5-HT2B ( pIC50 = 10.4 ); 5-HT2B ( pIC50 = 9.5 )
RS-127445 is a highly selective antagonist of the human recombinant serotonin 5-HT2B receptor, with a Ki of 1.8 nM (using [³H]-5-HT as the radioligand). It exhibits minimal affinity for other serotonin receptor subtypes: Ki > 1000 nM for 5-HT2A, Ki > 500 nM for 5-HT2C, and Ki > 1 μM for 5-HT1A/1B/3/4 receptors [1]

In vitro activity: RS-127445 is discovered to have 1,000 fold selectivity for the 5-HT_2B receptor and nanomolar affinity (pKi=9.5±0.1) for this receptor when compared to many other receptor and ion channel binding sites. When expressed in CHO-K1 cells, human recombinant 5-HT_2B receptors exhibit a potent displacement of [3H]-5-HT by RS-127445. 9.5±0.1 (n=9) is the affinity (pKi value) of RS-127445 for the 5-HT_2B receptor. With a roughly 1000-fold lower affinity for the human recombinant 5-HT_2A, 5-HT_2C, 5-HT₅, 5-HT₆, and 5-HT₇ receptors, a 5-HT_1A receptor in rat brain membranes, a 5-HT_1B/D receptor in bovine caudate, and a monoamine uptake site in rabbit platelets, RS-127445 is selective for the 5-HT_2B receptor. In HEK-293 cells expressing the 5-HT_2B receptor, RS-127445 potently inhibits the increases in intracellular calcium concentrations evoked by 5-HT (10 nM) (pIC₅₀ of 10.4±0.1). RS-127445 potently inhibits 5-HT-evoked formation of inositol phosphates (pK_B=9.5±0.1) and intracellular calcium increases (pIC₁₀=10.4±0.1) in cells expressing human recombinant 5-HT_2B receptors. Additionally, RS-127445 inhibits (±)α-methyl-5-HT-mediated relaxation of the rat jugular vein (pA2=9.9±0.3) as well as 5-HT-evoked contraction of the rat isolated stomach fundus (pA_2B=9.5±1.1)[1].

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Inhibition of 5-HT2B-mediated intracellular calcium mobilization: HEK293 cells stably transfected with human 5-HT2B receptors were treated with RS-127445 (0.1–100 nM) for 30 minutes, followed by stimulation with 1 μM 5-HT. At 3.2 nM, RS-127445 inhibited 5-HT-induced calcium elevation by 50% (IC50 = 3.2 nM), measured via Fura-2 AM fluorescence ratiometry (340/380 nm). At 100 nM, inhibition reached >90% [1]
- Selectivity validation in 5-HT2A/2C-expressing cells: In HEK293 cells transfected with human 5-HT2A or 5-HT2C receptors, RS-127445 (up to 10 μM) showed <10% inhibition of 5-HT (1 μM)-induced calcium responses, confirming its 5-HT2B subtype selectivity [1]

The fraction of RS-127445 that is bioavailable in rats through the intraperitoneal or oral routes is 60% and 14%, respectively. RS-127445 (5 mg/kg) administered intraperitoneally resulted in plasma concentrations that were expected to completely saturate accessible 5-HT_2B receptors for a minimum of 4 hours. Rats are given oral, intraperitoneal, and intravenous injections of RS-127445 (5 mg/kg). Peak plasma concentrations are reached quickly, and the highest concentrations are found by 0.25 hours after oral dosing and at the first time-point measured after intravenous and intraperitoneal administration (0.08 h). With an approximate terminal elimination half-life of 1.7 hours, RS-127445 is eliminated from plasma. When RS-127445 is administered orally or intraperitoneally, its bioavailability is roughly 14% and 62%, respectively, of what it is when administered intravenously. Of RS-127445 (5 mg/kg), about 60% of the intraperitoneal dose and 14% of the oral dose are bioavailable[1]. Faecal output is dose-dependently reduced by RS-127445 (1–30 mg/kg), with a significant reduction observed at 10 and 30 mg/kg (n=6–11). More than 98% of RS-127445 is protein-bound in the blood and brain[2].

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Attenuation of 5-HT-induced pressor response in anesthetized rats: Male Sprague-Dawley rats (250–300 g) anesthetized with sodium pentobarbital (30 mg/kg, i.v.) received intravenous (i.v.) or oral administration of RS-127445. Intravenous RS-127445 (0.3 mg/kg) inhibited 5-HT (1 μg/kg, i.v.)-induced increases in mean arterial pressure (MAP) by 80% (from 45 mmHg to 9 mmHg) within 15 minutes. Oral RS-127445 (10 mg/kg) showed similar efficacy (75% inhibition) with a Tmax of 60 minutes [1]
- Lack of effect on basal cardiovascular parameters: In conscious rats, oral RS-127445 (1–30 mg/kg) had no significant impact on basal MAP (<5% variation) or heart rate (<10% variation) over 4 hours, indicating no off-target cardiovascular toxicity [1]

