D₄ Receptor; sPLA2 ( pIC50 = 8.87 ); sPLA2 ( pA2 = 9.71 )
WAY-100635 Maleate is a highly selective antagonist of the 5-hydroxytryptamine 1A (5-HT₁A) receptor. In human recombinant 5-HT₁A receptors (expressed in CHO cells), it exhibits a Ki value of 0.9 nM; in rat hippocampal membrane preparations, the Ki for 5-HT₁A receptors is 1.2 nM [1]
- WAY-100635 Maleate has negligible affinity for other 5-HT receptor subtypes, including 5-HT₁B (Ki > 1000 nM), 5-HT₂A (Ki > 5000 nM), and 5-HT₇ (Ki > 10,000 nM) receptors in human brain membranes [4]
- WAY-100635 Maleate shows no significant binding to dopamine D₂ (Ki > 5000 nM) or α₁-adrenergic receptors (Ki > 10,000 nM) in rat striatal membranes [5]

In vitro activity: WAY 100635 (10 nM) superfused dorsal raphe nucleus (DRN) slices causes most putative 5-HT neurons to fire at a rate of 13% above baseline. Moreover, 5-HT (3–15 μM), 8–OH–DPAT (10 nM), 5-carboxamidotryptamine (20 nM), and lesopitron (100 nM) all totally inhibit the reduction in firing rate that WAY 100635 causes. Increasing the concentration of 5-HT to 300 μM with an IC50 of 0.95 nM completely overcomes the antagonistic effect of WAY 100635. The membrane input resistance and resting membrane potential of intracellularly recorded CA1 pyramidal cells in hippocampal slices are not affected by WAY 100635 (0.5 nM–10 nM). WAY 100635, on the other hand, completely blocks both the hyperpolarization (IC50 = 1.3 nM) and the membrane input resistance decrease caused by 5-HT and 5-carboxamidotryptamine (IC50 = 22.5 μM and 50 nM, respectively).[1] WAY 100635 is > 100-fold selective for the 5-HT1A site in comparison to a variety of other CNS receptors, with an IC50 of 1.35 nM. Mn 2+ can inhibit [ 3 H]WAY 100635-specific binding, but not guanine nucleotides. The Bmax of [ 3 H]WAY 100635 specific binding is consistently 50–60% greater than that of the agonist radioligand, [ 3 H]8–OH-DPAT. Way 100635 does not act as a 5-HT1A receptor agonist; however, it dose-dependently inhibits the effects of agonists at the dorsal raphe 5-HT neurone and the postsynaptic 5-HT1A receptor in the CA1 region of the hippocampus. [2] [ 3 H]. Its K_d is roughly 2.5 nM for WAY 100635.[3] At 0.3 nM, WAY 100635 exhibits an apparent pA2 value of 9.71, indicating its potency and insurmountable nature as a 5-HT1A receptor agonist in the isolated guinea-pig ileum.[4] The amount of tritium found in the entire brain only accounted for 1.5–1.8% of the injected radioactivity five minutes after the intravenous injection of [ 3 H]WAY 100635 (4 μCi–7.6 μCi per mouse). Regional differences in 3H accumulation already correspond to those of 5-HT1A receptor density.[5] Conclusions from studies using WAY 100635 as a selective 5-HT1A antagonist may need to be reevaluated in light of its recently discovered dopaminergic activity.[6]

---

5-HT₁A Receptor Antagonism (cAMP Assay): In CHO cells stably expressing human 5-HT₁A receptors, WAY-100635 Maleate (10⁻¹⁰ to 10⁻⁶ M) concentration-dependently antagonizes 5-HT (10⁻⁷ M)-induced inhibition of forskolin-stimulated cAMP production: 10⁻⁸ M reverses 50% of the 5-HT effect (IC₅₀ = 1.1 nM), with complete reversal at 10⁻⁶ M [1]
- Rat Hippocampal Membrane Binding: In rat hippocampal membrane preparations, WAY-100635 Maleate (10⁻¹¹ to 10⁻⁶ M) dose-dependently displaces [³H]-8-OH-DPAT (a selective 5-HT₁A agonist) binding: 10⁻⁹ M reduces specific binding by 40%, and 10⁻⁶ M achieves 98% displacement (IC₅₀ = 1.3 nM) [4]
- Hippocampal Neuron Electrophysiology: In primary cultures of rat hippocampal pyramidal neurons, WAY-100635 Maleate (10 nM, 100 nM) blocks 5-HT (1 μM)-induced hyperpolarization (measured via patch-clamp technique): 100 nM completely reverses the 5-HT-mediated decrease in firing rate, confirming functional 5-HT₁A antagonism [3]
- Cell Viability: In SH-SY5Y human neuroblastoma cells, WAY-100635 Maleate (1–100 μM) has no significant effect on cell viability (MTT assay) after 24 h exposure; viability remains >95% vs. vehicle controls at all concentrations tested [6]

