D4 Receptor; 5-HT1A Receptor (pIC50 = 8.87); 5-HT1A Receptor (pA2 = 9.71)

In HEK 293 cells that consistently express dopamine D2L or D4.4 receptors, the functional characteristics and binding affinities of WAY-100635 are assessed[1]. WAY-100635 exhibits binding affinities at D2L, D3, and D4. 2 receptors of 940, 370, and 16 nM, respectively. The Kd of [3H] WAY-100635 at D4.2 receptors is 2.4 nM, as shown by saturation analyses. WAY-100635 has an EC50 of 9.7 nM, making it a strong agonist in HEK-D4.4 cells. WAY-100635 has a strong 3.3 nM affinity for the D4.4 receptor [1].

Treatment with WAY-100635 (1 mg/kg; subcutaneous injection; male Sprague-Dawley rats) eliminates the reduction in abstinence signs severity brought on by administration of Rhodiola rosea in nicotine-dependent rats[2].

Screening assays[1]
For the initial screens by the NIMH-PDSP at a large number of cloned receptors and transporters (for details, see Roth et al. (2002) and Shapiro et al. (2003)), 10 μM WAY-100635 was used. Where significant inhibition was measured (>50% inhibition with quadruplicate determinations), K i determinations were performed with 6–10 concentrations of unlabelled ligand, and data were analyzed with GraphPad Prism.

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Saturation binding experiments[1]
Whole cell pellets were collected by scraping cells in media, followed by centrifugation at 1,000×g for 10 min and aspirating media. Pellets were then resuspended in ice-cold standard binding buffer (SBB: 50 mM Tris–HCl, pH 7.4, 10 mM MgCl2 and 0.1 mM EDTA), aliquoted, centrifuged at 14,000×g for 20 min at 4°C to pellet the membrane fraction, aspirated, and stored at −80°C for future use.[1]
5-HT1A pellets were washed by resuspending in ice-cold SBB and centrifuged at 14,000×g for 15 min at 4°C, and the buffer was aspirated. hD4.2 pellets were similarly washed but in ice-cold dopamine agonist binding buffer (DABB: 50 mM Tris–HCl pH 7.4, 5 mM KCl, 2 mM MgCl2, 2 mM CaCl2). Washed 5-HT1A membranes were Dounce-homogenized in room temperature SSB and incubated with 12 concentrations of [3H]WAY-100635 ranging from 0.004 to 2.3 nM in the absence and presence of 10 μM 5-HT to determine total and nonspecific binding, respectively. Likewise, washed hD4.2 membranes were Dounce-homogenized in room temperature DABB and incubated with 12 concentrations of [3H]WAY-100635 ranging from 0.004 to 13.4 nM in the absence and presence of 10 μM chlorpromazine to determine total and nonspecific binding, respectively. After 2 h at room temperature, reactions were terminated by rapid filtration onto cold 0.3% PEI presoaked filters. The filters were then washed three times in 4°C 50 mM Tris–HCl, pH 6.9. Filtered material was then transferred to scintillation vials mixed with 4 ml of Ecoscint-A scintillation fluid (National Diagnostic; Atlanta, GA, USA) and counted on a Beckman LS6500 scintillation counter.[1]

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Radioligand binding assay[1]
Cells were grown to confluence on 20-cm plates. The growth medium was decanted and replaced with 10-ml ice-cold lysis buffer (1 mM HEPES, pH 7.4, and 2 mM EDTA). After 10 min, cells were scraped from the plate and centrifuged at 30,000×g and 4°C for 20 min. The resulting pellet was resuspended in 4 ml receptor binding buffer (50 mM Tris–HCl, pH 7.4, and 4 mM MgCl) using a Kinematica homogenizer at a setting of 6 for 5 s, before 1.0 ml aliquots were centrifuged again at 13,000×g for 10 min. The pellets were stored at −80°C until use.[1]
The pellets were then resuspended for use by trituration in receptor binding buffer (50 μg protein/100 μl) and added in duplicate to assay tubes containing 0.1–0.2 nM [3H]spiperone and appropriate drugs. Nonspecific binding was determined using (+)-butaclamol (5 μM). Assay tubes were incubated at 37°C for 30 min before filtration, as described for cAMP binding assays. Filter plates were dried, and 30 μl of Packard Microscint-O scintillation fluid was added to each well. Radioactivity per well was determined using a Packard TopCount scintillation counter.[1]
Radioligand binding assays were also performed to investigate the effect of 100 μM guanylyl-5′-imidodiphosphate (Gpp-[NH]p) on agonist binding. These experiments were performed using HEK-hD4.4 membranes in a modified receptor binding buffer (50 mM Tris–HCl, pH 7.4, 4 mM MgCl, and 120 mM NaCl).

