human 5-HT1B Receptor (pKi = 8.85); human 5-HT1D Receptor (pKi = 8.31)
5-HT1B receptor and 5-HT1D receptor (serotonin receptor subtypes). GR 125,743 is a selective 5-HT1B/1D receptor antagonist.

For h5-HT1B, GR 125743 has a Kd of 0.61 nM[1].
Saturation binding experiments were performed with [3H]GR 125743, a selective 5-HT1B/1D ligand, to membrane preparations of COS-7 cells transiently expressing either the wt h 5-HT1B or the chimeric 5-HT1B/2A and 5-HT1B/2AΔ44 receptors, and with [3H]ketanserin to membranes of human embryonic kidney 293 cells stably expressing the wt h 5-HT2A receptor. The [3H]GR 125743 equilibrium dissociation constants of both chimera were similar to that of the wt h 5-HT1B receptor (Table 1). The maximal binding capacity of the wt h 5-HT1B receptor was 3- to 4-fold lower than that of the chimeric 5-HT1B/2A and 5-HT1B/2AΔ44 receptors and 3-fold higher than that of the wt h 5-HT2A receptor (Table 1). A series of nine 5-HT ligands was tested for inhibition of [3H]GR 125743 binding to wt h 5-HT1B, 5-HT1B/2A, and 5-HT1B/2AΔ44 receptors and compared to their binding affinities for the wt h 5-HT1D and h 5-HT2A receptors (Table 2). The chimeric 5-HT1B/2A receptor yielded a 21-fold increased binding affinity for the 5-HT2 antagonist ketanserin as compared to the wt h 5-HT1B receptor. This binding affinity is close (3-fold lower) to what is observed for the wt h 5-HT1D receptor but 87-fold lower than for the wt h 5-HT2A receptor (Table 2). The structurally related piperidine derivative ritanserin yielded an almost similar binding affinity as compared to the wt h 5-HT1B receptor, this value being 3- and 47-fold lower than for the wt h 5-HT1D and h 5-HT2A receptors, respectively. This ketanserin binding feature was lost when the last 44 amino acids of the 5-HT2A receptor-derived C-terminal portion were truncated in the chimeric 5-HT1B/2AΔ44 receptor. The binding affinities of the 5-HT antagonists GR 125743 and SB-224289 were not modified in either chimeric 5-HT receptor as compared to the parental h 5-HT1B receptor. The binding affinities of the agonists F 11356, zolmitriptan, 5-HT, and sumatriptan were slightly increased (1.6- to 6-fold) at both chimeric receptors as compared to the wt h 5-HT1B receptor. The 5-HT2 agonist DOI (1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane) was inactive at either the wt h 5-HT1B, h 5-HT1D, or chimeric 5-HT1B receptor [1].

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GR 125,743 binds to wild-type human h5-HT1B with a pKi of 8.85 and to h5-HT1D with a pKi of 8.31. For h5-HT1B, GR 125743 has a Kd of 0.61 nM. The compound demonstrates approximately 100-fold selectivity over other 5-HT receptor subtypes. Saturation binding experiments are performed with [3H]GR 125743, a selective 5-HT1B/1D ligand. In guinea-pig brain, ketanserin and ritanserin showed low affinity for [3H]GR 125,743 binding to striatal sites (Ki = 12600 and 369 nM), suggesting that 5-HT1B (rather than 5-HT1D) receptors are predominantly labelled in this tissue.

In conscious guinea pigs, GR 125743 (0.3 mg/kg; i.p.) dramatically lowers extracellular 5-HT in the frontal cortex [2].

