamma-methylaminopropyl)iminodibenzyl N-(3-methylaminopropyl)iminobibenzyl BDBM35229 DESIPRAMINE HYDROCHLORIDE CHEMBL72 Desmethylimipramine
- ChEMBL_1490338 (CHEMBL3532834) Inhibition of CYP2D6 (unknown origin) assessed as desipramine oxidation
- Radioligand Binding Assay (Ki) Compounds were evaluated the inhibition of [3H] nisoxetine binding to MDCK-Net6 cells, stably transfected with the human norepinephrine transporter (hNET). Data from wells containing 1 uM desipramine were used to define non-specific hNET binding. Total radioligand bound is defined by addition of binding buffer alone in the presence of [3H]nisoxetine (Perkin-Elmer). The radioligand binding reaction was initiated by addition of [3H]nisoxetine, and incubated for 2 h at 37 deg C. The KD value estimated for [3H]nisoxetine was 10 nM using intact whole cells. The inhibition constant (Ki) was calculated by the Cheng and Prusoff equation.
- Radioligand Binding Assay (Ki) and Norepinephrine Uptake Assay (IC50) Compounds were evaluated the inhibition of [3H] nisoxetine binding to MDCK-Net6 cells, stably transfected with the human norepinephrine transporter (hNET). Data from wells containing 1 uM desipramine were used to define non-specific hNET binding. Total radioligand bound is defined by addition of binding buffer alone in the presence of [3H]nisoxetine (Perkin-Elmer). The radioligand binding reaction was initiated by addition of [3H]nisoxetine, and incubated for 2 h at 37 deg C. The KD value estimated for [3H]nisoxetine was 10 nM using intact whole cells. The inhibition constant (Ki) was calculated by the Cheng and Prusoff equation. IC50 Values were obtained from inhibition of norepinephrine uptake in MDCK-Net6 cells, stably transfected with the human NET.
- Binding Assay Human norepinephrine transporter (NET) enriched membranes (5 μg) were incubated with 5 nM of radiolabeled [3H]-Nisoxetin in assay buffer containing 50 mM Tris-HCl, 120 mM NaCl, 5 mM KCl, pH 7.4.NSB (non specific binding) was measured by adding 10 μM desipramine. The binding of the test compound was measured in five different concentrations. After 60 min incubation at 4° C., binding reaction was terminated by filtering through Multiscreen GF/C (Millipore) presoaked in 0.5% polyethyleneimine in Vacuum Manifold Station, followed by 3 washes with ice-cold filtration buffer containing 50 mM Tris-HCl, 0.9% NaCl, pH 7.4.Filter plates were dried at 60° C. for 1 hour and 30 μl of scintillation cocktail were added to each well before radioactivity reading.
- Binding Assay Human norepinephrine transporter (NET) enriched membranes (5 μg) were incubated with 5 nM of radiolabeled [3H]-Nisoxetin in assay buffer containing 50 mM Tris-HCl, 120 mM NaCl, 5 mM KCl, pH 7.4.NSB (non specific binding) was measured by adding desipramine 10 μM. The binding of the test compound was measured at five different concentrations. After 60 min incubation at 4° C., binding reaction was terminated by filtering through Multiscreen GF/C (Millipore) presoaked in 0.5% polyethyleneimine in Vacuum Manifold Station, followed by 3 washes with ice-cold filtration buffer containing 50 mM Tris-HCl, 0.9% NaCl, pH 7.4.Filter plates were dried at 60° C. for 1 h and 30 μl of scintillation cocktail were added to each well before radioactivity reading.