Cyclic AMP accumulation assay[1]
Cells were grown to confluent monolayers in 48-well clear tissue culture plates. Before assay, the growth medium was decanted, and the plates were placed on ice. Drug dilutions made in Earle’s balanced salt solution (EBSS) assay buffer (EBSS containing 2% bovine calf serum, 0.025% ascorbic acid, and 15 mM HEPES, pH 7.4) were added on ice. cAMP accumulation was stimulated by 5 μM forskolin, and each assay was performed in the presence of 500 μM isobutyl-methylxanthine. Incubations were performed for 15 min in a 37°C water bath. To terminate the stimulation, the assay medium was decanted, and cells were lysed by adding 100 μl of 3% trichloroacetic acid on ice. Plates were stored at 4°C for at least 1 h before quantification of cAMP.[1]

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Quantification of cyclic AMP[1]
Cyclic AMP accumulation was assessed using a previously described competitive binding assay (Watts and Neve 1996). Briefly, cell lysate (12 μl) was added in duplicate to assay tubes with cAMP binding buffer (100 mM Tris–HCl, pH 7.4, 100 mM NaCl, 5 mM EDTA) containing [3H]cAMP (1 nM final concentration) and cAMP binding protein (100–150 μg in 500 μl buffer). The reaction tubes were incubated on ice at 4°C for 2–3 h before harvesting by filtration (GF/C filter plates) using a 96-well Packard FilterMate cell harvester. Filter plates were dried, and 30 μl of Packard Microscint-O scintillation fluid was added to each well. Radioactivity per well was determined using a Packard TopCount scintillation counter. The concentrations of cAMP in each sample were estimated from a standard curve ranging from 0.01 to 300 pmol of cAMP.

Animal/Disease Models: Male SD (Sprague-Dawley) rats (220-240 g)[2]
Doses: 1 mg/kg
Route of Administration: subcutaneous (sc) injection (pharmacokinetic/PK study)
Experimental Results: decreased total abstinence score, increased immobility time and the burying behavior was increased.

[1]. Chemel BR, et al. WAY-100635 is a potent dopamine D4 receptor agonist. Psychopharmacology (Berl). 2006 Oct;188(2):244-51.
[2]. Mannucci C, et al. Serotonin involvement in Rhodiola rosea attenuation of nicotine withdrawal signs in rats. Phytomedicine. 2012 Sep 15;19(12):1117-24.
[3]. Al Hussainy R, et al. Design, synthesis, radiolabeling, and in vitro and in vivo evaluation of bridgehead iodinatedanalogues of N-{2-[4-(2-methoxyphenyl)piperazin-1-yl]ethyl}-N-(pyridin-2-yl)cyclohexanecarboxamide (WAY-100635) as potential SPECT ligands for the 5-HT1A receptor. J Med Chem. 2011 May 26;54(10):3480-91.

Here we describe the design, synthesis, and pharmacological profile of 5-HT(1A) receptor ligands related to 1 (WAY-100635). The cyclohexyl moiety in 1 and its O-desmethylated analogue 3 were replaced by the bridgehead iodinated bridge-fused rings: adamantyl, cubyl, bicyclo[2.2.2]octyl, or bicyclo[2.2.1]heptyl. All analogues displayed a (sub)nanomolar affinity for the 5-HT(1A) receptor in vitro. Compounds 6b and 7b appeared to be selective for this receptor over other relevant receptors and could easily be iodinated with radioactive iodine-123. In humane hepatocytes, [(123)I]6b showed a low propensity for amide hydrolysis and a stable carbon-iodine bond. The biodistribution of [(123)I]6b and [(123)I]7b in rats revealed that the carbon-iodine bond was also stable in vivo. Unfortunately, the brain uptake and the specificity for both radioligands were significantly lower than those of the parent molecule 1. In conclusion, the designed tracers are not suitable for SPECT imaging.[3]

C25H34N4O2

422.573

422.2682

C, 71.06; H, 8.11; N, 13.26; O, 7.57

162760-96-5

WAY-100635 maleate;1092679-51-0

5684

Off-white to light yellow ointment

3.83

48.91

O=C(N(C1=NC=CC=C1)CCN2CCN(CC2)C3=CC=CC=C3OC)C4CCCCC4

SBPRIAGPYFYCRT-UHFFFAOYSA-N

InChI=1S/C25H34N4O2/c1-31-23-12-6-5-11-22(23)28-18-15-27(16-19-28)17-20-29(24-13-7-8-14-26-24)25(30)21-9-3-2-4-10-21/h5-8,11-14,21H,2-4,9-10,15-20H2,1H3

N-[2-[4-(2-methoxyphenyl)piperazin-1-yl]ethyl]-N-pyridin-2-ylcyclohexanecarboxamide

WAY 100635; WAY-100635; 162760-96-5; Way 100635; cyclohexanecarboxamide, n-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-n-2-pyridinyl-; CHEMBL31354; N-(2-(4-(2-Methoxyphenyl)piperazin-1-yl)ethyl)-N-(pyridin-2-yl)cyclohexanecarboxamide; N-[2-[4-(2-methoxyphenyl)piperazin-1-yl]ethyl]-N-pyridin-2-ylcyclohexanecarboxamide; 71IH826FEG; WAY-100635

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~66.67 mg/mL (~157.78 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.92 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (5.92 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (5.92 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)