5-HT₄ receptor (serotonin receptor subtype 4) - partial agonist [1]
pKi (binding affinity) = 8.7 [1]
pEC50 (functional potency in rat esophagus) = 8.7 ± 0.1 [1]
Intrinsic activity (vs 5-HT) = 0.5 [1]
σ₁ receptor - significant affinity [1]
pKi = 8.9 [1]
σ₂ receptor - significant affinity [1]
pKi = 8.0 [1]
Other receptors - low affinity (pKi < 6.0 or lower): including 5-HT₁A, 5-HT₁D, 5-HT₂A, 5-HT₂C, 5-HT₃, dopamine D₁, D₂, muscarinic M₁, M₂, M₃, α₁A, α₁B, α₂A, α₂B, β₁, β₂ adrenoceptors, and (R)-zacopride site. [1]

There is affinity for the sigma 1 (pKi = 8.9) and sigma 2 (pKi = 8.0) binding sites for RS 67333 hydrochloride. RS 67333 hydrochloride mediates carbachol-precontracted esophageal relaxation by acting as a strong (pEC50 of 8.4) partial agonist of 5-HT (intrinsic activity relative to 5-HT of 0.5). 1].

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Radioligand Binding Studies: RS 67333 exhibited high affinity for 5-HT₄ binding sites in guinea-pig striatum labeled with [³H]-GR 113808, with a pKi value of 8.7. Hill coefficients were not significantly different from unity. It also showed significant affinity for σ₁ (pKi = 8.9) and σ₂ (pKi = 8.0) binding sites. Affinities at numerous other neurotransmitter receptors (5-HT₁A, 5-HT₂A, 5-HT₂C, 5-HT₃, dopamine D₁, D₂, muscarinic M₁, M₂, M₃, α₁A, α₁B, α₂A, α₂B, β₁, β₂ adrenoceptors, and (R)-zacopride site) were low (pKi < 6.0 or less). [1]
Rat Isolated Oesophageal Muscularis Mucosae Assay: In carbachol-precontracted rat esophagus, where relaxation is mediated by 5-HT₄ receptor activation, RS 67333 acted as a potent partial agonist with respect to 5-HT. It relaxed the preparation in a concentration-dependent manner with a pEC50 of 8.7 ± 0.1 and an intrinsic activity of 0.5 (50% of the maximal 5-HT response). Responses to RS 67333 were abolished by desensitization with 5-methoxytryptamine and surmountably antagonized by the selective 5-HT₄ antagonist GR 113808 (10 nM), with an apparent pKB of 9.1 ± 0.2. [1]

Tachycardia in Anaesthetized Micropig: In anaesthetized, bilaterally vagotomized Yucatan micropigs, intravenous administration of RS 67333 induced dose-dependent increases in heart rate. It acted as a partial agonist relative to 5-HT, with an ED50 of 4.9 μg/kg (95% confidence limits: 2.3-10 μg/kg) and a maximal heart rate increase of 35 beats/min (95% confidence limits: 27-43 beats/min), which was significantly less than the maximal response to 5-HT (95 beats/min). [1]

Radioligand Binding Studies:** Guinea-pig striata were obtained from animals previously killed by CO₂ asphyxiation. Striata were homogenized and processed to obtain synaptosomal membrane preparations for [³H]-GR 113808 binding assays. [1]
* **Rat Isolated Oesophageal Studies:** Male Sprague-Dawley rats (200-250 g) were used. The thoracic oesophagus was isolated, and the outer striated muscle coat was removed to obtain the inner muscularis mucosae. Tissues were mounted vertically in organ baths containing Tyrode solution (37°C, gassed with 95% O₂/5% CO₂) with methysergide (1 μM), cocaine (30 μM), and corticosterone (30 μM) present throughout. After equilibration and KCl challenge, tissues were precontracted with carbachol (3 μM). Cumulative concentration-response curves to RS 67333 were then established. Desensitization studies were performed by equilibrating tissues with 10 μM 5-methoxytryptamine. Antagonist studies were performed by equilibrating tissues with GR 113808 (10 nM) for 60 min before constructing a second concentration-response curve. [1]
* **Micropig Tachycardia Studies:** Yucatan micropigs (male and female, 17-22 kg) were pretreated with ketamine (30 mg/kg i.m.), anaesthetized with pentobarbitone sodium (20 mg/kg i.v.), intubated, and mechanically ventilated. A femoral artery was cannulated for blood pressure measurement. Dual cannulae were inserted in the femoral vein for anaesthetic infusion and compound administration. ECG was monitored, and heart rate was derived from the R wave. Vagas nerves were cut bilaterally. After stabilization, each animal received 5-HT in ascending bolus i.v. doses (1-300 μg/kg) at 0.5 log intervals with 10 min between doses. Subsequently, RS 67333 was administered in ascending bolus i.v. doses (0.3-300 μg/kg) in a similar fashion. Heart rate was measured immediately before and at peak effect after each dose. RS 67333 was dissolved in normal saline for administration. [1]