- Norepinephrine Transporter Binding Assay 1. Each tube receives the following components: 25 ul drug or vehicle 25 ul [3H]-Nisoxetine 200 ul tissue suspension. 2. Initiate binding reaction with the addition of tissue, and incubate at room temperature for 1 hour. 3. Terminate binding reaction by rapid filtration of tube contents onto 0.1% PEI treated GF/C filters (TopCount). 4. Rinse the assay tubes once with ice cold 50 mM NaCl, then rapidly rinse the filters with 6×1 ml/tube of the same wash buffer. 5. Radioactivity trapped onto the filters is assessed using liquid scintillation spectrophotometer after soaking the filters for at least 1 hour in scintillation cocktail. Materials and Reagents 1. The receptor source is human recombinant/CHO. 2. [3H]-Nisoxetine, diluted to a concentration of 10 nM in assay buffer, is used as the radioligand. Thus, the final ligand concentration is 1 nM. 3. Non specific binding is defined as that remaining in the presence of 1×10−6M desipramine (MW=302.8) (Make fresh: in bag with dessicating rocks 1, dissolves in water 1E-3). 4. The reference compound for the assay is desipramine. Desimpramine is run at following final concentrations: 1×10−10, 2×10−10, 5×10−10, 1×10−9, 2×10−9, 5×10−9, 1×10−8, 2×10−8, 5×10−8, 1×10−7, 2×10−7, 5×10−7M 5. The positive control is any of the above run at the final concentrations of: 1×10−9, 5×10−9, 2×10−8 M. 6. The Kd for the receptor is 3 nM. Buffers MW (g/mole) g/1 L Incubation Buffer: 50 mM Tris, pH 7.4 121 6.06 5 mM KCl 74.6 0.38 120 mM NaCl 58.4 7.02 Wash Buffer: 50 mM NaCl 58.4 3.0
- Radioligand Binding Assay Compounds were dissolved in 100% DMSO at a concentration 100 times the desired highest assay concentration, serially diluted 1:3 in 100% DMSO, and 1.0 ul/well of each solution was dispensed to a Nunc polypropylene, round bottom, 384-well plate. 100% inhibition is defined with 1.0 ul/well of 10 mM desipramine dissolved in DMSO. 50 ul/well of a 2x membrane preparation (0.4 mg/ml in 50 mM Tris-Cl, pH 7.5, 120 mM NaCl, 5 mM KCl) and 50 ul/well of a 2x radioligand solution (4 nM [3H]nisoxetine in 50 mM Tris-Cl, pH 7.5, 120 mM NaCl, 5 mM KCl) were added to the well and the reaction incubated for 1 hour at room temperature. The contents of the assay plate were then transferred to a Millipore MultiscreenHTS GF/B filter plate which was pretreated with 0.5% PEI for at least one hour. The plate was vacuum filtered and washed with 7 washes of 100 ul/well 50 mM Tris-Cl, pH 7.5, 120 mM NaCl, 5 mM KCl chilled to 4 C.
- Radioligand Binding Assay Compounds were dissolved in 100% DMSO at a concentration 100 times the desired highest assay concentration, serially diluted 1:3 in 100% DMSO, and 1.0 ul/well of each solution was dispensed to a Nunc polypropylene, round bottom, 384-well plate. 100% inhibition is defined with 1.0 ul/well of 10 mM desipramine dissolved in DMSO. 50 ul/well of a 2x membrane preparation (0.4 mg/ml in 50 mM Tris-Cl, pH 7.5, 120 mM NaCl, 5 mM KCl) and 50 ul/well of a 2x radioligand solution (4 nM [3H]nisoxetine in 50 mM Tris-Cl, pH 7.5, 120 mM NaCl, 5 mM KCl) were added to the well and the reaction incubated for 1 hour at room temperature. The contents of the assay plate were then transferred to a Millipore MultiscreenHTS GF/B filter plate which was pretreated with 0.5% PEI for at least one hour. The plate was vacuum filtered and washed with 7 washes of 100 ul/well 50 mM Tris-Cl, pH 7.5, 120 mM NaCl, 5 mM KCl chilled to 4° C.
- Norepinephrine Transporter Binding Assay Brains from male Sprague-Dawley rats weighing 200-225 g (Taconic Labs, Germantown, N.Y.) were removed, frontal cortex dissected and rapidly frozen. Membranes were prepared by homogenizing tissues in 20 volumes (w/v) of 50 mM Tris containing 120 mM NaCl and 5 mM KCl, (pH 7.4 at 25° C.), using a Brinkman Polytron and centrifuged at 50,000xg for 10 min at 4° C. The resulting pellet was resuspended in buffer, recentrifuged and resuspended in buffer to a concentration of 80 mg/mL. Ligand binding experiments were conducted in assay tubes containing 0.5 mL buffer for 60 min at 0-4° C. Each tube contained 0.5 nM [3H]Nisoxetine (PerkinElmer Life Sciences, Boston, Mass.) and 8 mg frontal cortex tissue (original wet weight). Nonspecific binding was determined using 1 mM desipramine. Incubations were terminated by rapid filtration through Whatman GF/B filters, presoaked in 0.05% polyethylenimine, using a Brandel R48 filtering manifold (Brandel Instruments Gaithersburg, Md.). The filters were washed twice with 3 mL cold buffer and transferred to scintillation vials. Beckman Ready Value (3.0 mL) was added and the vials were counted using a Beckman 6000 liquid scintillation counter (Beckman Coulter Instruments, Fullerton, Calif.).
