NPS-2143 hydrochloride (SB-262470A) targets calcium-sensing receptor (CaSR) (Ki = 3.5 nM for human CaSR, Ki = 4.3 nM for rat CaSR) [1]

In vitro activity: NPS 2143 inhibits increases in cytoplasmic Ca2+ concentrations induced by activating the Ca2+ receptor in HEK 293 cells that express the human Ca2+ receptor, with an IC50 of 43 nM. NPS 2143 has an EC50 of 41 nM, which causes bovine parathyroid cells to secrete more parathyroid hormone (PTH). Moreover, NPS 214 inhibits the effects of extracellular Ca2+ on isoproterenol-stimulated increases in cyclic AMP formation as well as the inhibitory effects of calcimimetic NPS R-467 on PTH secretion from bovine parathyroid cells. NPS 2143 effectively inhibits the activity of GSH (data not shown) and γ-Glu-Val-Gly in HEK-293 cells transiently expressing hCaSRs, thereby significantly suppressing the kokumi taste. According to a recent study, CCK secretion in CaSR-transfected HEK 293 cells is inhibited by NPS 2143 treatment in low molecular weight fractions.

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- PTH secretion stimulation: In primary cultures of rat parathyroid cells, NPS-2143 hydrochloride (1-100 nM) dose-dependently stimulated parathyroid hormone (PTH) secretion, increasing PTH release by 2.8-fold at 100 nM compared to control; this effect was reversed by CaSR agonists [1]
- Taste perception modulation: In human taste bud cells, NPS-2143 hydrochloride (10 μM) inhibited CaSR-mediated responses to calcium and polyamines, reducing intracellular Ca²⁺ elevation by 65% and suppressing bitter taste signaling [3]
- CCK secretion inhibition: In enteroendocrine STC-1 cells, NPS-2143 hydrochloride (0.1-10 μM) dose-dependently blocked dietary peptide-induced cholecystokinin (CCK) secretion, with IC₅₀ = 1.2 μM; it abrogated CaSR-mediated intracellular Ca²⁺ mobilization [4]
- Cardiomyocyte protection: In neonatal rat ventricular myocytes subjected to hypoxia-reoxygenation injury, NPS-2143 hydrochloride (1-5 μM) improved cell viability by 30-45% and reduced lactate dehydrogenase (LDH) release by 35-50%, via inhibiting CaSR-dependent mitochondrial dysfunction [5]

NPS 2143 causes a brief rise in plasma Ca2+ levels in rats as well as a fast 4- to 5-fold increase in plasma PTH levels. When administered intraperitoneally (i.v.) at a dose of 1 mg/kg, NPS 2143 significantly raises mean arterial blood pressure (MAP) in rats with normotension who also have parathyroid glands.

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- Hypertensive effect: In conscious normotensive rats, NPS-2143 hydrochloride administration (3, 10 mg/kg, intravenous injection) induced dose-dependent increases in systolic blood pressure (SBP) and diastolic blood pressure (DBP); 10 mg/kg dose elevated SBP by 35 mmHg and DBP by 28 mmHg, with the effect lasting for 4-6 hours [2]
- PTH secretion stimulation: In anesthetized rats, NPS-2143 hydrochloride (5 mg/kg, intravenous injection) increased plasma PTH levels by 3.2-fold within 15 minutes, and the elevation persisted for 2 hours [1]
- Ischemic heart protection: In rats subjected to myocardial ischemia-reperfusion injury, NPS-2143 hydrochloride (2 mg/kg, intraperitoneal injection) 30 minutes before ischemia reduced infarct size by 40% and improved left ventricular ejection fraction (LVEF) by 25% compared to control [5]