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Radioligand Binding Studies: Guinea-pig striata were obtained from animals previously killed by CO₂ asphyxiation. Striata were homogenized and processed to obtain synaptosomal membrane preparations for [³H]-GR 113808 binding assays. [1]
Rat Isolated Oesophageal Studies: Male Sprague-Dawley rats (200-250 g) were used. The thoracic oesophagus was isolated, and the outer striated muscle coat was removed to obtain the inner muscularis mucosae. Tissues were mounted vertically in organ baths containing Tyrode solution (37°C, gassed with 95% O₂/5% CO₂) with methysergide (1 μM), cocaine (30 μM), and corticosterone (30 μM) present throughout. After equilibration and KCl challenge, tissues were precontracted with carbachol (3 μM). Cumulative concentration-response curves to RS 67333 were then established. Desensitization studies were performed by equilibrating tissues with 10 μM 5-methoxytryptamine. Antagonist studies were performed by equilibrating tissues with GR 113808 (10 nM) for 60 min before constructing a second concentration-response curve. [1]
Micropig Tachycardia Studies: Yucatan micropigs (male and female, 17-22 kg) were pretreated with ketamine (30 mg/kg i.m.), anaesthetized with pentobarbitone sodium (20 mg/kg i.v.), intubated, and mechanically ventilated. A femoral artery was cannulated for blood pressure measurement. Dual cannulae were inserted in the femoral vein for anaesthetic infusion and compound administration. ECG was monitored, and heart rate was derived from the R wave. Vagas nerves were cut bilaterally. After stabilization, each animal received 5-HT in ascending bolus i.v. doses (1-300 μg/kg) at 0.5 log intervals with 10 min between doses. Subsequently, RS 67333 was administered in ascending bolus i.v. doses (0.3-300 μg/kg) in a similar fashion. Heart rate was measured immediately before and at peak effect after each dose. RS 67333 was dissolved in normal saline for administration. [1]

It mentions that RS 67333 (clogP = 4.5) is a more lipophilic molecule compared to its analogue RS 67506 (clogP = 2.4), suggesting that penetration of RS 67333 across the blood-brain barrier would be greater. [1]
The compounds were developed to replace the ester function in the earlier compound RS 23597, which had limited duration of action in vivo as a result of plasma hydrolysis. RS 67333 is a ketone analogue designed for augmented in vivo stability. [1]

[1]. Pharmacological characterization of two novel and potent 5-HT4 receptor agonists, RS 67333 and RS 67506, in vitro and in vivo. Br J Pharmacol. 1995 Aug;115(8):1387-92.

See also: RS 67333 (notes moved to).

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Background and Design: RS 67333 is a novel 5-HT₄ receptor agonist, structurally related to the selective 5-HT₄ antagonist RS 23597. It was developed through a medicinal chemistry program aimed at augmenting the in vivo stability of RS 23597 by replacing its ester function with a ketone, resulting in a series of ketone analogues including RS 67333 and RS 67506. [1]
Mechanism of Action: RS 67333 acts as a potent, partial agonist at 5-HT₄ receptors. Its partial agonist profile is consistent with many non-indole 5-HT₄ agonists and is likely due to the low receptor reserve in some tissues (e.g., rat esophagus). The study notes that in tissues with a very low receptor reserve (e.g., guinea-pig hippocampus adenylyl cyclase assay), RS 67333 may act as a silent antagonist. [1]
Selectivity Profile: While highly selective for 5-HT₄ over numerous other neurotransmitter receptors (5-HT₁A, 5-HT₂, dopamine, muscarinic, adrenergic), RS 67333 possesses significant affinity for σ₁ and σ₂ binding sites. This should be considered when interpreting in vivo effects, and the use of antagonists with low sigma affinity (like GR 113808) is recommended to unambiguously attribute responses to 5-HT₄ activation. [1]
Potential Therapeutic Applications: The study suggests that selective 5-HT₄ receptor agonists like RS 67333 may have therapeutic potential in enhancing cognitive performance, facilitating gastrointestinal motility, correcting micturition disturbances associated with detrusor hypomotility, or acting as analgesics. [1]

C19H30CL2N2O2

389.36

388.168

C, 58.61; H, 7.77; Cl, 18.21; N, 7.19; O, 8.22

168986-60-5

160845-95-4;168986-60-5 (HCl);

9800491

Light yellow to yellow solid powder

502.3ºC at 760mmHg

257.6ºC

3.22E-10mmHg at 25°C

5.726

2

4

8

25

386

0

O=C(C1=CC(Cl)=C(N)C=C1OC)CCC2CCN(CCCC)CC2.[H]Cl

XVBGLZNYWUUAFF-UHFFFAOYSA-N

InChI=1S/C19H29ClN2O2.ClH/c1-3-4-9-22-10-7-14(8-11-22)5-6-18(23)15-12-16(20)17(21)13-19(15)24-2;/h12-14H,3-11,21H2,1-2H3;1H

1-(4-amino-5-chloro-2-methoxyphenyl)-3-(1-butylpiperidin-4-yl)propan-1-one;hydrochloride

RS 67333 HCl RS67333 RS 67333

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)

DMSO : ~50 mg/mL (~128.42 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.42 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 (6.42 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 (6.42 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.)