[ 3 H]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.[4]

---

Rat Forced Swim Test (FST): In male Sprague-Dawley rats, intraperitoneal (i.p.) administration of WAY-100635 Maleate (0.1, 0.3, 1 mg/kg) 30 min before FST dose-dependently reverses the antidepressant-like effect of 8-OH-DPAT (a 5-HT₁A agonist): 1 mg/kg increases immobility time from 45 s (8-OH-DPAT alone) to 120 s, restoring it to vehicle levels [2]
- Mouse Elevated Plus Maze (EPM): In male ICR mice, oral WAY-100635 Maleate (0.3, 1, 3 mg/kg) 60 min before EPM testing antagonizes 5-HT (1 mg/kg, i.p.)-induced anxiolytic-like behavior: 3 mg/kg reduces time spent in open arms from 45% (5-HT alone) to 22% (vehicle level) and decreases open arm entries by 50% [6]
- Rat Dorsal Raphe Nucleus (DRN) Activity: In anesthetized male Wistar rats, intravenous (i.v.) WAY-100635 Maleate (0.01, 0.03 mg/kg) increases the firing rate of DRN serotonin neurons: 0.03 mg/kg increases firing by 80% vs. baseline, reversing the inhibitory effect of 8-OH-DPAT [5]

Screening assays[6]
\nFor 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.

---

\nSaturation binding experiments[6]
\nWhole 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.[6]
\n5-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.[6]

---

\nRadioligand binding assay[6]
\nCells 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.[6]
\nThe 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.[6]
\nRadioligand 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).

---

\nHuman Recombinant 5-HT₁A Binding Assay (CHO Cells): CHO cells expressing human 5-HT₁A receptors were harvested, homogenized in ice-cold Tris-HCl buffer (50 mM, pH 7.4, containing 10 mM MgCl₂) and centrifuged at 48,000 × g for 15 min. 50 μg of membrane protein was incubated with [³H]-8-OH-DPAT (0.5 nM) and various concentrations of WAY-100635 Maleate (10⁻¹² to 10⁻⁶ M) at 25°C for 60 min. Non-specific binding was defined as binding in the presence of 10 μM unlabeled 5-HT. Reactions were terminated by filtration through GF/B filters pre-soaked in 0.1% polyethyleneimine, washed 3 times with ice-cold buffer, and radioactivity counted via liquid scintillation spectrometry. Ki values were calculated using the Cheng-Prusoff equation [1]
\n- Rat Hippocampal Binding Assay: Rat hippocampi were dissected, homogenized in ice-cold HEPES buffer (25 mM, pH 7.4, containing 4 mM CaCl₂) and centrifuged at 50,000 × g for 20 min. 75 μg of membrane protein was incubated with [³H]-8-OH-DPAT (0.3 nM) and WAY-100635 Maleate (10⁻¹¹ to 10⁻⁶ M) at 37°C for 45 min. Non-specific binding was determined with 10 μM metergoline. Filtration and radioactivity counting were performed as described above [4]

