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].

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[3H]WAY 100635 is demonstrated to bind specifically to 5-HT1A receptors in the brain after being given intravenously to mice. Additionally, WAY 100635 dose-dependently inhibits 8-OH-DPAT'scapacityto cause the "5-HT syndrome," hypothermia, hyperphagia, and an increase in plasma ACTH levels. This is achieved by inhibiting the firing of dorsal raphe 5-HT neurones. WAY 100635 produces anxiolytic-like effects in the mouse light/dark box anxiety model. In the delayed-matching-to-position model of rat short-term memory, WAY 100635 reverses the disruptive effects of 8-OH-DPAT on motor motivational performance but has no intrinsic effect on cognition.[2] In the anesthetized rat, WAY 100635 inhibits the inhibitory action of 8-OH-DPAT on dorsal raphe neuronal firing at doses that have no inherent inhibitory effect. In behavioral models, WAY 100635 does not directly cause any overt behavioral changes in rats or guinea pigs, but it effectively counteracts the behavioral syndrome caused by 8-OH-DPAT (minimum effective dose = 0.003 mg/kg s.c. and ID50 = 0.01 mg/kg s.c., respectively). With ID50 values of 0.01 mg/kg s.c., WAY 100635 also prevents the hypothermia that is brought on by 8-OH-DPAT in rats and mice.

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.

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Biodistribution Studies of [123I]6b and [123I]7b in Rat: General Procedure[3]
Animal experiments were performed on male Wistar rats weighing approximately 230 g. Approval for the applied animal protocol used was obtained from the National Council on Animal Care and the in-house Ethics Committee according to the guidelines of the law on animal experiments of The Netherlands. The solution of the radiolabeled compounds was prepared 1 day prior to these studies. Rats were anesthetized with 2% isoflurane and received an intravenous injection of 200 μL of the radiolabeled compounds in the tail vein. After different time intervals, the rats were sacrificed, under anesthesia, by cervical dislocation, and blood was collected from the decapitated body. Tissues of interest were removed, weighed, and measured for radioactivity using a LKB Wallac 1282 CompuGamma CS. For calculation of the injected dose, five aliquots of the injected solution were weighed and counted for radioactivity. Results were decay corrected and expressed as percentage of injected dose per gram of tissue ± standard deviation (% ID/g ± SD).[3]
[123I]6b[3]
Two groups of four rats were used. The first group received 200 μL (7 MBq, <20 ng) of the radiolabeled compound intravenously and was sacrificed at 45 min after injection. In the second group, rats received an intravenous administration of nonlabeled compound 1 (2 mg/kg) 5 min prior to the intravenous administration of [123I]6b (7 MBq). Rats were also sacrificed and dissected at 45 min after injection.[3]
[123I]7b[3]
Three groups of four rats were used. Each rat received an intravenous injection of 200 μL (7 MBq, <20 ng) of the radiolabeled compound. They were sacrificed at 15, 45, and 120 min after injection and processed as above.

[1]. WAY-100635 is a potent dopamine D4 receptor agonist. Psychopharmacology (Berl). 2006 Oct;188(2):244-51.

[2]. Serotonin involvement in Rhodiola rosea attenuation of nicotine withdrawal signs in rats. Phytomedicine. 2012 Sep 15;19(12):1117-24.

[3]. 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.

N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridyl)cyclohexanecarboxamide belongs to the piperazine class of compounds.

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Background and Objectives: WAY-100635 is a typical 5-HT1A receptor antagonist and has been widely used as a pharmacological probe to study the distribution and function of the 5-HT1A receptor. Our results indicate that WAY-100635 possesses potent inducing activity independent of its affinity for the 5-HT1A receptor. In this study, we evaluated the in vitro pharmacological properties of this compound on two D2-like receptor subtypes. Methods: The functional properties and binding affinity of WAY-100635 were evaluated in HEK 293 cells stably expressing dopamine D2L or D4.4 receptors. Results: Preliminary screening by the National Institute of Mental Health (NIMH) Psychoactive Drug Screening Program showed that WAY-100635 had binding affinities of 940 nM, 370 nM, and 16 nM for the D2L, D3, and D4.2 receptors, respectively. Subsequent saturation analysis showed that the dissociation constant (Kd) of [3H]WAY-100635 on the D4.2 receptor was 2.4 nM, only ten times higher than that on the 5-HT1A receptor. WAY-100635 and its major metabolite WAY-100634 were both potent agonists in HEK-D4.4 cells (EC50 values of 9.7 ± 2.2 nM and 0.65 ± 0.2 nM, respectively). WAY-100635 exhibited complete agonism, while WAY-100634 exhibited near-complete agonism. In HEK-D2L cells, WAY-100635 had a weak antagonistic effect on 300 nM quinpyrrole. Subsequent radioligand binding studies confirmed that WAY-100635 had a high affinity for the D4.4 receptor, but a weaker binding to the D2L receptor (3.3±0.6 nM and 420±11 nM, respectively). Conclusion: This study shows that WAY-100635 is not a “selective” 5-HT1A receptor antagonist as previously reported. Therefore, the conclusions of previous studies that regarded WAY-100635 as a selective 5-HT1A receptor antagonist may need to be re-evaluated. [1] Rhodiola rosea has been used in traditional medicine for hundreds of years to stimulate the nervous system, enhance physical and mental functions, and treat fatigue. It is known that taking Rhodiola rosea extract has antidepressant activity, but its mechanism of action is still unclear. Evidence from animal models and human studies suggests that nicotine can alleviate depressive symptoms, while nicotine withdrawal can induce depressive-like symptoms. We investigated the effects of Rhodiola rosea on nicotine withdrawal symptoms. Nicotine dependence was induced in animals by subcutaneous injection of nicotine (2 mg/kg, four times daily) for 14 days. Another group of animals received nicotine treatment for 14 days, followed by treatment with Rhodiola rosea extract and concurrent administration of the selective 5-HT receptor antagonist WAY 100635 (1 mg/kg). After nicotine withdrawal, behavioral parameters (motor activity, withdrawal symptoms, marble-bubble test), diencephalon serotonin metabolism, and serotonin receptor 1A expression were assessed. Results showed that the N group treated with Rhodiola rosea exhibited significantly increased 5-HT levels and a significant increase in serotonin receptor 1A, indicating that serotonin is involved in the beneficial effect of Rhodiola rosea in alleviating nicotine withdrawal symptoms. [2]

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This article describes the design, synthesis, and pharmacological properties of 5-HT(1A) receptor ligands associated with compound 1 (WAY-100635). The cyclohexyl moiety in compound 1 and its O-demethylated analog 3 is replaced by a bridged fused ring with an iodinated bridgehead: adamantyl, cuboalkyl, bicyclo[2.2.2]octyl, or bicyclo[2.2.1]heptyl. All analogs exhibit (sub)nanomolar affinity for the 5-HT(1A) receptor in vitro. Compounds 6b and 7b show higher selectivity for this receptor than other associated receptors and are readily iodinated with radioactive iodine-123. In human hepatocytes, [(123)I]6b amides show low hydrolytic tendency and stable carbon-iodine bonds. Biodistribution of [(123)I]6b and [(123)I]7b in rats indicates that carbon-iodine bonds are also stable in vivo. However, the brain uptake and specificity of these two radioligands are significantly lower than those of the parent molecule 1. In conclusion, the designed tracer is not suitable for SPECT imaging. [3]

C25H34N4O2

422.573

422.268

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

3.828

0

5

7

31

546

0

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.)