- NET Radioligand Binding Assay Compounds were dissolved in 100% DMSO at a concentration 100 times the desired highest assay concentration, serially diluted 1:3 in 100% DMSO, and 1.0 μl/well of each solution was dispensed to a Nunc polypropylene, round bottom, 384-well plate. 100% inhibition is defined with 1.0 μl/well of 10 mM desipramine dissolved in DMSO. 50 μl/well of a 2× membrane preparation (0.4 mg/ml in 50 mM Tris-Cl, pH 7.5, 120 mM NaCl, 5 mM KCl) and 50 μl/well of a 2× radioligand solution (4 nM [3H]nisoxetine in 50 mM Tris-Cl, pH 7.5, 120 mM NaCl, 5 mM KCl) were added to the well and the reaction incubated for 1 hour at room temperature. The contents of the assay plate were then transferred to a Millipore MultiscreenHTS GF/B filter plate which was pretreated with 0.5% PEI for at least one hour. The plate was vacuum filtered and washed with 7 washes of 100 μl/well 50 mM Tris-Cl, pH 7.5, 120 mM NaCl, 5 mM KCl chilled to 4° C. The filtration and washing were completed in less than 90 seconds. The plates were air-dried overnight, 12 μl/well of MicroScint scintillation fluid added, and the plates counted in a Trilux.
- Uptake Assay Protocol NET: This protocol was designed to measure inhibition of uptake by the human norepinephrine transporter. The reagents were human NET (HEK293F) cells, desipramine (Sigma), nomifensine, neurotransmitter transporter uptake assay kit (Molecular Devices), freestyle 293 expression medium (Invitrogen), 10× Hank's Balanced Salt Solution (HBSS; Invitrogen), 1 M HEPES (Mediatech), Biocoat poly-D-lysine 96-well, black, clear plates (Becton, Dickinson), and 500 μL polypropylene U-bottom 96-well plates (Fisher). The Assay Buffer (AB) was 1×HBSS and 0.02 M HEPES.The HEK293F cells were transfected with the human norepinephrine transporter and frozen in 1 mL aliquots at about 1E+07 cells/mL. On the day of the experiment, the cells were removed from −80° C. or liquid nitrogen and thawed in a room temperature water bath. The cells were dilute to about 1-2E+06 with Freestyle medium. A 1 mL sample (1:2 dilution) was prepared, and the cells were counted. The cells were spun at 1100 rpm for 5 minutes, and the medium was aspirated off. The cells were resuspend in medium at 1.5E+06 cells/mL for about 60,000 cells per well. 40 μL of cells were dispensed per well in the Biocoat plates. The plates were spun at 1100 rpm for 1 minute to improve homogeneity of the cell layer and were incubated at 37° C. for a minimum of 3 hours.
- Binding Assay Norepinephrine Transporter Binding Assay. Brains from male Sprague-Dawley rats weighing 200-225 g (Taconic Labs, Germantown, N.Y.) were removed, frontal cortex dissected and rapidly frozen. Membranes were prepared by homogenizing tissues in 20 volumes (w/v) of 50 mM Tris containing 120 mM NaCl and 5 mM KCl, (pH 7.4 at 25° C.), using a Brinkman Polytron and centrifuged at 50,000×g for 10 min at 4° C. The resulting pellet was resuspended in buffer, recentrifuged and resuspended in buffer to a concentration of 80 mg/mL. Ligand binding experiments were conducted in assay tubes containing 0.5 mL buffer for 60 min at 0-4° C. Each tube contained 0.5 nM [3H]Nisoxetine (PerkinElmer Life Sciences, Boston, Mass.) and 8 mg frontal cortex tissue (original wet weight). Nonspecific binding was determined using 1 mM desipramine. Incubations were terminated by rapid filtration through Whatman GF/B filters, presoaked in 0.05% polyethylenimine, using a Brandel R48 filtering manifold (Brandel Instruments Gaithersburg, Md.). The filters were washed twice with 3 mL cold buffer and transferred to scintillation vials. Beckman Ready Value (3.0 mL) was added and the vials were counted using a Beckman 6000 liquid scintillation counter (Beckman Coulter Instruments, Fullerton, Calif.). Each compound was tested with concentrations ranging from 0.01 nM to 100 mM for competition against binding of [3H]Nisoxetine, in at least three independent experiments, each performed in triplicate.