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The role of 5-HT(1B/1D) receptors in modulating extracellular 5-hydroxytryptamine (5-HT) levels in the guinea pig was investigated with the non-selective 5-HT(1B/1D) receptor inverse agonist, methiothepin, and the selective 5-HT(1B/1D) receptor partial agonists, GR 127935 (n-[4-methoxy-3-(4-methyl-1-piperizinyl)phenyl]-2'-methyl-4'-(5-me thyl-1,2,4-oxadiazole-3-yl)[1,1'-biphenyl]-4-carboxamide) and GR 125743 (n-[4-methoxy-3-(4-methyl-1-piperizinyl)phenyl]-3-methyl-4-(4-pyri dinyl)benzamide). Extracellular 5-HT levels were measured using the technique of brain microdialysis, in the frontal cortex of the freely moving guinea-pig. Extracellular 5-HT was tetrodotoxin sensitive and calcium dependent, and increased when perfused with a high concentration of K+. In addition, extracellular 5-HT levels were lowered by the 5-HT(1B/1D) receptor agonist, sumatriptan, and the 5-HT1A receptor agonist, 8-hydroxy-2-(di-n-propylamino)tetralin, while perfusion of the selective serotonin re-uptake inhibitor, paroxetine, increased 5-HT in a concentration-dependent manner. Perfusion of methiothepin, GR 127935 and GR 125743 into the frontal cortex caused significant but transient increases of extracellular 5-HT. However, systemic administration of methiothepin, GR 127935 and GR 125743, at 0.3 mg/kg i.p., produced significant decreases in extracellular 5-HT, to minima of 27 +/- 3%, 31 +/- 12% and 27 +/- 13% of basal, respectively. The increase of extracellular 5-HT, following 5-HT(1B/1D) receptor inverse and partial agonist perfusion into the frontal cortex, was probably a consequence of attenuation of an endogenous 5-HT tone at terminal 5-HT autoreceptors. The unexpected decrease in 5-HT levels following systemic administration may be a result of additional attenuation of endogenous 5-HT tone at cell body autoreceptors in the raphe. Such an increase in local 5-HT levels could then stimulate 5-HT1A receptors to inhibit cell firing and hence decrease 5-HT levels in the terminal regions. This was confirmed when co-administration of the 5-HT1A receptor antagonist, WAY 100635, significantly attenuated the GR 127935 decrease in 5-HT[2].

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In vivo, GR 125,743 (0.3 mg/kg; i.p.) produces significant decreases in extracellular 5-HT in the frontal cortex of conscious guinea pigs. The compound has been studied for potential therapeutic applications in the treatment of migraine, anxiety, and depression. It is used for the research of Parkinson's disease and cardiovascular diseases. Specific animal model data and detailed dosing regimens are available in the primary literature.

Expression of 5-HT receptors and radioligand binding experiments [1]
COS-7 cells (5 × 106 cells) were transfected with 10 μg of either plasmid pcDNA3/h 5-HT1B, pCR3.1/5-HT1B/2A, or pCR3.1/5-HT1B/2AΔ44 by electroporation as previously described. A human embryonic kidney 293 cell line stably expressing the h 5-HT2A receptor was grown in complete Dulbecco’s modified Eagle’s medium and selected on 1.25 mg/mL geneticin as described. Binding assays were performed with either 1.0 nM [3H] N-[4-methoxy-3-(4-methylpiperazin-1-yl)phenyl]-3-methyl-4-(4-pyridil)-benzamide (GR 125743) for 5-HT1B receptor binding or 1.0 nM [3H]ketanserin for 5-HT2A receptor binding. Incubation mixtures consisted of 0.4 mL of cell membrane, 0.05 mL of radioligand, and 0.05 mL of compound for inhibition or 10 μM 5-HT to determine non-specific binding. The reactions were performed as described. Data were analyzed graphically with inhibition curves and ic50 values were derived as the concentration of the compound producing 50% inhibition of specific radioligand binding. Inhibition constants Ki were calculated according to the equation Ki = ic50/(1 + C/Kd), with C the concentration and Kd the equilibrium dissociation constant of the radioligand. The Kd values were obtained from saturation binding experiments performed as described. Membrane protein levels were estimated with a dye-binding assay using the BioRad protein assay kit and BSA as a standard.