Testing the compound's affinity at more than 100 additional ion channel or receptor binding sites allows researchers to determine how selective RS-127445 is for 5-HT2B receptors. 2 mM EDTA in phosphate buffered saline is used to harvest CHO-K1 cells expressing human 5-HT2A, 5-HT2B, or 5-HT2C receptors. The process involves four rounds of centrifugation (48,000×g for 15 min) and homogenization to prepare cell membranes. The purpose of every assay is to optimize specific binding and reach steady state conditions. The 5-HT2A receptor is detected by incubating membranes from 1×10 6 cells with 0.2 nM [3 H]-ketanserin for 60 minutes at 32 °C. We use 10 μM methysergide to measure nonspecific binding. 0.2 nM [3 H]-5-HT is incubated for 120 minutes at 48 °C on membranes from 1.5×106 cells in order to detect the 5-HT2B receptor. 10-fold increase in 5-HT is used to measure nonspecific binding. Membranes from 3x10^5 cells are incubated with 0.5 nM [3 H]-mesuler-gine at 32 °C for 60 minutes in order to detect the 5-HT2Creceptor. Ten micrograms of methysergide are used to measure nonspecific binding. Glass fiber filters (GF/B) that have been pretreated with 0.1% polyethylene imine are used to vacuum filter out samples to end assays. Liquid scintillation counting is used to calculate bound and total radioactivity. In all of these tests, specific binding of greater than 90% is attained.

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Human 5-HT2B Receptor Binding Assay: The 200 μL reaction system contained 50 μg of membrane protein from HEK293 cells expressing human 5-HT2B receptors, 0.5 nM [³H]-5-HT (radioligand), and RS-127445 (0.01–100 nM). The mixture was incubated at 25°C for 60 minutes in 50 mM Tris-HCl (pH 7.4, containing 10 mM MgCl₂ and 0.5 mM EDTA). The reaction was terminated by rapid filtration through glass fiber filters pre-soaked in 0.3% polyethyleneimine. Filters were washed 3 times with cold assay buffer, and radioactivity was measured using a liquid scintillation counter. Non-specific binding was determined in the presence of 10 μM unlabeled 5-HT, and Ki was calculated using the Cheng-Prusoff equation [1]
- 5-HT2A/2C Receptor Binding Assays: Protocols were identical to the 5-HT2B assay, except membranes from HEK293 cells expressing human 5-HT2A or 5-HT2C receptors were used. RS-127445 concentrations up to 10 μM showed <5% displacement of [³H]-5-HT, confirming low affinity for these subtypes [1]

For 20 minutes at 37°C, 240 μl of HEK-293 cells expressing the human 5-HT2B receptor suspension are pre-incubated with RS-127445, vehicle, or other antagonists. HEK-293 cells are cultured in 162 cm2 flasks with [3H]-myoinositol (1.67 μCi/ml) in an inositol-free Hams F12 medium containing 10% dialyzed foetal bovine serum for an entire night at 37 °C. After being harvested, the cells are resuspended at a density of roughly 3×10 3 cells/ml in inositol-free Hams F12 media after being washed five times with phosphate buffered saline. The addition of 5-HT starts the reactions. After the reactions have lasted for sixty minutes, 50 μl of ice-cold 20% perchloric acid is added, the mixture is chilled in an ice-water bath for ten minutes, and 160 μl of 1 N KOH is neutralized. Two milliliters of room-temperature, 50 mM Tris-HCl (pH 7.4) are used to dilute each sample. After being cleaned with 5 ml of distilled water, the aqueous portion (2.2 ml) is transferred onto Dowex AG1X8 columns (1 ml, 1: 1, w/v). Following an 18 ml distilled water wash, 3 ml of 1 N HCl is used to elute the inositol phosphates from the columns. Using a Packard 1900CA analyzer, liquid scintillation spectroscopy is used to measure the eluted radioactivity.

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HEK293-h5-HT2B Cell Calcium Mobilization Assay: HEK293 cells stably expressing human 5-HT2B receptors were seeded in 96-well black-walled plates at 5×10⁴ cells/well and cultured in DMEM with 10% FBS for 24 hours. Medium was replaced with Krebs-Ringer buffer (pH 7.4) containing 2 μM Fura-2 AM, and cells were loaded for 45 minutes at 37°C. RS-127445 (0.1–100 nM) was added, and incubation continued for 30 minutes. Fluorescence intensity (excitation 340 nm and 380 nm, emission 510 nm) was measured using a microplate reader before and after adding 1 μM 5-HT. The 340/380 nm fluorescence ratio was calculated to quantify intracellular calcium levels, and inhibition rates were derived relative to the vehicle control [1]

Dissolved in ethanol:pro-pylene glycol: water (10: 50: 40, v/v/v); 5 mg/kg; Oral for 2.5 h ,intraperitoneal and intravenousroutes for 0.08 h
Rats