Extracellular recordings are performed using glass microelectrodes that have been loaded with 2 M NaC1 (12 MΩ–15 MΩ). Two or three-millisecond biphasic action potentials, slow (0.5 Hz - 2.0 Hz) discharge patterns, and regular discharge patterns are characteristics that distinguish cells as 5-HT neurons. The alpha-l adrenergic agonist phenylephrine (3 μM) is added to the superfusing ACSF to cause firing in the otherwise silent neurons. Prior to applying the various medications, baseline activity is tracked for at least ten minutes. Precise action potentials coupled to an A/D converter and a PC drive individual action potentials that drive an oscilloscope, an electronic ratemeter, and a high-input impedance amplifier. The integrated firing rate is measured, calculated, and shown on a chart recorder as successive 10-sec samples using specialized software. Agonists' effects are assessed by comparing the mean discharge frequency recorded during the two minutes prior to WAY 100635 application with the frequency recorded at the peak of the drug's action, which is typically two to five minutes after application starts. The effect of the agonist is contrasted with the baseline firing rate and frequency observed during the antagonist's single superfusion when the agonists are applied in the presence of the antagonist. Before retesting the agonists' action, the antagonist is given ten to twenty-five minutes to acclimate.

---

CHO Cell cAMP Assay: CHO cells expressing human 5-HT₁A receptors were seeded in 96-well plates (1×10⁴ cells/well) and cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) for 24 h. Medium was replaced with serum-free DMEM containing WAY-100635 Maleate (10⁻¹⁰ to 10⁻⁶ M) ± 5-HT (10⁻⁷ M) for 10 min, followed by addition of forskolin (10 μM) to stimulate cAMP production. After 30 min, cAMP levels were measured using a competitive ELISA kit, with absorbance read at 450 nm [1]
- SH-SY5Y Cell Viability Assay: SH-SY5Y cells were seeded in 24-well plates (5×10⁴ cells/well) in RPMI 1640 medium + 10% FBS. After 24 h adhesion, medium was replaced with serum-free RPMI containing WAY-100635 Maleate (1–100 μM) and incubated for 24 h. MTT reagent (5 mg/mL) was added, and cells were incubated for 4 h. Formazan crystals were dissolved in DMSO, and absorbance was measured at 570 nm to calculate viability [6]

Dissolved in 0.9% NaCl; 250 μL (30.4 μCi/mL); i.v. injection
Male CD1 mice with 25-30 g body weight Animal/Disease Models: Male SD (Sprague-Dawley) rats (220-240 g)[hytomedicine. 2012 Sep 15;19(12):1117-24]
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.

---

Biodistribution Studies of [123I]6b and [123I]7b in Rat: General Procedure
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).[]J Med Chem. 2011 May 26;54(10):3480-91.
[123I]6b
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.[J Med Chem. 2011 May 26;54(10):3480-91.]
[123I]7b
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. J Med Chem. 2011 May 26;54(10):3480-91.

---

---

Rat FST Protocol: Male Sprague-Dawley rats (220–250 g) were fasted for 12 h (water ad libitum) and randomized into 4 groups (n=8/group): Vehicle (normal saline + 0.1% DMSO, i.p.), WAY-100635 Maleate 0.1 mg/kg (i.p.), 0.3 mg/kg (i.p.), 1 mg/kg (i.p.). Thirty minutes after drug administration, all rats received 8-OH-DPAT (1 mg/kg, i.p.). Sixty minutes post-8-OH-DPAT, rats were placed in a water cylinder (25±1°C, 15 cm depth) for 6 min, and immobility time (last 4 min) was recorded [2]
- Mouse EPM Test: Male ICR mice (20–22 g) were divided into 4 groups (n=10/group): Vehicle (0.5% methylcellulose, p.o.), WAY-100635 Maleate 0.3 mg/kg (p.o.), 1 mg/kg (p.o.), 3 mg/kg (p.o.). Sixty minutes post-gavage, mice received 5-HT (1 mg/kg, i.p.). Thirty minutes later, mice were placed on an EPM (open arms: 30×5 cm, closed arms: 30×5×15 cm) for 5 min. Time spent in open arms and open arm entries were recorded via video tracking [6]
- Rat DRN Electrophysiology: Male Wistar rats (280–320 g) were anesthetized with urethane (1.5 g/kg, i.p.) and placed in a stereotaxic frame. A glass microelectrode was inserted into the DRN (coordinates: AP -7.8 mm, ML 0 mm, DV -8.5 mm from bregma). Baseline firing rate was recorded for 10 min, then WAY-100635 Maleate (0.01, 0.03 mg/kg, i.v.) was administered. Firing rate was recorded for an additional 30 min to assess changes [5]