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Two chimeric 5-hydroxytryptamine (5-HT) receptors were constructed by exchanging the C-terminal portion of the human (h) 5-HT(1B) receptor with the equivalent domain of the h 5-HT(2A) receptor (5-HT(1B/2A)) or with this domain truncated from its last 44 amino acids (5-HT(1B/2ADelta44)). The equilibrium dissociation constant of the radioligand [(3)H]GR 125743 was similar for both chimera compared to the wild-type (wt) h 5-HT(1B) receptor upon transient expression in COS-7 cells. Ketanserin binding affinity was 21-fold increased from pK(i): 5.79 (wt h 5-HT(1B) receptor) to pK(i): 7.11 at the 5-HT(1B/2A) chimeric receptor, this latter value being close to that of the wt h 5-HT(1D) receptor (pK(i): 7.62). This enhanced ketanserin binding affinity was lost when the last 44 C-terminal amino acids of the 5-HT(2A) receptor were deleted in the chimera 5-HT(1B/2ADelta44) (pK(i): 5.80). The binding affinities of the 5-HT antagonists ritanserin, GR 125743, and SB-224289 were not modified at either chimeric 5-HT receptor. The agonists F 11356, 5-HT, zolmitriptan, and sumatriptan yielded slightly increased (2- to 6-fold) binding affinities at both chimera as compared to the wt h 5-HT(1B) receptor. The present data suggest a role for the C-terminal intracellular receptor domain in modifying ketanserin/5-HT(1B) receptor interactions[1].

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5-HT1B/1D receptor binding affinity is assessed using in vitro radioligand binding assays with membrane preparations from cells expressing recombinant human 5-HT1B or 5-HT1D receptors, or from guinea-pig brain tissue. Competitive binding with [3H]GR 125743 or other selective ligands (e.g., [3H]5-carboxamidotryptamine) is measured. pKi and Kd values are calculated from saturation and displacement curves using nonlinear regression analysis (e.g., Scatchard analysis, Cheng-Prusoff equation). Saturation binding experiments are performed with [3H]GR 125743 to determine Bmax and Kd values.

Cellular activity is evaluated in cells expressing 5-HT1B or 5-HT1D receptors. Receptor-mediated signaling such as inhibition of forskolin-stimulated cAMP accumulation is measured using ELISA or HTRF-based assays. Antagonist activity is confirmed by blocking agonist (e.g., 5-HT, sumatriptan)-induced responses. Selectivity is evaluated by testing against other serotonin receptor subtypes (5-HT1A, 5-HT2A, 5-HT2C, 5-HT3, 5-HT4, 5-HT6, 5-HT7) in radioligand binding or functional assays. GR 125743 demonstrates approximately 100-fold selectivity over other 5-HT receptor subtypes.

Animal/Disease Models: Male Dunkin Hartley guinea pig (350-450 g) [2]
Doses: 0.3 mg/kg
Route of Administration: intraperitoneal (ip) injection
Experimental Results: A significant transient increase in extracellular 5-HT production.

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In vivo efficacy is studied in animal models of Parkinson's disease, cardiovascular disease, migraine, anxiety, and depression. GR 125,743 is administered via various routes including intraperitoneal (i.p.) injection. In conscious guinea pigs, GR 125743 (0.3 mg/kg; i.p.) dramatically lowers extracellular 5-HT in the frontal cortex. Microdialysis is used to measure extracellular 5-HT levels in brain regions. Behavioral assays may include tests for anxiety (elevated plus maze, open field), depression (forced swim test, tail suspension test), motor function (rotarod, locomotor activity), and pain responses. Cardiovascular parameters such as blood pressure and heart rate may be measured in relevant models. Specific protocols are detailed in the primary literature.