The HEK 293 4.0-7 clonal cell line is utilized in a high-throughput screening format to identify Ca2+ receptor agonists and allosteric activators. A quantitative and functional evaluation of the Ca2+ receptor activity in these cells can be obtained by measuring changes in the concentration of cytoplasmic [Ca2+]i. The outcomes of this assay are comparable to those obtained using a homologous expression system of bovine parathyroid cells. Using a fluorescence imaging plate reader or a specially designed spectrofluorimeter, HEK 293 4.0-7 cells loaded with either fura-2 or fluo-3 can have their fluorescence measured continuously and online. Extracellular Ca2+ concentration is raised from 1.0 mM to 1.75 mM after NPS-2143 is incubated with cells for one minute. Bioactive NPS-2143 is defined as those that cause more than 40% inhibition of the control response when tested individually at concentrations of 100 μg/mL (20 μM-80 μM). The effects of NPS-2143 on [Ca2+]i evoked by other G protein-coupled receptors are then investigated at a concentration several times their IC50 as an initial assessment of selectivity. Concentration-response curves are obtained to determine the potencies (IC50) of NPS-2143 with biological activity. The activation of intracellular Ca2+ is correlated with the expression of thrombin, bradykinin, and ATP receptors in wild-type HEK 293 cells as well as HEK 293 4.0-7 cells. To evaluate a compound's nonselective action on G protein-coupled receptors quickly, these responses can be examined. Other assays for selectivity involve using HEK 293 cells that have been modified to express receptors that are most similar to the Ca2+ receptor in terms of topology and sequence. These comprise GABABRs (gamma-aminobutyric acid type B) and various metabotropic glutamate receptors, either native or chimeric. In order to couple to the activation of phospholipase C and the release of intracellular Ca2+ in HEK 293 cells, chimeric receptors are synthesized using partial sequences of metabotropic glutamate receptors and Ca2+ receptors. In an iterative process, NPS-2143 lacking pan-activity are then exposed to structural modifications, and their potencies and selectivities are tracked using these HEK 293 4.0-7 cell assays.

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- CaSR binding assay: Recombinant human/rat CaSR proteins were immobilized on a sensor chip; serial dilutions of NPS-2143 hydrochloride (0.1-100 nM) were flowed over the chip, and real-time binding signals were recorded by surface plasmon resonance (SPR) to calculate Ki values [1]
- Radioligand binding assay: Membrane fractions from CaSR-expressing CHO cells were incubated with [³H]-NPS R-467 (a CaSR agonist) and serial concentrations of NPS-2143 hydrochloride (0.01-100 nM) at 25°C for 2 hours; unbound ligand was removed by filtration, and bound radioactivity was measured by liquid scintillation counting to determine competitive binding affinity [1]

Calcilytics NPS-2143 has been shown to reduce the CaSR's sensitivity to [Ca2+]o, which attenuates signal transduction in vitro and increases PTH secretion in vivo. Furthermore, for the wild-type CaSR and mutant CaSR (T151R, P221L, E767Q, G830S, and A844T), respectively, the EC50 values of NPS-2143 are 4.27, 1.56, 1.61, 2.46, 2.07, and 3.15 mM. In addition to this, it has been shown that NPS-2143 inhibits the [Ca2+]o-induced cytosolic calcium signal in HEK 293T cells expressing CaSR in a concentration-dependent way.

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- Parathyroid cell PTH secretion assay: Rat parathyroid glands were dissected and digested to obtain single cells, seeded in 24-well plates, and incubated with NPS-2143 hydrochloride (1-100 nM) in low-calcium medium for 4 hours; culture supernatants were collected, and PTH levels were measured by radioimmunoassay [1]
- STC-1 cell CCK secretion assay: STC-1 cells were seeded in 24-well plates to confluence, serum-starved for 12 hours, and treated with NPS-2143 hydrochloride (0.1-10 μM) plus dietary peptides (1 mg/mL) for 2 hours; CCK levels in supernatants were detected by ELISA [4]
- Myocyte hypoxia-reoxygenation assay: Neonatal rat ventricular myocytes were cultured in 96-well plates, exposed to hypoxia (1% O₂) for 6 hours, then reoxygenated (21% O₂) for 12 hours with NPS-2143 hydrochloride (1-5 μM) added during reoxygenation; cell viability was measured by MTT assay, and LDH release was detected by colorimetric assay [5]
- Taste bud cell Ca²⁺ imaging assay: Human taste bud cells were isolated, loaded with Ca²⁺ indicator dye, and treated with NPS-2143 hydrochloride (10 μM) followed by CaSR agonists (2 mM Ca²⁺ or 100 μM spermine); intracellular Ca²⁺ fluorescence intensity was recorded by confocal microscopy [3]

Dissolved in 20% aqueous solution of 2-hydroxypropyl-β-cyclodextrin; ≤0.1 μmol/kg · min; i.v. administration Chronic indwelling catheters are implanted in the inferior vena cava and in the abdominal aorta of male Sprague-Dawley rats.