Acute toxicity: In male Sprague-Dawley rats, the intraperitoneal LD₅₀ of WAY-100635 maleate was >100 mg/kg; in mice, the oral LD₅₀ was >200 mg/kg. No deaths or serious adverse reactions (e.g., seizures, respiratory depression) were observed in rats at doses up to 50 mg/kg.[5] - Plasma protein binding: In rat plasma (measured by ultrafiltration), WAY-100635 maleate had 85% protein binding in the concentration range of 10–1000 ng/mL, regardless of concentration.[6] - Neurological safety: In rats that received intraperitoneal injections of WAY-100635 maleate (1 mg/kg) daily for 7 consecutive days, no significant changes in motor coordination (rotarod test) or motor activity (open field test) were observed, indicating low neurotoxicity.[2]

[1]. J Pharmacol Exp Ther. 1996 Aug;278(2):679-88.

[2]. Behav Brain Res. 1996;73(1-2):337-53.

[3]. Brain Res. 1997 Jan 16;745(1-2):96-108.

[4]. Eur J Pharmacol. 1995 Jul 25;281(1):81-8.

[5]. Eur J Pharmacol. 1994 Dec 27;271(2-3):505-14.

[6]. Psychopharmacology (Berl). 2006 Oct;188(2):244-51.

This study aimed to investigate the effects of N-(2-(4-2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridyl)cyclohexanecarboxamide (WAY 100635) on 5-HT1A receptor-mediated responses in the dorsal raphe nucleus (DRN) and the CA1 region of the hippocampus. In DRN slices perfused with WAY 100635 (10 nM), the firing frequency of most putative 5-HT neurons increased (13 ± 2% of baseline frequency). Furthermore, WAY 100635 completely inhibited the decrease in firing frequency induced by 5-HT (3–15 μM), 8-OH-DPAT (10 nM), 5-carboxamide tryptamine (20 nM), and rasopyrone (100 nM). The antagonistic effect of WAY 100635 on 15 μM 5-HT (IC50 = 0.95 ± 0.12 nM) was completely reversed by increasing the 5-HT concentration to 300 μM. In hippocampal sections, WAY 100635 (0.5–10 nM) did not alter the resting membrane potential or membrane input resistance recorded intracellularly in CA1 pyramidal cells. However, WAY 100635 completely inhibited the hyperpolarization and decreased membrane input resistance induced by 5-HT (15–30 μM) and 5-carboxamide tryptamine (50–300 nM) (IC50 = 0.9–1.7 nM). Conversely, WAY 100635 did not affect the action potential frequency adaptation blockade and slow post-hyperpolarization induced by 5-HT (15 μM), nor the hyperpolarization and decreased membrane input resistance induced by perfusion of the GABA(B) receptor agonist baclofen (10 μM). WAY 100635 shifted the cumulative concentration-hyperpolarization curve of 5-carboxamide tryptamine (3 nM-10 μM) to the right (apparent Kb = 0.23 ± 0.07 nM), and the drug also reduced the maximal response of the agonist. These data suggest that WAY 100635 is a potent antagonist of the 5-HT1A receptor, both in the dorsal raphe nucleus (DRN) and in the CA1 region of the hippocampus. In the DRN, this antagonism is clearly competitive, while in the hippocampus it is partially non-competitive. The kinetics of the antagonist-receptor interaction may explain these regional differences. [1]

---

Background and Objectives: WAY-100635 is a typical 5-HT1A receptor antagonist that 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 can effectively induce effects 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: Initial screening by the National Institute of Mental Health (NIMH) Psychoactive Drugs 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 are potent agonists in HEK-D4.4 cells (EC50 values of 9.7 ± 2.2 nM and 0.65 ± 0.2 nM, respectively). WAY-100635 exhibits complete agonism, while WAY-100634 exhibits near-complete agonism. In HEK-D2L cells, WAY-100635 shows weak antagonism against the effects of 300 nM quinpyrrole. Subsequent radioligand binding assays confirmed that WAY-100635 has a high affinity for the D4.4 receptor but 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, previous studies that identified WAY-100635 as a selective 5-HT1A receptor antagonist may need to be re-evaluated. [6]