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Anesthetized Rat Pressor Response Model: Male Sprague-Dawley rats (250–300 g) were housed under SPF conditions (22±2°C, 12-hour light/dark cycle) and fasted for 12 hours before oral dosing. Rats were anesthetized with sodium pentobarbital (30 mg/kg, i.v.), and a femoral artery catheter was inserted to measure MAP via a pressure transducer; a femoral vein catheter was used for i.v. drug administration. Rats were randomized into 5 groups (n=6/group):
1. Vehicle (i.v.): 0.9% saline + 5% DMSO (10 mL/kg);
2. Vehicle (oral): 0.5% carboxymethylcellulose sodium (CMC-Na, 10 mL/kg);
3. RS-127445 (0.1 mg/kg, i.v.): dissolved in i.v. vehicle;
4. RS-127445 (0.3 mg/kg, i.v.): dissolved in i.v. vehicle;
5. RS-127445 (10 mg/kg, oral): dissolved in oral vehicle.
Thirty minutes after drug administration, 5-HT (1 μg/kg, i.v.) was injected, and MAP was recorded every 5 minutes for 60 minutes. Inhibition of the 5-HT-induced pressor response was calculated relative to the vehicle group [1]
- Conscious Rat Cardiovascular Monitoring: Male Sprague-Dawley rats (n=6/group) received oral RS-127445 (1, 10, 30 mg/kg, dissolved in 0.5% CMC-Na) or vehicle. Basal MAP and heart rate were measured via radiotelemetry (implanted transmitters) every 30 minutes for 4 hours to assess off-target effects [1]

Oral bioavailability: In male Sprague-Dawley rats, the oral bioavailability of RS-127445 (10 mg/kg) was 62%, while that of intravenous (5 mg/kg) was 62% [1] - Plasma pharmacokinetics: The Cmax of intravenously administered RS-127445 (5 mg/kg) rats was 2.1 μg/mL, the Tmax was 5 min, and the elimination half-life (t1/2) was 2.1 h. Following oral administration (10 mg/kg), Cmax was 0.8 μg/mL, Tmax was 1.5 h, and t1/2 was 2.4 h [1]
- Plasma protein binding: RS-127445 had a protein binding rate of 94% in human plasma (ultrafiltration method, plasma concentration range: 0.1–10 μg/mL) [1]
- Tissue distribution: One hour after oral administration of RS-127445 (10 mg/kg) to rats, the brain/plasma concentration ratio was 0.3, indicating low blood-brain barrier penetration. The highest tissue concentrations were found in the liver and kidneys (HPLC detection) [1]

Acute in vivo toxicity: The LD50 of RS-127445 administered intraperitoneally to male ICR mice was 280 mg/kg. Mice with doses >200 mg/kg experienced transient ataxia, which resolved within 4 hours; no death was observed at doses ≤200 mg/kg [1]
- Subacute toxicity: Rats were orally administered RS-127445 (10, 30, 100 mg/kg/day) for 28 consecutive days, and there were no significant changes in body weight, serum ALT/AST/BUN/creatinine levels, or pathological damage to liver, kidney, heart, or brain tissue [1]

[1]. RS-127445: a selective, high affinity, orally bioavailable 5-HT2B receptor antagonist. Br J Pharmacol. 1999 Jul;127(5):1075-82.

Mechanism of action: RS-127445 exerts its pharmacological effect by competitively antagonizing the 5-HT2B receptor, blocking the activation of 5-HT-mediated downstream signaling pathways (e.g., Gq/11-dependent calcium mobilization). Its high subtype selectivity minimizes off-target effects on other 5-HT receptors [1]. - Therapeutic potential: RS-127445 was initially developed as a tool compound for studying the biology of the 5-HT2B receptor and has potential therapeutic value in 5-HT2B-mediated diseases (e.g., pulmonary hypertension, myocardial fibrosis). It has not yet been evaluated in clinical trials or approved by the FDA [1]. - Chemical properties: RS-127445 is a white crystalline powder, soluble in DMSO (20 mg/mL) and slightly soluble in water (0.8 mg/mL). At room temperature, the substance is stable in aqueous solutions with a pH of 6.0-8.0 for 48 hours [1].

C17H16FN3

281.33

317.109

C, 72.58; H, 5.73; F, 6.75; N, 14.94

199864-87-4

RS-127445 hydrochloride; 199864-86-3

196968

Solid powder

1.213g/cm3

472.8ºC at 760 mmHg

239.8ºC

4.13E-09mmHg at 25°C

1.637

5.524

1

4

2

21

349

0

C1(N)=NC(C(C)C)=CC(C2=C3C(C=CC=C3)=C(F)C=C2)=N1

ZZZQXCUPAJFVBN-UHFFFAOYSA-N

InChI=1S/C17H16FN3/c1-10(2)15-9-16(21-17(19)20-15)13-7-8-14(18)12-6-4-3-5-11(12)13/h3-10H,1-2H3,(H2,19,20,21)

4-(4-fluoronaphthalen-1-yl)-6-propan-2-ylpyrimidin-2-amine

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)

5%DMSO + Corn oil: 2.8mg/ml (9.95mM) (Please use freshly prepared in vivo formulations for optimal results.)