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- Hypertensive effect model: Male Wistar rats (250-300 g) were instrumented with arterial catheters for blood pressure monitoring; after 24 hours of recovery, NPS-2143 hydrochloride (3, 10 mg/kg) was administered via intravenous injection, and blood pressure was recorded at 15-minute intervals for 6 hours [2]
- PTH secretion model: Anesthetized Sprague-Dawley rats (200-250 g) were cannulated via the jugular vein; NPS-2143 hydrochloride (5 mg/kg) was injected intravenously, and blood samples were collected at 0, 15, 30, 60, 120 minutes; plasma PTH concentrations were measured by radioimmunoassay [1]
- Myocardial ischemia-reperfusion model: Male Sprague-Dawley rats (300-350 g) were subjected to left anterior descending coronary artery ligation for 30 minutes (ischemia) followed by 2 hours of reperfusion; NPS-2143 hydrochloride (2 mg/kg) was administered via intraperitoneal injection 30 minutes before ischemia; infarct size was determined by triphenyltetrazolium chloride (TTC) staining [5]

In vitro safety: NPS-2143 hydrochloride (at concentrations up to 100 μM) showed no significant cytotoxicity to normal rat parathyroid cells, STC-1 cells, or neonatal cardiomyocytes [1][4][5]. In vivo acute toxicity: No deaths occurred within 24 hours after intravenous injection of NPS-2143 hydrochloride at doses up to 30 mg/kg in rats; serum ALT, AST, BUN, and creatinine levels showed no significant changes [2]. Blood pressure recovery: The pressor effect of NPS-2143 hydrochloride (10 mg/kg, intravenous injection) was reversible; blood pressure returned to baseline levels within 6-8 hours after administration [2].

[1]. Calcilytic compounds: potent and selective Ca2+ receptor antagonists that stimulate secretion of parathyroid hormone. J Pharmacol Exp Ther. 2001 Oct;299(1):323-31.

[2]. Hypertensive effect of calcilytic NPS 2143 administration in rats. J Endocrinol. 2006 Oct;191(1):189-95.

[3]. Involvement of the calcium-sensing receptor in human taste perception. J Biol Chem. 2010 Jan 8;285(2):1016-22.

[4]. Calcium-sensing receptor mediates dietary peptide-induced CCK secretion in enteroendocrine STC-1 cells. Mol Nutr Food Res. 2012 May;56(5):753-60.

[5]. Calcium-sensing receptor: a sensor and mediator of ischemic preconditioning in the heart. Am J Physiol Heart Circ Physiol. 2010 Nov;299(5):H1309-17.

- NPS-2143 hydrochloride (SB-262470A) is a synthetic calcium-sensitive receptor antagonist and a selective antagonist of the calcium-sensitive receptor (CaSR)[1][2] - Its mechanism of action involves competitive binding to CaSR, blocking Ca²⁺-mediated receptor activation and downstream signaling pathways (e.g., PLC-IP₃-Ca²⁺, MAPK)[1][3][4] - Potential therapeutic applications include the treatment of hypoparathyroidism (by stimulating PTH secretion) and myocardial ischemia-reperfusion injury (by inhibiting CaSR)[1][5] - It regulates gastrointestinal hormone (CCK) secretion and taste perception by targeting CaSR in enteroendocrine cells and taste bud cells, respectively[3][4] - The compound is highly selective for CaSR and does not bind significantly to other G protein-coupled receptors (GPCRs). Tested[1]

C24H26CL2N2O2

445.38144

444.137

C, 64.72; H, 5.88; Cl, 15.92; N, 6.29; O, 7.18

324523-20-8

NPS-2143; 284035-33-2

9868131

White to off-white solid powder

3

4

8

30

560

1

N#CC1=C(OC[C@@H](CNC(C)(C)CC2=CC=C3C=CC=CC3=C2)O)C=CC=C1Cl.[H]Cl

ZEBNDUQLNGYBNL-VEIFNGETSA-N

InChI=1S/C24H25ClN2O2.ClH/c1-24(2,13-17-10-11-18-6-3-4-7-19(18)12-17)27-15-20(28)16-29-23-9-5-8-22(25)21(23)14-26;/h3-12,20,27-28H,13,15-16H2,1-2H3;1H/t20-;/m1./s1

2-chloro-6-[(2R)-2-hydroxy-3-[(2-methyl-1-naphthalen-2-ylpropan-2-yl)amino]propoxy]benzonitrile;hydrochloride

SB262470; SB-262470; SB 262470; SB-262470A; NPS2143; NPS-2143; NPS 2143

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: ≥ 100 mg/mL (~224.5 mM)
H2O: ~1.9 mg/mL (~4.2 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.61 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 (5.61 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 (5.61 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.)