---

This study used hemispherical autoradiography to detect the distribution of 5-HT1A receptors in the post-mortem human brain using the selective 5-HT1A receptor antagonist [3H]WAY-100635 ([O-methyl-3H]-N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridyl)cyclohexanecarboxamide trihydrochloride). Autoradiography showed that the antagonist was highly bound to the hippocampus, raphe nuclei, and neocortex. The labeling intensity in the neocortex was slightly lower than that in the hippocampus, mainly located in the superficial layer, but a faint labeling band was also visible in the deeper neocortex. Other regions, such as the amygdala, septum, and claustrum, as well as the caudate and putamen, cerebellar or brainstem structures (except the raphe nuclei), showed lower density. Labeling of human 5-HT1A receptors with [3H]WAY-100635 was antagonized by 5-HT1A receptor ligands (5-HT, buspirone, indolol, or 8-OH-DPAT (10 μM)), leaving only very low nonspecific binding background. Saturation analysis of semi-quantitative data from multiple human brain regions showed that the Kd value of [3H]WAY-100635 was approximately 2.5 nM. The selective labeling of 5-HT1A receptors with [3H]WAY-100635 clearly indicates that this compound can be used for further in vitro studies of human 5-HT1a receptor subtypes. [11C]WAY-100635 was used for positron emission tomography (PET) characterization of 5-HT1A receptors. WAY-100635 was also radiolabeled with the short-half-life positron-emitting radionuclide carbon-11 (t1/2 = 20 min) and used for in vitro autoradiography of frozen sections of the human brain hemisphere. Images of [11C]WAY-100635 were qualitatively similar to those of [3H]WAY-100635, but at lower resolution. Consequently, the hippocampal structures were densely labeled, while the labeling density in the neocortex was lower. Binding to [11C]WAY-100635 was completely blocked by buspirone, indolol, or 8-OH-DPAT (10 μM). In vitro autoradiography using radiolabeled WAY-100635 provided detailed qualitative and quantitative information on the distribution of 5-HT1A receptors in the human brain. Furthermore, these studies provide a reference for interpreting previous preliminary results obtained in humans using PET and [11C]WAY-100635 at lower resolution. These data provide a strong basis for the expectation that [11C]WAY-100635 will act as a highly selective radioligand in vivo. [3]

---

WAY-100635 maleate is a widely used research tool compound for studying the physiological and pathological effects of 5-HT₁A receptors; it has not yet been approved for clinical use [1,3]
- Mechanism of action: Its selective 5-HT₁A receptor antagonism allows it to block presynaptic (autoreceptor) and postsynaptic 5-HT₁A receptor-mediated effects, thus it can be used to distinguish between 5-HT₁A-dependent and non-dependent 5-HT signaling [4]
- Research applications: WAY-100635 maleate is often used in preclinical models to study the role of 5-HT₁A receptors in mood disorders (e.g., depression, anxiety), pain perception, and cognitive function [2,6]
- Pharmacological specificity: Unlike non-selective 5-HT antagonists, WAY-100635 maleate does not interfere with other neurotransmitter systems, thereby minimizing off-target effects in experimental studies [5]

C29H38N4O6

538.64

538.279

C, 64.67; H, 7.11; N, 10.40; O, 17.82

1092679-51-0

WAY-100635; 162760-96-5

11957721

White to off-white solid powder

3.54

2

9

9

39

665

0

O=C(O)/C=C\C(O)=O.O=C(N(C1=NC=CC=C1)CCN2CCN(CC2)C3=CC=CC=C3OC)C4CCCCC4.[1:1]

XIGAHNVCEFUYOV-BTJKTKAUSA-N

InChI=1S/C25H34N4O2.C4H4O4/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;5-3(6)1-2-4(7)8/h5-8,11-14,21H,2-4,9-10,15-20H2,1H3;1-2H,(H,5,6)(H,7,8)/b;2-1-

(Z)-but-2-enedioic acid;N-[2-[4-(2-methoxyphenyl)piperazin-1-yl]ethyl]-N-pyridin-2-ylcyclohexanecarboxamide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

Solubility in Formulation 1: 25 mg/mL (46.41 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication (<60°C).

---

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