GR 125,743 has a molecular formula of C24H26N4O2 and a molecular weight of 402.49 g/mol. Purity: ≥98% by HPLC. CAS Number: 148547-33-5. The compound is a selective 5-HT1B/1D receptor antagonist. For h5-HT1B, GR 125743 has a Kd of 0.61 nM. Detailed pharmacokinetic parameters including half-life, oral bioavailability, volume of distribution, and plasma protein binding are available from the primary literature. The compound is soluble in DMSO and should be stored at -20°C for long-term stability.

Safety data for GR 125,743 indicate it is a research compound for laboratory use only. As a serotonin receptor antagonist, it may have central nervous system effects. Specific toxicological profiles including acute toxicity, organ toxicity, genotoxicity, and reproductive toxicity are not extensively published. Standard laboratory safety practices should be followed during handling, including the use of personal protective equipment and adequate ventilation. The compound is for research use only and not for human therapeutic applications without appropriate regulatory approval.

[1]. Induction of a high-affinity ketanserin binding site at the 5-Hydroxytryptamine(1B) receptor by modification of its carboxy-terminal intracellular portion. Characterization of human serotonin 1D and 1B receptors using [3H]-GR-125743, a nov.

[2]. The role of 5-HT1B/1D receptors in the modulation of 5-hydroxytryptamine levels in the frontal cortex of the conscious guinea pig. European Journal of Pharmacology. 1997 May 12;326(1):23-30.

In summary, we report here that the binding affinity of ketoserin to the human 5-HT1B receptor can be modulated by exchanging the C-terminal intracellular portion of the human 5-HT1B receptor with the corresponding portion of the human 5-HT2A receptor. These data suggest that the extracellular domain may play an important role in ligand binding of the 5-HT1B receptor. This concept may contribute to the development of three-dimensional models of G protein-coupled receptors. [1]

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GR 125,743 is a research tool compound for studying 5-HT1B/1D receptor biology. It is not approved for clinical use. The compound has been investigated for Parkinson's disease, cardiovascular disease, migraine, anxiety, and depression. [3H]GR 125743 has been developed as a radiolabelled derivative for receptor binding studies. In guinea-pig brain, [3H]GR 125,743 binding predominantly labels 5-HT1B receptors (rather than 5-HT1D) in striatal tissue. GR 125743 is a valuable pharmacological probe for serotonin receptor research, particularly for understanding the role of 5-HT1B/1D receptors in neurotransmitter regulation, vasoconstriction, and behavioral processes.

C25H28N4O2

416.53

416.221

C, 72.09; H, 6.78; N, 13.45; O, 7.68

148547-33-5

148547-33-5;

5311130

White to off-white solid powder

1.2±0.1 g/cm3

541.2±50.0 °C at 760 mmHg

281.1±30.1 °C

0.0±1.4 mmHg at 25°C

1.623

2.97

1

5

5

31

574

0

GNOXPYACARZYMW-UHFFFAOYSA-N

InChI=1S/C25H28N4O2/c1-18-16-20(4-6-22(18)19-8-10-26-11-9-19)25(30)27-21-5-7-24(31-3)23(17-21)29-14-12-28(2)13-15-29/h4-11,16-17H,12-15H2,1-3H3,(H,27,30)

N-[4-methoxy-3-(4-methylpiperazin-1-yl)phenyl]-3-methyl-4-pyridin-4-ylbenzamide

GR-125,743; GR125,743; GR 125743; 148547-33-5; GR 125,743; GR125743; GR-125743; N-[4-methoxy-3-(4-methylpiperazin-1-yl)phenyl]-3-methyl-4-pyridin-4-ylbenzamide; N-(4-METHOXY-3-(4-METHYLPIPERAZIN-1-YL)PHENYL)-3-METHYL-4-(PYRIDIN-4-YL)BENZAMIDE; N-(4-methoxy-3-(4-methylpiperazin-1-yl)phenyl)-3-methyl-4-pyridin-4-ylbenzamide; GR 125,743

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

DMSO : ~100 mg/mL (~240.08 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.00 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 (6.00 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 (6.00 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.)