- Assay for Dopamine Reuptake Inhibition Uptake inhibition assay for the dopamine transporter was conducted in rat brain synaptosomes as described elsewhere with minor modifications (Rothman et al., Synapse 39, 32-41 (2001)). Freshly removed caudate was homogenized in 10% ice-cold sucrose with 12 strokes of a hand-held Potter-Elvehjem homogenizer followed by centrifugation at 1000×g for 10 min. The supernatants were saved on ice and used immediately. Transporter activity was assessed using 5 nM [3H]dopamine. The assay buffer was Krebs-phosphate buffer containing 154.4 mM NaCl, 2.9 mM KCl, 1.1 mM CaCl2, 0.83 mM MgCl2, 5 mM glucose, 1 mg/mL ascorbic acid, and 50 μM pargyline. The selectivity of the uptake assay for DAT was optimized by including 100 nM citalopram and 100 nM desipramine as blockers of SERT and NET in the sucrose solution and assay buffer. Uptake inhibition assays were conducted at 25° C. and were initiated by adding 100 μl of tissue to 900 μL assay buffer containing test drug and [3H]dopamine. Test drugs were diluted in assay buffer containing 1 mg/mL bovine serum albumin. Nonspecific uptake was measured by incubating in the presence of 10 μM indatraline. The reactions were stopped after 15 minutes by rapid vacuum filtration with a cell harvester (BRANDEL) over GF/B filters (Whatman) presoaked in wash buffer maintained at 25° C. (10 mM Tris-HCl, pH 7.4/150 mM NaCl). Filters were rinsed with 6 mL wash buffer and retained tritium was quantified by a MicroBeta liquid scintillation counter (PerkinElmer) after overnight extraction in 0.6 mL of liquid scintillation cocktail (Cytoscint, ICN). The data from three experiments were pooled and fit to a dose-response curve equation (using Kaleidagraph), to yield an Emax and EC50 value.
- Norepinephrine Transporter Binding Assay Norepinephrine Transporter Binding Assay. Brains from male Sprague-Dawley rats weighing 200-225 g (Taconic Labs, Germantown, N.Y.) were removed, frontal cortex dissected and rapidly frozen. Membranes were prepared by homogenizing tissues in 20 volumes (w/v) of 50 mM Tris containing 120 mM NaCl and 5 mM KCl, (pH 7.4 at 25° C.), using a Brinkman Polytron and centrifuged at 50,000×g for 10 min at 4° C. The resulting pellet was resuspended in buffer, recentrifuged and resuspended in buffer to a concentration of 80 mg/mL. Ligand binding experiments were conducted in assay tubes containing 0.5 mL buffer for 60 min at 0-4° C. Each tube contained 0.5 nM [3H]Nisoxetine (PerkinElmer Life Sciences, Boston, Mass.) and 8 mg frontal cortex tissue (original wet weight). Nonspecific binding was determined using 1 mM desipramine. Incubations were terminated by rapid filtration through Whatman GF/B filters, presoaked in 0.05% polyethylenimine, using a Brandel R48 filtering manifold (Brandel Instruments Gaithersburg, Md.). The filters were washed twice with 3 mL cold buffer and transferred to scintillation vials. Beckman Ready Value (3.0 mL) was added and the vials were counted using a Beckman 6000 liquid scintillation counter (Beckman Coulter Instruments, Fullerton, Calif.). Each compound was tested with concentrations ranging from 0.01 nM to 100 mM for competition against binding of [3H]Nisoxetine, in at least three independent experiments, each performed in triplicate. Data were analyzed by using GraphPad Prism software (San Diego, Calif.) [11; Zou et. al. J. Med. Chem. 2006, 49, 6391-6399]. The results of the in vitro assays, grouped by functionality into the amides and amines are presented in Tables 1 and 2, respectively. All compounds were tested as racemic mixtures.